9419023 Willis During the previous period of NSF support, this laboratory genetically identified the Pseudomonas syringae pv. syringae gacA gene as the cognate response regulator for the lemA environmental sensor. Within bean pathogenic P. syringae pv. syringae strains such as B728a, this regulatory pair controls lesion formation, and the production of the toxin syringomycin and the production of protease. Based on the PI's studies and the work of others, it is now apparent that the lemA/gacA regulatory pair are conserved within several species of Pseudomonas. Functional homologs have been identified within P. fluorescens, P. vindiflava, P. cichorii, and P. aeruginosa. The number of systems for which lemA/gacA regulatory system appears to be operative is increasing, indicative of a fundamental global regulatory network in these bacteria. Dr. Todd Kitten, during his research funded by the NSF Plant Biology Program, isolated a number of extragenic suppressors within NPS3136, a lemAI::Tn5 derivative of B728a. From a screen of 43,000 colonies of EMS mutagenized NPS3136, he identified 53 mutants that were restored for protease production. Dr. Kitten is currently analyzing cosmids carrying wild-type genes that restore either protease or syringomycin production to SUP27. The PI will continue his genetic study of lesion formation on bean in two ways. First, his laboratory will screen a genomic library of the suppressor mutant SUP27 for cosmid clones that restore lesion formation to NUVS1. Restoring clones will be analyzed for copy number or extragenic suppression. Second, it will continue its characterization of the lesion-defective mutant KW3815. This strain is defective in lesion formation but not protease or syringomycin production. KW3815 was isolated from a screen of over 6000 Tn5-generated mutants of B728a for loss of lesion formation on bean pods. The PI has established that the Tn5 insertion is causal to the lesion minus phenotype of KW3815 and has isolated a restoring clone from a B728a gen omic library. The restoring gene will be sequenced and reporter fusions constructed to determine if the mutated gene in this strain is a member of the lemA/gacA regulon. In his continuing analysis of the lemA/gacA regulon, the PI will use reporter fusions to identify additional gacA-regulated genes. His lab will introduce random Tn5-lacZ insertions into a B728a gacA mutant and screen for the production of (-galactosidase on plates containing X-GAL. Blue and white colonies will be separated and subsequently transformed via plate mating with a cosmid clone containing the wild-type gacA gene. Those colonies that express (-galactosidase (turn blue) in the presence of a functional gacA gene will be assumed to contain an insert in a gene that is positively regulated by lemA/gacA. Those colonies that lose the ability to produce (-galactosidase will be assumed to contain an insertion within a down regulated gene. All gacA regulated insertion mutants will be screened for loss of protease or syringomycin production and a reduction in lesion forming ability on bean. Using a DNA fragment containing the promoter region of the lemA-regulated gene tblA as a probe, Southwestern analysis revealed a DNA, binding protein that is regulated by the lemA and gacA genes. As part of this project, the PI will isolate the DNA binding protein using biotin labeled probe DNA and streptavidin linked to paramagnetic beads. Antiserum will be prepared to the purified protein and will be used to identify the gene encoding the DNA binding protein from a (gt11 expression library. %%% This research helps to explain how a bacterium that causes disease in plants responds to signals produced by the plant. Understanding this process could help prevent diseases in plants. ***
