Irina S. Kulichevskaya

Phylogenetic Analysis and In Situ Identification of Bacteria Community Composition in an Acidic Sphagnum Peat Bog

ABSTRACT The Bacteria community composition in an acidic Sphagnum peat bog (pH 3.9 to 4.5) was characterized by a combination of 16S rRNA gene clone library analysis, rRNA-targeted fluorescence in situ hybridization (FISH), and cultivation. Among 84 environmental 16S rRNA gene clones, a set of only 16 cloned sequences was closely related (≥95% similarity) to taxonomically described organisms. Main groups of clones were affiliated with the Acidobacteria (24 clones), Alphaproteobacteria (20), Verrucomicrobia (13), Actinobacteria (8), Deltaproteobacteria (4), Chloroflexi (3), and Planctomycetes (3). The proportion of cells that hybridized with oligonucleotide probes specific for members of the domains Bacteria (EUB338-mix) and Archaea (ARCH915 and ARC344) accounted for only 12 to 22% of the total cell counts. Up to 24% of the EUB338-positive cells could be assigned by FISH to specific bacterial phyla. Alphaproteobacteria and Planctomycetes were the most numerous bacterial groups (up to 1.3 × 107 and 1.1 × 107 cells g−1 peat, respectively). In contrast to conventional plating techniques, a novel biofilm-mediated enrichment approach allowed us to isolate some representatives of predominant Bacteria groups, such as Acidobacteria and Planctomycetes. This novel strategy has great potential to enable the isolation of a significant proportion of the peat bog bacterial diversity.

Substrate-induced growth and isolation of Acidobacteria from acidic Sphagnum peat.

Fluorescence in situ hybridization (FISH) was applied to estimate the population size of the poorly characterized phylum Acidobacteria in acidic peat sampled from nine different Sphagnum-dominated wetlands of Northern Russia. The cell numbers of these bacteria in oxic peat layers ranged from 0.4 × 106 to 1.3 × 107 cells per g of wet peat, comprising up to 4% of total bacterial cells. Substrate-induced growth of acidobacteria was observed after amendment of peat samples with glucose, pectin, xylan, starch, ethanol and methanol, while weak or no response was obtained for acetate, pyruvate, mannitol and cellobiose. Using low-nutrient media and FISH-mediated monitoring of the isolation procedure, we succeeded in obtaining nine strains of acidobacteria in pure cultures. These strains belonged to subdivisions 1 and 3 of the Acidobacteria and represented strictly aerobic, heterotrophic organisms. Except for methanol, the substrate utilization patterns of these isolates matched the results obtained in our substrate-amendment experiments with native peat. All strains were also capable of utilizing galacturonic acid, a characteristic component of the cell wall in Sphagnum spp, which is released during moss decomposition. Most isolates from subdivision 1 were truly acidophilic organisms with the growth optimum at pH 3.5–4.5, while the isolates from subdivision 3 grew optimally at pH 5.5–6.5. Another important phenotypic trait of novel strains was their capability of active growth at low temperatures. Both acidophily and low-temperature growth are consistent with the occurrence of acidobacteria in cold and acidic northern wetlands.

Bryobacter aggregatus gen. nov., sp. nov., a peat-inhabiting, aerobic chemo-organotroph from subdivision 3 of the Acidobacteria.

Bryobacter aggregatus gen. nov., sp. nov. is proposed to accommodate three strains of slowly growing, chemo-organotrophic bacteria isolated from acidic Sphagnum peat bogs. These bacteria were strictly aerobic, Gram-negative, colourless, non-motile coccoids or short rods that multiplied by normal cell division and formed irregularly shaped cell aggregates. Strains MPL3(T), MPL1011 and MOB76 were acidotolerant, mesophilic organisms capable of growth at pH 4.5-7.2 and between 4 and 33 degrees C (optimum growth at pH 5.5-6.5 and 22-28 degrees C). The preferred growth substrates were sugars, some heteropolysaccharides and galacturonic and glucuronic acids, which are released during decomposition of Sphagnum moss. The major fatty acids were iso-C(15 : 0), C(16 : 0) and summed feature 3 (iso-C(15 : 0) 2-OH and/or C(16 : 1)omega7c); the major quinones were MK-9 and MK-10. The DNA G+C content was 55.5-56.5 mol%. Strains MPL3(T), MPL1011 and MOB76 possessed nearly identical 16S rRNA gene sequences and belonged to the phylum Acidobacteria. They represent the first taxonomically characterized members of acidobacterial subdivision 3 and display only 81.7-86.7 % 16S rRNA gene sequence similarity to other members of the Acidobacteria with validly published names. Therefore, strains MPL3(T), MPL1011 and MOB76 are classified as representatives of a novel species in a new genus, for which the name Bryobacter aggregatus gen. nov., sp. nov. is proposed; strain MPL3(T) (=ATCC BAA-1390(T) =DSM 18758(T)) is the type strain of Bryobacter aggregatus.

