Supplementary Note. Potential functional consequences of pseudogenes: further examples.
PipD in SPI-5 is mutated in Paratyphi A. In serovar Dublin, a mutation in pipB reduces the intestinal secretory and inflammatory response induced in the bovine ligated ileal loop but has no effect on the outcome of intraperotenal injection in mice, suggesting that this gene contributes to enteric but not to systemic salmonellosis 1.
Shortage of available iron limits growth inside host cells so iron acquisition increases pathogenicity. However, iron toxicity is a concern in other environments. fhuA and fhuE, which are mutated in SPA and Typhi, are involved in the import of conjugated Fe(III) into the cell, often captured from carrier proteins in the host. FhuA is a receptor for phage, and a transporter for siderophore antibiotics. fhuE is also a pseudogene in Shigella flexneri, which is another recently evolved host-specialist invasive pathogen 2,3. rhlB is a pseudogene in SPA; in Typhimurium, mutants in this gene cannot accurately sense the iron level and/or may accumulate iron to a greater extent than wild-type bacteria 4. SufS and sufD are also mutated in SPA. Mutants in these genes have a defect in using ferrioxamine B as an iron source 5. Ferrioxamine B would be transported by fhuE, which is a mutant in SPA. Two mutated genes located in SPI-2 are part of the iron responsive Fur regulon: ttrB is a mutant in SPA and ttrS is mutated in Typhi. The ttr cluster encodes a gene for tetrathionate as a terminal respiratory electron acceptor 6. The evolution of Paratyphi A and Typhi has involved the disabling of a number of Fe(III) transport and responsive systems; these mutations likely have a profound effect on the response of these serovars to iron in the host or in the environment.
NarW is mutated in both SPA and Typhi. In Typhi it is fused with narV. These genes are part of a cluster that is induced in Typhimurium when inside epithelial cells. Although the roles of narW and narV are not yet known, a mutant in the first gene in the cluster, narZ, is known to decrease oral virulence by ten fold in the mouse
WcaK is mutated in both serovars. This gene is involved in sugar nucleotide precursors for colonic acid synthesis, or M antigen, an extracellular polysaccharide. Other members of the same cluster are mutated in Typhi (wcaD, wcaA), and in SPA (wcaJ, galF). Disrupting the production of this structure appears have been important in the evolution of Paratyphi A and Typhi as there are multiple lesions in both serovars. FliB, required for methylation of lysines in flagellin, is mutated in both serovars, which may also affect antigenicity.
DacD, penicillin binding protein 6b, is mutated in both serovars. Proteins in this family are involved in synthesizing and remodeling peptidoglycan pentapeptide muropeptides, the structural component of the bacterial cell wall 7. Two other related proteins are mutated in Typhi, pbpG and STY2118. The gene mpl, encoding murein peptide ligase, also involved in peptidoglycan metabolism, is mutated in SPA 8.
A few of the pseudogenes in SPA can be correlated with a specific long established phenotype that has been used to distinguish this serovar from other Salmonella. For example, Paratyphi A cannot metabolize D-tartrate, and the sequence reveals a mutation in a transporter for tartrate. Some Paratyphi B strains also do not metabolize tartrate due to a different mutation in the same gene 10. Paratyphi A lacks lysine decarboxylase activity; cadC, the gene that encodes a protein for activation of the lysine decarboxylase operon is a pseudogene. Interestingly, cadC is also a common target for mutation in enteroinvasive members of the Shigella/E. coli complex where the inability to synthesize lysine decarboxylase is thought to be an adaptive mutation 11. UhpT, uhpB, and uhpC, which are part of a glucose-6-phosphate utilization system known to be induced intracellularly in Shigella 12, are all pseudogenes in SPA. Conversely, Paratyphi A remains competent in other metabolic functions whose absence is a characteristic of Typhi where there are pseudogenes in araH, L-arabinose transport system permease, speC, ornithine decarboxylase, and in the dulcitol/galactitol operon, which are likely to be responsible for these known metabolic differences.
Among the 173 pseudogenes found in SPA some are in genes that appear to have a homologue that might take over all or part of the lost function. These include possible functional paralogues of the fhuA and fhuE genes, which are members of one of at least three iron transport systems, the others of which are intact. Similarly, hyaA and dacD are pseudogenes that may have functional homologues and are among only a handful of genes mutated in both SPA and Typhi. Other pseudogenes that may have functional paralogues or redundant systems include ccmH, dmsC, mgtA, proQ, sapB, and sdaC in SPA, and dmsA, hyaF, sthB, and ygbK in Typhi.
