Targeting methanotrophs and isolation of a novel psychrophilic Methylobacter species from a terrestrial Arctic alkaline methane seep in Lagoon Pingo, Central Spitsbergen (78° N).

The microbial diversity associated with terrestrial groundwater seepage through permafrost soils is tightly coupled to the geochemistry of these fluids. Terrestrial alkaline methane seeps from Lagoon Pingo, Central Spitsbergen (78°N) in Norway, with methane-saturated and oxygen-limited groundwater discharge providing a potential habitat for methanotrophy. Here, we report on the microbial community's comparative analyses and distribution patterns at two sites close to Lagoon Pingo's methane emission source. To target methane-oxidizing bacteria from this system, we analysed the microbial community pattern of replicate samples from two sections near the main methane seepage source. DNA extraction, metabarcoding and subsequent sequencing of 16S rRNA genes revealed microbial communities where the major prokaryotic phyla were Pseudomonadota (42-47%), Gemmatimonadota (4-14%) and Actinobacteriota (7-11%). Among the Pseudomonadota, members of the genus Methylobacter were present at relative abundances between 1.6 and 4.7%. Enrichment targeting the methane oxidising bacteria was set up using methane seep sediments as inoculum and methane as the sole carbon and energy source, and this resulted in the isolation of a novel psychrophilic methane oxidizer, LS7-T4AT. The optimum growth temperature for the isolate was 13 °C and the pH optimum was 8.0. The morphology of cells was short rods, and TEM analysis revealed intracytoplasmic membranes arranged in stacks, a distinctive feature for Type I methanotrophs in the family Methylomonadaceae of the class Gammaproteobacteria. The strain belongs to the genus Methylobacter based on high 16S rRNA gene similarity to the psychrophilic species of Methylobacter psychrophilus Z-0021T (98.95%), the psychrophilic strain Methylobacter sp. strain S3L5C (99.00%), and the Arctic mesophilic species of Methylobacter tundripaludum SV96T (99.06%). The genome size of LS7-T4AT was 4,338,157 bp with a G + C content of 47.93%. The average nucleotide identities (ANIb) of strain LS7-T4AT to 10 isolated strains of genus Methylobacter were between 75.54 and 85.51%, lower than the species threshold of 95%. The strain LS7-T4AT represents a novel Arctic species, distinct from other members of the genus Methylobacter, for which the name Methylobacter svalbardensis sp. nov. is proposed. The type of strain is LS7-T4AT (DSMZ:114308, JCM:39463).

Antibiotic susceptibility of marine Planctomycetes.

Antimicrobials are naturally produced by microbes and therefore have always been present in their environment, as well as accompanying resistance mechanisms. The antibiotic resistance profile of environmental species is particularly relevant since genetic determinants of resistance can spread through horizontal gene transfer and reach clinically important species. The phylum Planctomycetes comprises Gram-negative bacteria characterised by unusual features and appear to be ubiquitously distributed. Members of this group have recently been characterised as producers of bioactive compounds, namely antimicrobials, but their antibiotic susceptibility profile has been scarcely studied. In this study, the antibiotic susceptibility profile of six phylogenetically distinct strains of Planctomycetes was assessed. All strains showed resistance to beta-lactams, aminoglycosides and glycopeptides. Our results showed that antibiotics which target protein synthesis or DNA replication, with the exception of aminoglycosides, were the most effective against the tested strains. The highest efficacy was observed for chloramphenicol, clindamycin and ciprofloxacin. The highest level of antimicrobial resistance was observed in the uncharacterised novel taxon Planctomyces sp. strain FF15 which was only susceptible to erythromycin and ciprofloxacin.

Simple models combining competition, defence and resource availability have broad implications in pelagic microbial food webs.