Zavarzinella formosa gen. nov., sp. nov., a Novel Stalked, Gemmata-like Planctomycete from a Siberian Peat Bog

An aerobic, pink-pigmented, budding and rosette-forming bacterium was isolated from an acidic Sphagnum peat bog and designated strain A10(T). The 16S rRNA gene sequence analysis showed that strain A10(T) was a member of the order Planctomycetales and belonged to a phylogenetic lineage defined by the genus Gemmata, with 90 % sequence similarity to that of Gemmata obscuriglobus, the only taxonomically described organism of this group. Ellipsoid-shaped cells of strain A10(T) were uniformly covered with crateriform pits and possessed long (up to 10-15 mum) and unusually thick (0.5-0.7 mum) stalks of a unique ultrastructure. Thin sections revealed a complex intracellular membrane system compartmentalizing the cells. Strain A10(T) was a moderately acidophilic, mesophilic organism capable of growth at pH values between 3.8 and 7.2 (with an optimum at pH 5.5-6.0) and at temperatures between 10 and 30 degrees C (with an optimum at 20-25 degrees C). The major fatty acids were C(18 : 0), C(18 : 1)omega5c and C(16 : 1)omega5c and the major quinone was MK-6. Cells of strain A10(T) contained high amounts of bound saturated and monounsaturated C(26)-C(32) (omega-1) hydroxy fatty acids. The G+C content of the DNA was 62.5 mol%. The unique cell morphology, the capability of growth in acidic conditions and a number of chemotaxonomic and genotypic characteristics served to differentiate strain A10(T) from G. obscuriglobus. Based on these data, the novel isolate should be considered as representing a novel genus and species of planctomycetes, for which the name Zavarzinella formosa gen. nov., sp. nov. is proposed The type strain is A10(T) (=DSM 19928(T)=VKM B-2478(T)).

Bryocella elongata gen. nov., sp. nov., a member of subdivision 1 of the Acidobacteria isolated from a methanotrophic enrichment culture, and emended description of Edaphobacter aggregans Koch et al. 2008

An aerobic, pink-pigmented, chemo-organotrophic bacterium, designated strain SN10(T), was isolated from a methanotrophic enrichment culture obtained from an acidic Sphagnum peat. This isolate was represented by Gram-negative, non-motile rods that multiply by normal cell division and form rosettes. Strain SN10(T) is an obligately acidophilic, mesophilic bacterium capable of growth at pH 3.2-6.6 (with an optimum at pH 4.7-5.2) and at 6-32 °C (with an optimum at 20-24 °C). The preferred growth substrates are sugars and several heteropolysaccharides of plant and microbial origin, such as pectin, lichenan, fucoidan and gellan gum. While not being capable of growth on C(1) compounds, strain SN10(T) can develop in co-culture with exopolysaccharide-producing methanotrophs by utilization of their capsular material. The major fatty acids determined in strain SN10(T) using the conventional lipid extraction procedure are iso-C(15:0) and C(16:1)ω7c. Upon hydrolysis of total cell material, substantial amounts of the uncommon membrane-spanning lipid 13,16-dimethyl octacosanedioic acid (isodiabolic acid) were also detected. The polar lipids are two phosphohexoses, phosphatidylethanolamine, phosphatidylglycerol and several phospholipids of unknown structure. The major quinone is MK-8. Pigments are carotenoids. The G+C content of the DNA is 60.7 mol%. Strain SN10(T) forms a separate lineage within subdivision 1 of the phylum Acidobacteria and displays 94.0-95.4% 16S rRNA gene sequence similarity to members of the genera Edaphobacter and Granulicella, 93.0-93.7% similarity to members of the genus Terriglobus and 92.2-92.3 % similarity to the type strains of Telmatobacter bradus and Acidobacterium capsulatum. Therefore, strain SN10(T) is classified within a novel genus and species, for which the name Bryocella elongata gen. nov., sp. nov. is proposed. Strain SN10(T) (=LMG 25276(T) =DSM 22489(T)) is the type strain of Bryocella elongata. An emended description of Edaphobacter aggregans Koch et al. 2008 is also given.

Detection of representatives of the Planctomycetes in Sphagnum peat bogs by molecular and cultivation approaches

By means of fluorescence in situ hybridization with 16S rRNA-targeted oligonucleotide probes (FISH), it has been shown that members of the phylum Planctomycetes represent a numerically significant bacterial group in boreal Sphagnum peat bogs. The population size of planctomycetes in oxic layers of the peat bog profile was in the range of 0.4–2.0 × 107 cells per g of wet peat, comprising 4 to 13% of the total bacterial cell number. A novel effective approach that combined a traditional cultivation technique with FISH-mediated monitoring of the target organism during the isolation procedure has been developed for the isolation of planctomycetes. Using this approach, we succeeded in isolating several peat-inhabiting planctomycetes in a pure culture. Sequencing of the 16S rRNA genes from two of these isolates, strains A10 and MPL7, showed that they belonged to the planctomycete lineages defined by the genera Gemmata and Planctomyces, respectively. The 16S rRNA gene sequence similarity between strains A10 and MPL7 and the phylogenetically closest organisms, namely, Gemmata obscuriglobus and Planctomyces limnophilus, was only 90%. These results suggest that the indigenous planctomycetes inhabiting Sphagnum peat bogs are so far unknown organisms.

