MODIFIED STRAIN OF SALMONELLA ENTERICA TYPHI

Abstract

The present invention relates to the modification of a live attenuated strain of Salmonella enterica serovar Typhi, wherein its natural surface-exposed polysaccharide and flagellin antigens may be converted to, or augmented by, those from other strains of Salmonella, including S. enterica serovars Paratyphi, Typhimurium and Enteritidis. The present invention also relates to modified strains of Salmonella enterica serovar Typhi being suitable for use as components of a vaccine for enteric fever and salmonellosis.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. .sctn. 371 to International Application No. PCT/EP2020/052298, filed Jan. 30, 2020, which application claims priority EP Application No. 19154550.8, filed Jan. 30, 2019, the disclosures of which are incorporated herein by reference in their entirety.

FIELD OF INVENTION

[0002] The present invention relates to the modification of an attenuated strain of Salmonella enterica serovar Typhi, such that its natural surface-exposed polysaccharide and flagellin antigens are converted to, or augmented by, those from other strains of Salmonella, including S. enterica serovars Paratyphi, Typhimurium and Enteritidis. Such a modification utilises the long history of safe use of strains of S. typhi in humans as a typhoid vaccine, to deliver homologous antigens from other members of the genus Salmonella as components of vaccines for enteric fever and Salmonellosis.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0003] Accompanying this filing is a Sequence Listing entitled, "Sequence-Listing_ST25" created on Feb. 4, 2022 and having 72,739 bytes of data, machine formatted on IBM-PC, MS-Windows operating system. The sequence listing is hereby incorporated by reference in its entirety for all purposes.

BACKGROUND

[0004] Enteric fever is exclusive to humans and is caused by two serovars of Salmonella enterica: Typhi and Paratyphi, the latter comprising serovars A, B and C. Typhoid fever is estimated to have caused 21.7 million illnesses leading to 217,000 deaths in 2000, with 5.4 million cases of paratyphoid fever annually (Crump et al. 2004, Bull. World Health Organ. 82: 346-353). Typhoid and paratyphoid fevers are very similar infections of the reticuloendothelial system, intestinal lymphoid tissue and gallbladder, leading to acute febrile illnesses. Blood culture or serological tests are required to differentiate them. Outbreaks of typhoid fever are frequent in sub-Saharan Africa and Asia, with S. paratyphi A responsible for up to 50% of enteric fever cases in Asia; enteric fever is also endemic in Latin America (Crump & Mintz 2010, Clin. Infectious Dis. 50: 241-246). S. paratyphi A is the most abundant strain causing paratyphoid fever globally, with several reports showing it causing an increasing number of the total enteric fever cases (Fangtham & Wilde 2008, Int. J. Travel Med. 15: 344-350).

[0005] All licensed injected typhoid vaccines use the Vi capsular polysaccharide antigen purified from S. typhi, and are single-dose with boosting recommended every 2-3 years (Martin 2012, Curr. Opin. Infect. Dis. 25: 489-499). The main adverse event is pain at the injection site. The only live attenuated typhoid vaccine is S. typhi Ty21a (Vivotif.RTM.), developed by chemical mutagenesis of S. typhi Ty2 and administered orally in 3-4 doses, with boosting required after 5-7 years (Martin 2012, Curr. Opin. Infect. Dis. 25: 489-499). Ty21a is very safe and well tolerated. In a comparative clinical study of injected vaccine Typherix.RTM. versus Vivotif.RTM., only the latter was found to generate immune responses that mimic the natural infection (Kantele et al. 2013, Plos One 8: e60583).

[0006] The Vi antigen is not present in S. paratyphi A or B (but is expressed by S. paratyphi C), so injected Vi vaccines are ineffective against the two most prevalent S. paratyphi strains. Ty21a has been proven to confer cross-protection against S. paratyphi B in field studies (Levine et al. 2007, Clin. Infectious Dis. 45: S24-S28). However, field studies using Ty21a showed little or no cross-protection against S. paratyphi A, despite the generation of cross-reactive antibody responses (Wahid et al. 2012, Clin. & Vaccine Immunol. 19: 825-834).

[0007] To try to address the short duration of protection and lack of memory response of Vi vaccines, Vi polysaccharide has been conjugated to carrier proteins in a new generation of Vi glycoconjugate vaccines. Carrier proteins include Pseudomonas aeruginosa exotoxin, tetanus and diphtheria toxoids (Martin 2012, Curr. Opin. Infect. Dis. 25: 489-499). Injectable conjugates of O-antigens purified from S. paratyphi A have also been developed, primarily O2 conjugated to tetanus toxoid (O2-TT), to diphtheria toxoid (O2-DT) and to a detoxified mutant of the diphtheria toxin (O2-CRM.sub.197), co-administered with Vi conjugated to the same carrier protein as enteric fever vaccines targeting S. typhi and S. paratyphi A (Martin et al. 2016, Vaccine 34: 2900-2902).

[0008] The live attenuated approach to enteric fever vaccine development has significant advantages over injectable Vi vaccines: longer duration of protection, generation of immunological memory, closer immunological profile to the natural infection and elimination of needles. In addition to the licensed chemically mutagenised typhoid vaccine strain Ty21a, other specifically mutated live vaccine strains of S. typhi have been evaluated in clinical trials: CVD 906 and CVD 908 (.DELTA.aroC, .DELTA.aroD); CVD 906-htrA and CVD 908-htrA (.DELTA.aroC, .DELTA.aroD, .DELTA.htrA); CVD 909 (.DELTA.aroC, .DELTA.aroD, .DELTA.htrA and constitutive expression of Vi); M01ZHO9 (.DELTA.aroC, .DELTA.ssaV); Ty800 (.DELTA.phoP, .DELTA.phoQ); .chi.3927 (.DELTA.cya, .DELTA.crp) (Tennant & Levine 2015, Vaccine 33: C36-C41) and .chi.4073 (.DELTA.cya, .DELTA.crp, .DELTA.cdt) (Paterson & Maskell 2010, Hum. Vaccines 6: 379-384). It is reasonable to expect a degree of cross-protection from these specifically mutated S. typhi strains to S. paratyphi B as is the case for Ty21a.

[0009] Attenuated strains of S. paratyphi A have also been produced, including .DELTA.phoPQ mutants tested pre-clinically (Roland et al. 2010, Vaccine 28: 3679-3687), and CVD 1902 (.DELTA.guaBA, .DELTA.clpX) which has been evaluated in a clinical trial (Tennant & Levine 2015, Vaccine 33: C36-C41). A combination of CVD 909 and CVD 1902 is in clinical development as a vaccine targeting S. typhi and S. paratyphi A (Martin et al. 2016, Vaccine 34: 2900-2902). However, this strategy requires the clinical evaluation of S. paratyphi A, which does not have the long history of safe use of S. typhi.

[0010] Non-typhoidal Salmonella (NTS) cause gastroenteritis, with symptoms including diarrhoea and fever. The increase in cases of an invasive form of non-typhoidal Salmonella (iNTS), predominantly in Africa, is an important public health issue. The strains responsible for the vast majority of iNTS cases are S. enterica serovars Typhimurium and Enteritidis, and multidrug resistant isolates are of particular concern (MacLennan & Levine 2013, Expert Rev. Anti Infect. Ther. 11:443-446). iNTS strains cause a significantly more severe form of the disease, with prolonged symptoms and shedding of bacteria lasting for several weeks. There are currently no vaccines for NTS approved for human use.

[0011] The benefits of live attenuated vaccines include the induction of mucosal and cell-mediated immune responses, in addition to systemic antibody responses, and the duration of these responses can be longer than those from injected subunit vaccines as descried above for typhoid. Attenuated S. typhi strains have been administered to millions of people as experimental and licensed vaccines with an excellent record of safety and immunogenicity. This serovar also lacks the ability to persist in environmental reservoirs due to its exclusivity to humans, thus increasing its biosafety. Therefore, there are several reasons why it is advantageous to use live attenuated S. typhi as a vector for delivery of homologous antigens from other serovars of S. enterica, rather than attenuating the wild-type strains where the effect of the attenuating mutations may not be predictable. For example, the S. typhi vaccine candidate ZH9 carrying mutations in the genes aroC and ssaV has been shown to be safe and well tolerated in multiple clinical trials (Lyon et al. 2010, Vaccine 28: 3602-3608), whereas the same mutations introduced into S. typhimurium resulted in prolonged shedding in stools (Hindle et al. 2002, Infect. Immun. 70: 3457-3467).

[0012] The three most important surface antigens of the S. enterica serovars for the induction of protective immunity are lipopolysaccharide O-antigens, flagella (H-antigens) and Vi. The table below summarises the antigenic compositions of the principle enteric fever and iNTS strains following the Kauffmann-White-Le Minor scheme classification scheme (Grimont & Weill 2007, Antigenic formulae of the Salmonella serovars, 9.sup.th Edition).

TABLE-US-00001 S. enterica Flagella (H-antigens) serovar O-antigens Vi Phase 1 Phase 2 Typhi 9, 12 Vi d -- Paratyphi A 1, 2, 12 -- a -- Paratyphi B 1, 4, [5], 12 -- b 1.2 Paratyphi C 6, 7 Vi c 1.5 Typhimurium 1, 4, [5], 12 -- i 1.2 Enteritidis 1, 9, 12 -- g,m -- [ ] indicates antigens exceptionally found in wild-type strains.

[0013] Salmonella lipopolysaccharides consists of lipid A linked to the KDO (3-deoxy-D -manno-octulosonic acid) terminus of a conserved core region, which is then linked to a variable, repeated O-antigen trisaccharide. In S. typhi, S. paratyphi A, S. paratyphi B, S. typhimurium and S. enteritidis this repeated O-antigen is O12, a triglyceride of mannose (Man), rhamnose (Rha) and galactose (Gal). In S. paratyphi A, a branch of paratose (Par; 3,6-dideoxy-D-ribo-hexose) from the C-3 of Man confers serogroup specificity: O2 (FIG. 1). In S. paratyphi B and S. typhimurium the C-3 Man has a diglyceride of abequose (Abe; 3,6-dideoxy-D -xylo-hexose) conferring the specificity O4. S. typhi and S. enteritidis have tyvelose (Tyv; 3,6-dideoxy-D-arabino-hexose) on the C-3 Man, conferring the specificity O9. FIG. 2 shows the biosynthetic pathway resulting in either Abe, Par or Tyv and representing the O-antigen differences between the serovars. S. typhi and S. paratyphi C additionally express the Vi capsular polysaccharide antigen.

[0014] Except for the flagella produced by S. typhi Ty21a, flagellin is not a component of any current licensed vaccine for an S. enterica infection. Flagellin is an important pathogen-associated molecular pattern (PAMP) that is recognised by toll-like receptor 5 (TLR5) and is highly immunogenic, making it an important component of a live vaccine for S. enterica. The flagella filament of S. enterica is composed of approximately 20,000 flagellin (FliC or FljB) proteins with a terminal cap encoded by fliD (Haiko & Westerlund-Wikstrom 2013, Biology 2: 1242-1267). S. typhi and S. paratyphi A are generally monotypic for flagellin, expressing only FliC.

[0015] There is a particular need in the art for improved vaccines directed toward S. enterica serovars Paratyphi A, B, C, Typhimurium and Enteritidis.

SUMMARY OF INVENTION

[0016] In a first aspect, the present invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain is modified to express the lipopolysaccharide O2 O-antigens and the flagella proteins of Salmonella enterica serovar Paratyphi A.

[0017] In a second aspect, the present invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain is modified to express the lipopolysaccharide O4 O-antigens and the flagella proteins of Salmonella enterica serovar Paratyphi B and Salmonella enterica serovar Typhimurium.

[0018] In a third aspect, the present invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain is further modified to contain a functional fepE gene, such that long O-antigen chains are generated, preferably wherein the O-antigen chains are 100 repeated units of the trisaccharide backbone in length.

[0019] In a fourth aspect, the present invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain is modified to either constitutively express the gtrC gene (encoding rhamnose acetyltransferase), or alternatively, wherein said strain is modified to express the gtrC gene in trans.

[0020] In a fifth aspect, the present invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain has its native fliC gene (SEQ ID NO: 1) substituted with the fliC gene of Salmonella enterica serovar Paratyphi A (SEQ ID NO: 2), Salmonella enterica serovar Paratyphi B (SEQ ID NO: 3), Salmonella enterica serovar Paratyphi C (SEQ ID NO: 5), Salmonella enterica serovar Typhimurium (SEQ ID NO: 7) and Salmonella enterica serovar Enteritidis (SEQ ID NO: 9), such that the conferred serotype is altered from an Hd serotype to a Ha, Hb, Hc, Hi and Hg,m serotype respectively.

[0021] In a sixth aspect, the present invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein the fljBA locus, controlling expression of the fljB gene of Salmonella enterica serovar Paratyphi B (SEQ ID NO: 4), Salmonella enterica serovar Paratyphi C (SEQ ID NO: 6) and Salmonella enterica serovar Typhimurium (SEQ ID NO: 8) are inserted into the chromosome of Salmonella enterica serovar Typhi or expressed in trans.

[0022] In a seventh aspect, the present invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain expresses the atypical variants of flagellin of Salmonella enterica serovar Paratyphi A, Salmonella enterica serovar Paratyphi B, Salmonella enterica serovar Paratyphi C, Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Enteritidis.

[0023] In an eighth aspect, the present invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain has inserted a second copy of the tviA gene (SEQ ID NO: 10).

[0024] The present invention further includes a vaccine comprising one or more said modified strains for use in enhancing immunogenicity against Salmonella enterica serovar Paratyphi A, Paratyphi B, Paratyphi C, Typhimurium and Enteritidis.

DESCRIPTION OF FIGURES

[0025] FIG. 1 illustrates the structure of the S. paratyphi O-antigen chain linked to the core region (Micoli et al. 2012, Plos One 7: e47039). Par (.alpha.-D-Par) is replaced by Tyv in S. typhi and S. enteritidis, and by Abe in S. paratyphi B and S. typhimurium.

[0026] FIG. 2 illustrates the biosynthetic pathways of O-antigen-related CDP-linked sugars in Salmonella enterica serovars: Typhi and Enteritidis (terminating with CDP-tyvelose); Paratyphi A (terminating with CDP-paratose); Paratyphi B and Typhimurium (terminating with CDP-abequose). Enzymes involved at each step are indicated. Adapted from Reeves et al. 2013 Plos One 8: e69306.

[0027] FIG. 3 illustrates part of the wild-type O-antigen locus from S. typhi ZH9 that has been modified by mutation of the wild-type rfbE cistron (SEQ ID NO: 12): either by deletion of the majority of the rfbE cistron to generate S. Typhi ZH9PL2 or replacement of the rfbE cistron with spacer DNA comprising the wbdR cistron (SEQ ID NO: 13) maintaining the original reading frame in S. typhi ZH9W.

[0028] FIG. 4 shows micrographs of S. typhi ZH9 and derivative strains ZH9PL2 and ZH9W probed with A. an anti-S. typhi LPS mAb, and B. an anti-S. paratyphi A LPS mAb; left column images are phase contrast and right column images are immuno-fluorescence micrographs.

[0029] FIG. 5 shows a silver-stained polyacrylamide gel of LPS preparations from S. typhi ZH9 and derivative strains ZH9PL2 and ZH9W, indicating the short and long O-antigen chains.

[0030] FIG. 6 illustrates part of the wild-type O-antigen locus from S. typhi ZH9 modified by replacement of the rfbS (SEQ ID NO: 14) and rfbE cistrons with rfbJ (SEQ ID NO: 15).

[0031] FIG. 7 illustrates the plasmid pBAD2fepE with the P.sub.araBAD promoter (SEQ ID NO: 17) used to express the fepE cistron.

[0032] FIG. 8 shows a silver-stained polyacrylamide gel of LPS preparations from S. typhi ZH9 and ZH9(pBAD2fepE) induced by the addition of arabinose, indicating the short, long and very long O-antigen chains.

[0033] FIG. 9 shows micrographs of S. Typhi ZH9 and derivative strains ZH9PF probed with A. an anti-S. typhi flagellin antiserum, and B. an anti-S. Paratyphi A LPS flagellin antiserum; left column images are phase contrast and right column images are immuno-fluorescence micrographs.

[0034] FIG. 10 illustrates the flagella phase variation in S. enterica (from Bonifield and Hughes 2003, J. Bacteriol. 185: 567-3574).

[0035] FIG. 11 illustrates the plasmid pBRT4tviA with the P.sub.ssaG promoter (SEQ ID NO: 16) used to express the tviA cistron.

[0036] FIG. 12 shows micrographs of S. typhi ZH9 derivative strain ZH9PA probed with A. anti-S. typhi and anti-S. paratyphi A LPS mAbs, and B. anti-S. typhi and anti-S. paratyphi A flagellin antisera; left images in each pair are phase contrast and right images are immuno-fluorescence micrographs.

[0037] FIG. 13 shows endpoint titers of antibodies specific for Typhi LPS (O:9), Paratyphi LPs (O:2), Typhi flagellin (H:d) and Paratyphi flagellin (H:a) in mouse sera following a single subcutaneous immunization of mice with ZH9 alone (1.times.10.sup.8 cfu/mouse), ZH9PA alone (1.times.10.sup.8 cfu/mouse) or a combination of ZH9 and ZH9PA (0.5.times.10.sup.8 cfu +0.5.times.10.sup.8 cfu ZH9PA/mouse).

DETAILED DESCRIPTION

[0038] The following description is presented to enable any person skilled in the art to make and use the present invention. Various modifications to the disclosed embodiments will be readily apparent to those skilled in the art.

[0039] In a first aspect, the present invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain may be modified to express the lipopolysaccharide O2 O-antigens and the flagella proteins of Salmonella enterica serovar Paratyphi A.

[0040] The term `live attenuated strain` in the context of the present invention refers to the alteration of said strain to reduce its pathogenicity, rendering it harmless to the host, whilst maintaining its viability. This method is commonly used in the development of vaccines due to its ability to elicit a highly specific immune response whilst maintaining an acceptable safety profile. Development of attenuated live bacterial vaccines may involve a number of methods, examples include, but are not limited to; passing the pathogens under in vitro conditions until virulence is lost, chemical mutagenesis and genetic engineering techniques.

[0041] It is envisaged that the lipopolysaccharide O9 O-antigens of Salmonella enterica serovar Typhi may be replaced with the O2 O-antigens of Salmonella enterica serovar Paratyphi A.

[0042] The S. paratyphi A O-antigen biosynthetic pathway involves the precursor CDP-4-keto-3,6-dideoxy-D-glucose being converted to CDP-Par by the CDP-paratose synthase, RfbS. In addition to rfbS (previously called prt), S. typhi has a functional rfbE gene (previously called tyv) encoding CDP-paratose 2-epimerase, which converts CDP-Par to CDP-Tyv (FIG. 2).

[0043] The present invention involves the inactivation of the chromosomal rfbE in Salmonella enterica serovar Typhi (FIG. 3). It was found, surprisingly, that inactivation of rfbE resulted in the standard long LPS of S. typhi, known to be highly immunogenic from clinical studies, being altered to instead have the chemical composition of that of S. paratyphi A. It is a surprising finding that such a biochemical modification should result in the microorganism having the ability to produce long LPS of the S. paratyphi type. In one embodiment, the inactivation may result from the deletion of rfbE. The deletion may be either a partial or full deletion.

[0044] The rfbE inactivation prevents Tyv from being synthesised, resulting in Par being attached to Man. This alteration in the biochemical pathway introduces the Salmonella enterica serovar Paratyphi A O2 O-antigen (FIGS. 1 and 4).

[0045] In a preferred embodiment, the modification to inactivate the rfbE gene retains non-coding DNA without disrupting the expression of downstream (non-rfbE) coding sequences.

[0046] In a preferred embodiment, the deletion of the rfbE cistron is accompanied by the insertion of a non-coding spacer region intended to maintain the correct reading frame.

[0047] The spacer region may be any suitable non-coding DNA sequence which retains the correct reading frame when inserted. Preferably, the spacer region of DNA is the cistron of the Escherichia coli gene wbdR which results in the production of long LPS.

[0048] It is a surprising finding that the modifications disclosed herein result in a long LPS with the S. paratyphi A O2 O-antigen characteristic. This has benefits in vaccine production, allowing live attenuated strains of S. typhi, and/or derivatives thereof, to be produced, offering additional protection against S. paratyphi A. The vaccine therefore has benefits over conventional vaccines which protect only against S. typhi.

[0049] The term `spacer region of DNA` in the context of the present invention refers to a region of non-coding DNA located between genes. The term `cistron` refers to a section of DNA which encodes for a specific polypeptide in protein synthesis. The insertion of a spacer region of DNA may involve the transformation of an electrocompetent plasmid with a replacement cassette. See Example 1 for further details.

[0050] Where the methods herein described involve the use of a plasmid, said plasmid will ideally have an origin of replication selected from pMB1, ColEI, p15A, pSC101 and RK2. The plasmid may contain an antibiotic resistance gene selected from .beta.-lactamase (bla), kanamycin phosphotransferase (kan), tetracycline efflux protein (tetA) or chloramphenicol acetyltransferase (cat). Ideally the antibiotic resistance gene will be excised prior to or shortly after transformation into the live bacterial vector strain, for example by a mechanism such as `X-mark` (Cranenburgh & Leckenby 2012, WO2012/001352). A plasmid maintenance system may be required to prevent plasmid loss. These may include mechanisms to place a native chromosomal gene under a heterologous promoter such as the `Operator-Repressor Titration for Vaccines` (ORT-VAC; Garmory et al. 2005, Infect. Immun. 73: 2005-2011) or `oriSELECT` (Cranenburgh 2005, WO 2005/052167) systems, neither of which require an additional selectable marker gene to be present on the plasmid. Alternatively, a selectable marker gene will be used that is not an antibiotic resistance gene, such as a gene to complement a host cell mutation (Degryse 1991, Mol. Gen. Genet. 227: 49-51).

[0051] Preferably, the spacer region of DNA is the cistron of the Escherichia coli gene wbdR. Other non-functional genes of Salmonella enterica serovar Typhi of approximately the same length as the rfbE cistron may also be used for this purpose. It is preferable that the chosen spacer DNA used for this purpose will be approximately 50-2000 base pairs in length as well as lacking a terminator sequence. The use of this spacer region results in the inactivation of rfbEwithout causing any downstream effects (SEQ ID NO: 20) and effectively changing Salmonella enterica serovar Typhi LPS to Salmonella enterica serovar Paratyphi A.

[0052] The inventors have shown that deletion of rfbE whilst maintaining the original reading frame (via the use of a spacer region of DNA) is a crucial requirement of the above process.

[0053] Preferably, the resulting lipopolysaccharide O2 O-antigens of Salmonella enterica serovar Paratyphi A are at least equivalent in length to the lipopolysaccharide O9 O-antigens of Salmonella enterica serovar Typhi. It is preferable that the resulting lipopolysaccharide will be 16-35 O-antigen repeat units in length, a range which constitutes a `long` lipopolysaccharide species. A person skilled in the art will understand the desirability of the presence of O-antigen repeat units in triggering an immunogenic reaction.

[0054] It is envisaged that the present invention may also include the live attenuated strain, according to above, wherein said strain may have its native fliC gene replaced with the fliC gene of Salmonella enterica serovar Paratyphi A, such that the conferred serotype is altered from an Hd serotype to a Ha serotype, where `serotype` refers to a distinct variation within the bacterial species.

[0055] The Phase 1 flagellum of S. typhi is essential for motility and invasion, and confers the serotype Hd. The filament consists of the flagellum protein FliC, with a FliD cap. The inventors have discovered that replacing the fliC on the S. Typhi chromosome with that of S. paratyphi A results in the conversion from the Hd to the Ha serotype of functional flagella.

[0056] Chromosomal replacement may be used to achieve the above substitution. The substitution may be a full or partial replacement. In the context of a partial replacement, it is preferable that the replacement of the amino acids in positions 176-414 is carried out. The latter may involve the transformation of an electrocompetent plasmid with a replacement cassette. See Example 2 for further details. Alternatively, the substituted fliC gene may be expressed in trans from a plasmid or additional chromosomal location.

[0057] An additional embodiment of the present invention is the live attenuated strain described above wherein the strain may be further modified to contain a functional fepE gene, such that long O-antigen chains are generated, preferably wherein the O-antigen chains are 100 repeated units of the trisaccharide backbone in length.

[0058] The fepE gene encodes the length regulator of very long O-antigen chains, wherein `very long` is taken to mean more than 100 repeated units of the trisaccharide backbone. S. typhi does not possess these long O-antigen chains due to a mutation introducing a stop codon into the gene (SEQ ID NO: 21). S. typhi may be manipulated into expressing these long O-antigen chains via a number of methods; the natural promoter of fepE may be replaced with an alternative promoter, for example P.sub.araBAD, the chromosomal mutation of fepE in S. typhi may be repaired ora functional copy of fepE (SEQ ID NO: 11) may be inserted elsewhere in the S. typhi chromosome. For vaccine applications, an in vivo-induced promoter or a constitutive promoter may be utilised, examples of such promoters include P.sub.pagC, P.sub.nirB, P.sub.ssaG, P.sub.sifA, P.sub.sifB, P.sub.sseA, P.sub.sseG P.sub.sseJ, P.sub.lac, P.sub.tac, P.sub.trc and lambda P.sub.L/P.sub.R.

[0059] A `promoter` refers to a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding or non-coding sequence. A promoter may also be a regulatory DNA sequence that affects the binding of RNA polymerase at the transcription initiation site. For the purposes of defining the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence may be found a transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase.

[0060] Promoters can be constitutively active (wherein `active` means transcription is `on`), spatially restricted or inducible. As used herein `spatially restricted` refers to a promoter that is only active in a specific subset of cells or cellular compartment of a multicellular organism. A spatially restricted promoter can thus be used to activate the expression of a nucleic acid in a particular tissue or cell type of a multicellular organism.

[0061] As used herein an Inducible promoter' refers to a promoter that enables the temporal and/or spatial activation of transcription in response to external physical or environmental stimuli. Inducible promoters include those activated by the presence of specific small molecules that alleviate transcriptional repression. For example, transcription from such an inducible promoter may be regulated by a `repressor protein`. As used herein, `repressor protein` refers to a polypeptide that binds to and occupies the inducible promoter to prevent transcription initiation. When bound to the promoter, said repressor protein can prevent binding or recruitment of RNA polymerase or associated co-factors to the transcription initiation site to prevent the activation of transcription. However, upon binding its relevant small molecule, or encountering its relevant physical or environmental stimulus, the repressor protein can no longer bind to the promoter sequence, and thus transcriptional repression is relieved. Where the above examples include in vivo-induced promoters for expression of cistrons encoding enzymes involved in O-antigen biosynthesis, or for expression of alternative fliC cistrons or TviA, such promoters include but are not limited to: P.sub.pagC, P.sub.nirB P.sub.ssaG, P.sub.sifA P.sub.sifB, P.sub.sseA, P.sub.sseG and P.sub.sseJ (Dunstan et al. 1999, Infect. Immun. 67: 5133-5141; Xu et al. 2010, Infect. Immun. 78: 4828-4838; Kroger et al. 2013, Cell Host & Microbe 14: 683-695). Other promoters of use include lambda P.sub.L and P.sub.R the temperature-induced lambda repressor cl including its thermo-labile mutant repressor c1857 (Love et al. 1996, Gene 176:49-53; SEQ ID NO: 24 & 25) and promoters that are constitutive in Salmonella in the absence of the Lac! repressor such as P.sub.lac, P.sub.tac and P.sub.trc (Terpe 2006, Appl. Microbiol. Biotechnol. 72: 211-222). In some embodiments, the functional variants include those having similar or modified sequences to P.sub.pagC, P.sub.nirB, P.sub.ssaG, P.sub.sifA, P.sub.sifB, P.sub.sseA, P.sub.sseG P.sub.sseJ and lambda P.sub.L/P.sub.R, and similar or substantially identical promoter activity as the wild-type promoter from which the variant is derived, particularly with respect to its ability to induce expression in vivo. Similar modified sequences may include having at least about 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the wild-type sequence of any of P.sub.pagC, P.sub.nirB, P.sub.ssaG, P.sub.sifA, P.sub.sifB, P.sub.sseA, P.sub.sseG, P.sub.sseJ and lambda P.sub.L/P.sub.R.

[0062] Preferably, the introduction of these long O-antigen chains may be beneficial in inducing an LPS-specific immune response. There may be an additional benefit where the LPS is naturally very long such as from expression of fepE.

[0063] It is further envisaged that the live attenuated strain described above may be modified to constitutively express gtrC or to express gtrC in trans.

