Anastasia A. Ivanova

High Diversity of Planctomycetes in Soils of Two Lichen-Dominated Sub-Arctic Ecosystems of Northwestern Siberia

A wide variety of terrestrial ecosystems in tundra have a ground vegetation cover composed of reindeer lichens (genera Cladonia and Cetraria). The microbial communities of two lichen-dominated ecosystems typical of the sub-arctic zone of northwestern Siberia, that is a forested tundra soil and a shallow acidic peatland, were examined in our study. As revealed by molecular analyses, soil and peat layers just beneath the lichen cover were abundantly colonized by bacteria from the phylum Planctomycetes. Highest abundance of planctomycetes detected by fluorescence in situ hybridization was in the range 2.2–2.7 × 107 cells per gram of wet weight. 16S rRNA gene fragments from the Planctomycetes comprised 8–13% of total 16S rRNA gene reads retrieved using Illumina pair-end sequencing from the soil and peat samples. Lichen-associated assemblages of planctomycetes displayed unexpectedly high diversity, with a total of 89,662 reads representing 1723 operational taxonomic units determined at 97% sequence identity. The soil of forested tundra was dominated by uncultivated members of the family Planctomycetaceae (53–71% of total Planctomycetes-like reads), while sequences affiliated with the Phycisphaera-related group WD2101 (recently assigned to the order Tepidisphaerales) were most abundant in peat (28–51% of total reads). Representatives of the Isosphaera–Singulisphaera group (14–28% of total reads) and the lineages defined by the genera Gemmata (1–4%) and Planctopirus–Rubinisphaera (1–3%) were present in both habitats. Two strains of Singulisphaera-like bacteria were isolated from studied soil and peat samples. These planctomycetes displayed good tolerance of low temperatures (4–15°C) and were capable of growth on a number of polysaccharides, including lichenan, a characteristic component of lichen-derived phytomass.

Paludisphaera borealis gen. nov., sp. nov., a hydrolytic planctomycete from northern wetlands, and proposal of Isosphaeraceae 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 : 1ω9 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 Paludisphaera.

Identification of microbial populations driving biopolymer degradation in acidic peatlands by metatranscriptomic analysis.

Northern peatlands play a crucial role in the global carbon balance, serving as a persistent sink for atmospheric CO2 and a global carbon store. Their most extensive type, Sphagnum‐dominated acidic peatlands, is inhabited by microorganisms with poorly understood degradation capabilities. Here, we applied a combination of barcoded pyrosequencing of SSU rRNA genes and Illumina RNA‐Seq of total RNA (metatranscriptomics) to identify microbial populations and enzymes involved in degrading the major components of Sphagnum‐derived litter and exoskeletons of peat‐inhabiting arthropods: cellulose, xylan, pectin and chitin. Biopolymer addition to peat induced a threefold to fivefold increase in bacterial cell numbers. Functional community profiles of assembled mRNA differed between experimental treatments. In particular, pectin and xylan triggered increased transcript abundance of genes involved in energy metabolism and central carbon metabolism, such as glycolysis and TCA cycle. Concurrently, the substrate‐induced activity of bacteria on these two biopolymers stimulated grazing of peat‐inhabiting protozoa. Alveolata (ciliates) was the most responsive protozoa group as confirmed by analysis of both SSU rRNA genes and SSU rRNA. A stimulation of alphaproteobacterial methanotrophs on pectin was consistently shown by rRNA and mRNA data. Most likely, their significant enrichment was due to the utilization of methanol released during the degradation of pectin. Analysis of SSU rRNA and total mRNA revealed a specific response of Acidobacteria and Actinobacteria to chitin and pectin, respectively. Relatives of Telmatobacter bradus were most responsive among the Acidobacteria, while the actinobacterial response was primarily affiliated with Frankiales and Propionibacteriales. The expression of a wide repertoire of carbohydrate‐active enzymes (CAZymes) corresponded well to the detection of a highly diverse peat‐inhabiting microbial community, which is dominated by yet uncultivated bacteria.

Comparative Genomics of Four Isosphaeraceae Planctomycetes: A Common Pool of Plasmids and Glycoside Hydrolase Genes Shared by Paludisphaera borealis PX4T, Isosphaera pallida IS1BT, Singulisphaera acidiphila DSM 18658T, and Strain SH-PL62