Acidicapsa borealis gen. nov., sp. nov. and Acidicapsa ligni sp. nov., subdivision 1 Acidobacteria from Sphagnum peat and decaying wood

Two strains of subdivision 1 Acidobacteria, a pink-pigmented bacterium KA1(T) and a colourless isolate WH120(T), were obtained from acidic Sphagnum peat and wood under decay by the white-rot fungus Hyploma fasciculare, respectively. Cells of these isolates were Gram-negative-staining, non-motile, short rods, which were covered by large polysaccharide capsules and occurred singly, in pairs, or in short chains. Strains KA1(T) and WH120(T) were strictly aerobic mesophiles that grew between 10 and 33 °C, with an optimum at 22-28 °C. Both isolates developed under acidic conditions, but strain WH120(T) was more acidophilic (pH growth range 3.5-6.4; optimum, 4.0-4.5) than strain KA1(T) (pH growth range 3.5-7.3; optimum , 5.0-5.5). The preferred growth substrates were sugars. In addition, the wood-derived isolate WH120(T) grew on oxalate, lactate and xylan, while the peat-inhabiting acidobacterium strain KA1(T) utilized galacturonate, glucuronate and pectin. The major fatty acids were iso-C(15:0) and iso-C(17:1)ω8c; the cells also contained significant amounts of 13,16-dimethyl octacosanedioic acid. The quinone was MK-8. The DNA G+C contents of strains KA1(T) and WH120(T) were 54.1 and 51.7 mol%, respectively. Strains KA1(T) and WH120(T) displayed 97.8% 16S rRNA gene sequence similarity to each other. The closest recognized relatives were Acidobacterium capsulatum and Telmatobacter bradus (93.4-94.3% 16S rRNA gene sequence similarity). These species differed from strains KA1(T) and WH120(T) by their ability to grow under anoxic conditions, the absence of capsules, presence of cell motility and differing fatty acid composition. Based on these differences, the two new isolates are proposed as representing a novel genus, Acidicapsa gen. nov., and two novel species. Acidicapsa borealis gen. nov., sp. nov. is the type species for the new genus with strain KA1(T) (=DSM 23886(T)=LMG 25897(T)=VKM B-2678(T)) as the type strain. The name Acidicapsa ligni sp. nov. is proposed for strain WH120(T) (=LMG 26244(T)=VKM B-2677(T)=NCCB 100371(T)).

Analysis of the bacterial community developing in the course of Sphagnum moss decomposition

Slow degradation of organic matter in acidic Sphagnum peat bogs suggests a limited activity of organotrophic microorganisms. Monitoring of the Sphagnum debris decomposition in a laboratory simulation experiment showed that this process was accompanied by a shift in the water color to brownish due to accumulation of humic substances and by the development of a specific bacterial community with a density of 2.4 × 107 cells ml−1. About half of these organisms are metabolically active and detectable with rRNA-specific oligonucleotide probes. Molecular identification of the components of this microbial community showed the numerical dominance of bacteria affiliated with the phyla Alphaproteobacteria, Actinobacteria, and Planctomycetes. The population sizes of the Firmicutes and Bacteroidetes, which are believed to be the main agents of bacterially-mediated decomposition in eutrophic wetlands, were low. The numbers of planctomycetes increased at the final stage of Sphagnum decomposition. The representative isolates of the Alphaproteobacteria were able to utilize galacturonic acid, the only low-molecular-weight organic compound detected in the water samples; the representatives of the Planctomycetes were able to decompose some heteropolysaccharides, which points to the possible functional role of these groups of microorganisms in the community under study. Thus, the composition of the bacterial community responsible for Sphagnum decomposition in acidic and low-mineral oligotrophic conditions seems to be fundamentally different from that of the bacterial community which decomposes plant debris in eutrophic ecosystems at neutral pH.

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.

Methylocystis bryophila sp. nov., a facultatively methanotrophic bacterium from acidic Sphagnum peat, and emended description of the genus Methylocystis (ex Whittenbury et al. 1970) Bowman et al. 1993

A novel species is proposed for two facultatively methanotrophic representatives of the genus Methylocystis, strains H2s(T) 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 H2s(T) 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 aerotolerant nitrogenase. Strains H2s(T) and S284 grow between pH 4.2 and 7.6 (optimum pH 6.0-6.5) and at 8-37 °C (optimum 25-30 °C). The major fatty acids are C18 : 1ω8c, C18 : 1ω7c and C16 : 1ω7c; the major quinone is Q-8. The DNA G+C content is 62.0-62.3 mol%. Strains H2s(T) 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 H2s(T) and S284 are classified as members of a novel species, for which the name Methylocystis bryophila sp. nov. is proposed; strain H2s(T) ( = DSM 21852(T)  = VKM B-2545(T)) is the type strain.