Stable-isotope probing implicates Methylophaga spp and novel Gammaproteobacteria in marine methanol and methylamine metabolism

Abstract

The metabolism of one-carbon (C 1 ) compounds in the marine environment affects global warming, seawater ecology and atmospheric chemistry. Despite their global significance, marine microorganisms that consume C 1 compounds in situ remain poorly characterized. Stable-isotope probing (SIP) is an ideal tool for linking the function and phylogeny of methylotrophic organisms by the metabolism and incorporation of stable-isotope-labelled substrates into nucleic acids. By combining DNA-SIP and time-series sampling, we characterized the organisms involved in the assimilation of methanol and methylamine in coastal sea water (Plymouth, UK). Labelled nucleic acids were analysed by denaturing gradient gel electrophoresis (DGGE) and clone libraries of 16S rRNA genes. In addition, we characterized the functional gene complement of labelled nucleic acids with an improved primer set targeting methanol dehydrogenase (mxaF) and newly designed primers for methylamine dehydrogenase (mauA). Predominant DGGE phylotypes, 16S rRNA, methanol and methylamine dehydrogenase gene sequences, and cultured isolates all implicated Methylophaga spp, moderately halophilic marine methylotrophs, in the consumption of both methanol and methylamine. Additionally, an mxaF sequence obtained from DNA extracted from sea water clustered with those detected in 13 C-DNA, suggesting a predominance of Methylophaga spp among marine methylotrophs. Unexpectedly, most predominant 16S rRNA and functional gene sequences from 13 C-DNA were clustered in distinct substrate-specific clades, with 16S rRNA genes clustering with sequences from the Gammaproteobacteria. These clades have no cultured representatives and reveal an ecological adaptation of particular uncultured methylotrophs to specific C 1 compounds in the coastal marine environment.

Transcriptomic analysis of a marine bacterial community enriched with dimethylsulfoniopropionate

“…An important advantage of the experimental design was that the 30 min incubation time limited the possibility for uptake of DMSP metabolites by non-DMSP-degrading bacteria. For other methodologies that have been developed to link microbial activities to specific taxa, such as MAR-FISH (Vila et al, 2004;Malmstrom et al, 2004a) and stable isotope probing (Neufeld et al, 2007), longer incubation times are required. However, the short incubation time may have biased transcript retrieval against oligotrophic taxa that respond slowly to changing environmental conditions, such as SAR11 cells that have few regulatory systems for environmental cues (Giovannoni et al, 2005).…”

Targeted metagenomics of active microbial populations with stable-isotope probing

“…The DNA-SIP technique involves incubating an environmental sample with a stableisotope-labeled substrate. Although 13 C-labeled substrates have been most commonly used, other 60 isotopes have also been used, including 15 N and multiple forms of labelled water (e.g., H 2 18 O or 2 H 2 O). Importantly, incubation conditions enable assimilation of the target substrate and incorporation of isotope into genomes (and lipids, proteins, and RNA) of active microorganisms during replication and cellular metabolism.…”

“…Examples demonstrating the wide-range of applications of DNA-SIP include methane assimilation in landfill cover soil [13] and peatlands [14], methanol 70 assimilation in coastal sea water [15] and soda lake sediments [16] studies have focused on pmoA and mmoX for methanotrophs [13], mxaF and mauA for methylotrophs [15], arrA for bacterial respiration of As(V) [17], nirK and nirS for denitrifiers Despite the widespread use of DNA-SIP for targeting active microorganisms, the limitations of this method include the potential for cross-feeding of label from primary 85 consumers to other microbes, low recovery of heavy DNA, non-availability and high costs associated with the purchase of isotopically labeled substrates, and the requirement for relatively high concentrations of substrate for efficient labeling of DNA [35]. In order to help circumvent these weaknesses, careful experimental design with different incubation times can help detect and minimize cross-feeding [36,37].…”

Active methylotrophs in the sediments of Lonar Lake, a saline and alkaline ecosystem formed by meteor impact

“…Denaturing gradient gel electrophoresis (DGGE) and clone library analyses based on 16S rRNA genes were performed using PCR products amplified with primer sets GC341F/907R (Muyzer et al, 1998) and 27F/1492R (Weisburg et al, 1991), respectively. PCR amplifications were also carried out with primers specific for the functional genes mxaF, 1003f and 1555r (Neufeld et al, 2007c); pmoA, A189f and mb661r (Costello and Lidstrom, 1999); mmoX, 206F and 886R (Hutchens et al, 2004); and mauA, mauAf1 and mauAr1 (Neufeld et al, 2007c). All PCR reactions were carried out in a total volume of 50 ml in 0.5 ml tubes.…”

“…One pathway by which methylamine is used by methylotrophic bacteria contains methylamine dehydrogenase, but alternative pathways may also be present (Anthony, 1982;Latypova et al, 2009). Some of the marine methylotrophs involved in the metabolism of methylamine have been identified using PCR primers targeting the structural gene (mauA) encoding the small subunit of methylamine dehydrogenase (Neufeld et al, 2007c). Relatively few studies have focused on isolation of methylotrophs from saline and alkaline environments (Khmelenina et al, 1996;Sorokin et al, 2000;Doronina et al, 2001Doronina et al, , 2003aKaluzhnaya et al, 2001), and the active organisms (Lin et al, 2004(Lin et al, , 2005 and enzymes involved are poorly characterized.…”