The family Isosphaeraceae accommodates stalk-free planctomycetes with spherical cells, which can be assembled in short chains, long filaments, or aggregates. These bacteria inhabit a wide variety of terrestrial environments, among those the recently described Paludisphaera borealis PX4T that was isolated from acidic boreal wetlands. Here, we analyzed its finished genome in comparison to those of three other members of the Isosphaeraceae: Isosphaera pallida IS1BT, Singulisphaera acidiphila DSM 18658T, and the uncharacterized planctomycete strain SH-PL62. The complete genome of P. borealis PX4T consists of a 7.5 Mb chromosome and two plasmids, 112 and 43 kb in size. Annotation of the genome sequence revealed 5802 potential protein-coding genes of which 2775 could be functionally assigned. The genes encoding metabolic pathways common for chemo-organotrophic bacteria, such as glycolysis, citrate cycle, pentose-phosphate pathway, and oxidative phosphorylation were identified. Several genes involved in the synthesis of peptidoglycan as well as N-methylated ornithine lipids were present in the genome of P. borealis PX4T. A total of 26 giant genes with a size >5 kb were detected. The genome encodes a wide repertoire of carbohydrate-active enzymes (CAZymes) including 44 glycoside hydrolases (GH) and 83 glycosyltransferases (GT) affiliated with 21 and 13 CAZy families, respectively. The most-represented families are GH5, GH13, GH57, GT2, GT4, and GT83. The experimentally determined carbohydrate utilization pattern agrees well with the genome-predicted capabilities. The CAZyme repertoire in P. borealis PX4T is highly similar to that in the uncharacterized planctomycete SH-PL62 and S. acidiphila DSM 18658T, but different to that in the thermophile I. pallida IS1BT. The latter strain has a strongly reduced CAZyme content. In P. borealis PX4T, many of its CAZyme genes are organized in clusters. Contrary to most other members of the order Planctomycetales, all four analyzed Isosphaeraceae planctomycetes have plasmids in numbers varying from one to four. The plasmids from P. borealis PX4T display synteny to plasmids from other family members, providing evidence for their common evolutionary origin.

Planctomicrobium piriforme gen. nov., sp. nov., a stalked planctomycete from a littoral wetland of a boreal lake.

An aerobic, budding, non-pigmented and rosette-forming bacterium was isolated from a littoral wetland of a boreal lake located in Valaam Island, northern Russia, and designated strain P3(T). Ellipsoidal to pear-shaped cells of this bacterium were covered with crateriform pits and possessed stalks suggesting a planctomycete morphotype. 16S rRNA gene sequence analysis confirmed that strain P3(T) was a member of the order Planctomycetales and belonged to a phylogenetic lineage defined by the genus Planctomyces , with 89 and 86% sequence similarity to Planctomyces brasiliensis and Planctomyces maris , respectively. Strain P3(T) was a mildly acidophilic, mesophilic organism capable of growth at pH values between pH 4.2 and 7.1 (with an optimum at pH 6.0-6.5) and at temperatures between 10 and 30 °C (optimum at 20-28 °C). Most sugars, a number of polysaccharides and several organic acids were the preferred growth substrates. Compared with Planctomyces brasiliensis and Planctomyces maris , which require NaCl for growth, strain P3(T) was salt-sensitive and did not develop at NaCl concentrations above 0.5% (w/v). The major fatty acids were C16 : 0 and C16 : 1ω7c; the cells also contained significant amounts of C18 : 1ω7c and C18 : 0. The major intact polar lipids were diacylglycerol-O-(N,N,N-trimethyl)homoserine (DGTS) lipids; the major neutral lipids were long-chain 1,(ω-1)-diols and C31 : 9 hydrocarbon. The quinone was MK-6, and the G+C content of the DNA was 59.0 mol%. Strain P3(T) differed from Planctomyces brasiliensis and Planctomyces maris by cell morphology, substrate utilization pattern and a number of physiological characteristics. Based on these data, the novel isolate should be considered as representing a novel genus and species of planctomycetes, for which the name Planctomicrobium piriforme gen. nov., sp. nov., is proposed. The type strain is P3(T) ( =DSM 26348(T) =VKM B-2887(T)).

Phylogeny of β-xylanases from Planctomycetes

Here, we present the results of a computational analysis of a group of hypothetical GH10 endo-β-xylanases from the Planctomycetes, a bacterial phylum with poorly characterized functional capabilities. These proteins are encoded in all analyzed genomes of heterotrophic Planctomycetes and form a phylogenetically distinct and tight cluster. In addition, we determined nucleotide sequences for endo-β-xylanase genes from five strains of Isosphaera-Singulisphaera group of the Planctomycetes. The trees constructed for the 16S rRNA genes and the inferred amino acid sequences of endo-β-xylanases were highly congruent, thus suggesting the vertical transfer of endo-β-xylanase genes and their functional importance in Planctomycetes.

A novel filamentous planctomycete of the Isosphaera-Singulisphaera group isolated from a Sphagnum peat bog

Planctomycetes are common inhabitants of northern wetlands. A significant proportion of these bacteria revealed in peat with the Planctomycetes-specific probes PLA46 and PLA886 is represented by filamentous forms which have not been cultured under laboratory conditions. In the present work, one of such organisms was isolated from a Sphagnum peat bog in a monoculture. The organism had large spherical cells assembled in long filaments. It could grow only in associations with bacterial satellites; attempts to isolate it in pure culture were unsuccessful. The organism was identified by PCR amplification, cloning, and subsequent analysis of its 16S rRNA gene fragment. Comparative sequence analysis revealed its affiliation with the Isosphaera-Singulisphaera group within the order Planctomycetales. The nucleotide sequence of the 16S rRNA gene of the new organism exhibited 94–96% similarity to those of the unicellular peat-inhabiting planctomycete Singulisphaera acidiphila and uncharacterized filamentous planctomycete “Nostocoida limicola III” from activated sludge. The new planctomycete utilized heteropolysaccharides of microbial origin as growth substrates and could grow at the low pH and temperatures typical of the northern wetlands.

