Publications by authors named "Elizaveta M Rivkina"

14 Publications

  • Page 1 of 1

Engineering of Thermal Stability in a Cold-Active Oligo-1,6-Glucosidase from with Unusual Amino Acid Content.

Biomolecules 2021 08 17;11(8). Epub 2021 Aug 17.

Department of Bioengineering, Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia.

A gene coding for a novel putative amylase, oligo-1,6-glucosidase from a psychrotrophic bacterium from Siberian permafrost soil was cloned and expressed in . The amino acid sequence of the predicted protein EsOgl and its 3D model displayed several features characteristic for the cold-active enzymes while possessing an unusually high number of proline residues in the loops-a typical feature of thermophilic enzymes. The activity of the purified recombinant protein was tested with -nitrophenyl α-D-glucopyranoside as a substrate. The enzyme displayed a plateau-shaped temperature-activity profile with the optimum at 25 °C and a pronounced activity at low temperatures (50% of maximum activity at 5 °C). To improve the thermal stability at temperatures above 40 °C, we have introduced proline residues into four positions of EsOgl by site-directed mutagenesis according to "the proline rule". Two of the mutants, S130P and A109P demonstrated a three- and two-fold increased half-life at 45 °C. Moreover, S130P mutation led to a 60% increase in the catalytic rate constant. Combining the mutations resulted in a further increase in stability transforming the temperature-activity profile to a typical mesophilic pattern. In the most thermostable variant A109P/S130P/E176P, the half-life at 45 °C was increased from 11 min (wild-type) to 129 min.
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http://dx.doi.org/10.3390/biom11081229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8392543PMC
August 2021

Genomic reconstruction of fossil and living microorganisms in ancient Siberian permafrost.

Microbiome 2021 05 17;9(1):110. Epub 2021 May 17.

Princeton University, B88, Guyot Hall, Princeton, NJ, 08544, USA.

Background: Total DNA (intracellular, iDNA and extracellular, eDNA) from ancient permafrost records the mixed genetic repository of the past and present microbial populations through geological time. Given the exceptional preservation of eDNA under perennial frozen conditions, typical metagenomic sequencing of total DNA precludes the discrimination between fossil and living microorganisms in ancient cryogenic environments. DNA repair protocols were combined with high throughput sequencing (HTS) of separate iDNA and eDNA fraction to reconstruct metagenome-assembled genomes (MAGs) from ancient microbial DNA entrapped in Siberian coastal permafrost.

Results: Despite the severe DNA damage in ancient permafrost, the coupling of DNA repair and HTS resulted in a total of 52 MAGs from sediments across a chronosequence (26-120 kyr). These MAGs were compared with those derived from the same samples but without utilizing DNA repair protocols. The MAGs from the youngest stratum showed minimal DNA damage and thus likely originated from viable, active microbial species. Many MAGs from the older and deeper sediment appear related to past aerobic microbial populations that had died upon freezing. MAGs from anaerobic lineages, including Asgard archaea, however exhibited minimal DNA damage and likely represent extant living microorganisms that have become adapted to the cryogenic and anoxic environments. The integration of aspartic acid racemization modeling and metaproteomics further constrained the metabolic status of the living microbial populations. Collectively, combining DNA repair protocols with HTS unveiled the adaptive strategies of microbes to long-term survivability in ancient permafrost.

Conclusions: Our results indicated that coupling of DNA repair protocols with simultaneous sequencing of iDNA and eDNA fractions enabled the assembly of MAGs from past and living microorganisms in ancient permafrost. The genomic reconstruction from the past and extant microbial populations expanded our understanding about the microbial successions and biogeochemical alterations from the past paleoenvironment to the present-day frozen state. Furthermore, we provided genomic insights into long-term survival mechanisms of microorganisms under cryogenic conditions through geological time. The combined strategies in this study can be extrapolated to examine other ancient non-permafrost environments and constrain the search for past and extant extraterrestrial life in permafrost and ice deposits on Mars. Video abstract.
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http://dx.doi.org/10.1186/s40168-021-01057-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8130349PMC
May 2021

Structural and Biochemical Characterization of a Cold-Active PMGL3 Esterase with Unusual Oligomeric Structure.

