The Succession of the Cellulolytic Microbial Community from the Soil during Oat Straw Decomposition.

The process of straw decomposition is dynamic and is accompanied by the succession of the microbial decomposing community, which is driven by poorly understood interactions between microorganisms. Soil is a complex ecological niche, and the soil microbiome can serve as a source of potentially active cellulolytic microorganisms. Here, we performed an experiment on the de novo colonization of oat straw by the soil microbial community by placing nylon bags with sterilized oat straw in the pots filled with chernozem soil and incubating them for 6 months. The aim was to investigate the changes in decomposer microbiota during this process using conventional sequencing techniques. The bacterial succession during straw decomposition occurred in three phases: the early phase (first month) was characterized by high microbial activity and low diversity, the middle phase (second to third month) was characterized by low activity and low diversity, and the late phase (fourth to sixth months) was characterized by low activity and high diversity. Analysis of amplicon sequencing data revealed three groups of co-changing phylotypes corresponding to these phases. The early active phase was abundant in the cellulolytic members from Pseudomonadota, Bacteroidota, Bacillota, and Actinobacteriota for bacteria and Ascomycota for fungi, and most of the primary phylotypes were gone by the end of the phase. The second intermediate phase was marked by the set of phylotypes from the same phyla persisting in the community. In the mature community of the late phase, apart from the core phylotypes, non-cellulolytic members from Bdellovibrionota, Myxococcota, Chloroflexota, and Thermoproteota appeared. Full metagenome sequencing of the microbial community from the end of the middle phase confirmed that major bacterial and fungal members of this consortium had genes of glycoside hydrolases (GH) connected to cellulose and chitin degradation. The real-time analysis of the selection of these genes showed that their representation varied between phases, and this occurred under the influence of the host, and not the GH family factor. Our findings demonstrate that soil microbial community may act as an efficient source of cellulolytic microorganisms and that colonization of the cellulolytic substrate occurs in several phases, each characterized by its own taxonomic and functional profile.

Microbial Composition on Abandoned and Reclaimed Mining Sites in the Komi Republic (North Russia).

Restoration of anthropogenically disturbed soils is an urgent problem in modern ecology and soil biology. Restoration processes in northern environments are especially important, due to the small amounts of fertile land and low levels of natural succession. We analyzed the soil microbiota, which is one of the indicators of the succession process is the soil. Samples were obtained from three disturbed soils (self-overgrown and reclaimed quarries), and two undisturbed soils (primary and secondary forests). Primary Forest soil had a well-developed soil profile, and a low pH and TOC (total organic carbon) amount. The microbial community of this soil had low richness, formed a clear remote cluster in the beta-diversity analysis, and showed an overrepresentation of Geobacter (Desulfobacteriota). Soil formation in clay and limestone abandoned quarries was at the initial stage, and was caused by both a low rate of mineral profile formation and severe climatic conditions in the region. Microbial communities of these soils did not have specific abundant taxa, and included a high amount of sparse taxa. Differences in taxa composition were correlated with abiotic factors (ammonium concentration), which, in turn, can be explained by the parent rock properties. Limestone quarry reclaimed by topsoil coverage resulted in an adaptation of the top soil microbiota to a novel parent rock. According to the CCA analysis, the microbial composition of samples was connected with pH, TOC and ammonium nitrogen concentration. Changes in pH and TOC were connected with ASVs from Chloroflexota, Gemmatimonadota and Patescibacteria. ASVs from Gemmatimonadota also were correlated with a high ammonium concentration.

Microevolution, speciation and macroevolution in rhizobia: Genomic mechanisms and selective patterns.

Nodule bacteria (rhizobia), N2-fixing symbionts of leguminous plants, represent an excellent model to study the fundamental issues of evolutionary biology, including the tradeoff between microevolution, speciation, and macroevolution, which remains poorly understood for free-living organisms. Taxonomically, rhizobia are extremely diverse: they are represented by nearly a dozen families of α-proteobacteria (Rhizobiales) and by some ß-proteobacteria. Their genomes are composed of core parts, including house-keeping genes (hkg), and of accessory parts, including symbiotically specialized (sym) genes. In multipartite genomes of evolutionary advanced fast-growing species (Rhizobiaceae), sym genes are clustered on extra-chromosomal replicons (megaplasmids, chromids), facilitating gene transfer in plant-associated microbial communities. In this review, we demonstrate that in rhizobia, microevolution and speciation involve different genomic and ecological mechanisms: the first one is based on the diversification of sym genes occurring under the impacts of host-induced natural selection (including its disruptive, frequency-dependent and group forms); the second one-on the diversification of hkgs under the impacts of unknown factors. By contrast, macroevolution represents the polyphyletic origin of super-species taxa, which are dependent on the transfer of sym genes from rhizobia to various soil-borne bacteria. Since the expression of newly acquired sym genes on foreign genomic backgrounds is usually restricted, conversion of resulted recombinants into the novel rhizobia species involves post-transfer genetic changes. They are presumably supported by host-induced selective processes resulting in the sequential derepression of nod genes responsible for nodulation and of nif/fix genes responsible for symbiotic N2 fixation.

