Comparative Genomics of Salmonella enterica Serovar Typhi Strains Ty2 and CT18

Abstract

We present the 4.8-Mb complete genome sequence of Salmonella enterica serovar Typhi strain Ty2, a human-specific pathogen causing typhoid fever. A comparison with the genome sequence of recently isolated S. enterica serovar Typhi strain CT18 showed that 29 of the 4,646 predicted genes in Ty2 are unique to this strain, while 84 genes are unique to CT18. Both genomes contain more than 200 pseudogenes; 9 of these genes in CT18 are intact in Ty2, while 11 intact CT18 genes are pseudogenes in Ty2. A half-genome interreplichore inversion in Ty2 relative to CT18 was confirmed. The two strains exhibit differences in prophages, insertion sequences, and island structures. While CT18 carries two plasmids, one conferring multiple drug resistance, Ty2 has no plasmids and is sensitive to antibiotics.

Salmonella enterica serovar Typhi is a human-specific pathogen causing enteric typhoid fever, a severe infection of the reticuloendothelial system (8, 10, 14). Although difficult to estimate, it is thought that at least 16 million cases and 500,000 deaths occur each year around the world (8). The early administration of antibiotic treatment has proven to be highly effective in eliminating infections, but indiscriminate use of antibiotics has led to the emergence of multidrug-resistant strains of S. enterica serovar Typhi (13, 31). Since typhoid is becoming difficult to treat with conventional drugs, information about the whole genome sequence and genes of S. enterica serovar Typhi will help to reveal more specific targets for drugs aimed at disease treatment and vaccines for prevention. Although S. enterica serovar Typhi can grow in laboratory media and survive in other hosts, such as experimental mice, humans are the only known natural hosts. The mouse model is a poor representive of the human disease. Experimental infections of chimpanzees or human volunteers have been the only way to relate bacterial genetic characteristics with pathogenic effects. Consequently, what is known about S. enterica serovar Typhi pathogenicity has been largely extrapolated from studies of S. enterica serovar Typhimurium infections in mice.

In this work, we present the genome sequence of the well-studied pathogenic strain Ty2. This strain was the foundation for vaccine development and was the parent of mutant strains Ty21a and CVD908 and their derivatives, used in trials of live attenuated vaccines (16). Isolated before the emergence of drug resisitance in the 1970s, it contains no plasmids. In contrast, recently isolated S. enterica serovar Typhi strain CT18 (25) carries multiple drug resistance cassettes on a large plasmid and contains a second large plasmid closely related to pMT1 of Yersinia pestis. The genome sequence of CT18 was recently determined at the Sanger Centre, Hinxton, United Kingdom (GenBank accession number AL513382), and we have used this sequence to perform a detailed comparison of the two genomes.

MATERIALS AND METHODS

Bacterial strain.

S. enterica serovar Typhi strain Ty2 was obtained from K. Sanderson (Salmonella Genetic Stock Center isolate 2666) and was redeposited at the American Type Culture Collection under accession number 700931. The strain was cultured and DNA was kindly prepared by R. A. Welch, University of Wisconsin.

Sequencing.

A whole-genome shotgun library was prepared using nebulization to produce random fragments by mechanically shearing genomic DNA (21). Fragments in the size range of 1 to 2.5 kb were prepared by agarose gel electrophoresis, ends were repaired, and the fragments were cloned into the M13Janus vector (4). Random phage clones were isolated, and purified DNAs were prepared as templates for sequencing reactions by using dye terminator chemistry. Some templates were prepared by PCR directly from M13Janus clones. Sequence data were collected with ABI377 and 3700 automated sequencers, and data were assembled by using Seqman II, Genome Edition (DNASTAR, Madison, Wis.). For the finishing steps, clonal and genomic PCR techniques provided templates for primer walking to link contigs, add complementary reads, and increase coverage. The final coverage was 9.8-fold. The contig order and assembly structure were independently confirmed by using a whole-genome optical map of recognition sites for restriction enzyme NheI (18).

Annotation and analysis.

