Phenazines are heteroaromatic compounds consist of a central pyrazine ring fused with two benzenes. Various functional groups linked to the dibenzopyrasine core cause differences in chemical, physical and biological properties of phenazines. The interest to these substances does not reduce in several decades. New biological activities and practical applications discovered in recent years force the researchers to study all aspects of phenazines synthesis, degradation and mechanisms of their action. In this study, we demonstrated the involvement of coxA gene product (cytochrome c oxidase, su I) in phenazines biosynthesis in P. chlororaphis subsp. aurantiaca. Overlap PCR was used to knockout coxAgene and derived mutants were analyzed for their ability to grow on rich and minimal culture media, as well as for the phenazines production level. We showed that the product of coxA gene is necessary for the phenazines production in rich growth media. At the same time CoxA protein seems has no effect on phenazines production in M9 minimal salts medium. CoxA protein is one of the core proteins of large transmembrane protein complex cytochrome c oxidase found in bacteria, archaea, and mitochondria of eukaryotes. We demonstrated that the knockout of even one subunit of this complex multiunit protein leads to a significant decrease (to trace concentrations) or complete suppression of phenazine antibiotics production on rich PCA-medium in P. chlororaphis subsp. aurantiaca.

Phenazines are heteroaromatic compounds consist of a central pyrazine ring fused with two benzenes. Various functional groups linked to the dibenzopyrasine core cause differences in chemical, physical and biological properties of phenazines. The interest to these substances does not reduce in several decades. New biological activities and practical applications discovered in recent years force the researchers to study all aspects of phenazines synthesis, degradation and mechanisms of their action.In this study, we demonstrated the involvement of coxA gene product (cytochrome c oxidase, su I) in phenazines biosynthesis in P. chlororaphis subsp. aurantiaca. Overlap PCR was used to knockout coxAgene and derived mutants were analyzed for their ability to grow on rich and minimal culture media, as well as for the phenazines production level. We showed that the product of coxA gene is necessary for the phenazines production in rich growth media. At the same time CoxA protein seems has no effect on phenazines production in M9 minimal salts medium. CoxA protein is one of the core proteins of large transmembrane protein complex cytochrome c oxidase found in bacteria, archaea, and mitochondria of eukaryotes. We demonstrated that the knockout of even one subunit of this complex multiunit protein leads to a signi cant decrease (to trace concentrations) or complete suppression of phenazine antibiotics production on rich PCA-medium in P. chlororaphis subsp. aurantiaca.

Proteomic analysis is a highly effective method for bacteria identification and the elucidation of protein's content in prokaryotic cells at different growth conditions. To our knowledge this approach is hardly ever used for characterization of producers of biologically active substances. The understanding of the changes in protein profile in mutant strains capable of biologically active substances overproduction helps to recognize the biochemical and molecular basis of metabolic changes which lead to overproduction. So that, proteomic analysis could be especially useful for optimization the producer's creation techniques.The purpose of current research was to carry out proteomic profiling of bacteria P. chlororaphis subsp. aurantiaca mutant strains capable of overproduction of phenazine antibiotics. Microbiological and biochemical methods were used for these aims.In current research a proteomic analysis of strains of P. chlororaphis subsp. aurantiaca producing phenazines was carried out. An early (during log-phase) onset of expression of individual genes of phz-operon which codes enzymes for phenazines synthesis was demonstrated. It was also found that the wild type strain has the highest level of PhzO protein. The gene encoding this protein is located outside the phz-operon. We weren't able to establish the correlation among PhzO protein content and concentration of the derivatives for which appearance PhzO is responsible. A general tendency of producer strains towards the accumulation of enzymes and proteins of the antioxidant defense system was revealed. Producer strains also demonstrate a significant increase in the concentration of proteins involved in DNA repair as well as chaperones involved in the native protein conformation maintenance.