In food webs, interactions between competition and defence control the partitioning of limiting resources. As a result, simple models of these interactions contain links between biogeochemistry, diversity, food web structure and ecosystem function. Working at hierarchical levels, these mechanisms also produce self-similarity and therefore suggest how complexity can be generated from repeated application of simple underlying principles. Reviewing theoretical and experimental literature relevant to the marine photic zone, we argue that there is a wide spectrum of phenomena, including single cell activity of prokaryotes, microbial biodiversity at different levels of resolution, ecosystem functioning, regional biogeochemical features and evolution at different timescales; that all can be understood as variations over a common principle, summarised in what has been termed the 'Killing-the-Winner' (KtW) motif. Considering food webs as assemblages of such motifs may thus allow for a more integrated approach to aquatic microbial ecology.

Disentangling the complexity of permafrost soil by using high resolution profiling of microbial community composition, key functions and respiration rates.

Thawing permafrost can stimulate microbial activity, leading to faster decomposition of formerly preserved organic matter and CO2 release. Detailed knowledge about the vertical distribution of the responsible microbial community that is changing with increasing soil depth is limited. In this study, we determined the microbial community composition from cores sampled in a high Arctic heath at Svalbard, Norway; spanning from the active layer (AL) into the permafrost layer (PL). A special aim has been on identifying a layer of recently thawed soil, the transition zone (TZ), which might provide new insights into the fate of thawing permafrost. A unique sampling strategy allowed us to observe a diverse and gradually shifting microbial community in the AL, a Bacteroidetes dominated community in the TZ and throughout the PL, a community strongly dominated by a single Actinobacteria family (Intrasporangiaceae). The contrasting abundances of these two taxa caused a community difference of about 60%, just within 3 cm from TZ to PL. We incubated subsamples at about 5°C and measured highest CO2 production rates under aerobic incubations, yet contrasting for five different layers and correlating to the microbial community composition. This high resolution strategy provides new insights on how microbial communities are structured in permafrost and a better understanding of how they respond to thaw.

Diversity patterns and isolation of Planctomycetes associated with metalliferous deposits from hydrothermal vent fields along the Valu Fa Ridge (SW Pacific).

The microbial diversity associated with diffuse venting deep-sea hydrothermal deposits is tightly coupled to the geochemistry of the hydrothermal fluids. Previous 16S rRNA gene amplicon sequencing (metabarcoding) of marine iron-hydroxide deposits along the Arctic Mid Ocean Ridge, revealed the presence of diverse bacterial communities associated with these deposits (Storesund and Øvreås in Antonie van Leeuwenhoek 104:569-584, 2013). One of the most abundant and diverse phyla detected was the enigmatic Planctomycetes. Here we report on the comparative analyses of the diversity and distribution patterns of Planctomycetes associated with metalliferous deposits from two diffuse-flow hydrothermal vent fields (Mariner and Vai Lili) from the Valu Fa Ridge in the Southwestern Pacific. Metabarcoding of 16S rRNA genes showed that the major prokaryotic phyla were Proteobacteria (51-73% of all 16S rRNA gene reads), Epsilonbacteraeota (0.5-19%), Bacteriodetes (5-17%), Planctomycetes (0.4-11%), Candidatus Latescibacteria (0-5%) and Marine Benthic Group E (Hydrothermarchaeota) (0-5%). The two different sampling sites differed considerably in overall community composition. The abundance of Planctomycetes also varied substantially between the samples and the sites, with the majority of the sequences affiliated with uncultivated members of the classes Planctomycetacia and Phycisphaerae, and other deep branching lineages. Seven different strains affiliated with the order Planctomycetales were isolated, mostly from the Vai Lili samples, where also the highest Planctomycetales diversity was seen. Most of the isolates were affiliated with the genera Gimesia, Rhodopirellula and Blastopirellula. One isolate was only distantly related to known cultured, but uncharacterized species within the Pir4 group. This study shows that the deep-sea Planctomycetes represent a very heterogeneous group with a high phylogenetic diversity and a substantial potential for novel organism discovery in these deep ocean environments.

The Relative Abundance and Transcriptional Activity of Marine Sponge-Associated Microorganisms Emphasizing Groups Involved in Sulfur Cycle.