When the genome of S. enterica serovar Typhi strain CT18 was published, our sequence was completely enclosed in a single contig, but many ambiguities remained and the sequence was not annotated. We made use of the CT18 sequence to accelerate our analysis. Open reading frames (ORFs) in Ty2 were defined by using GeneMark.hmm (1) and compared with CT18 ORFs by using BLAST. Most of the differences were identified from the ORF comparisons and then were checked in detail by using Lasergene DNA analysis tools (DNASTAR). We compared the Ty2 genome with the CT18 genome for all ambiguities and conflicts. When our primary trace data were adequate and consistent with the CT18 sequence at a confidence level of over 70%, we corrected the ambiguities and conflicts to match the CT18 consensus sequence. In total, 2,739 of 4,742 total ambiguities and conflicts were corrected by this process, leaving 2,003 in the Ty2 sequence due to insufficient evidence tosupport such changes or sound data confirming about 450 real differences between the two genomes. Every sequence difference was carefully inspected, and any ambiguity that could indicate a Ty2 feature different from CT18 was resolved. All Ty2 annotations that were different from CT18 were based on sound data. Ty2 ORFs were assigned identifiers in the form t0001.

Nucleotide sequence accession number.

The Ty2 sequence contains cross-references to the equivalent genes in the CT18 sequence and is available at GenBank under accession number AE014613.

RESULTS AND DISCUSSION

The genome of Ty2 consists of a single, circular chromosome of 4,791,961 bp with an average G+C content of 52.05%. Figure 1 shows the circular genome map of Ty2. Base pair 1 was chosen to correspond to Escherichia coli K-12 minute zero (3). The replication origin and terminus were determined according to their homologies to those in K-12. They are near kb 3750 and 1544, respectively. The C/G distribution switches polarity at both the origin and the terminus. Ty2 has seven rRNA operons, with five copies in one replichore and two in the other (Fig. 1). However, the rRNA operons are located in opposite replichores in Ty2 and CT18 versus K-12 and S. enterica serovar Typhimurium (20, 23, 32). The Ty2 genome is distinguished from that of CT18 by a major interreplichore inversion that spans the terminus of replication and almost half of the Ty2 genome (Fig. 1 and 2). This rearrangement leaves the two replichores slightly uneven in size. The boundaries of the half-genome inversion lie within rRNA operons at the locations of rrnG and rrnH, by comparison with the arrangement in E. coli K-12; both operons were rrnG-rrnH hybrids in Ty2 and were highly likely to have mediated the inversion through homologous recombination (20). Although this recombination did not change the rRNA organization, such genome rearrangements by homologous recombination at rRNA genes often result in distinguishable ribotypes. Variations in ribotypes seem to be linked to host adaptation (32). Besides this major inversion, there is a small inverted region that is also translocated (Fig. 1, green segment in sixth circle).

FIG. 1.

Circular genome map of Ty2. The Ty2 genome has 4,545 ORFs and pseudogenes, 4,516 of which are shared with CT18 (outer circle, blue) and 29 of which are unique (pink). Arrowheads within the second circle show the locations and orientations of rRNA operons (red) and tRNAs (turquoise) (not drawn to scale). The third circle shows insertion element distributions: blue, IS200; red, other IS elements. The fourth circle shows the scale in base pairs. The fifth circle shows the C/G skew, calculated for each sliding window of 10 kb along the genome. The sixth and seventh (innermost) circles show the CT18 and Ty2 genome comparison: blue (and above the axis in CT18) indicates colinear regions, red (and below the axis in CT18) indicates inverted regions, green indicates a region that is translocated and inverted again within the half-genome inversion region, and yellow indicates unique regions. The map was created with GenVision (DNASTAR).

FIG. 2.

Comparative linear maps of Ty2 and CT18 genomes. The region inverted in Ty2 relative to CT18 is indicated by crossed lines. Black segments are unique regions (islands), lettered for cross-reference with Table 1. Tick marks outside the genome bars indicate the corresponding locations where islands are positioned in the other genome.

A comparison of Ty2 with CT18 reveals that more than 98% of the genome sequence is shared in these two S. enterica serovar Typhi strains. Apart from the inversion, the organization of the two genomes is quite similar, unlike the extensive rearrangements seen in a comparison of two Y. pestis strains, KIM and CO92 (7). For Ty2, we predicted 4,339 ORFs, 206 pseudogenes, and 101 RNA genes by comparison with the CT18 annotations (GenBank accession number AL513382). They average 910 bp in length and cover 88% of the genome. While 4,195 ORFs and pseudogenes are identical to those in CT18, 282 differ only in single point mutations from CT18. Together, they account for 97% of the total, a finding consistent with the results of a whole-genome nucleotide comparison. Seven Ty2 ORFs have insertions or deletions relative to CT18 ORFs that are multiples of 3 bp, resulting in longer or shorter proteins without disruption of the reading frames. Ty2 has relatively few insertion sequences (IS), having 26 copies of IS200F and only 3 copies (1 each) of other IS elements: IS1230B, IS285, and IS1351. In addition to these elements, CT18 contains three copies of IS1 (Table 1).

