Uneven genotypic diversity of Escherichia coli in fecal sources limits the performance of a library-dependent method of microbial source tracking on the southwestern French Atlantic coast.

To develop a library-dependent method of tracking fecal sources of contamination of beaches on the Atlantic coast of southwestern France, a library of 6368 Escherichia coli isolates was constructed from samples of feces, from 40 known human or animal sources collected in the vicinity of Arcachon Bay in 2010, and in French Basque Country, Landes, and Béarn, between 2017 and 2018. Different schemes of source identification were tested: use of the complete or filtered reference library; characterization of the isolates by genotypic or proteomic profiling based on ERIC-PCR or MALDI-TOF mass spectrometry, respectively; isolate by isolate assignment using either classifiers based on the Pearson similarity or SVM (support vector machine). With the exception of one source identification scheme, which was discarded since it used self-assignment, all tested schemes resulted in low rates of correct classification (<35%) and significant rates of incorrect classification (>15%). The heterogeneous coverage of E. coli genotypic diversity between sources and the uneven distribution of E. coli genotypes in the library likely explain the difficulties encountered in identifying the sources of fecal contamination. Shannon diversity index of sources ranged from 0 for several wildlife species sampled once to 3.03 for sewage treatment plant effluents sampled on various occasions, showing discrepancies between sources. The uneven genotypic composition of the library was attested by the value of the Pielou index (0.54), the high proportion of nondiscriminatory genotypes (>91% of the isolates), and the very low proportion of discriminatory genotypes (<3%). Since efforts made to constitute such a library are not affordable for routine analyses, the results question the relevance of developing such a method for identifying sources of fecal contamination on such a coastline.

Metal contaminations impact archaeal community composition, abundance and function in remote alpine lakes.

Using the 16S rRNA and mcrA genes, we investigated the composition, abundance and activity of sediment archaeal communities within 18 high-mountain lakes under contrasted metal levels from different origins (bedrock erosion, past-mining activities and atmospheric depositions). Bathyarchaeota, Euryarchaeota and Woesearchaeota were the major phyla found at the meta-community scale, representing 48%, 18.3% and 15.2% of the archaeal community respectively. Metals were equally important as physicochemical variables in explaining the assemblage of archaeal communities and their abundance. Methanogenesis appeared as a process of central importance in the carbon cycle within sediments of alpine lakes as indicated by the absolute abundance of methanogen 16S rRNA and mcrA gene transcripts (105 to 109 copies g-1 ). We showed that methanogen abundance and activity were significantly reduced with increasing concentrations of Pb and Cd, two indicators of airborne metal contaminations. Considering the ecological importance of methanogenesis in sediment habitats, these metal contaminations may have system wide implications even in remote area such as alpine lakes. Overall, this work was pioneer in integrating the effect of long-range atmospheric depositions on archaeal communities and indicated that metal contamination might significantly compromise the contribution of Archaea to the carbon cycling of the mountain lake sediments.

Complete genome sequence of the aerobic marine methanotroph Methylomonas methanica MC09.

Methylomonas methanica MC09 is a mesophilic, halotolerant, aerobic, methanotrophic member of the Gammaproteobacteria, isolated from coastal seawater. Here we present the complete genome sequence of this strain, the first available from an aerobic marine methanotroph.

Acetate repression of methane oxidation by supplemental Methylocella silvestris in a peat soil microcosm.

Methylocella spp. are facultative methanotrophs that grow on methane and multicarbon substrates, such as acetate. Acetate represses transcription of methane monooxygenase of Methylocella silvestris in laboratory culture. DNA stable-isotope probing (DNA-SIP) using (13)C-methane and (12)C-acetate, carried out with Methylocella-spiked peat soil, showed that acetate also repressed methane oxidation by Methylocella in environmental samples.

Substrate-specific clades of active marine methylotrophs associated with a phytoplankton bloom in a temperate coastal environment.

