Cultivating Uncultured Bacteria from Northern Wetlands: Knowledge Gained and Remaining Gaps

Northern wetlands play a key role in the global carbon budget, particularly in the budgets of the greenhouse gas methane. These ecosystems also determine the hydrology of northern rivers and represent one of the largest reservoirs of fresh water in the Northern Hemisphere. Sphagnum-dominated peat bogs and fens are the most extensive types of northern wetlands. In comparison to many other terrestrial ecosystems, the bacterial diversity in Sphagnum-dominated wetlands remains largely unexplored. As demonstrated by cultivation-independent studies, a large proportion of the indigenous microbial communities in these acidic, cold, nutrient-poor, and water-saturated environments is composed of as-yet-uncultivated bacteria with unknown physiologies. Most of them are slow-growing, oligotrophic microorganisms that are difficult to isolate and to manipulate in the laboratory. Yet, significant breakthroughs in cultivation of these elusive organisms have been made during the last decade. This article describes the major prerequisites for successful cultivation of peat-inhabiting microbes, gives an overview of the currently captured bacterial diversity from northern wetlands and discusses the unique characteristics of the newly discovered organisms

Facultative methanotrophy: false leads, true results, and suggestions for future research

Methanotrophs are a group of phylogenetically diverse microorganisms characterized by their ability to utilize methane as their sole source of carbon and energy. Early studies suggested that growth on methane could be stimulated with the addition of some small organic acids, but initial efforts to find facultative methanotrophs, i.e., methanotrophs able to utilize compounds with carbon–carbon bonds as sole growth substrates were inconclusive. Recently, however, facultative methanotrophs in the genera Methylocella , Methylocapsa , and Methylocystis have been reported that can grow on acetate, as well as on larger organic acids or ethanol for some species. All identified facultative methanotrophs group within the Alphaproteobacteria and utilize the serine cycle for carbon assimilation from formaldehyde. It is possible that facultative methanotrophs are able to convert acetate into intermediates of the serine cycle (e.g. malate and glyoxylate), because a variety of acetate assimilation pathways convert acetate into these compounds (e.g. the glyoxylate shunt of the tricarboxylic acid cycle, the ethylmalonyl‐CoA pathway, the citramalate cycle, and the methylaspartate cycle). In this review, we summarize the history of facultative methanotrophy, describe scenarios for the basis of facultative methanotrophy, and pose several topics for future research in this area.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/86841/1/fml2315.pd

Abundance, Diversity, and Depth Distribution of Planctomycetes in Acidic Northern Wetlands

Members of the bacterial phylum Planctomycetes inhabit various aquatic and terrestrial environments. In this study, fluorescence in situ hybridization (FISH) was applied to assess the abundance and depth distribution of these bacteria in nine different acidic wetlands of Northern Russia. Planctomycetes were most abundant in the oxic part of the wetland profiles. The respective cell numbers were in the range 1.1–6.7 × 107 cells g−1 of wet peat, comprising 2–14% of total bacterial cells, and displaying linear correlation to the peat water pH. Most peatland sites showed a sharp decline of planctomycete abundance with depth, while in two particular sites this decline was followed by a second population maximum in an anoxic part of the bog profile. Oxic peat layers were dominated by representatives of the Isosphaera–Singulisphaera group, while anoxic peat was inhabited mostly by Zavarzinella- and Pirellula-like planctomycetes. Phylogenetically related bacteria of the candidate division OP3 were detected in both oxic and anoxic peat layers with cell densities of 0.6–4.6 × 106 cells g−1 of wet peat

Draft genome sequence of Methyloferula stellata AR4, an obligate methanotroph possessing only a soluble methane monooxygenase

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

Abundant Trimethylornithine Lipids and Specific Gene Sequences Are Indicative of Planctomycete Importance at the Oxic/Anoxic Interface in <i>Sphagnum</i>-Dominated Northern Wetlands

Northern wetlands make up a substantial terrestrial carbon sink and are often dominated by decay-resistant Sphagnum mosses.Recent studies have shown that planctomycetes appear to be involved in degradation of Sphagnum-derived debris. Novel trimethylornithine(TMO) lipids have recently been characterized as abundant lipids in various Sphagnum wetland planctomyceteisolates, but their occurrence in the environment has not yet been confirmed. We applied a combined intact polar lipid (IPL) andmolecular analysis of peat cores collected from two northern wetlands (Saxnäs Mosse [Sweden] and Obukhovskoye [Russia]) inorder to investigate the preferred niche and abundance of TMO-producing planctomycetes. TMOs were present throughout theprofiles of Sphagnum bogs, but their concentration peaked at the oxic/anoxic interface, which coincided with a maximum abundanceof planctomycete-specific 16S rRNA gene sequences. The sequences detected at the oxic/anoxic interface were affiliatedwith the Isosphaera group, while sequences present in the anoxic peat layers were related to an uncultured planctomycete group.Pyrosequencing-based analysis identified Planctomycetes as the major bacterial group at the oxic/anoxic interface at the Obukhovskoyepeat (54% of total 16S rRNA gene sequence reads), followed by Acidobacteria (19% reads), while in the Saxnäs Mossepeat, Acidobacteria were dominant (46%), and Planctomycetes contributed to 6% of the total reads. The detection of abundantTMO lipids in planctomycetes isolated from peat bogs and the lack of TMO production by cultures of acidobacteria suggest thatplanctomycetes are the producers of TMOs in peat bogs. The higher accumulation of TMOs at the oxic/anoxic interface and thechange in the planctomycete community with depth suggest that these IPLs could be synthesized as a response to changing redoxconditions at the oxic/anoxic interface