TABLE 1.

Regions of differences between Ty2 and CT18

Fig. 2 location	tRNA	Ty2			CT18
		Coordinates	Features	Length (bp)	Features	Length (bp)
a		754495-754496		0	IS1 (STY2419-STY2420)	769
b	Asn	956415-978952	Restriction-modification module (t0870-t0872)	22,536		0
c		1092785-1092786		0	Part of phage (STY2040-STY2077)	23,992
d		1489236-1490494	yneH; glutaminase (t1446)	1,258		0
e		1542863-1542864	narV and narW fused (t1490)	0	narV and narW intact	746
f		1933051-1933052		0	Part of phage (STY1048-STY1073)	19,969
g		2654902-2654903		0	ORFs (STY311 and STY312)	972
h	ssrA	2734849-2745476	Part of phage (t2647-t2656)	10,626		0
i	Gly	3039580-3045437	Hypervariable region; phage? (t2947-t2954)	5,858	Hypervariable region; phage? (STY3188-STY3193)	6,270
j		3407317-3407836	Spacer tRNAIle-tRNAAla	519	Spacer tRNAGlu	353
k		3732787-3734141	Spacer tRNAGlu	1,354	Spacer tRNAIle-tRNAAla	585
m		3973595-3973596		0	IS1 (STY4124-STY4125)	770
n		4442808-4442809		0	STY4580-STY4582	1,059
o		4501860-4501861		0	IS1 (STY4657-STY4658)	770

Ty2 prophages.

There are 29 ORFs unique to Ty2, whereas 84 are unique to CT18 (Table 2), many of them associated with putative prophages. Like the CT18 genome, the Ty2 genome contains seven regions that are prophage-like. However, they are not all identical. The relationships between the prophage regions of the two genomes are shown by the diagram in Fig. 3. Four prophages are present in identical locations in both genomes relative to the adjacent nonphage genes (Table 3). The prophage integrated near tRNA^Phe in Ty2 is probably a remnant prophage; the homologous region in CT18 is not annotated as a prophage in that genome. As shown in Fig. 3, two Ty2 prophages are composed of recombined parts of CT18 prophages (or vice versa), and both genomes also have parts of prophages that are unique to those strains (Table 1). Similar observations for the prophages of O157:H7 suggested that the modular nature of prophage genomes makes a significant contribution to strain variation (24, 27).

TABLE 2.

