In silico identification and experimental validation of PmrAB targets in Salmonella typhimurium by regulatory motif detection.
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.
Marchal
Kathleen
K
ESAT-SCD, Katholieke Universiteit Leuven, Kasteelpark Arenberg 10, 3001 Leuven-Heverlee, Belgium. Kathleen.Marchal@esat.kuleuven.ac.be
De Keersmaecker
Sigrid
S
Monsieurs
Pieter
P
van Boxel
Nadja
N
Lemmens
Karen
K
Thijs
Gert
G
Vanderleyden
Jos
J
De Moor
Bart
B
D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000032:analysis* / Q000378:metabolism; D017930:Genes, Reporter; D016680:Genome, Bacterial; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D012045:Regulatory Sequences, Nucleic Acid; D012486:Salmonella typhimurium / Q000235:genetics* / Q000472:pathogenicity; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; C409039:PmrB protein, bacteria; D014157:Transcription Factors; C081902:pmrA protein, Bacteria