ABSTRACT
Planctomycetes of the genus Singulisphaera are common inhabitants of soils and peatlands. Although described members of this genus are characterized as possessing hydrolytic capabilities, the ability to degrade chitin has not yet been reported for these bacteria. In this study, a novel Singulisphaera representative, strain Ch08, was isolated from a chitinolytic enrichment culture obtained from a boreal fen in Northern European Russia. The 16S rRNA gene sequence of this isolate displayed 98.2% similarity to that of Singulisphaera acidiphila MOB10T. Substrate utilization tests confirmed that strain Ch08 is capable of growth on amorphous chitin. The complete genome of strain Ch08 determined in this study was 10.85 Mb in size and encoded two predicted chitinases, which were only distantly related to each other and affiliated with the glycoside hydrolase family GH18. One of these chitinases had a close homologue in the genome of S. acidiphila MOB10T. The experimental verification of S. acidiphila MOB10T growth on amorphous chitin was also positive. Transcriptome analysis performed with glucose- and chitin-growth cells of strain Ch08 showed upregulation of the predicted chitinase shared by strain Ch08 and S. acidiphila MOB10T. The gene encoding this protein was expressed in Escherichia coli, and the endochitinase activity of the recombinant enzyme was confirmed. The ability to utilize chitin, a major constituent of fungal cell walls and arthropod exoskeletons, appears to be one of the previously unrecognized ecological functions of Singulisphaera-like planctomycetes.
ABSTRACT
Sulphate-reducing bacteria, primarily Desulfovibrio, are responsible for the active generation of H2S in swine production waste. The model species for sulphate reduction studies, Desulfovibrio vulgaris strain L2, was previously isolated from swine manure characterized by high rates of dissimilatory sulphate reduction. The source of electron acceptors in low-sulphate swine waste for the high rate of H2S formation remains uncertain. Here, we demonstrate the ability of the L2 strain to use common animal farming supplements including L-lysine-sulphate, gypsum and gypsum plasterboards as electron acceptors for H2S production. Genome sequencing of strain L2 revealed the presence of two megaplasmids and predicted resistance to various antimicrobials and mercury, which was confirmed in physiological experiments. Most of antibiotic resistance genes (ARG) are carried by two class 1 integrons located on the chromosome and on the plasmid pDsulf-L2-2. These ARGs, predicted to confer resistance to beta-lactams, aminoglycosides, lincosamides, sulphonamides, chloramphenicol and tetracycline, were probably laterally acquired from various Gammaproteobacteria and Firmicutes. Resistance to mercury is likely enabled by two mer operons also located on the chromosome and on pDsulf-L2-2 and acquired via horizontal gene transfer. The second megaplasmid, pDsulf-L2-1, encoded nitrogenase, catalase and type III secretion system suggesting close contact of the strain with intestinal cells in the swine gut. The location of ARGs on mobile elements allows us to consider D. vulgaris strain L2 as a possible vector transferring antimicrobials resistance determinants between the gut microbiote and microbial communities in environmental biotopes.
ABSTRACT
[This corrects the article DOI: 10.3389/fmicb.2018.02775.].
ABSTRACT
Large areas in the northern hemisphere are covered by extensive wetlands, which represent a complex mosaic of raised bogs, eutrophic fens, and aapa mires all in proximity to each other. Aapa mires differ from other types of wetlands by their concave surface, heavily watered by the central part, as well as by the presence of large-patterned string-flark complexes. In this paper, we characterized microbial diversity patterns in the surface peat layers of the neighboring string and flark structures located within the mire site in the Vologda region of European North Russia, using 16S rRNA gene sequencing. The microbial communities in raised strings were clearly distinct from those in submerged flarks. Strings were dominated by the Alpha- and Gammaproteobacteria. Other abundant groups were the Acidobacteriota, Bacteroidota, Verrucomicrobiota, Actinobacteriota, and Planctomycetota. Archaea accounted for only 0.4% of 16S rRNA gene sequences retrieved from strings. By contrast, they comprised about 22% of all sequences in submerged flarks and mostly belonged to methanogenic lineages. Methanotrophs were nearly absent. Other flark-specific microorganisms included the phyla Chloroflexi, Spirochaetota, Desulfobacterota, Beijerinckiaceae- and Rhodomicrobiaceae-affiliated Alphaproteobacteria, and uncultivated groups env.OPS_17 and vadinHA17 of the Bacteroidota. Such pattern probably reflects local anaerobic conditions in the submerged peat layers in flarks.