The Structure of Stable Cellulolytic Consortia Isolated from Natural Lignocellulosic Substrates.

Recycling plant matter is one of the challenges facing humanity today and depends on efficient lignocellulose degradation. Although many bacterial strains from natural substrates demonstrate cellulolytic activities, the CAZymes (Carbohydrate-Active enZYmes) responsible for these activities are very diverse and usually distributed among different bacteria in one habitat. Thus, using microbial consortia can be a solution to rapid and effective decomposition of plant biomass. Four cellulolytic consortia were isolated from enrichment cultures from composting natural lignocellulosic substrates-oat straw, pine sawdust, and birch leaf litter. Enrichment cultures facilitated growth of similar, but not identical cellulose-decomposing bacteria from different substrates. Major components in all consortia were from Proteobacteria, Actinobacteriota and Bacteroidota, but some were specific for different substrates-Verrucomicrobiota and Myxococcota from straw, Planctomycetota from sawdust and Firmicutes from leaf litter. While most members of the consortia were involved in the lignocellulose degradation, some demonstrated additional metabolic activities. Consortia did not differ in the composition of CAZymes genes, but rather in axillary functions, such as ABC-transporters and two-component systems, usually taxon-specific and associated with CAZymes. Our findings show that enrichment cultures can provide reproducible cellulolytic consortia from various lignocellulosic substrates, the stability of which is ensured by tight microbial relations between its components.

Microbiomes of different ages in Rendzic Leptosols in the Crimean Peninsula.

Rendzic Leptosols are intrazonal soils formed on limestone bedrock. The specialty of these soils is that parent rock material is more influential in shaping soil characteristics than zonal factors such as climate, especially during soil formation. Unlike fast evolving Podzols due to their leaching regime, Leptosols do not undergo rapid development due to the nature of the limestone. Little is known how microbiome reflects this process, so we assessed microbiome composition of Rendzic Leptosols of different ages, arising from disruption and subsequent reclamation. The mountains and foothills that cover much of the Crimean Peninsula are ideal for this type of study, as the soils were formed on limestone and have been subjected to anthropogenic impacts through much of human history. Microbiomes of four soil sites forming a chronosequence, including different soil horizons, were studied using sequencing of 16S rRNA gene libraries and quantitative PCR. Dominant phyla for all soil sites were Actinobacteria, Proteobacteria, Acidobacteria, Bacteroidetes, Thaumarchaeota, Planctomycetes, Verrucomicrobia and Firmicutes. Alpha diversity was similar across sites and tended to be higher in topsoil. Beta diversity showed that microbiomes diverged according to the soil site and the soil horizon. The oldest and the youngest soils had the most similar microbiomes, which could have been caused by their geographic proximity. Oligotrophic bacteria from Chitinophagaceae, Blastocatellaceae and Rubrobacteriaceae dominated the microbiome of these soils. The microbiome of 700-year old soil was the most diverse. This soil was from the only study location with topsoil formed by plant litter, which provided additional nutrients and could have been the driving force of this differentiation. Consistent with this assumption, this soil was abundant in copiotrophic bacteria from Proteobacteria and Actinobacteria phyla. The microbiome of 50-year old Leptosol was more similar to the microbiome of benchmark soil than the microbiome of 700-year old soil, especially by weighted metrics. CCA analysis, in combination with PERMANOVA, linked differences in microbiomes to the joint change of all soil chemical parameters between soil horizons. Local factors, such as parent material and plant litter, more strongly influenced the microbiome composition in Rendzic Leptosols than soil age.

The difference between cellulolytic 'culturomes' and microbiomes inhabiting two contrasting soil types.

