| Summary: | 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. === Graduation date: 1999
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