Oxidation of methanethiol (MT) is a significant step in the sulfur cycle. MT is an intermediate of metabolism of globally significant organosulfur compounds including dimethylsulfoniopropionate (DMSP) and dimethylsulfide (DMS), which have key roles in marine carbon and sulfur cycling. In aerobic bacteria, MT is degraded by a MT oxidase (MTO). The enzymatic and genetic basis of MT oxidation have remained poorly characterized. Here, we identify for the first time the MTO enzyme and its encoding gene (mtoX) in the DMS-degrading bacterium Hyphomicrobium sp. VS. We show that MTO is a homotetrameric metalloenzyme that requires Cu for enzyme activity. MTO is predicted to be a soluble periplasmic enzyme and a member of a distinct clade of the Selenium-binding protein (SBP56) family for which no function has been reported. Genes orthologous to mtoX exist in many bacteria able to degrade DMS, other one-carbon compounds or DMSP, notably in the marine model organism Ruegeria pomeroyi DSS-3, a member of the Rhodobacteraceae family that is abundant in marine environments. Marker exchange mutagenesis of mtoX disrupted the ability of R. pomeroyi to metabolize MT confirming its function in this DMSP-degrading bacterium. In R. pomeroyi, transcription of mtoX was enhanced by DMSP, methylmercaptopropionate and MT. Rates of MT degradation increased after pre-incubation of the wild-type strain with MT. The detection of mtoX orthologs in diverse bacteria, environmental samples and its abundance in a range of metagenomic data sets point to this enzyme being widely distributed in the environment and having a key role in global sulfur cycling.

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Dimethylsulphide (DMS) has an important role in the global sulphur cycle and atmospheric chemistry. Microorganisms using DMS as sole carbon, sulphur or energy source, contribute to the cycling of DMS in a wide variety of ecosystems. The diversity of microbial populations degrading DMS in terrestrial environments is poorly understood. Based on cultivation studies, a wide range of bacteria isolated from terrestrial ecosystems were shown to be able to degrade DMS, yet it remains unknown whether any of these have important roles in situ. In this study, we identified bacteria using DMS as a carbon and energy source in terrestrial environments, an agricultural soil and a lake sediment, by DNA stable isotope probing (SIP). Microbial communities involved in DMS degradation were analysed by denaturing gradient gel electrophoresis, high-throughput sequencing of SIP gradient fractions and metagenomic sequencing of phi29-amplified community DNA. Labelling patterns of time course SIP experiments identified members of the Methylophilaceae family, not previously implicated in DMS degradation, as dominant DMS-degrading populations in soil and lake sediment. Thiobacillus spp. were also detected in 13C-DNA from SIP incubations. Metagenomic sequencing also suggested involvement of Methylophilaceae in DMS degradation and further indicated shifts in the functional profile of the DMS-assimilating communities in line with methylotrophy and oxidation of inorganic sulphur compounds. Overall, these data suggest that unlike in the marine environment where gammaproteobacterial populations were identified by SIP as DMS degraders, betaproteobacterial Methylophilaceae may have a key role in DMS cycling in terrestrial environments.

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Sludge age or mean cell residence time (MCRT) plays a crucial role in design and operation of wastewater treatment plants. The change in performance, for example micropollutant removal, associated with changes in MCRT is often attributed to changes in microbial diversity. We operated four identical laboratory-scale sequencing batch reactors (two test and two control) in parallel for 212 days. Sludge age was decreased gradually (from 10.4to 2.6 days) in experimental reactors whereas it was kept constant (10.4 days) in control reactors. The reactor performance and biomass changed in a manner consistent with our understanding of the effect of sludge age on a reactors performance: the effluent quality and biomass declined with decreasing MCRT. The composition of the bacterial and ammonia-oxidizing bacterial communities in four reactors was analyzed using denaturing gradient gel electrophoresis (DGGE), and similarities in band patterns were measured using the Dice coefficient. The overall similarity between the communities in reactors run at different sludge ages was indistinguishable from the similarity in communities in reactors run at identical sludge ages. This was true for both the general bacterial communities and putative AOB communities. The number of detectable bands in DGGE profiles was also unaffected by sludge age (p approximately 0.5 in both cases). Initially, the detectable diversity of activated sludge communities in all four reactors clustered with time, regardless of their designation or sludge age; however, these clusters were only weakly supported by bootstrap analysis. However, after 135 days, a sludge age specific clustering was observed in the bacterial community but not the putative ammonia-oxidizing bacterial community. The mean self-similarity of each reactor decreased, variance increased, and the number of detectable bands in DGGE profiles decreased over time in all reactors. The changes observed with time are consistent with ecological drift. Sludge age has a subtler and slower effect than we anticipated. However, we postulate that sludge age may be more evident in the taxa occurring below the detection limit of DGGE. New sequencing technology may help us address this hypothesis.

Microbial degradation is a major determinant of the fate of pollutants in the environment. para-Nitrophenol (PNP) is an EPAlisted priority pollutant with a wide environmental distribution, but little is known about the microorganisms that degrade it in the environment. We studied the diversity of active PNP-degrading bacterial populations in river water using a novel functional marker approach coupled with [ 13 C 6 ]PNP stable isotope probing (SIP). Culturing together with culture-independent terminal restriction fragment length polymorphism analysis of 16S rRNA gene amplicons identified Pseudomonas syringae to be the major driver of PNP degradation in river water microcosms. This was confirmed by SIP-pyrosequencing of amplified 16S rRNA. Similarly, functional gene analysis showed that degradation followed the Gram-negative bacterial pathway and involved pnpA from Pseudomonas spp. However, analysis of maleylacetate reductase (encoded by mar), an enzyme common to late stages of both Gram-negative and Gram-positive bacterial PNP degradation pathways, identified a diverse assemblage of bacteria associated with PNP degradation, suggesting that mar has limited use as a specific marker of PNP biodegradation. Both the pnpA and mar genes were detected in a PNP-degrading isolate, P. syringae AKHD2, which was isolated from river water. Our results suggest that PNP-degrading cultures of Pseudomonas spp. are representative of environmental PNP-degrading populations.

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