Vitaly V. Kadnikov

Characterization of Melioribacter roseus gen. nov., sp. nov., a novel facultatively anaerobic thermophilic cellulolytic bacterium from the class Ignavibacteria, and a proposal of a novel bacterial phylum Ignavibacteriae

A novel moderately thermophilic, facultatively anaerobic chemoorganotrophic bacterium strain P3M-2(T) was isolated from a microbial mat developing on the wooden surface of a chute under the flow of hot water (46°C) coming out of a 2775-m-deep oil exploration well (Tomsk region, Russia). Strain P3M-2(T) is a moderate thermophile and facultative anaerobe growing on mono-, di- or polysaccharides by aerobic respiration, fermentation or by reducing diverse electron acceptors [nitrite, Fe(III), As(V)]. Its closest cultivated relative (90.8% rRNA gene sequence identity) is Ignavibacterium album, the only chemoorganotrophic member of the phylum Chlorobi. New genus and species Melioribacter roseus are proposed for isolate P3M-2(T) . Together with I. album, the new organism represents the class Ignavibacteria assigned to the phylum Chlorobi. The revealed group includes a variety of uncultured environmental clones, the 16S rRNA gene sequences of some of which have been previously attributed to the candidate division ZB1. Phylogenetic analysis of M. roseus and I. album based on their 23S rRNA and RecA sequences confirmed that these two organisms could represent an even deeper, phylum-level lineage. Hence, we propose a new phylum Ignavibacteriae within the Bacteroidetes-Chlorobi group with a sole class Ignavibacteria, two families Ignavibacteriaceae and Melioribacteraceae and two species I. album and M. roseus. This proposal correlates with chemotaxonomic data and phenotypic differences of both organisms from other cultured representatives of Chlorobi. The most essential differences, supported by the analyses of complete genomes of both organisms, are motility, facultatively anaerobic and obligately organotrophic mode of life, the absence of chlorosomes and the apparent inability to grow phototrophically.

Microbial community structure in methane hydrate-bearing sediments of freshwater Lake Baikal.

Gas hydrates in marine sediments have been known for many years but recently hydrates were found in the sediments of Lake Baikal, the largest freshwater basin in the world. Marine gas hydrates are associated with complex microbial communities involved in methanogenesis, methane oxidation, sulfate reduction and other biotransformations. However, the contribution of microorganisms to the formation of gas hydrates remains poorly understood. We examined the microbial communities in the hydrate-bearing sediments and water column of Lake Baikal using pyrosequencing of 16S rRNA genes. Aerobic methanotrophic bacteria dominated the water sample collected at the lake floor in the hydrate-bearing site. The shallow sediments were dominated by Archaea. Methanogens of the orders Methanomicrobiales and Methanosarcinales were abundant, whereas representatives of archaeal lineages known to perform anaerobic oxidation of methane, as well as sulfate-reducing bacteria, were not found. Affiliation of archaea to methanogenic rather than methane-oxidizing lineages was supported by analysis of the sequences of the methyl coenzyme M reductase gene. The deeper sediments located at 85-90 cm depth close to the hydrate were dominated by Bacteria, mostly assigned to Chloroflexi, candidate division JS1 and Caldiserica. Overall, our results are consistent with the biological origin of methane hydrates in Lake Baikal.

Genome sequence and analysis of methylotrophic yeast Hansenula polymorpha DL1

BackgroundHansenula polymorpha DL1 is a methylotrophic yeast, widely used in fundamental studies of methanol metabolism, peroxisome biogenesis and function, and also as a microbial cell factory for production of recombinant proteins and metabolic engineering towards the goal of high temperature ethanol production.ResultsWe have sequenced the 9 Mbp H. polymorpha DL1 genome and performed whole-genome analysis for the H. polymorpha transcriptome obtained from both methanol- and glucose-grown cells. RNA-seq analysis revealed the complex and dynamic character of the H. polymorpha transcriptome under the two studied conditions, identified abundant and highly unregulated expression of 40% of the genome in methanol grown cells, and revealed alternative splicing events. We have identified subtelomerically biased protein families in H. polymorpha, clusters of LTR elements at G + C-poor chromosomal loci in the middle of each of the seven H. polymorpha chromosomes, and established the evolutionary position of H. polymorpha DL1 within a separate yeast clade together with the methylotrophic yeast Pichia pastoris and the non-methylotrophic yeast Dekkera bruxellensis. Intergenome comparisons uncovered extensive gene order reshuffling between the three yeast genomes. Phylogenetic analyses enabled us to reveal patterns of evolution of methylotrophy in yeasts and filamentous fungi.ConclusionsOur results open new opportunities for in-depth understanding of many aspects of H. polymorpha life cycle, physiology and metabolism as well as genome evolution in methylotrophic yeasts and may lead to novel improvements toward the application of H. polymorpha DL-1 as a microbial cell factory.