Paratyphi A is also unique among Salmonella genomes investigated so far, in that it is missing XapABR involved in metabolism of xanthosine 13. In Typhi, the named candidates for recent deletion include the dgoRKAT cluster, encoding genes in galactonate metabolism, which is also partly deleted in Paratyphi A. DgoA, is present in Paratyphi A but has sustained a point mutation. The acrE gene fragment pseudogene in Typhi is one edge of a deletion that includes two other genes including envR, encoding a possible transcriptional repressor of the acrEF operon. Other genes deleted in Typhi but present in all or almost all other Salmonella include IdnKDO, an operon for L-idonate catabolism 14. A truncation in Typhi of fdnG, in one of the two formate dehydrogenase clusters, is accompanied by the deletion of fdnHI. The orthologue of STM1572, which is a porin closely related to OmpC/NmpC, is deleted in Typhi but no other Salmonella, to date.
1. Wood, M.W. et al. Identification of a pathogenicity island required for Salmonella enteropathogenicity. Mol Microbiol 29, 883-91. (1998).
2. Wei, J. et al. Complete genome sequence and comparative genomics of Shigella flexneri serotype 2a strain 2457T. Infect Immun 71, 2775-86 (2003).
3. Jin, Q. et al. Genome sequence of Shigella flexneri 2a: insights into pathogenicity through comparison with genomes of Escherichia coli K12 and O157. Nucleic Acids Res 30, 4432-41 (2002).
4. Bernstein, J.A., Lin, P.H., Cohen, S.N. & Lin-Chao, S. Global analysis of Escherichia coli RNA degradosome function using DNA microarrays. Proc Natl Acad Sci U S A 101, 2758-63 (2004).
5. Patzer, S.I. & Hantke, K. SufS is a NifS-like protein, and SufD is necessary for stability of the [2Fe-2S] FhuF protein in Escherichia coli. J Bacteriol 181, 3307-9 (1999).
6. Price-Carter, M., Tingey, J., Bobik, T.A. & Roth, J.R. The alternative electron acceptor tetrathionate supports B12-dependent anaerobic growth of Salmonella enterica serovar typhimurium on ethanolamine or 1,2-propanediol. J Bacteriol 183, 2463-75. (2001).
7. Nelson, D.E., Ghosh, A.S., Paulson, A.L. & Young, K.D. Contribution of membrane-binding and enzymatic domains of penicillin binding protein 5 to maintenance of uniform cellular morphology of Escherichia coli. J Bacteriol 184, 3630-9 (2002).
8. Uehara, T. & Park, J.T. Identification of MpaA, an amidase in Escherichia coli that hydrolyzes the gamma-D-glutamyl-meso-diaminopimelate bond in murein peptides. J Bacteriol 185, 679-82 (2003).
9. Liu, D., Verma, N.K., Romana, L.K. & Reeves, P.R. Relationships among the rfb regions of Salmonella serovars A, B, and D. J Bacteriol 173, 4814-9. (1991).
10. Malorny, B., Bunge, C. & Helmuth, R. Discrimination of d-tartrate-fermenting and -nonfermenting Salmonella enterica subsp. enterica isolates by genotypic and phenotypic methods. J Clin Microbiol 41, 4292-7 (2003).
11. Casalino, M., Latella, M.C., Prosseda, G. & Colonna, B. CadC is the preferential target of a convergent evolution driving enteroinvasive Escherichia coli toward a lysine decarboxylase-defective phenotype. Infect Immun 71, 5472-9 (2003).
12. Runyen-Janecky, L.J. & Payne, S.M. Identification of chromosomal Shigella flexneri genes induced by the eukaryotic intracellular environment. Infect Immun 70, 4379-88 (2002).
13. Seeger, C., Poulsen, C. & Dandanell, G. Identification and characterization of genes (xapA, xapB, and xapR) involved in xanthosine catabolism in Escherichia coli. J Bacteriol 177, 5506-16 (1995).
14. Bausch, C., Ramsey, M. & Conway, T. Transcriptional organization and regulation of the L-idonic acid pathway (GntII system) in Escherichia coli. J Bacteriol 186, 1388-97 (2004).