Transposon mutagenesis identifies genes critical for growth of Pseudomonas nitroreducens TX1 on octylphenol polyethoxylates

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 Tn5-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.