Genomic analysis of Caldithrix abyssi, the thermophilic anaerobic bacterium of the novel bacterial phylum Calditrichaeota

The genome of Caldithrix abyssi, the first cultivated representative of a phylum-level bacterial lineage, was sequenced within the framework of Genomic Encyclopedia of Bacteria and Archaea (GEBA) project. The genomic analysis revealed mechanisms allowing this anaerobic bacterium to ferment peptides or to implement nitrate reduction with acetate or molecular hydrogen as electron donors. The genome encoded five different [NiFe]- and [FeFe]-hydrogenases, one of which, group 1 [NiFe]-hydrogenase, is presumably involved in lithoheterotrophic growth, three other produce H2 during fermentation, and one is apparently bidirectional. The ability to reduce nitrate is determined by a nitrate reductase of the Nap family, while nitrite reduction to ammonia is presumably catalyzed by an octaheme cytochrome c nitrite reductase εHao. The genome contained genes of respiratory polysulfide/thiosulfate reductase, however, elemental sulfur and thiosulfate were not used as the electron acceptors for anaerobic respiration with acetate or H2, probably due to the lack of the gene of the maturation protein. Nevertheless, elemental sulfur and thiosulfate stimulated growth on fermentable substrates (peptides), being reduced to sulfide, most probably through the action of the cytoplasmic sulfide dehydrogenase and/or NAD(P)-dependent [NiFe]-hydrogenase (sulfhydrogenase) encoded by the genome. Surprisingly, the genome of this anaerobic microorganism encoded all genes for cytochrome c oxidase, however, its maturation machinery seems to be non-operational due to genomic rearrangements of supplementary genes. Despite the fact that sugars were not among the substrates reported when C. abyssi was first described, our genomic analysis revealed multiple genes of glycoside hydrolases, and some of them were predicted to be secreted. This finding aided in bringing out four carbohydrates that supported the growth of C. abyssi: starch, cellobiose, glucomannan and xyloglucan. The genomic analysis demonstrated the ability of C. abyssi to synthesize nucleotides and most amino acids and vitamins. Finally, the genomic sequence allowed us to perform a phylogenomic analysis, based on 38 protein sequences, which confirmed the deep branching of this lineage and justified the proposal of a novel phylum Calditrichaeota.The work conducted by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported under Contract No. DE-AC02-05CH11231. OS and MSG were supported by the Russian Science Foundation (RSF, grant 14-24-00155). EB-O and SG were supported by the RSF grant 14-24-00165. IK, NC, AL, and MM were supported by the Russian Foundation for Basic Research grant 14-04-00503.http://www.frontiersin.orgam2017Biochemistr

Desulfonatronospira sulfatiphila sp. Nov., and Desulfitispora elongata sp. nov., two novel haloalkaliphilic sulfidogenic bacteria from soda lakes

Two novel haloalkaliphilic bacteria with dissimilatory sulfidogenic metabolism were recovered from syntrophic associations obtained from anaerobic sediments of hypersaline soda lakes in Kulunda Steppe (Altai, Russia). Strain ASO3-2T was a member of a sulfidogenic syntrophic association oxidizing acetate at extremely haloalkaline conditions, and was isolated in pure culture using formate as electron donor and sulfate as electron acceptor. It was identified as representing a novel member of the genus Desulfonatronospira within the Deltaproteobacteria. In contrast to the two known species of this genus, the novel isolate was able to grow with formate as electron donor and sulfate, as well as with sulfite, as electron acceptor. Strain Acr1T was a minor component in a soda lake syntrophic association converting benzoate to methane and acetate. It became dominant in a subculture fed with crotonate. While growing on crotonate, strain Acr1T formed unusually long cells filled with polyhydroxyalkanoate-like granules. Its metabolism was limited to fermentation of crotonate and pyruvate and the ability to utilize thiosulfate and sulfur/polysulfide as electron acceptor. Strain Acr1T was identified as representing a novel member of the genus Desulfitispora in the class Clostridia. Both isolates were obligately haloalkaliphilic with extreme salt tolerance. On the basis of phenotypic and phylogenetic analyses, the novel sulfidogenic isolates from soda lakes are proposed to represent two novel species: Desulfonatronospira sulfatiphila sp. nov. (ASO3-2T=DSM 100427=UNIQEM U993T) and Desulfitispora elongata sp. nov. (Acr1T=DSM 29990=UNIQEM U994T).Accepted Author ManuscriptBT/Environmental Biotechnolog

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 five years via a 2.7 km deep borehole 3P, drilled down to a Palaeozoic basement. The borehole water discharges with a temperature of ca. 50oC. 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 five 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

Diversity and Metabolic Potential of the Terrestrial Mud Volcano Microbial Community with a High Abundance of Archaea Mediating the Anaerobic Oxidation of Methane

Terrestrial mud volcanoes (TMVs) are important natural sources of methane emission. The microorganisms inhabiting these environments remain largely unknown. We studied the phylogenetic composition and metabolic potential of the prokaryotic communities of TMVs located in the Taman Peninsula, Russia, using a metagenomic approach. One of the examined sites harbored a unique community with a high abundance of anaerobic methane-oxidizing archaea belonging to ANME-3 group (39% of all 16S rRNA gene reads). The high number of ANME-3 archaea was confirmed by qPCR, while the process of anaerobic methane oxidation was demonstrated by radioisotopic experiments. We recovered metagenome-assembled genomes (MAGs) of archaeal and bacterial community members and analyzed their metabolic capabilities. The ANME-3 MAG contained a complete set of genes for methanogenesis as well as of ribosomal RNA and did not encode proteins involved in dissimilatory nitrate or sulfate reduction. The presence of multiheme c-type cytochromes suggests that ANME-3 can couple methane oxidation with the reduction of metal oxides or with the interspecies electron transfer to a bacterial partner. The bacterial members of the community were mainly represented by autotrophic, nitrate-reducing, sulfur-oxidizing bacteria, as well as by fermentative microorganisms. This study extends the current knowledge of the phylogenetic and metabolic diversity of prokaryotes in TMVs and provides a first insight into the genomic features of ANME-3 archaea.</jats:p