ABSTRACT
Two strains of filamentous, colorless sulfur bacteria were isolated from bacterial fouling in the outflow of hydrogen sulfide-containing waters from a coal mine (Thiothrix sp. Ku-5) and on the seashore of the White Sea (Thiothrix sp. AS). Metagenome-assembled genome (MAG) A52 was obtained from a sulfidic spring in the Volgograd region, Russia. Phylogenetic analysis based on the 16S rRNA gene sequences showed that all genomes represented the genus Thiothrix. Based on their average nucleotide identity and digital DNA-DNA hybridization data these new isolates and the MAG represent three species within the genus Thiothrix with the proposed names Thiothrix subterranea sp. nov. Ku-5T, Thiothrix litoralis sp. nov. AST, and "Candidatus Thiothrix anitrata" sp. nov. A52. The complete genome sequences of Thiothrix fructosivorans QT and Thiothrix unzii A1T were determined. Complete genomes of seven Thiothrix isolates, as well as two MAGs, were used for pangenome analysis. The Thiothrix core genome consisted of 1,355 genes, including ones for the glycolysis, the tricarboxylic acid cycle, the aerobic respiratory chain, and the Calvin cycle of carbon fixation. Genes for dissimilatory oxidation of reduced sulfur compounds, namely the branched SOX system (SoxAXBYZ), direct (soeABC) and indirect (aprAB, sat) pathways of sulfite oxidation, sulfur oxidation complex Dsr (dsrABEFHCEMKLJONR), sulfide oxidation systems SQR (sqrA, sqrF), and FCSD (fccAB) were found in the core genome. Genomes differ in the set of genes for dissimilatory reduction of nitrogen compounds, nitrogen fixation, and the presence of various types of RuBisCO.
ABSTRACT
Planctomycetes of the family Gemmataceae are strictly aerobic chemo-organotrophs that display a number of hydrolytic capabilities. A member of this family, Telmatocola sphagniphila SP2T, is the first described planctomycete with experimentally proven ability for growth on cellulose. In this study, the complete genome sequence of strain SP2T was obtained and the genome-encoded determinants of its cellulolytic potential were analyzed. The T. sphagniphila SP2T genome was 6.59 Mb in size and contained over 5200 potential protein-coding genes. The search for enzymes that could be potentially involved in cellulose degradation identified a putative cellulase that contained a domain from the GH44 family of glycoside hydrolases. Homologous enzymes were also revealed in the genomes of two other Gemmataceae planctomycetes, Zavarzinella formosa A10T and Tuwongella immobilis MBLW1T. The gene encoding this predicted cellulase in strain SP2T was expressed in E. coli and the hydrolytic activity of the recombinant enzyme was confirmed in tests with carboxymethyl cellulose but not with crystalline cellulose, xylan, mannan or laminarin. This is the first experimentally characterized cellulolytic enzyme from planctomycetes.
Subject(s)
Escherichia coli , Planctomycetales , Planctomycetales/geneticsABSTRACT
Microbial life can be supported at subzero temperatures in permafrost up to several million years old. Genome analysis of strains isolated from permafrost provides a unique opportunity to study microorganisms that have not previously come into contact with the human population. Acinetobacter lwoffii is a typical soil bacterium that has been increasingly reported as hospital pathogens associated with bacteremia. In order to identify the specific genetic characteristics of ancient permafrost-conserved strains of A. lwoffii and their differences from present-day clinical isolates, we carried out a genome-wide analysis of five strains of A. lwoffii isolated from permafrost aged from 15 thousand to 1.8 million years. Surprisingly, we did not identify chromosomal genetic determinants that distinguish permafrost strains from clinical A. lwoffii isolates and strains from other natural habitats. Phylogenetic analysis based on whole genome sequences showed that permafrost strains do not form a separate cluster and some of them are most closely related to clinical isolates. The genomes of clinical and permafrost strains contain similar mobile elements and prophages, which indicates an intense horizontal transfer of genetic material. Comparison of plasmids of modern and permafrost strains showed that plasmids from the modern strains are enriched with antibiotic resistance genes, while the content of genes for resistance to heavy metals and arsenic is nearly the same. The thawing of permafrost caused by global warming could release new potentially pathogenic strains of Acinetobacter.