Marine microorganisms that consume one-carbon (C(1)) compounds are poorly described, despite their impact on global climate via an influence on aquatic and atmospheric chemistry. This study investigated marine bacterial communities involved in the metabolism of C(1) compounds. These communities were of relevance to surface seawater and atmospheric chemistry in the context of a bloom that was dominated by phytoplankton known to produce dimethylsulfoniopropionate. In addition to using 16S rRNA gene fingerprinting and clone libraries to characterize samples taken from a bloom transect in July 2006, seawater samples from the phytoplankton bloom were incubated with (13)C-labeled methanol, monomethylamine, dimethylamine, methyl bromide, and dimethyl sulfide to identify microbial populations involved in the turnover of C(1) compounds, using DNA stable isotope probing. The [(13)C]DNA samples from a single time point were characterized and compared using denaturing gradient gel electrophoresis (DGGE), fingerprint cluster analysis, and 16S rRNA gene clone library analysis. Bacterial community DGGE fingerprints from (13)C-labeled DNA were distinct from those obtained with the DNA of the nonlabeled community DNA and suggested some overlap in substrate utilization between active methylotroph populations growing on different C(1) substrates. Active methylotrophs were affiliated with Methylophaga spp. and several clades of undescribed Gammaproteobacteria that utilized methanol, methylamines (both monomethylamine and dimethylamine), and dimethyl sulfide. rRNA gene sequences corresponding to populations assimilating (13)C-labeled methyl bromide and other substrates were associated with members of the Alphaproteobacteria (e.g., the family Rhodobacteraceae), the Cytophaga-Flexibacter-Bacteroides group, and unknown taxa. This study expands the known diversity of marine methylotrophs in surface seawater and provides a comprehensive data set for focused cultivation and metagenomic analyses in the future.

Novel uncultured Epsilonproteobacteria dominate a filamentous sulphur mat from the 13 degrees N hydrothermal vent field, East Pacific Rise.

Rapid growth of microbial sulphur mats have repeatedly been observed during oceanographic cruises to various deep-sea hydrothermal vent sites. The microorganisms involved in the mat formation have not been phylogenetically characterized, although the production of morphologically similar sulphur filaments by a Arcobacter strain coastal marine has been documented. An in situ collector deployed for 5 days at the 13 degrees N deep-sea hydrothermal vent site on the East Pacific Rise (EPR) was rapidly colonized by a filamentous microbial mat. Microscopic and chemical analyses revealed that the mat consisted of a network of microorganisms embedded in a mucous sulphur-rich matrix. Molecular surveys based on 16S rRNA gene and aclB genes placed all the environmental clone sequences within the Epsilonproteobacteria. Although few 16S rRNA gene sequences were affiliated with that of cultured organisms, the majority was related to uncultured representatives of the Arcobacter group (< or = 95% sequence similarity). A probe designed to target all of the identified lineages hybridized with more than 95% of the mat community. Simultaneous hybridizations with the latter probe and a probe specific to Arcobacter spp. confirmed the numerical dominance of Arcobacter-like bacteria. This study provides the first example of the prevalence and ecological significance of free-living Arcobacter at deep-sea hydrothermal vents.

Uncultured Archaea in a hydrothermal microbial assemblage: phylogenetic diversity and characterization of a genome fragment from a euryarchaeote.

The polychaete Alvinella pompejana lives in organic tubes on the walls of active hydrothermal chimneys along the East Pacific Rise. To examine the diversity of the archaeal community associated with the polychaete tubes, we constructed libraries by direct PCR amplification and cloning of 16S rRNA genes. Almost half of the sequences of the 16S rRNA gene libraries clustered with uncultured archaeal groups. In an effort to access genomic information from uncultured archaeal members we further constructed a fosmid library from the same DNA source. One of the clones, Alv-FOS5, was sequenced completely. Its sequence analysis revealed an incomplete rRNA operon and 32 predicted ORFs. Seventeen of these ORFs have been assigned putative functions, including transcription and translation, cellular processes and signalling, transport systems and metabolic pathways. Phylogenetic analyses of the 16S rRNA gene suggested that Alv-FOS5 formed a new lineage related to members of Deep-Sea Hydrothermal Vent Euryarchaeota group II. Phylogenetic analyses of predicted proteins revealed the existence of likely cases of horizontal gene transfer, both between Crenarchaeota and Euryarchaeota and between Archaea and Bacteria. This study is the first step in using genomics to reveal the physiology of an as yet uncultured group of archaea from deep-sea hydrothermal vents.