ORFs unique to each strain

Strain	ORF	Product
Ty2	t0869	Hypothetical protein
	t0870	Hypothetical protein
	t0871	Type III restriction-modification system restriction subunit
	t0872	Type III restriction-modification system methylation subunit
	t1446	Putative glutaminase
	t2647	Putative prophage integrase
	t2648	Phage P4 psu protein
	t2649	Phage P4 delta protein
	t2650	Phage P4 sid protein
	t2651	Phage P4 DNA-binding protein
	t2652	Phage P4 cI protein
	t2653	Phage P4 epsilon protein
	t2654	Phage P4 ORF151
	t2655	Phage P4 ORF106
	t2656	Phage P4 alpha protein
	t2718	Hypothetical protein
	t2720	Hypothetical protein
	t2722	Hypothetical protein
	t2724	Hypothetical protein
	t2947	Hypothetical protein
	t2948	Hypothetical protein
	t2949	Hypothetical protein
	t2950	Hypothetical protein
	t2951	Hypothetical protein
	t2952	Hypothetical protein
	t2953	Possible integrase
	t2954	Hypothetical protein
	t3166	Hypothetical protein
	t3172	Putative transcriptional repressor (DeoR family)
CT18a	STY0311	Hypothetical protein
	STY0312	Probable secreted protein
	STY1048	Putative bacteriophage protein
	STY1049	Putative bacteriophage protein
	STY1050	Putative bacteriophage protein
	STY1051	Putative bacteriophage protein
	STY1052	Putative bacteriophage protein
	STY1053	Putative bacteriophage protein
	STY1054	Putative bacteriophage protein
	STY1055	Putative bacteriophage protein
	STY1056	Putative bacteriophage protein
	STY1057	Putative bacteriophage protein
	STY1058	Hypothetical prophage protein
	STY1059	Putative bacteriophage protein
	STY1060	Putative bacteriophage protein
	STY1061	Putative bacteriophage protein
	STY1062	Putative bacteriophage protein
	STY1063	Putative bacteriophage protein
	STY1064	Putative bacteriophage protein
	STY1065	Putative secreted protein
	STY1066	Putative bacteriophage protein
	STY1067	Putative bacteriophage protein
	STY1068	Putative bacteriophage protein
	STY1069	Putative bacteriophage protein
	STY1070	Putative bacteriophage protein
	STY1071	Putative bacteriophage protein
	STY1072	Hypothetical prophage protein
	STY1073	Hypothetical prophage protein
	STY1485	Respiratory nitrate reductase 2 gamma chain; NarV
	STY1641	Alternative bacteriophage tail fiber C terminus
	STY20074A	Restriction alleviation and modification enhancement protein
	STY2012	Phage recombinase (pseudogene)
	STY2040	Putative bacteriophage protein
	STY2041	Putative bacteriophage protein
	STY2042	Hypothetical protein
	STY2043	Putative bacteriophage protein
	STY2044	Putative endolysin
	STY2045	Putative bacteriophage protein
	STY2046	Hypothetical protein
	STY2047	Putative membrane protein
	STY2048	Putative bacteriophage protein
	STY2049	Hypothetical protein
	STY2050	Hypothetical protein
	STY2051	Putative bacteriophage protein
	STY2052	Hypothetical protein
	STY2053	Putative bacteriophage cohesive ends
	STY2054	Cell-killing toxin-antitoxin system
	STY2054A	Host cell-killing modulation protein
	STY2055	Hypothetical protein
	STY2056	Putative transposase
	STY2057	Hypothetical protein
	STY2058	Putative membrane protein
	STY2059	Putative bacteriophage protein
	STY2060	Hypothetical protein
	STY2061	Conserved hypothetical protein
	STY2062	Putative DNA replication protein
	STY2063	Conserved hypothetical protein
	STY2064	Conserved hypothetical protein
	STY2065	Putative cro repressor
	STY2066	Putative regulator
	STY2067	Conserved hypothetical protein
	STY2068	Conserved hypothetical protein
	STY2069	Hypothetical protein
	STY2070	Putative cell division inhibitor protein
	STY2071	Putative bacteriophage protein
	STY2072	Putative bacteriophage protein
	STY2073	Exodeoxyribonuclease VIII
	STY2074	Conserved hypothetical protein
	STY2075	Conserved hypothetical protein
	STY2076	Putative excisionase
	STY2077	Putative integrase
	STY2419	Insertion element IS1 protein InsB
	STY2420	Insertion element IS1 protein InsA
	STY3188	Hypothetical protein
	STY3189	Hypothetical protein
	STY3191	Hypothetical protein
	STY3192	Hypothetical protein
	STY3193	Possible integrase
	STY4124	Insertion element IS1 protein InsA
	STY4125	Insertion element IS1 protein InsB
	STY4580	Putative membrane protein
	STY4582	Possible exported protein
	STY4657	Insertion element IS1 protein InsB
	STY4658	Insertion element IS1 protein

^aData for CT18 are from GenBank accession number AL513382.

FIG. 3.

Diagram of prophage regions in Ty2 and CT18. Open rectangles outlined in black, cognate prophage and prophage segments; gray rectangles, unique prophage segments; open rectangle outlined in gray, segment not annotated as phage in CT18; small black rectangles, tRNAs near phage integration sites. The diagram is not drawn to scale.

TABLE 3.

