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With advances in DNA sequencing and miniaturized molecular biology workflows, rapid and affordable sequencing of single-cell genomes has become a reality. Compared to 16S rRNA gene surveys and shotgun metagenomics, large-scale application of single-cell genomics to whole microbial communities provides an integrated snapshot of community composition and function, directly links mobile elements to their hosts, and enables analysis of population heterogeneity of the dominant community members. To that end, we sequenced nearly 500 single-cell genomes from a low diversity hot spring sediment sample from Dewar Creek, British Columbia, and compared this approach to 16S rRNA gene amplicon and shotgun metagenomics applied to the same sample. We found that the broad taxonomic profiles were similar across the three sequencing approaches, though several lineages were missing from the 16S rRNA gene amplicon dataset, likely the result of primer mismatches. At the functional level, we detected a large array of mobile genetic elements present in the single-cell genomes but absent from the corresponding same species metagenome-assembled genomes. Moreover, we performed a single-cell population genomic analysis of the three most abundant community members, revealing differences in population structure based on mutation and recombination profiles. While the average pairwise nucleotide identities were similar across the dominant species-level lineages, we observed differences in the extent of recombination between these dominant populations. Most intriguingly, the creek's Hydrogenobacter sp. population appeared to be so recombinogenic that it more closely resembled a sexual species than a clonally evolving microbe. Together, this work demonstrates that a randomized single-cell approach can be useful for the exploration of previously uncultivated microbes from community composition to population structure.

The recent leveraging of genome-resolved metagenomics has generated an enormous number of genomes from novel uncultured microbial lineages yet left many clades undescribed. Here, we present a global analysis of genomes belonging to Binatota (UBP10), a globally distributed, yet-uncharacterized bacterial phylum. All orders in Binatota encoded the capacity for aerobic methylotrophy using methanol, methylamine, sulfomethanes, and chloromethanes as the substrates. Methylotrophy in Binatota was characterized by order-specific substrate degradation preferences, as well as extensive metabolic versatility, i.e., the utilization of diverse sets of genes, pathways, and combinations to achieve a specific metabolic goal. The genomes also encoded multiple alkane hydroxylases and monooxygenases, potentially enabling growth on a wide range of alkanes and fatty acids. Pigmentation is inferred from a complete pathway for carotenoids (lycopene, β- and γ-carotenes, xanthins, chlorobactenes, and spheroidenes) production. Further, the majority of genes involved in bacteriochlorophyll a, c, and d biosynthesis were identified, although absence of key genes and failure to identify a photosynthetic reaction center preclude proposing phototrophic capacities. Analysis of 16S rRNA databases showed the preferences of Binatota to terrestrial and freshwater ecosystems, hydrocarbon-rich habitats, and sponges, supporting their potential role in mitigating methanol and methane emissions, breakdown of alkanes, and their association with sponges. Our results expand the lists of methylotrophic, aerobic alkane-degrading, and pigment-producing lineages. We also highlight the consistent encountering of incomplete biosynthetic pathways in microbial genomes, a phenomenon necessitating careful assessment when assigning putative functions based on a set-threshold of pathway completion.IMPORTANCE A wide range of microbial lineages remain uncultured, yet little is known regarding their metabolic capacities, physiological preferences, and ecological roles in various ecosystems. We conducted a thorough comparative genomic analysis of 108 genomes belonging to the Binatota (UBP10), a globally distributed, yet-uncharacterized bacterial phylum. We present evidence that members of the order Binatota specialize in methylotrophy and identify an extensive repertoire of genes and pathways mediating the oxidation of multiple one-carbon (C1) compounds in Binatota genomes. The occurrence of multiple alkane hydroxylases and monooxygenases in these genomes was also identified, potentially enabling growth on a wide range of alkanes and fatty acids. Pigmentation is inferred from a complete pathway for carotenoids production. We also report on the presence of incomplete chlorophyll biosynthetic pathways in all genomes and propose several evolutionary-grounded scenarios that could explain such a pattern. Assessment of the ecological distribution patterns of the Binatota indicates preference of its members to terrestrial and freshwater ecosystems characterized by high methane and methanol emissions, as well as multiple hydrocarbon-rich habitats and marine sponges.