[0064] Particular S. enterica serovars are acetylated on the rhamnose on the O-antigen, a feature which has been demonstrated as important for O2 O-antigen specificity in S. paratyphi A. The family 2 gtr operon (SEQ ID NO: 22) encodes the rhamnose acetyltransferase GtrC in S. typhi and S. paratyphi A. To achieve a greater and more consistent level of rhamnose acetylation it may be desirable to make gtrC constitutively expressed, for example, either on a plasmid or from an additional chromosomal locus. Alternatively, the native family 2 gtr operon promoter responsible for phase variation can be replaced with a constitutive promoter or one that is conditionally expressed in vivo.

[0065] It is further envisaged that the live attenuated strain described above may be further modified to contain an additional copy of the tviA gene under the control of a phagosomally induced promoter.

[0066] The Vi capsular polysaccharide antigen contributes to the virulence of S. typhi but is naturally down-regulated upon invasion of the liver and spleen (Janis et al. 2011, Infect. Immun. 79: 2481-2488). Regulation of Vi expression is carried out by the positive transcriptional regulator TviA.

[0067] The insertion of a second copy of the tviA gene into S. typhi may induce immune responses against Vi and enhance the anti-flagellin response. The second copy may be inserted into the S. typhi in trans, either on a plasmid (FIG. 11) or integrated into the S. typhi chromosome, such that it is under the control of a phagosomally induced promoter. Examples of appropriate phagosomally induced promoters include; P.sub.pagC, P.sub.nirB, P.sub.ssaG, P.sub.sifA, P.sub.sifB, P.sub.sseA, P.sub.sseG, P.sub.sseJ.

[0068] A further embodiment of the present invention may be a vaccine comprising the live attenuated strains herein disclosed, for use in enhancing immunogenicity against S. paratyphi A and for use in the treatment or prevention of enteric fever and salmonellosis. The vaccine may contain a single live attenuated strain or combine more than one live attenuated strain, for example, the vaccine may contain ZH9 and/or one of its derivative strains; ZH9PA, ZH9PL2, ZH9W or ZH9PF. For example, combinations may include ZH9 +ZH9PL2, ZH9 +ZH9W, ZH9 +ZH9PF, preferably the combination is ZH9 +ZH9PA.

[0069] The term `immunogenicity` refers to the ability of a particular substance to provoke an immune response.

[0070] The term `vaccine` may be taken to comprise a number of additional elements in addition to the attenuated live strain herein disclosed. The attenuated live strain may be present in a composition together with any other suitable adjuvant, diluent or excipient. Examples of suitable adjuvants, diluents or excipients include, but are not limited to; disodium hydrogen phosphate, soya peptone, potassium dihydrogen phosphate, ammonium chloride, sodium chloride, magnesium sulphate, calcium chloride, sucrose, sterile saline and sterile water.

[0071] The vaccine may be administered by any appropriate route, preferably orally or intranasal routes; however the former is the preferred route of administration.

[0072] The vaccine strain or strains will preferentially be lyophilised by a process such as freeze-drying and will be stored in sachets for later rehydration and oral administration to young children. Alternatively, they may be dispensed into enterically coated capsules for oral administration to older children and adults. For the encapsulated formulation, the lyophilised Salmonella will ideally be mixed with a bile-adsorbing resin such as cholestyramine to enhance survival when released from the capsule into the small intestine (Edwards and Slater 2009, Vaccine 27: 3897-3903).

[0073] The skilled person will appreciated that the vaccine may contain the aforementioned live attenuated strains (e.g. ZH9 and ZH9PA) of Salmonella entertica serovar Typhi ata density of 10.sup.8, 10.sup.9, .sub.1010, 10.sup.11 or 10.sup.12 colony-forming units per dose. The dosing regime may involve a single dose or multiple doses, ideally the vaccine will be administered in 1-3 doses.

[0074] In a second aspect, the present invention provides for a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain is modified to express the lipopolysaccharide O4 O-antigens and the flagella proteins of Salmonella enterica serovar Paratyphi B and Salmonella enterica serovar Typhimurium.

[0075] To achieve the above modification, the gene rfbJ (previously called abe), encoding CDP-abequose synthase from S. paratyphi B or S. typhimurium, can be inserted to replace rfbS with or without the simultaneous replacement of rfbE (FIG. 6), as RfbJ is at the equivalent step in the S. typhi/Paratyphi A biosynthetic pathway as CDP-paratose synthase and enables the conversion of CDP-4-keto-3,6-dideoxy-D-glucose to CDP-Abequose (FIG. 2). This would result in the O-antigen repeats containing Abe instead of Tyv, giving the O4 specificity of S. paratyphi B and S. typhimurium. Preferably, rfbS is inactivated via a mutation resulting in a partial or total deletion of the cistron, for example, using Xer-cise wherein a lambda-Red-mediated recombineering approach is used to inactivate the rfbS cistron, followed by Xer recombination to remove the selectable marker gene.

[0076] Alternatively to the replacement method described above, and rfbJ may be expressed in trans, either from a plasmid or alternative chromosomal locus, leading to a mixture of O4 and O9 O-antigens, designed to induce antibody responses to S. typhi, S. paratyphi B and S. typhimurium.

[0077] The invention further intends the live attenuated strain, according to the second aspect of the present invention, may have its native fliC gene replaced with the fliC gene of Salmonella enterica serovar Paratyphi B and/or Salmonalla enterica serovar Typhimurium, such that the conferred serotype is altered from an Hd serotype to a Hb and Hi serotype respectively.

[0078] It is further envisaged that the live attenuated strain, according to the second aspect of the present invention, may have the flijBA locus of Salmonella enterica serovar Paratyphi B and Salmonella enterica serovar Typhimurium inserted into the chromosome of Salmonella enterica serovar Typhi or expressed in trans.

[0079] Several serovars of S. enterica (including S. paratyphi B and C, and S. Typhimurium) have an additional antigenically distinct flagellin gene fljB, which is subject to phase variation such that flagella composed of either FliC or FljB is produced (FIG. 10). Co-transcribed with fljB is fljA, a flagellum-specific sigma factor that represses the fliC gene (Bonifield and Hughes 2003, J. Bacteriol. 185: 567-3574). The fljBA promoter is flanked by the Hin recombinase recognition sites hixL and hixR. Together with enhancer proteins Fis and HU, Hin mediates a reversible DNA inversion between the hix sites such that in one orientation the fljBA promoter transcribes the fljBA operon (SEQ ID NO: 23) producing FljB flagellin and repressing fliC expression via FIjA, thus generating flagella filaments composed of FljB. In the opposite orientation there is no expression of the fljBA operon, enabling the production of flagellin consisting of fliC. S. typhi does not express Phase 2 flagella due to deletion of fljB and hin, so has flagella comprised of FliC only (McClelland et al. 2004, Nature Genetics 36: 1268-1274).

[0080] It is envisaged that the fljBA locus of S. paratyphi B, S. paratyphi C or S. typhimurium may be inserted into the chromosome of S. typhi or expressed in trans from a plasmid, thus introducing the phase-variable flagella phenotype of the desired serovar. Alternatively, one or both of the hix sites flanking the native promoter of the fljBA operon, or the hin recombinase gene, may be mutated to prevent DNA inversion, leading to constitutive expression such that flagella filaments are comprised of only FljB. The latter approach may be coupled with the pre-described modification of the S. typhi fliC.

[0081] It is further envisaged that the live attenuated strain, according to the second aspect of the present invention, may be further modified to include the additional modifications previously described regarding fepE, gtrC and tviA expression.

[0082] A further intended application of the present invention is a vaccine comprising the live attenuated strain, according to the second aspect of the present invention, for use in enhancing immunogenicity against Salmonella enterica serovar Paratyphi B and/or Salmonella enterica serovar Typhimurium.

[0083] In a third aspect, the present invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain is further modified to contain a functional fepE gene, such that long O-antigen chains are generated, preferably wherein the O-antigen chains are 100 repeated units of the trisaccharide backbone in length. The method by which this effect may be achieved, and further details regarding this aspect of the invention, have been previously outlined on pages 12 and 13 of the present application.

[0084] In a fourth aspect, the present invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain is modified to either constitutively express the gtrC gene, or alternatively, wherein said strain is modified to express the gtrC gene in trans. The method by which this effect may be achieved, and further details regarding this aspect of the invention, have been previously outlined on page 14 and 15 of the present application.

[0085] In a fifth aspect, the present invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain has its native fliC gene substituted with the fliC gene of Salmonella enterica serovar Paratyphi A, Salmonella enterica serovar Paratyphi B, Salmonella enterica serovar Paratyphi C, Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Enteritidis, such that the conferred serotype is altered from an Hd serotype to a Ha, Hb, Hc, Hi and Hg,m serotype respectively. The method by which this effect may be achieved, and further details regarding this aspect of the invention, have been previously outlined on page 12 and 17 of the present application.

[0086] In a sixth aspect, the present invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein the fljBA locus of Salmonella enterica serovar Paratyphi B, Salmonella enterica serovar Paratyphi C and Salmonella enterica serovar Typhimurium are inserted into the chromosome of Salmonella enterica serovar Typhi or expressed in trans. The method by which this effect may be achieved, and further details regarding this aspect of the invention, have been previously outlined on pages 17 and 18 of the present application.

[0087] In a seventh aspect, the present invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain expresses the atypical variants of flagellin of Salmonella enterica serovar Paratyphi A, Salmonella enterica serovar Paratyphi B, Salmonella enterica serovar Paratyphi C, Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Enteritidis.

[0088] S. typhi isolates are subject to phase variation: one such isolate expresses a variant of FljB called Hz66 from a linear plasmid called pBSSB1 (Baker et al. 2007, Plos Pathogens 3: e59), another flagella variant is Hj which has a 261 bp in-frame deletion of the central region of the Hd fliC gene (Frankel et al. 1989, EMBO J. 8: 3149-3152).

[0089] For Hz66 this can be achieved by the inclusion of the pBSSB1 plasmid in the S. typhi-derived vaccine strain. Alternatively, FljBz.sup.66 or Hj may be expressed from a chromosomal location which may be the location of the deleted fljB gene, the native chromosomal location of fliC (thereby replacing it with a variant), or expressed on a plasmid from its native promoter or from a phagosomally induced promoter such as P.sub.ssaG. The amino acid sequences of the Hz66 and Hj are described in Schreiber et al. 2015, Nature 5: 7947.

[0090] In an eighth aspect, the present invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain has inserted a second copy of the tviA gene. The method by which this effect may be achieved, and further details regarding this aspect of the invention, have been previously outlined on page 15 of the present application.

[0091] It is envisaged that the live attenuated strains herein disclosed may be administered in isolation or in combination (e.g. ZH9 and ZH9PA), in the form of a vaccine, to give the subject a broad protection against a variety of S. enterica serovars, specifically, Salmonella enterica serovar Paratyphi A and B, and Salmonella enterica serovar Typhimurium.

[0092] The table lists S. typhi ZH9 and its derivative strains altered for LPS and flagellin:

TABLE-US-00002 Strain name O-antigen H-antigen ZH9 O9 Hd ZH9PL2 O2 (short) Hd ZH9W O2 Hd ZH9PF O9 Ha ZH9PA O2 Ha

[0093] The invention will now be illustrated in the following examples with reference to the accompanying drawings.

EXAMPLE 1

[0094] To construct S. typhi ZH9 expressing S. paratyphi A LPS, the rfbE gene was deleted in two different ways. In one method of deletion, a spacer cistron wbdR was synthesised flanked with .about.700 bp of DNA homologous to rfbE upstream gene rfbS and downstream gene rfbX to create a deletion cassette. A Notl restriction site at the 3' end of the spacer cistron was included to clone a dif-flanked antibiotic resistance marker gene cat gene amplified with primers designed with Notl restriction site. The cat gene was amplified from pBRT1N plasmid synthetically generated with 5Notldifcat and 3Notldifcat primers designed with dif sequences. E. coli TOP10 cells were used for cloning operations to generate the pUCpW_difCAT plasmid. Chromosomal replacement of the rfbE gene with the spacer gene was carried out as described in Bloor and Cranenburgh 2006 (Appl. Environ. Microbiol. 72: 2520-2525). Briefly, S. typhi ZH9 was first transformed with a pLGBEK plasmid coding for .lamda. Red gene functions for integration of linear DNA. Electrocompetent ZH9(pLGBEK) was then transformed with the deletion cassette linearised using Sall and Sacl restriction enzymes (SEQ ID NO: 26), and transformants were selected on LB-aro (LB medium containing aromatic amino acids and precursors of aromatic amino acid biosynthesis) agar plates supplemented with 20 .mu.g/ml chloramphenicol. Single colonies were isolated and cultured overnight in LB-aro broth in the absence of antibiotics. Xer recombination deleted the cat gene to generate chloramphenicol-sensitive colonies of ZH9W.

[0095] In the other method of deletion, the dif-flanked antibiotic resistance gene cat was amplified with the primers rfbEdelF and rfbEdelR designed with homologous sequences to rfbS and rfbX genes respectively. The difCAT cassette was amplified from the synthetic pMKtetORTDAP plasmid to generate the PL deletion cassette (SEQ ID NO: 27). Electrocompetent ZH9(pLGBEK) were then transformed with the amplified DNA sequence and transformants were selected on LB-aro agar plates supplemented with 20 .mu.g/ml chloramphenicol. Single colonies were isolated and cultured overnight in LB-aro broth in the absence of antibiotics. Xer recombination deleted the cat gene to generate chloramphenicol-sensitive colonies of ZH9PL2.

EXAMPLE 2

[0096] To construct S. typhi ZH9 expressing S. paratyphi A flagellin, the native fliC gene was replaced with the S. paratyphi A fliC. The replacement cassette was synthesised with S. paratyphi A fliC flanked with .about.700 bp of DNA homologous to the gene fliD upstream of fliC, and a pseudogene downstream of fliC. A Notl restriction site at the 3' end of S. paratyphi A fliC was included to clone a dif-flanked antibiotic resistance marker gene cat gene amplified with primers designed with Notl restriction site. The cat gene was amplified from pBRT1 N plasmid synthetically generated with 5Notldifcat and 3Notldifcat primers designed with dif sequences. TOP10 E. coli cells were used for cloning operations to generate pUCpF_difCAT plasmid. Chromosomal replacement of S. typhi FliC gene with S. paratyphi A FliC was carried out as described in Example 1: electrocompetent ZH9(pLGBEK) was transformed with the pUCpF_difCAT replacement cassette excised using Sall and Sacl restriction enzymes (SEQ ID NO: 28), and transformants were selected on LB-aro agar plates supplemented with 20 .mu.g/ml chloramphenicol. Single colonies were isolated and cultured overnight in LB-aro mix broth in the absence of antibiotic. Xer recombination resulted in the deletion of the cat gene to generate chloramphenicol-sensitive colonies of ZH9PF.

[0097] To construct S. typhi ZH9 expressing both S. paratyphi A LPS and Flagellin, electrocompetent ZH9PF (pLGBEK) was then transformed with the replacement cassette generated from the pUCpW_difCAT cassette excised using Sall and Sacl restriction enzymes, and transformants were selected on LB-aro agar plates supplemented with 20 .mu.g/ml chloramphenicol. Single colonies were isolated and cultured overnight in LB-aro broth in the absence of antibiotic. Xer recombination resulted in the deletion of the cat gene to generate chloramphenicol-sensitive colonies of ZH9PA.

EXAMPLE 3

[0098] To construct the medium copy-number expression plasmid pBRT4tviA (FIG. 11; SEQ ID NO: 29), primers 5tviANdel and 3tviASall were used to amplify the tviA gene of S. typhi ZH9 from chromosomal DNA. The PCR product and expression plasmid pBRT4c were digested using Ndel and Sall and ligated to generate the precursor plasmid pBRT4tviAc. An E. coli pepA mutant strain was used for cloning operations. The pBRT4tviAc plasmid was transformed into S. typhi ZH9, and transformants were selected on LB-aro agar plates supplemented with 20 .mu.g/ml chloramphenicol. Single colonies were isolated and cultured overnight in LB-aro mix broth in the absence of antibiotic. Xer recombination resulted in the deletion of the cat gene to generate chloramphenicol-sensitive colonies of ZH9(pBRT4tviA). pBRT4tviA has tviA under the control of the phagosome-induced SPI-2 promoter P.sub.ssaG, which is not active in nutrient broths such as LB or TB. PCN (Phosphate Carbon Nitrogen minimal medium) with a low concentration of inorganic phosphate was used to induce P.sub.ssaG.

EXAMPLE 4

[0099] To construct the medium copy number expression plasmid pBAD2fepE (SEQ ID NO: 30), primers fepE5_pBAD and fepE3_pBAD_pSC were used to amplify the fepE gene of S. typhimurium WT05 (aroC, ssaV) from chromosomal DNA. The PCR product and expression plasmid pBAD2 were digested using Ndel and Sall and ligated to generate the plasmid pBAD2fepE. pBAD2fepE has fepE under the control of arabinose promoter, which is not active in nutrient broths such as LB or TB and require the addition of arabinose at 0.02% to induce the expression of the length regulator of very long (VL) 0 antigen chains. E. coli NEB5-alpha was used for cloning operations. The pBAD2fepE plasmid was transformed into S. typhi ZH9, and transformants were selected on LB-aro agar plates supplemented with 50 .mu.g/ml kanamycin. Single colonies of ZH9 (pBAD2fepE) were isolated and cultured overnight in LB-aro broth supplemented with 50 .mu.g/ml kanamycin and 1:1000 of 20% arabinose to induce expression of LPS with very long (VL) O-antigen chains.

EXAMPLE 5

[0100] For immunofluorescence microscopy, S. typhi ZH9 and its derivative strains ZH9W, ZH9PL, ZH9PF and ZH9PA were cultured for 18 hours in LB-aro broth at 37.degree. C. and 200 r.p.m. A volume of each culture equivalent to an optical density of A600 =1 was collected and washed in PBS. Pellets were resuspended in 10 .mu.l of PBS with 1 pl of primary antibody and incubated for 10 minutes at ambient temperature. LPS analysis was carried out by staining ZH9, ZH9W, ZH9PL and ZH9PA with one of the following primary antibodies: anti-S. typhi LPS monoclonal antibody B348M (Genetex), anti-S. paratyphi A LPS monoclonal antibody (Bio-rad), O:9 antiserum (SSI) and O:2 antiserum (SSI). Flagellin analysis was carried out by staining ZH9, ZH9PF and ZH9PA with the following primary antibodies: H:d antiserum (SSI) and H:a antiserum (SSI). Bacterial cells primary stained were then washed in PBS and pellets were resuspended in 10 .mu.l of PBS with 1 .mu.l of secondary antibody conjugated to Dylight 488 fluorochrome. After 10 minutes incubation at room temperature, cells were washed in PBS and a small volume were applied on a microscope slide to be visualised using a fluorescent microscope (Zeiss Axiophot) with attached Zeiss Axiocam camera. Fluorescence imaging demonstrated the conversion of the O9 to the O2 serotype of LPS in ZH9W and ZH9PL (FIG. 4), and the conversion of the Hd flagellin serotype to Ha in ZH9PF (FIG. 9), with both modifications introduced into ZH9PA (FIG. 12).

[0101] To analyse LPS O-antigen length, ZH9 and ZH9(pBAD2fepE) pre-cultures were used to inoculate LB-aro broth and grown at 37.degree. C. and 200 r.p.m., with ZH9(pBAD2fepE) supplemented with 50 .mu.g/ml kanamycin and induced by adding 0.02% arabinose. When exponential phase was reached, 4 mL of each culture was lysed to prepare LPS using an LPS extraction kit (Intron Biotechnology). LPS samples were run on an SDS-PAGE gel and silver-stained. FIG. 5 shows that the replacement of rfbE gene by the spacer gene wbdR is required to express LPS with long O-antigen chains. ZH9(pBAD2fepE) expressed LPS with the very long O-antigen chains not seen in ZH9 (FIG. 8).

EXAMPLE 6

[0102] To assess the immunogenicity of the ZH9PA strain and to confirm retained immunogenicity of both ZH9 and ZH9PA strains when co-administered, an immunogenicity study was conducted in mice. Balb/c animals were immunized via a single subcutaneous immunization with ZH9 alone (1.times.10.sup.8 cfu/ mouse), ZH9PA alone (1.times.10.sup.8cfu/mouse) or combination of the two ZH9+ZH9PA (0.5.times.10.sup.8 cfu ZH9 +0.5.times.10.sup.8 cfu ZH9PA/mouse). Pre-immune serum samples were collected prior to immunization and terminal serum samples were collected 35 days after immunization. All samples were centrifuged for serum isolation and serum stored at -80.degree. C.

[0103] The sera were used to run in house standardized ELISA assays aimed at assessing the titers specific against Salmonella typhi LPS (O:9) and flagellin (H:d), S. paratyphi A LPS (O:2) and flagellin (H:a). Briefly, half-area ELISA plates (Corning) were coated with the following specific antigens (all provided by The Native Antigen Company), diluted in 50 mM carbonate/bicarbonate buffer (pH 9.6):

TABLE-US-00003 Antigen Coating dilution/concentration S. Typhi LPS 1:500 S. Paratyphi SLAV LPS 1:500 S. Typhi flagellin 0.2 .mu.g/mL S. Paratyphi SLAV flagellin 0.5 .mu.g/mL

[0104] Plates were incubated at 4.degree. C. overnight (.about.16h). The day after, plates were washed with PBS+0.05% Tween-20 before blocking with Pierce.TM. Protein-Free (PBS) blocking buffer for 1 hour at 37.degree. C. After washing plates as before, mouse sera from the oral immunogenicity study were added for 1 additional hour at 37.degree. C. A standard curve was also generated for each antigen by using serovar-specific reagents as follows:

TABLE-US-00004 Reagent Working dilution Monoclonal antibody anti-S. Typhi LPS 1:1000 Monoclonal antibody anti-S. Paratyphi A 1:250 LPS Rabbit antiserum S. Typhi flagellin 1:50 Rabbit antiserum S. Paratyphi A flagellin 1:200

[0105] Reacting sera and standard curve for S. typhi and Paratyphi A LPS were detected using a secondary goat anti-mouse antibody conjugated directly to horse radish peroxidase (HRP), whilst standard curve for S. typhi and Paratyphi A flagellin with HRP-conjugated goat anti-rabbit antibody. After washing plates as above, secondary antibodies were added at 1:2000 dilution in Pierce.TM. Protein-Free (PBS) blocking buffer and incubated at room temperature for 1 hour. Positive sera were revealed using 3,3',5,5'-Tetramethylbenzidine (TMB) substrate and measured using a spectrophotometer at 450nM. Absorbance (OD) was plotted using a 4 parametric logistic curve of the positive control; then end point titers were determined as logarithm of the dilution value at which the serial dilution curve of each mouse serum met OD equal 1.

[0106] FIG. 13 shows that the endpoint titers of antibodies specific for Typhi LPS (O:9), Paratyphi LPS (O:2), Typhi flagellin (H:d) and Paratyphi A flagellin (H:a) display no significant differences following immunization with ZH9/ZH9PA alone and the combination of the two. Accordingly, this demonstrates that when the strains are co-administered that immunogenicity is retained.

[0107] Sequences used throughout the specification and forming part of the description:

TABLE-US-00005 (S. Typhi fliC cistron) SEQ ID NO: 1 atggcacaagtcattaatacaaacagcctgtcgctgttgacccagaataacctgaacaaatcccagtccgca ctgggcactgctatcgagcgtttgtcttccggtctgcgtatcaacagcgcgaaagacgatgcggcaggacag gcgattgctaaccgttttaccgcgaacatcaaaggtctgactcaggcttcccgtaacgctaacgacggtatctc cattgcgcagaccactgaaggcgcgctgaacgaaatcaacaacaacctgcagcgtgtgcgtgaactggcg gttcagtctgcgaatggtactaactcccagtctgacctcgactccatccaggctgaaatcacccagcgcctga acgaaatcgaccgtgtatccggccagactcagttcaacggcgtgaaagtcctggcgcaggacaacaccct gaccatccaggttggtgccaacgacggtgaaactatcgatattgatttaaaagaaatcagctctaaaacactg ggacttgataagcttaatgtccaagatgcctacaccccgaaagaaactgctgtaaccgttgataaaactacct ataaaaatggtacagatcctattacagcccagagcaatactgatatccaaactgcaattggcggtggtgcaa cgggggttactggggctgatatcaaatttaaagatggtcaatactatttagatgttaaaggcggtgcttctgct- gg tgtttataaagccacttatgatgaaactacaaagaaagttaatattgatacgactgataaaactccgttggcaa ctgcggaagctacagctattcggggaacggccactataacccacaaccaaattgctgaagtaacaaaaga gggtgttgatacgaccacagttgcggctcaacttgctgcagcaggggttactggcgccgataaggacaatac tagccttgtaaaactatcgtttgaggataaaaacggtaaggttattgatggtggctatgcagtgaaaatgggcg acgatttctatgccgctacatatgatgagaaaacaggtgcaattactgctaaaaccactacttatacagatggt actggcgttgctcaaactggagctgtgaaatttggtggcgcaaatggtaaatctgaagttgttactgctaccga- t ggtaagacttacttagcaagcgaccttgacaaacataacttcagaacaggcggtgagcttaaagaggttaat acagataagactgaaaacccactgcagaaaattgatgctgccttggcacaggttgatacacttcgttctgacct gggtgcggttcagaaccgtttcaactccgctatcaccaacctgggcaataccgtaaataacctgtcttctgccc gtagccgtatcgaagattccgactacgcaaccgaagtctccaacatgtctcgcgcgcagattctgcagcagg ccggtacctccgttctggcgcaggcgaaccaggttccgcaaaacgtcctctctttactgcgttaa (S. Paratyphi A fliC cistron) SEQ ID NO: 2 atggcacaagtcattaatacaaacagcctgtcgctgttgacccagaataacctgaacaaatcccagtccgct ctgggcaccgctatcgagcgtctgtcttccggtctgcgtatcaacagcgcgaaagacgatgcggcaggtcag gcaattgctaaccgtttcaccgcgaacatcaaaggtctgactcaggcttcccgtaacgctaacgacggtatctc cattgcgcagaccactgaaggcgcgctgaacgaaatcaacaacaacctgcagcgtgtgcgtgaactggcg gttcagtctgctaacagcaccaactcccagtctgacctcgactccatccaggctgaaatcacccagcgcctga acgaaatcgaccgtgtatccggtcagactcagttcaacggcgtgaaagtcctggcgcaggacaacaccctg accatccaggttggtgccaacaacggtgaaaccattgatatcgatctgaaacagatcaactctcagaccctg ggtctggatacgctgaatgtgcagaaaaaatatgatgtgaagagcgaagcggtcacgccttcggctacatta agcactactgcacttgatggtgctggcctcaaaaccggaaccggttctacaactgatactggttcaattaagga tggtaaggtttactataacagcacctctaaaaattattatgttgaagtagaatttaccgatgcgaccgatcaaa- c caacaaaggcggattctataaagttaatgttgctgatgatggtgcagtcacaatgactgcggctaccaccaaa gaggctacaactcctacaggtattactgaagttactcaagtccaaaaacctgtggctgctccagctgctatcca ggctcagttgactgctgcccatgtgaccggcgctgatactgctgaaatggttaagatgtcttatacggataaaa acggtaagactattgatggcggtttcggtgttaaagttggggctgatatttatgctgcaacaaaaaataaagat ggatcgttcagcattaacaccactgaatataccgataaagacggcaacactaaaactgcactaaaccaact gggtggcgcagacggtaaaactgaagttgtttctatcgacggtaaaacctacaatgccagcaaagccgctg gtcacaactttaaagcacagccagagctggctgaagcggctgctgcaaccaccgaaaacccgctggctaa aattgatgccgcgctggcgcaggttgatgcgctgcgttctgacttgggtgcggttcagaaccgtttcaactccg- c tatcaccaacctgggcaataccgtaaataacctgtcttctgcccgtagccgtatcgaagattccgactacgcga ccgaagtttccaacatgtctcgcgcgcagatcctgcagcaggccggtacctccgttctggcgcaggcgaacc aggttccgcaaaacgtcctctctttactgcgttaa (S. Paratyphi B fliC cistron) SEQ ID NO: 3 atggcacaagtcattaatacaaacagcctgtcgctgttgacccagaataacctgaacaaatcccagtccgct ctgggcaccgctatcgagcgtctgtcttctggtctgcgtatcaacagcgcgaaagacgatgcggcaggtcag gcgattgctaaccgttttaccgcgaacatcaaaggtctgactcaggcttcccgtaacgctaacgacggtatctc cattgcgcagaccactgaaggcgcgctgaacgaaatcaacaacaacctgcagcgtgtgcgtgaactggcg gttcagtctgctaacagcaccaactcccagtctgacctcgactccatccaggctgaaatcacccagcgcctga acgaaatcgaccgtgtatccggccagactcagttcaacggcgtgaaagtcctggcgcaggacaacaccct gaccatccaggttggcgcgaacgacggtgaaactatcgatatcgatctgaagcagatcaactctcagaccct gggtctggatactttaagtgtacaggatgcctatacgccaaaaggtaccgctgttaccagagatgttaccacct ataaaaatggtggtactactcttacagcacctaacgcagcagcaattgataccgctttaggtacgactggtgc ggcgggtactgcggctgtgaaatttaaagacggtaactacttcgttgaggtgaccggtacaactaaagatggt ctgtatgaagcgacagttgatgcagctggcgcggtgacaatgaccgcaaataaagcaacagtaactgggg ctagtacagttactgaaaaccaaattgtagacgctgttacaccgacgccagttgatacagtcgcagcagctac tgcattgaccaatgcaggtgtgacaggtgcgacaggtaataccagcttggttaaaatgtcatttgaagataaa aatggcaaagttactgatgcgggttacgcgcttaaagttggaaatgattattatgccgctgattacgatgaaaa aactggtgagataaaagctaaaactgtaaattatactgacgctactggtgcgacaaaaaccggtgctgtgaa atttggcggtgcgaatggtaaaactgaagttgtgaccaccgttgatggtaatacttatcaggctagtgatgtaa- a agggcataatttccagagtggtggcgctttaagcgaggctgtaaccactaaaactgaaaacccgctggctaa aattgatgccgcgctggcgcaagttgatgcgctgcgttctgacttgggtgcggttcagaaccgtttcaactccg- c tatcaccaacctgggcaataccgtaaacaacctgtctgaagcccgtagccgtatcgaagattccgactacgc gaccgaagtctccaacatgtcccgcgcgcagattctgcagcaggccggtacctccgttctggcgcaggcga accaggttccgcaaaacgtcctctctttactgcgttaa (S. Paratyphi B fljB cistron) SEQ ID NO: 4 atggcacaagtaatcaacactaacagtctgtcgctgctgacccagaataacctgaacaaatcccagtccgc actgggcaccgctatcgagcgtctgtcttctggtctgcgtatcaacagcgcgaaagacgatgcggcaggtca ggcaattgctaaccgtttcaccgcgaacatcaaaggcctgactcaggcttcccgtaacgctaacgacggtatc tccattgcgcagaccactgaaggcgcgctgaacgaaatcaacaacaacctgcagcgtgtgcgtgaactgg cggttcagtctgctaacagcactaactcccagtctgacctcgactctatccaggctgaaattacccagcgtctg aacgaaatcgaccgtgtatccggccagactcagttcaacggcgtgaaagtcctggcgcaggacaacaccc tgaccatccaggttggtgccaacgacggtgaaactatcgatatcgatctgaagcagatcaactctcagaccct gggtctggactcactgaacgtgcagaaagcgtatgatgtgaaagatacagcagtaacaacgaaagcttatg ccaataatggtactacactggatgtatcgggtcttgatgatgcagctattaaagcggctacggggggtacgaat ggtacggcttctgtaaccggtggtgcggttaaatttgacgcagataataacaagtactttgttactattggtgg- ctt tactggtgctgatgccgccaaaaatggcgattatgaagttaacgttgctactgacggtacagtaacccttgcgg ctggcgcaactaaaaccacaatgcctgctggtgcgacaactaaaacagaagtacaggagttaaaagatac accggcagttgtttcagcagatgctaaaaatgccttaattgctggcggcgttgacgctaccgatgctaatggcg ctgagttggtcaaaatgtcttataccgataaaaatggtaagacaattgaaggcggttatgcgcttaaagctggc gataagtattacgccgcagattacgatgaagcgacaggagcaattaaagctaaaaccacaagttatactgct gctgacggcactaccaaaacagcagctaaccaactgggtggcgtagacggtaaaaccgaagtcgttactat cgacggtaaaacctacaatgccagcaaagccgctggtcatgatttcaaagcacaaccagagctggcggaa gcagccgctaaaaccaccgaaaacccgctgcagaaaattgatgccgcgctggcgcaggtggatgcgctg cgctctgatctgggtgcggtacaaaaccgtttcaactccgctatcaccaacctgggcaataccgtaaacaacc tgtctgaagcgcgtagccgtatcgaagattccgactacgcgaccgaagtttccaacatgtctcgcgcgcagat tctgcagcaggccggtacttccgttctggcgcaggctaaccaggtcccgcagaacgtgctgtctctgttacgtt- a a (S. Paratyphi C fliC cistron) SEQ ID NO: 5 atggcacaagtcattaatacaaacagcctgtcgctgttgacccagaataacctgaacaaatcccagtctgctc tgggtaccgctatcgagcgtctgtcttccggtctgcgtatcaacagcgcgaaagacgatgcggcaggtcagg cgattgctaaccgtttcaccgcgaacatcaaaggtctgactcaggcttcccgtaacgctaacgacggtatctcc attgcgcagaccactgaaggcgcgctgaacgaaatcaacaacaacctgcagcgtgtgcgtgaactggcgg ttcagtctgctaacagcaccaactcccagtctgacctcgactccatccaggctgaaatcacccagcgtctgaa cgaaatcgaccgtgtatccggtcagactcagttcaacggcgtgaaagtcctggcgcaggacaacactctga ccatccaggttggtgccaacgacggtgaaactatcgatatcgatctgaagcagatcaactctcagaccctgg gcctagatacgctgaatgtgcagaaaaaatatgatgtgagcgatactgctgtagctgcttcctattccgactcg aaacagaatattgctgttcctgataaaacagctattactgcaaaaattggtgcagcaaccagtggtggtgctgg tataaaagcagatattagctttaaagatggcaagtattacgcgactgtcagtggatacgatgatgccgcagat acagataaaaatggaacctatgaagtcactgttgccgcagatacaggagcagttacttttgcgactacacca acagtggttgacttaccaactgatgcaaaagcagtttcaaaagttcaacagaatgatactgaaatagcagca acaaatgcgaaagctgcattaaaagctgcaggagttgcagatgcagaagctgatacagctactttagtgaa aatgtcttatacagataataatggcaaagttattgatggtgggttcgcatttaagacctccggtggttattatg- cag catctgttgataaatctggcgcagctagcttgaaagttactagctacgttgacgctaccactggtaccgaaaaa actgctgcgaataaattaggtggcgcagacggtaaaaccgaagttgttactatcgacggtaaaacctacaat gccagcaaagccgctgggcacaacttcaaagcacagccagagctggcggaagcggctgctacaaccact gaaaacccgctgcagaaaattgatgctgctttggcgcaggtggatgcgctgcgttctgacctgggtgcggttc

agaaccgtttcaactccgctatcaccaacctgggcaataccgtaaataacctgtcttctgcccgtagccgtatc gaagattccgactacgcgaccgaagtttccaacatgtctcgcgcgcagattctgcagcaggccggtacctcc gttctggcgcaggcgaaccaggttccgcaaaacgtcctctctttactgcgttaa (S. Paratyphi C fljB cistron) SEQ ID NO: 6 atggcacaagtaatcaacactaacagtctgtcgctgctgacccagaataacctgaacaaatcccagtccgc actgggcaccgctatcgagcgtctgtcttccggtctgcgtatcaacagcgcgaaagacgatgcggcaggtca ggcgattgctaaccgttttaccgcgaacatcaaaggtctgactcaggcttcccgtaacgctaacgacggtattt ctattgcgcagaccactgaaggcgcgctgaacgaaatcaacaacaacctgcagcgtgtgcgtgaactggc ggttcagtctgctaacagcactaactcacagtctgacctcgactctatccaggctgaaatcacccagcgtctga acgaaatcgaccgtgtatccggtcagactcagttcaacggcgtgaaagtcctggcgcaggacaacaccctg accatccaggttggtgccaacgacggtgaaactatcgatatcgatctgaagcagatcaactctcagaccctg ggtctggactcactgaacgtgcagaaagcgtatgatgtgaaagatacagcagtaacaacgaaagcttatgc caataatggtactacactggatgtatcgggtcttgatgatacagctatcaaagcggctataggtggtacgactg gtacggctgctgtaaccggtagtgcggttaaatttgacgcagataataacaagtactttgttactattggtggc- ttt actggtgctgatgccgccaaaaatggcgattatgaagttaacgttgctactgacggtacagtaacccttgcgg ctggcgcaactaaaaccacaatgcctgctggtgcgacaactaaaacagaagtacaggagttaaaagatac accggcagttgtttcagcagatgctaaaaatgccttaattgctggcggcgttgacgctaccgatgctaatggcg ctgagttggtcaaaatgtcttataccgataaaaatggtaagacaattgaaggcggttatgcgcttaaagctggc gataagtattacgccgcagattacgatgaagcgacaggagcaattaaagctaaaaccacaagttatactgct gctgacggcactaccaaaacagcagctaaccaactgggtggcgtagacggtaaaaccgaagtcgttactat cgacggtaaaacctacaatgccagcaaagccgctggtcatgatttcaaagcacaaccagagctggcggaa gcagccgctaaaaccaccgaaaacccgctgcagaaaattgatgccgcgctggcgcaggtggatgcgctg cgctctgatctgggtgcggtacaaaaccgtttcaactccgctatcaccaacctgggcaataccgtaaacaacc tgtctgaagcgcgtagccgtatcgaagattccgactacgcgaccgaagtttccaacatgtctcgcgcgcagat tctgcagcaggccggtacttccgttctggcgcaggctaaccaggtcccgcagaacgtgctgtctctgttacgtt- a a (S. Typhimurium fliC cistron) SEQ ID NO: 7 atggcacaagtcattaatacaaacagcctgtcgctgttgacccagaataacctgaacaaatcccagtccgct ctgggcaccgctatcgagcgtctgtcttccggtctgcgtatcaacagcgcgaaagacgatgcggcaggtcag gcgattgctaaccgttttaccgcgaacatcaaaggtctgactcaggcttcccgtaacgctaacgacggtatctc cattgcgcagaccactgaaggcgcgctgaacgaaatcaacaacaacctgcagcgtgtgcgtgaactggcg gttcagtctgctaacagcaccaactcccagtctgacctcgactccatccaggctgaaatcacccagcgcctga acgaaatcgaccgtgtatccggccagactcagttcaacggcgtgaaagtcctggcgcaggacaacaccct gaccatccaggttggtgccaacgacggtgaaactatcgatatcgatctgaagcagatcaactctcagaccct gggtctggatacgctgaatgtgcaacaaaaatataaggtcagcgatacggctgcaactgttacaggatatgc cgatactacgattgctttagacaatagtacttttaaagcctcggctactggtcttggtggtactgaccagaaaa- tt gatggcgatttaaaatttgatgatacgactggaaaatattacgccaaagttaccgttacggggggaactggta aagatggctattatgaagtttccgttgataagacgaacggtgaggtgactcttgctggcggtgcgacttccccg cttacaggtggactacctgcgacagcaactgaggatgtgaaaaatgtacaagttgcaaatgctgatttgacag aggctaaagccgcattgacagcagcaggtgttaccggcacagcatctgttgttaagatgtcttatactgataat aacggtaaaactattgatggtggtttagcagttaaggtaggcgatgattactattctgcaactcaaaataaaga- t ggttccataagtattaatactacgaaatacactgcagatgacggtacatccaaaactgcactaaacaaactg ggtggcgcagacggcaaaaccgaagttgtttctattggtggtaaaacttacgctgcaagtaaagccgaaggt cacaactttaaagcacagcctgatctggcggaagcggctgctacaaccaccgaaaacccgctgcagaaa attgatgctgctttggcacaggttgacacgttacgttctgacctgggtgcggtacagaaccgtttcaactccgc- ta ttaccaacctgggcaacaccgtaaacaacctgacttctgcccgtagccgtatcgaagattccgactacgcga ccgaagtttccaacatgtctcgcgcgcagattctgcagcaggccggtacctccgttctggcgcaggcgaacc aggttccgcaaaacgtcctctctttactgcgttaa (S. Typhimurium fljB cistron) SEQ ID NO: 8 atggcacaagtaatcaacactaacagtctgtcgctgctgacccagaataacctgaacaaatcccagtccgc actgggcaccgctatcgagcgtctgtcttctggtctgcgtatcaacagcgcgaaagacgatgcggcaggtca ggcgattgctaaccgtttcaccgcgaacatcaaaggtctgactcaggcttcccgtaacgctaacgacggtatc tccattgcgcagaccactgaaggcgcgctgaacgaaatcaacaacaacctgcagcgtgtgcgtgaactgg cggttcagtctgctaacagcaccaactcccagtctgacctcgactccatccaggctgaaatcacccagcgcct gaacgaaatcgaccgtgtatccggccagactcagttcaacggcgtgaaagtcctggcgcaggacaacacc ctgaccatccaggttggcgccaacgacggtgaaactatcgatatcgatctgaagcagatcaactctcagacc ctgggtctggactcactgaacgtgcagaaagcgtatgatgtgaaagatacagcagtaacaacgaaagcttat gccaataatggtactacactggatgtatcgggtcttgatgatgcagctattaaagcggctacgggtggtacgaa tggtacggcttctgtaaccggtggtgcggttaaatttgacgcagataataacaagtactttgttactattggtg- gct ttactggtgctgatgccgccaaaaatggcgattatgaagttaacgttgctactgacggtacagtaacccttgcg gctggcgcaactaaaaccacaatgcctgctggtgcgacaactaaaacagaagtacaggagttaaaagata caccggcagttgtttcagcagatgctaaaaatgccttaattgctggcggcgttgacgctaccgatgctaatggc gctgagttggtcaaaatgtcttataccgataaaaatggtaagacaattgaaggcggttatgcgcttaaagctgg cgataagtattacgccgcagattacgatgaagcgacaggagcaattaaagctaaaactacaagttatactgc tgctgacggcactaccaaaacagcggctaaccaactgggtggcgtagacggtaaaaccgaagtcgttacta tcgacggtaaaacctacaatgccagcaaagccgctggtcatgatttcaaagcacaaccagagctggcgga agcagccgctaaaaccaccgaaaacccgctgcagaaaattgatgccgcgctggcgcaggtggatgcgct gcgctctgatctgggtgcggtacaaaaccgtttcaactctgctatcaccaacctgggcaataccgtaaacaat ctgtctgaagcgcgtagccgtatcgaagattccgactacgcgaccgaagtttccaacatgtctcgcgcgcag attctgcagcaggccggtacttccgttctggcgcaggctaaccaggtcccgcagaacgtgctgtctctgttacg- t taa (S. Enteritidis fliC cistron) SEQ ID NO: 9 atggcacaagtcattaatacaaacagcctgtcgctgttgacccagaataacctgaacaaatctcagtcctcac tgagttccgctattgagcgtctgtcctctggtctgcgtatcaacagcgcgaaagacgatgcggcaggccaggc gattgctaaccgcttcacttctaatatcaaaggtctgactcaggcttcccgtaacgctaacgacggcatttcta- tt gcgcagaccactgaaggtgcgctgaatgaaatcaacaacaacctgcagcgtgtgcgtgagttgtctgttcag gccactaacgggactaactctgattccgatctgaaatctatccaggatgaaattcagcaacgtctggaagaa atcgatcgcgtttctaatcagactcaatttaacggtgttaaagtcctgtctcaggacaaccagatgaaaatcca ggttggtgctaacgatggtgaaaccattaccatcgatctgcaaaaaattgatgtgaaaagccttggccttgatg ggttcaatgttaatgggccaaaagaagcgacagtgggtgatctgaaatccagcttcaagaatgttacgggtta cgacacctatgcagcgggtgccgataaatatcgtgtagatattaattccggtgctgtagtgactgatgcagcag caccggataaagtatatgtaaatgcagcaaacggtcagttaacaactgacgatgcggaaaataacactgcg gttgatctctttaagaccactaaatctactgctggtaccgctgaagccaaagcgatagctggtgccattaaagg tggtaaggaaggagatacctttgattataaaggcgtgacttttactattgatacaaaaactggtgatgacggta atggtaaggtttctactaccatcaatggtgaaaaagttacgttaactgtcgctgatattgccactggcgcgacg- g atgttaatgctgctaccttacaatcaagcaaaaatgtttatacatctgtagtgaacggtcagtttacttttgat- gata aaaccaaaaacgagagtgcgaaactttctgatttggaagcaaacaatgctgttaagggcgaaagtaaaatt acagtaaatggggctgaatatactgctaacgccacgggtgataagatcaccttagctggcaaaaccatgttta ttgataaaacagcttctggcgtaagtacattaatcaatgaagacgctgccgcagccaagaaaagtaccgcta acccactggcttcaattgattctgcattgtcaaaagtggacgcagttcgttcttctctgggggcaattcaaaac- cg ttttgattcagccattaccaaccttggcaatacggtaaccaatctgaactccgcgcgtagccgtatcgaagatg ctgactatgcaacggaagtttctaatatgtctaaagcgcagattctgcagcaggctggtacttccgttctggcg- c aggctaaccaggttccgcaaaacgtcctctctttactgcgttaa (S. Typhi tviA cistron) SEQ ID NO: 10 Atgaggtttcatcatttctggcctccgaatgatatctatttcggggttggagctgctggcattattgaagaagt- gtc actgataacaaatgacagaaattatttgtttgtgaacctaaatcgctacagcctgttaaatgccctgaattttt- tca cgcgaatgagtgatattaataaaataatcgttatcatttcaagttcgcgactaatgccccttgcacgtttttgg- ttga cagagtgcaaaaatgttattgctgttttcgatgcggcaacatcagtccaggatattatcagaaatgtcagtcaa- c accaaagtggtgaaaagatcttgacggagcagagagattatcgtttcagaattaaccgtaaggatatagtaa agatgaaatatttcctttcggaaagtggtatggaagagcttcaggatagatttatgaactcatcatcgactatg- ta tcgctggagaaaagaattggcagtaaaatttggagtacgtgagccgcgctatctgttattgccggattcagtta- c tttactgtaa (S. Typhimurium fepE) SEQ ID NO: 11 Atgccatctcttaatgtaaaacaagaaaaaaatcagtcatttgcaggttattcactgccgcccgccaacagtc atgaaatcgatttgtttagccttatagaggtgttatggcaggcgaaacgtcgtattcttgctaccgttttcgcc- tttgc gtgcgtggggttgcttctgtcctttctgctgccgcaaaaatggaccagccaggcgattgtcacaccggcggagt

cggtacagtggcaggggctggagagaacgttgaccgcgctgcgcgtgttggatatggaggtaagcgttgatc ggggcagcgtatttaatctgtttattaaaaagtttagctcgccctcgctgctggaagaatatcttcgttcttct- ccgt atgtcatggatcaattaaaaggcgcgcaaatagacgagcaggatcttcaccgggcgattgtcctgctgagcg aaaaaatgaaagcggtggacagtaatgtcggcaagaaaaatgaaacgtcgttattcacgtcgtggacattg agttttaccgcgccgacgcgggaagaagcgcaaaaagtgctggctggctatattcagtacatctccgatatcg tcgtgaaagagacgctggaaaatattcgtaaccagctggaaatcaaaacccgctatgagcaggaaaagct ggcgatggatcgggtgcgtctcaaaaatcagcttgatgccaatattcaacgtcttcattattcgctggaaatcg- c caacgccgccggtattaagagaccggtttacagcaatggtcaggcggtaaaagatgatccggatttttctattt ctctcggcgcggatggtatttcccgcaaactggaaattgaaaaaggggtaacggacgtggccgagatcgac ggtgatttgcgtaaccgtcaataccatgttgaacaactggcggcaatgaatgtgagtgacgtgaagtttacccc gtttaaatatcaactgtcgccgtctctgccagtgaaaaaagatggcccgggtaaagccatcattattatcctgg- c ggcgttgattggcggtatgatggcctgcggcggcgtattactgcgtcacgcgatggtctcgcgtaaaatggaa aacgcgctggcgatagatgaacggttagtctga (S. Typhi rfbE cistron) SEQ ID NO: 12 Atgaagcttttaattaccggtggatgtggcttccttgggagtaatcttgcctcctttgctttaagtcaagggat- tgatt taattgtattcgataatctatcacgtaaaggtgcaacagataatttacattggttatcctccttaggaaacttt- gagt ttgtacatggtgatattcgcaacaaaaatgatgttacaagattaataactaagtatatgcctgatagctgtttt- cat cttgcaggtcaagtggcaatgactacatctattgacaatccttgtatggattttgaaattaatgtaggtggaac- ttt aaatttacttgaggcagtacggcagtataattcaaattgtaatataatttattcatcaacaaataaagtatacg- gc gatcttgagcaatataaatacaatgaaacagaaactagatacacttgtgtagataagcctaatggatatgatg agagcacacaattagatttccactcaccatatggttgttcaaaaggtgctgcagaccaatacatgcttgattat- g caaggatttttggtttgaatacagtggtgttcaggcattcatcaatgtatggtgggagacagtttgctacttat- gatc aaggctgggtaggttggttttgtcaaaaagcggttgaaattaaaaatggtattaataaacccttcactatttct- ggt aatggtaagcaagttagggatgttttgcatgctgaagatatgatttcgttatatttcactgccttggcaaatgt- atca aaaattagggggaacgcttttaatattggtggtaccattgtcaacagcctatcattacttgaattattcaaatt- gctt gaagattattgcaacatagatatgaggttcactaatttacctgtaagggaaagtgatcagcgtgtttttgttgc- ag atattaaaaaaatcactaatgcaattgactggagcccgaaagtctcggcaaaagatggtgtccagaaaatgt atgattggactagttctatatga (E. coli O157:H7 wbdR cistron) SEQ ID NO: 13 Atgaatttgtatggtatttttggtgctggaagttatggtagagaaacaatacccattctaaatcaacaaataaa- g caagaatgtggttctgactatgctctggtttttgtggatgatgttttggcaggaaagaaagttaatggttttga- agtg ctttcaaccaactgctttctaaaagccccttatttaaaaaagtattttaatgttgctattgctaatgataagat- acga cagagagtgtctgagtcaatattattacacggggttgaaccaataactataaaacatccaaatagcgttgttta- t gatcatactatgataggtagtggcgctattatttctccctttgttacaatatctactaatactcatatagggag- gtttttt catgcaaacatatactcatacgttgcacatgattgtcaaataggagactatgttacatttgctcctggggctaa- at gtaatggatatgttgttattgaagacaatgcatatataggctcgggtgcagtaattaagcagggtgttcctaat- cg cccacttattattggcgcgggagccattataggtatgggggctgttgtcactaaaagtgttcctgccggtataa- ct gtgtgcggaaatccagcaagagaaatgaaaagatcgccaacatctatttaa (S. Typhi rfbS cistron) SEQ ID NO: 14 Atgaaaattctaataatgggagcgtttgggttccttggatcacgacttacatcctacttcgaaagtcgacatac- tg tgattggcttagcaaggaagaggaacaatgaagctaccataaataatattatttacacgacagaaaataattg gatcgaaaaaatactagaatttgaaccgaatattattattaacactattgcttgctatggaagacataacgaac ctgcaacagctttaatagaaagcaatattcttatgcctatcagagtattagaatctatctcatcacttgatgca- gta ttcataaattgtggaacatcactgccaccaaatacgagtttatatgcatatactaaacaaaaagcaaatgaact cgccgccgccattatagataaagtttgcggtaaatatatagagttaaaattggagcatttctatggagcttttg- at ggagacgataagtttaccagtatggttattagacgttgtttaagtaaccagccagtaaagttaacatctggttt- gc aacagagagatttcttgtatataaaagatctactaacagcgttcgattgtattataagtaatgttaataatttc- ccc aaatttcatagtattgaagttggtagtggagaggcgatatcaattcgtgaatatgtagatactgttaaaaatat- ca caaaaagcaattctataattgaatttggcgtggtcaaagaaagagtaaatgaattgatgtatagttgtgctgat- a tagcagaacttgaaaaaataggatggaaaagagagttctctcttgttgatgcattaactgaaataattgaaga ggaagggaaatga (S. Typhimurium rfbJ cistron) SEQ ID NO: 15 Atgacctttttgaaagaatatgtaattgtcagtggggcttccggctttattggtaagcatttactcgaagcgct- aaa aaaatcggggatttcagttgtcgcaatcactcgagatgtaataaaaaataatagtaatgcattagctaatgtta- g atggtgcagttgggataatatcgaattattagtcgaggagttatcaattgattctgcattaattggtatcattc- atttg gcaacagaatatgggcataaaacatcatctctcataaatattgaagatgcaaatgttataaaaccattaaagc ttcttgatttggcaataaaatatcgggcggatatctttttaaatacagatagtttttttgccaagaaagatttt- aattat caacatatgcggccttatataattactaaaagacactttgatgaaattgggcattattatgctaatatgcatga- ca tttcatttgtaaacatgcgattagagcatgtatatgggcctggggatggtgaaaataaatttattccatacatt- atc gactgcttaaataaaaaacagagttgcgtgaaatgtacaacaggcgaacagataagagactttatttttgtag atgatgtggtaaatgcttatttaactatattagaaaatagaaaagaagtaccttcatatactgagtatcaagtt- gg aactggtgctggggtaagtttgaaagattttctggtttatttgcaaaatactatgatgccaggttcatcgagta- tattt gaatttggtgcgatagagcaaagagataatgaaataatgttctctgtagcaaataataaaaatttaaaagcaa tgggctggaaaccaaatttcgattataaaaaaggaattgaagaactactgaaacggttatga (S. Typhimurium P.sub.ssaG promoter region) SEQ ID NO: 16 Tattgccatcgcggatgtcgcctgtcttatctaccatcataaacatcatttgcctatggctcacgacagtatag- gc aatgccgttttttatattgctaattgtttcgccaatcaacgcaaaagtatggcgattgctaaagccgtctccct- ggg cggtagattagccttaaccgcgacggtaatgactcattcatactggagtggtagtttgggactacagcctcatt- t attagagcgtcttaatgatattacctatggactaatgagttttactcgcttcggtatggatgggatggcaatga- cc ggtatgcaggtcagcagcccattatatcgtttgctggctcaggtaacgccagaacaacgtgcgccggagtaat cgttttcaggtatataccggatgttcattgctttctaaattttgctatgttgccagtatccttacgatgtattt- attttaagg aaaagc (E. coli araC repressor and P.sub.araBAD promoter) SEQ ID NO: 17 ttatgacaacttgacggctacatcattcactttttcttcacaaccggcacggaactcgctcgggctggccccgg- t gcattttttaaatacccgcgagaaatagagttgatcgtcaaaaccaacattgcgaccgacggtggcgatagg catccgggtggtgctcaaaagcagcttcgcctggctgatacgttggtcctcgcgccagcttaagacgctaatcc ctaactgctggcggaaaagatgtgacagacgcgacggcgacaagcaaacatgctgtgcgacgctggcga tatcaaaattgctgtctgccaggtgatcgctgatgtactgacaagcctcgcgtacccgattatccatcggtgga- t ggagcgactcgttaatcgcttccatgcgccgcagtaacaattgctcaagcagatttatcgccagcagctccga atagcgcccttccccttgcccggcgttaatgatttgcccaaacaggtcgctgaaatgcggctggtgcgcttcat- c cgggcgaaagaaccccgtattggcaaatattgacggccagttaagccattcatgccagtaggcgcgcggac gaaagtaaacccactggtgataccattcgcgagcctccggatgacgaccgtagtgatgaatctctcctggcg ggaacagcaaaatatcacccggtcggcaaacaaattctcgtccctgatttttcaccaccccctgaccgcgaat ggtgagattgagaatataacctttcattcccagcggtcggtcgataaaaaaatcgagataaccgttggcctca atcggcgttaaacccgccaccagatgggcattaaacgagtatcccggcagcaggggatcattttgcgcttca gccatacttttcatactcccgccattcagagaagaaaccaattgtccatattgcatcagacattgccgtcactg- c gtcttttactggctcttctcgctaaccaaaccggtaaccccgcttattaaaagcattctgtaacaaagcgggac- c aaagccatgacaaaaacgcgtaacaaaagtgtctataatcacggcagaaaagtccacattgattatttgcac ggcgtcacactttgctatgccatagcatttttatccataagattagcggatcctacctgacgctttttatcgca- actc tctactgtttctccatacccgtttttttgggctagcgaattgaggaggagatataca (S. Paratyphi A rfbE pseudogene) SEQ ID NO: 18 Ttgaagaggaagagaaatgaagctttaattaccggtggatgtggcttccttgggagtaatcttgcctcctttgc- tt taagtcaagggattgatttaattgtattcgataatctatcacgtaaaggtgcaacagataatttacattggtta- tcct ccttaggaaactttgagtttgtacatggtgatattcgcaacaaaaatgatgttacaagattaataactaagtat- at gcctgatagctgttttcatcttgcaggtcaagtggcaatgactacatctattgacaatccttgtatggattttg- aaatt aatgtaggtggaactttaaatttacttgaggcagtacggcagtataattcaaattgtaatataatttattcatc- aac