Phage regions similar in Ty2 and CT18

ORFs		tRNA	Comments
Ty2	CT18
t1346-t1397	STY1591-STY1642
t1867-t1996	STY1011-STY1047
t1897-t1929	STY2013-STY2039
t2657-t2674	STY2879-STY2897	ssrA	tmRNA insertion site; contains iro operon
t3400-t3435	STY3657-STY3703	Trp
t4294-t4338	STY4600-STY4645		Contains sopE; virulence factor
t4358-t4371	STY4667-STY4680	Phe	Possible phage remnant
t4517-t4529	STY4821-STY4832	Leu	Part of large island; contains serine/threonine kinases

Neither the CT18 genome nor the Ty2 genome contains the Gifsy-1 phage identified in S. enterica serovar Typhimurium LT2 (9), and only parts of Gifsy-2 are represented (the leftmost CT18 region in Fig. 3; genes t1904 to t1919 in Ty2). In both genomes, these regions are incorporated into putative prophages different from Gifsy-2 and different from each other. Gifsy-2 in LT2 encodes two proteins important for survival in macrophages, SodC (superoxide dismutase) and MsgA (function unknown). In Ty2 and CT18, the Gifsy-like regions do not contain genes for either of these, but both genes are present elsewhere in nonphage regions that are homologous in the two genomes. The previously identified LT2 phages Fels-1 and Fels-2 are not present in Ty2, although there are some regions of homology where Ty2 prophage genes are similar, as is the case for CT18 (22). Virulence-related genes iroBCDEN (Ty2 ORFs t2668 to t2672, encoding an iron uptake and storage system) and sopE (t4303, encoding an invasion-associated, type III-secreted effector protein) are located in cognate prophages in the two genomes.

Other differences.

Like the phage and IS differences, several lineage-specific islands were found by strain comparison (Table 1). It was not possible to ascertain whether these differences resulted from insertions into one genome or deletions from the other. CT18 ORFs STY0311 and STY0312 are not present in Ty2. The products of these ORFs are not similar to any protein in the database, but the product of STY0312 has characteristics of a secreted protein, as do the products of STY0314 and STY0316 nearby. Since secreted products often interact with extrabacterial targets, these ORFs may be related to pathogenicity. t1446 in Ty2 encodes a homolog of glutaminase (EC.3.5.1.2) that is encoded by yneH in LT2. This enzyme has roles in glutamate and nitrogen metabolism. CT18 has no equivalent gene. A 22.5-kb island at tRNA^Asn in Ty2 carries t0871 and t0872, whose products are similar to a restriction enzyme and the C-terminal portion of a modification methylase enzyme of a type III system that probably is not functional, since only 173 out of 525 amino acids (aa) remain. An intact hsdRMS locus encodes the conserved Salmonella and E. coli type I restriction-modification system elsewhere in the genome.

The rRNA loci rrnG and rrnH mediated the large genomic inversion noted above. Recombination between these loci leaves hybrid rrn regions with spacer tRNAs different from those in the analogous K-12 loci, as identified by the DNA sequences flanking the rRNA genes. We found tRNA^Glu at Ty2 rrnH (versus tRNA^Ile-tRNA^Ala in K-12) and the converse at rrnG. Although CT18 is colinear with K-12 in this part of the genome, it has the same spacers as Ty2 in rrnH and rrnG, suggesting that two events have occurred: an inversion followed by a reinversion restoring colinearity with K-12. Near the origin of replication, intrareplichore inversions in both the CT18 and the Ty2 genomes have resulted in hybrid rrn loci with tRNA^Ile-tRNA^Ala in CT18 and tRNA^Glu in Ty2 at one hybrid site and the converse at the other site. In this region, the two genomes are colinear, so the difference in spacers again can be explained by a second recombination event. In fact, only rrnB is intact in both genomes. It is interesting that unusual rrn loci with apparently recombined spacer tRNA segments also appear among the annotated genes in Shigella flexneri strain 301 (GenBank accession number AF386526). While these events are admittedly difficult to reconstruct, it is known that rrn-mediated rearrangements are common in S. enterica serovar Typhi (19) and have been used to distinguish Typhi isolates by ribotyping (23).

Differences among pseudogenes in CT18 and Ty2.

Perhaps the most significant difference between Ty2 and CT18 is found in the selective silencing of gene functions in the form of pseudogenes. In CT18, there are 204 verified pseudogenes; 9 of these are intact genes in Ty2 (Table 4). On the other hand, Ty2 has its own unique set of 11 pseudogenes as well as 195 in common with CT18 (Table 5). Seven Ty2-specific pseudogenes result from frameshifts, while four result from disruptions by point mutations that create internal stop codons. One of the pseudogenes in CT18 (STY2012), which encodes a partial phage recombinase and which is located at a phage insertion point, is entirely lost in Ty2. A Ty2 gene (t0235) that encodes a putative chitinase has an internal stop codon and is annotated as a pseudogene in our Ty2 GenBank entry. Chitinase, related to lysozyme, is an enzyme that disrupts cell membranes by digestion of peptidoglycan linkages. In the corresponding CT18 annotation (ORF STY0257), although the DNA sequence for this gene is identical to that in the Ty2 annotation, it is stated that the stop codon is translationally suppressed by insertion of a tryptophan residue. While there has been a report of this mode of suppression at a low efficiency, we know of no evidence to support it in this particular case.