High-throughput 16S rRNA sequencing was performed to compare the microbiomes inhabiting two contrasting soil types-sod-podzolic soil and chernozem-and the corresponding culturome communities of potentially cellulolytic bacteria cultured on standard Hutchinson media. For each soil type, soil-specific microorganisms have been identified: for sod-podzolic soil-Acidothermus, Devosia, Phenylobacterium and Tumebacillus, and for chernozem soil-Sphingomonas, Bacillus and Blastococcus. The dynamics of differences between soil types for bulk soil samples and culturomes varied depending on the taxonomic level of the corresponding phylotypes. At high taxonomic levels, the number of common taxa between soil types increased more slowly for bulk soil than for culturome. Differences between soil-specific phylotypes were detected in bulk soil at a low taxonomic level (genus, species). A total of 13 phylotypes were represented both in soil and in culturome. No relationship was shown between the abundance of these phylotypes in soil and culturome.

Search for Ancestral Features in Genomes of Rhizobium leguminosarum bv. viciae Strains Isolated from the Relict Legume Vavilovia formosa.

Vavilovia formosa is a relict leguminous plant growing in hard-to-reach habitats in the rocky highlands of the Caucasus and Middle East, and it is considered as the putative closest living relative of the last common ancestor (LCA) of the Fabeae tribe. Symbionts of Vavilovia belonging to Rhizobium leguminosarum bv. viciae compose a discrete group that differs from the other strains, especially in the nucleotide sequences of the symbiotically specialised (sym) genes. Comparison of the genomes of Vavilovia strains with the reference group composed of R. leguminosarum bv. viciae strains isolated from Pisum and Vicia demonstrated that the vavilovia strains have a set of genomic features, probably indicating the important stages of microevolution of the symbiotic system. Specifically, symbionts of Vavilovia (considered as an ancestral group) demonstrated a scattered arrangement of sym genes (>90 kb cluster on pSym), with the location of nodT gene outside of the other nod operons, the presence of nodX and fixW, and the absence of chromosomal fixNOPQ copies. In contrast, the reference (derived) group harboured sym genes as a compact cluster (<60 kb) on a single pSym, lacking nodX and fixW, with nodT between nodN and nodO, and possessing chromosomal fixNOPQ copies. The TOM strain, obtained from nodules of the primitive "Afghan" peas, occupied an intermediate position because it has the chromosomal fixNOPQ copy, while the other features, the most important of which is presence of nodX and fixW, were similar to the Vavilovia strains. We suggest that genome evolution from the ancestral to the derived R. leguminosarum bv. viciae groups follows the "gain-and-loss of sym genes" and the "compaction of sym cluster" strategies, which are common for the macro-evolutionary and micro-evolutionary processes. The revealed genomic features are in concordance with a relict status of the vavilovia strains, indicating that V. formosa coexists with ancestral microsymbionts, which are presumably close to the LCA of R. leguminosarum bv. viciae.

Rhizobia Isolated from the Relict Legume Vavilovia formosa Represent a Genetically Specific Group within Rhizobium leguminosarum biovar viciae.

Twenty-two rhizobia strains isolated from three distinct populations (North Ossetia, Dagestan, and Armenia) of a relict legume Vavilovia formosa were analysed to determine their position within Rhizobium leguminosarum biovar viciae (Rlv). These bacteria are described as symbionts of four plant genera Pisum, Vicia, Lathyrus, and Lens from the Fabeae tribe, of which Vavilovia is considered to be closest to its last common ancestor (LCA). In contrast to biovar viciae, bacteria from Rhizobium leguminosarum biovar trifolii (Rlt) inoculate plants from the Trifolieae tribe. Comparison of house-keeping (hkg: 16S rRNA, glnII, gltA, and dnaK) and symbiotic (sym: nodA, nodC, nodD, and nifH) genes of the symbionts of V. formosa with those of other Rlv and Rlt strains reveals a significant group separation, which was most pronounced for sym genes. A remarkable feature of the strains isolated from V. formosa was the presence of the nodX gene, which was commonly found in Rlv strains isolated from Afghanistan pea genotypes. Tube testing of different strains on nine plant species, including all genera from the Fabeae tribe, demonstrated that the strains from V. formosa nodulated the same cross inoculation group as the other Rlv strains. Comparison of nucleotide similarity in sym genes suggested that their diversification within sym-biotypes of Rlv was elicited by host plants. Contrariwise, that of hkg genes could be caused by either local adaptation to soil niches or by genetic drift. Long-term ecological isolation, genetic separation, and the ancestral position of V. formosa suggested that symbionts of V. formosa could be responsible for preserving ancestral genotypes of the Rlv biovar.

Extra-slow-growing Tardiphaga strains isolated from nodules of Vavilovia formosa (Stev.) Fed.

