Impacts of Natural Selection on Evolution of Core and Symbiotically Specialized (sym) Genes in the Polytypic Species Neorhizobium galegae.

Nodule bacteria (rhizobia) represent a suitable model to address a range of fundamental genetic problems, including the impacts of natural selection on the evolution of symbiotic microorganisms. Rhizobia possess multipartite genomes in which symbiotically specialized (sym) genes differ from core genes in their natural histories. Diversification of sym genes is responsible for rhizobia microevolution, which depends on host-induced natural selection. By contrast, diversification of core genes is responsible for rhizobia speciation, which occurs under the impacts of still unknown selective factors. In this paper, we demonstrate that in goat's rue rhizobia (Neorhizobium galegae) populations collected at North Caucasus, representing two host-specific biovars orientalis and officianalis (N2-fixing symbionts of Galega orientalis and G. officinalis), the evolutionary mechanisms are different for core and sym genes. In both N. galegae biovars, core genes are more polymorphic than sym genes. In bv. orientalis, the evolution of core genes occurs under the impacts of driving selection (dN/dS > 1), while the evolution of sym genes is close to neutral (dN/dS ≈ 1). In bv. officinalis, the evolution of core genes is neutral, while for sym genes, it is dependent on purifying selection (dN/dS < 1). A marked phylogenetic congruence of core and sym genes revealed using ANI analysis may be due to a low intensity of gene transfer within and between N. galegae biovars. Polymorphism in both gene groups and the impacts of driving selection on core gene evolution are more pronounced in bv. orientalis than in bv. officianalis, reflecting the diversities of their respective host plant species. In bv. orientalis, a highly significant (P0 < 0.001) positive correlation is revealed between the p-distance and dN/dS values for core genes, while in bv. officinalis, this correlation is of low significance (0.05 < P0 < 0.10). For sym genes, the correlation between p-distance and dN/dS values is negative in bv. officinalis but is not revealed in bv. orientalis. These data, along with the functional annotation of core genes implemented using Gene Ontology tools, suggest that the evolution of bv. officinalis is based mostly on adaptation for in planta niches while in bv. orientalis, evolution presumably depends on adaptation for soil niches. New insights into the tradeoff between natural selection and genetic diversity are presented, suggesting that gene nucleotide polymorphism may be extended by driving selection only in ecologically versatile organisms capable of supporting a broad spectrum of gene alleles in their gene pools.

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.

RIAM: A Universal Accessible Protocol for the Isolation of High Purity DNA from Various Soils and Other Humic Substances.

A single universal open protocol RIAM (named after Research Institute for Agricultural Microbiology) for the isolation of high purity DNA from different types of soils and other substrates (high and low in humic, clay content, organic fertilizer, etc.) is proposed. The main features of the RIAM protocol are the absence of the sorption-desorption stage on silica columns, the use of high concentrations of phosphate in buffers, which prevents DNA sorption on minerals, and DNA precipitation using CTAB. The performance of RIAM was compared with a reference commercial kit and showed very good results in relation to the purity and quantity of DNA, as well as the absence of inhibitory activity on PCR. In all cases, the RIAM ensured the isolation of DNA in quantities much greater than the commercial kit without the effect of PCR inhibition up to 50 ng DNA per reaction in a volume of 15 µL. The latter circumstance along with the ability of the protocol to extract low molecular weight DNA fractions makes the method especially suitable for those cases where quantitative assessments, detection of minor components of soil microbiota, and completeness of isolation of all DNA fractions are required.

Be aware of the allele-specific bias and compositional effects in multi-template PCR.