ABSTRACT
Bacillus velezensis strain BZR 277 is a rhizobacterium isolated from the rhizoplane of a winter oilseed rape plant from the Krasnodar region in Russia. This study presents the genome sequence of the rhizobacterium Bacillus velezensis BZR 277, which exhibited high antagonistic activity against the root-knot nematode Meloidogyne incognita Kof. The data on the genome sequence can help to identify pathways for the biosynthesis of metabolites responsible for nematicidal activity in order to improve crop protection against plant-parasitic nematodes.
ABSTRACT
Food products may be a source of Salmonella, one of the main causal agents of food poisoning, especially after the emergence of strains resistant to antimicrobial preparations. The present work dealt with investigation of the occurrence of resistance to antimicrobial preparations among S. enterica strains isolated from food. The isolates belonged to 11 serovars, among which Infantis (28%), Enteritidis (19%), and Typhimurium (13.4%) predominated. The isolates were most commonly resistant to trimethoprim/sulfamethoxazole (n = 19, 59.38%), cefazolin (n = 15, 46.86%), tetracycline (n = 13, 40.63%), and amikacin (n = 9, 28.13%). Most of the strains (68.75%) exhibited multiple resistance to commonly used antibiotics. High-throughput sequencing was used to analyse three multidrug-resistant strains (resistant to six or more antibiotics). Two of them (SZL 30 and SZL 31) belonged to S. Infantis, while one strain belonged to S. Typhimurium (SZL 38). Analysis of the genomes of the sequenced strains revealed the genes responsible for antibiotic resistance. In the genomes of strains SZL 30 and SZL 31 the genes of antibiotic resistance were shown to be localized mostly in integrons within plasmids, while most of the antibiotic resistance genes of strain SZL 38 were localized in a chromosomal island (17,949 nt). Genomes of the Salmonella strains SZL 30, SZL 31, and SZL 38 were shown to contain full-size pathogenicity islands: SPI-1, SPI-2, SPI-4, SPI-5, SPI-9, SPI-11, SPI-13, SPI-14, and CS54. Moreover, the genome of strain SZL 38 was also found to contain the full-size pathogenicity islands SPI-3, SPI-6, SPI-12, and SPI-16. The emergence of multidrug-resistant strains of various Salmonella serovars indicates that further research on the transmission pathways for these genetic determinants and monitoring of the distribution of these microorganisms are necessary.
ABSTRACT
Phycisphaera-like WD2101 'soil group' is one of the as-yet-uncultivated phylogenetic clades within the phylum Planctomycetes. Members of this clade are commonly detected in various terrestrial habitats. This study shows that WD2101 represented one of the major planctomycete groups in 10 boreal peatlands, comprising up to 76% and 36% of all Planctomycetes-affiliated 16S rRNA gene reads in raised bogs and eutrophic fens respectively. These types of peatlands displayed clearly distinct intra-group diversity of WD2101-affiliated planctomycetes. The first isolate of this enigmatic planctomycete group, strain M1803, was obtained from a humic lake surrounded by Sphagnum peat bogs. Strain M1803 displayed 89.2% 16S rRNA gene similarity to Tepidisphaera mucosa and was represented by motile cocci that divided by binary fission and grew under micro-oxic conditions. The complete 7.19 Mb genome of strain M1803 contained an array of genes encoding Planctomycetal type bacterial microcompartment organelle likely involved in l-rhamnose metabolism, suggesting participation of M1803-like planctomycetes in polysaccharide degradation in peatlands. The corresponding cellular microcompartments were revealed in ultrathin cell sections. Strain M1803 was classified as a novel genus and species, Humisphaera borealis gen. nov., sp. nov., affiliated with the formerly recognized WD2101 'soil group'.