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

Lonar Lake is a unique saline and alkaline ecosystem formed by meteor impact in the Deccan basalts in India around 52,000 years ago. To investigate the role of methylotrophy in the cycling of carbon in this unusual environment, stable-isotope probing (SIP) was carried out using the one-carbon compounds methane, methanol and methylamine. Denaturing gradient gel electrophoresis fingerprinting analyses performed with heavy (13)C-labelled DNA retrieved from sediment microcosms confirmed the enrichment and labelling of active methylotrophic communities. Clone libraries were constructed using PCR primers targeting 16S rRNA genes and functional genes. Methylomicrobium, Methylophaga and Bacillus spp. were identified as the predominant active methylotrophs in methane, methanol and methylamine SIP microcosms, respectively. Absence of mauA gene amplification in the methylamine SIP heavy fraction also indicated that methylamine metabolism in Lonar Lake sediments may not be mediated by the methylamine dehydrogenase enzyme pathway. Many gene sequences retrieved in this study were not affiliated with extant methanotrophs or methylotrophs. These sequences may represent hitherto uncharacterized novel methylotrophs or heterotrophic organisms that may have been cross-feeding on methylotrophic metabolites or biomass. This study represents an essential first step towards understanding the relevance of methylotrophy in the soda lake sediments of an unusual impact crater structure.

Life without light: microbial diversity and evidence of sulfur- and ammonium-based chemolithotrophy in Movile Cave.

Microbial diversity in Movile Cave (Romania) was studied using bacterial and archaeal 16S rRNA gene sequence and functional gene analyses, including ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), soxB (sulfate thioesterase/thiohydrolase) and amoA (ammonia monooxygenase). Sulfur oxidizers from both Gammaproteobacteria and Betaproteobacteria were detected in 16S rRNA, soxB and RuBisCO gene libraries. DNA-based stable-isotope probing analyses using 13C-bicarbonate showed that Thiobacillus spp. were most active in assimilating CO2 and also implied that ammonia and nitrite oxidizers were active during incubations. Nitrosomonas spp. were detected in both 16S rRNA and amoA gene libraries from the 'heavy' DNA and sequences related to nitrite-oxidizing bacteria Nitrospira and Candidatus 'Nitrotoga' were also detected in the 'heavy' DNA, which suggests that ammonia/nitrite oxidation may be another major primary production process in this unique ecosystem. A significant number of sequences associated with known methylotrophs from the Betaproteobacteria were obtained, including Methylotenera, Methylophilus and Methylovorus, supporting the view that cycling of one-carbon compounds may be an important process within Movile Cave. Other sequences detected in the bacterial 16S rRNA clone library included Verrucomicrobia, Firmicutes, Bacteroidetes, alphaproteobacterial Rhodobacterales and gammaproteobacterial Xanthomonadales. Archaeal 16S rRNA sequences retrieved were restricted within two groups, namely the Deep-sea Hydrothermal Vent Euryarchaeota group and the Miscellaneous Crenarchaeotic group. No sequences related to known sulfur-oxidizing archaea, ammonia-oxidizing archaea, methanogens or anaerobic methane-oxidizing archaea were detected in this clone library. The results provided molecular biological evidence to support the hypothesis that Movile Cave is driven by chemolithoautotrophy, mainly through sulfur oxidation by sulfur-oxidizing bacteria and reveal that ammonia- and nitrite-oxidizing bacteria may also be major primary producers in Movile Cave.

