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

<i>Paludisphaera borealis</i> gen. nov., sp. nov., a hydrolytic planctomycete from northern wetlands, and proposal of <i>Isosphaeraceae</i> fam. nov.

Two isolates of aerobic, budding, pink-pigmented bacteria, designated strains PX4T and PT1, were isolated from a boreal Sphagnum peat bog and a forested tundra wetland. Cells of these strains were non-motile spheres that occurred singly or in short chains. Novel isolates were capable of growth at pH values between 3.5 and 6.5 (optimum at pH?5.0–5.5) and at temperatures between 6 and 30?°C (optimum at 15–25?°C). Most sugars and a number of polysaccharides including pectin, xylan, lichenin and Phytagel were used as growth substrates. The major fatty acids were C16?:?0, C18?:?1?9 and C18?:?0; the major polar lipids were phosphocholine and trimethylornithine. The quinone was menaquinone-6, and the G+C content of the DNA was 66?mol%. Strains PX4T and PT1 were members of the order Planctomycetales and displayed 93–94?% 16S rRNA gene sequence similarity to Aquisphaera giovannonii, 91–92?% to species of the genus Singulisphaera and 90–91?% to Isosphaera pallida. The two novel strains, however, differed from members of these genera by cell morphology, substrate utilization pattern and a number of physiological characteristics. Based on these data, the novel isolates should be considered as representing a novel genus and species of planctomycetes, for which the name Paludisphaera borealis gen. nov., sp. nov., is proposed. The type strain is PX4T (?=?DSM 28747T?=?VKM B-2904T). We also suggest the establishment of a novel family, Isosphaeraceae fam. nov., to accommodate stalk-free planctomycetes with spherical cells, which can be assembled in short chains, long filaments or shapeless aggregates. This family includes the genera Isosphaera, Aquisphaera, Singulisphaera and Paludisphaer

<i>Telmatocola sphagniphila</i> 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 C16:1?5c, C18:1?5c, C16:0, and C18:0. Characteristic lipids are the n-C31 polyunsaturated alkene (9–10 double bonds) and C30:1/C32:1 (?-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. no

Fatty Acid and Hopanoid Adaption to Cold in the Methanotroph Methylovulum psychrotolerans

Three strains of aerobic psychrotolerant methanotrophic bacteria Methylovulum psychrotolerans, isolated from geographically remote low-temperature environments in Northern Russia, were grown at three different growth temperatures, 20, 10 and 4°C and were found to be capable of oxidizing methane at all temperatures. The three M. psychrotolerans strains adapted their membranes to decreasing growth temperature by increasing the percent of unsaturated fatty acid (FAs), both for the bulk and intact polar lipid (IPL)-bound FAs. Furthermore, the ratio of βOH-C16:0 to n-C16:0 increased as growth temperature decreased. The IPL head group composition did not change as an adaption to temperature. The most notable hopanoid temperature adaptation of M. psychrotolerans was an increase in unsaturated hopanols with decreasing temperature. As the growth temperature decreased from 20 to 4°C, the percent of unsaturated M. psychrotolerans bulk-FAs increased from 79 to 89 % while the total percent of unsaturated hopanoids increased from 27 to 49 %. While increased FA unsaturation in response to decreased temperature is a commonly observed response in order to maintain the liquid-crystalline character of bacterial membranes, hopanoid unsaturation upon cold exposition has not previously been described. In order to investigate the mechanisms of both FA and hopanoid cold-adaption in M. psychrotolerans we identified genes in the genome of M. psychrotolerans that potentially code for FA and hopanoid desaturases. The unsaturation of hopanoids represents a novel membrane adaption to maintain homeostasis upon cold adaptation

Microbiology of wetlands

Wetlands are ecologically as well as economically important systems due to their high productivity, their nutrient (re)cycling capacities, and their prominent contribution to global greenhouse gas emissions. Being on the transition between terrestrial and—aquatic ecosystems, wetlands are buffers for terrestrial run off thereby preventing eutrophication of inland as well as coastal waters. The close proximity of oxic–anoxic conditions, often created by wetland plant roots, facilitates the simultaneous activity of aerobic as well as anaerobic microbial communities. Input of nutrients and fast recycling due to active aerobes and anaerobes makes these systems highly productive and therefore attractive for humans as well as many other organisms. Wetlands globally are under high pressure due to anthropogenic activities as well as climate change. Changes of land-use as well as altered hydrology due to climate change will lead to disturbance and loss of these habitats. However, the diversity and functioning of microbial communities in wetland systems is highly underexplored in comparison to soils and aquatic ecosystems.

