Evgeny E. Andronov

[Macro- and microevolution of bacteria in symbiotic systems].

Using the examples of diverse interactions among prokaryotes and eukaryotes, the relationships between molecular and population mechanisms of evolution of symbiotic bacteria are addressed. Their circulation in host-environment systems activates microevolutionary factors that direct combinative or reductive genome evolution in facultative, ecologically obligatory, and genetically obligatory symbioses. It is shown on the example of symbiosis of rhizobia with legumes, that due to intensive systemic intra-genome rearrangements and horizontal gene transfer, two types of gene systems evolve in these bacteria: (1) controlling the pathogenesis-like processes of host recognition and penetration and (2) responsible for mutualistic interactions that are related to nitrogen fixation and its transfer to the host. The evolution of gene systems of type 1 is directed by individual (Darwinian, frequency-dependent) selection, which is responsible for gene-for-gene interactions between the partners. In the evolution of the type 2 systems, group (interdeme, kin) selection plays the key role, being responsible for the development of bacterial traits beneficial for the host. It is shown that evolution of mutualism can be described in terms of biological altruism, whose regularities are common for intraspecific and interspecific relationships. Macroevolutionary rearrangements of bacterial genomes result from the structural changes in their populations, wherein various selection modes are combined with stochastic processes (genetic drift, population waves) induced in the symbiotic systems.

Comparative Analysis of Prokaryotic Communities Associated with Organic and Conventional Farming Systems

One of the most important challenges in agriculture is to determine the effectiveness and environmental impact of certain farming practices. The aim of present study was to determine and compare the taxonomic composition of the microbiomes established in soil following long-term exposure (14 years) to a conventional and organic farming systems (CFS and OFS accordingly). Soil from unclared forest next to the fields was used as a control. The analysis was based on RT-PCR and pyrosequencing of 16S rRNA genes of bacteria and archaea. The number of bacteria was significantly lower in CFS than in OFS and woodland. The highest amount of archaea was detected in woodland, whereas the amounts in CFS and OFS were lower and similar. The most common phyla in the soil microbial communities analyzed were Proteobacteria (57.9%), Acidobacteria (16.1%), Actinobacteria (7.9%), Verrucomicrobia (2.0%), Bacteroidetes (2.7%) and Firmicutes (4.8%). Woodland soil differed from croplands in the taxonomic composition of microbial phyla. Croplands were enriched with Proteobacteria (mainly the genus Pseudomonas), while Acidobacteria were detected almost exclusively in woodland soil. The most pronounced differences between the CFS and OFS microbiomes were found within the genus Pseudomonas, which significantly (p<0,05) increased its number in CFS soil compared to OFS. Other differences in microbiomes of cropping systems concerned minor taxa. A higher relative abundance of bacteria belonging to the families Oxalobacteriaceae, Koribacteriaceae, Nakamurellaceae and genera Ralstonia, Paenibacillus and Pedobacter was found in CFS as compared with OFS. On the other hand, microbiomes of OFS were enriched with proteobacteria of the family Comamonadaceae (genera Hylemonella) and Hyphomicrobiaceae, actinobacteria from the family Micrococcaceae, and bacteria of the genera Geobacter, Methylotenera, Rhizobium (mainly Rhizobium leguminosarum) and Clostridium. Thus, the fields under OFS and CFS did not differ greatly for the composition of the microbiome. These results, which were also confirmed by cluster analysis, indicated that microbial communities in the field soil do not necessarily differ largely between conventional and organic farming systems.

Microvirga ossetica sp. nov., a species of rhizobia isolated from root nodules of the legume species Vicia alpestris Steven