During the last decades, our knowledge about the activity of sponge-associated microorganisms and their contribution to biogeochemical cycling has gradually increased. Functional groups involved in carbon and nitrogen metabolism are well documented, whereas knowledge about microorganisms involved in the sulfur cycle is still limited. Both sulfate reduction and sulfide oxidation has been detected in the cold water sponge Geodia barretti from Korsfjord in Norway, and with specimens from this site, the present study aims to identify extant versus active sponge-associated microbiota with focus on sulfur metabolism. Comparative analysis of small subunit ribosomal RNA (16S rRNA) gene (DNA) and transcript (complementary DNA (cDNA)) libraries revealed profound differences. The transcript library was predominated by Chloroflexi despite their low abundance in the gene library. An opposite result was found for Acidobacteria. Proteobacteria were detected in both libraries with representatives of the Alpha- and Gammaproteobacteria related to clades with presumably thiotrophic bacteria from sponges and other marine invertebrates. Sequences that clustered with sponge-associated Deltaproteobacteria were remotely related to cultivated sulfate-reducing bacteria. The microbes involved in sulfur cycling were identified by the functional gene aprA (adenosine-5'-phosphosulfate reductase) and its transcript. Of the aprA sequences (DNA and cDNA), 87 % affiliated with sulfur-oxidizing bacteria. They clustered with Alphaproteobacteria and with clades of deep-branching Gammaproteobacteria. The remaining sequences clustered with sulfate-reducing Archaea of the phylum Euryarchaeota. These results indicate an active role of yet uncharacterized Bacteria and Archaea in the sponge's sulfur cycle.

Prokaryotic Community Diversity Along an Increasing Salt Gradient in a Soda Ash Concentration Pond.

The effect of salinity on prokaryotic community diversity in Abijata-Shalla Soda Ash Concentration Pond system was investigated by using high-throughput 16S rRNA gene 454 pyrosequencing. Surface water and brine samples from five sites spanning a salinity range of 3.4 % (Lake Abijata) to 32 % (SP230F, crystallizer pond) were analyzed. Overall, 33 prokaryotic phyla were detected, and the dominant prokaryotic phyla accounted for more than 95 % of the reads consisting of Planctomycetes, Bacteroidetes, candidate division TM7, Deinococcus-Thermus, Firmicutes, Actinobacteria, Proteobacteria, and Euryarchaeota. Diversity indices indicated that operational taxonomic unit (OTU) richness decreases drastically with increasing salinity in the pond system. A total of 471 OTUs were found at 3.4 % salinity whereas 49 OTUs were detected in pond SP211 (25 % salinity), and only 19 OTUs in the crystallization pond at 32 % salinity (SP230F). Along the salinity gradient, archaeal community gradually replaced bacterial community. Thus, archaeal community accounted for 0.4 % in Lake Abijata while 99.0 % in pond SP230F. This study demonstrates that salinity appears to be the key environmental parameter in structuring the prokaryotic communities of haloalkaline environments. Further, it confirmed that the prokaryotic diversity in Lake Abijata is high and it harbors taxa with low or no phylogenetic similarities to existing prokaryotic taxa and thus represents novel microorganisms.

Correlating microbial community profiles with geochemical data in highly stratified sediments from the Arctic Mid-Ocean Ridge.