Biomolecules 2021 01 5;11(1). Epub 2021 Jan 5.

Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, 119071 Moscow, Russia.

The gene coding for a novel cold-active esterase PMGL3 was previously obtained from a Siberian permafrost metagenomic DNA library and expressed in . We elucidated the 3D structure of the enzyme which belongs to the hormone-sensitive lipase (HSL) family. Similar to other bacterial HSLs, PMGL3 shares a canonical α/β hydrolase fold and is presumably a dimer in solution but, in addition to the dimer, it forms a tetrameric structure in a crystal and upon prolonged incubation at 4 °C. Detailed analysis demonstrated that the crystal tetramer of PMGL3 has a unique architecture compared to other known tetramers of the bacterial HSLs. To study the role of the specific residues comprising the tetramerization interface of PMGL3, several mutant variants were constructed. Size exclusion chromatography (SEC) analysis of D7N, E47Q, and K67A mutants demonstrated that they still contained a portion of tetrameric form after heat treatment, although its amount was significantly lower in D7N and K67A compared to the wild type. Moreover, the D7N and K67A mutants demonstrated a 40 and 60% increase in the half-life at 40 °C in comparison with the wild type protein. values of these mutants were similar to that of the wt PMGL3. However, the catalytic constants of the E47Q and K67A mutants were reduced by ~40%.
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http://dx.doi.org/10.3390/biom11010057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7824956PMC
January 2021

Insights into community of photosynthetic microorganisms from permafrost.

FEMS Microbiol Ecol 2020 11;96(12)

Soil Cryology Laboratory, Institute of Physicochemical and Biological Problems in Soil Science, Institutskaya Street, Bldg. 2, Pushchino, Russia.

This work integrates cultivation studies of Siberian permafrost and analyses of metagenomes from different locations in the Arctic with the aim of obtaining insights into the community of photosynthetic microorganisms in perennially frozen deposits. Cyanobacteria and microalgae have been described in Arctic aquatic and surface soil environments, but their diversity and ability to withstand harsh conditions within the permafrost are still largely unknown. Community structure of photosynthetic organisms in permafrost sediments was explored using Arctic metagenomes available through the MG-RAST. Sequences affiliated with cyanobacteria represented from 0.25 to 3.03% of total sequences, followed by sequences affiliated with Streptophyta (algae and vascular plants) 0.01-0.45% and Chlorophyta (green algae) 0.01-0.1%. Enrichment and cultivation approaches revealed that cyanobacteria and green algae survive in permafrost and they could be revived during prolonged incubation at low light intensity. Among photosynthetic microorganisms isolated from permafrost, the filamentous Oscillatoria-like cyanobacteria and unicellular green algae of the genus Chlorella were dominant. Our findings suggest that permafrost cyanobacteria and green algae are expected to be effective members of the re-assembled community after permafrost thawing and soil collapse.
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http://dx.doi.org/10.1093/femsec/fiaa229DOI Listing
November 2020

Crystal structure of PMGL2 esterase from the hormone-sensitive lipase family with GCSAG motif around the catalytic serine.

PLoS One 2020 28;15(1):e0226838. Epub 2020 Jan 28.

Department of Enzyme Engineering, Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia.

