A primary motivation for determining genome sequences of microbial pathogens is to understand the bases of their pathogenicity. Proper regulation of virulence genes can be as essential to pathogenicity as is possession of these genes, so predicting regulatory networks from genome sequences is a high priority. A plausible but poorly tested assumption underlies many of these predictions. Specifically, the presence in two species of both a conserved regulatory protein and of conserved target genes with candidate upstream binding sites is presumed to imply that their regulatory relationship has been conserved. The two major goals of this project are to test this bioinformatic assumption and, in so doing, better characterize a major bacterial regulatory network (regulon). The leucine-responsive regulatory protein (Lrp) is conserved among many Gram-negative bacteria, and in E. coil affects expression of as many as 400 genes. Recent evidence indicates that many of these genes are preferentially expressed during transition to stationary phase, and may play a role in pathogenicity in related organisms. Three hypotheses will be tested: * The hypothesis that species with conserved Irp genes have conserved Lrp function, to be tested by determining whether Lrp levels vary comparably in these species, and by assessing the extent to which Lrp orthologs are interchangeable. The Irp genes to be tested are from Proteus mirabilis (98% identical to the E. coil protein) V. cholerae (92%), and P. multocida (75%). * The hypothesis that species with highly-conserved Lrp orthologs show a conserved pattern of regulation, to be tested by using microarrays to analyze the effects of Irp mutation in E. coil O157:H7, V. cholerae, and P. multocida. E. coil O157:H7 Lrp is identical to that of E. coil K-12, but the former is a pathogen with -25% more genes, some of which may belong to the Lrp regulon. * The hypothesis that Irp mutations have analogous effects on the virulence of different pathogenic bacteria, to be tested by determining the effects of a Irp null allele in an animal model for V. cholerae.