Caldanaerobacter uzonensis sp. nov., an anaerobic, thermophilic, heterotrophic bacterium isolated from a hot spring

An anaerobic thermophilic bacterium, strain K67T, was isolated from a terrestrial hot spring of Uzon Caldera, Kamchatka Peninsula. Analysis of the 16S rRNA gene sequence revealed that the novel isolate belongs to the genusCaldanaerobacter, with 95 % 16S rRNA gene sequence similarity toCaldanaerobacter subterraneussubsp.subterraneusSEBR 7858T, suggesting that it represents a novel species of the genusCaldanaerobacter. Strain K67Twas characterized as an obligate anaerobe, a thermophile (growth at 50–75 °С; optimum 68–70 °C), a neutrophile (growth at pH25 °C 4.8–8.0; optimum pH25 °C 6.8) and an obligate organotroph (growth by fermentation of various sugars, peptides and polysaccharides). Major fermentation products were acetate, H2and CO2; ethanol, lactate andl-alanine were formed in smaller amounts. Thiosulfate stimulated growth and was reduced to hydrogen sulfide. Nitrate, sulfate, sulfite and elemental sulfur were not reduced and did not stimulate growth. Thus, according to the strain's phylogenetic position and phenotypic novelties (lower upper limit of temperature range for growth, the ability to grow on arabinose, the inability to reduce elemental sulfur and the formation of alanine as a minor fermentation product), the novel speciesCaldanaerobacter uzonensissp. nov. is proposed, with the type strain K67T(=DSM 18923T=VKM В-2408T).</jats:p

PCR-Based Identification of Hyperthermophilic Archaea of the Family Thermococcaceae

A method for rapid detection and identification of hyperthermophilic archaea of the family Thermococcaceae based on PCR amplification of 16S rRNA gene fragments with primers TcPc 173F (5′-TCCCCCATAGGYCTGRGGTACTGGAAGGTC-3′) and TcPc 589R (5′-GCCGTGRGATTTCGCCAGGGACTTACGGGC-3′) was developed and used for identification of new isolates

Diversity, Methane Oxidation Activity, and Metabolic Potential of Microbial Communities in Terrestrial Mud Volcanos of the Taman Peninsula

Microbial communities of terrestrial mud volcanoes are involved in aerobic and anaerobic methane oxidation, but the biological mechanisms of these processes are still understudied. We have investigated the taxonomic composition, rates of methane oxidation, and metabolic potential of microbial communities in five mud volcanoes of the Taman Peninsula, Russia. Methane oxidation rates measured by the radiotracer technique varied from 2.0 to 460 nmol CH4 cm−3 day−1 in different mud samples. This is the first measurement of high activity of microbial methane oxidation in terrestrial mud volcanos. 16S rRNA gene amplicon sequencing has shown that Bacteria accounted for 65–99% of prokaryotic diversity in all samples. The most abundant phyla were Pseudomonadota, Desulfobacterota, and Halobacterota. A total of 32 prokaryotic genera, which include methanotrophs, sulfur or iron reducers, and facultative anaerobes with broad metabolic capabilities, were detected in relative abundance >5%. The most highly represented genus of aerobic methanotrophs was Methyloprofundus reaching 36%. The most numerous group of anaerobic methanotrophs was ANME-2a-b (Ca. Methanocomedenaceae), identified in 60% of the samples and attaining relative abundance of 54%. The analysis of the metagenome-assembled genomes of a community with high methane oxidation rate indicates the importance of CO2 fixation, Fe(III) and nitrate reduction, and sulfide oxidation. This study expands current knowledge on the occurrence, distribution, and activity of microorganisms associated with methane cycle in terrestrial mud volcanoes

Complete Genome Sequence of the Anaerobic, Protein-Degrading Hyperthermophilic Crenarchaeon Desulfurococcus kamchatkensis▿ †

Desulfurococcus kamchatkensis is an anaerobic organotrophic hyperthermophilic crenarchaeon isolated from a terrestrial hot spring. Its genome consists of a single circular chromosome of 1,365,223 bp with no extrachromosomal elements. A total of 1,474 protein-encoding genes were annotated, among which 205 are exclusive for D. kamchatkensis. The search for a replication origin site revealed a single region coinciding with a global extreme of the nucleotide composition disparity curve and containing a set of crenarchaeon-type origin recognition boxes. Unlike in most archaea, two genes encoding homologs of the eukaryotic initiator proteins Orc1 and Cdc6 are located distantly from this site. A number of mobile elements are present in the genome, including seven transposons representing IS607 and IS200/IS605 families and multiple copies of miniature inverted repeat transposable elements. Two large clusters of regularly interspaced repeats are present; none of the spacer sequences matches known archaeal extrachromosomal elements, except one spacer matches the sequence of a resident gene of D. kamchatkensis. Many of the predicted metabolic enzymes are associated with the fermentation of peptides and sugars, including more than 30 peptidases with diverse specificities, a number of polysaccharide degradation enzymes, and many transporters. Consistently, the genome encodes both enzymes of the modified Embden-Meyerhof pathway of glucose oxidation and a set of enzymes needed for gluconeogenesis. The genome structure and content reflect the organism's nutritionally diverse, competitive natural environment, which is periodically invaded by viruses and other mobile elements