Identification of active methylotrophic bacteria inhabiting surface sediment of a marine estuary.

Methylotrophs play an essential role in the global carbon cycle due to their participation in methane oxidation and C1 metabolism. Despite this important biogeochemical role, marine and estuarine microorganisms that consume C1 compounds are poorly characterized. In this study, we investigated the diversity of active methylotrophs and methanotrophs in sediment from the Colne Estuary (Brightlingsea, UK). Aerobic surface sediment samples were examined for the presence of C1 -utilizing communities using DNA stable-isotope probing (DNA-SIP) with (13) C-labelled methane, methanol and monomethylamine. Active methylotrophic bacteria were confirmed after DNA-SIP and denaturing gradient gel electrophoresis analyses. Clone libraries of 16S rRNA gene amplicons revealed the presence of methylotrophic bacteria affiliated with Methylophaga spp. in methanol and monomethylamine incubations. The addition of marine ammonium mineral salts medium to the microcosms increased the rate of substrate metabolism in DNA-SIP incubations, although nutrient addition did not affect the active populations contributing (13) C-labelled DNA. The (13) CH4 SIP incubations indicated the predominant activity of type I methanotrophs and microarray hybridization of amplified particulate methane monooxygenase (pmoA) genes confirmed the role of type Ia methanotrophs in SIP incubations. Type II methanotrophs (i.e. Methylocystis and Methylosinus) were only detected in the original sediment and in the unlabelled DNA fractions, which indicated that type II methanotrophs were not actively involved in C1 compound assimilation in DNA-SIP incubations with estuarine surface sediment samples.

Thermophilic lifestyle for an uncultured archaeon from hydrothermal vents: evidence from environmental genomics.

We present a comparative analysis of two genome fragments isolated from a diverse and widely distributed group of uncultured euryarchaea from deep-sea hydrothermal vents. The optimal activity and thermostability of a DNA polymerase predicted in one fragment were close to that of the thermophilic archaeon Thermoplasma acidophilum, providing evidence for a thermophilic way of life of this group of uncultured archaea.

Construction of a shuttle vector for, and spheroplast transformation of, the hyperthermophilic archaeon Pyrococcus abyssi.

Our understanding of the genetics of species of the best-studied hyperthermophilic archaea, Pyrococcus spp., is presently limited by the lack of suitable genetic tools, such as a stable cloning vector and the ability to select individual transformants on plates. Here we describe the development of a reliable host-vector system for the hyperthermophilic archaeon Pyrococcus abyssi. Shuttle vectors were constructed based on the endogenous plasmid pGT5 from P. abyssi strain GE5 and the bacterial vector pLitmus38. As no antibiotic resistance marker is currently available for Pyrococcus spp., we generated a selectable auxotrophic marker. Uracil auxotrophs resistant to 5-fluoorotic acid were isolated from P. abyssi strain GE9 (devoid of pGT5). Genetic analysis of these mutants revealed mutations in the pyrE and/or pyrF genes, encoding key enzymes of the pyrimidine biosynthetic pathway. Two pyrE mutants exhibiting low reversion rates were retained for complementation experiments. For that purpose, the pyrE gene, encoding orotate phosphoribosyltransferase (OPRTase) of the thermoacidophilic crenarchaeote Sulfolobus acidocaldarius, was introduced into the pGT5-based vector, giving rise to pYS2. With a polyethylene glycol-spheroplast method, we could reproducibly transform P. abyssi GE9 pyrE mutants to prototrophy, though with low frequency (10(2) to 10(3) transformants per micro g of pYS2 plasmid DNA). Transformants did grow as well as the wild type on minimal medium without uracil and showed comparable OPRTase activity. Vector pYS2 proved to be very stable and was maintained at high copy number under selective conditions in both Escherichia coli and P. abyssi.