Lipases comprise a large class of hydrolytic enzymes which catalyze the cleavage of the ester bonds in triacylglycerols and find numerous biotechnological applications. Previously, we have cloned the gene coding for a novel esterase PMGL2 from a Siberian permafrost metagenomic DNA library. We have determined the 3D structure of PMGL2 which belongs to the hormone-sensitive lipase (HSL) family and contains a new variant of the active site motif, GCSAG. Similar to many other HSLs, PMGL2 forms dimers in solution and in the crystal. Our results demonstrated that PMGL2 and structurally characterized members of the GTSAG motif subfamily possess a common dimerization interface that significantly differs from that of members of the GDSAG subfamily of known structure. Moreover, PMGL2 had a unique organization of the active site cavity with significantly different topology compared to the other lipolytic enzymes from the HSL family with known structure including the distinct orientation of the active site entrances within the dimer and about four times larger size of the active site cavity. To study the role of the cysteine residue in GCSAG motif of PMGL2, the catalytic properties and structure of its double C173T/C202S mutant were examined and found to be very similar to the wild type protein. The presence of the bound PEG molecule in the active site of the mutant form allowed for precise mapping of the amino acid residues forming the substrate cavity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0226838PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6986724PMC
April 2020

Draft Genome Sequence of Microbacterium sp. Gd 4-13, Isolated from Gydanskiy Peninsula Permafrost Sediments of Marine Origin.

Microbiol Resour Announc 2019 Oct 3;8(40). Epub 2019 Oct 3.

Institute of Physicochemical and Biological Problems in Soil Science, Russian Academy of Sciences, Pushchino, Russia.

Here, we report the draft genome sequence of sp. strain Gd 4-13, isolated from late Pleistocene permafrost of marine origin located on the Gydanskiy Peninsula. Genome sequence analysis was performed to understand strain survivability mechanisms under permafrost conditions and to expand biotechnology applications.
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http://dx.doi.org/10.1128/MRA.00889-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6776773PMC
October 2019

Predominance of Anaerobic, Spore-Forming Bacteria in Metabolically Active Microbial Communities from Ancient Siberian Permafrost.

Appl Environ Microbiol 2019 08 18;85(15). Epub 2019 Jul 18.

Princeton University, Princeton, New Jersey, USA.

The prevalence of microbial life in permafrost up to several million years (Ma) old has been well documented. However, the long-term survivability, evolution, and metabolic activity of the entombed microbes over this time span remain underexplored. We integrated aspartic acid (Asp) racemization assays with metagenomic sequencing to characterize the microbial activity, phylogenetic diversity, and metabolic functions of indigenous microbial communities across a ∼0.01- to 1.1-Ma chronosequence of continuously frozen permafrost from northeastern Siberia. Although Asp in the older bulk sediments (0.8 to 1.1 Ma) underwent severe racemization relative to that in the youngest sediment (∼0.01 Ma), the much lower d-Asp/l-Asp ratio (0.05 to 0.14) in the separated cells from all samples suggested that indigenous microbial communities were viable and metabolically active in ancient permafrost up to 1.1 Ma. The microbial community in the youngest sediment was the most diverse and was dominated by the phyla and In contrast, microbial diversity decreased dramatically in the older sediments, and anaerobic, spore-forming bacteria within became overwhelmingly dominant. In addition to the enrichment of sporulation-related genes, functional genes involved in anaerobic metabolic pathways such as fermentation, sulfate reduction, and methanogenesis were more abundant in the older sediments. Taken together, the predominance of spore-forming bacteria and associated anaerobic metabolism in the older sediments suggest that a subset of the original indigenous microbial community entrapped in the permafrost survived burial over geological time. Understanding the long-term survivability and associated metabolic traits of microorganisms in ancient permafrost frozen millions of years ago provides a unique window into the burial and preservation processes experienced in general by subsurface microorganisms in sedimentary deposits because of permafrost's hydrological isolation and exceptional DNA preservation. We employed aspartic acid racemization modeling and metagenomics to determine which microbial communities were metabolically active in the 1.1-Ma permafrost from northeastern Siberia. The simultaneous sequencing of extracellular and intracellular genomic DNA provided insight into the metabolic potential distinguishing extinct from extant microorganisms under frozen conditions over this time interval. This in-depth metagenomic sequencing advances our understanding of the microbial diversity and metabolic functions of extant microbiomes from early Pleistocene permafrost. Therefore, these findings extend our knowledge of the survivability of microbes in permafrost from 33,000 years to 1.1 Ma.
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http://dx.doi.org/10.1128/AEM.00560-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6643238PMC
August 2019

Methanogens in the Antarctic Dry Valley permafrost.

