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Genetic evidence that the gacA gene encodes the cognate response regulator for the lemA sensor in Pseudomonas syringae.

ABSTRACT: Mutational analysis of the bean-pathogenic Pseudomonas syringae pv. syringae strain B728a has led to the genetic identification of the gacA gene as encoding the response regulator for the unlinked lemA sensor kinase. The analysis of a collection of spontaneous mutants of P. syringae pv. syringae suggested that the gacA gene was involved in lesion formation and the production of protease and syringomycin. The gacA gene originally was identified as a regulator of extracellular antibiotic production by Pseudomonas fluorescens, and the predicted GacA protein is a member of the FixJ family of bacterial response regulators. The sequence of the putative B728a GacA protein revealed 92% identity with the P. fluorescens GacA protein. An insertional mutation within the P. syringae pv. syringae gacA gene abrogated lesion formation on beans, production of extracellular protease, and production of the toxin syringomycin, the same phenotypes affected by a lemA mutation. DNA sequence analysis identified the P. syringae pv. syringae uvrC gene immediately downstream of the gacA gene, an arrangement conserved in P. fluorescens and Escherichia coli. The gacA insertional mutant was sensitive to UV, presumably because of polarity on transcription of the downstream uvrC gene. Southwestern (DNA-protein) analysis revealed that the lemA and gacA genes were required for the full expression of a DNA binding activity.

SUBMITTER: Rich JJ

PROVIDER: S-EPMC197202 | biostudies-other | 1994 Dec

REPOSITORIES: biostudies-other

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Genetic evidence that the gacA gene encodes the cognate response regulator for the lemA sensor in Pseudomonas syringae.

Rich J J JJ Kinscherf T G TG Kitten T T Willis D K DK

Journal of bacteriology 19941201 24

Mutational analysis of the bean-pathogenic Pseudomonas syringae pv. syringae strain B728a has led to the genetic identification of the gacA gene as encoding the response regulator for the unlinked lemA sensor kinase. The analysis of a collection of spontaneous mutants of P. syringae pv. syringae suggested that the gacA gene was involved in lesion formation and the production of protease and syringomycin. The gacA gene originally was identified as a regulator of extracellular antibiotic productio ...[more]

PMID: 8002569

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Project description:BackgroundThe CpxA sensor kinase-CpxR response regulator two-component regulatory system is a sentinel of bacterial envelope integrity. Integrating diverse signals, it can alter the expression of a wide array of components that serve to shield the envelope from damage and to promote bacterial survival. In bacterial pathogens such as Yersinia pseudotuberculosis, this also extends to pathogenesis. CpxR is thought to dimerize upon phosphorylation by the sensor kinase CpxA. This phosphorylation enables CpxR binding to specific DNA sequences where it acts on gene transcription. As Cpx pathway activation is dependent on protein-protein interactions, we performed an interaction analysis of CpxR and CpxA from Y. pseudotuberculosis.ResultsCpxR full-length and truncated versions that either contained or lacked a putative internal linker were all assessed for their ability to homodimerize and interact with CpxA. Using an adenylate cyclase-based bacterial two hybrid approach, full-length CpxR readily engaged with CpxA. The CpxR N-terminus could also homodimerize with itself and with a full-length CpxR. A second homodimerization assay based upon the ?cI repressor also demonstrated that the CpxR C-terminus could homodimerize. While the linker was not specifically required, it enhanced CpxR homodimerization. Mutagenesis of cpxR identified the aspartate at residue 51, putative N-terminal coiled-coil and C-terminal winged-helix-turn-helix domains as mediators of CpxR homodimerization. Scrutiny of CpxA full-length and truncated versions revealed that dimerization involved the N-terminus and an internal dimerization and histidine phosphotransfer domain.ConclusionsThis interaction analysis mapped regions of CpxR and CpxA that were responsible for interactions with self or with each other. When combined with other physiological and biochemical tests both hybrid-based assays can be useful in dissecting molecular contacts that may underpin Cpx pathway activation and repression.

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Genetic evidence that the gacA gene encodes the cognate response regulator for the lemA sensor in Pseudomonas syringae.

Publications

Genetic evidence that the gacA gene encodes the cognate response regulator for the lemA sensor in Pseudomonas syringae.

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