aaataaagtatacggcgatcttgagcaatataaatacaatgaaacagaaactagatacacttgtgtagataa gcctaatggatatgatgagagcacacaattagatttccactcaccatatggttgttcaaaaggtgctgcagacc aatacatgcttgattatgcaaggatttttggtttgaatacagtggtgttcaggcattcatcaatgtatggtggg- aga cagtttgctacttatgatcaaggctgggtaggttggttttgtcaaaaagcggttgaaattaaaaatggtattaa- ta aacccttcactatttctggtaatggtaagcaagttagggatgttttgcatgctgaagatatgatttcgttatat- ttcac tgccttggcaaatgtatcaaaaattagggggaacgcttttaatattggtggtaccattgtcaacagcctatcat- ta cttgaattattcaaattgcttgaagattattgcaacatagatatgaggttcactaatttacctgtaagggaaag- tga tcagcgtgtttttgttgcagatattaaaaaaatcactaatgcaattgactggagcccgaaagtctcggcaaaag atggtgtccagaaaatgtatgattggactagttctatatga (S. Typhi rfbE locus with a partial deletion of rfbE) SEQ ID NO: 19 tcacgacttacatcctacttcgaaagtcgacatactgtgattggcttagcaaggaagaggaacaatgaagcta ccataaataatattatttacacgacagaaaataattggatcgaaaaaatactagaatttgaaccgaatattatt- a ttaacactattgcttgctatggaagacataacgaacctgcaacagctttaatagaaagcaatattcttatgcct- at cagagtattagaatctatctcatcacttgatgcagtattcataaattgtggaacatcactgccaccaaatacga- g tttatatgcatatactaaacaaaaagcaaatgaactcgccgccgccattatagataaagtttgcggtaaatata- t agagttaaaattggagcatttctatggagcttttgatggagacgataagtttaccagtatggttattagacgtt- gttt aagtaaccagccagtaaagttaacatctggtttgcaacagagagatttcttgtatataaaagatctactaacag cgttcgattgtattataagtaatgttaataatttccccaaatttcatagtattgaagttggtagtggagaggcg- atat caattcgtgaatatgtagatactgttaaaaatatcacaaaaagcaattctataattgaatttggcgtggtcaaa- g aaagagtaaatgaattgatgtatagttgtgctgatatagcagaacttgaaaaaataggatggaaaagagagt tctctcttgttgatgcattaactgaaataattgaagaggaagggaaatgaaaagcttggtaccgagctcggatc cactagtaacggccgccagtgtgctggaattcgccctttaagcggccgcatttaacataatatacattatgcgc accgcggccgcggaaagggcgaattctgcagatatccatcacactggcggccgctcgagcatgcatctag agtgaggaaactgaggttggttagaattccaagacatcttattattgccgcttcctcttggctttcaaagataa- taa ttgccggtgttcagttagtaagtgttaaatttcttttagaaattcttggcgaagaatcatacgctgtatttact- cttttaa ctggattattggtctggtttagcattgcagatattgggattggtagtagtctacaaaattatatatctgagttg- aaag ctgatagaaaatcatatgatgcatatatcaaggccgcagttcatattctattcgcatccttaatcattttaagc- tcta cattattcttcttatcagataaattatcgtcactatatcttacttcatttagcgatgaattgaaaaacaactca- ggaa gttatttttttatagcaagtatattatttatattcatcggcgttgggagtgtggtctataaaatattatttgcg- gaactgtt agggtggaaagctaatataattaatgcattatcttatcttttaggttttttagatgtagttgcgatccattatt- taatgcc agattcgagtattaccttcgctttagtagcattgtatgctccggtagcaatactgcccattatatatatatcgt- ttcgg tatatatatgttcttaaagcgaaagtaaactttaacacctataaattattactatcacgttcatcagggtttct- gatttt ttcgtccttatcgataatagttttacaaactgattatattgtgatgtctcagaaattatctgctgcagatatta- taaaat atactgtaacgatgaaaatatttggtttaatgttttttatttatactgcggtattacaagcattatggccagta- tgtgct (S. Typhi rfbE locus with rfbE disrupted by wbdR) SEQ ID NO: 20 aggcttgactacagagcatttagattatgtagttagcaagtttgaagagttctttggtttgaatttctaatttt- gaggg ggggggattcccctctatgatttcatgaaaattctaataatgggagcgtttgggttccttggatcacgacttac- atc ctacttcgaaagtcgacatactgtgattggcttagcaaggaagaggaacaatgaagctaccataaataatatt atttacacgacagaaaataattggatcgaaaaaatactagaatttgaaccgaatattattattaacactattgc- tt gctatggaagacataacgaacctgcaacagctttaatagaaagcaatattcttatgcctatcagagtattagaa tctatctcatcacttgatgcagtattcataaattgtggaacatcactgccaccaaatacgagtttatatgcata- tac taaacaaaaagcaaatgaactcgccgccgccattatagataaagtttgcggtaaatatatagagttaaaattg gagcatttctatggagcttttgatggagacgataagtttaccagtatggttattagacgttgtttaagtaacca- gcc agtaaagttaacatctggtttgcaacagagagatttcttgtatataaaagatctactaacagcgttcgattgta- tta taagtaatgttaataatttccccaaatttcatagtattgaagttggtagtggagaggcgatatcaattcgtgaa- tat gtagatactgttaaaaatatcacaaaaagcaattctataattgaatttggcgtggtcaaagaaagagtaaatg aattgatgtatagttgtgctgatatagcagaacttgaaaaaataggatggaaaagagagttctctcttgttgat- g cattaactgaaataattgaagaggaagggaaatgaatttgtatggtatttttggtgctggaagttatggtagag- a aacaatacccattctaaatcaacaaataaagcaagaatgtggttctgactatgctctggtttttgtggatgatg- ttt tggcaggaaagaaagttaatggttttgaagtgctttcaaccaactgctttctaaaagccccttatttaaaaaag- t attttaatgttgctattgctaatgataagatacgacagagagtgtctgagtcaatattattacacggggttgaa- cc aataactataaaacatccaaatagcgttgtttatgatcatactatgataggtagtggcgctattatttctccct- ttgtt acaatatctactaatactcatatagggaggttttttcatgcaaacatatactcatacgttgcacatgattgtca- aat aggagactatgttacatttgctcctggggctaaatgtaatggatatgttgttattgaagacaatgcatatatag- gct cgggtgcagtaattaagcagggtgttcctaatcgcccacttattattggcgcgggagccattataggtatgggg gctgttgtcactaaaagtgttcctgccggtataactgtgtgcggaaatccagcaagagaaatgaaaagatcgc caacatctatttaatgcggccgcatttaacataatatacattatgcgcaccgcggccgccagtgtgaggatcct gtttctgcccgcgaaagcgggcataattaaagaatgaaatattttttataattaaaagatgaagctgacgtgag gaaactgaggttggttagaattccaagacatcttattattgccgcttcctcttggctttcaaagataataattg- ccg gtgttcagttagtaagtgttaaatttcttttagaaattcttggcgaagaatcatacgctgtatttactctttta- actggat tattggtctggtttagcattgcagatattgggattggtagtagtctacaaaattatatatctgagttgaaagct- gata gaaaatcatatgatgcatatatcaaggccgcagttcatattctattcgcatccttaatcattttaagctctaca- ttatt cttcttatcagataaattatcgtcactatatcttacttcatttagcgatgaattgaaaaacaactcaggaagtt- attttt ttatagcaagtatattatttatattcatcggcgttgggagtgtggtctataaaatattatttgcggaactgtta- gggtg gaaagctaatataattaatgcattatcttatcttttaggttttttagatgtagttgcgatccattatttaatgc- cagattc gagtattaccttcgctttagtagcattgtatgctccggtagcaatactgcccattatatatatatcgtttcggt- atatat atgttcttaaagcgaaagtaaactttaacacctataaattattactatcacgttcatcagggtttctgattttt- tcgtcc ttatcgataatagttttacaaactgattatattgtgatgtctcagaaattatctgctgcagatattataaaata- tactgt aacgatgaaaatatttggtttaatgttttttatttatactgcggtattacaagcattatggccagtatgtgctg- aattac gagtgaaaatgcagtggagaaagctgcatagaatcattttcctaaatattattggtggggtattttttattggt- cttg gtacgttatttatttatgttttaaaggattatatctatagcataattgctaacggtatagattataatattagt- ggggttg tttttgttttactggctgtgtattttagtataagagtttggtgtgatacatttgctatgttacttcaaagtatg- aaccaatta aaaattctttggctcatagttccgtgtcaggcattaattggtggtgtgactcaatggtattttgcagagcatta- tgga atagttggtattttatacggactaattttatcgttctcgctaactgttttttggggattgccagtgtattatat- gtataaga gtaaaaggctagcataatatgaaggtatcattttgtatcccaacgtataatcgagtaaaattcattgaagacct- t cttgaaagtattaataatcaatcttctcactccttaattgtagaagtatgt (S. Typhi fepE pseudogene) SEQ ID NO: 21 Atgccatctcttaatgtaaaacaagagaaaaatcagtcatttgcaggttactcactgccgcccgccaacagtc atgaaatcgatttgtttagccttatagaggtgttatggcaggcgaaacgtcgtattcttgctaccgttttcgcc- tttgc gtgcgtggggttgcttctgtcctttctgctgccgcaaaaatggaccagccaggcgattgtcacaccggcggagt cggtacagtagcaggggctggagagaacgttgaccgcgctgcgcgtgttggatatggaggtaagcgttgatc gggccagcgtatttaatctgtttattaaaaagtttagctcgccctcgctgctggaagaatatcttcgttcttct- ccgta tgtcatggatcaattaaaaggcgcgcaaatagacgagcaggatcttcaccgggcgattgtcgtgctgagcga aaaaatgaaagcggtggacagcaatgccggcaagaaaaatgaaacgtcgttattcacgtcgtggacgctg agttttaccgcgccgacgcgggaagaagcgcaaaaagtgttggctggctatattcagtacatctccgatatcg tcgtgaaagagacgctggaaaatattcgtaaccagctggaaatcaaaacccgctacgagcaggaaaagct ggcgatggatcgggtgcgtctcaaaaatcagcttgatgccaatattcaacgtcttcattattcgctggaaatcg- c caacgccgctggcattaagagaccggtttacagtaatggtcaggcggtaaaagatgatccggatttttctattt- c cctcggcgcggatggtatttcccgcaaactggaaattgaaaaaggggtaacggacgtggccgagatcgac ggtgatttgcgtaaccgtcaatactatgttgaacaactggcggcaatgaatgtgagcgacgtgaagtttacccc

gtttaaatatcaactgtcgccgtctctgccagtgaaaaaagatggcccgggtaaagcgatcattattatcctgg cggcgttgattggcggtatgatggcctgcggcggcgtattactgcgtcacgcgatggtctcgcgtaaaatgga aaacgcgctggcgatagatgaacggttagtctga (S. Typhi family 2 gtr operon) SEQ ID NO: 22 actgcactgacggtggcggttgaaacgctgaaggcataaagatcttcactctcccggccgatgtcctgataac tcatctccagcgcaaacagtcgaatgatattgcgctcgatctcgtcgtacagggggtctgaagcttcttcacca attatggctcaaaagtgccgttacgattgcgcggagttgccagttcaaaactgcctgttggggctttaatggct- ttt tgccggaaccatttttacggtttgcctcaacatcctgagccagatgggaatcaagttcagcagacagggtaga gtcggttaaatacttgattaatggcgttaagatgccatctttgcccgttaatgcctggccggacctgaagggct- tt aagtgctttgtcgaaatcgaagggatgggacatgtgccattcttttttattttatgttactaaaattatacaga- attttt aacgctccccctctccccagcacttccaccctttcaagtacctctctctgaaaaaacatgcaaagccttgtaag acgatgtaaagctttacatgtcccgtttttattccaagacgcttggcaatcagcaataccaattgatcgataac- at cgatcaatatattaaaactcaatagcttaaaactattaaaaatacaattattgatcgcttatatcgatcaaacc- aa tttgtagtgctacactccagacctttctgaatcggctaattttcataatgttgaagttattcgctaagtacaca- tcgat cggtgttcttaacacgctcattcattggggagtatttgctttctgtatgtatgggatgcatacgcatcaggcgc- tga cgaacttttccgattttgttatcgctgtatcgttcagcttctatgctaatgcgcgcttcacctttaatgccagc- actact gcaattctctacatgatgtatatgggattcatgggaacactgagcgctgttgttggatggatggctgaccaatg- tt ctttgccaccattggttaccctcatcactttctcggcaattagcctggtatgcggctttatctattccagattc- attatct tcagggataaaaatgaaaatctctcttgtcgtcctggtttttaacgaagaagacacgataccgattttctatag- aa cggtacatgagtttaatgaacttgaaaaatataaagttgagattatttttattaatgacggaagtaaagatgtg- ac ggagtcaataattaaaataatagccgtatctgatccactcgtcattccgttttcgtttacacgaaacttcggga- at gatgcaagatgcacaaccattttattaatctttttttaaattgaggtaatttaagttggaacacttaaaataca- gac ctgatatagatggattacgcgcaatagcggttttatctgtggtaatattccattatttcccatcattattgccg- ggtgg gtttgttggagtagatatattctttgtgatatctggataccttataacatcaataatattaaaatctgcatcaa- acaa atcattttcataccttgatttctataaaaggagagtgcttagaatatttccagcattatccatagttcttgtat- catgtct tattgttggttggatttatttattccaggatgattacaaattacttggtaagcatOtttagtggctcattcttt- atatcaa actttactctttggagtgagtctggctattttgattcaaaatcataccttaaacctttactacatttgtggtcg- ctggga attgaagagcaattttatataatatggccagtagttatattgctatgctttagaagcaaaaaccataacagaaa- c atagtattatcatgcgcaactatatttataattagctatgcgattagcatttttacaatggcatctgatggcgg- agct aattactactctcccgcatcaagattttgggagttaatggctggagcgattatatccacattgagatttatagg- aat aaacacttcgttatcaaaattaatgtccctgttaggaattatactaatcgcattatcaataaccatgatagatg- aa aagatgtcatttcctggatatatagcaataatcccaatacttggcgcctctcttataatagcatctaatggtaa- tga tttagttgtgtcgaaattgcttagtgttaggcctgttgttttctttggtcttattagctatcctctttatttgt- ggcattggcct atttattcattctatcgttcaatatttgctggctcaccagactaccatgaattaactcttcttttattattatc- gttctttttg gcgatattaacttattatttaattgaaaaaccactgagaaattccagaagtaaatatatcacagcaatattatt- ag cattatctgtatttgggacgggtttaattggcgcatttatttttcatataaatggagttaaagacagggaaatc- aata aatcagcaagtgaatatgcttctgttactgacgtgtacaattattataaatatggagaactactccgtggaggg- a tatgtcactcagtacaacttactgctgccatatccaatggatgtataaaaaatggcaagcataatatatttatc- att ggtgattcttatgcggcggctcttttcaatgggctttctcattatatagataataaaggttctgattatataat- aagcc aaatgacagatggtaatgctcctcctctatttgttgacggtaaagatgatttacagagaagtgtcatcactcta- aa caataatagaattaatgaaattaaacgtgttcagcctgaggtggttctgctgacatggtcagttcgaggaacaa atggagtacatgataaaaagttagcaattgatgcgttatcattaaccattaaaaaaattaaagaggcatcccct gactcaaggattgttttcattggaccagtcccggaatggaatgcaaatttagttaaaataatatctaactacct- ga gtgagtttaaaaaaactccaccattgtatatgacatatggattaaatagtgaaataagcgagtgggactcttac- t ttagtaacaatgttccaaaaatgggaattgaatatatatcagcatacaaagcattatgtaacgaaagtggatgt cttacaagagttggtaatggtcctgattttatcactgccgttgattggggacatttaacaaagcctggttctga- tttc ctttttaataaaattggaaataaaataatcaaatagataggctgttactattacatataaatccaatatggaac- at gccagtcatactgtgtaactgccactatattaacggtgatcgctcaggcggtcaccgaactcgataataaagc gaa (S. Typhimurium hin-fljBA locus) SEQ ID NO: 23 tgttgtaatttttattttaattcattcgtttttttatgcggcttgccggaaaatatctgtataaggtagatacg- ccaatac caaaaataatagctagttgctgccgaggatggcctttctctaatagccgactaatctgttcctgttcatgtttg- ttga tcgcccgagggcgccctcccagtcgtccttgcgctctggcggcagccagtccggcaagggttcgctcgacaa ttaattctcgctccatctcggccagtgctgacattacatgaaaaaagaatcgccccatcgcgctactggtatca atactatcggttaaagaatggaagtgagctccacgttcatgtaattctgatattaacgccaccaggtttttcac- gc tgcggcccagtctgtctaatttccagacgacaagagtatcgcctttatttacatactttaacgctcgtttcagg- ccg gggcggtttgcaatcttgccactgatacggtcctcaaaaatgcggtcacaatttgcactagtaagcgcattacg ctgtaaatcgatattttggtcaattgttgacacccgaatatacccaatagtagccatgattttctcctttacat- caga taaggaagaattttagtcgcttttctcatggaggattgctttatcaaaaaccttccaaaaggaaaattttatgg- ca caagtaatcaacactaacagtctgtcgctgctgacccagaataacctgaacaaatcccagtccgcactgggc accgctatcgagcgtctgtcttctggtctgcgtatcaacagcgcgaaagacgatgcggcaggtcaggcgattg ctaaccgtttcaccgcgaacatcaaaggtctgactcaggcttcccgtaacgctaacgacggtatctccattgcg cagaccactgaaggcgcgctgaacgaaatcaacaacaacctgcagcgtgtgcgtgaactggcggttcagt ctgctaacagcaccaactcccagtctgacctcgactccatccaggctgaaatcacccagcgcctgaacgaa atcgaccgtgtatccggccagactcagttcaacggcgtgaaagtcctggcgcaggacaacaccctgaccat ccaggttggcgccaacgacggtgaaactatcgatatcgatctgaagcagatcaactctcagaccctgggtct ggactcactgaacgtgcagaaagcgtatgatgtgaaagatacagcagtaacaacgaaagcttatgccaata atggtactacactggatgtatcgggtcttgatgatgcagctattaaagcggctacgggtggtacgaatggtacg gcttctgtaaccggtggtgcggttaaatttgacgcagataataacaagtactttgttactattggtggctttac- tggt gctgatgccgccaaaaatggcgattatgaagttaacgttgctactgacggtacagtaacccttgcggctggcg caactaaaaccacaatgcctgctggtgcgacaactaaaacagaagtacaggagttaaaagatacaccgg cagttgtttcagcagatgctaaaaatgccttaattgctggcggcgttgacgctaccgatgctaatggcgctgag- t tggtcaaaatgtcttataccgataaaaatggtaagacaattgaaggcggttatgcgcttaaagctggcgataa gtattacgccgcagattacgatgaagcgacaggagcaattaaagctaaaactacaagttatactgctgctga cggcactaccaaaacagcggctaaccaactgggtggcgtagacggtaaaaccgaagtcgttactatcgac ggtaaaacctacaatgccagcaaagccgctggtcatgatttcaaagcacaaccagagctggcggaagcag ccgctaaaaccaccgaaaacccgctgcagaaaattgatgccgcgctggcgcaggtggatgcgctgcgctct gatctgggtgcggtacaaaaccgtttcaactctgctatcaccaacctgggcaataccgtaaacaatctgtctga agcgcgtagccgtatcgaagattccgactacgcgaccgaagtttccaacatgtctcgcgcgcagattctgca gcaggccggtacttccgttctggcgcaggctaaccaggtcccgcagaacgtgctgtctctgttacgttaattta- tt tcgttttattcagccccgtgaattcggggctttttcatttagcatagatgaatatatatttatggaatgtatgg- ctgtaa atgatatttcctacgggcgagaagctgaaatatggccgcgggattattctatgcttgctcgtcgagttcaattt- cta cgttttaatgatatccctgttcgattggtgagtaataatgcccggataatcacaggctacattgcgaagtttaa- tcc gaaggaaaatttgattctggcttcggataaacctaaaggaaataagcgcattgaagttaaactagagtctctg gcaattcttgaagaattatcaggtaatgacgcttttaatctttcgctggtgccggctgacggatttaatcttca- gca atatactccatcaagaagagattatttctcgatttgcaataagtgctataaacagggagtcggtatcaaaatct- a tatgaagtatggacaggttttgactggcaaaacgacaggcgtaaatgcgtgtcaggttggtgtgaggacatcc aatggcaatcatatgcaagttatgtttgactgggtgagcaggatcacgtcttcggactacgctgaataacgcct acggtaataaaaaattccgtgagaaaagtaaaacttagggggctaccggaggggacctaatgaacggag gtcatggaaggtattcatcgtgccagactcttgctcttgtcagaagaaggtaaaagta (Bacteriophage lambda tandem P.sub.R and P.sub.L promoters) SEQ ID NO: 24 Acgttaaatctatcaccgcaagggataaatatctaacaccgtgcgtgttgactattttacctctggcggtgata- at ggttgcatgtactaaggaggttgtatggaacaacgcataaccctgaaagattatgcaatgcgctttgggcaaa

ccaagacagctaaagatctctcacctaccaaacaatgcccccctgcaaaaaataaattcatataaaaaaca tacagataaccatctgcggtgataaattatctctggcggtgttgacataaataccactggcggtgatactgagc acatcagcaggacgcactgaccaccatgaaggtgacgctcttaaaaattaagccctgaagaagggcagca ttcaaagcagaaggctttggggtgtgtgatacgaaacgaagcattgggatctatcgatgcatgccatggtacc cgggagctcgaattaattctagaaataattttgtttaactttaagaaggagatata (Bacteriophage lambda thermo-labile repressor cl857 cistron) SEQ ID NO: 25 Atgagcacaaaaaagaaaccattaacacaagagcagcttgaggacgcacgtcgccttaaagcaatttatg aaaaaaagaaaaatgaacttggcttatcccaggaatctgtcgcagacaagatggggatggggcagtcagg cgttggtgctttatttaatggcatcaatgcattaaatgcttataacgccgcattgcttgcaaaaattctcaaag- ttag cgttgaagaatttagcccttcaatcgccagagaaatctacgagatgtatgaagcggttagtatgcagccgtca cttagaagtgagtatgagtaccctgttttttctcatgttcaggcagggatgttctcacctaagcttagaacctt- tacc aaaggtgatgcggagagatgggtaagcacaaccaaaaaagccagtgattctgcattctggcttgaggttga aggtaattccatgaccgcaccaacaggctccaagccaagctttcctgacggaatgttaattctcgttgaccctg agcaggctgttgagccaggtgatttctgcatagccagacttgggggtgatgagtttaccttcaagaaactgatc agggatagcggtcaggtgtttttacaaccactaaacccacagtacccaatgatcccatgcaatgagagttgttc cgttgtggggaaagttatcgctagtcagtggcctgaagagacgtttggctaa (pUCpW_difCAT rfbE deletion cassette) SEQ ID NO: 26 tcgacatactgtgattggcttagcaaggaagaggaacaatgaagctaccataaataatattatttacacgaca gaaaataattggatcgaaaaaatactagaatttgaaccgaatattattattaacactattgcttgctatggaag- a cataacgaacctgcaacagctttaatagaaagcaatattcttatgcctatcagagtattagaatctatctcatc- a cttgatgcagtattcataaattgtggaacatcactgccaccaaatacgagtttatatgcatatactaaacaaaa- a gcaaatgaactcgccgccgccattatagataaagtttgcggtaaatatatagagttaaaattggagcatttcta- t ggagcttttgatggagacgataagtttaccagtatggttattagacgttgtttaagtaaccagccagtaaagtt- aa catctggtttgcaacagagagatttcttgtatataaaagatctactaacagcgttcgattgtattataagtaat- gtta ataatttccccaaatttcatagtattgaagttggtagtggagaggcgatatcaattcgtgaatatgtagatact- gtt aaaaatatcacaaaaagcaattctataattgaatttggcgtggtcaaagaaagagtaaatgaattgatgtata gttgtgctgatatagcagaacttgaaaaaataggatggaaaagagagttctctcttgttgatgcattaactgaa ataattgaagaggaagggaaatgaatttgtatggtatttttggtgctggaagttatggtagagaaacaataccc attctaaatcaacaaataaagcaagaatgtggttctgactatgctctggtttttgtggatgatgttttggcagg- aaa gaaagttaatggttttgaagtgctttcaaccaactgctttctaaaagccccttatttaaaaaagtattttaatg- ttgct attgctaatgataagatacgacagagagtgtctgagtcaatattattacacggggttgaaccaataactataaa acatccaaatagcgttgtttatgatcatactatgataggtagtggcgctattatttctccctttgttacaatat- ctacta atactcatatagggaggttttttcatgcaaacatatactcatacgttgcacatgattgtcaaataggagactat- gtt acatttgctcctggggctaaatgtaatggatatgttgttattgaagacaatgcatatataggctcgggtgcagt- aa ttaagcagggtgttcctaatcgcccacttattattggcgcgggagccattataggtatgggggctgttgtcact- aa aagtgttcctgccggtataactgtgtgcggaaatccagcaagagaaatgaaaagatcgccaacatctatttaa tgcggccgcatttaacataatatacattatgcgcaccgcccgaacaccactcgccacaaaaaaccgccgga acgtccaaaagtacgggttttgctgcccgcaaacgggctgttctggtgttgctagtttgttatcagaatcgcag- at ccggcttcagccggtttgccggctgaaagcgctatttcttccagaattgccatgattttttccccacgggaggc- gt cactggctcccgtgttgtcggcagctttgattcgataagcagcatcgcctgtttcaggctgtctatgggccggc- c aaatcagtaagttggcagcatcacccgacgcactttgcgccgaataaatacctgtgacggaagatcacttcg cagaataaataaatcctggtgtccctgttgataccgggaagccctgggccaacttttggcgaaaatgagacgt tgatcggcacgtaagaggttccaactttcaccataatgaaataagatcactaccgggcgtattttttgagttat- cg agattttcaggagctaaggaagctaaaatggagaaaaaaatcactggatataccaccgttgatatatcccaa tggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctgga- t attacggcctttttaaagaccgtaaagaaaaataagcacaagttttatccggcctttattcacattcttgcccg- cct gatgaatgctcatccggaattccgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcaccctt gttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgacgatttccggcag- tt tctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgaga- at atgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaaacgtggccaatatggacaactt- cttcg cccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattcaggttcat catgccgtttgtgatggcttccatgtcggcagaatgcttaatgaattacaacagtactgcgatgagtggcaggg cggggcgtaatttttttaaggcagttattggtgcccttaaacgcctggttgctacgcctgaataagtgataata- ag cggatcctaggatggtgttaagcgggcggttttgagatgtaaactcgcccatttaacataatatacattatgcg- c accgcggccgccagtgtgaggatcctgtttctgcccgcgaaagcgggcataattaaagaatgaaatatttttta taattaaaagatgaagctgacgtgaggaaactgaggttggttagaattccaagacatcttattattgccgcttc- c tcttggctttcaaagataataattgccggtgttcagttagtaagtgttaaatttcttttagaaattcttggcga- agaat catacgctgtatttactcttttaactggattattggtctggtttagcattgcagatattgggattggtagtagt- ctacaa aattatatatctgagttgaaagctgatagaaaatcatatgatgcatatatcaaggccgcagttcatattctatt- cg catccttaatcattttaagctctacattattcttcttatcagataaattatcgtcactatatcttacttcattt- agcgatga attgaaaaacaactcaggaagttatttttttatagcaagtatattatttatattcatcggcgttgggagtgtgg- tctat aaaatattatttgcggaactgttagggtggaaagctaatataattaatgcattatcttatcttttaggtttttt- agatgt agttgcgatccattatttaatgccagattcgagtattaccttcgctttagtagcattgtatgctccggtagcaa- tact gcccattatatatatatcgtttcggtatatatatgttcttaaagcgaaagtaaactttaacacctataaattat- tacta tcacgttcatcagggtttctgattttttcgtccttatcgataatagttttacaaactgattatattgtgatgtc- tcagaaat tatctggagct (PL rfbE deletion cassette) SEQ ID NO: 27 aataggatggaaaagagagttctctcttgttgatgcattaactgaaataattgaagaggaagggaaatgaaa agcttggtaccgagctcggatccactagtaacggccgccagtgtgctggaattcgccctttaagcggccgcat ttaacataatatacattatgcgcaccatccgcttattatcacttattcaggcgtagcaccaggcgtttaagggc- ac caataactgccttaaaaaaattacgccccgccctgccactcatcgcagtactgttgtaattcattaagcattct- gc cgacatggaagccatcacaaacggcatgatgaacctgaatcgccagcggcatcagcaccttgtcgccttgc gtataatatttgcccatggtgaaaacgggggcgaagaagttgtccatattggccacgtttaaatcaaaactggt gaaactcacccagggattggctgagacgaaaaacatattctcaataaaccctttagggaaataggccaggtt ttcaccgtaacacgccacatcttgcgaatatatgtgtagaaactgccggaaatcgtcgtggtattcactccaga gcgatgaaaacgtttcagtttgctcatggaaaacggtgtaacaagggtgaacactatcccatatcaccagctc accgtctttcattgccatacggaattccggatgagcattcatcaggcgggcaagaatgtgaataaaggccgg ataaaacttgtgcttatttttctttacggtctttaaaaaggccgtaatatccagctgaacggtctggttatagg- tacat tgagcaactgactgaaatgcctcaaaatgttctttacgatgccattgggatatatcaacggtggtatatccagt- g atttttttctccattttagcttccttagctcctgaaaatctcgataactcaaaaaatacgcccggtagtgatct- tatttc attatggtgaaagttggaacctcttacgtgccgatcaacgtctcattttcgccaaaagttggcccagggcttcc- c ggtatcaacagggacaccaggatttatttattctgcgaagtgatcttccgtcacaggtatttattcgaagacga- a agggatgcaggagtcgcataagggatttaacataatatacattatgcgcaccgcggccgcggaaagggcg aattctgcagatatccatcacactggcggccgctcgagcatgcatctagagtgaggaaactgaggttggttag aattccaagacatcttattattgccgcttcctcttggctttcaaa (pUCpF_difCAT fliC replacement cassette) SEQ ID NO: 28 cattccctgaggggcgtcggttacggtattgctctgacgctcaatgtcgatgccgtttacgttcagcttcgcgt- tttc tgctttcaccagctcttgcatattgccggtattggtggtgctgtcataagcgagtagatcgttaagttttgtat- cgcct tccaccgtgatcttcatcgtattgtcggtaccgctattggcggtaagcaccaactggaattcgttctctttgac- ctta acgatactggcggcgataccgctgtcggcgtcattaatggcgtcacggatcgcctccatggaggtgtcgccttt atccagcttaatttccagcggctctttacgtcccggctgttcaattttaattgtccgggatgtgaccgacgtat- cgc ccaactgctctttggtggttgcgaaggtggtttttgtcgccagcgactgcgcggcggcaagctgggttacgcta- a tcttataagtccctgcggcagcgcctgcggtagtactgactttgaggtcctctgtcgtgctggacgccacggta- g