FEMS Microbiol Ecol 2018 08;94(8)

Institute of Physicochemical and Biological Problems in Soil Science, Russian Academy of Sciences, Pushchino, 142290, Russia.

Polar permafrost is at the forefront of climate change, yet only a few studies have enriched the native methane-producing microbes that might provide positive feedbacks to climate change. Samples Ant1 and Ant2, collected in Antarctic Miers Valley from permafrost sediments, with and without biogenic methane, respectively, were evaluated for methanogenic activity and presence of methanogens. After a one-year incubation of both samples under anaerobic conditions, methane production was observed only at room temperature in microcosm Ant1 with CO2/H2 (20/80) as carbon and energy sources and was monitored during the subsequent 10 years. The concentration of methane in the headspace of microcosm Ant1 changed from 0.8% to a maximum of 45%. Archaeal 16S rRNA genes from microcosm Ant1 were related to psychrotolerant Methanosarcina lacustris. Repeated efforts at achieving a pure culture of this organism were unsuccessful. Metagenomic reads obtained for the methane-producing microcosm Ant1 were assembled and resulted in a 99.84% complete genome affiliated with the genus Methanosarcina. The metagenome assembled genome contained cold-adapted enzymes and pathways suggesting that the novel uncultured Methanosarcina sp. Ant1 is adapted to sub-freezing conditions in permafrost. This is the first methanogen genome reported from the 15 000 years old permafrost of the Antarctic Dry Valleys.
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http://dx.doi.org/10.1093/femsec/fiy109DOI Listing
August 2018

Draft Genome Sequence of Antarctic Methanogen Enriched from Dry Valley Permafrost.

Genome Announc 2016 Dec 8;4(6). Epub 2016 Dec 8.

Department of Microbiology, University of Tennessee, Knoxville, Tennessee, USA

A genomic reconstruction belonging to the genus Methanosarcina was assembled from metagenomic data from a methane-producing enrichment of Antarctic permafrost. This is the first methanogen genome reported from permafrost of the Dry Valleys and can help shed light on future climate-affected methane dynamics.
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http://dx.doi.org/10.1128/genomeA.01362-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5146446PMC
December 2016

New member of the hormone-sensitive lipase family from the permafrost microbial community.

Bioengineered 2017 Jul 18;8(4):420-423. Epub 2016 Oct 18.

b Institute of Physicochemical and Biological Problems in Soil Science , Russian Academy of Sciences , Pushchino , Russia.

Siberian permafrost is a unique environment inhabited with diverse groups of microorganisms. Among them, there are numerous producers of biotechnologically relevant enzymes including lipases and esterases. Recently, we have constructed a metagenomic library from a permafrost sample and identified in it several genes coding for potential lipolytic enzymes. In the current work, properties of the recombinant esterases obtained from this library are compared with the previously characterized lipase from Psychrobacter cryohalolentis and other representatives of the hormone-sensitive lipase family.
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http://dx.doi.org/10.1080/21655979.2016.1230571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5553336PMC
July 2017

Expression and characterization of a new esterase with GCSAG motif from a permafrost metagenomic library.

FEMS Microbiol Ecol 2016 May 28;92(5):fiw046. Epub 2016 Feb 28.

Institute of Physicochemical and Biological Problems in Soil Science, Russian Academy of Sciences, Institutskaya str., 2, 142290, Pushchino, Moscow Region, Russia.

