Molecular genetic analysis of determinants defining synthesis of 2,4-diacetylphloroglucinol by Pseudomonas brassicacearum BIM B-446 bacteria

Abstract

Based on a full-sized sequence of the genome of Pseudomonas brassicacearum BIM В-446 bacteria, we determined the nucleotide sequence of the locus encoding the synthesis of the 2,4-diacetylphloroglucinol antibiotic. It was shown in the limits of a nucleotide sequence with 9087 bp size to be localized at open reading frames homologous (96–99% identical residues) to structural (phlA, phlC, phlB, phlD, phlE, and phlI) and regulatory (phlF, phlG, and phlH) genes of Pseudomonas brassicacearum and Pseudomonas fluorescens bacteria, which determine the production of 2,4-diacetylphloroglucinol. It emerged that closely related phl-operons differ by their environment. Thus, different genes were localized on the 3'-end. It was determined that inactivation of the phlA gene in P. brassicacearum BIM bacteria caused a loss of the ability to synthesize antibiotic and inhibited the growth of the phytopathogenic Fusarium culmorum, F. oxysporum, and Botrytis cinerea fungi; the antimicrobial activity was also decreased toward with fungal (Alternaria alternate) and bacterial pathogens (Pseudomonas syringae and Pectobacterium carotovorum). During inactivation of the phlF regulatory gene, which determines the synthesis of the phl-operon transcription repressor, the production of 2,4-diacetylphloroglucinol was increased. In contrast to the wild-type bacteria, phlF-mutants synthesized an antibiotic that was found in cultural liquid after 12 h of cultivation; its content reached the maximum in medium with saccharose as the carbon source.