acttaaataaatccgctttatttaacgcggtatttgccgtctggaatttttctaatgcgcttttcaatgtgcca- taggc ggttagctttgccgaattcgcgctctgctgtttggtaattggcgttaagcgtcctttttcgttctttgtcaggt- ctgtcaa caactggtctaacggtaagtttgatcccacacctaatgatgaaattgaagccatgccttcttcctttttgattg- caa acagtagttaagcgcgttatcggcaatctggaggcaaagtttaatgataattttgcaaaaataatgcgcggaat aatgatgcataaagcggctatttcgccgcctaagaaaaagatcgggggaagtgaaaaattttctaaagttcg aaattcaggtgccgatacaagggttacggtgagaaaccgtgggcaacagcccaataacatcaagttgtaatt gataaggaaaagatcatggcacaagtcattaatacaaacagcctgtcgctgttgacccagaataacctgaa caaatcccagtccgctctgggcaccgctatcgagcgtctgtcttccggtctgcgtatcaacagcgcgaaagac gatgcggcaggtcaggcaattgctaaccgtttcaccgcgaacatcaaaggtctgactcaggcttcccgtaac gctaacgacggtatctccattgcgcagaccactgaaggcgcgctgaacgaaatcaacaacaacctgcagc gtgtgcgtgaactggcggttcagtctgctaacagcaccaactcccagtctgacctcgactccatccaggctga aatcacccagcgcctgaacgaaatcgaccgtgtatccggtcagactcagttcaacggcgtgaaagtcctgg cgcaggacaacaccctgaccatccaggttggtgccaacaacggtgaaaccattgatatcgatctgaaacag atcaactctcagaccctgggtctggatacgctgaatgtgcagaaaaaatatgatgtgaagagcgaagcggtc acgccttcggctacattaagcactactgcacttgatggtgctggcctcaaaaccggaaccggttctacaactg atactggttcaattaaggatggtaaggtttactataacagcacctctaaaaattattatgttgaagtagaattt- acc gatgcgaccgatcaaaccaacaaaggcggattctataaagttaatgttgctgatgatggtgcagtcacaatga ctgcggctaccaccaaagaggctacaactcctacaggtattactgaagttactcaagtccaaaaacctgtgg ctgctccagctgctatccaggctcagttgactgctgcccatgtgaccggcgctgatactgctgaaatggttaag atgtcttatacggataaaaacggtaagactattgatggcggtttcggtgttaaagttggggctgatatttatgc- tgc aacaaaaaataaagatggatcgttcagcattaacaccactgaatataccgataaagacggcaacactaaa actgcactaaaccaactgggtggcgcagacggtaaaactgaagttgtttctatcgacggtaaaacctacaat gccagcaaagccgctggtcacaactttaaagcacagccagagctggctgaagcggctgctgcaaccaccg aaaacccgctggctaaaattgatgccgcgctggcgcaggttgatgcgctgcgttctgacttgggtgcggttcag aaccgtttcaactccgctatcaccaacctgggcaataccgtaaataacctgtcttctgcccgtagccgtatcga agattccgactacgcgaccgaagtttccaacatgtctcgcgcgcagatcctgcagcaggccggtacctccgtt ctggcgcaggcgaaccaggttccgcaaaacgtcctctctttactgcgttaatgcggccgcatttaacataatat acattatgcgcaccgcccgaacaccactcgccacaaaaaaccgccggaacgtccaaaagtacgggttttg ctgcccgcaaacgggctgttctggtgttgctagtttgttatcagaatcgcagatccggcttcagccggtttgcc- gg ctgaaagcgctatttcttccagaattgccatgattttttccccacgggaggcgtcactggctcccgtgttgtcg- gc agctttgattcgataagcagcatcgcctgtttcaggctgtctatgggccggccaaatcagtaagttggcagcat- c acccgacgcactttgcgccgaataaatacctgtgacggaagatcacttcgcagaataaataaatcctggtgtc cctgttgataccgggaagccctgggccaacttttggcgaaaatgagacgttgatcggcacgtaagaggttcc aactttcaccataatgaaataagatcactaccgggcgtattttttgagttatcgagattttcaggagctaagga- a gctaaaatggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacattttg aggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaagacc- gt aaagaaaaataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaat- tc cgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagca- a actgaaacgttttcatcgctctggagtgaataccacgacgatttccggcagtttctacacatatattcgcaaga- tg tggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagccaat- ccct gggtgagtttcaccagttttgatttaaacgtggccaatatggacaacttcttcgcccccgttttcaccatgggc- aa atattatacgcaaggcgacaaggtgctgatgccgctggcgattcaggttcatcatgccgtttgtgatggcttcc- a tgtcggcagaatgcttaatgaattacaacagtactgcgatgagtggcagggcggggcgtaatttttttaaggca gttattggtgcccttaaacgcctggttgctacgcctgaataagtgataataagcggatcctaggatggtgttaa- g cgggcggttttgagatgtaaactcgcccatttaacataatatacattatgcgcaccgcggccgccagtgtgagg atccccggcgattgattcaccgacacgtggtacacaatcaaggcagcgaaagctgccttttttaattccggag cctgtgtaatgaaagaaatcaccgtcactgaacctgcctttgtcacccgcttttcctgttctggctcggcctgt- cgc gaccattgttgtaagggctggaaaatcacgctggataagacgacggttaaaaagtatctcgccagtaaagac acgacgattcgtaccatcgcgcaagaccatattattctgctgaaaaagaacaataatcattggggggaaatta aactgccttcggcgctgggaagttgcccttatctggatgaggaccgtttgtgccgggtacaaaaacgttaggc gcaaaggcattaagtcatacctgttcctctttcccacgggcgcaccatacctataaaaatgaggtacgtaactc cctgagtcttgcctgtccggaggtaacgtcccgcattttaaacgatcctgacgcaatggcgctcggcgaaaaa acaatcattcagcagacattcaatactgcgccgttattctcaccgcagcaaaagttactcaatctgttttgcct- ga gtctgatcaaccatgccaacagcagtacggaaacggcgctctatgggttgattaaattcgtcatgtatgcacat aaatttgccaaaattgatgatgccgcgctgggtgaactggaacaggtgtatgccgcgttacttgagcagttgca gaccggcgtgctggcgcaggaattgatgaatatcgcgccggacagcaaggtaaaaacctcgctggtattgc agatgcagaactatttccgctcgctcccgcttagtcgtggcagtgttatcctcgatcactatatccagtgtctt- ctgc gggtgctgacggcggaagagggcgtttcaatggagcagaaggttagcgatattgagtcctcattagcgcgct gtttacaggcggatgagcagcagaagaactgggctttcagaaatttaattctctataaaatttgggaaaataat- t tccccaaccagccgaatg (tviA expression plasmid pBRT4tviA) SEQ ID NO: 29 aacatcgatattgccatcgcggatgtcgcctgtcttatctaccatcataaacatcatttgcctatggctcacga- ca gtataggcaatgccgttttttatattgctaattgtttcgccaatcaacgcaaaagtatggcgattgctaaagcc- gtc tccctgggcggtagattagccttaaccgcgacggtaatgactcattcatactggagtggtagtttgggactaca gcctcatttattagagcgtcttaatgatattacctatggactaatgagttttactcgcttcggtatggatggga- tggc aatgaccggtatgcaggtcagcagcccattatatcgtttgctggctcaggtaacgccagaacaacgtgcgcc ggagtaatcgttttcaggtatataccggatgttcattgctttctaaattttgctatgttgccagtatccttacg- atgtattt attttaaggaaaagccatatgaggtttcatcatttctggcctccgaatgatatctatttcggggttggagctgc- tgg cattattgaagaagtgtcactgataacaaatgacagaaattatttgtttgtgaacctaaatcgctacagcctgt- ta aatgccctgaattttttcacgcgaatgagtgatattaataaaataatcgttatcatttcaagttcgcgactaat- gcc ccttgcacgtttttggttgacagagtgcaaaaatgttattgctgttttcgatgcggcaacatcagtccaggata- ttat cagaaatgtcagtcaacaccaaagtggtgaaaagatcttgacggagcagagagattatcgtttcagaattaa ccgtaaggatatagtaaagatgaaatatttcctttcggaaagtggtatggaagagcttcaggatagatttatga actcatcatcgactatgtatcgctggagaaaagaattggcagtaaaatttggagtacgtgagccgcgctatctg ttattgccggattcagttactttactgtaatgtcgacataaaacgaaaggctcagtcgaaagactgggcctttc- gt tttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccgggagcggatttgaacgttgcgaa- g caacggcccggagggtggcgggcaggacgcccgccataaactgccaggcatcaaattaagcagaaggc catcctgacggatggccttttctgcagataaaaggatctaggtgaagatcctttttgataatctcatgaccaaa- at cccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctt- ttt ttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagag ctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagcc- g tagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggc- t gctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcgg tcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacc tacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcg gcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtc gggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgc cagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatccc- ctgattc tgtggataaccgtattaccgcctttgagtgagctgataccgctttttcgtgacattcagttcgctgcgctcacg- gct ctggcagtgaatgggggtaaatggcactacaggcgccttttatggattcatgcaaggaaactacccataatac aagaaaagcccgtcacgggcttctcagggcgttttatggcgggtctgctatgtggtgctatctgactttttgct- gtt cagcagttcctgccctctgattttccagtctgaccacttcggattatcccgtgacaggtcattcagactggcta- atg cacccagtaaggcagcggtatcatcaacaggcttacccgtcttactgtcaaccagacccgccaggataagc aatccggcagactggtacagagcatggtcacgggctttacgggcggctctggcttcggctcgcttttctgcctg- t atcaggttcatgagcggccgcggcgcgccagcttatcattgataagcttcttgaactctttatcactgataaag- a cgcgtcatagacagcctgaaacaggcgatgctgcttatcgaatcaaagctgccgacaacacgggagccag tgacgcctcccgtggggaaaaaatcatggcaattctggaagaaatagcgctttcagccggcaaaccggctg

aagccggatctgcgattctgataacaaactagcaacaccagaacagcccgtttgcgggcagcaaaacccg tacttttggacgttccggcggttttttgtggcgagtggtgttcgggcggtgcgcgcaagatccattatgttaaa- cgg gcgagtttacatctcaaaaccgcccgcttaacaccattcatgagcggccgccagtgtgctggaattcggcttca tgattttttattcaacgaagagtt (fepE expression plasmid pBAD2fepE) SEQ ID NO: 30 gtgcctgtcaaatggacgaagcagggattctgcaaaccctatgctactccgtcaagccgtcaattgtctgattc gttaccaattatgacaacttgacggctacatcattcactttttcttcacaaccggcacggaactcgctcgggct- g gccccggtgcattttttaaatacccgcgagaaatagagttgatcgtcaaaaccaacattgcgaccgacggtgg cgataggcatccgggtggtgctcaaaagcagcttcgcctggctgatacgttggtcctcgcgccagcttaagac gctaatccctaactgctggcggaaaagatgtgacagacgcgacggcgacaagcaaacatgctgtgcgacg ctggcgatatcaaaattgctgtctgccaggtgatcgctgatgtactgacaagcctcgcgtacccgattatccat- c ggtggatggagcgactcgttaatcgcttccatgcgccgcagtaacaattgctcaagcagatttatcgccagca gctccgaatagcgcccttccccttgcccggcgttaatgatttgcccaaacaggtcgctgaaatgcggctggtgc gcttcatccgggcgaaagaaccccgtattggcaaatattgacggccagttaagccattcatgccagtaggcg cgcggacgaaagtaaacccactggtgataccattcgcgagcctccggatgacgaccgtagtgatgaatctct cctggcgggaacagcaaaatatcacccggtcggcaaacaaattctcgtccctgatttttcaccaccccctgac cgcgaatggtgagattgagaatataacctttcattcccagcggtcggtcgataaaaaaatcgagataaccgtt ggcctcaatcggcgttaaacccgccaccagatgggcattaaacgagtatcccggcagcaggggatcattttg cgcttcagccatacttttcatactcccgccattcagagaagaaaccaattgtccatattgcatcagacattgcc- gt cactgcgtcttttactggctcttctcgctaaccaaaccggtaaccccgcttattaaaagcattctgtaacaaag- cg ggaccaaagccatgacaaaaacgcgtaacaaaagtgtctataatcacggcagaaaagtccacattgattat ttgcacggcgtcacactttgctatgccatagcatttttatccataagattagcggatcctacctgacgcttttt- atcg caactctctactgtttctccatacccgtttttttgggctagcgaattgaggaggagatatacaTatgccatctc- tta atgtaaaacaagaaaaaaatcagtcatttgcaggttattcactgccgcccgccaacagtcatgaaatcgattt gtttagccttatagaggtgttatggcaggcgaaacgtcgtattcttgctaccgttttcgcctttgcgtgcgtgg- ggtt gcttctgtcctttctgctgccgcaaaaatggaccagccaggcgattgtcacaccggcggagtcggtacagtgg caggggctggagagaacgttgaccgcgctgcgcgtgttggatatggaggtaagcgttgatcggggcagcgt atttaatctgtttattaaaaagtttagctcgccctcgctgctggaagaatatcttcgttcttctccgtatgtca- tggatc aattaaaaggcgcgcaaatagacgagcaggatcttcaccgggcgattgtcctgctgagcgaaaaaatgaa agcggtggacagtaatgtcggcaagaaaaatgaaacgtcgttattcacgtcgtggacattgagttttaccgcg ccgacgcgggaagaagcgcaaaaagtgctggctggctatattcagtacatctccgatatcgtcgtgaaaga gacgctggaaaatattcgtaaccagctggaaatcaaaacccgctatgagcaggaaaagctggcgatggat cgggtgcgtctcaaaaatcagcttgatgccaatattcaacgtcttcattattcgctggaaatcgccaacgccgc cggtattaagagaccggtttacagcaatggtcaggcggtaaaagatgatccggatttttctatttctctcggcg- c ggatggtatttcccgcaaactggaaattgaaaaaggggtaacggacgtggccgagatcgacggtgatttgc gtaaccgtcaataccatgttgaacaactggcggcaatgaatgtgagtgacgtgaagtttaccccgtttaaatat caactgtcgccgtctctgccagtgaaaaaagatggcccgggtaaagccatcattattatcctggcggcgttgat tggcggtatgatggcctgcggcggcgtattactgcgtcacgcgatggtctcgcgtaaaatggaaaacgcgct ggcgatagatgaacggttagtctgaGtcgacctgcaggcatgcaagcttggctgttttggcggatgagagaa gattttcagcctgatacagattaaatcagaacgcagaagcggtctgataaaacagaatttgcctggcggcagt agcgcggtggtcccacctgaccccatgccgaactcagaagtgaaacgccgtagcgccgatggtagtgtgg ggtctccccatgcgagagtagggaactgccaggcatcaaataaaacgaaaggctcagtcgaaagactgg gcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccgggagcggatttgaa- cgt tgcgaagcaacggcccggagggtggcgggcaggacgcccgccataaactgccaggcatcaaattaagc agaaggccatcctgacggatggcctttttgcgtttctacaaactctttttgtttatttttctaaatacattcaa- atatgta tccgctcatgtggccggcccggcctaggaaagccacgttgtgtctcaaaatctctgatgttacattgcacaaga taaaaatatatcatcatgaacaataaaactgtctgcttacataaacagtaatacaaggggtgttatgagccata ttcaacgggaaacgtcttgctcgaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgg- g ctcgcgataatgtcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagttgttt ctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacgga atttatgcctcttccgaccatcaagcattttatccgtactcctgatgatgcatggttactcaccactgcgatcc- ccg ggaaaacagcattccaggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttc ctgcgccggttgcattcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggc- gcaat cacgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtc- t ggaaagaaatgcataagcttttgccattctcaccggattcagtcgtcactcatggtgatttctcacttgataac- ctt atttttgacgaggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatct- t gccatcctatggaactgcctcggtgagttttctccttcattacagaaacggctttttcaaaaatatggtattga- taat cctgatatgaataaattgcagtttcatttgatgctcgatgagtttttctaatcagaattggttaattggttgta- acactg gcagagcattacgctgacttgacgggacggcggctttgttgaataaatcgaacttttcctaggccgggccggc cacatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggat cttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtt- tgtt tgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgt ccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaa- t cctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccgg ataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctaca ccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggaca ggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctgg tatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcg- gagc ctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacttttacgg- ttcct ggccttttgctggccttttgctcacatgtatggtgttaagcgggcggttttgagatgtaaactcgcccgtttaa- cata atggatcttgcgcgcaccgcccgaacaccactcgccacaaaaaaccgccggaacgtccaaaagtacggg ttttgctgcccgcaaacgggctgttctggtgttgctagtttgttatcagaatcgcagatccggcttcagccggt- ttgc cggctgaaagcgctatttcttccagaattgccatgattttttccccacgggaggcgtcactggctcccgtgttg- tc ggcagctttgattcgataagcagcatcgcctgtttcaggctgtctatgacatgttctttcctgcgttatcccca- attgt gagcgctcacaatttgctgcggtaagtcgcataaaaaccattcttcataattcaatccatttactatgttatgt- tctg ag