Gram-stain-negative strains V5/3MT, V5/5K, V5/5M and V5/13 were isolated from root nodules of Vicia alpestris plants growing in the North Ossetia region (Caucasus). Sequencing of the partial 16S rRNA gene (rrs) and four housekeeping genes (dnaK, gyrB, recA and rpoB) showed that the isolates from V. alpestris were most closely related to the species Microvirga zambiensis (order Rhizobiales, family Methylobacteriaceae) which was described for the single isolate from root nodule of Listia angolensis growing in Zambia. Sequence similarities between the Microvirga-related isolates and M. zambiensis WSM3693T ranged from 98.5 to 98.7 % for rrs and from 79.7 to 95.8 % for housekeeping genes. Cellular fatty acids of the isolates V5/3MT, V5/5K, V5/5M and V5/13 included important amounts of C18 : 1ω7c (54.0-67.2 %), C16 : 0 (6.0-7.8 %), C19 : 0 cyclo ω8c (3.1-10.2 %), summed feature 2 (comprising one or more of iso-C16 : 1 I, C14 : 0 3-OH and unknown ECL 10.938, 5.8-22.5 %) and summed feature 3 (comprising C16 : 1ω7c and/or iso-C15 : 02-OH, 2.9-4.0 %). DNA-DNA hybridization between the isolate V5/3MT and M. zambiensis WSM3693T revealed DNA-DNA relatedness of 35.3 %. Analysis of morphological and physiological features of the novel isolates demonstrated their unique phenotypic profile in comparison with reference strains from closely related species of the genus Microvirga. On the basis of genotypic and phenotypic analysis, a novel species named Microvirga ossetica sp. nov. is proposed. The type strain is V5/3MT (=LMG 29787T=RCAM 02728T). Three additional strains of the species are V5/5K, V5/5M and V5/13.

Recent results from NA61/SHINE

Abstract The NA61/SHINE fixed-target experiment at the CERN SPS studies the onset of deconfinement and searches for the critical point of strongly interacting matter by measuring hadron production as a function of the collision energy and the colliding system size. This contribution summarises recent results on hadron spectra and fluctuations, in particular new results on charged kaon production in 7 Be+ 9 Be collisions. Also an overview of the proposed future program of NA61/SHINE is presented.

The Evolutionary Moulding in plant-microbial symbiosis: matching population diversity of rhizobial nodA and legume NFR5 genes

We propose the Evolutionary Moulding hypothesis that population diversities of partners in nitrogen-fixing rhizobium-legume symbiosis are matched, and tested it in nucleotide polymorphism of symbiotic genes encoding two components of the plant-bacteria signalling system. The first component is the rhizobial nodA acyltransferase involved in the fatty acid tail decoration of Nod factor (rhizobia signalling molecule). The second component is the plant NFR5 receptor, putatively required for Nod-factor binding. We collected three wild growing legume species together with soil samples adjacent to the roots (soil pool) from one large 25-year fallow: Vicia sativa, Lathyrus pratensis and Trifolium hybridum nodulated by one of the two Rhizobium leguminosarum biovars (viciae and trifolii). For each plant species we prepared three pools for DNA extraction: the plant pool (30 plant indiv.), the nodule pool (90 nodules) and the soil pool (30 samples). NFR5 gene libraries from the plant pool and nodA gene libraries from nodule and soil pools were sequenced by Sanger technology and High-throughput pyrosequencing, respectively. Analysis of the data demonstrated concordance in population diversities of one symbiotic partner (rhizobia) the second partner (legume host), in line with the Evolutionary Moulding hypothesis. This effect was evinced by the following observations for each plant species: (1) significantly increased diversity in the nodule nodA popset (set of gene sequences derived from the nodule population) compared to the soil popset; (2) a monotonic relationship between the diversity in the plant NFR5 gene popset and the nodule rhizobial nodA gene popset; and (3) higher topological similarity of the NFR5 gene tree with the nodA gene tree of the nodule popset, than with the nodA gene tree of the soil popset. Both nonsynonymous diversity and Tajima’s D were increased in the nodule popsets compared to the soil popsets, consistent with relaxation of negative selection and/or admixture of balancing selection underlying the Evolutionary Moulding effect. We propose that the observed genetic concordance arises from the selection of particular characteristics of the nodule nodA genes by the host plant.

The preservation of microbial DNA in archived soils of various genetic types

This study is a comparative analysis of samples of archived (stored for over 70–90 years) and modern soils of two different genetic types–chernozem and sod-podzolic soils. We revealed a reduction in biodiversity of archived soils relative to their modern state. Particularly, long-term storage in the museum exerted a greater impact on the microbiomes of sod-podzolic soils, while chernozem samples better preserved the native community. Thus, the persistence of microbial DNA in soil is largely determined by the physico-chemical characteristics that differ across soil types. Chernozems create better conditions for the long-term DNA preservation than sod-podzolic soils. This results in supposedly higher levels of biodiversity conservation in the microbiomes of chernozem with preservation of major microbial taxa dominant in the modern (control) soil samples, which makes archived chernozems a promising object for paleosoil studies.

Does the Miocene-Pliocene relict legume Oxytropis triphylla form nitrogen-fixing nodules with a combination of bacterial strains?

