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In silico identification and experimental validation of PmrAB targets in Salmonella typhimurium by regulatory motif detection.

Marchal K, De Keersmaecker S, Monsieurs P, van Boxel N, Lemmens K, Thijs G, Vanderleyden J, De Moor B - Genome Biol. (2004)

Bottom Line: Site-directed mutagenesis of the PmrA-binding site (PmrA box) in yibD revealed specific sequence requirements.We demonstrated the efficiency of our procedure by recovering most of the known PmrAB-dependent targets and by identifying unknown targets that we were able to validate experimentally.We also pinpointed directions for further research that could help elucidate the S. typhimurium virulence pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: ESAT-SCD, Katholieke Universiteit Leuven, Kasteelpark Arenberg 10, 3001 Leuven-Heverlee, Belgium. Kathleen.Marchal@esat.kuleuven.ac.be

ABSTRACT

Background: The PmrAB (BasSR) two-component regulatory system is required for Salmonella typhimurium virulence. PmrAB-controlled modifications of the lipopolysaccharide (LPS) layer confer resistance to cationic antibiotic polypeptides, which may allow bacteria to survive within macrophages. The PmrAB system also confers resistance to Fe3+-mediated killing. New targets of the system have recently been discovered that seem not to have a role in the well-described functions of PmrAB, suggesting that the PmrAB-dependent regulon might contain additional, unidentified targets.

Results: We performed an in silico analysis of possible targets of the PmrAB system. Using a motif model of the PmrA binding site in DNA, genome-wide screening was carried out to detect PmrAB target genes. To increase confidence in the predictions, all putative targets were subjected to a cross-species comparison (phylogenetic footprinting) using a Gibbs sampling-based motif-detection procedure. As well as the known targets, we detected additional targets with unknown functions. Four of these were experimentally validated (yibD, aroQ, mig-13 and sseJ). Site-directed mutagenesis of the PmrA-binding site (PmrA box) in yibD revealed specific sequence requirements.

Conclusions: We demonstrated the efficiency of our procedure by recovering most of the known PmrAB-dependent targets and by identifying unknown targets that we were able to validate experimentally. We also pinpointed directions for further research that could help elucidate the S. typhimurium virulence pathway.

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Refined consensus of the PmrA box. (a) Alignment of all experimentally verified PmrA sites ([15] or this work) in S. typhimurium [1]. PmrA sites in the orthologs of these respective experimentally verified genes are also displayed if these PmrA motif instances deviated from the PmrA motif in S. typhimurium. (b) An adapted motif model of the PmrA site was built (represented by its logo) on the basis of the sequences represented in (a).
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Figure 4: Refined consensus of the PmrA box. (a) Alignment of all experimentally verified PmrA sites ([15] or this work) in S. typhimurium [1]. PmrA sites in the orthologs of these respective experimentally verified genes are also displayed if these PmrA motif instances deviated from the PmrA motif in S. typhimurium. (b) An adapted motif model of the PmrA site was built (represented by its logo) on the basis of the sequences represented in (a).

Mentions: On the basis of the instances of the PmrA motif in experimentally verified PmrAB targets of Salmonella (verified previously or validated in this study), a PmrA consensus was built (Figure 4). The motif consensus of PmrA was converted into a regular expression (A/C)(C/T)T(A/T)A(T/G/A) N5NTT(A/T)A(T/A/G). To construct this regular expression we only considered the two conserved half-sites, because the PmrA motif is believed to be a dyad [15]. We preferred the part between the conserved half-sites of the regular expression to be represented as degenerate (that is, N5). Indeed, the observed degree of conservation in the intermediate part of the motif model (Figure 4b) is probably related to the restricted sample size of the training set rather than being an intrinsic property of the motif. Promising motifs (indicated in bold in Table 1) are, therefore, motifs that match this regular expression and thus contain nucleotides that occur in the conserved half-sites of one of the experimentally verified examples. Promising targets for which the putative PmrA motif was also conserved in species other than Salmonella were mig-13, yrbF, yjgD, ybdO, yejG, lasT and ybdN. Promising targets only present in S. typhimurium and/or S. typhi were STM1269 (aroQ), STM1273, sseJ and lpfA. Note that this listing is just based on an arbitrary selection criterion, that is, a preliminary PmrA motif consensus that will be improved as more PmrAB targets become experimentally validated. As well as the targets mentioned above, Table 1 contains other targets that are of interest because their annotation relates to the PmrAB system (such as yncD).