Sequence CWU 1

1

3011521DNASalmonella typhi 1atggcacaag tcattaatac aaacagcctg tcgctgttga cccagaataa cctgaacaaa 60tcccagtccg cactgggcac tgctatcgag cgtttgtctt ccggtctgcg tatcaacagc 120gcgaaagacg atgcggcagg acaggcgatt gctaaccgtt ttaccgcgaa catcaaaggt 180ctgactcagg cttcccgtaa cgctaacgac ggtatctcca ttgcgcagac cactgaaggc 240gcgctgaacg aaatcaacaa caacctgcag cgtgtgcgtg aactggcggt tcagtctgcg 300aatggtacta actcccagtc tgacctcgac tccatccagg ctgaaatcac ccagcgcctg 360aacgaaatcg accgtgtatc cggccagact cagttcaacg gcgtgaaagt cctggcgcag 420gacaacaccc tgaccatcca ggttggtgcc aacgacggtg aaactatcga tattgattta 480aaagaaatca gctctaaaac actgggactt gataagctta atgtccaaga tgcctacacc 540ccgaaagaaa ctgctgtaac cgttgataaa actacctata aaaatggtac agatcctatt 600acagcccaga gcaatactga tatccaaact gcaattggcg gtggtgcaac gggggttact 660ggggctgata tcaaatttaa agatggtcaa tactatttag atgttaaagg cggtgcttct 720gctggtgttt ataaagccac ttatgatgaa actacaaaga aagttaatat tgatacgact 780gataaaactc cgttggcaac tgcggaagct acagctattc ggggaacggc cactataacc 840cacaaccaaa ttgctgaagt aacaaaagag ggtgttgata cgaccacagt tgcggctcaa 900cttgctgcag caggggttac tggcgccgat aaggacaata ctagccttgt aaaactatcg 960tttgaggata aaaacggtaa ggttattgat ggtggctatg cagtgaaaat gggcgacgat 1020ttctatgccg ctacatatga tgagaaaaca ggtgcaatta ctgctaaaac cactacttat 1080acagatggta ctggcgttgc tcaaactgga gctgtgaaat ttggtggcgc aaatggtaaa 1140tctgaagttg ttactgctac cgatggtaag acttacttag caagcgacct tgacaaacat 1200aacttcagaa caggcggtga gcttaaagag gttaatacag ataagactga aaacccactg 1260cagaaaattg atgctgcctt ggcacaggtt gatacacttc gttctgacct gggtgcggtt 1320cagaaccgtt tcaactccgc tatcaccaac ctgggcaata ccgtaaataa cctgtcttct 1380gcccgtagcc gtatcgaaga ttccgactac gcaaccgaag tctccaacat gtctcgcgcg 1440cagattctgc agcaggccgg tacctccgtt ctggcgcagg cgaaccaggt tccgcaaaac 1500gtcctctctt tactgcgtta a 152121488DNASalmonella Paratyphi 2atggcacaag tcattaatac aaacagcctg tcgctgttga cccagaataa cctgaacaaa 60tcccagtccg ctctgggcac cgctatcgag cgtctgtctt ccggtctgcg tatcaacagc 120gcgaaagacg atgcggcagg tcaggcaatt gctaaccgtt tcaccgcgaa catcaaaggt 180ctgactcagg cttcccgtaa cgctaacgac ggtatctcca ttgcgcagac cactgaaggc 240gcgctgaacg aaatcaacaa caacctgcag cgtgtgcgtg aactggcggt tcagtctgct 300aacagcacca actcccagtc tgacctcgac tccatccagg ctgaaatcac ccagcgcctg 360aacgaaatcg accgtgtatc cggtcagact cagttcaacg gcgtgaaagt cctggcgcag 420gacaacaccc tgaccatcca ggttggtgcc aacaacggtg aaaccattga tatcgatctg 480aaacagatca actctcagac cctgggtctg gatacgctga atgtgcagaa aaaatatgat 540gtgaagagcg aagcggtcac gccttcggct acattaagca ctactgcact tgatggtgct 600ggcctcaaaa ccggaaccgg ttctacaact gatactggtt caattaagga tggtaaggtt 660tactataaca gcacctctaa aaattattat gttgaagtag aatttaccga tgcgaccgat 720caaaccaaca aaggcggatt ctataaagtt aatgttgctg atgatggtgc agtcacaatg 780actgcggcta ccaccaaaga ggctacaact cctacaggta ttactgaagt tactcaagtc 840caaaaacctg tggctgctcc agctgctatc caggctcagt tgactgctgc ccatgtgacc 900ggcgctgata ctgctgaaat ggttaagatg tcttatacgg ataaaaacgg taagactatt 960gatggcggtt tcggtgttaa agttggggct gatatttatg ctgcaacaaa aaataaagat 1020ggatcgttca gcattaacac cactgaatat accgataaag acggcaacac taaaactgca 1080ctaaaccaac tgggtggcgc agacggtaaa actgaagttg tttctatcga cggtaaaacc 1140tacaatgcca gcaaagccgc tggtcacaac tttaaagcac agccagagct ggctgaagcg 1200gctgctgcaa ccaccgaaaa cccgctggct aaaattgatg ccgcgctggc gcaggttgat 1260gcgctgcgtt ctgacttggg tgcggttcag aaccgtttca actccgctat caccaacctg 1320ggcaataccg taaataacct gtcttctgcc cgtagccgta tcgaagattc cgactacgcg 1380accgaagttt ccaacatgtc tcgcgcgcag atcctgcagc aggccggtac ctccgttctg 1440gcgcaggcga accaggttcc gcaaaacgtc ctctctttac tgcgttaa 148831488DNASalmonella paratyphi 3atggcacaag tcattaatac aaacagcctg tcgctgttga cccagaataa cctgaacaaa 60tcccagtccg ctctgggcac cgctatcgag cgtctgtctt ctggtctgcg tatcaacagc 120gcgaaagacg atgcggcagg tcaggcgatt gctaaccgtt ttaccgcgaa catcaaaggt 180ctgactcagg cttcccgtaa cgctaacgac ggtatctcca ttgcgcagac cactgaaggc 240gcgctgaacg aaatcaacaa caacctgcag cgtgtgcgtg aactggcggt tcagtctgct 300aacagcacca actcccagtc tgacctcgac tccatccagg ctgaaatcac ccagcgcctg 360aacgaaatcg accgtgtatc cggccagact cagttcaacg gcgtgaaagt cctggcgcag 420gacaacaccc tgaccatcca ggttggcgcg aacgacggtg aaactatcga tatcgatctg 480aagcagatca actctcagac cctgggtctg gatactttaa gtgtacagga tgcctatacg 540ccaaaaggta ccgctgttac cagagatgtt accacctata aaaatggtgg tactactctt 600acagcaccta acgcagcagc aattgatacc gctttaggta cgactggtgc ggcgggtact 660gcggctgtga aatttaaaga cggtaactac ttcgttgagg tgaccggtac aactaaagat 720ggtctgtatg aagcgacagt tgatgcagct ggcgcggtga caatgaccgc aaataaagca 780acagtaactg gggctagtac agttactgaa aaccaaattg tagacgctgt tacaccgacg 840ccagttgata cagtcgcagc agctactgca ttgaccaatg caggtgtgac aggtgcgaca 900ggtaatacca gcttggttaa aatgtcattt gaagataaaa atggcaaagt tactgatgcg 960ggttacgcgc ttaaagttgg aaatgattat tatgccgctg attacgatga aaaaactggt 1020gagataaaag ctaaaactgt aaattatact gacgctactg gtgcgacaaa aaccggtgct 1080gtgaaatttg gcggtgcgaa tggtaaaact gaagttgtga ccaccgttga tggtaatact 1140tatcaggcta gtgatgtaaa agggcataat ttccagagtg gtggcgcttt aagcgaggct 1200gtaaccacta aaactgaaaa cccgctggct aaaattgatg ccgcgctggc gcaagttgat 1260gcgctgcgtt ctgacttggg tgcggttcag aaccgtttca actccgctat caccaacctg 1320ggcaataccg taaacaacct gtctgaagcc cgtagccgta tcgaagattc cgactacgcg 1380accgaagtct ccaacatgtc ccgcgcgcag attctgcagc aggccggtac ctccgttctg 1440gcgcaggcga accaggttcc gcaaaacgtc ctctctttac tgcgttaa 148841521DNASalmonella paratyphi 4atggcacaag taatcaacac taacagtctg tcgctgctga cccagaataa cctgaacaaa 60tcccagtccg cactgggcac cgctatcgag cgtctgtctt ctggtctgcg tatcaacagc 120gcgaaagacg atgcggcagg tcaggcaatt gctaaccgtt tcaccgcgaa catcaaaggc 180ctgactcagg cttcccgtaa cgctaacgac ggtatctcca ttgcgcagac cactgaaggc 240gcgctgaacg aaatcaacaa caacctgcag cgtgtgcgtg aactggcggt tcagtctgct 300aacagcacta actcccagtc tgacctcgac tctatccagg ctgaaattac ccagcgtctg 360aacgaaatcg accgtgtatc cggccagact cagttcaacg gcgtgaaagt cctggcgcag 420gacaacaccc tgaccatcca ggttggtgcc aacgacggtg aaactatcga tatcgatctg 480aagcagatca actctcagac cctgggtctg gactcactga acgtgcagaa agcgtatgat 540gtgaaagata cagcagtaac aacgaaagct tatgccaata atggtactac actggatgta 600tcgggtcttg atgatgcagc tattaaagcg gctacggggg gtacgaatgg tacggcttct 660gtaaccggtg gtgcggttaa atttgacgca gataataaca agtactttgt tactattggt 720ggctttactg gtgctgatgc cgccaaaaat ggcgattatg aagttaacgt tgctactgac 780ggtacagtaa cccttgcggc tggcgcaact aaaaccacaa tgcctgctgg tgcgacaact 840aaaacagaag tacaggagtt aaaagataca ccggcagttg tttcagcaga tgctaaaaat 900gccttaattg ctggcggcgt tgacgctacc gatgctaatg gcgctgagtt ggtcaaaatg 960tcttataccg ataaaaatgg taagacaatt gaaggcggtt atgcgcttaa agctggcgat 1020aagtattacg ccgcagatta cgatgaagcg acaggagcaa ttaaagctaa aaccacaagt 1080tatactgctg ctgacggcac taccaaaaca gcagctaacc aactgggtgg cgtagacggt 1140aaaaccgaag tcgttactat cgacggtaaa acctacaatg ccagcaaagc cgctggtcat 1200gatttcaaag cacaaccaga gctggcggaa gcagccgcta aaaccaccga aaacccgctg 1260cagaaaattg atgccgcgct ggcgcaggtg gatgcgctgc gctctgatct gggtgcggta 1320caaaaccgtt tcaactccgc tatcaccaac ctgggcaata ccgtaaacaa cctgtctgaa 1380gcgcgtagcc gtatcgaaga ttccgactac gcgaccgaag tttccaacat gtctcgcgcg 1440cagattctgc agcaggccgg tacttccgtt ctggcgcagg ctaaccaggt cccgcagaac 1500gtgctgtctc tgttacgtta a 152151506DNASalmonella paratyphi 5atggcacaag tcattaatac aaacagcctg tcgctgttga cccagaataa cctgaacaaa 60tcccagtctg ctctgggtac cgctatcgag cgtctgtctt ccggtctgcg tatcaacagc 120gcgaaagacg atgcggcagg tcaggcgatt gctaaccgtt tcaccgcgaa catcaaaggt 180ctgactcagg cttcccgtaa cgctaacgac ggtatctcca ttgcgcagac cactgaaggc 240gcgctgaacg aaatcaacaa caacctgcag cgtgtgcgtg aactggcggt tcagtctgct 300aacagcacca actcccagtc tgacctcgac tccatccagg ctgaaatcac ccagcgtctg 360aacgaaatcg accgtgtatc cggtcagact cagttcaacg gcgtgaaagt cctggcgcag 420gacaacactc tgaccatcca ggttggtgcc aacgacggtg aaactatcga tatcgatctg 480aagcagatca actctcagac cctgggccta gatacgctga atgtgcagaa aaaatatgat 540gtgagcgata ctgctgtagc tgcttcctat tccgactcga aacagaatat tgctgttcct 600gataaaacag ctattactgc aaaaattggt gcagcaacca gtggtggtgc tggtataaaa 660gcagatatta gctttaaaga tggcaagtat tacgcgactg tcagtggata cgatgatgcc 720gcagatacag ataaaaatgg aacctatgaa gtcactgttg ccgcagatac aggagcagtt 780acttttgcga ctacaccaac agtggttgac ttaccaactg atgcaaaagc agtttcaaaa 840gttcaacaga atgatactga aatagcagca acaaatgcga aagctgcatt aaaagctgca 900ggagttgcag atgcagaagc tgatacagct actttagtga aaatgtctta tacagataat 960aatggcaaag ttattgatgg tgggttcgca tttaagacct ccggtggtta ttatgcagca 1020tctgttgata aatctggcgc agctagcttg aaagttacta gctacgttga cgctaccact 1080ggtaccgaaa aaactgctgc gaataaatta ggtggcgcag acggtaaaac cgaagttgtt 1140actatcgacg gtaaaaccta caatgccagc aaagccgctg ggcacaactt caaagcacag 1200ccagagctgg cggaagcggc tgctacaacc actgaaaacc cgctgcagaa aattgatgct 1260gctttggcgc aggtggatgc gctgcgttct gacctgggtg cggttcagaa ccgtttcaac 1320tccgctatca ccaacctggg caataccgta aataacctgt cttctgcccg tagccgtatc 1380gaagattccg actacgcgac cgaagtttcc aacatgtctc gcgcgcagat tctgcagcag 1440gccggtacct ccgttctggc gcaggcgaac caggttccgc aaaacgtcct ctctttactg 1500cgttaa 150661521DNASalmonella paratyphi 6atggcacaag taatcaacac taacagtctg tcgctgctga cccagaataa cctgaacaaa 60tcccagtccg cactgggcac cgctatcgag cgtctgtctt ccggtctgcg tatcaacagc 120gcgaaagacg atgcggcagg tcaggcgatt gctaaccgtt ttaccgcgaa catcaaaggt 180ctgactcagg cttcccgtaa cgctaacgac ggtatttcta ttgcgcagac cactgaaggc 240gcgctgaacg aaatcaacaa caacctgcag cgtgtgcgtg aactggcggt tcagtctgct 300aacagcacta actcacagtc tgacctcgac tctatccagg ctgaaatcac ccagcgtctg 360aacgaaatcg accgtgtatc cggtcagact cagttcaacg gcgtgaaagt cctggcgcag 420gacaacaccc tgaccatcca ggttggtgcc aacgacggtg aaactatcga tatcgatctg 480aagcagatca actctcagac cctgggtctg gactcactga acgtgcagaa agcgtatgat 540gtgaaagata cagcagtaac aacgaaagct tatgccaata atggtactac actggatgta 600tcgggtcttg atgatacagc tatcaaagcg gctataggtg gtacgactgg tacggctgct 660gtaaccggta gtgcggttaa atttgacgca gataataaca agtactttgt tactattggt 720ggctttactg gtgctgatgc cgccaaaaat ggcgattatg aagttaacgt tgctactgac 780ggtacagtaa cccttgcggc tggcgcaact aaaaccacaa tgcctgctgg tgcgacaact 840aaaacagaag tacaggagtt aaaagataca ccggcagttg tttcagcaga tgctaaaaat 900gccttaattg ctggcggcgt tgacgctacc gatgctaatg gcgctgagtt ggtcaaaatg 960tcttataccg ataaaaatgg taagacaatt gaaggcggtt atgcgcttaa agctggcgat 1020aagtattacg ccgcagatta cgatgaagcg acaggagcaa ttaaagctaa aaccacaagt 1080tatactgctg ctgacggcac taccaaaaca gcagctaacc aactgggtgg cgtagacggt 1140aaaaccgaag tcgttactat cgacggtaaa acctacaatg ccagcaaagc cgctggtcat 1200gatttcaaag cacaaccaga gctggcggaa gcagccgcta aaaccaccga aaacccgctg 1260cagaaaattg atgccgcgct ggcgcaggtg gatgcgctgc gctctgatct gggtgcggta 1320caaaaccgtt tcaactccgc tatcaccaac ctgggcaata ccgtaaacaa cctgtctgaa 1380gcgcgtagcc gtatcgaaga ttccgactac gcgaccgaag tttccaacat gtctcgcgcg 1440cagattctgc agcaggccgg tacttccgtt ctggcgcagg ctaaccaggt cccgcagaac 1500gtgctgtctc tgttacgtta a 152171488DNASalmonella typhimurium 7atggcacaag tcattaatac aaacagcctg tcgctgttga cccagaataa cctgaacaaa 60tcccagtccg ctctgggcac cgctatcgag cgtctgtctt ccggtctgcg tatcaacagc 120gcgaaagacg atgcggcagg tcaggcgatt gctaaccgtt ttaccgcgaa catcaaaggt 180ctgactcagg cttcccgtaa cgctaacgac ggtatctcca ttgcgcagac cactgaaggc 240gcgctgaacg aaatcaacaa caacctgcag cgtgtgcgtg aactggcggt tcagtctgct 300aacagcacca actcccagtc tgacctcgac tccatccagg ctgaaatcac ccagcgcctg 360aacgaaatcg accgtgtatc cggccagact cagttcaacg gcgtgaaagt cctggcgcag 420gacaacaccc tgaccatcca ggttggtgcc aacgacggtg aaactatcga tatcgatctg 480aagcagatca actctcagac cctgggtctg gatacgctga atgtgcaaca aaaatataag 540gtcagcgata cggctgcaac tgttacagga tatgccgata ctacgattgc tttagacaat 600agtactttta aagcctcggc tactggtctt ggtggtactg accagaaaat tgatggcgat 660ttaaaatttg atgatacgac tggaaaatat tacgccaaag ttaccgttac ggggggaact 720ggtaaagatg gctattatga agtttccgtt gataagacga acggtgaggt gactcttgct 780ggcggtgcga cttccccgct tacaggtgga ctacctgcga cagcaactga ggatgtgaaa 840aatgtacaag ttgcaaatgc tgatttgaca gaggctaaag ccgcattgac agcagcaggt 900gttaccggca cagcatctgt tgttaagatg tcttatactg ataataacgg taaaactatt 960gatggtggtt tagcagttaa ggtaggcgat gattactatt ctgcaactca aaataaagat 1020ggttccataa gtattaatac tacgaaatac actgcagatg acggtacatc caaaactgca 1080ctaaacaaac tgggtggcgc agacggcaaa accgaagttg tttctattgg tggtaaaact 1140tacgctgcaa gtaaagccga aggtcacaac tttaaagcac agcctgatct ggcggaagcg 1200gctgctacaa ccaccgaaaa cccgctgcag aaaattgatg ctgctttggc acaggttgac 1260acgttacgtt ctgacctggg tgcggtacag aaccgtttca actccgctat taccaacctg 1320ggcaacaccg taaacaacct gacttctgcc cgtagccgta tcgaagattc cgactacgcg 1380accgaagttt ccaacatgtc tcgcgcgcag attctgcagc aggccggtac ctccgttctg 1440gcgcaggcga accaggttcc gcaaaacgtc ctctctttac tgcgttaa 148881521DNASalmonella typhimurium 8atggcacaag taatcaacac taacagtctg tcgctgctga cccagaataa cctgaacaaa 60tcccagtccg cactgggcac cgctatcgag cgtctgtctt ctggtctgcg tatcaacagc 120gcgaaagacg atgcggcagg tcaggcgatt gctaaccgtt tcaccgcgaa catcaaaggt 180ctgactcagg cttcccgtaa cgctaacgac ggtatctcca ttgcgcagac cactgaaggc 240gcgctgaacg aaatcaacaa caacctgcag cgtgtgcgtg aactggcggt tcagtctgct 300aacagcacca actcccagtc tgacctcgac tccatccagg ctgaaatcac ccagcgcctg 360aacgaaatcg accgtgtatc cggccagact cagttcaacg gcgtgaaagt cctggcgcag 420gacaacaccc tgaccatcca ggttggcgcc aacgacggtg aaactatcga tatcgatctg 480aagcagatca actctcagac cctgggtctg gactcactga acgtgcagaa agcgtatgat 540gtgaaagata cagcagtaac aacgaaagct tatgccaata atggtactac actggatgta 600tcgggtcttg atgatgcagc tattaaagcg gctacgggtg gtacgaatgg tacggcttct 660gtaaccggtg gtgcggttaa atttgacgca gataataaca agtactttgt tactattggt 720ggctttactg gtgctgatgc cgccaaaaat ggcgattatg aagttaacgt tgctactgac 780ggtacagtaa cccttgcggc tggcgcaact aaaaccacaa tgcctgctgg tgcgacaact 840aaaacagaag tacaggagtt aaaagataca ccggcagttg tttcagcaga tgctaaaaat 900gccttaattg ctggcggcgt tgacgctacc gatgctaatg gcgctgagtt ggtcaaaatg 960tcttataccg ataaaaatgg taagacaatt gaaggcggtt atgcgcttaa agctggcgat 1020aagtattacg ccgcagatta cgatgaagcg acaggagcaa ttaaagctaa aactacaagt 1080tatactgctg ctgacggcac taccaaaaca gcggctaacc aactgggtgg cgtagacggt 1140aaaaccgaag tcgttactat cgacggtaaa acctacaatg ccagcaaagc cgctggtcat 1200gatttcaaag cacaaccaga gctggcggaa gcagccgcta aaaccaccga aaacccgctg 1260cagaaaattg atgccgcgct ggcgcaggtg gatgcgctgc gctctgatct gggtgcggta 1320caaaaccgtt tcaactctgc tatcaccaac ctgggcaata ccgtaaacaa tctgtctgaa 1380gcgcgtagcc gtatcgaaga ttccgactac gcgaccgaag tttccaacat gtctcgcgcg 1440cagattctgc agcaggccgg tacttccgtt ctggcgcagg ctaaccaggt cccgcagaac 1500gtgctgtctc tgttacgtta a 152191518DNASalmonella enteritidis 9atggcacaag tcattaatac aaacagcctg tcgctgttga cccagaataa cctgaacaaa 60tctcagtcct cactgagttc cgctattgag cgtctgtcct ctggtctgcg tatcaacagc 120gcgaaagacg atgcggcagg ccaggcgatt gctaaccgct tcacttctaa tatcaaaggt 180ctgactcagg cttcccgtaa cgctaacgac ggcatttcta ttgcgcagac cactgaaggt 240gcgctgaatg aaatcaacaa caacctgcag cgtgtgcgtg agttgtctgt tcaggccact 300aacgggacta actctgattc cgatctgaaa tctatccagg atgaaattca gcaacgtctg 360gaagaaatcg atcgcgtttc taatcagact caatttaacg gtgttaaagt cctgtctcag 420gacaaccaga tgaaaatcca ggttggtgct aacgatggtg aaaccattac catcgatctg 480caaaaaattg atgtgaaaag ccttggcctt gatgggttca atgttaatgg gccaaaagaa 540gcgacagtgg gtgatctgaa atccagcttc aagaatgtta cgggttacga cacctatgca 600gcgggtgccg ataaatatcg tgtagatatt aattccggtg ctgtagtgac tgatgcagca 660gcaccggata aagtatatgt aaatgcagca aacggtcagt taacaactga cgatgcggaa 720aataacactg cggttgatct ctttaagacc actaaatcta ctgctggtac cgctgaagcc 780aaagcgatag ctggtgccat taaaggtggt aaggaaggag atacctttga ttataaaggc 840gtgactttta ctattgatac aaaaactggt gatgacggta atggtaaggt ttctactacc 900atcaatggtg aaaaagttac gttaactgtc gctgatattg ccactggcgc gacggatgtt 960aatgctgcta ccttacaatc aagcaaaaat gtttatacat ctgtagtgaa cggtcagttt 1020acttttgatg ataaaaccaa aaacgagagt gcgaaacttt ctgatttgga agcaaacaat 1080gctgttaagg gcgaaagtaa aattacagta aatggggctg aatatactgc taacgccacg 1140ggtgataaga tcaccttagc tggcaaaacc atgtttattg ataaaacagc ttctggcgta 1200agtacattaa tcaatgaaga cgctgccgca gccaagaaaa gtaccgctaa cccactggct 1260tcaattgatt ctgcattgtc aaaagtggac gcagttcgtt cttctctggg ggcaattcaa 1320aaccgttttg attcagccat taccaacctt ggcaatacgg taaccaatct gaactccgcg 1380cgtagccgta tcgaagatgc tgactatgca acggaagttt ctaatatgtc taaagcgcag 1440attctgcagc aggctggtac ttccgttctg gcgcaggcta accaggttcc gcaaaacgtc 1500ctctctttac tgcgttaa 151810540DNASalmonella typhi 10atgaggtttc atcatttctg gcctccgaat gatatctatt tcggggttgg agctgctggc 60attattgaag aagtgtcact gataacaaat gacagaaatt atttgtttgt gaacctaaat 120cgctacagcc tgttaaatgc cctgaatttt ttcacgcgaa tgagtgatat taataaaata 180atcgttatca tttcaagttc gcgactaatg ccccttgcac gtttttggtt gacagagtgc 240aaaaatgtta ttgctgtttt cgatgcggca acatcagtcc aggatattat cagaaatgtc 300agtcaacacc aaagtggtga aaagatcttg acggagcaga gagattatcg tttcagaatt 360aaccgtaagg atatagtaaa gatgaaatat ttcctttcgg aaagtggtat ggaagagctt 420caggatagat ttatgaactc atcatcgact atgtatcgct ggagaaaaga attggcagta 480aaatttggag tacgtgagcc gcgctatctg ttattgccgg attcagttac tttactgtaa 540111137DNASalmonella typhimurium 11atgccatctc ttaatgtaaa acaagaaaaa aatcagtcat ttgcaggtta ttcactgccg 60cccgccaaca gtcatgaaat cgatttgttt agccttatag aggtgttatg gcaggcgaaa 120cgtcgtattc ttgctaccgt tttcgccttt gcgtgcgtgg ggttgcttct gtcctttctg 180ctgccgcaaa aatggaccag ccaggcgatt gtcacaccgg cggagtcggt acagtggcag 240gggctggaga gaacgttgac cgcgctgcgc gtgttggata tggaggtaag cgttgatcgg 300ggcagcgtat ttaatctgtt tattaaaaag