TABLE 4.

Genes that were pseudogenes in CT18^a but intact in Ty2

ORF		Gene	Product
Ty2	CT18
t2448	STY0453	ybaD	Conserved hypothetical protein
t2319	STY0590	fimI	Fimbrin-like protein FimI
t1254	STY1735	ttrS	Sensor kinase TtrS protein
t1023	STY1987	sopE2	Putative invasion-associated secreted protein
t0757	STY2328	wcaA	Putative glycosyltransferase
t0589	STY2504		Putative transcriptional regulator
t0425	STY2668		Phosphoenolpyruvate-protein phosphotransferase
t3035	STY3280		Bacteriocin
t3695	STY3955	torC	Cytochrome c-type protein

^aFor CT18 mutation details, see Parkhill et al. (25).

TABLE 5.

Genes that were pseudogenes in Ty2 but intact in CT18

ORF		Gene	Product	Mutation
Ty2	CT18
t2523	STY0371	stbC	Outer membrane fimbrial usher protein	Internal stop codon
t2474	STY0423	aroM	AroM protein	Frameshift
t1913	STY1027		Hypothetical bacteriophage protein	Internal stop codon
t1549	STY1423		Putative exported protein	Frameshift
t1490	STY1485	narV	Respiratory nitrate reductase 2 gamma chain	Deletion fuses two ORFs into one
	STY1486	narW	Respiratory nitrate reductase 2 delta chain	Deletion fuses two ORFs into one
t1429	STY1553		Putative d-mannonate oxidoreductase	Internal stop codon
t1183	STY1810	astA	Arginine N-succinyltransferase	Frameshift
t1166	STY1829		Putative regulatory protein	Frameshift
t0706	STY2379	stcC or yehB	Putative outer membrane usher protein	Frameshift
t2688	STY2913	gabP	GabA permease (4-aminobutyrate transport carrier)	Internal stop codon
t4529	STY4832		Bacteriophage P4 DNA primase	Frameshift

Table 6 shows genes in Ty2 that have changes causing their coding frames to diverge from those in CT18. For example, gltX, encoding glutamyl-tRNA synthetase, is intact in Ty2, but in CT18, a frameshifting deletion results in a predicted protein three residues longer at the C terminus. Since gltX is an essential gene, the extra residues presumably do not affect the synthetic activity appreciably. Some of the pseudogenes listed in Table 4 to 6 also may have a role in pathogenesis. In addition to the loss of 7 out of 12 fimbrial operons in both strains, Ty2 has lost 2 more (stcC and stbC) but has gained fimI. Three other genes that are intact in Ty2 may be associated with pathogenicity. TtrS is a sensor for tetrathionate, an alternative electron acceptor in vitamin B₁₂-dependent anaerobic growth, and may be important for intracellular survival. SopE2 is secreted by a type III mechanism into host cells, where it is involved in actin rearrangements (12), a common first step in bacterial attack or invasion of host cells. WcaA is thought to be a glycosyltransferase involved in the synthesis of colanic acid, which is secreted to form the exopolysaccharide capsule providing protection from, for example, dehydration, acid stress, and osmotic stress, conditions encountered both inside and outside the human host. This capsule is also implicated in another pathogenic mechnism, biofilm formation (6, 28).

TABLE 6.

ORFs whose products have divergent C-terminal sequences

ORF		Gene	Product	Comments
Ty2	CT18
t0080	STY0089	yaaU	Putative major facilitator superfamily transport protein	59 aa longer; stop codon further downstream in Ty2
t1445	STY1536		Putative aldehyde dehydrogenase	87 aa longer; insertion of 1,260 bp in Ty2
t1379	STY1609		Hypothetical protein	103 aa longer; stop codon further downstream in Ty2
t0442	STY2654	gltX	Glutamyl-tRNA synthetase	3 aa longer; frameshift in Ty2
t2825	STY3049	rpoS	RNA polymerase sigma subunit transposase	54 aa longer; frameshift in Ty2
t3447	STY3706	tnpA		18 aa longer; stop codon further downstream in Ty2
t3828	STY4105	sapB	Putative autotransporter	9 aa longer; frameshift in Ty2
t4435	STY4740	sgaB	Putative phosphotransferase system IIB protein	77 aa longer; frameshift in Ty2

NR-Z and RpoS.