High-throughput sequencing of amplicon libraries is the most widespread and one of the most effective ways to study the taxonomic structure of microbial communities, even despite growing accessibility of whole metagenome sequencing. Due to the targeted amplification, the method provides unparalleled resolution of communities, but at the same time perturbs initial community structure thereby reducing data robustness and compromising downstream analyses. Experimental research of the perturbations is largely limited to comparative studies on different PCR protocols without considering other sources of experimental variation related to characteristics of the initial microbial composition itself. Here we analyse these sources and demonstrate how dramatically they effect the relative abundances of taxa during the PCR cycles. We developed the mathematical model of the PCR amplification assuming the heterogeneity of amplification efficiencies and considering the compositional nature of data. We designed the experiment-five consecutive amplicon cycles (22-26) with 12 replicates for one real human stool microbial sample-and estimated the dynamics of the microbial community in line with the model. We found the high heterogeneity in amplicon efficiencies of taxa that leads to the non-linear and substantial (up to fivefold) changes in relative abundances during PCR. The analysis of possible sources of heterogeneity revealed the significant association between amplicon efficiencies and the energy of secondary structures of the DNA templates. The result of our work highlights non-trivial changes in the dynamics of real-life microbial communities due to their compositional nature. Obtained effects are specific not only for amplicon libraries, but also for any studies of metagenome dynamics.

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.

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.

Stable and artifact-resistant decoding of 3D hand trajectories from ECoG signals using the generalized additive model.

OBJECTIVE: The key criterion for reliability of brain-computer interface (BCI) devices is their stability and robustness in natural environments in the presence of spurious signals and artifacts. APPROACH: To improve stability and robustness, a generalized additive model (GAM) is proposed for BCI decoder identification. Together with partial least squares (PLS), GAM can be applied to treat high-dimensional data and it is compatible with real-time applications. For evaluation of prediction quality, along with standard criteria such as Pearson correlation, root mean square error (RMSE), mean absolute error (MAE), additional criteria, mean absolute differential error (MADE) and dynamic time warping (DTW) distance, are chosen. These criteria reflect the smoothness and dissimilarity of the predicted and observed signals in the presence of phase desynchronization. MAIN RESULTS: The efficiency of the GAM-PLS model is tested on the publicly available database of simultaneous recordings of the continuous three-dimensional hand trajectories and epidural electrocorticogram signals of the Japanese macaque. GAM-PLS outperforms the generic PLS and improves the evaluation criteria: 22% (Pearson correlation), 8% (RMSE), 13% (MAE), 31% (MADE), 20% (DTW). SIGNIFICANCE: Motor-related BCIs are systems to improve the quality of life of individuals with severe motor disabilities. The improvement of the reliability of the BCI decoder is an important step toward real-life applications of BCI technologies.

Iterative N-way partial least squares for a binary self-paced brain-computer interface in freely moving animals.

In this paper a tensor-based approach is developed for calibration of binary self-paced brain-computer interface (BCI) systems. In order to form the feature tensor, electrocorticograms, recorded during behavioral experiments in freely moving animals (rats), were mapped to the spatial-temporal-frequency space using the continuous wavelet transformation. An N-way partial least squares (NPLS) method is applied for tensor factorization and the prediction of a movement intention depending on neuronal activity. To cope with the huge feature tensor dimension, an iterative NPLS (INPLS) algorithm is proposed. Computational experiments demonstrated the good accuracy and robustness of INPLS. The algorithm does not depend on any prior neurophysiological knowledge and allows fully automatic system calibration and extraction of the BCI-related features. Based on the analysis of time intervals preceding the BCI events, the calibration procedure constructs a predictive model of control. The BCI system was validated by experiments in freely moving animals under conditions close to those in a natural environment.

Classification of multi-modal data in a self-paced binary BCI in freely moving animals.

The goal of the present article is to compare different classifiers using multi-modal data analysis in a binary self-paced BCI. Individual classifiers were applied to multi-modal neuronal data which was projected to a low dimensional space of latent variables using the Iterative N-way Partial Least Squares algorithm. To create a multi-way feature array, electrocorticograms (ECoG) recorded from animal brains were mapped to the spatial-temporal-frequency space using continuous wavelet transformation. To compare the classifiers BCI experiments were simulated. For this purpose we used 9 recordings from behavioral experiments previously recorded in rats free to move in a nature like environment.