Hendrik Schäfer

Identification of and Spatio-Temporal Differences between Microbial Assemblages from Two Neighboring Sulfurous Lakes: Comparison by Microscopy and Denaturing Gradient Gel Electrophoresis

ABSTRACT The microbial assemblages of Lake Cisó and Lake Vilar (Banyoles, northeast Spain) were analyzed in space and time by microscopy and by performing PCR-denaturing gradient gel electrophoresis (DGGE) and sequence analysis of 16S rRNA gene fragments. Samples obtained from different water depths and at two different times of the year (in the winter during holomixis and in the early spring during a phytoplankton bloom) were analyzed. Although the lakes have the same climatic conditions and the same water source, the limnological parameters were different, as were most of the morphologically distinguishable photosynthetic bacteria enumerated by microscopy. The phylogenetic affiliations of the predominant DGGE bands were inferred by performing a comparative 16S rRNA sequence analysis. Sequences obtained from Lake Cisó samples were related to gram-positive bacteria and to members of the divisionProteobacteria. Sequences obtained from Lake Vilar samples were related to members of theCytophaga-Flavobacterium-Bacteroides phylum and to cyanobacteria. Thus, we found that like the previously reported differences between morphologically distinct inhabitants of the two lakes, there were also differences among the community members whose morphologies did not differ conspicuously. The changes in the species composition from winter to spring were also marked. The two lakes both contained sequences belonging to phototrophic green sulfur bacteria, which is consistent with microscopic observations, but these sequences were different from the sequences of cultured strains previously isolated from the lakes. Euryarchaeal sequences (i.e., methanogen- and thermoplasma-related sequences) also were present in both lakes. These euryarchaeal group sequences dominated the archaeal sequences in Lake Cisó but not in Lake Vilar. In Lake Vilar, a new planktonic population related to the crenarchaeota produced the dominant archaeal band. The phylogenetic analysis indicated that new bacterial and archaeal lineages were present and that the microbial diversity of these assemblages was greater than previously known. We evaluated the correspondence between the abundances of several morphotypes and DGGE bands by comparing microscopy and sequencing results. Our data provide evidence that the sequences obtained from the DGGE fingerprints correspond to the microorganisms that are actually present at higher concentrations in the natural system.

Denaturing gradient gel electrophoresis in marine microbial ecology

Publisher Summary Genetic fingerprinting techniques are excellently suited to comparison of large numbers of samples. Genetic fingerprinting of microbial communities provides banding patterns or profiles that reflect the genetic diversity of the community. Denaturing gradient gel electrophoresis (DGGE) of PCRamplified gene fragments is one of the genetic fingerprinting techniques used in microbial ecology. In DGGE, similar-sized DNA fragments are separated in a gradient of DNA denaturants according to differences in sequence. A variant of DGGE, temperature gradient gel electrophoresis (TGGE), makes use of a temperature gradient to separate gene fragments. DGGE is relatively easy to perform and is especially suited to the analysis of multiple samples. Since its introduction into microbial ecology, it has been adapted in many laboratories as a convenient tool for the assessment of microbial diversity in natural samples. It has been shown by several studies that the approach is reproducible and sensitive. Other new fingerprinting techniques, such as automated T-RFLP might be more sensitive but identification of predominant community members still requires cloning and sequencing of PCR products. A potential future development in PCR-DGGE fingerprinting might be to use fluorescently labelled PCR primers, which might (1) make staining of gels unnecessary, and (2) make it possible to add intra-lane standards with a different fluorochrome, facilitating gel-to-gel comparisons.

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

The metabolism of one-carbon (C1) compounds in the marine environment affects global warming, seawater ecology and atmospheric chemistry. Despite their global significance, marine microorganisms that consume C1 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 13C-DNA, suggesting a predominance of Methylophaga spp among marine methylotrophs. Unexpectedly, most predominant 16S rRNA and functional gene sequences from 13C-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 C1 compounds in the coastal marine environment.

The response of the microbial community of marine sediments to organic carbon input under anaerobic conditions.