Genome analysis of Fimbriiglobus ruber SP5T, a planctomycete with confirmed chitinolytic capability

ABSTRACT Members of the bacterial order Planctomycetales have often been observed in associations with Crustacea. The ability to degrade chitin, however, has never been reported for any of the cultured planctomycetes although utilization of N-acetylglucosamine (GlcNAc) as a sole carbon and nitrogen source is well recognized for these bacteria. Here, we demonstrate the chitinolytic capability of a member of the family Gemmataceae, Fimbriiglobus ruber SP5T, which was isolated from a peat bog. As revealed by metatranscriptomic analysis of chitin-amended peat, the pool of 16S rRNA reads from F. ruber increased in response to chitin availability. Strain SP5T displayed only weak growth on amorphous chitin as a sole source of carbon but grew well with chitin as a source of nitrogen. The genome of F. ruber SP5T is 12.364 Mb in size and is the largest among all currently determined planctomycete genomes. It encodes several enzymes putatively involved in chitin degradation, including two chitinases affiliated with the glycoside hydrolase (GH) family GH18, GH20 family β-N-acetylglucosaminidase, and the complete set of enzymes required for utilization of GlcNAc. The gene encoding one of the predicted chitinases was expressed in Escherichia coli, and the endochitinase activity of the recombinant enzyme was confirmed. The genome also contains genes required for the assembly of type IV pili, which may be used to adhere to chitin and possibly other biopolymers. The ability to use chitin as a source of nitrogen is of special importance for planctomycetes that inhabit N-depleted ombrotrophic wetlands. IMPORTANCE Planctomycetes represent an important part of the microbial community in Sphagnum-dominated peatlands, but their potential functions in these ecosystems remain poorly understood. This study reports the presence of chitinolytic potential in one of the recently described peat-inhabiting members of the family Gemmataceae, Fimbriiglobus ruber SP5T. This planctomycete uses chitin, a major constituent of fungal cell walls and exoskeletons of peat-inhabiting arthropods, as a source of nitrogen in N-depleted ombrotrophic Sphagnum-dominated peatlands. This study reports the chitin-degrading capability of representatives of the order Planctomycetales.

Distinct diversity patterns of Planctomycetes associated with the freshwater macrophyte Nuphar lutea (L.) Smith

Members of the phylum Planctomycetes were originally described as freshwater bacteria. Most recent studies, however, address planctomycete diversity in other environments colonized by these microorganisms, including marine and terrestrial ecosystems. This study was initiated in order to revisit the specific patterns of planctomycete diversity in freshwater habitats using cultivation-independent approaches. The specific focus was made on planctomycetes associated with Nuphar lutea (L.) Smith, an emergent macrophyte with floating leaves, which is widespread in the Holarctic. As revealed by Illumina pair-end sequencing of 16S rRNA gene fragments, the bacterial assemblages colonizing floating leaf blades of waterlilies sampled from two different boreal lakes displayed similar composition but were distinct from the planktonic bacterial communities. 16S rRNA gene fragments from the Planctomycetes comprised 0.1–1 and 1–2.2% of total 16S rRNA gene reads retrieved from water samples and plant leaves, respectively. Planktonic planctomycetes were mostly affiliated with the class Planctomycetaceae (77–97%), while members of the Phycisphaerae were less abundant (3–22%). The relative proportion of the latter group, however, increased by 13–45% on leaves of N. lutea. The Phycisphaera-related group WD2101, Pirellula-like planctomycetes, as well as Gemmata, Zavarzinella and Planctopirus species were the most abundant groups of planctomycetes associated with plant leaves, which may suggest their involvement in the degradation of plant-derived organic matter.

Metatranscriptomics reveals the hydrolytic potential of peat-inhabiting Planctomycetes

Members of the phylum Planctomycetes are common inhabitants of northern Sphagnum-dominated wetlands. Evidence is accumulating that, in these environments, some planctomycetes may be involved in degrading polymeric organic matter. The experimental data, however, remain scarce due to the low number of characterized representatives of this phylum. In a previous study, we used metatranscriptomics to assess the activity response of peat-inhabiting microorganisms to biopolymers abundantly present in native peat. The community responses to cellulose, xylan, pectin, and chitin availability were analysed relative to unamended controls. Here, we re-analysed these metatranscriptomes and retrieved a total of 1,602,783 rRNA and 35,522 mRNA sequences affiliated with the Planctomycetes. Each of the four polymers induced specific planctomycete responses. These were most pronounced on chitin. The two groups with increased 16S rRNA transcript pools were Gemmata- and Phycisphaera-like planctomycetes. Among uncultivated members of the Planctomycetaceae, two increased transcript pools were detected in pectin-amended samples and belonged to Pirellula-like bacteria. The analysis of taxonomically assigned mRNA reads confirmed the specific response of Gemmata-related planctomycetes to chitin amendment suggesting the presence of chitinolytic capabilities in these bacteria.