Microbial communities and their associated metabolic activity in marine sediments have a profound impact on global biogeochemical cycles. Their composition and structure are attributed to geochemical and physical factors, but finding direct correlations has remained a challenge. Here we show a significant statistical relationship between variation in geochemical composition and prokaryotic community structure within deep-sea sediments. We obtained comprehensive geochemical data from two gravity cores near the hydrothermal vent field Loki's Castle at the Arctic Mid-Ocean Ridge, in the Norwegian-Greenland Sea. Geochemical properties in the rift valley sediments exhibited strong centimeter-scale stratigraphic variability. Microbial populations were profiled by pyrosequencing from 15 sediment horizons (59,364 16S rRNA gene tags), quantitatively assessed by qPCR, and phylogenetically analyzed. Although the same taxa were generally present in all samples, their relative abundances varied substantially among horizons and fluctuated between Bacteria- and Archaea-dominated communities. By independently summarizing covariance structures of the relative abundance data and geochemical data, using principal components analysis, we found a significant correlation between changes in geochemical composition and changes in community structure. Differences in organic carbon and mineralogy shaped the relative abundance of microbial taxa. We used correlations to build hypotheses about energy metabolisms, particularly of the Deep Sea Archaeal Group, specific Deltaproteobacteria, and sediment lineages of potentially anaerobic Marine Group I Archaea. We demonstrate that total prokaryotic community structure can be directly correlated to geochemistry within these sediments, thus enhancing our understanding of biogeochemical cycling and our ability to predict metabolisms of uncultured microbes in deep-sea sediments.

Microbial community structure and functioning in marine sediments associated with diffuse hydrothermal venting assessed by integrated meta-omics.

Deep-sea hydrothermal vents are unique environments on Earth, as they host chemosynthetic ecosystems fuelled by geochemical energy with chemolithoautotrophic microorganisms at the basis of the food webs. Whereas discrete high-temperature venting systems have been studied extensively, the microbiotas associated with low-temperature diffuse venting are not well understood. We analysed the structure and functioning of microbial communities in two diffuse venting sediments from the Jan Mayen vent fields in the Norwegian-Greenland Sea, applying an integrated 'omics' approach combining metatranscriptomics, metaproteomics and metagenomics. Polymerase chain reaction-independent three-domain community profiling showed that the two sediments hosted highly similar communities dominated by Epsilonproteobacteria, Deltaproteobacteria and Gammaproteobacteria, besides ciliates, nematodes and various archaeal taxa. Active metabolic pathways were identified through transcripts and peptides, with genes of sulphur and methane oxidation, and carbon fixation pathways highly expressed, in addition to genes of aerobic and anaerobic (nitrate and sulphate) respiratory chains. High expression of chemotaxis and flagella genes reflected a lifestyle in a dynamic habitat rich in physico-chemical gradients. The major metabolic pathways could be assigned to distinct taxonomic groups, thus enabling hypotheses about the function of the different prokaryotic and eukaryotic taxa. This study advances our understanding of the functioning of microbial communities in diffuse hydrothermal venting sediments.

Multidrug-resistant Enterococcus faecium strains enter the Norwegian marine environment through treated sewage.

This study aimed to understand the antibiotic resistance prevalence among Enterococcus spp. from raw and treated sewage in Bergen city, Norway. In total, 517 Enterococcus spp. isolates were obtained from raw and treated sewage from five sewage treatment plants (STPs) over three sampling occasions, with Enterococcus faecium as the most prevalent (n = 492) species. E. faecium strains (n = 307) obtained from the influent samples, showed the highest resistance against quinupristin/dalfopristin (67.8%). We observed reduced susceptibility to erythromycin (30.6%) and tetracycline (6.2%) in these strains. E. faecium strains (n = 185) obtained from the effluent samples showed highest resistance against quinupristin/dalfopristin (68.1%) and reduced susceptibility to erythromycin (24.9%) and tetracycline (8.6%). We did not detect resistance against last-resort antibiotics, such as linezolid, vancomycin, and tigecycline in any of the strains. Multidrug-resistant (MDR) E. faecium strains were detected in both influent (2.3%) and effluent (2.2%) samples. Whole genome sequencing of the Enterococcus spp. strains (n = 25) showed the presence of several antibiotic resistance genes, conferring resistance against aminoglycosides, tetracyclines, and macrolides, as well as several virulence genes and plasmid replicons. Two sequenced MDR strains from the effluents belonged to the hospital-associated clonal complex 17 and carried multiple virulence genes. Our study demonstrates that clinically relevant MDR Enterococcus spp. strains are entering the marine environment through treated sewage.

Quantifying temporal change in biodiversity: challenges and opportunities.