tttagctcgc cctcgctgct ggaagaatat 360cttcgttctt ctccgtatgt catggatcaa ttaaaaggcg cgcaaataga cgagcaggat 420cttcaccggg cgattgtcct gctgagcgaa aaaatgaaag cggtggacag taatgtcggc 480aagaaaaatg aaacgtcgtt attcacgtcg tggacattga gttttaccgc gccgacgcgg 540gaagaagcgc aaaaagtgct ggctggctat attcagtaca tctccgatat cgtcgtgaaa 600gagacgctgg aaaatattcg taaccagctg gaaatcaaaa cccgctatga gcaggaaaag 660ctggcgatgg atcgggtgcg tctcaaaaat cagcttgatg ccaatattca acgtcttcat 720tattcgctgg aaatcgccaa cgccgccggt attaagagac cggtttacag caatggtcag 780gcggtaaaag atgatccgga tttttctatt tctctcggcg cggatggtat ttcccgcaaa 840ctggaaattg aaaaaggggt aacggacgtg gccgagatcg acggtgattt gcgtaaccgt 900caataccatg ttgaacaact ggcggcaatg aatgtgagtg acgtgaagtt taccccgttt 960aaatatcaac tgtcgccgtc tctgccagtg aaaaaagatg gcccgggtaa agccatcatt 1020attatcctgg cggcgttgat tggcggtatg atggcctgcg gcggcgtatt actgcgtcac 1080gcgatggtct cgcgtaaaat ggaaaacgcg ctggcgatag atgaacggtt agtctga 1137121017DNASalmonella typhi 12atgaagcttt taattaccgg tggatgtggc ttccttggga gtaatcttgc ctcctttgct 60ttaagtcaag ggattgattt aattgtattc gataatctat cacgtaaagg tgcaacagat 120aatttacatt ggttatcctc cttaggaaac tttgagtttg tacatggtga tattcgcaac 180aaaaatgatg ttacaagatt aataactaag tatatgcctg atagctgttt tcatcttgca 240ggtcaagtgg caatgactac atctattgac aatccttgta tggattttga aattaatgta 300ggtggaactt taaatttact tgaggcagta cggcagtata attcaaattg taatataatt 360tattcatcaa caaataaagt atacggcgat cttgagcaat ataaatacaa tgaaacagaa 420actagataca cttgtgtaga taagcctaat ggatatgatg agagcacaca attagatttc 480cactcaccat atggttgttc aaaaggtgct gcagaccaat acatgcttga ttatgcaagg 540atttttggtt tgaatacagt ggtgttcagg cattcatcaa tgtatggtgg gagacagttt 600gctacttatg atcaaggctg ggtaggttgg ttttgtcaaa aagcggttga aattaaaaat 660ggtattaata aacccttcac tatttctggt aatggtaagc aagttaggga tgttttgcat 720gctgaagata tgatttcgtt atatttcact gccttggcaa atgtatcaaa aattaggggg 780aacgctttta atattggtgg taccattgtc aacagcctat cattacttga attattcaaa 840ttgcttgaag attattgcaa catagatatg aggttcacta atttacctgt aagggaaagt 900gatcagcgtg tttttgttgc agatattaaa aaaatcacta atgcaattga ctggagcccg 960aaagtctcgg caaaagatgg tgtccagaaa atgtatgatt ggactagttc tatatga 101713666DNAEscherichia coli 13atgaatttgt atggtatttt tggtgctgga agttatggta gagaaacaat acccattcta 60aatcaacaaa taaagcaaga atgtggttct gactatgctc tggtttttgt ggatgatgtt 120ttggcaggaa agaaagttaa tggttttgaa gtgctttcaa ccaactgctt tctaaaagcc 180ccttatttaa aaaagtattt taatgttgct attgctaatg ataagatacg acagagagtg 240tctgagtcaa tattattaca cggggttgaa ccaataacta taaaacatcc aaatagcgtt 300gtttatgatc atactatgat aggtagtggc gctattattt ctccctttgt tacaatatct 360actaatactc atatagggag gttttttcat gcaaacatat actcatacgt tgcacatgat 420tgtcaaatag gagactatgt tacatttgct cctggggcta aatgtaatgg atatgttgtt 480attgaagaca atgcatatat aggctcgggt gcagtaatta agcagggtgt tcctaatcgc 540ccacttatta ttggcgcggg agccattata ggtatggggg ctgttgtcac taaaagtgtt 600cctgccggta taactgtgtg cggaaatcca gcaagagaaa tgaaaagatc gccaacatct 660atttaa 66614840DNASalmonella typhi 14atgaaaattc taataatggg agcgtttggg ttccttggat cacgacttac atcctacttc 60gaaagtcgac atactgtgat tggcttagca aggaagagga acaatgaagc taccataaat 120aatattattt acacgacaga aaataattgg atcgaaaaaa tactagaatt tgaaccgaat 180attattatta acactattgc ttgctatgga agacataacg aacctgcaac agctttaata 240gaaagcaata ttcttatgcc tatcagagta ttagaatcta tctcatcact tgatgcagta 300ttcataaatt gtggaacatc actgccacca aatacgagtt tatatgcata tactaaacaa 360aaagcaaatg aactcgccgc cgccattata gataaagttt gcggtaaata tatagagtta 420aaattggagc atttctatgg agcttttgat ggagacgata agtttaccag tatggttatt 480agacgttgtt taagtaacca gccagtaaag ttaacatctg gtttgcaaca gagagatttc 540ttgtatataa aagatctact aacagcgttc gattgtatta taagtaatgt taataatttc 600cccaaatttc atagtattga agttggtagt ggagaggcga tatcaattcg tgaatatgta 660gatactgtta aaaatatcac aaaaagcaat tctataattg aatttggcgt ggtcaaagaa 720agagtaaatg aattgatgta tagttgtgct gatatagcag aacttgaaaa aataggatgg 780aaaagagagt tctctcttgt tgatgcatta actgaaataa ttgaagagga agggaaatga 84015900DNASalmonella typhimurium 15atgacctttt tgaaagaata tgtaattgtc agtggggctt ccggctttat tggtaagcat 60ttactcgaag cgctaaaaaa atcggggatt tcagttgtcg caatcactcg agatgtaata 120aaaaataata gtaatgcatt agctaatgtt agatggtgca gttgggataa tatcgaatta 180ttagtcgagg agttatcaat tgattctgca ttaattggta tcattcattt ggcaacagaa 240tatgggcata aaacatcatc tctcataaat attgaagatg caaatgttat aaaaccatta 300aagcttcttg atttggcaat aaaatatcgg gcggatatct ttttaaatac agatagtttt 360tttgccaaga aagattttaa ttatcaacat atgcggcctt atataattac taaaagacac 420tttgatgaaa ttgggcatta ttatgctaat atgcatgaca tttcatttgt aaacatgcga 480ttagagcatg tatatgggcc tggggatggt gaaaataaat ttattccata cattatcgac 540tgcttaaata aaaaacagag ttgcgtgaaa tgtacaacag gcgaacagat aagagacttt 600atttttgtag atgatgtggt aaatgcttat ttaactatat tagaaaatag aaaagaagta 660ccttcatata ctgagtatca agttggaact ggtgctgggg taagtttgaa agattttctg 720gtttatttgc aaaatactat gatgccaggt tcatcgagta tatttgaatt tggtgcgata 780gagcaaagag ataatgaaat aatgttctct gtagcaaata ataaaaattt aaaagcaatg 840ggctggaaac caaatttcga ttataaaaaa ggaattgaag aactactgaa acggttatga 90016466DNASalmonella typhimurium 16tattgccatc gcggatgtcg cctgtcttat ctaccatcat aaacatcatt tgcctatggc 60tcacgacagt ataggcaatg ccgtttttta tattgctaat tgtttcgcca atcaacgcaa 120aagtatggcg attgctaaag ccgtctccct gggcggtaga ttagccttaa ccgcgacggt 180aatgactcat tcatactgga gtggtagttt gggactacag cctcatttat tagagcgtct 240taatgatatt acctatggac taatgagttt tactcgcttc ggtatggatg ggatggcaat 300gaccggtatg caggtcagca gcccattata tcgtttgctg gctcaggtaa cgccagaaca 360acgtgcgccg gagtaatcgt tttcaggtat ataccggatg ttcattgctt tctaaatttt 420gctatgttgc cagtatcctt acgatgtatt tattttaagg aaaagc 466171231DNAEscherichia coli 17ttatgacaac ttgacggcta catcattcac tttttcttca caaccggcac ggaactcgct 60cgggctggcc ccggtgcatt ttttaaatac ccgcgagaaa tagagttgat cgtcaaaacc 120aacattgcga ccgacggtgg cgataggcat ccgggtggtg ctcaaaagca gcttcgcctg 180gctgatacgt tggtcctcgc gccagcttaa gacgctaatc cctaactgct ggcggaaaag 240atgtgacaga cgcgacggcg acaagcaaac atgctgtgcg acgctggcga tatcaaaatt 300gctgtctgcc aggtgatcgc tgatgtactg acaagcctcg cgtacccgat tatccatcgg 360tggatggagc gactcgttaa tcgcttccat gcgccgcagt aacaattgct caagcagatt 420tatcgccagc agctccgaat agcgcccttc cccttgcccg gcgttaatga tttgcccaaa 480caggtcgctg aaatgcggct ggtgcgcttc atccgggcga aagaaccccg tattggcaaa 540tattgacggc cagttaagcc attcatgcca gtaggcgcgc ggacgaaagt aaacccactg 600gtgataccat tcgcgagcct ccggatgacg accgtagtga tgaatctctc ctggcgggaa 660cagcaaaata tcacccggtc ggcaaacaaa ttctcgtccc tgatttttca ccaccccctg 720accgcgaatg gtgagattga gaatataacc tttcattccc agcggtcggt cgataaaaaa 780atcgagataa ccgttggcct caatcggcgt taaacccgcc accagatggg cattaaacga 840gtatcccggc agcaggggat cattttgcgc ttcagccata cttttcatac tcccgccatt 900cagagaagaa accaattgtc catattgcat cagacattgc cgtcactgcg tcttttactg 960gctcttctcg ctaaccaaac cggtaacccc gcttattaaa agcattctgt aacaaagcgg 1020gaccaaagcc atgacaaaaa cgcgtaacaa aagtgtctat aatcacggca gaaaagtcca 1080cattgattat ttgcacggcg tcacactttg ctatgccata gcatttttat ccataagatt 1140agcggatcct acctgacgct ttttatcgca actctctact gtttctccat acccgttttt 1200ttgggctagc gaattgagga ggagatatac a 1231181032DNASalmonella Paratyphi 18ttgaagagga agagaaatga agctttaatt accggtggat gtggcttcct tgggagtaat 60cttgcctcct ttgctttaag tcaagggatt gatttaattg tattcgataa tctatcacgt 120aaaggtgcaa cagataattt acattggtta tcctccttag gaaactttga gtttgtacat 180ggtgatattc gcaacaaaaa tgatgttaca agattaataa ctaagtatat gcctgatagc 240tgttttcatc ttgcaggtca agtggcaatg actacatcta ttgacaatcc ttgtatggat 300tttgaaatta atgtaggtgg aactttaaat ttacttgagg cagtacggca gtataattca 360aattgtaata taatttattc atcaacaaat aaagtatacg gcgatcttga gcaatataaa 420tacaatgaaa cagaaactag atacacttgt gtagataagc ctaatggata tgatgagagc 480acacaattag atttccactc accatatggt tgttcaaaag gtgctgcaga ccaatacatg 540cttgattatg caaggatttt tggtttgaat acagtggtgt tcaggcattc atcaatgtat 600ggtgggagac agtttgctac ttatgatcaa ggctgggtag gttggttttg tcaaaaagcg 660gttgaaatta aaaatggtat taataaaccc ttcactattt ctggtaatgg taagcaagtt 720agggatgttt tgcatgctga agatatgatt tcgttatatt tcactgcctt ggcaaatgta 780tcaaaaatta gggggaacgc ttttaatatt ggtggtacca ttgtcaacag cctatcatta 840cttgaattat tcaaattgct tgaagattat tgcaacatag atatgaggtt cactaattta 900cctgtaaggg aaagtgatca gcgtgttttt gttgcagata ttaaaaaaat cactaatgca 960attgactgga gcccgaaagt ctcggcaaaa gatggtgtcc agaaaatgta tgattggact 1020agttctatat ga 1032191843DNASalmonella typhi 19tcacgactta catcctactt cgaaagtcga catactgtga ttggcttagc aaggaagagg 60aacaatgaag ctaccataaa taatattatt tacacgacag aaaataattg gatcgaaaaa 120atactagaat ttgaaccgaa tattattatt aacactattg cttgctatgg aagacataac 180gaacctgcaa cagctttaat agaaagcaat attcttatgc ctatcagagt attagaatct 240atctcatcac ttgatgcagt attcataaat tgtggaacat cactgccacc aaatacgagt 300ttatatgcat atactaaaca aaaagcaaat gaactcgccg ccgccattat agataaagtt 360tgcggtaaat atatagagtt aaaattggag catttctatg gagcttttga tggagacgat 420aagtttacca gtatggttat tagacgttgt ttaagtaacc agccagtaaa gttaacatct 480ggtttgcaac agagagattt cttgtatata aaagatctac taacagcgtt cgattgtatt 540ataagtaatg ttaataattt ccccaaattt catagtattg aagttggtag tggagaggcg 600atatcaattc gtgaatatgt agatactgtt aaaaatatca caaaaagcaa ttctataatt 660gaatttggcg tggtcaaaga aagagtaaat gaattgatgt atagttgtgc tgatatagca 720gaacttgaaa aaataggatg gaaaagagag ttctctcttg ttgatgcatt aactgaaata 780attgaagagg aagggaaatg aaaagcttgg taccgagctc ggatccacta gtaacggccg 840ccagtgtgct ggaattcgcc ctttaagcgg ccgcatttaa cataatatac attatgcgca 900ccgcggccgc ggaaagggcg aattctgcag atatccatca cactggcggc cgctcgagca 960tgcatctaga gtgaggaaac tgaggttggt tagaattcca agacatctta ttattgccgc 1020ttcctcttgg ctttcaaaga taataattgc cggtgttcag ttagtaagtg ttaaatttct 1080tttagaaatt cttggcgaag aatcatacgc tgtatttact cttttaactg gattattggt 1140ctggtttagc attgcagata ttgggattgg tagtagtcta caaaattata tatctgagtt 1200gaaagctgat agaaaatcat atgatgcata tatcaaggcc gcagttcata ttctattcgc 1260atccttaatc attttaagct ctacattatt cttcttatca gataaattat cgtcactata 1320tcttacttca tttagcgatg aattgaaaaa caactcagga agttattttt ttatagcaag 1380tatattattt atattcatcg gcgttgggag tgtggtctat aaaatattat ttgcggaact 1440gttagggtgg aaagctaata taattaatgc attatcttat cttttaggtt ttttagatgt 1500agttgcgatc cattatttaa tgccagattc gagtattacc ttcgctttag tagcattgta 1560tgctccggta gcaatactgc ccattatata tatatcgttt cggtatatat atgttcttaa 1620agcgaaagta aactttaaca cctataaatt attactatca cgttcatcag ggtttctgat 1680tttttcgtcc ttatcgataa tagttttaca aactgattat attgtgatgt ctcagaaatt 1740atctgctgca gatattataa aatatactgt aacgatgaaa atatttggtt taatgttttt 1800tatttatact gcggtattac aagcattatg gccagtatgt gct 1843203142DNASalmonella typhi 20aggcttgact acagagcatt tagattatgt agttagcaag tttgaagagt tctttggttt 60gaatttctaa ttttgagggg gggggattcc cctctatgat ttcatgaaaa ttctaataat 120gggagcgttt gggttccttg gatcacgact tacatcctac ttcgaaagtc gacatactgt 180gattggctta gcaaggaaga ggaacaatga agctaccata aataatatta tttacacgac 240agaaaataat tggatcgaaa aaatactaga atttgaaccg aatattatta ttaacactat 300tgcttgctat ggaagacata acgaacctgc aacagcttta atagaaagca atattcttat 360gcctatcaga gtattagaat ctatctcatc acttgatgca gtattcataa attgtggaac 420atcactgcca ccaaatacga gtttatatgc atatactaaa caaaaagcaa atgaactcgc 480cgccgccatt atagataaag tttgcggtaa atatatagag ttaaaattgg agcatttcta 540tggagctttt gatggagacg ataagtttac cagtatggtt attagacgtt gtttaagtaa 600ccagccagta aagttaacat ctggtttgca acagagagat ttcttgtata taaaagatct 660actaacagcg ttcgattgta ttataagtaa tgttaataat ttccccaaat ttcatagtat 720tgaagttggt agtggagagg cgatatcaat tcgtgaatat gtagatactg ttaaaaatat 780cacaaaaagc aattctataa ttgaatttgg cgtggtcaaa gaaagagtaa atgaattgat 840gtatagttgt gctgatatag cagaacttga aaaaatagga tggaaaagag agttctctct 900tgttgatgca ttaactgaaa taattgaaga ggaagggaaa tgaatttgta tggtattttt 960ggtgctggaa gttatggtag agaaacaata cccattctaa atcaacaaat aaagcaagaa 1020tgtggttctg actatgctct ggtttttgtg gatgatgttt tggcaggaaa gaaagttaat 1080ggttttgaag tgctttcaac caactgcttt ctaaaagccc cttatttaaa aaagtatttt 1140aatgttgcta ttgctaatga taagatacga cagagagtgt ctgagtcaat attattacac 1200ggggttgaac caataactat aaaacatcca aatagcgttg tttatgatca tactatgata 1260ggtagtggcg ctattatttc tccctttgtt acaatatcta ctaatactca tatagggagg 1320ttttttcatg caaacatata ctcatacgtt gcacatgatt gtcaaatagg agactatgtt 1380acatttgctc ctggggctaa atgtaatgga tatgttgtta ttgaagacaa tgcatatata 1440ggctcgggtg cagtaattaa gcagggtgtt cctaatcgcc cacttattat tggcgcggga 1500gccattatag gtatgggggc tgttgtcact aaaagtgttc ctgccggtat aactgtgtgc 1560ggaaatccag caagagaaat gaaaagatcg ccaacatcta tttaatgcgg ccgcatttaa 1620cataatatac attatgcgca ccgcggccgc cagtgtgagg atcctgtttc tgcccgcgaa 1680agcgggcata attaaagaat gaaatatttt ttataattaa aagatgaagc tgacgtgagg 1740aaactgaggt tggttagaat tccaagacat cttattattg ccgcttcctc ttggctttca 1800aagataataa ttgccggtgt tcagttagta agtgttaaat ttcttttaga aattcttggc 1860gaagaatcat acgctgtatt tactctttta actggattat tggtctggtt tagcattgca 1920gatattggga ttggtagtag tctacaaaat tatatatctg agttgaaagc tgatagaaaa 1980tcatatgatg catatatcaa ggccgcagtt catattctat tcgcatcctt aatcatttta 2040agctctacat tattcttctt atcagataaa ttatcgtcac tatatcttac ttcatttagc 2100gatgaattga aaaacaactc aggaagttat ttttttatag caagtatatt atttatattc 2160atcggcgttg ggagtgtggt ctataaaata ttatttgcgg aactgttagg gtggaaagct 2220aatataatta atgcattatc ttatctttta ggttttttag atgtagttgc gatccattat 2280ttaatgccag attcgagtat taccttcgct ttagtagcat tgtatgctcc ggtagcaata 2340ctgcccatta tatatatatc gtttcggtat atatatgttc ttaaagcgaa agtaaacttt 2400aacacctata aattattact atcacgttca tcagggtttc tgattttttc gtccttatcg 2460ataatagttt tacaaactga ttatattgtg atgtctcaga aattatctgc tgcagatatt 2520ataaaatata ctgtaacgat gaaaatattt ggtttaatgt tttttattta tactgcggta 2580ttacaagcat tatggccagt atgtgctgaa ttacgagtga aaatgcagtg gagaaagctg 2640catagaatca ttttcctaaa tattattggt ggggtatttt ttattggtct tggtacgtta 2700tttatttatg ttttaaagga ttatatctat agcataattg ctaacggtat agattataat 2760attagtgggg ttgtttttgt tttactggct gtgtatttta gtataagagt ttggtgtgat 2820acatttgcta tgttacttca aagtatgaac caattaaaaa ttctttggct catagttccg 2880tgtcaggcat taattggtgg tgtgactcaa tggtattttg cagagcatta tggaatagtt 2940ggtattttat acggactaat tttatcgttc tcgctaactg ttttttgggg attgccagtg 3000tattatatgt ataagagtaa aaggctagca taatatgaag gtatcatttt gtatcccaac 3060gtataatcga gtaaaattca ttgaagacct tcttgaaagt attaataatc aatcttctca 3120ctccttaatt gtagaagtat gt 3142211137DNASalmonella typhi 21atgccatctc ttaatgtaaa acaagagaaa aatcagtcat ttgcaggtta ctcactgccg 60cccgccaaca gtcatgaaat cgatttgttt agccttatag aggtgttatg gcaggcgaaa 120cgtcgtattc ttgctaccgt tttcgccttt gcgtgcgtgg ggttgcttct gtcctttctg 180ctgccgcaaa aatggaccag ccaggcgatt gtcacaccgg cggagtcggt acagtagcag 240gggctggaga gaacgttgac cgcgctgcgc gtgttggata tggaggtaag cgttgatcgg 300gccagcgtat ttaatctgtt tattaaaaag tttagctcgc cctcgctgct ggaagaatat 360cttcgttctt ctccgtatgt catggatcaa ttaaaaggcg cgcaaataga cgagcaggat 420cttcaccggg cgattgtcgt gctgagcgaa aaaatgaaag cggtggacag caatgccggc 480aagaaaaatg aaacgtcgtt attcacgtcg tggacgctga gttttaccgc gccgacgcgg 540gaagaagcgc aaaaagtgtt ggctggctat attcagtaca tctccgatat cgtcgtgaaa 600gagacgctgg aaaatattcg taaccagctg gaaatcaaaa cccgctacga gcaggaaaag 660ctggcgatgg atcgggtgcg tctcaaaaat cagcttgatg ccaatattca acgtcttcat 720tattcgctgg aaatcgccaa cgccgctggc attaagagac cggtttacag taatggtcag 780gcggtaaaag atgatccgga tttttctatt tccctcggcg cggatggtat ttcccgcaaa 840ctggaaattg aaaaaggggt aacggacgtg gccgagatcg acggtgattt gcgtaaccgt 900caatactatg ttgaacaact ggcggcaatg aatgtgagcg acgtgaagtt taccccgttt 960aaatatcaac tgtcgccgtc tctgccagtg aaaaaagatg gcccgggtaa agcgatcatt 1020attatcctgg cggcgttgat tggcggtatg atggcctgcg gcggcgtatt actgcgtcac 1080gcgatggtct cgcgtaaaat ggaaaacgcg ctggcgatag atgaacggtt agtctga 1137223394DNASalmonella typhi 22actgcactga cggtggcggt tgaaacgctg aaggcataaa gatcttcact ctcccggccg 60atgtcctgat aactcatctc cagcgcaaac agtcgaatga tattgcgctc gatctcgtcg 120tacagggggt ctgaagcttc ttcaccaatt atggctcaaa agtgccgtta cgattgcgcg 180gagttgccag ttcaaaactg cctgttgggg ctttaatggc tttttgccgg aaccattttt 240acggtttgcc tcaacatcct gagccagatg ggaatcaagt tcagcagaca gggtagagtc 300ggttaaatac ttgattaatg gcgttaagat gccatctttg cccgttaatg cctggccgga 360cctgaagggc tttaagtgct ttgtcgaaat cgaagggatg ggacatgtgc cattcttttt 420tattttatgt tactaaaatt atacagaatt tttaacgctc cccctctccc cagcacttcc 480accctttcaa gtacctctct ctgaaaaaac atgcaaagcc ttgtaagacg atgtaaagct 540ttacatgtcc cgtttttatt ccaagacgct tggcaatcag caataccaat tgatcgataa 600catcgatcaa tatattaaaa ctcaatagct taaaactatt aaaaatacaa ttattgatcg 660cttatatcga tcaaaccaat ttgtagtgct acactccaga cctttctgaa tcggctaatt 720ttcataatgt tgaagttatt cgctaagtac acatcgatcg gtgttcttaa cacgctcatt 780cattggggag tatttgcttt ctgtatgtat gggatgcata cgcatcaggc gctgacgaac 840ttttccgatt ttgttatcgc tgtatcgttc agcttctatg ctaatgcgcg cttcaccttt 900aatgccagca ctactgcaat tctctacatg atgtatatgg gattcatggg aacactgagc 960gctgttgttg gatggatggc tgaccaatgt tctttgccac cattggttac cctcatcact 1020ttctcggcaa ttagcctggt atgcggcttt atctattcca gattcattat cttcagggat 1080aaaaatgaaa atctctcttg tcgtcctggt ttttaacgaa gaagacacga taccgatttt 1140ctatagaacg gtacatgagt ttaatgaact tgaaaaatat aaagttgaga ttatttttat 1200taatgacgga agtaaagatg tgacggagtc aataattaaa ataatagccg tatctgatcc 1260actcgtcatt ccgttttcgt ttacacgaaa cttcgggaat gatgcaagat gcacaaccat 1320tttattaatc tttttttaaa ttgaggtaat ttaagttgga acacttaaaa tacagacctg 1380atatagatgg attacgcgca atagcggttt tatctgtggt aatattccat tatttcccat

1440cattattgcc gggtgggttt gttggagtag atatattctt tgtgatatct ggatacctta 1500taacatcaat aatattaaaa tctgcatcaa acaaatcatt ttcatacctt gatttctata 1560aaaggagagt gcttagaata tttccagcat tatccatagt tcttgtatca tgtcttattg 1620ttggttggat ttatttattc caggatgatt acaaattact tggtaagcat gtttttagtg 1680gctcattctt tatatcaaac tttactcttt ggagtgagtc tggctatttt gattcaaaat 1740cataccttaa acctttacta catttgtggt cgctgggaat tgaagagcaa ttttatataa 1800tatggccagt agttatattg ctatgcttta gaagcaaaaa ccataacaga aacatagtat 1860tatcatgcgc aactatattt ataattagct atgcgattag catttttaca atggcatctg 1920atggcggagc taattactac tctcccgcat caagattttg ggagttaatg gctggagcga 1980ttatatccac attgagattt ataggaataa acacttcgtt atcaaaatta atgtccctgt 2040taggaattat actaatcgca ttatcaataa ccatgataga tgaaaagatg tcatttcctg 2100gatatatagc aataatccca atacttggcg cctctcttat aatagcatct aatggtaatg 2160atttagttgt gtcgaaattg cttagtgtta ggcctgttgt tttctttggt cttattagct 2220atcctcttta tttgtggcat tggcctattt attcattcta tcgttcaata tttgctggct 2280caccagacta ccatgaatta actcttcttt tattattatc gttctttttg gcgatattaa 2340cttattattt aattgaaaaa ccactgagaa attccagaag taaatatatc acagcaatat 2400tattagcatt atctgtattt gggacgggtt taattggcgc atttattttt catataaatg 2460gagttaaaga cagggaaatc aataaatcag caagtgaata tgcttctgtt actgacgtgt 2520acaattatta taaatatgga gaactactcc gtggagggat atgtcactca gtacaactta 2580ctgctgccat atccaatgga tgtataaaaa atggcaagca taatatattt atcattggtg 2640attcttatgc ggcggctctt ttcaatgggc tttctcatta tatagataat aaaggttctg 2700attatataat aagccaaatg acagatggta atgctcctcc tctatttgtt gacggtaaag 2760atgatttaca gagaagtgtc atcactctaa acaataatag aattaatgaa attaaacgtg 2820ttcagcctga ggtggttctg ctgacatggt cagttcgagg aacaaatgga gtacatgata 2880aaaagttagc aattgatgcg ttatcattaa ccattaaaaa aattaaagag gcatcccctg 2940actcaaggat tgttttcatt ggaccagtcc cggaatggaa tgcaaattta gttaaaataa 3000tatctaacta cctgagtgag tttaaaaaaa ctccaccatt gtatatgaca tatggattaa 3060atagtgaaat aagcgagtgg gactcttact ttagtaacaa tgttccaaaa atgggaattg 3120aatatatatc agcatacaaa gcattatgta acgaaagtgg atgtcttaca agagttggta 3180atggtcctga ttttatcact gccgttgatt ggggacattt aacaaagcct ggttctgatt 3240tcctttttaa taaaattgga aataaaataa tcaaatagat aggctgttac tattacatat 3300aaatccaata tggaacatgc cagtcatact gtgtaactgc cactatatta acggtgatcg 3360ctcaggcggt caccgaactc gataataaag cgaa 3394232940DNASalmonella typhimurium 23tgttgtaatt tttattttaa ttcattcgtt tttttatgcg gcttgccgga aaatatctgt 60ataaggtaga tacgccaata ccaaaaataa tagctagttg ctgccgagga tggcctttct 120ctaatagccg actaatctgt tcctgttcat gtttgttgat cgcccgaggg cgccctccca 180gtcgtccttg cgctctggcg gcagccagtc cggcaagggt tcgctcgaca attaattctc 240gctccatctc ggccagtgct gacattacat gaaaaaagaa tcgccccatc gcgctactgg 300tatcaatact atcggttaaa gaatggaagt gagctccacg ttcatgtaat tctgatatta 360acgccaccag gtttttcacg ctgcggccca gtctgtctaa tttccagacg acaagagtat 420cgcctttatt tacatacttt aacgctcgtt tcaggccggg gcggtttgca atcttgccac 480tgatacggtc ctcaaaaatg cggtcacaat ttgcactagt aagcgcatta cgctgtaaat 540cgatattttg gtcaattgtt gacacccgaa tatacccaat agtagccatg attttctcct 600ttacatcaga taaggaagaa ttttagtcgc ttttctcatg gaggattgct ttatcaaaaa 660ccttccaaaa ggaaaatttt atggcacaag taatcaacac taacagtctg tcgctgctga 720cccagaataa cctgaacaaa tcccagtccg cactgggcac cgctatcgag cgtctgtctt 780ctggtctgcg tatcaacagc gcgaaagacg atgcggcagg tcaggcgatt gctaaccgtt 840tcaccgcgaa catcaaaggt ctgactcagg cttcccgtaa cgctaacgac ggtatctcca 900ttgcgcagac cactgaaggc gcgctgaacg aaatcaacaa caacctgcag cgtgtgcgtg 960aactggcggt tcagtctgct aacagcacca actcccagtc tgacctcgac tccatccagg 1020ctgaaatcac ccagcgcctg aacgaaatcg accgtgtatc cggccagact cagttcaacg 1080gcgtgaaagt cctggcgcag gacaacaccc tgaccatcca ggttggcgcc aacgacggtg 1140aaactatcga tatcgatctg aagcagatca actctcagac cctgggtctg gactcactga 1200acgtgcagaa agcgtatgat gtgaaagata cagcagtaac aacgaaagct tatgccaata 1260atggtactac actggatgta tcgggtcttg atgatgcagc tattaaagcg gctacgggtg 1320gtacgaatgg tacggcttct gtaaccggtg gtgcggttaa atttgacgca gataataaca 1380agtactttgt tactattggt ggctttactg gtgctgatgc cgccaaaaat ggcgattatg 1440aagttaacgt tgctactgac ggtacagtaa cccttgcggc tggcgcaact aaaaccacaa 1500tgcctgctgg tgcgacaact aaaacagaag tacaggagtt aaaagataca ccggcagttg 1560tttcagcaga tgctaaaaat gccttaattg ctggcggcgt tgacgctacc gatgctaatg 1620gcgctgagtt ggtcaaaatg tcttataccg ataaaaatgg taagacaatt gaaggcggtt 1680atgcgcttaa agctggcgat aagtattacg ccgcagatta cgatgaagcg acaggagcaa 1740ttaaagctaa aactacaagt tatactgctg ctgacggcac taccaaaaca gcggctaacc 1800aactgggtgg cgtagacggt aaaaccgaag tcgttactat cgacggtaaa acctacaatg 1860ccagcaaagc cgctggtcat gatttcaaag cacaaccaga gctggcggaa gcagccgcta 1920aaaccaccga aaacccgctg cagaaaattg atgccgcgct ggcgcaggtg gatgcgctgc 1980gctctgatct gggtgcggta caaaaccgtt tcaactctgc tatcaccaac ctgggcaata 2040ccgtaaacaa tctgtctgaa gcgcgtagcc gtatcgaaga ttccgactac gcgaccgaag 2100tttccaacat gtctcgcgcg cagattctgc agcaggccgg tacttccgtt ctggcgcagg 2160ctaaccaggt cccgcagaac gtgctgtctc tgttacgtta atttatttcg ttttattcag 2220ccccgtgaat tcggggcttt ttcatttagc atagatgaat atatatttat ggaatgtatg 2280gctgtaaatg atatttccta cgggcgagaa gctgaaatat ggccgcggga ttattctatg 2340cttgctcgtc gagttcaatt tctacgtttt aatgatatcc ctgttcgatt ggtgagtaat 2400aatgcccgga taatcacagg ctacattgcg aagtttaatc cgaaggaaaa tttgattctg 2460gcttcggata aacctaaagg aaataagcgc attgaagtta aactagagtc tctggcaatt 2520cttgaagaat tatcaggtaa tgacgctttt aatctttcgc tggtgccggc tgacggattt 2580aatcttcagc aatatactcc atcaagaaga gattatttct cgatttgcaa taagtgctat 2640aaacagggag tcggtatcaa aatctatatg aagtatggac aggttttgac tggcaaaacg 2700acaggcgtaa atgcgtgtca ggttggtgtg aggacatcca atggcaatca tatgcaagtt 2760atgtttgact gggtgagcag gatcacgtct tcggactacg ctgaataacg cctacggtaa 2820taaaaaattc cgtgagaaaa gtaaaactta gggggctacc ggaggggacc taatgaacgg 2880aggtcatgga aggtattcat cgtgccagac tcttgctctt gtcagaagaa ggtaaaagta 294024493DNABacteriophage lambda 24acgttaaatc tatcaccgca agggataaat atctaacacc gtgcgtgttg actattttac 60ctctggcggt gataatggtt gcatgtacta aggaggttgt atggaacaac gcataaccct 120gaaagattat gcaatgcgct ttgggcaaac caagacagct aaagatctct cacctaccaa 180acaatgcccc cctgcaaaaa ataaattcat ataaaaaaca tacagataac catctgcggt 240gataaattat ctctggcggt gttgacataa ataccactgg cggtgatact gagcacatca 300gcaggacgca ctgaccacca tgaaggtgac gctcttaaaa attaagccct gaagaagggc 360agcattcaaa gcagaaggct ttggggtgtg tgatacgaaa cgaagcattg ggatctatcg 420atgcatgcca tggtacccgg gagctcgaat taattctaga aataattttg tttaacttta 480agaaggagat ata 49325714DNABacteriophage lambda 25atgagcacaa aaaagaaacc attaacacaa gagcagcttg aggacgcacg tcgccttaaa 60gcaatttatg aaaaaaagaa aaatgaactt ggcttatccc aggaatctgt cgcagacaag 120atggggatgg ggcagtcagg cgttggtgct ttatttaatg gcatcaatgc attaaatgct 180tataacgccg cattgcttgc aaaaattctc aaagttagcg ttgaagaatt tagcccttca 240atcgccagag aaatctacga gatgtatgaa gcggttagta tgcagccgtc acttagaagt 300gagtatgagt accctgtttt ttctcatgtt caggcaggga tgttctcacc taagcttaga 360acctttacca aaggtgatgc ggagagatgg gtaagcacaa ccaaaaaagc cagtgattct 420gcattctggc ttgaggttga aggtaattcc atgaccgcac caacaggctc caagccaagc 480tttcctgacg gaatgttaat tctcgttgac cctgagcagg ctgttgagcc aggtgatttc 540tgcatagcca gacttggggg tgatgagttt accttcaaga aactgatcag ggatagcggt 600caggtgtttt tacaaccact aaacccacag tacccaatga tcccatgcaa tgagagttgt 660tccgttgtgg ggaaagttat cgctagtcag tggcctgaag agacgtttgg ctaa 714263659DNAArtificial SequencepUCpW_difCAT rfbE deletion cassette 26tcgacatact gtgattggct tagcaaggaa gaggaacaat gaagctacca taaataatat 60tatttacacg acagaaaata attggatcga aaaaatacta gaatttgaac cgaatattat 120tattaacact attgcttgct atggaagaca taacgaacct gcaacagctt taatagaaag 180caatattctt atgcctatca gagtattaga atctatctca tcacttgatg cagtattcat 240aaattgtgga acatcactgc caccaaatac gagtttatat gcatatacta aacaaaaagc 300aaatgaactc gccgccgcca ttatagataa agtttgcggt aaatatatag agttaaaatt 360ggagcatttc tatggagctt ttgatggaga cgataagttt accagtatgg ttattagacg 420ttgtttaagt aaccagccag taaagttaac atctggtttg caacagagag atttcttgta 480tataaaagat ctactaacag cgttcgattg tattataagt aatgttaata atttccccaa 540atttcatagt attgaagttg gtagtggaga ggcgatatca attcgtgaat atgtagatac 600tgttaaaaat atcacaaaaa gcaattctat aattgaattt ggcgtggtca aagaaagagt 660aaatgaattg atgtatagtt gtgctgatat agcagaactt gaaaaaatag gatggaaaag 720agagttctct cttgttgatg cattaactga aataattgaa gaggaaggga aatgaatttg 780tatggtattt ttggtgctgg aagttatggt agagaaacaa tacccattct aaatcaacaa 840ataaagcaag aatgtggttc tgactatgct ctggtttttg tggatgatgt tttggcagga 900aagaaagtta atggttttga agtgctttca accaactgct ttctaaaagc cccttattta 960aaaaagtatt ttaatgttgc tattgctaat gataagatac gacagagagt gtctgagtca 1020atattattac acggggttga accaataact ataaaacatc caaatagcgt tgtttatgat 1080catactatga taggtagtgg cgctattatt tctccctttg ttacaatatc tactaatact 1140catataggga ggttttttca tgcaaacata tactcatacg ttgcacatga ttgtcaaata 1200ggagactatg ttacatttgc tcctggggct aaatgtaatg gatatgttgt tattgaagac 1260aatgcatata taggctcggg tgcagtaatt aagcagggtg ttcctaatcg cccacttatt 1320attggcgcgg gagccattat aggtatgggg gctgttgtca ctaaaagtgt tcctgccggt 1380ataactgtgt gcggaaatcc agcaagagaa atgaaaagat cgccaacatc tatttaatgc 1440ggccgcattt aacataatat acattatgcg caccgcccga acaccactcg ccacaaaaaa 1500ccgccggaac gtccaaaagt acgggttttg ctgcccgcaa acgggctgtt ctggtgttgc 1560tagtttgtta tcagaatcgc agatccggct tcagccggtt tgccggctga aagcgctatt 1620tcttccagaa ttgccatgat tttttcccca cgggaggcgt cactggctcc cgtgttgtcg 1680gcagctttga ttcgataagc agcatcgcct gtttcaggct gtctatgggc cggccaaatc 1740agtaagttgg cagcatcacc cgacgcactt tgcgccgaat aaatacctgt gacggaagat 1800cacttcgcag aataaataaa tcctggtgtc cctgttgata ccgggaagcc ctgggccaac 1860ttttggcgaa aatgagacgt tgatcggcac gtaagaggtt ccaactttca ccataatgaa 1920ataagatcac taccgggcgt attttttgag ttatcgagat tttcaggagc taaggaagct 1980aaaatggaga aaaaaatcac tggatatacc accgttgata tatcccaatg gcatcgtaaa 2040gaacattttg aggcatttca gtcagttgct caatgtacct ataaccagac cgttcagctg 2100gatattacgg cctttttaaa gaccgtaaag aaaaataagc acaagtttta tccggccttt 2160attcacattc ttgcccgcct gatgaatgct catccggaat tccgtatggc aatgaaagac 2220ggtgagctgg tgatatggga tagtgttcac ccttgttaca ccgttttcca tgagcaaact 2280gaaacgtttt catcgctctg gagtgaatac cacgacgatt tccggcagtt tctacacata 2340tattcgcaag atgtggcgtg ttacggtgaa aacctggcct atttccctaa agggtttatt 2400gagaatatgt ttttcgtctc agccaatccc tgggtgagtt tcaccagttt tgatttaaac 2460gtggccaata tggacaactt cttcgccccc gttttcacca tgggcaaata ttatacgcaa 2520ggcgacaagg tgctgatgcc gctggcgatt caggttcatc atgccgtttg tgatggcttc 2580catgtcggca gaatgcttaa tgaattacaa cagtactgcg atgagtggca gggcggggcg 2640taattttttt aaggcagtta ttggtgccct taaacgcctg gttgctacgc ctgaataagt 2700gataataagc ggatcctagg atggtgttaa gcgggcggtt ttgagatgta aactcgccca 2760tttaacataa tatacattat gcgcaccgcg gccgccagtg tgaggatcct gtttctgccc 2820gcgaaagcgg gcataattaa agaatgaaat attttttata attaaaagat gaagctgacg 2880tgaggaaact gaggttggtt agaattccaa gacatcttat tattgccgct tcctcttggc 2940tttcaaagat aataattgcc ggtgttcagt tagtaagtgt taaatttctt ttagaaattc 3000ttggcgaaga atcatacgct gtatttactc ttttaactgg attattggtc tggtttagca 3060ttgcagatat tgggattggt agtagtctac aaaattatat atctgagttg aaagctgata 3120gaaaatcata tgatgcatat atcaaggccg cagttcatat tctattcgca tccttaatca 3180ttttaagctc tacattattc ttcttatcag ataaattatc gtcactatat cttacttcat 3240ttagcgatga attgaaaaac aactcaggaa gttatttttt tatagcaagt atattattta 3300tattcatcgg cgttgggagt gtggtctata aaatattatt tgcggaactg ttagggtgga 3360aagctaatat aattaatgca ttatcttatc ttttaggttt tttagatgta gttgcgatcc 3420attatttaat gccagattcg agtattacct tcgctttagt agcattgtat gctccggtag 3480caatactgcc cattatatat atatcgtttc ggtatatata tgttcttaaa gcgaaagtaa 3540actttaacac ctataaatta ttactatcac gttcatcagg gtttctgatt ttttcgtcct 3600tatcgataat agttttacaa actgattata ttgtgatgtc tcagaaatta tctggagct 3659271305DNAArtificial SequencePL rfbE deletion cassette 27aataggatgg aaaagagagt tctctcttgt tgatgcatta actgaaataa ttgaagagga 60agggaaatga aaagcttggt accgagctcg gatccactag taacggccgc cagtgtgctg 120gaattcgccc tttaagcggc cgcatttaac ataatataca ttatgcgcac catccgctta 180ttatcactta ttcaggcgta gcaccaggcg tttaagggca ccaataactg ccttaaaaaa 240attacgcccc gccctgccac tcatcgcagt actgttgtaa ttcattaagc attctgccga 300catggaagcc atcacaaacg gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt 360cgccttgcgt ataatatttg cccatggtga aaacgggggc gaagaagttg tccatattgg 420ccacgtttaa atcaaaactg gtgaaactca cccagggatt ggctgagacg aaaaacatat 480tctcaataaa ccctttaggg aaataggcca ggttttcacc gtaacacgcc acatcttgcg 540aatatatgtg tagaaactgc cggaaatcgt cgtggtattc actccagagc gatgaaaacg 600tttcagtttg ctcatggaaa acggtgtaac aagggtgaac actatcccat atcaccagct 660caccgtcttt cattgccata cggaattccg gatgagcatt catcaggcgg gcaagaatgt 720gaataaaggc cggataaaac ttgtgcttat ttttctttac ggtctttaaa aaggccgtaa 780tatccagctg aacggtctgg ttataggtac attgagcaac tgactgaaat gcctcaaaat 840gttctttacg atgccattgg gatatatcaa cggtggtata tccagtgatt tttttctcca 900ttttagcttc cttagctcct gaaaatctcg ataactcaaa aaatacgccc ggtagtgatc 960ttatttcatt atggtgaaag ttggaacctc ttacgtgccg atcaacgtct cattttcgcc 1020aaaagttggc ccagggcttc ccggtatcaa cagggacacc aggatttatt tattctgcga 1080agtgatcttc cgtcacaggt atttattcga agacgaaagg gatgcaggag tcgcataagg 1140gatttaacat aatatacatt atgcgcaccg cggccgcgga aagggcgaat tctgcagata 1200tccatcacac tggcggccgc tcgagcatgc atctagagtg aggaaactga ggttggttag 1260aattccaaga catcttatta ttgccgcttc ctcttggctt tcaaa 1305284915DNAArtificial SequencepUCpF_difCAT fliC replacement cassette 28cattccctga ggggcgtcgg ttacggtatt gctctgacgc tcaatgtcga tgccgtttac 60gttcagcttc gcgttttctg ctttcaccag ctcttgcata ttgccggtat tggtggtgct 120gtcataagcg agtagatcgt taagttttgt atcgccttcc accgtgatct tcatcgtatt 180gtcggtaccg ctattggcgg taagcaccaa ctggaattcg ttctctttga ccttaacgat 240actggcggcg ataccgctgt cggcgtcatt aatggcgtca cggatcgcct ccatggaggt 300gtcgccttta tccagcttaa tttccagcgg ctctttacgt cccggctgtt caattttaat 360tgtccgggat gtgaccgacg tatcgcccaa ctgctctttg gtggttgcga aggtggtttt 420tgtcgccagc gactgcgcgg cggcaagctg ggttacgcta atcttataag tccctgcggc 480agcgcctgcg gtagtactga ctttgaggtc ctctgtcgtg ctggacgcca cggtagactt 540aaataaatcc gctttattta acgcggtatt tgccgtctgg aatttttcta atgcgctttt 600caatgtgcca taggcggtta gctttgccga attcgcgctc tgctgtttgg taattggcgt 660taagcgtcct ttttcgttct ttgtcaggtc tgtcaacaac tggtctaacg gtaagtttga 720tcccacacct aatgatgaaa ttgaagccat gccttcttcc tttttgattg caaacagtag 780ttaagcgcgt tatcggcaat ctggaggcaa agtttaatga taattttgca aaaataatgc 840gcggaataat gatgcataaa gcggctattt cgccgcctaa gaaaaagatc gggggaagtg 900aaaaattttc taaagttcga aattcaggtg ccgatacaag ggttacggtg agaaaccgtg 960ggcaacagcc caataacatc aagttgtaat tgataaggaa aagatcatgg cacaagtcat 1020taatacaaac agcctgtcgc tgttgaccca gaataacctg aacaaatccc agtccgctct 1080gggcaccgct atcgagcgtc tgtcttccgg tctgcgtatc aacagcgcga aagacgatgc 1140ggcaggtcag gcaattgcta accgtttcac cgcgaacatc aaaggtctga ctcaggcttc 1200ccgtaacgct aacgacggta tctccattgc gcagaccact gaaggcgcgc tgaacgaaat 1260caacaacaac ctgcagcgtg tgcgtgaact ggcggttcag tctgctaaca gcaccaactc 1320ccagtctgac ctcgactcca tccaggctga aatcacccag cgcctgaacg aaatcgaccg 1380tgtatccggt cagactcagt tcaacggcgt gaaagtcctg gcgcaggaca acaccctgac 1440catccaggtt ggtgccaaca acggtgaaac cattgatatc gatctgaaac agatcaactc 1500tcagaccctg ggtctggata cgctgaatgt gcagaaaaaa tatgatgtga agagcgaagc 1560ggtcacgcct tcggctacat taagcactac tgcacttgat ggtgctggcc tcaaaaccgg 1620aaccggttct acaactgata ctggttcaat taaggatggt aaggtttact ataacagcac 1680ctctaaaaat tattatgttg aagtagaatt taccgatgcg accgatcaaa ccaacaaagg 1740cggattctat aaagttaatg ttgctgatga tggtgcagtc acaatgactg cggctaccac 1800caaagaggct acaactccta caggtattac tgaagttact caagtccaaa aacctgtggc 1860tgctccagct gctatccagg ctcagttgac tgctgcccat gtgaccggcg ctgatactgc 1920tgaaatggtt aagatgtctt atacggataa aaacggtaag actattgatg gcggtttcgg 1980tgttaaagtt ggggctgata tttatgctgc aacaaaaaat aaagatggat cgttcagcat 2040taacaccact gaatataccg ataaagacgg caacactaaa actgcactaa accaactggg 2100tggcgcagac ggtaaaactg aagttgtttc tatcgacggt aaaacctaca atgccagcaa 2160agccgctggt cacaacttta aagcacagcc agagctggct gaagcggctg ctgcaaccac 2220cgaaaacccg ctggctaaaa ttgatgccgc gctggcgcag gttgatgcgc tgcgttctga 2280cttgggtgcg gttcagaacc gtttcaactc cgctatcacc aacctgggca ataccgtaaa 2340taacctgtct tctgcccgta gccgtatcga agattccgac tacgcgaccg aagtttccaa 2400catgtctcgc gcgcagatcc tgcagcaggc cggtacctcc gttctggcgc aggcgaacca 2460ggttccgcaa aacgtcctct ctttactgcg ttaatgcggc cgcatttaac ataatataca 2520ttatgcgcac cgcccgaaca ccactcgcca caaaaaaccg ccggaacgtc caaaagtacg 2580ggttttgctg cccgcaaacg ggctgttctg gtgttgctag tttgttatca gaatcgcaga 2640tccggcttca gccggtttgc cggctgaaag cgctatttct tccagaattg ccatgatttt 2700ttccccacgg gaggcgtcac tggctcccgt gttgtcggca gctttgattc gataagcagc 2760atcgcctgtt tcaggctgtc tatgggccgg ccaaatcagt aagttggcag catcacccga 2820cgcactttgc gccgaataaa tacctgtgac ggaagatcac ttcgcagaat aaataaatcc 2880tggtgtccct gttgataccg ggaagccctg ggccaacttt tggcgaaaat gagacgttga 2940tcggcacgta agaggttcca actttcacca taatgaaata agatcactac cgggcgtatt 3000ttttgagtta tcgagatttt caggagctaa ggaagctaaa atggagaaaa aaatcactgg 3060atataccacc gttgatatat cccaatggca tcgtaaagaa cattttgagg catttcagtc 3120agttgctcaa tgtacctata accagaccgt tcagctggat attacggcct ttttaaagac 3180cgtaaagaaa aataagcaca agttttatcc ggcctttatt cacattcttg cccgcctgat 3240gaatgctcat ccggaattcc gtatggcaat gaaagacggt gagctggtga tatgggatag 3300tgttcaccct tgttacaccg ttttccatga gcaaactgaa acgttttcat cgctctggag 3360tgaataccac gacgatttcc ggcagtttct acacatatat tcgcaagatg tggcgtgtta 3420cggtgaaaac ctggcctatt tccctaaagg gtttattgag aatatgtttt tcgtctcagc 3480caatccctgg gtgagtttca ccagttttga tttaaacgtg gccaatatgg acaacttctt 3540cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct 3600ggcgattcag gttcatcatg