Members of the bacterial candidate phylum WPS-2 (or Eremiobacterota) are abundant in several dry, bare soil environments. In a bare soil deposited by an extinct iron-sulfur spring, we found that WPS-2 comprised up to 24% of the bacterial community and up to 108 cells per g of soil based on 16S rRNA gene sequencing and quantification. A single genus-level cluster (Ca. Rubrimentiphilum) predominated in bare soils but was less abundant in adjacent forest. Nearly complete genomes of Ca. Rubrimentiphilum were recovered as single amplified genomes (SAGs) and metagenome-assembled genomes (MAGs). Surprisingly, given the abundance of WPS-2 in bare soils, the genomes did not indicate any capacity for autotrophy, phototrophy, or trace gas metabolism. Instead, they suggest a predominantly aerobic organoheterotrophic lifestyle, perhaps based on scavenging amino acids, nucleotides, and complex oligopeptides, along with lithotrophic capacity on thiosulfate. Network analyses of the entire community showed that some species of Chloroflexi, Actinobacteria, and candidate phylum AD3 (or Dormibacterota) co-occurred with Ca. Rubrimentiphilum and may represent ecological or metabolic partners. We propose that Ca. Rubrimentiphilum act as efficient heterotrophic scavengers. Combined with previous studies, these data suggest that the phylum WPS-2 includes bacteria with diverse metabolic capabilities.

Copper-containing membrane monooxygenases (CuMMOs) are encoded by xmoCAB(D) gene clusters and catalyze the oxidation of methane, ammonia, or some short-chain alkanes and alkenes. In a metagenome constructed from an oilsands tailings pond we detected an xmoCABD gene cluster with

Recent discoveries suggest that the candidate superphyla Patescibacteria and DPANN constitute a large fraction of the phylogenetic diversity of Bacteria and Archaea. Their small genomes and limited coding potential have been hypothesized to be ancestral adaptations to obligate symbiotic lifestyles. To test this hypothesis, we performed cell-cell association, genomic, and phylogenetic analyses on 4,829 individual cells of Bacteria and Archaea from 46 globally distributed surface and subsurface field samples. This confirmed the ubiquity and abundance of Patescibacteria and DPANN in subsurface environments, the small size of their genomes and cells, and the divergence of their gene content from other Bacteria and Archaea. Our analyses suggest that most Patescibacteria and DPANN in the studied subsurface environments do not form specific physical associations with other microorganisms. These data also suggest that their unusual genomic features and prevalent auxotrophies may be a result of ancestral, minimal cellular energy transduction mechanisms that lack respiration, thus relying solely on fermentation for energy conservation.

The genomes of the aerobic methanotrophs "Methyloterricola oryzae" strain 73aT and Methylomagnum ishizawai strain 175 were sequenced. Both strains were isolated from rice plants. Methyloterricola oryzae strain 73aT represents the first isolate of rice paddy cluster I, and strain 175 is the second representative of the recently described genus Methylomagnum.

The genome sequences of Methylobacter marinus A45, Methylobacter sp. strain BBA5.1, and Methylomarinum vadi IT-4 were obtained. These aerobic methanotrophs are typical members of coastal and hydrothermal vent marine ecosystems.

Analysis of the increasing wealth of metagenomic data collected from diverse environments can lead to the discovery of novel branches on the tree of life. Here we analyse 5.2 Tb of metagenomic data collected globally to discover a novel bacterial phylum ('Candidatus Kryptonia') found exclusively in high-temperature pH-neutral geothermal springs. This lineage had remained hidden as a taxonomic 'blind spot' because of mismatches in the primers commonly used for ribosomal gene surveys. Genome reconstruction from metagenomic data combined with single-cell genomics results in several high-quality genomes representing four genera from the new phylum. Metabolic reconstruction indicates a heterotrophic lifestyle with conspicuous nutritional deficiencies, suggesting the need for metabolic complementarity with other microbes. Co-occurrence patterns identifies a number of putative partners, including an uncultured Armatimonadetes lineage. The discovery of Kryptonia within previously studied geothermal springs underscores the importance of globally sampled metagenomic data in detection of microbial novelty, and highlights the extraordinary diversity of microbial life still awaiting discovery.