Growing concern about biodiversity loss underscores the need to quantify and understand temporal change. Here, we review the opportunities presented by biodiversity time series, and address three related issues: (i) recognizing the characteristics of temporal data; (ii) selecting appropriate statistical procedures for analysing temporal data; and (iii) inferring and forecasting biodiversity change. With regard to the first issue, we draw attention to defining characteristics of biodiversity time series--lack of physical boundaries, uni-dimensionality, autocorrelation and directionality--that inform the choice of analytic methods. Second, we explore methods of quantifying change in biodiversity at different timescales, noting that autocorrelation can be viewed as a feature that sheds light on the underlying structure of temporal change. Finally, we address the transition from inferring to forecasting biodiversity change, highlighting potential pitfalls associated with phase-shifts and novel conditions.

Diversity of Planctomycetes in iron-hydroxide deposits from the Arctic Mid Ocean Ridge (AMOR) and description of Bythopirellula goksoyri gen. nov., sp. nov., a novel Planctomycete from deep sea iron-hydroxide deposits.

Planctomycetes form a deep branching and distinct phylum of the domain Bacteria, and represent a fascinating group due to their unusual features such as intracellular compartmentalization and lack of peptidoglycan in their cell walls. The phylum Planctomycetes was described already in 1924, but still the diversity of this phylum represents an enigma and unexploited resource. In this study the diversity of the phylum Planctomycetes in low temperature iron-hydroxide deposits at the Mohns Ridge, a part of the Arctic Mid Ocean Ridge (AMOR), was characterised by descriptive analysis of 16S rRNA gene sequences in combination with isolation of planctomycetes strains. The 16S rRNA gene sequences were affiliated with three order within the phylum Planctomycetes namely the (i)Planctomycetales, (ii) "Candidatus Brocadiales" and (iii) Phycisphaerae in addition to sequences affiliating to hitherto unknown Planctomycetes. The majority of the sequences were affiliated with the CCM11a group (Phycisphaerae), and with the Pir4 group (Planctomycetaceae). Two strains from the order Planctomycetales were isolated. One strain (Plm2) showed high similarity to the previously isolated Planctomyces maris (99 % 16S rRNA sequence identity). The other strain (Pr1d) belonged to the Pir4 group, and showed highest identity with Rhodopirellula baltica (86 %), Blastopirellula marina (86 %) and Pirellula staleyi (85 %). Based on its physiological and biochemical properties, strain Pr1d(T) is considered to represent a new genus of the order Planctomycetales. We propose to classify the novel planctomycete in a new genus and species, Bythoypirellula goksoyri gen. nov., sp. nov., the type strain being Pr1d(T).

The mystery of the ice cold rose-Microbiome of an Arctic winter frost flower.

Under very cold conditions, delicate ice-crystal structures called frost flowers emerge on the surface of newly formed sea ice. These understudied, ephemeral structures include saline brine, organic material, inorganic nutrients, and bacterial and archaeal communities in their brine channels. Hitherto, only a few frost flowers have been studied during spring and these have been reported to be dominated by Rhizobia or members of the SAR11 clade. Here we report on the microbiome of frost flowers sampled during the winter and polar night in the Barents Sea. There was a distinct difference in community profile between the extracted DNA and RNA, but both were dominated by members of the SAR11 clade (78% relative abundance and 41.5% relative activity). The data further suggested the abundance and activity of Cand. Nitrosopumilus, Nitrospinia, and Nitrosomonas. Combined with the inference of marker genes based on the 16S rRNA gene data, this indicates that sulfur and nitrogen cycling are likely the major metabolism in these ephemeral structures.

Sewage-based surveillance shows presence of Klebsiella pneumoniae resistant against last resort antibiotics in the population in Bergen, Norway.