In silico identification and experimental validation of PmrAB targets in Salmonella typhimurium by regulatory motif detection.

Marchal K, De Keersmaecker S, Monsieurs P, van Boxel N, Lemmens K, Thijs G, Vanderleyden J, De Moor B - Genome Biol. (2004)

Refined consensus of the PmrA box. (a) Alignment of all experimentally verified PmrA sites ([15] or this work) in S. typhimurium [1]. PmrA sites in the orthologs of these respective experimentally verified genes are also displayed if these PmrA motif instances deviated from the PmrA motif in S. typhimurium. (b) An adapted motif model of the PmrA site was built (represented by its logo) on the basis of the sequences represented in (a).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC395753&req=5

Figure 4: Refined consensus of the PmrA box. (a) Alignment of all experimentally verified PmrA sites ([15] or this work) in S. typhimurium [1]. PmrA sites in the orthologs of these respective experimentally verified genes are also displayed if these PmrA motif instances deviated from the PmrA motif in S. typhimurium. (b) An adapted motif model of the PmrA site was built (represented by its logo) on the basis of the sequences represented in (a).
Mentions: On the basis of the instances of the PmrA motif in experimentally verified PmrAB targets of Salmonella (verified previously or validated in this study), a PmrA consensus was built (Figure 4). The motif consensus of PmrA was converted into a regular expression (A/C)(C/T)T(A/T)A(T/G/A) N5NTT(A/T)A(T/A/G). To construct this regular expression we only considered the two conserved half-sites, because the PmrA motif is believed to be a dyad [15]. We preferred the part between the conserved half-sites of the regular expression to be represented as degenerate (that is, N5). Indeed, the observed degree of conservation in the intermediate part of the motif model (Figure 4b) is probably related to the restricted sample size of the training set rather than being an intrinsic property of the motif. Promising motifs (indicated in bold in Table 1) are, therefore, motifs that match this regular expression and thus contain nucleotides that occur in the conserved half-sites of one of the experimentally verified examples. Promising targets for which the putative PmrA motif was also conserved in species other than Salmonella were mig-13, yrbF, yjgD, ybdO, yejG, lasT and ybdN. Promising targets only present in S. typhimurium and/or S. typhi were STM1269 (aroQ), STM1273, sseJ and lpfA. Note that this listing is just based on an arbitrary selection criterion, that is, a preliminary PmrA motif consensus that will be improved as more PmrAB targets become experimentally validated. As well as the targets mentioned above, Table 1 contains other targets that are of interest because their annotation relates to the PmrAB system (such as yncD).

Bottom Line: Site-directed mutagenesis of the PmrA-binding site (PmrA box) in yibD revealed specific sequence requirements.We demonstrated the efficiency of our procedure by recovering most of the known PmrAB-dependent targets and by identifying unknown targets that we were able to validate experimentally.We also pinpointed directions for further research that could help elucidate the S. typhimurium virulence pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: ESAT-SCD, Katholieke Universiteit Leuven, Kasteelpark Arenberg 10, 3001 Leuven-Heverlee, Belgium. Kathleen.Marchal@esat.kuleuven.ac.be

ABSTRACT

Background: The PmrAB (BasSR) two-component regulatory system is required for Salmonella typhimurium virulence. PmrAB-controlled modifications of the lipopolysaccharide (LPS) layer confer resistance to cationic antibiotic polypeptides, which may allow bacteria to survive within macrophages. The PmrAB system also confers resistance to Fe3+-mediated killing. New targets of the system have recently been discovered that seem not to have a role in the well-described functions of PmrAB, suggesting that the PmrAB-dependent regulon might contain additional, unidentified targets.

Results: We performed an in silico analysis of possible targets of the PmrAB system. Using a motif model of the PmrA binding site in DNA, genome-wide screening was carried out to detect PmrAB target genes. To increase confidence in the predictions, all putative targets were subjected to a cross-species comparison (phylogenetic footprinting) using a Gibbs sampling-based motif-detection procedure. As well as the known targets, we detected additional targets with unknown functions. Four of these were experimentally validated (yibD, aroQ, mig-13 and sseJ). Site-directed mutagenesis of the PmrA-binding site (PmrA box) in yibD revealed specific sequence requirements.

Conclusions: We demonstrated the efficiency of our procedure by recovering most of the known PmrAB-dependent targets and by identifying unknown targets that we were able to validate experimentally. We also pinpointed directions for further research that could help elucidate the S. typhimurium virulence pathway.

Show MeSH