Characterization Of Genes In The Cyclohexanol Operon Of Polaromonas Sp. Strain Js666

Abstract

Polaromonas sp. strain JS666 is the only isolate capable of growth through the aerobic oxidation of cis-dichloroethene (cDCE). Study of the JS666 genome, along with previous results from transcriptomics and proteomics studies, have suggested the involvement of a cyclohexanone monooxygenase gene (chmo, chnB, Bpro_5565) in the process. Specifically, CHMO was hypothesized to catalyze the epoxidation of cDCE. We successfully inactivated the chmo gene through use of a suicide vector, and the resulting chmo-knockout strain (KO) was incapable of growth on cyclohexanone (CYHX), cDCE, ethanol (EtOH) and cyclohexanol. The overexpressed CHMO in Escherichia coli showed activity on CYHX; nonetheless, no activity was confirmed with cDCE. This suggests that CHMO is not involved in the first step of cDCE degradation, however, differences in cellular environments (i.e. pH, protein folding and posttranslational modification) between the E. coli and JS666 strains could have contributed to the outcome. Genes in the same operon with chmo, namely the adjacent, upstream hydrolase (chnC, Bpro_5566) and two adjacent, downstream alcohol dehydrogenases (chnD and chnA, Bpro_5563 and Bpro_5564, respectively) may serve important roles in cDCE degradation. When CYHX or EtOH was supplied as co-substrate, wild-type JS666 (WT) quickly exhibited cDCE degradation and sustained it through multiple additions, but not when succinate or acetate was co-administered, suggesting that CYHX and EtOH each elevate expression of proteins involved in cDCE metabolism. ChnD overexpressed in E. coli showed activity not only on EtOH and cyclohexanol, but also on 2,2dichloroacetaldehyde (DCAL), which is a hydroxylation product of cDCE by cyctochrome P450 (Bpro_5301), reducing it to 2,2-dichloroethanol. This suggests that ChnD is involved in cDCE degradation - not as a first step, but in subsequent steps. Changes in proteome levels during cDCE-degradation were explored using a quantitative shotgun proteomics technique, Isobaric Tag for Relative and Absolute Quantitation (iTRAQ). iTRAQ studies showed that genes adjacent to chmo were still translated in the KO, but with reduced protein abundance, particularly with respect to downstream genes. Given that we demonstrated ChnD (and possibly ChnA) acts on alcohol, and given that the KO lost ability to degrade alcohol and cDCE, it is therefore reasonable to propose that the lower abundance of ChnD (and presumably ChnA) in KO is the explanation for the differing behaviors of KO vs. WT strains. Through iTRAQ studies, we found that cDCE increased the relative abundances of proteins involved in glyoxylate metabolism. Several proteins that may be involved in cDCE-degradation pathways were also identified, such as an aldehyde dehydrogenase (Bpro_3952) and enzymes that are hypothetically involved in glyoxal metabolism. This also supports a hypothesis of a cDCE-degradation pathway involving glyoxal formation from hydrolysis of cDCE-epoxide. Collectively, data gathered from these studies suggest possible roles of genes in the cyclohexanol operon and update our understanding on multiple pathways of cDCE-biodegradation in JS666. iv