Genomic Analysis of Melioribacter roseus, Facultatively Anaerobic Organotrophic Bacterium Representing a Novel Deep Lineage within Bacteriodetes/Chlorobi Group

Melioribacter roseus is a moderately thermophilic facultatively anaerobic organotrophic bacterium representing a novel deep branch within Bacteriodetes/Chlorobi group. To better understand the metabolic capabilities and possible ecological functions of M. roseus and get insights into the evolutionary history of this bacterial lineage, we sequenced the genome of the type strain P3M-2T. A total of 2838 open reading frames was predicted from its 3.30 Mb genome. The whole proteome analysis supported phylum-level classification of M. roseus since most of the predicted proteins had closest matches in Bacteriodetes, Proteobacteria, Chlorobi, Firmicutes and deeply-branching bacterium Caldithrix abyssi, rather than in one particular phylum. Consistent with the ability of the bacterium to grow on complex carbohydrates, the genome analysis revealed more than one hundred glycoside hydrolases, glycoside transferases, polysaccharide lyases and carbohydrate esterases. The reconstructed central metabolism revealed pathways enabling the fermentation of complex organic substrates, as well as their complete oxidation through aerobic and anaerobic respiration. Genes encoding the photosynthetic and nitrogen-fixation machinery of green sulfur bacteria, as well as key enzymes of autotrophic carbon fixation pathways, were not identified. The M. roseus genome supports its affiliation to a novel phylum Ignavibateriae, representing the first step on the evolutionary pathway from heterotrophic ancestors of Bacteriodetes/Chlorobi group towards anaerobic photoautotrophic Chlorobi.

The 203 kbp mitochondrial genome of the phytopathogenic fungus Sclerotinia borealis reveals multiple invasions of introns and genomic duplications.

Here we report the complete sequence of the mitochondrial (mt) genome of the necrotrophic phytopathogenic fungus Sclerotinia borealis, a member of the order Helotiales of Ascomycetes. The 203,051 bp long mtDNA of S. borealis represents one of the largest sequenced fungal mt genomes. The large size is mostly determined by the presence of mobile genetic elements, which include 61 introns. Introns contain a total of 125,394 bp, are scattered throughout the genome, and are found in 12 protein-coding genes and in the ribosomal RNA genes. Most introns contain complete or truncated ORFs that are related to homing endonucleases of the LAGLIDADG and GIY-YIG families. Integrations of mobile elements are also evidenced by the presence of two regions similar to fragments of inverton-like plasmids. Although duplications of some short genome regions, resulting in the appearance of truncated extra copies of genes, did occur, we found no evidences of extensive accumulation of repeat sequences accounting for mitochondrial genome size expansion in some other fungi. Comparisons of mtDNA of S. borealis with other members of the order Helotiales reveal considerable gene order conservation and a dynamic pattern of intron acquisition and loss during evolution. Our data are consistent with the hypothesis that horizontal DNA transfer has played a significant role in the evolution and size expansion of the S. borealis mt genome.

Draft genome sequence of the first acid-tolerant sulfate-reducing deltaproteobacterium Desulfovibrio sp. TomC having potential for minewater treatment

The sulfidogenic bacterium Desulfovibrio sp. TomC was isolated from acidic waste at the abandoned gold ore mining site in the Martaiga gold ore belt, Western Siberia. This bacterium, being the first reported acid-tolerant gram-negative sulfate-reducer of the order Deltaproteobacteria, is able to grow at pH as low as 2.5 and is resistant to high concentrations of metals. The draft 5.3 Mb genome sequence of Desulfovibrio sp. TomC has been established and provides the genetic basis for application of this microorganism in bioreactors and other bioremediation schemes for the treatment of metal-containing wastewater.

Composition of the Microbial Communities of Bituminous Constructions at Natural Oil Seeps at the Bottom of Lake Baikal

Microbial communities of two bituminous constructions at the bottom of Lake Baikal in the region of natural oil seeps at a depth of 900 m have been investigated. Construction 8 contained biodegraded hydrocarbons, and construction 3, through which oil seeped, contained material that experienced biodegradation to a lesser degree. The composition of the microbial communities was studied by means of pyrosequencing of 16S rRNA gene fragments. Most of the bacterial 16S rRNA gene sequences identified in both bituminous constructions were attributed to proteobacteria, along with which Actinobacteria, Acidobacteria, Bacteroidetes, and TM7 were revealed. About 40% of the bacterial sequences in bituminous construction 3 belonged to representatives of uncultured groups within the classes Alphaproteobacteria and Betaproteobacteria and the phylum Bacteroidetes. The 16S rRNA gene sequences of archaea belonged to aceticlastic and hydrogenotrophic methanogens of the orders Methanosarcinales, Methanomicrobiales, and Methanobacteriales. The 16S rRNA genes of various groups of bacteria carrying out aerobic biodegradation of aromatic compounds and n-alkanes were found; their compositions differed between the constructions. Neither known groups of denitrifying betaproteobacteria nor known groups of sulfate-reducing deltaproteobacteria capable of carrying out anaerobic degradation of n-alkanes were found, which agrees with the low content of nitrate and sulfate in the water. In the anaerobic zone of bituminous constructions, the processes of biodegradation of hydrocarbons are probably carried out in the absence of alternative electron acceptors by the syntrophic community, including deltaproteobacteria of the genus Syntrophus and methanogenic archaea.