Eleven extra-slow-growing strains were isolated from nodules of the relict legume Vavilovia formosa growing in North Ossetia (Caucasus) and Armenia. All isolates formed a single rrs cluster together with the type strain Tardiphaga robiniae LMG 26467(T), while the sequencing of the 16S-23S rDNA intergenic region (ITS) and housekeeping genes glnII, atpD, dnaK, gyrB, recA and rpoB divided them into three groups. North Ossetian isolates (in contrast to the Armenian ones) were clustered separately from the type strain LMG 26467(T). However, all isolates were classified as T. robiniae because the DNA-DNA relatedness between them and the type strain LMG 26467(T) was 69.6% minimum. Two symbiosis-related genes (nodM and nodT) were amplified in all isolated Tardiphaga strains. It was shown that the nodM gene phylogeny is similar to that of ITS and housekeeping genes. The presence of the other symbiosis-related genes in described Tardiphaga strains, which is recently described genus of rhizobia, as well as their ability to form nodules on any plants are under investigation.

Bosea vaviloviae sp. nov., a new species of slow-growing rhizobia isolated from nodules of the relict species Vavilovia formosa (Stev.) Fed.

The Gram-negative, rod-shaped slow-growing strains Vaf-17, Vaf-18(T) and Vaf-43 were isolated from the nodules of Vavilovia formosa plants growing in the hard-to-reach mountainous region of the North Ossetian State Natural Reserve (north Caucasus, Russian Federation). The sequencing of 16S rDNA (rrs), ITS region and five housekeeping genes (atpD, dnaK, recA, gyrB and rpoB) showed that the isolated strains were most closely related to the species Bosea lathyri (class Alphaproteobacteria, family Bradyrhizobiaceae) which was described for isolates from root nodules of Lathyrus latifolius. However the sequence similarity between the isolated strains and the type strain B. lathyri LMG 26379(T) for the ITS region was 90 % and for the housekeeping genes it was ranged from 92 to 95 %. All phylogenetic trees, except for the rrs-dendrogram showed that the isolates from V. formosa formed well-separated clusters within the Bosea group. Differences in phenotypic properties of the B. lathyri type strain and the isolates from V. formosa were studied using the microassay system GENIII MicroPlate BioLog. Whole-cell fatty acid analysis showed that the strains Vaf-17, Vaf-18(T) and Vaf-43 had notable amounts of C16:0 (4.8-6.0 %), C16:0 3-OH (6.4-6.6 %), C16:1 ω5c (8.8-9.0 %), C17:0 cyclo (13.5-13.9 %), C18:1 ω7c (43.4-45.4 %), C19:0 cyclo ω8c (10.5-12.6 %) and Summed Feature (SF) 3 (6.4-8.0 %). The DNA-DNA relatedness between the strains Vaf-18(T) and B. lathyri LMG 26379(T) was 24.0 %. On the basis of genotypic and phenotypic analysis a new species Bosea vaviloviae sp. nov. (type strain RCAM 02129(T) = LMG 28367(T) = Vaf-18(T)) is proposed.

Genetic diversity of rhizobia isolated from nodules of the relic species Vavilovia formosa (Stev.) Fed.

Sixteen bacterial strains were isolated from root nodules of Vavilovia formosa plants originated from the North Ossetian State Natural Reserve (Caucasus, Russia). Phylogenetic analysis of these strains was performed using partial 16S rRNA gene and internally transcribed spacer (ITS) sequences. The results showed that the isolates belong to three families of root nodule bacteria. Twelve of them were related to the genus Rhizobium (family Rhizobiaceae) but four strains can be most probably identified as Phyllobacterium-related (family Phyllobacteriaceae), Bosea- and Rhodopseudomonas-related (family Bradyrhizobiaceae). Amplified fragment length polymorphism clustering was congruent with ITS phylogeny but displayed more variability for Rhizobium isolates, which formed a single group at the level of 30 % similarity. We expect that the isolates obtained can belong to new taxa at genus, species or subspecies levels. The results of PCR amplification of the nodulation genes nodC and nodX showed their presence in all Rhizobium isolates and one Rhodopseudomonas-related isolate. The nodC gene sequences of V. formosa isolates were closely related to those of the species Rhizobium leguminosarum bv. viciae but formed separate clusters and did not intermingle with any reference strains. The presence of the nodX gene, which is necessary for nodulation of Afghan peas (Pisum sativum L.) originated from the Middle East, allows the speculation that these wild-type pea cultivars may be the closest existing relatives of V. formosa. Thus, the studies of genetic diversity and symbiotic genes of V. formosa microsymbionts provide the primary information about their phylogeny and contribute to the conservation of this relict leguminous species.