Cyanobacterial biomass was added to anaerobic sediment to simulate the natural input of complex organic substrate that occurs in nature after algae blooms. Sediments were incubated at 0 degree C, 8 degrees C and 24 degrees C for 13 days. Community dynamics were measured by fluorescence in situ hybridisation (FISH), denaturing gradient gel electrophoresis (DGGE), and sequencing of 16S rDNA PCR products. Metabolic changes were followed by the analysis of total carbon mineralisation, sulfate reduction, and ammonium production rates. The addition of organic material resulted in significant changes in the composition of the microbial community at all temperatures tested. Sulfate reduction was the main mineralisation process detected. However, not sulfate-reducers but rather members of the Cytophaga-Flavobacterium phylogenetic cluster showed the highest increase in the bacterial cells as detected by FISH. We conclude that these organisms play an important role in the anaerobic decomposition of complex organic material perhaps because they are the main catalysts of macromolecule hydrolysis and fermentation. The molecular methods also indicated a stimulation of ribosome synthesis. The detection of a large number of rRNA-rich cells belonging to the Cytophaga-Flavobacterium phylogenetic cluster further supports the importance of their role in the degradation of complex organic material in anaerobic marine sediments. Their detection in high numbers in the field may indicate recent deposition events.

Microbial degradation of dimethylsulphide and related C1-sulphur compounds: organisms and pathways controlling fluxes of sulphur in the biosphere

Dimethylsulphide (DMS) plays a major role in the global sulphur cycle. It has important implications for atmospheric chemistry, climate regulation, and sulphur transport from the marine to the atmospheric and terrestrial environments. In addition, DMS acts as an info-chemical for a wide range of organisms ranging from micro-organisms to mammals. Micro-organisms that cycle DMS are widely distributed in a range of environments, for instance, oxic and anoxic marine, freshwater and terrestrial habitats. Despite the importance of DMS that has been unearthed by many studies since the early 1970s, the understanding of the biochemistry, genetics, and ecology of DMS-degrading micro-organisms is still limited. This review examines current knowledge on the microbial cycling of DMS and points out areas for future research that should shed more light on the role of organisms degrading DMS and related compounds in the biosphere.

Carnitine metabolism to trimethylamine by an unusual Rieske-type oxygenase from human microbiota

Significance Metabolism of l-carnitine, a compound abundant in human diet, to trimethylamine by human microbiota has been shown to promote atherosclerosis and subsequent development of heart disease. However, the underpinning molecular and biochemical mechanisms remain unknown. In this study, we reveal that a previously unidentified Rieske-type protein is responsible for carnitine transformation to trimethylamine from human microbiota. Knowledge gained in our study provides the opportunity not only to explore Rieske protein inhibitors in preventing trimethylamine formation in animal studies and clinical trials, but also for its use as a functional genetic marker to better understand human microbiota and their dynamics in our health and disease in future epidemiological studies and dietary interventions. Dietary intake of l-carnitine can promote cardiovascular diseases in humans through microbial production of trimethylamine (TMA) and its subsequent oxidation to trimethylamine N-oxide by hepatic flavin-containing monooxygenases. Although our microbiota are responsible for TMA formation from carnitine, the underpinning molecular and biochemical mechanisms remain unclear. In this study, using bioinformatics approaches, we first identified a two-component Rieske-type oxygenase/reductase (CntAB) and associated gene cluster proposed to be involved in carnitine metabolism in representative genomes of the human microbiota. CntA belongs to a group of previously uncharacterized Rieske-type proteins and has an unusual “bridging” glutamate but not the aspartate residue, which is believed to facilitate intersubunit electron transfer between the Rieske center and the catalytic mononuclear iron center. Using Acinetobacter baumannii as the model, we then demonstrate that cntAB is essential in carnitine degradation to TMA. Heterologous overexpression of cntAB enables Escherichia coli to produce TMA, confirming that these genes are sufficient in TMA formation. Site-directed mutagenesis experiments have confirmed that this unusual “bridging glutamate” residue in CntA is essential in catalysis and neither mutant (E205D, E205A) is able to produce TMA. Taken together, the data in our study reveal the molecular and biochemical mechanisms underpinning carnitine metabolism to TMA in human microbiota and assign the role of this novel group of Rieske-type proteins in microbial carnitine metabolism.

Isolation of Methylophaga spp. from marine dimethylsulfide-degrading enrichment cultures and identification of polypeptides induced during growth on dimethylsulfide.

ABSTRACT Dimethylsulfide (DMS)-degrading enrichment cultures were established from samples of coastal seawater, nonaxenic Emiliania huxleyi cultures, and mixed marine methyl halide-degrading enrichment cultures. Bacterial populations from a broad phylogenetic range were identified in the mixed DMS-degrading enrichment cultures by denaturing gradient gel electrophoresis (DGGE). Sequences of dominant DGGE bands were similar to those of members of the genera Methylophaga and Alcanivorax. Several closely related Methylophaga strains were obtained that were able to grow on DMS as the carbon and energy source. Roseobacter-related populations were detected in some of the enrichment cultures; however, none of the Roseobacter group isolates that were tested were able to grow on DMS. Oxidation of DMS by Methylophaga sp. strain DMS010 was not affected by addition of the inhibitor chloroform or methyl tert-butyl ether, suggesting that DMS metabolism may occur by a route different from those described for Thiobacillus species and other unidentified marine isolates. Addition of DMS and methanethiol to whole-cell suspensions of strain DMS010 induced oxygen uptake when strain DMS010 was grown on DMS but not in cells grown on methanol. The apparent Kms of strain DMS010 for DMS and for methanethiol were 2.1 and 4.6 μM, respectively, when grown on DMS. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the biomass of strain DMS010 and analysis of peptide bands by mass spectrometry techniques and N-terminal sequencing provided the first insight into the identity of polypeptides induced during growth on DMS. These included XoxF, a homolog of the large subunit of methanol dehydrogenase for which a biological role has not been identified previously.