The aim of this study was to understand the prevalence of antibiotic resistance in Klebsiella pneumoniae present in the population in Bergen city, Norway using city-scale sewage-based surveillance, as well as the potential spread of K. pneumoniae into the marine environment through treated sewage. From a total of 30 sewage samples collected from five different sewage treatment plants (STPs), 563 presumptive K. pneumoniae isolates were obtained on Simmons Citrate Agar with myo-Inositol (SCAI) plates, and 44 presumptive K. pneumoniae isolates on SCAI plates with cefotaxime. Colistin resistance was observed in 35 isolates, while cefotaxime resistance and tigecycline resistance was observed in only five isolates each, out of 563 presumptive K. pneumoniae isolates. All 44 isolates obtained on cefotaxime-containing plates were multidrug-resistant, with 25% (n = 11) showing resistance against tigecycline. Clinically important acquired antibiotic resistance genes (ARGs), like blaCTX-M-14, blaCTX-M-15, qnrS1, aac(3)-IIe, tet(A), and sul1, were detected in several sequenced Klebsiella spp. isolates (n = 53). All sequenced colistin-resistant isolates (n = 13) had a mutation in the mgrB gene with nucleotide substitution at position C88T creating a premature stop codon. All sequenced tigecycline-resistant isolates (n = 4) harbored a Tet(A) variant with 22 amino acid (aa) substitutions compared to the reference protein. The sequenced K. pneumoniae isolates (n = 44) belonged to 22 different sequence types (STs) with ST730 (29.5%) as most prevalent, followed by pathogenic ST307 (11.4%). Virulence factors, including aerobactin (iutA), enterobactin (entABCDEFS and fepABCDG), salmochelin (iro), and yersiniabactin (ybt) were detected in several sequenced K. pneumoniae isolates, suggesting pathogenicity potential. Heavy metal resistance genes were common in sequenced K. pneumoniae isolates (n = 44) with silver (silABCEFPRS) and copper (pcoABDRS) resistance genes present in 79.5% of the isolates. Sewage-based surveillance can be a useful tool for understanding antibiotic resistance in pathogens present within a population and to provide up-to date information on the current resistance situation. Our study presents a framework for population-based surveillance of resistance in K. pneumoniae.

Seasonality of the bacterial and archaeal community composition of the Northern Barents Sea.

The Barents Sea is a transition zone between the Atlantic and the Arctic Ocean. The ecosystem in this region is highly variable, and a seasonal baseline of biological factors is needed to monitor the effects of global warming. In this study, we report the results from the investigations of the bacterial and archaeal community in late winter, spring, summer, and early winter along a transect through the northern Barents Sea into the Arctic Ocean east of Svalbard using 16S rRNA metabarcoding. Winter samples were dominated by members of the SAR11 clade and a community of nitrifiers, namely Cand. Nitrosopumilus and LS-NOB (Nitrospinia), suggest a prevalence of chemoautotrophic metabolisms. During spring and summer, members of the Gammaproteobacteria (mainly members of the SAR92 and OM60(NOR5) clades, Nitrincolaceae) and Bacteroidia (mainly Polaribacter, Formosa, and members of the NS9 marine group), which followed a succession based on their utilization of different phytoplankton-derived carbon sources, prevailed. Our results indicate that Arctic marine bacterial and archaeal communities switch from carbon cycling in spring and summer to nitrogen cycling in winter and provide a seasonal baseline to study the changes in these processes in response to the effects of climate change.

Diversity and metabolic energy in bacteria.

Why are some groups of bacteria more diverse than others? We hypothesize that the metabolic energy available to a bacterial functional group (a biogeochemical group or 'guild') has a role in such a group's taxonomic diversity. We tested this hypothesis by looking at the metacommunity diversity of functional groups in multiple biomes. We observed a positive correlation between estimates of a functional group's diversity and their metabolic energy yield. Moreover, the slope of that relationship was similar in all biomes. These findings could imply the existence of a universal mechanism controlling the diversity of all functional groups in all biomes in the same way. We consider a variety of possible explanations from the classical (environmental variation) to the 'non-Darwinian' (a drift barrier effect). Unfortunately, these explanations are not mutually exclusive, and a deeper understanding of the ultimate cause(s) of bacterial diversity will require us to determine if and how the key parameters in population genetics (effective population size, mutation rate, and selective gradients) vary between functional groups and with environmental conditions: this is a difficult task.

