Systematic Unraveling of the Unsolved Pathway of Nicotine Degradation in Pseudomonas

Abstract

Microorganisms such as Pseudomonas putida play important roles in the mineralization of organic wastes and toxic compounds. To comprehensively and accurately elucidate key processes of nicotine degradation in Pseudomonas putida, we measured differential protein abundance levels with MS-based spectral counting in P. putida S16 grown on nicotine or glycerol, a non-repressive carbon source. In silico analyses highlighted significant clustering of proteins involved in a functional pathway in nicotine degradation. The transcriptional regulation of differentially expressed genes was analyzed by using quantitative reverse transcription-PCR. We observed the following key results: (i) The proteomes, containing 1,292 observed proteins, provide a detailed view of enzymes involved in nicotine metabolism. These proteins could be assigned to the functional groups of transport, detoxification, and amino acid metabolism. There were significant differences in the cytosolic protein patterns of cells growing in a nicotine medium and those in a glycerol medium. (ii) The key step in the conversion of 3-succinoylpyridine to 6-hydroxy-3-succinoylpyridine was catalyzed by a multi-enzyme reaction consisting of a molybdopeterin binding oxidase ( spmA), molybdopterin dehydrogenase ( spmB), and a (2Fe-2S)-binding ferredoxin ( spmC) with molybdenum molybdopterin cytosine dinucleotide as a cofactor. (iii) The gene of a novel nicotine oxidoreductase ( nicA2) was cloned, and the recombinant protein was characterized. The proteins and functional pathway identified in the current study represent attractive targets for degradation of environmental toxic compounds.

Author Summary

Pseudomonas putida strains are among the microorganisms that have acquired the capability to use toxic and xenobiotic compounds, such as nicotine, for growth. Although nicotine degradation by Pseudomonas was first discovered more than 50 years ago, the underlying molecular mechanisms remain unclear. In the last few years, we have made significant efforts to identify the key genes for the hydroxylation of 3-succinoylpyridine (SP) through genomic library screening and purification of wild-type enzymes. However, these efforts did not result in identifying any genes related to SP hydroxylation. In this study, by using comparative genetic analysis, we report the identification of 3 key genes, spmA, spmB and spmC from P. putida S16. The heterotrimeric enzyme encoded by these genes requires molybdopterin-cytosine dinucleotide as a cofactor. The proteomes of strain S16 grown on nicotine or glycerol contain 1,292 observed proteins, and provide a detailed view of enzymes involved in nicotine degradation. Our comparative analysis of the proteomic profiles of nicotine grown versus glycerol grown bacterial cells reveals a wide range of cellular processes and functions related to nicotine catabolism.