Genetics of the glutamate-mediated methylamine utilization pathway in the facultative methylotrophic beta-proteobacterium Methyloversatilis universalis FAM5

The ability of some microbial species to oxidize monomethylamine via glutamate‐mediated pathways was proposed in the 1960s; however, genetic determinants of the pathways have never been described. In the present study we describe a gene cluster essential for operation of the N‐methylglutamate pathway in the methylotrophic beta‐proteobacterium Methyloversatilis universalis FAM5. Four major polypeptides from protein fractions displaying high activities of N‐methylglutamate synthetase, N‐methylglutamate dehydrogenase and γ‐glutamylmethylamide synthetase were selected for mass spectrometry‐based identification. The activities of enzymes were associated with the presence of peptides identified as ferredoxin‐dependent glutamate synthase (GltB2), large subunit of putative heterotetrameric sarcosine oxidase (SoxA) and glutamine synthetase type III (GSIII) respectively. A gene cluster (8.3 kb) harbouring gltB2, soxA and gsIII‐like genes was amplified from M. universalis FAM5, sequenced and assembled. Two partial and six complete open reading frames arranged in the order soxBDAG‐gsIII‐gltB132 were identified and subjected to mutational analysis, functional and metabolic profiling. We demonstrated that gltB‐like and sox‐like genes play a key role in methylamine utilization and encode N‐methylglutamate synthetase and N‐methylglutamate dehydrogenase respectively. Metabolic, enzymatic and mutational analyses showed that the gsIII‐like gene encodes γ‐glutamylmethylamide synthetase; however, this enzyme is not essential for oxidation of methylamine.

Analysis of Methane Monooxygenase Genes in Mono Lake Suggests That Increased Methane Oxidation Activity May Correlate with a Change in Methanotroph Community Structure

ABSTRACT Mono Lake is an alkaline hypersaline lake that supports high methane oxidation rates. Retrieved pmoA sequences showed a broad diversity of aerobic methane oxidizers including the type I methanotrophs Methylobacter (the dominant genus), Methylomicrobium, and Methylothermus, and the type II methanotroph Methylocystis. Stratification of Mono Lake resulted in variation of aerobic methane oxidation rates with depth. Methanotroph diversity as determined by analysis of pmoA using new denaturing gradient gel electrophoresis primers suggested that variations in methane oxidation activity may correlate with changes in methanotroph community composition.

Phylogenetic and functional gene analysis of the bacterial and archaeal communities associated with the surface microlayer of an estuary.

The surface microlayer (SML) is the thin biogenic film found at the surface of a water body. The SML is poorly understood but has been shown to be important in biogeochemical cycling and sea–air gas exchange. We sampled the SML of the Blyth estuary at two sites (salinities 21 and 31 psu) using 47 mm polycarbonate membranes. DNA was extracted from the SML and corresponding subsurface water (0.4 m depth) and microbial (bacteria and archaea) community analysis was performed using denaturing gradient gel electrophoresis of 16S rRNA gene PCR amplicons. The diversity of bacterial functional genes that encode enzyme subunits for methane monooxygenase (pmoA and mmoX) and carbon monoxide dehydrogenase (coxL) was assessed using PCR, clone library construction and restriction fragment length polymorphism (RFLP) analysis. Methanotroph genes were present only in low copy numbers and pmoA was detected only in subsurface samples. Diversity of mmoX genes was low and most of the clone sequences detected were similar to those of mmoX from Methylomonas spp. Interestingly, some sequences detected in the SML were different from those detected in the subsurface. RFLP analysis of coxL clone libraries indicated a high diversity of carbon monoxide (CO)-utilizing bacteria in the estuary. The habitats of the closely related coxL sequences suggest that CO-utilizing bacteria in the estuary are recruited from both marine and freshwater/terrestrial inputs. In contrast, methanotroph recruitment appears to occur solely from freshwater input into the estuary.