ccgtttgtga tggcttccat gtcggcagaa tgcttaatga 3660attacaacag tactgcgatg agtggcaggg cggggcgtaa tttttttaag gcagttattg 3720gtgcccttaa acgcctggtt gctacgcctg aataagtgat aataagcgga tcctaggatg 3780gtgttaagcg ggcggttttg agatgtaaac tcgcccattt aacataatat acattatgcg 3840caccgcggcc gccagtgtga ggatccccgg cgattgattc accgacacgt ggtacacaat 3900caaggcagcg aaagctgcct tttttaattc cggagcctgt gtaatgaaag aaatcaccgt 3960cactgaacct gcctttgtca cccgcttttc ctgttctggc tcggcctgtc gcgaccattg 4020ttgtaagggc tggaaaatca cgctggataa gacgacggtt aaaaagtatc tcgccagtaa 4080agacacgacg attcgtacca tcgcgcaaga ccatattatt ctgctgaaaa agaacaataa 4140tcattggggg gaaattaaac tgccttcggc gctgggaagt tgcccttatc tggatgagga 4200ccgtttgtgc cgggtacaaa aacgttaggc gcaaaggcat taagtcatac ctgttcctct 4260ttcccacggg cgcaccatac ctataaaaat gaggtacgta actccctgag tcttgcctgt 4320ccggaggtaa cgtcccgcat tttaaacgat cctgacgcaa tggcgctcgg cgaaaaaaca 4380atcattcagc agacattcaa tactgcgccg ttattctcac cgcagcaaaa gttactcaat 4440ctgttttgcc tgagtctgat caaccatgcc aacagcagta cggaaacggc gctctatggg 4500ttgattaaat tcgtcatgta tgcacataaa tttgccaaaa ttgatgatgc cgcgctgggt 4560gaactggaac aggtgtatgc cgcgttactt gagcagttgc agaccggcgt gctggcgcag 4620gaattgatga atatcgcgcc ggacagcaag gtaaaaacct cgctggtatt gcagatgcag 4680aactatttcc gctcgctccc gcttagtcgt ggcagtgtta tcctcgatca ctatatccag 4740tgtcttctgc gggtgctgac ggcggaagag ggcgtttcaa tggagcagaa ggttagcgat 4800attgagtcct cattagcgcg ctgtttacag gcggatgagc agcagaagaa ctgggctttc 4860agaaatttaa ttctctataa aatttgggaa aataatttcc ccaaccagcc gaatg 4915292931DNAArtificial SequencetviA expression plasmid pBRT4tviA 29aacatcgata ttgccatcgc ggatgtcgcc tgtcttatct accatcataa acatcatttg 60cctatggctc acgacagtat aggcaatgcc gttttttata ttgctaattg tttcgccaat 120caacgcaaaa gtatggcgat tgctaaagcc gtctccctgg gcggtagatt agccttaacc 180gcgacggtaa tgactcattc atactggagt ggtagtttgg gactacagcc tcatttatta 240gagcgtctta atgatattac ctatggacta atgagtttta ctcgcttcgg tatggatggg 300atggcaatga ccggtatgca ggtcagcagc ccattatatc gtttgctggc tcaggtaacg 360ccagaacaac gtgcgccgga gtaatcgttt tcaggtatat accggatgtt cattgctttc 420taaattttgc tatgttgcca gtatccttac gatgtattta ttttaaggaa aagccatatg 480aggtttcatc atttctggcc tccgaatgat atctatttcg gggttggagc tgctggcatt 540attgaagaag tgtcactgat aacaaatgac agaaattatt tgtttgtgaa cctaaatcgc 600tacagcctgt taaatgccct gaattttttc acgcgaatga gtgatattaa taaaataatc 660gttatcattt caagttcgcg actaatgccc cttgcacgtt tttggttgac agagtgcaaa 720aatgttattg ctgttttcga tgcggcaaca tcagtccagg atattatcag aaatgtcagt 780caacaccaaa gtggtgaaaa gatcttgacg gagcagagag attatcgttt cagaattaac 840cgtaaggata tagtaaagat gaaatatttc ctttcggaaa gtggtatgga agagcttcag 900gatagattta tgaactcatc atcgactatg tatcgctgga gaaaagaatt ggcagtaaaa 960tttggagtac gtgagccgcg ctatctgtta ttgccggatt cagttacttt actgtaatgt 1020cgacataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc 1080ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca 1140acggcccgga gggtggcggg caggacgccc gccataaact gccaggcatc aaattaagca 1200gaaggccatc ctgacggatg gccttttctg cagataaaag gatctaggtg aagatccttt 1260ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc 1320ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 1380tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 1440ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag 1500tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc 1560tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 1620actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 1680cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat 1740gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 1800tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 1860ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 1920ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 1980cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg 2040cctttgagtg agctgatacc gctttttcgt gacattcagt tcgctgcgct cacggctctg 2100gcagtgaatg ggggtaaatg gcactacagg cgccttttat ggattcatgc aaggaaacta 2160cccataatac aagaaaagcc cgtcacgggc ttctcagggc gttttatggc gggtctgcta 2220tgtggtgcta tctgactttt tgctgttcag cagttcctgc cctctgattt tccagtctga 2280ccacttcgga ttatcccgtg acaggtcatt cagactggct aatgcaccca gtaaggcagc 2340ggtatcatca acaggcttac ccgtcttact gtcaaccaga cccgccagga taagcaatcc 2400ggcagactgg tacagagcat ggtcacgggc tttacgggcg gctctggctt cggctcgctt 2460ttctgcctgt atcaggttca tgagcggccg cggcgcgcca gcttatcatt gataagcttc 2520ttgaactctt tatcactgat aaagacgcgt catagacagc ctgaaacagg cgatgctgct 2580tatcgaatca aagctgccga caacacggga gccagtgacg cctcccgtgg ggaaaaaatc 2640atggcaattc tggaagaaat agcgctttca gccggcaaac cggctgaagc cggatctgcg 2700attctgataa caaactagca acaccagaac agcccgtttg cgggcagcaa aacccgtact 2760tttggacgtt ccggcggttt tttgtggcga gtggtgttcg ggcggtgcgc gcaagatcca 2820ttatgttaaa cgggcgagtt tacatctcaa aaccgcccgc ttaacaccat tcatgagcgg 2880ccgccagtgt gctggaattc ggcttcatga ttttttattc aacgaagagt t 2931305188DNAArtificial SequencefepE expression plasmid pBAD2fepE 30gtgcctgtca aatggacgaa gcagggattc tgcaaaccct atgctactcc gtcaagccgt 60caattgtctg attcgttacc aattatgaca acttgacggc tacatcattc actttttctt 120cacaaccggc acggaactcg ctcgggctgg ccccggtgca ttttttaaat acccgcgaga 180aatagagttg atcgtcaaaa ccaacattgc gaccgacggt ggcgataggc atccgggtgg 240tgctcaaaag cagcttcgcc tggctgatac gttggtcctc gcgccagctt aagacgctaa 300tccctaactg ctggcggaaa agatgtgaca gacgcgacgg cgacaagcaa acatgctgtg 360cgacgctggc gatatcaaaa ttgctgtctg ccaggtgatc gctgatgtac tgacaagcct 420cgcgtacccg attatccatc ggtggatgga gcgactcgtt aatcgcttcc atgcgccgca 480gtaacaattg ctcaagcaga tttatcgcca gcagctccga atagcgccct tccccttgcc 540cggcgttaat gatttgccca aacaggtcgc tgaaatgcgg ctggtgcgct tcatccgggc 600gaaagaaccc cgtattggca aatattgacg gccagttaag ccattcatgc cagtaggcgc 660gcggacgaaa gtaaacccac tggtgatacc attcgcgagc ctccggatga cgaccgtagt 720gatgaatctc tcctggcggg aacagcaaaa tatcacccgg tcggcaaaca aattctcgtc 780cctgattttt caccaccccc tgaccgcgaa tggtgagatt gagaatataa cctttcattc 840ccagcggtcg gtcgataaaa aaatcgagat aaccgttggc ctcaatcggc gttaaacccg 900ccaccagatg ggcattaaac gagtatcccg gcagcagggg atcattttgc gcttcagcca 960tacttttcat actcccgcca ttcagagaag aaaccaattg tccatattgc atcagacatt 1020gccgtcactg cgtcttttac tggctcttct cgctaaccaa accggtaacc ccgcttatta 1080aaagcattct gtaacaaagc gggaccaaag ccatgacaaa aacgcgtaac aaaagtgtct 1140ataatcacgg cagaaaagtc cacattgatt atttgcacgg cgtcacactt tgctatgcca 1200tagcattttt atccataaga ttagcggatc ctacctgacg ctttttatcg caactctcta 1260ctgtttctcc atacccgttt ttttgggcta gcgaattgag gaggagatat acatatgcca 1320tctcttaatg taaaacaaga aaaaaatcag tcatttgcag gttattcact gccgcccgcc 1380aacagtcatg aaatcgattt gtttagcctt atagaggtgt tatggcaggc gaaacgtcgt 1440attcttgcta ccgttttcgc ctttgcgtgc gtggggttgc ttctgtcctt tctgctgccg 1500caaaaatgga ccagccaggc gattgtcaca ccggcggagt cggtacagtg gcaggggctg 1560gagagaacgt tgaccgcgct gcgcgtgttg gatatggagg taagcgttga tcggggcagc 1620gtatttaatc tgtttattaa aaagtttagc tcgccctcgc tgctggaaga atatcttcgt 1680tcttctccgt atgtcatgga tcaattaaaa ggcgcgcaaa tagacgagca ggatcttcac 1740cgggcgattg tcctgctgag cgaaaaaatg aaagcggtgg acagtaatgt cggcaagaaa 1800aatgaaacgt cgttattcac gtcgtggaca ttgagtttta ccgcgccgac gcgggaagaa 1860gcgcaaaaag tgctggctgg ctatattcag tacatctccg atatcgtcgt gaaagagacg 1920ctggaaaata ttcgtaacca gctggaaatc aaaacccgct atgagcagga aaagctggcg 1980atggatcggg tgcgtctcaa aaatcagctt gatgccaata ttcaacgtct tcattattcg 2040ctggaaatcg ccaacgccgc cggtattaag agaccggttt acagcaatgg tcaggcggta 2100aaagatgatc cggatttttc tatttctctc ggcgcggatg gtatttcccg caaactggaa 2160attgaaaaag gggtaacgga cgtggccgag atcgacggtg atttgcgtaa ccgtcaatac 2220catgttgaac aactggcggc aatgaatgtg agtgacgtga agtttacccc gtttaaatat 2280caactgtcgc cgtctctgcc agtgaaaaaa gatggcccgg gtaaagccat cattattatc 2340ctggcggcgt tgattggcgg tatgatggcc tgcggcggcg tattactgcg tcacgcgatg 2400gtctcgcgta aaatggaaaa cgcgctggcg atagatgaac ggttagtctg agtcgacctg 2460caggcatgca agcttggctg ttttggcgga tgagagaaga ttttcagcct gatacagatt 2520aaatcagaac gcagaagcgg tctgataaaa cagaatttgc ctggcggcag tagcgcggtg 2580gtcccacctg accccatgcc gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg 2640gggtctcccc atgcgagagt agggaactgc caggcatcaa ataaaacgaa aggctcagtc 2700gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac 2760aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg 2820acgcccgcca taaactgcca ggcatcaaat taagcagaag gccatcctga cggatggcct 2880ttttgcgttt ctacaaactc tttttgttta tttttctaaa tacattcaaa tatgtatccg 2940ctcatgtggc cggcccggcc taggaaagcc acgttgtgtc tcaaaatctc tgatgttaca 3000ttgcacaaga taaaaatata tcatcatgaa caataaaact gtctgcttac ataaacagta 3060atacaagggg tgttatgagc catattcaac gggaaacgtc ttgctcgagg ccgcgattaa 3120attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat 3180caggtgcgac aatctatcga ttgtatggga agcccgatgc gccagagttg tttctgaaac 3240atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga 3300cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt 3360tactcaccac tgcgatcccc gggaaaacag cattccaggt attagaagaa tatcctgatt 3420caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg 3480tttgtaattg tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa 3540tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg 3600aacaagtctg gaaagaaatg cataagcttt tgccattctc accggattca gtcgtcactc 3660atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg 3720atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc 3780tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc 3840ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa tcagaattgg 3900ttaattggtt gtaacactgg cagagcatta cgctgacttg acgggacggc ggctttgttg 3960aataaatcga acttttccta ggccgggccg gccacatgac caaaatccct taacgtgagt 4020tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt 4080tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt 4140gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc 4200agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg 4260tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg 4320ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt 4380cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac 4440tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg 4500acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg 4560gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat 4620ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt 4680tacggttcct ggccttttgc tggccttttg ctcactttta cggttcctgg ccttttgctg 4740gccttttgct cacatgtatg gtgttaagcg ggcggttttg agatgtaaac tcgcccgttt 4800aacataatgg atcttgcgcg caccgcccga acaccactcg ccacaaaaaa ccgccggaac 4860gtccaaaagt acgggttttg ctgcccgcaa acgggctgtt ctggtgttgc tagtttgtta 4920tcagaatcgc agatccggct tcagccggtt tgccggctga aagcgctatt tcttccagaa 4980ttgccatgat tttttcccca cgggaggcgt cactggctcc cgtgttgtcg gcagctttga 5040ttcgataagc agcatcgcct gtttcaggct gtctatgaca tgttctttcc tgcgttatcc 5100ccaattgtga gcgctcacaa tttgctgcgg taagtcgcat aaaaaccatt cttcataatt 5160caatccattt actatgttat gttctgag 5188

Claims

1. A live attenuated strain of Salmonella enterica serovar Typhi wherein said strain is modified to express the lipopolysaccharide O2 O-antigens and the flagella proteins of Salmonella enterica serovar Paratyphi A.

2. The live attenuated strain of claim 1, wherein the modification of said strain includes where the native rfbE gene is inactivated.

3. The live attenuated strain of claim 1, wherein the modification of said strain includes where the native rfbE gene is deleted.

4. The live attenuated strain of claim 1, wherein the native rfbE gene is inactivated by the insertion of a spacer region of DNA, preferably wherein the spacer region of DNA is the cistron of the Escherichia coli gene wbdR.

5. The live attenuated strain of claim 1 wherein the resulting lipopolysaccharide O2 O-antigens are at least equivalent in length to the lipopolysaccharide O9 O-antigens of Salmonella enterica serovar Typhi.

6. The live attenuated strain of claim 1, wherein said strain has its native fliC gene substituted with the fliC gene of Salmonella enterica serovar Paratyphi A, or wherein the substituted fliC gene is expressed in trans, such that the conferred serotype is altered from an Hd serotype to a Ha serotype.

7. The live attenuated strain of claim 1, wherein said strain is modified to contain a functional fepE gene, such that long O-antige chains are generated, preferably wherein the O-antigen chains are 100 repeated units of the trisaccharide backbone in length and/or wherein said strain is further modified to constitutively express gtrC, or wherein the gtrC gene is expressed in trans, and/or wherein said strain is further modified to contain an additional copy of the tviA gene under the control of a phagosomally induced promoter.

8. A vaccine comprising the live attenuated strain of claim 1, for use in enhancing immunogenicity against Salmonella enterica serovar Paratyphi A and for use in the treatment or prevention of enteric fever and salmonellosis.

9. The vaccine according to claim 1, wherein said strain is selected from the group consisting of ZH9, ZH9PA and a combination of ZH9 and ZH9PA.

10. The vaccine according to claim 9, wherein the vaccine further comprises an additional live attenuated Salmonella strain.

11. A live attenuated strain of Salmonella enterica serovar Typhi wherein said strain is modified to express the lipopolysaccharide O4 O-antigens and the flagella proteins of Salmonella enterica serovar Paratyphi B and Salmonella enterica serovar Typhimurium.

12. The live attenuated strain of claim 11, wherein the modification of said strain includes the replacement of the native rfbS gene with the rfbJ gene of Salmonella enterica serovar Paratyphi B and/or Salmonella enterica serovar Typhimurium, or wherein the modification of said strain includes the inactivation of the native rfbS gene with the concurrent trans expression of the rfbJ gene of Salmonella enterica serovar Paratyphi B and/or Salmonella enterica serovar Typhimurium.

13. The live attenuated strain of claim 11, wherein said strain has its native fliC gene replaced with the fliC gene of Salmonella enterica serovar Paratyphi B and/or Salmonella enterica serovar Typhimurium, or wherein the substituted fliC gene is expressed in trans, such that the conferred serotype is altered from an Hd serotype to a Hb and Hi serotype respectively.

14. The live attenuated strain of claim 11, wherein the fljBA locus of Salmonella enterica serovar Paratyphi B and Salmonella enterica serovar Typhimurium are inserted into the chromosome of Salmonella enterica serovar Typhi or expressed in trans.

15. The live attenuated strain of claim 11, wherein the hix sites flanking the native promoter of the fIjBA operon, or the hin recombinase gene, are modified to achieve constitutive expression.

16. The live attenuated strain of claim 11, wherein the strain is further modified according to claim 7.

17. A vaccine comprising the live attenuated strain of claim 11, for use in enhancing immunogenicity against Salmonella enterica serovar Paratyphi B and/or Salmonella enterica serovar Typhimurium and for use in the treatment or prevention of enteric fever and salmonellosis.