Subject(s)
Bacteria , Soil , Bacteria/genetics , Bacterial Typing Techniques , DNA, Bacterial , Fatty Acids , Phylogeny , Planctomycetes , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , Soil MicrobiologyABSTRACT
Large areas in Northern Russia are covered by extensive mires, which represent a complex mosaic of ombrotrophic raised bogs, minerotrophic and eutrophic fens, all in a close proximity to each other. In this paper, we compared microbial diversity patterns in the surface peat layers of the neighbouring raised bogs and eutrophic fens that are located within two geographically remote mire sites in Vologda region using 16S rRNA gene sequencing. Regardless of location, the microbial communities in raised bogs were highly similar to each other but were clearly distinct from those in eutrophic fens. Bogs were dominated by the Acidobacteria (30%-40% of total 16S rRNA gene reads), which belong to the orders Acidobacteriales and Bryobacterales. Other bog-specific bacteria included the Phycisphaera-like group WD2101 and the families Isosphaeraceae and Gemmataceae of the Planctomycetes, orders Opitutales and Pedosphaerales of the Verrucomicrobia and a particular group of alphaproteobacteria within the Rhizobiales. In contrast, fens hosted Anaerolineae-affiliated Chloroflexi, Vicinamibacteria- and Blastocatellia-affiliated Acidobacteria, Rokubacteria, uncultivated group OM190 of the Planctomycetes and several groups of betaproteobacteria. The Patescibacteria were detected in both types of wetlands but their relative abundance was higher in fens. A number of key parameters that define the distribution of particular bacterial groups in mires were identified.
ABSTRACT
A novel actinobacterium, strain K3-2T, was isolated in pure culture from a thawing ancient ice wedge at Mammoth Mountain (Eastern Siberia, Russia). Colonies of strain K3-2T were yellowish orange; cells had the fine structure typical of Gram-positive bacteria, were non-motile short rods and were non-spore-forming. Strain K3-2T was mesophilic (optimum growth at 28 °Ð¡), but capable of growing at 4 °Ð¡. The cell-wall peptidoglycan of strain K3-2T contained lysine (the diagnostic diamino acid), glutamic acid, alanine, ornithine, glycine and serine. The polar lipids were phosphatidylglycerol, lysophosphatidylserine, three unidentified phospholipids and glycolipids. The major fatty acids were anteiso-C15â:â0 and C16â:â0. The only menaquinone detected was MK-8(H4). 16S rRNA gene analysis indicated that strain K3-2T belongs to the genus Serinibacter. The closest taxonomically described relatives were Serinibacter salmoneus Kis4-28T and Serinibacter tropicus PS-14-7T, with 97.20 and 97.20â% 16 s rRNA gene sequence similarity, respectively. The average nucleotide identity value of the whole genome sequence between strain K3-2T and S. salmoneus Kis4-28T was 78.9â%. DNA-DNA relatedness values between strain K3-2T and S. salmoneus DSM 21801T (=Kis4-28T) and S. tropicus VKPM Ac 2044T (=PS-14-7T) were 41 and 47â%. Thus, strain K3-2T represents a novel species of the genus Serinibacter for which the name Serinibacter arcticus sp. nov. is proposed. The type strain is K3-2T (DSM 103859T=VKM ÐÑ-2719T).
Subject(s)
Actinobacteria/classification , Ice , Phylogeny , Actinobacteria/isolation & purification , Bacterial Typing Techniques , Base Composition , Cell Wall/chemistry , DNA, Bacterial/genetics , Fatty Acids/chemistry , Glycolipids/chemistry , Nucleic Acid Hybridization , Peptidoglycan/chemistry , Phospholipids/chemistry , Pigmentation , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , Siberia , Vitamin K 2/analogs & derivatives , Vitamin K 2/chemistryABSTRACT
The endophytic strains Bacillus amyloliquefaciens V417 and V167 were isolated from cultured grape plants. We sequenced the complete genomes of these strains to reveal their potential beneficial properties for plant growth promotion and control of fungal pathogens. Genes responsible for the synthesis of antimicrobial compounds and siderophores were identified.