As a result of construction and screening of a metagenomic library prepared from a permafrost-derived microcosm, we have isolated a novel gene coding for a putative lipolytic enzyme that belongs to the hormone-sensitive lipase family. It encodes a polypeptide of 343 amino acid residues whose amino acid sequence displays maximum likelihood with uncharacterized proteins from Sphingomonas species. A putative catalytic serine residue of PMGL2 resides in a new variant of a recently discovered GTSAG sequence in which a Thr residue is replaced by a Cys residue (GCSAG). The recombinant PMGL2 was produced in Escherichia coli cells and purified by Ni-affinity chromatography. The resulting protein preferably utilizes short-chain p-nitrophenyl esters (C4 and C8) and therefore is an esterase. It possesses maximum activity at 45°C in slightly alkaline conditions and has limited thermostability at higher temperatures. Activity of PMGL2 is stimulated in the presence of 0.25-1.5 M NaCl indicating the good salt tolerance of the new enzyme. Mass spectrometric analysis demonstrated that N-terminal methionine in PMGL2 is processed and cysteine residues do not form a disulfide bond. The results of the study demonstrate the significance of the permafrost environment as a unique genetic reservoir and its potential for metagenomic exploration.
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http://dx.doi.org/10.1093/femsec/fiw046DOI Listing
May 2016

Desulfovibrio arcticus sp. nov., a psychrotolerant sulfate-reducing bacterium from a cryopeg.

Int J Syst Evol Microbiol 2012 Jan 11;62(Pt 1):33-37. Epub 2011 Feb 11.

Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region, 142290, Russia.

A psychrotolerant sulfate-reducing bacterium, designated B15(T), was isolated from supercooled water brine from within permafrost of the Varandey Peninsula, on the southern coast of the Barents Sea. Cells were Gram-negative, motile vibrions (3.0-4.0×0.4-0.5 µm) with a single polar flagellum. The isolate was positive for desulfoviridin as a bisulfite reductase. Strain B15(T) grew at -2 to 28 °C (optimum 24 °C) and with 0-2.0% NaCl (optimum 0.2%). The isolate used H(2) plus acetate, formate, ethanol, lactate, pyruvate and choline as electron donors and used sulfate, sulfite, thiosulfate, elemental sulfur, DMSO and Fe(3+) as electron acceptors. Pyruvate and lactate were not fermented in the absence of sulfate. The G+C content of genomic DNA was 55.2 mol%. Analysis of the 16S rRNA gene sequence showed that the isolate belonged to the genus Desulfovibrio. Its closest relatives were Desulfovibrio idahonensis CY1(T) (98.8% 16S rRNA gene sequence similarity) and Desulfovibrio mexicanus Lup1(T) (96.5%). On the basis of genotypic, phenotypic and phylogenetic characteristics, the isolate represents a novel species, for which the name Desulfovibrio arcticus sp. nov. is proposed; the type strain is B15(T) (=VKM B-2367(T)=DSM 21064(T)).
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http://dx.doi.org/10.1099/ijs.0.021451-0DOI Listing
January 2012

Methanobacterium veterum sp. nov., from ancient Siberian permafrost.

Int J Syst Evol Microbiol 2010 Feb 4;60(Pt 2):455-459. Epub 2009 Aug 4.

Institute of Physicochemical and Biological Problems in Soil Sciences, Russian Academy of Sciences, Pushchino, 142290 Moscow Region, Russian Federation.

A methanogenic archaeon, strain MK4(T), was isolated from ancient permafrost after long-term selective anaerobic cultivation. The cells were rods, 2.0-8.0 microm long and 0.40-0.45 microm wide, and stained Gram-negative. Optimal growth was observed at 28 degrees C and pH 7.0-7.2 and in 0.05 M NaCl. The isolate used H(2) plus CO(2), methylamine plus H(2) and methanol plus H(2) as sources for growth and methanogenesis. Phylogenetic analysis of the 16S rRNA gene sequence of the strain showed close affinity with Methanobacterium bryantii (similarity >99 % to the type strain). On the basis of the level of DNA-DNA hybridization (62 %) between strain MK4(T) and Methanobacterium bryantii VKM B-1629(T) and phenotypic and phylogenetic differences, strain MK4(T) was assigned to a novel species of the genus Methanobacterium, Methanobacterium veterum sp. nov., with the type strain MK4(T) (=DSM 19849(T) =VKM B-2440(T)).
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http://dx.doi.org/10.1099/ijs.0.011205-0DOI Listing
February 2010
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