A Winter-to-Summer Transition of Bacterial and Archaeal Communities in Arctic Sea Ice.

The Arctic is warming 2-3 times faster than the global average, leading to a decrease in Arctic sea ice extent, thickness, and associated changes in sea ice structure. These changes impact sea ice habitat properties and the ice-associated ecosystems. Sea-ice algal blooms provide various algal-derived carbon sources for the bacterial and archaeal communities within the sea ice. Here, we detail the transition of these communities from winter through spring to early summer during the Norwegian young sea ICE (N-ICE2015) expedition. The winter community was dominated by the archaeon Candidatus Nitrosopumilus and bacteria belonging to the Gammaproteobacteria (Colwellia, Kangiellaceae, and Nitrinocolaceae), indicating that nitrogen-based metabolisms, particularly ammonia oxidation to nitrite by Cand. Nitrosopumilus was prevalent. At the onset of the vernal sea-ice algae bloom, the community shifted to the dominance of Gammaproteobacteria (Kangiellaceae, Nitrinocolaceae) and Bacteroidia (Polaribacter), while Cand. Nitrosopumilus almost disappeared. The bioinformatically predicted carbohydrate-active enzymes increased during spring and summer, indicating that sea-ice algae-derived carbon sources are a strong driver of bacterial and archaeal community succession in Arctic sea ice during the change of seasons. This implies a succession from a nitrogen metabolism-based winter community to an algal-derived carbon metabolism-based spring/ summer community.

Evolution of temperature optimum in Thermotogaceae and the prediction of trait values of uncultured organisms.

Quantitative characterization of the mode and rate of phenotypic evolution is rarely applied to prokaryotes. Here, we present an analysis of temperature optimum (T (opt)) evolution in the thermophilic family Thermotogaceae, which has a large number of cultured representatives. We use log-rate-interval analysis to show that T (opt) evolution in Thermotogaceae is consistent with a Brownian motion (BM) evolutionary model. The properties of the BM model are used to a establish confidence intervals on the unknown phenotypic trait value of an uncultured organism, given its distance to a close relative with known trait value. Cross-validation by bootstrapping indicates that the predictions are robust.

A Novel Moderately Thermophilic Facultative Methylotroph within the Class Alphaproteobacteria.

Methylotrophic bacteria (non-methanotrophic methanol oxidizers) consuming reduced carbon compounds containing no carbon-carbon bonds as their sole carbon and energy source have been found in a great variety of environments. Here, we report a unique moderately thermophilic methanol-oxidising bacterium (strain LS7-MT) that grows optimally at 55 °C (with a growth range spanning 30 to 60 °C). The pure isolate was recovered from a methane-utilizing mixed culture enrichment from an alkaline thermal spring in the Ethiopia Rift Valley, and utilized methanol, methylamine, glucose and a variety of multi-carbon compounds. Phylogenetic analysis of the 16S rRNA gene sequences demonstrated that strain LS7-MT represented a new facultatively methylotrophic bacterium within the order Hyphomicrobiales of the class Alphaproteobacteria. This new strain showed 94 to 96% 16S rRNA gene identity to the two methylotroph genera, Methyloceanibacter and Methyloligella. Analysis of the draft genome of strain LS7-MT revealed genes for methanol dehydrogenase, essential for methanol oxidation. Functional and comparative genomics of this new isolate revealed genomic and physiological divergence from extant methylotrophs. Strain LS7-MT contained a complete mxaF gene cluster and xoxF1 encoding the lanthanide-dependent methanol dehydrogenase (XoxF). This is the first report of methanol oxidation at 55 °C by a moderately thermophilic bacterium within the class Alphaproteobacteria. These findings expand our knowledge of methylotrophy by the phylum Proteobacteria in thermal ecosystems and their contribution to global carbon and nitrogen cycles.