Carbaryl as a Carbon and Nitrogen Source: an Inducible Methylamine Metabolic Pathway at the Biochemical and Molecular Levels in Pseudomonas sp. Strain C5pp

Abstract

Carbaryl is the most widely used carbamate family pesticide, and its persistent nature causes it to pollute both soil and water ecosystems. Microbes maintain the Earth’s biogeochemical cycles by metabolizing various compounds present in the matter, including xenobiotics, as a sole source of carbon, nitrogen, and energy. Soil isolatePseudomonassp. strain C5pp metabolizes carbaryl efficiently as the carbon source. Periplasmic carbaryl hydrolase catalyzes the conversion of carbaryl to 1-naphthol and methylamine. 1-Naphthol was further used as a carbon source via gentisate, whereas the metabolic fate of methylamine is not known. Here, we demonstrate that strain C5pp showed efficient growth on carbaryl when supplied as a carbon and nitrogen source, suggesting that the methylamine generated was used as the nitrogen source. Genes involved in the methylamine metabolism were annotated and characterized at the biochemical and molecular level. Transcriptional and enzyme activity studies corroborate that the γ-glutamylmethylamide/N-methylglutamate (GMA/NMG) pathway is involved in the metabolism of carbaryl and methylamine as a nitrogen source. Compared to carbaryl, methylamine was found to be an effective inducer for the metabolic and transporter genes. Strain C5pp also harbored genes involved in sarcosine metabolism that were cotranscribed and induced by sarcosine. The presence of inducible pathways for metabolism of carbaryl as a nitrogen and carbon source helps in complete and efficient mineralization of carbaryl by strain C5pp, thereby maintaining the biogeochemical cycles.IMPORTANCEThe degradation of xenobiotics plays a significant role in the environment to maintain ecological systems as well as to prevent the imbalance of biogeochemical cycles via carbon-nitrogen cycling. Carbaryl is the most widely used pesticide from the carbamate family.Pseudomonassp. strain C5pp, capable of utilizing carbaryl as a carbon and nitrogen source for its growth, subsequently helps in complete remediation of carbaryl. Thus, it maintains the ecosystem by balancing the biogeochemical cycles. The metabolic versatility and genetic diversity of strain C5pp for the transformation of contaminants like carbaryl and 1-naphthol into less harmful products make it a suitable candidate from the perspective of bioremediation.

Crystal structures of γ-glutamylmethylamide synthetase provide insight into bacterial metabolism of oceanic monomethylamine

“…The Km of RhGmaS for MMA was the lowest among the tested ammonia analogs, while the Km for NH4Cl is much higher ( Table 1), suggesting that MMA is likely the natural substrate of RhGmaS. This result is consistent with the previous reports that GmaS prefers MMA as its substrate rather than ammonia (4,19,20,24). To investigate whether the N-terminal His-tag would affect the kinetic properties of the recombinant RhGmaS, we used thrombin to cut off the Histag, and measured the kinetic parameters of RhGmaS without His-tag ( Fig.…”

“…GmaS is more specific to MMA, whereas the natural substrate of GS enzymes is ammonia ( 19 , 20 , 25 ). This is consistent with the results of sequence analysis, showing that GmaS lacks key ammonia-binding residues that are conserved in GS enzymes ( 4 , 21 , 24 , 26 ). GS enzymes can be divided into three distinct types, GSI, GSII, and GSIII ( 27 , 28 ), and the crystal structures of GS have been determined ( 29 , 30 , 31 ).…”

“…Amine donors, such as hydroxylamine and ethylamine, can replace ammonia as substrate for some GS enzymes ( 20 , 24 , 30 ). To determine whether Rh GmaS can catalyze different ammonia analogs, we analyzed the substrate specificity of Rh GmaS ( Table 1 ).…”

“…Sequence analysis indicates that GmaS is closely related to, but distinct from, the glutamine synthetase (GS) family, one of the oldest and most ubiquitously existing families of enzymes in biota ( 4 , 23 , 24 ). GmaS is more specific to MMA, whereas the natural substrate of GS enzymes is ammonia ( 19 , 20 , 25 ).…”

Variability in Assembly of Degradation Operons for Naphthalene and its derivative, Carbaryl, Suggests Mobilization through Horizontal Gene Transfer

“…Besides HGT events and assembly of genes in the form of operons, Pseudomonas sp. C5pp shows cellular level adaptation by compartmentalization of enzymes to tackle the toxicity of 1-naphthol [125,126]. This adaptation helps in degradation of higher concentration of Carbaryl (1% tested so far).…”