Abstract Four pairs of strains were isolated from four individual root nodules collected from different plants of the Miocene-Pliocene relict legume Oxytropis triphylla growing in the Baikal region (one nodule – one pair of strains). Identification of these strains by the 16S rRNA gene sequencing showed that one strain of each pair was fast-growing and phylogenetically closest to the type strains Phyllobacterium endophyticum PEPV15T (98.8–99.5% 16S rDNA similarity) and Phyllobacterium bourgognense STM 201T (98.8–99.3% 16S rDNA similarity). The other strain was slow-growing and closely related to the type strains Bosea vestrisii 34635T and Bosea eneae 34614T (99.5–99.8% 16S rDNA similarity). Results of the atpD gene sequencing suggested that Phyllobacterium-related isolates most probably belong to a new species of Phyllobacterium. It is known that none of the currently described strains of P. endophyticum, P. bourgognense (family Phyllobacteriaceae), B. vestrisii and B. eneae (family Bradyrhizobiaceae) can induce root nodules, but several symbiotic genes were found in the representatives of these two genera. Thus we hypothesize that the pairs of strains isolated from O. triphylla root nodules can be co-microsymbionts having complementary sets of symbiotic genes and their simultaneous presence in roots is required for efficient nodulation of the host plant. The whole genome sequencing of the studied strains is in progress to find and compare symbiotic genes.

Characteristics of Natural Selection in Populations of Nodule Bacteria (Rhizobium leguminosarum) Interacting With Different Host Plants

Using high throughput sequencing of the nodA gene, we studied the population dynamics of Rhizobium leguminosarum (bv. viciae, bv. trifolii) in rhizospheric and nodular subpopulations associated with the leguminous plants representing different cross-inoculation groups (Vicia sativa, Lathyrus pratensis of the vetch/vetchling/pea group and Trifolium hybridum of the clover group). The “rhizosphere → nodules” transitions result in either an increase or decrease in the frequencies of 10 of the 23 operational taxonomic units (OTUs) (which were identified with 95% similarity) depending on the symbiotic specificity and phylogenetic positions of OTUs. Statistical and bioinformatical analysis of the population structures suggest that the type of natural selection responsible for these changes may be diversifying at the whole-population level and frequency-dependent at the OTU-specific level, ensuring the divergent evolution of rhizobia interacting with different host species.

Investigation of the core microbiome in main soil types from the East European plain

The main goal of modern microbial ecology is to determine the key factors influencing the global diversity of microorganisms. Because of their complexity, soil communities are largely underexplored in this context. We studied soil genesis (combination of various soil-forming processes, specific to a particular soil type) that is driven by microbial activity. To investigate the interrelation between soil type and microbial diversity, we analyzed six soil types that are common in Russia, the Crimea, and Kazakhstan using 16S rDNA pyrosequencing. Soils of different types varied in the taxonomic composition of microbial communities. Their core microbiomes comprised 47 taxa within the orders Solirubrobacteriales and Hyphomicrobiaceae and the Gaiellaceae family. Two species from Bradyrhizobiaceae and Solirubrobactriaceae were present in all samples, whereas most other taxa were soil-type specific. Multiple resampling analysis revealed that two random soil samples from the same soil type shared more taxa than two samples from different types. The differences in community composition were mostly affected by the variation in pH values and exchangeable potassium content. The results show that data on the soil microbiome could be used for soil identification and clarification of their taxonomic position.

Divergent Evolution of Symbiotic Bacteria: Rhizobia of the Relic Legume Vavilovia formosa Form an Isolated Group within Rhizobium leguminosarum bv. viciae

Comparative sequence analysis of symbiotic genes (nodA, nodC, nodD, nifH), which are elements of accessory component of the rhizobial genome, demonstrated that the strains of Rhizobium leguminosarum bv. viciae, isolated from the nodules of a relic legume, Vavilovia formosa, the closest relative of hypothetical common ancestor of the tribe Fabeae, represented a group separated from the strains of R. leguminosarum bv. viciae, isolated from other representatives of this tribe (Vicia, Lathyrus, Pisum, Lens). No isolation was observed relative to the genes representing the core component of the rhizobial genome (16S rDNA, ITS, glnII) or relative to host specificity of the rhizobia. The data obtained suggest that sequence divergence of symbiotic genes marks the initial stage of sympatric speciation, which can be classified as the isolation of the relic “vaviloviae” symbiotype, a possible evolutionary precursor of the “viciae” biotype.