Download The rfb cluster, which encodes functions involved in assembling the

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.
The rfb cluster, which encodes functions involved in assembling the O-antigen, has a precise triplication
in Paratyphi A, similar to repeats in this region reported for other Paratyphi A strains 9.
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.
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