Draft Genome Sequence of Sclerotinia borealis, a Psychrophilic Plant Pathogenic Fungus.

ABSTRACT Sclerotinia borealis is a necrotrophic phytopathogenic fungus notable for its wide host range and environmental persistence. It grows at low temperatures, causing snow mold disease of crop plants. To understand the molecular mechanisms of its pathogenesis and adaptation to the psychrophilic lifestyle, we determined the 39.3-Mb draft genome sequence of S. borealis F-4128.

Stable and Variable Parts of Microbial Community in Siberian Deep Subsurface Thermal Aquifer System Revealed in a Long-Term Monitoring Study

The goal of this work was to study the diversity of microorganisms inhabiting a deep subsurface aquifer system in order to understand their functional roles and interspecies relations formed in the course of buried organic matter degradation. A microbial community of a deep subsurface thermal aquifer in the Tomsk Region, Western Siberia was monitored over the course of 5 years via a 2.7 km deep borehole 3P, drilled down to a Palaeozoic basement. The borehole water discharges with a temperature of ca. 50°C. Its chemical composition varies, but it steadily contains acetate, propionate, and traces of hydrocarbons and gives rise to microbial mats along the surface flow. Community analysis by PCR-DGGE 16S rRNA genes profiling, repeatedly performed within 5 years, revealed several dominating phylotypes consistently found in the borehole water, and highly variable diversity of prokaryotes, brought to the surface with the borehole outflow. The major planktonic components of the microbial community were Desulfovirgula thermocuniculi and Methanothermobacter spp. The composition of the minor part of the community was unstable, and molecular analysis did not reveal any regularity in its variations, except some predominance of uncultured Firmicutes. Batch cultures with complex organic substrates inoculated with water samples were set in order to enrich prokaryotes from the variable part of the community. PCR-DGGE analysis of these enrichments yielded uncultured Firmicutes, Chloroflexi, and Ignavibacteriae. A continuous-flow microaerophilic enrichment culture with a water sample amended with acetate contained Hydrogenophilus thermoluteolus, which was previously detected in the microbial mat developing at the outflow of the borehole. Cultivation results allowed us to assume that variable components of the 3P well community are hydrolytic organotrophs, degrading buried biopolymers, while the constant planktonic components of the community degrade dissolved fermentation products to methane and CO2, possibly via interspecies hydrogen transfer. Occasional washout of minor community components capable of oxygen respiration leads to the development of microbial mats at the outflow of the borehole where residual dissolved fermentation products are aerobically oxidized. Long-term community analysis with the combination of molecular and cultivation techniques allowed us to characterize stable and variable parts of the community and propose their environmental roles.

Effect of metal concentration on the microbial community in acid mine drainage of a polysulfide ore deposit

The composition of microbial communities of acid mine drainage (AMD) in two wells drilled in the terrace of the Sherlovaya Gora open-cast polymetallic ores mine (Eastern Siberia) was studied. While drainage water filling two wells, ShG14-1 and ShG14-8, had similar values of pH (2.6), Eh (447–494 mV), and temperature (6.5°C), the water in the first well contained more metals and sulfate. The water in ShG14-1 and ShG14-8 contained, respectively, 1898 and 434 mg/L of iron, 734 and 49 mg/L of manganese, 81 and 7 mg/L of copper, 3597 and 787 mg/L of zinc, and 15990 and 3632 mg/L of sulfate. Molecular analysis of the microbial communities was performed using pyrosequencing of the 16S rRNA gene fragments. The ShG14-8 microbial community included such bacterial taxa typically found in AMD sites as Gallionella (38.8% of total 16S rRNA gene sequences), Ferrovum (4.4%), Acidiphilium (9.1%), Acidisphaera (8.2%), Acidithiobacillus (7.2%), and Leptospirillum (4.6%). In the ShG14-1 sample with higher content of metals, strict acidophiles Acidithiobacillus (16.0%) and Leptospirillum (25.4%) were more abundant, while Gallionella, Ferrovum, Acidiphilium and Acidisphaera were almost absent. Ferrimicrobium (16.8%) and Sulfobacillus (1.4%) were detected in ShG14-1 but not in ShG14-8. Thus, the increase in concentration of metals in the acid mine drainage water under the same value of total acidity substantially altered the composition of the microbial community, preventing the development of “moderate” alpha- and beta-proteobacterial acidophiles, so that the community was dominated by the bacteria characteristic of the extremely acidic drainage waters.