Enterobacteriaceae represents a family of bacteria that exist in intimate relationships with their hosts. These bacteria can be extracellular commensals or intracellular pathogens that cause a range of diseases including urinary tract, respiratory and gastrointestinal tract infections. We determined the distribution of the Rcs sensor kinases, RcsC and RcsD, within Enterobacteriaceae by BLAST and architectural analyses. Organisms that have RcsC- and RcsD-like proteins (amino acid similarity over at least >80% of the full-length sequence) are primarily associated with gut colonization or infection. An exception is the plant pathogen E. carotovora
, which causes potato rot. We speculate that this species uses the Rcs pathway differently in plants and/or it colonizes the gut of an animal. Yersinia pestis
and Yersinia enterocolitica
both contain RcsD proteins that are truncated, raising the possibility that these organisms use a modified version of the pathway. Additional phylogenetic analysis of RcsC and RcsD with PSI-BLAST (Altschul et al., 1997
) revealed that the putative periplasmic domains of RcsC in Erwinia
spp. shared only 33–43% identity with the S. enterica
RcsC periplasmic domain (amino acids 39–312). Only the closely related Escherichia
spp. had RcsC and RcsD (amino acids 39–309) periplasmic domains with > 80% identity to the corresponding domains in S. enterica
. As the periplasmic domains are predicted to initiate signalling by sensing change in the bacterial environment, these results suggest that different bacteria may use the Rcs pathway to respond to different signals.
Pathogens are exposed to AMPs throughout infection, and genes that confer resistance to AMPs often are important for survival within the host (Ernst et al., 2001
). For instance, the PhoQ/PhoP sensor kinase system is required for AMP resistance and is activated by polymyxin B (Gunn and Miller, 1996
; Bader et al., 2003
). However, phoP
mutant bacteria are exquisitely sensitive to polymyxin B (Gunn and Miller, 1996
), whereas mutants in the Rcs pathway are only moderately sensitive (). This is consistent with the notion that the PhoQ/PhoP system serves as a master regulator of AMP resistance, whereas the Rcs pathway modulates bacterial resistance to AMPs. For example, the RcsC/RcsD/RcsB system may regulate responses to specific classes or concentrations of AMPs encountered within host tissues. Alternatively, if the Rcs pathway is one of multiple activators of particular AMP-resistance genes, significant levels of AMP-resistance proteins may still accumulate in an Rcs null mutant.
The RcsB-binding partner, RcsA, contributes to neither polymyxin B resistance in vitro
nor infection in mice ( and ). Therefore, Rcs-controlled genes that contribute to AMP resistance should be regulated independently of RcsA. A custom-designed high-fidelity oligonucleotide microarray was used to identify Rcs-regulated, RcsA-independent genes in polymyxin B at 37°C. This screen identified 21 loci and strains harbouring mutations in four of the loci were sensitive to polymyxin B exposure (). The remaining genes may not play a role in AMP resistance or may function in redundant pathways such that polymyxin B sensitivity would only be observed in strains with deletions in multiple genes. Previous studies using microarray screens to identify Rcs-regulated genes have been performed in E. coli
K12 by isolating total RNA from bacteria incubated at either 20°C (Hagiwara et al., 2003
) or 30°C (Ferrieres and Clarke, 2003
). The Rcs pathway was activated in these screens with either zinc treatment (Hagiwara et al., 2003
) or exogenously expressed djlA
(Ferrieres and Clarke, 2003
). While these studies revealed interesting Rcs-regulated genes, they were performed in non-pathogenic E. coli
and neither study focused on RcsA-independent genes. Of the 21 Rcs-activated, RcsA-independent genes identified in our microarray screen (), 16 were found in a previous Salmonella
microarray analysis of polymyxin B-induced genes (Bader et al., 2003
). The Bader et al
. microarray analysis focused on genes regulated by the PhoQ/PhoP two-component signalling system, a master regulator of AMP-resistance genes. It is not clear whether the genes reported here or in previous studies (Bader et al., 2003
; Ferrieres and Clarke, 2003
; Hagiwara et al., 2003
) are exclusively Rcs- (or PhoP-) dependent or whether they are regulated by both pathways. One possibility is that Rcs indirectly regulates expression of some genes by affecting the PhoQ/PhoP signalling system. If this were the case, it would suggest cross-talk between the RcsC/RcsD/RcsB and PhoQ/PhoP systems and underscores the complexity of bacterial signalling.