Genome sequences of Methylobacter luteus, Methylobacter whittenburyi, Methylosarcina fibrata, Methylomicrobium agile, and Methylovulum miyakonense were generated. The strains represent aerobic methanotrophs typically isolated from various terrestrial ecosystems.

Methylohalobius crimeensis strain 10Ki is a moderately halophilic aerobic methanotroph isolated from a hypersaline lake in the Crimean Peninsula, Ukraine. This organism has the highest salt tolerance of any cultured methanotroph. Here, we present a draft genome sequence of this bacterium.

Methyloferula stellata AR4 is an aerobic acidophilic methanotroph, which, in contrast to most known methanotrophs but similar to Methylocella spp., possesses only a soluble methane monooxygenase. However, it differs from Methylocella spp. by its inability to grow on multicarbon substrates. Here, we report the draft genome sequence of this bacterium.

Using hybrid packing materials in biofiltration systems takes advantage of both the inorganic and organic properties offered by the medium including structural stability and a source of available nutrients, respectively. In this study, hybrid mixtures of compost with either lava rock or biochar in four different mixture ratios were compared against 100% compost in a methane biofilter with active aeration at two ports along the height of the biofilter. Biochar outperformed lava rock as a packing material by providing the added benefit of participating in sorption reactions with CH 4 . This study provides evidence that a 7:1 volumetric mixture of biochar and compost can successfully remove up to 877 g CH 4 /m 3 ·d with empty-bed residence times of 82.8 min. Low-affinity methanotrophs were responsible for the CH 4 removal in these systems (K M(app) ranging from 5.7 to 42.7 µM CH 4 ). Sequencing of 16S rRNA gene amplicons indicated that Gammaproteobacteria methanotrophs, especially members of the genus Methylobacter , were responsible for most of the CH 4 removal. However, as the compost medium was replaced with more inert medium, there was a decline in CH 4 removal efficiency coinciding with an increased dominance of Alphaproteobacteria methanotrophs like Methylocystis and Methylocella . As a biologically-active material, compost served as the sole source of nutrients and inoculum for the biofilters which greatly simplified the operation of the system. Higher elimination capacities may be possible with higher compost content such as a 1:1 ratio of either biochar or lava rock, while maintaining the empty-bed residence time at 82.8 min. [ABSTRACT FROM AUTHOR]

Chthonomonas calidirosea T49T is a low-abundance, carbohydrate-scavenging, and thermophilic soil bacterium with a seemingly disorganized genome. We hypothesized that the C. calidirosea genome would be highly responsive to local selection pressure, resulting in the divergence of its genomic content, genome organization, and carbohydrate utilization phenotype across environments. We tested this hypothesis by sequencing the genomes of four C. calidirosea isolates obtained from four separate geothermal fields in the Taupō Volcanic Zone, New Zealand. For each isolation site, we measured physicochemical attributes and defined the associated microbial community by 16S rRNA gene sequencing. Despite their ecological and geographical isolation, the genome sequences showed low divergence (maximum, 1.17%). Isolate-specific variations included single-nucleotide polymorphisms (SNPs), restriction-modification systems, and mobile elements but few major deletions and no major rearrangements. The 50-fold variation in C. calidirosea relative abundance among the four sites correlated with site environmental characteristics but not with differences in genomic content. Conversely, the carbohydrate utilization profiles of the C. calidirosea isolates corresponded to the inferred isolate phylogenies, which only partially paralleled the geographical relationships among the sample sites. Genomic sequence conservation does not entirely parallel geographic distance, suggesting that stochastic dispersal and localized extinction, which allow for rapid population homogenization with little restriction by geographical barriers, are possible mechanisms of C. calidirosea distribution. This dispersal and extinction mechanism is likely not limited to C. calidirosea but may shape the populations and genomes of many other low-abundance free-living taxa. IMPORTANCE: This study compares the genomic sequence variations and metabolisms of four strains of Chthonomonas calidirosea, a rare thermophilic bacterium from the phylum Armatimonadetes. It additionally compares the microbial communities and chemistry of each of the geographically distinct sites from which the four C. calidirosea strains were isolated. C. calidirosea was previously reported to possess a highly disorganized genome, but it was unclear whether this reflected rapid evolution. Here, we show that each isolation site has a distinct chemistry and microbial community, but despite this, the C. calidirosea genome is highly conserved across all isolation sites. Furthermore, genomic sequence differences only partially paralleled geographic distance, suggesting that C. calidirosea genotypes are not primarily determined by adaptive evolution. Instead, the presence of C. calidirosea may be driven by stochastic dispersal and localized extinction. This ecological mechanism may apply to many other low-abundance taxa.