ABSTRACT
An isolate of strictly aerobic, pale-pink pigmented bacteria, strain AF10T, was obtained from an organic soil layer in forested tundra, Nadym region, West Siberia. Cells of strain AF10T were Gram-negative, non-motile rods that produced an amorphous extracellular polysaccharide-like substance and formed large cell aggregates in old cultures. These bacteria were chemoorganotrophic, mildly acidophilic and psychrotolerant, and grew between pH 3.5 and 7.0 (optimum, pH 4.5-5.0) and at temperatures between 2 and 30 °C. The preferred growth substrates were sugars and some polysaccharides. The major fatty acids were iso-C15â:â0, C16â:â0, C16â:â1∆9 c and 13,16-dimethyl octacosanedioic acid. The genome of strain AF10T was 6.14 Mbp in size and encoded a wide repertoire of carbohydrate active enzymes. The genomic DNA G+C content was 59.8 mol%. Phylogenetic analysis indicated that strain AF10T is a member of the genus Granulicella, family Acidobacteriaceae, but displays 94.4-98.0â% 16S rRNA gene sequence similarity to currently described members of this genus. On the basis of phenotypic, chemotaxonomic, phylogenetic and genomic analyses, we propose to classify this bacterium as representing a novel species of the genus Granulicella, Granulicellasibirica sp. nov. Strain AF10T (=DSM 104461T=VKM B-3276T) is the type strain.
Subject(s)
Acidobacteria/classification , Forests , Phylogeny , Soil Microbiology , Tundra , Acidobacteria/isolation & purification , Bacterial Typing Techniques , Base Composition , DNA, Bacterial/genetics , Fatty Acids/chemistry , Pigmentation , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , SiberiaABSTRACT
Candidate bacterial phylum BRC1 has been identified in a broad range of mostly organic-rich oxic and anoxic environments through molecular analysis of microbial communities. None of the members of BRC1 have been cultivated and only a few draft genome sequences have been obtained from metagenomes or as a result of single-cell sequencing. We have reconstructed complete genome of BRC1 bacterium, BY40, from metagenome of the microbial community of a deep subsurface thermal aquifer in the Tomsk Region of the Western Siberia, Russia, and used it for metabolic reconstruction and comparison with existing genomic data. Analysis of 3.3Mb genome of BY40 bacterium revealed numerous glycoside hydrolases that could enable utilization of carbohydrates, including enzymes of chitin-degradation pathway. The bacterium lacks flagellar machinery but the twitching motility is encoded. The reconstructed central metabolism revealed pathways enabling the fermentation of organic substrates, as well as their complete oxidation through aerobic and anaerobic respiration. Phylogenetic analysis using BY40 genome supported the phylum level classification of BRC1 lineage. Based on phylogenetic and genomic analyses, the novel bacterium is proposed to be classified as Candidatus Sumerlaea chitinivorans, within a candidate phylum Sumerlaeota.
Subject(s)
Bacteria/classification , Groundwater/microbiology , Metagenome , Phylogeny , Bacteria/genetics , Bacteria/isolation & purification , Bacterial Typing Techniques , Genome, Bacterial , RNA, Ribosomal, 16S/genetics , Russia , Water MicrobiologyABSTRACT
Members of the Acidobacteria are among the most efficient colonizers of acidic terrestrial habitats but the key traits underlying their environmental fitness remain to be understood. We analyzed indigenous assemblages of Acidobacteria in a lichen-covered acidic (pH 4.1) soil of forested tundra dominated by uncultivated members of subdivision 1. An isolate of these bacteria with cells occurring within saccular chambers, strain SBC82T, was obtained. The genome of strain SBC82T consists of a 7.11-Mb chromosome and four megaplasmids, and encodes a wide repertoire of enzymes involved in degradation of chitin, cellulose, and xylan. Among those, four secreted chitinases affiliated with the glycoside hydrolase family GH18 were identified. Strain SBC82T utilized amorphous chitin as a source of carbon and nitrogen; the respective enzyme activities were detected in tests with synthetic substrates. Chitinolytic capability was also confirmed for another phylogenetically related acidobacterium isolated from a Sphagnum peat bog, strain CCO287. As revealed by metatranscriptomic analysis of chitin-amended peat, 16S rRNA reads from these acidobacteria increased in response to chitin availability. Strains SBC82T and CCO287 were assigned to a novel genus and species, Acidisarcina polymorpha gen. nov., sp. nov. Members of this genus colonize acidic soils and peatlands and specialize in degrading complex polysaccharides.
