Enrichment, isolation and characterization of phenazine-1-carboxylic acid (PCA)-degrading bacteria under aerobic and anaerobic conditions

Phenazines are a large class of nitrogen-containing aromatic heterocyclic compounds produced and secreted by bacteria from phylogenetically diverse taxa under aerobic and anaerobic conditions. Phenazine-1-carboxylic acid (PCA) is regarded as a ‘core’ phenazine because it is transformed to other phenazine derivatives. Due to their important roles in ecological fitness, biocontrol of plant pathogens, infection in cystic fibrosis and potential in anticancer treatments, understanding the fate of phenazine compounds is prudent. Only seven bacterial species are known to degrade phenazines and all of them are aerobic. Hence, the aim of this study is to enrich, isolate and characterize additional bacteria with the ability to degrade phenazines aerobically and anaerobically. In this study, the isolation of a PCA-degrading Rhodanobacter sp. PCA2 belonging to Grammaproteobacteria is reported. Characterization studies revealed that strain PCA2 is also capable of transforming other phenazines including phenazine, pyocyanin and 1-hydroxyphenazine. The sequencing, annotation and analysis of the genome of strain PCA2 revealed that genes (ubiD and the homolog of the MFORT_16269 gene) involved in PCA degradation were plasmid borne. Studies on abundance and expression of the homolog of MFORT_16269 gene via qPCR and RT-qPCR showed its involvement in PCA degradation by strain PCA2. Furthermore, results from LC-MS analysis together with proteomics indicated that strain PCA2 degraded PCA via decarboxylation and cleavage of aromatic and nitrogen-containing rings, potentially catalysed by UbiD, UbiX, phenylpropionate dioxygenase, biphenyl-2,3-diol 1,2-dioxygenase, amidohydrolase and nitroreductase. Three intermediates including phenazine, (4Z)-2-hydroxy-5-{[(1Z)-6-(hydroxyamino)cyclohexa -2,4-dien-1-ylidene]carbamoyl} penta-2,4-dienoic acid (HCCPD) and phenylhydroxylamine are reported. This study also reports the isolation of the first known anaerobic PCA degrading bacterium. Enterobacteriaceae were highly enriched after anaerobic incubation with PCA as a carbon, nitrogen and energy source and iron(III) as a terminal electron acceptor. Ultimately a Morganella morganii strain was isolated and shown to oxidise PCA whilst reducing iron(III)