Differences between the loci for nitrate reductase Z (NR-Z) and RpoS in Ty2 and CT18 may seem to indicate significant differences in their abilities to thrive in anaerobic conditions, but a detailed investigation yielded more questions than answers. NR-A and NR-Z complexes provide electron transport during anaerobic respiration. CT18 has intact genes in both loci. In Ty2, the NR-A locus is intact, but the Z components encoded by narW and narV are fused by an in-frame deletion. Since both gene products are essential for a functional Z complex, this pathway is probably inactivated in Ty2, although the consequences are unclear. However, S. enterica serovar Typhi mutants deficient in anaerobic respiration are also less capable of intracellular replication (5), a factor which would compromise virulence.

The structure and function of the Z complex are understood, but its role is obscure. The narZYWV genes (product, NR-Z) are homologs of the genes of the NR-A locus, narGHIJ, which are active in anaerobic respiration. The four genes encode the alpha, beta, delta, and gamma components, respectively. By analogy with NR-A, the alpha component contains the catalytic site and requires a molybdenum cofactor for activity. The beta component contains an iron-sulfur center and transfers electrons from the gamma component to the alpha component. Both alpha and beta are cytoplasmic proteins. Paired gamma chains form a membrane anchor, and heme-iron centers are embedded in the membrane. Electrons are accepted by the gamma chains from quinone and then transferred to the beta protein, which in turn transfers them to the alpha subunits, where the reduction reaction is completed. The delta protein is not actually part of the complex but is essential for activity; it is thought to be important in assembling the complex. In Ty2 NarW, the C-terminal 54 aa out of 236 are deleted, and in NarV, aa 1 to 194 out of 225 are lost, including all of the transmembrane segments that form the membrane anchor.

NR-Z was thought to be expressed constitutively at a low level and was known not to be induced during anaerobic growth (11). Recently, the locus in S. enterica serovar Typhimurium was shown to be induced by carbon starvation (stationary phase) and to be RpoS dependent (34). NR-Z is also essential for starvation-induced heat and acid tolerance. NR-Z expression is not induced during anaerobic growth but is actually repressed by Fnr, the nitrogen-sensing regulator. Active hybrid enzymes mixing NR-A and NR-Z subunits have been obtained (2); therefore, it is possible that the NR-A membrane anchor tethers the NR-Z enzymes and that NarJ replaces the mutated NarW, if they are expressed under the same conditions as NR-Z.

However, rpoS is also abnormal in Ty2 (although it is intact in CT18). The alternative sigma factor encoded by this gene regulates more than 30 genes in the stress response. In Ty2, a frameshift at aa 312 replaces the last 12 aa of the wild-type product with 74 different aa. This change was discovered when mutations were introduced into Ty2 in efforts to attenuate its virulence for the creation of a safe vaccine strain, called Ty21a (29) or CVD908 (17). Subsequently, attenuation proved to be due partly to a mutant RpoS which had not been deliberately introduced. The mutation was later shown to be present in the Ty2 parent strain as well (30). Ty21a survived starvation and other stresses poorly, an advantage in a vaccine strain, and these deficiencies were complemented by an introduced wild-type RpoS. Unfortunately, no equivalent data exist for Ty2; therefore, we cannot be sure that other mutations in Ty21a, either engineered or accidental, have influenced the stress response of Ty21a or determine whether there is any direct effect on the virulence of Ty2.

We obtained Ty2 for sequencing from a Salmonella archive to minimize possible effects of repeated passaging. However, variant rpoS genes have been identified in archived stocks of Salmonella and have also been observed in E. coli K-12 W3110 (35). Some spontaneous mutants that arise during prolonged starvation are associated with a growth advantage (38). These observations suggest that mutation of rpoS under extreme stress itself may be a stress response, permitting the selection of a more efficient transcription factor that can improve fitness to respond to adverse conditions.