ABSTRACT
Members of the bacterial order Planctomycetales have often been observed in associations with Crustacea. The ability to degrade chitin, however, has never been reported for any of the cultured planctomycetes although utilization of N-acetylglucosamine (GlcNAc) as a sole carbon and nitrogen source is well recognized for these bacteria. Here, we demonstrate the chitinolytic capability of a member of the family Gemmataceae, Fimbriiglobus ruber SP5T, which was isolated from a peat bog. As revealed by metatranscriptomic analysis of chitin-amended peat, the pool of 16S rRNA reads from F. ruber increased in response to chitin availability. Strain SP5T displayed only weak growth on amorphous chitin as a sole source of carbon but grew well with chitin as a source of nitrogen. The genome of F. ruber SP5T is 12.364 Mb in size and is the largest among all currently determined planctomycete genomes. It encodes several enzymes putatively involved in chitin degradation, including two chitinases affiliated with the glycoside hydrolase (GH) family GH18, GH20 family ß-N-acetylglucosaminidase, and the complete set of enzymes required for utilization of GlcNAc. The gene encoding one of the predicted chitinases was expressed in Escherichia coli, and the endochitinase activity of the recombinant enzyme was confirmed. The genome also contains genes required for the assembly of type IV pili, which may be used to adhere to chitin and possibly other biopolymers. The ability to use chitin as a source of nitrogen is of special importance for planctomycetes that inhabit N-depleted ombrotrophic wetlands.IMPORTANCE Planctomycetes represent an important part of the microbial community in Sphagnum-dominated peatlands, but their potential functions in these ecosystems remain poorly understood. This study reports the presence of chitinolytic potential in one of the recently described peat-inhabiting members of the family Gemmataceae, Fimbriiglobus ruber SP5T This planctomycete uses chitin, a major constituent of fungal cell walls and exoskeletons of peat-inhabiting arthropods, as a source of nitrogen in N-depleted ombrotrophic Sphagnum-dominated peatlands. This study reports the chitin-degrading capability of representatives of the order Planctomycetales.
Subject(s)
Chitin/metabolism , Genome, Bacterial , Planctomycetales/genetics , Chitinases/analysis , Planctomycetales/metabolism , RNA, Bacterial/analysis , RNA, Ribosomal, 16S/analysis , Russia , Soil , WetlandsABSTRACT
BACKGROUND: The multigene family encoding the 5S rRNA, one of the most important structurally-functional part of the large ribosomal subunit, is an obligate component of all eukaryotic genomes. 5S rDNA has long been a favored target for cytological and phylogenetic studies due to the inherent peculiarities of its structural organization, such as the tandem arrays of repetitive units and their high interspecific divergence. The complex polyploid nature of the genome of bread wheat, Triticum aestivum, and the technically difficult task of sequencing clusters of tandem repeats mean that the detailed organization of extended genomic regions containing 5S rRNA genes remains unclear. This is despite the recent progress made in wheat genomic sequencing. Using pyrosequencing of BAC clones, in this work we studied the organization of two distinct 5S rDNA-tagged regions of the 5BS chromosome of bread wheat. RESULTS: Three BAC-clones containing 5S rDNA were identified in the 5BS chromosome-specific BAC-library of Triticum aestivum. Using the results of pyrosequencing and assembling, we obtained six 5S rDNA- containing contigs with a total length of 140,417 bp, and two sets (pools) of individual 5S rDNA sequences belonging to separate, but closely located genomic regions on the 5BS chromosome. Both regions are characterized by the presence of approximately 70-80 copies of 5S rDNA, however, they are completely different in their structural organization. The first region contained highly diverged short-type 5S rDNA units that were disrupted by multiple insertions of transposable elements. The second region contained the more conserved long-type 5S rDNA, organized as a single tandem array. FISH using probes specific to both 5S rDNA unit types showed differences in the distribution and intensity of signals on the chromosomes of polyploid wheat species and their diploid progenitors. CONCLUSION: A detailed structural organization of two closely located 5S rDNA-tagged genomic regions on the 5BS chromosome of bread wheat has been established. These two regions differ in the organization of both 5S rDNA and the neighboring sequences comprised of transposable elements, implying different modes of evolution for these regions.
