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.

Trifluralin-induced disorganization of microtubular cytoskeleton alters the development of roots in Hordeum vulgare L.

The extensive use of herbicides in agriculture becomes an important factor in environmental pollution, especially in case of slowly degradable compounds. Some agents act on plants during a long period of time, even if a very low concentration of the herbicide remains in the soil. Here, we investigated the toxicological effect of a low concentration of dinitroaniline herbicide, trifluralin, on growing seedlings of Hordeum vulgare L. Trifluralin in concentration of 1 microg/ml inhibited root growth. The mitotic activity of meristematic cells was suppressed due to the retardation of metaphase progression--alteration that can be caused by cytoskeleton disorder. Using antibodies to alpha-tubulin, we investigated the distribution of microtubules in root meristem cells. During all stages of mitosis, the highly regular system of microtubular cytoskeleton observed in control cells was slightly disorganized. An examination of root structure using light and electron microscopy demonstrated that the cell walls did not form normally during cell division that led to the appearance of large multinucleated cells. Also, the premature (pathological) cell differentiation was induced by trifluralin. A part of differentiating cells showed intracellular structural changes that are consistent with programmed cell death. It seems that the development of alterations in trifluralin-treated roots was due to the microtubular cytoskeleton disorganization.