Transposon Mutagenesis Identifies Genes Critical for Growth of Pseudomonas nitroreducens TX1 on Octylphenol Polyethoxylates

Abstract

ABSTRACT Pseudomonas nitroreducens TX1 is of special interest because of its ability to utilize 0.05% to 20% octylphenol polyethoxylates (OPEO n ) as a sole source of carbon. In this study, a library containing 30,000 Tn 5 -insertion mutants of the wild-type strain TX1 was constructed and screened for OPEO n utilization, and 93 mutants were found to be unable to grow on OPEO n . In total, 42 separate disrupted genes were identified, and the proteins encoded by the genes were then classified into various categories, namely, information storage and processing (14.3%), cellular processes and signaling (28.6%), metabolism (35.7%), and unknown proteins (21.4%). The individual deletion of genes encoding isocitrate lyase ( aceA ), malate synthase ( aceB ), and glycolate dehydrogenase ( glcE ) was carried out, and the requirement for aceA and aceB but not glcE confirmed the role of the glyoxylate cycle in OPEO n degradation. Furthermore, acetaldehyde dehydrogenase and acetyl-coenzyme A (acetyl-CoA) synthetase activity levels were 13.2- and 2.1-fold higher in TX1 cells grown on OPEO n than in TX1 cells grown on succinate, respectively. Growth of the various mutants on different carbon sources was tested, and based on these findings, a mechanism involving exoscission to liberate acetaldehyde from the end of the OPEO n chain during degradation is proposed for the breakdown of OPEO n . IMPORTANCE Octylphenol polyethoxylates belong to the alkylphenol polyethoxylate (APEO n ) nonionic surfactant family. Evidence based on the analysis of intermediate metabolites suggested that the primary biodegradation of APEO n can be achieved by two possible pathways for the stepwise removal of the C 2 ethoxylate units from the end of the chain. However, direct evidence for these hypotheses is still lacking. In this study, we described the use of transposon mutagenesis to identify genes critical to the catabolism of OPEO n by P. nitroreducens TX1. The exoscission of the ethoxylate chain leading to the liberation of acetaldehyde is proposed. Isocitrate lyase and malate synthase in glyoxylate cycle are required in the catabolism of ethoxylated surfactants. Our findings also provide many gene candidates that may help elucidate the mechanisms in stress responses to ethoxylated surfactants by bacteria.