Subject(s)
Chromosomes, Plant , DNA, Plant , DNA, Ribosomal , Triticum/genetics , Bread , Chromosomes, Artificial, Bacterial , DNA, Ribosomal Spacer , Genome, Plant , In Situ Hybridization, Fluorescence , Multigene Family , Sequence Alignment , Sequence Analysis, DNAABSTRACT
A new fold-type IV branched-chain amino acid aminotransferase VMUT0738 from the hyperthermophilic Crenarchaeon Vulcanisaeta moutnovskia was successfully expressed in Escherichia coli. Purified VMUT0738 showed activity toward numerous aliphatic and aromatic l-amino acids and 2-oxo acids at optimal pH 8.0. Distinguishing features of the VMUT0738 compared with typical BCAT are the absence of activity toward acidic substrates, high activity toward basic ones, and low but detectable activity toward the (R)-enantiomer of α-methylbenzylamine (0.0076U/mg) The activity of VMUT0738 increases with a rise in the temperature from 60°C to 90°C. VMUT0738 showed high thermostability (after 24h incubation at 70°C the enzyme lost only 27% of the initial activity) and the resistance to organic solvents. The sequence alignment revealed two motifs (V/I)xLDxR and PFG(K/H)YL characteristic of BCATs from species of the related genera Vulcanisaeta, Pyrobaculum and Thermoproteus that might be responsible for the unique substrate recognition profile of the enzyme.
Subject(s)
Archaeal Proteins/metabolism , Thermoproteaceae/enzymology , Transaminases/metabolism , Amino Acid Sequence , Archaeal Proteins/genetics , Cloning, Molecular , Enzyme Stability , Genes, Archaeal , Hydrogen-Ion Concentration , Kinetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Structural Homology, Protein , Substrate Specificity , Temperature , Thermoproteaceae/genetics , Transaminases/geneticsABSTRACT
Anaerobic enrichment from sediments of hypersaline alkaline lakes in Wadi el Natrun (Egypt) with chitin resulted in the isolation of a fermentative haloalkaliphilic bacterium, strain ACht6-1, growing exclusively with insoluble chitin as the substrate in a sodium carbonate-based medium at pH 8.5-10.5 and total Na(+) concentrations from 0.4 to 1.75 M. The isolate had a Gram-negative cell wall and formed lipid cysts in old cultures. The chitinolytic activity was associated with cells. Analysis of the 4.4 Mb draft genome identified pathways for chitin utilization, particularly, secreted chitinases linked to the cell surface, as well as genes for the hydrolysis of other polysaccharides and fermentation of sugars, while the genes needed for aerobic and anaerobic respiration were absent. Adaptation to a haloalkaliphilic lifestyle was reflected by the gene repertoire encoding sodium rather than proton-dependent membrane-bound ion pumps, including the Rnf-type complex, oxaloacetate decarboxylase, V-type ATPase, and pyrophosphatase. The phylogenetic analysis using 16S rRNA gene and ribosomal proteins indicated that ACht6-1 forms a novel deep lineage at the class level within the bacterial candidate division TG3. Based on phylogenetic, phenotypic and genomic analyses, the novel chitinolytic bacterium is described as Chitinispirillum alkaliphilum gen. nov., sp. nov., within a novel class Chitinispirillia that could be included into the phylum Fibrobacteres.