A two-component regulatory system controlling antibiotic production by Pseudomonas flourescens Pf-5 Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/s7526g132

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  • Tn5 mutagenesis of apdA (for antibiotic production) and deletion of gacA (for global antibiotic and cyanide) resulted in the same pleiotropic phenotype in Pseudomonas fluorescens (i.e. production of an array of secondary metabolites including the antibiotics pyrrolnitrin, pyoluteorin, and 2,4-diacetylphloroglucinol as well as a tryptophan-side-chain oxidase, hydrogen cyanide, and an extracellular protease was abolished). The apdA and gacA loci were identified and cloned from the genome of Pf-5. Nucleotide sequencing of the apdA and gacA loci was used to identify the open reading frames for these genes. The deduced amino acid sequences for apdA and gacA exhibited similarity to sensor kinase (ApdA) and response regulator (GacA) proteins that comprise two-component regulatory systems. The C-terminal domain of GacA containing the putative helix-turn-helix DNA-binding motif was fused to the glutathione S-transferase protein. The glutathione S-transferase GacA C-terminal fusion protein was used in a cycle selection procedure that was designed to identify GacA binding sites from a complex pool of DNA fragments. Although a putative binding site for GacA was identified using the cycle selection procedure, the results were inconclusive due to several inconsistencies in the DNA-binding assay. The upstream region of one gene, which codes for a putative porin, was identified as a putative binding site for GacA by the cycle selection procedure. Studies initiated to determine whether gacA regulates transcription of this putative porin gene have been unsuccessful, so it remains unclear whether this gene is regulated by GacA. Also, asymptotic limits to biological control of Rhizoctonia damping-off of cotton were observed with the biological control agent P. fluorescens Pf-5.
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