Interestingly, the genomic region between rpoS and mutS is highly plastic in members of the family Enterobacteriaceae (15), with many rearrangements in this interval. In S. enterica serovar Typhimurium, the expression of the Spv proteins, necessary for bacterial survival inside invaded host cells, is under RpoS control. The Spv genes, carried on a plasmid in serovar Typhimurium, are not present in serovar Typhi, and little is known about how different host tissue invasion mechanisms may be in serovar Typhi or how dependent on RpoS they may be. In fact, the extended RpoS protein in Ty2 retains intact both the RNA polymerase core binding site and the putative DNA binding site that together bring about enhanced binding of the polymerase to the DNA template. Stationary-phase (starvation) responses are under the control of RpoE (a different alternative sigma factor) (36), as are responses to other stimuli important in virulence, such as oxidative stress imposed by macrophage defenses. It is possible that these two transcription factors have some overlapping effects, making it very difficult to predict the effect of this particular mutant RpoS. It is interesting that S. flexneri 2a strain 2457T also has mutations in the narZYWV locus as well as a mutant rpoS (unpublished data). A frameshift changes the C-terminal 30 aa and truncates the transcript, whereas another 2a strain, 301, has also lost narZ but retains rpoS intact. There is much to be learned yet.

Clearly, while genome sequencing has revealed many genes with potentially important contributions to pathogenicity, discovering the details and deciphering the message, if any, in individual sets of pseudogenes will require extensive reseach in many laboratories, each with a specific expertise. A more appropriate model system is also needed. Newer techniques, such as microarray analysis of gene expression, have begun to provide the next level of information about genes involved in pathogenesis. Of the many knockout mutations already constructed in K-12 strain MG1655 in our laboratory, rpoS mutations should be very informative. A set of characterized mutations in an isogenic background should help to unravel the complex aspects of the stress response and provide clues as to how NR-Z may be involved.

Differences in pseudogene content between CT18 and Ty2 fall into no discernible pattern or functional relationship. These differences may have arisen due to variations in stresses applied by human host defense systems and may contribute subtle effects to the complex mechanisms of pathogenesis used by these two strains. They may also reflect a need to adjust the balance of metabolic capabilities to optimize virulence, perhaps achievable by more than one possible combination of genes.When analyzing pseudogenes, as emphasized by McClelland et al. (22), investigators should be aware that pseudogenes may be identified with confidence only when an intact homolog is found in a closely related (and sequenced) genome, and even then, annotation criteria vary. Some, but not all, annotations designate genes disrupted by IS elements as pseudogenes. It is often unclear whether small differences in protein structure, such as the three extra residues in GltX mentioned above, will eliminate function. Criteria based strictly on gene structure are not appropriate for secreted or surface proteins, such as those of fimbriae, whose genes are normally more variable than the conserved genes nearby (27), with the obvious advantage of evading the host immune system or potentially increasing the ability of the bacterium to attach to and affect host tissues.

How can we account for the accumulation of pseudogenes? Although mutagenic processes such as transposase induction are triggered by the kinds of stresses that bacteria undergo in repeated passages in the laboratory, strains that have been used in laboratories for the longest periods, E. coli K-12 and S. enterica serovar Typhimurium strain LT2, have almost 1 order of magnitude fewer pseudogenes than strains of intracellular pathogens that have also been used in laboratories for long periods, Y. pestis KIM, S. flexneri 2457T, and Ty2. The largest numbers of pseudogenes have been observed primarily in host-adapted pathogens that grow intracellularly and are thought to result from an adaptive process (33). In S. enterica serovar Typhi, adaptive changes have limited the host range to humans and (presumably) inactivated metabolic functions that are not needed for intracellular growth (26, 37) or survival in the intestine. These differences, as well as any of the far more numerous differences between S. enterica serovar Typhi and serovar Typhimurium strains, may underlie disease characteristics that are overtly or subtly distinctive of the pathogenic potential of the strains. It is important that each difference is examined with expert knowledge to identify the genetic variables that may yield valuable information through experimental evaluation. This goal may still be costly and difficult to carry out without an animal model that reproduces the human disease accurately and has reasonable costs. Inspection of every pseudogene for the possibility of residual or altered activity is not a trivial task; in many cases, even this initial test is not immediately possible, since researchers may have no idea of the function of encoded proteins or of which parts of encoded proteins may be essential for function.