Cytochrome c oxidase is one of the key enzymes providing the ability to produce phenazines in Pseudomonas chlororaphis subsp. aurantiaca.

Phenazines are heteroaromatic compounds consisting of a central pyrazine ring fused with two benzenes. Different functional groups attached to the dibenzopyrasin core cause differences in the chemical, physical, and biological properties of phenazines. Interest in these compounds has not diminished for decades. New biological activities and practical applications discovered in recent years force researchers to investigate all aspects of the synthesis, degradation, and mechanisms of action of phenazines. In this study, we have demonstrated the involvement of the coxA gene product (cytochrome c oxidase, su I) in the production of phenazines in P. chlororaphis subsp. aurantiaca. Overlap PCR was used to knock out the coxA gene and the resulting mutants were screened for their ability to grow on rich and minimal culture media and for phenazine production. The reintroduction of the full-length coxA gene into the B-162/coxA strains was used to further confirm the role of this gene product in the ability to produce phenazines. We were able to show that the product of the coxA gene is necessary for phenazine production in rich growth media. At the same time, the CoxA protein does not seem to have any effect on phenazine production in M9 minimal salt medium. We could show that knocking down even one subunit of the cytochrome c oxidase complex leads to a significant reduction (to trace concentrations) or complete suppression of phenazine antibiotic production on rich PCA medium in P. chlororaphis subsp. aurantiaca.

Genome analysis of Pseudomonas chlororaphis subsp. aurantiaca mutant strains with increased production of phenazines.

Genomes of three strains-phenazine producers-Pseudomonas chlororaphis subsp. aurantiaca (B-162 (wild type), mutant strain B-162/255, and its derivative B-162/17) were sequenced and compared. Comparison of a wild-type strain and B-162/255 mutant genomes revealed 32 mutations. 19 new mutations were detected in the genome of B-162/17. Further bioinformatics analysis allowed us to predict mutant protein functions and secondary structures of five gene products, mutations which might potentially influence phenazine synthesis and secretion in Pseudomonas bacteria. These genes encode phenylalanine hydroxylase transcriptional activator PhhR, type I secretion system permease/ATPase, transcriptional regulator MvaT, GacA response regulator, and histidine kinase. Amino acid substitutions were found in domains of studied proteins. One deletion in an intergenic region could affect a potential transcription factor binding site that participates in the regulation of gene that encodes ABC transporter.