is one of the array-identified genes that contributes to polymyxin B resistance (). Real-time quantitative PCR analyses confirm that ydeI
RNA accumulation is induced by the Rcs pathway independently of RcsA (), and is dependent on the sigma factor RpoS (). However, ydeI
RNA accumulates in the absence of RpoS, although at much lower levels than in wild type (). One explanation for this observation is that RcsB can recruit a second sigma factor, such as RpoD (sigma70), to the ydeI
promoter but the second sigma factor is much less efficient than RpoS at activating ydeI
transcription. In addition to ydeI
, other genes identified in our screen are RpoS-regulated. These genes include ecnB
(our unpublished data), yeaG
(Ibanez-Ruiz et al., 2000
(Bader et al., 2003
(Lacour and Landini, 2004
(Weber et al., 2005
). The 5′ untranslated region of ydeI
reveals a degenerate −35 region, consistent with regulation by RpoS (Typas and Hengge, 2006
). A recent report suggests that the Rcs pathway in E. coli
promotes RpoS protein accumulation by blocking the activity of an RpoS repressor, LrhA (Peterson et al., 2006
). It is possible that RcsB promotes ydeI
transcription via RpoS by inhibiting LrhA. However, as RpoS is a global sigma factor, specific transcription factors, such as RcsB, may determine the activation of specific genes under certain conditions (Hengge-Aronis, 2002
). For instance, RcsB, a proven DNA-binding protein and transcriptional regulator (Wehland and Bernhard, 2000
), may determine the transcription of ydeI
in the presence of AMPs by binding to the promoter of ydeI
and recruiting RpoS. Alternatively, an unidentified RcsB-activated transcription factor may bind the ydeI
promoter and recruit RpoS. Thus, the mechanism of transcriptional activation for ydeI
and other polymyxin B-induced genes after induction of the Rcs pathway remains to be determined.
In competitive infections, ydeI
null mutants were out-competed by wild-type S.
Typhimurium in 129Sv6 mice. After i.p. inoculation, ydeI
mutants had only a subtle deep tissue phenotype, comparable to that of Rcs mutant strains ( and ). However, after oral inoculation, 10- to 100-fold more wild type than ydeI
mutant bacteria were recovered from both intestinal and deep tissues (). The dependence of phenotype severity on inoculation route may be informative. Oral inoculation delivers the bacteria into the stomach. S.
Typhimurium must survive transit through to the small intestine where the bacteria breach the intestinal wall and access deep tissues, including the MLNs, spleen and liver. In contrast, i.p. inoculation by-passes the gastrointestinal tract and results in the rapid distribution of bacteria to the spleen and liver via blood-borne phagocytes. Several S.
Typhimurium mutants are attenuated for virulence after oral but not i.p. inoculation. For instance, mutants lacking a functional Salmonella
pathogenicity island-1 (SPI1) type three secretion system (T3SS) are out-competed by wild type 10-fold in the MLN and spleen only after oral inoculation (Galan and Curtiss, 1989
; Jones and Falkow, 1994
; Baumler et al., 1997
). SPI1 mediates epithelial cell invasion, suggesting that invasion of intestinal epithelial cells is important for bacterial access to deep tissues (Baumler et al., 1997
). These experiments were performed in a mouse model of acute infection (BALB/c, Nramp1−/−
mice). In a model of persistent infection (129Sv6, Nramp1+/+
mutants show a similar pattern in that they are strongly out-competed by wild type in deep tissues only after oral inoculation (). We think it unlikely that YdeI regulates the SPI1 T3SS because cell invasion defects were not observed for ydeI
mutants in tissue culture experiments where SPI1 mutant (invA
) strains were used as a control (data not shown). Perhaps YdeI is needed after oral but not i.p. inoculation because it plays a role in traversing the intestinal barrier to allow for colonization of deep tissues, an event that may occur repeatedly during persistent infection as the bacteria re-seed the Peyer’s Patches or MLNs from the gastrointestinal tract.
The molecular mechanism of YdeI is unknown. YdeI is a putative periplasmic protein that could interact directly with the periplasmic loops of either RcsC or RcsD to regulate Rcs signal transduction. It is possible that YdeI is activated by the Rcs pathway to regulate another signalling pathway, such as PhoQ/PhoP or PmrA/PmrB. Alternatively, YdeI could contribute to the stability of the cell envelope by directly or indirectly modifying outer membrane proteins, lipids, or polysaccharides. By understanding the mechanisms of YdeI and other genes regulated by the Rcs pathway we will gain insights into how pathogens adapt to changes in their host microenvironments.