Novel methylotrophic bacteria isolated from the River Thames (London, UK)

Enrichment and elective culture for methylotrophs from sediment of the River Thames in central London yielded a diversity of pure cultures representing several genera of Gram-negative and Gram-positive bacteria, which were mainly of organisms not generally regarded as typically methylotrophic. Substrates leading to successful isolations included methanol, monomethylamine, dimethylamine, trimethylamine, methanesulfonate and dimethylsulfone. Several isolates were studied in detail and shown by their biochemical and morphological properties and 16S rRNA gene sequencing to be Sphingomonas melonis strain ET35, Mycobacterium fluoranthenivorans strain DSQ3, Rhodococcus erythropolis strain DSQ4, Brevibacterium casei strain MSQ5, Klebsiella oxytoca strains MMA/F and MMA/1, Pseudomonas mendocina strain TSQ4, and Flavobacterium sp. strains MSA/1 and MMA/2. The results show that facultative methylotrophy is present across a wide range of Bacteria, suggesting that turnover of diverse C1-compounds is of much greater microbiological and environmental significance than is generally thought. The origins of the genes encoding the enzymes of methylotrophy in diverse heterotrophs need further study, and could further our understanding of the phylogeny and antiquity of methylotrophic systems

Telmatocola sphagniphila gen. nov., sp. nov., a Novel Dendriform Planctomycete from Northern Wetlands

Members of the phylum Planctomycetes are common inhabitants of northern wetlands. We used barcoded pyrosequencing to survey bacterial diversity in an acidic (pH 4.0) Sphagnum peat sampled from the peat bog Obukhovskoye, European North Russia. A total of 21189 bacterial 16S rRNA gene sequences were obtained, of which 1081 reads (5.1%) belonged to the Planctomycetes. Two-thirds of these sequences affiliated with planctomycete groups for which characterized representatives have not yet been available. Here, we describe two organisms from one of these previously uncultivated planctomycete groups. One isolate, strain OB3, was obtained from the peat sample used in our molecular study, while another strain, SP2T (=DSM 23888T = VKM B-2710T), was isolated from the peat bog Staroselsky moss. Both isolates are represented by aerobic, budding, pink-pigmented, non-motile, spherical cells that are arranged in unusual, dendriform-like structures during growth on solid media. These bacteria are moderately acidophilic and mesophilic, capable of growth at pH 4.0–7.0 (optimum pH 5.0–5.5) and at 6–30°C (optimum 20–26°C). The preferred growth substrates are various heteropolysaccharides and sugars, the latter being utilized only if provided in low concentrations (≤0.025%). In contrast to other described planctomycetes, strains SP2T and OB3 possess weak cellulolytic potential. The major fatty acids are C16:1ω5c, C18:1ω5c, C16:0, and C18:0. Characteristic lipids are the n-C31 polyunsaturated alkene (9–10 double bonds) and C30:1/C32:1 (ω-1) hydroxy fatty acids. The G + C content of the DNA is 58.5–59.0 mol%. Strains SP2T and OB3 share identical 16S rRNA gene sequences, which exhibit only 86 and 87% similarity to those of Gemmata obscuriglobus and Zavarzinella formosa. Based on the characteristics reported here, we propose to classify these novel planctomycetes as representatives of a novel genus and species, Telmatocola sphagniphila gen. nov., sp. nov

Methanobactin and MmoD work in concert to act as the ‘copper‐switch’ in methanotrophs

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/100314/1/emi12150.pd

Trace-gas metabolic versatility of the facultative methanotroph Methylocella silvestris

The climate-active gas methane is generated both by biological processes and by thermogenic decomposition of fossil organic material, which forms methane and short-chain alkanes, principally ethane, propane and butane1, 2. In addition to natural sources, environments are exposed to anthropogenic inputs of all these gases from oil and gas extraction and distribution. The gases provide carbon and/or energy for a diverse range of microorganisms that can metabolize them in both anoxic3 and oxic zones. Aerobic methanotrophs, which can assimilate methane, have been considered to be entirely distinct from utilizers of short-chain alkanes, and studies of environments exposed to mixtures of methane and multi-carbon alkanes have assumed that disparate groups of microorganisms are responsible for the metabolism of these gases. Here we describe the mechanism by which a single bacterial strain, Methylocella silvestris, can use methane or propane as a carbon and energy source, documenting a methanotroph that can utilize a short-chain alkane as an alternative to methane. Furthermore, during growth on a mixture of these gases, efficient consumption of both gases occurred at the same time. Two soluble di-iron centre monooxygenase (SDIMO) gene clusters were identified and were found to be differentially expressed during bacterial growth on these gases, although both were required for efficient propane utilization. This report of a methanotroph expressing an additional SDIMO that seems to be uniquely involved in short-chain alkane metabolism suggests that such metabolic flexibility may be important in many environments where methane and short-chain alkanes co-occur