Strain K22T is the type species of the recently- described genus Pyrinomonas, in subdivision 4 of the phylum Acidobacteria (Int J Syst Evol Micr. 2014; 64(1):220–7). It was isolated from geothermally-heated soil from Mt. Ngauruhoe, New Zealand, using low-nutrient medium. P. methylaliphatogenes K22T has a chemoheterotrophic metabolism; it can hydrolyze a limited range of simple carbohydrates and polypeptides. Its cell membrane is dominated by iso-branching fatty acids, and up to 40 % of its lipid content is membrane-spanning and ether lipids. It is obligately aerobic, thermophilic, moderately acidophilic, and non-spore-forming. The 3,788,560 bp genome of P. methylaliphatogenes K22T has a G + C content of 59.36 % and contains 3,189 protein-encoding and 55 non-coding RNA genes. Genomic analysis was consistent with nutritional requirements; in particular, the identified transporter classes reflect the oligotrophic nature of this strain. Electronic supplementary material The online version of this article (doi:10.1186/s40793-015-0099-5) contains supplementary material, which is available to authorized users.

When cells of a type II methanotrophic bacterium (Methylocystis strain LR1) were starved of methane, both the K(m(app)) and the V(max(app)) for methane decreased. The specific affinity (a(o)(s)) remained nearly constant. Therefore, the decreased K(m(app)) in starved cells was probably not an adjustment to better utilize low-methane concentrations.

Methanotrophic bacteria in an organic soil were enriched on gaseous mixing ratios of <275 parts per million of volume (ppmv) of methane (CH4). After 4 years of growth and periodic dilution (>10(20) times the initial soil inoculum), a mixed culture was obtained which displayed an apparent half-saturation constant [Km(app)] for CH4 of 56 to 186 nM (40 to 132 ppmv). This value was the same as that measured in the soil itself and about 1 order of magnitude lower than reported values for pure cultures of methane oxidizers. However, the Km(app) increased when the culture was transferred to higher mixing ratios of CH4 (1,000 ppmv, or 1%). Denaturing gradient gel electrophoresis of the enrichment grown on <275 ppmv of CH4 revealed a single gene product of pmoA, which codes for a subunit of particulate methane monooxygenase. This suggested that only one methanotroph species was present. This organism was isolated from a sample of the enrichment culture grown on 1% CH4 and phylogenetically positioned based on its 16S rRNA, pmoA, and mxaF gene sequences as a type II strain of the Methylocystis/Methylosinus group. A coculture of this strain with a Variovorax sp., when grown on <275 ppmv of CH4, had a Km(app) (129 to 188 nM) similar to that of the initial enrichment culture. The data suggest that the affinity of methanotrophic bacteria for CH4 varies with growth conditions and that the oxidation of atmospheric CH4 observed in this soil is carried out by type II methanotrophic bacteria which are similar to characterized species.