Acidobacteria

Acidobacteria are one of the globally distributed and highly diverse phyla of the domain Bacteria. These microorganisms inhabit a wide variety of terrestrial and aquatic habitats and are particularly abundant in acidic soils, peatlands and mineral iron-rich environments. Owing to the difficulties in cultivating Acidobacteria, the taxonomically described diversity within this phylum remains limited. All characterised representatives are Gram-negative, nonspore-forming bacteria that display a variety of cell morphologies. Most characterised acidobacteria are chemoheterotrophs, although photoheterotrophic members have also been described. Cells of these bacteria contain a number of characteristic lipids, which may be responsible for their environmental adaptations. Genomes of acidobacteria are up to 10 Mbp in size and encode a wide repertoire of carbohydrate-active enzymes involved in breakdown, utilisation and biosynthesis of diverse carbohydrates. Their functional role in the environment includes the decomposition of various biopolymers and participation in the global cycling of carbon, iron and hydrogen

Methylocystis bryophila sp. nov., a Novel Facultatively Methanotrophic Bacterium from Acidic Sphagnum Peat, and Emended Description of the Genus Methylocystis (ex Whittenbury et al. 1970) Bowman et al. 1993

A new species is proposed for two facultatively methanotrophic representatives of the genus Methylocystis, strains H2sT and S284, which were isolated from an acidic (pH 4.3) Sphagnum peat bog lake (Teufelssee, Germany) and an acidic (pH 3.8) peat bog (European North Russia), respectively. Cells of strains H2sT and S284 are aerobic, Gram-negative, non-motile, curved coccoids or short rods that contain an intracytoplasmic membrane system typical of type II methanotrophs. They possess both a soluble and a particulate methane monooxygenase (MMO); the latter is represented by two isozymes, pMMO1 and pMMO2. The preferred growth substrates are methane and methanol. In the absence of C1 substrates, however, these methanotrophs are capable of slow growth on acetate. Atmospheric nitrogen is fixed by means of an aero-tolerant nitrogenase. Strains H2sT and S284 develop between pH 4.2 and 7.6 (optimum pH 6.0-6.5), and at temperatures between 8 and 37°C (optimum 25-30°C). The major fatty acids are C18:1w8c, C18:1w7c, and C16:1w7c; the major quinone is Q-8. The DNA G+C content is 62.0-62.3 mol%. Strains H2sT and S284 share identical 16S rRNA gene sequences, which displayed 96.6-97.3% similarity to sequences of other taxonomically characterized members of the genus Methylocystis. Therefore, strains H2sT and S284 are classified as a novel species, for which the name Methylocystis bryophila sp. nov. is proposed. Strain H2sT (=DSM 21852T = VKM B-2545T) is the type strain of Methylocystis bryophila.

Diversity of the active methanotrophic community in acidic peatlands as assessed by mRNA and SIP-PLFA analyses

The active methanotroph community was investigated for the first time in heather (Calluna)-covered moorlands and Sphagnum/Eriophorum-covered UK peatlands. Direct extraction of mRNA from these soils facilitated detection of expression of methane monooxygenase genes, which revealed that particulate methane monooxygenase and not soluble methane monooxygenase was probably responsible for CH4 oxidation in situ, because only pmoA transcripts (encoding a subunit of particulate methane monooxygenase) were readily detectable. Differences in methanotroph community structures were observed between the Calluna-covered moorland and Sphagnum/Eriophorum-covered gully habitats. As with many other Sphagnum-covered peatlands, the Sphagnum/Eriophorum-covered gullies were dominated by Methylocystis. Methylocella and Methylocapsa-related species were also present. Methylobacter-related species were found as demonstrated by the use of a pmoA-based diagnostic microarray. In Calluna-covered moorlands, in addition to Methylocella and Methylocystis, a unique group of peat-associated type I methanotrophs (Gammaproteobacteria) and a group of uncultivated type II methanotrophs (Alphaproteobacteria) were also found. The pmoA sequences of the latter were only distantly related to Methylocapsa and also to the RA-14 group of methanotrophs, which are believed to be involved in oxidation of atmospheric concentrations of CH4. Soil samples were also labelled with (CH4)-C-13, and subsequent analysis of the C-13-labelled phospholipid fatty acids (PLFAs) showed that 16:1 omega 7, 18:1 omega 7 and 18:1 omega 9 were the major labelled PLFAs. The presence of C-13-labelled 18:1 omega 9, which was not a major PLFA of any extant methanotrophs, indicated the presence of novel methanotrophs in this peatland