[Show abstract][Hide abstract] ABSTRACT: A metagenomic analysis of the dynamic changes of the composition of the intestinal microbiome of five participants of the MARS-500 experiment was performed. DNA samples were isolated from the feces of the participants taken just before the experiment, upon 14, 30, 210, 363 and 510 days of isolation in the experimental module, and two weeks upon completion of the experiment. The taxonomic composition of the microbiome was analyzed by pyrosequencing of 16S rRNA gene fragments. Both the taxonomic and functional gene content of the microbiome of one participant were analyzed by whole metagenome sequencing using the SOLiD technique. Each participant had a specific microbiome that could be assigned to one of three recognized enterotypes. Two participants had enterotype I microbiomes characterized by the prevalence of Bacteroides, while the microbiomes of two others, assigned to type II, were dominated by Prevotella. One participant had a microbiome of mixed type. It was found that (1) changes in the taxonimic composition of the microbiomes occurred in the course of the ex-periment, but the enterotypes remained the same; (2) significant changes in the compositions of the microbiomes occurred just 14-30 days after the beginning of the experiment, presumably indicating the influence of stress factors in the first stage of the experiment; (3) a tendency toward a reversion of the microbiomes to their initial composition was observed two weeks after the end of the experiment, but complete recovery was not achieved. The metagenomic analysis of the microbiome of one of the participants showed that in spite of variations in the taxonomic compositions of microbiomes, the "functional" genetic composition was much more stable for most of the functional gene categories. Probably in the course of the experiment the taxonomic composition of the gut microbiome was adaptively changed to reflect the individual response to the experimental conditions. A new, balanced taxonomic composition of the microbiome was formed to ensure a stable gene content of the commu-nity as a whole without negative consequences for the health of the participants. KEYWORDS metagenomics, intestinal microbiota, stressful influences, enterotypes. INTRODUCTION Metagenomic studies of the human microbiome con-ducted within the framework of large-scale interna-tional research programs [1–3] are aimed at shedding light on the role microorganisms play in human life, developing diagnostic techniques, and preventing and treating various diseases. the taxonomic and genetic composition of the microbiota inhabiting the intestines
[Show abstract][Hide abstract] ABSTRACT: A metagenomic analysis of the dynamic changes of the composition of the intestinal microbiome of five participants of the MARS-500 experiment was performed. DNA samples were isolated from the feces of the participants taken just before the experiment, upon 14, 30, 210, 363 and 510 days of isolation in the experimental module, and two weeks upon completion of the experiment. The taxonomic composition of the microbiome was analyzed by pyrosequencing of 16S rRNA gene fragments. Both the taxonomic and functional gene content of the microbiome of one participant were analyzed by whole metagenome sequencing using the SOLiD technique. Each participant had a specific microbiome that could be assigned to one of three recognized enterotypes. Two participants had enterotype I microbiomes characterized by the prevalence of Bacteroides, while the microbiomes of two others, assigned to type II, were dominated by Prevotella. One participant had a microbiome of mixed type. It was found that (1) changes in the taxonimic composition of the microbiomes occurred in the course of the experiment, but the enterotypes remained the same; (2) significant changes in the compositions of the microbiomes occurred just 14-30 days after the beginning of the experiment, presumably indicating the influence of stress factors in the first stage of the experiment; (3) a tendency toward a reversion of the microbiomes to their initial composition was observed two weeks after the end of the experiment, but complete recovery was not achieved. The metagenomic analysis of the microbiome of one of the participants showed that in spite of variations in the taxonomic compositions of microbiomes, the "functional" genetic composition was much more stable for most of the functional gene categories. Probably in the course of the experiment the taxonomic composition of the gut microbiome was adaptively changed to reflect the individual response to the experimental conditions. A new, balanced taxonomic composition of the microbiome was formed to ensure a stable gene content of the community as a whole without negative consequences for the health of the participants.
[Show abstract][Hide abstract] ABSTRACT: Synechocystis sp. PCC 6803 is a model organism for the study of photosynthetic processes. Methods to genetically manipulate this bacterium are essential to investigate these processes and to evaluate potential biotechnological applications. We developed a vector for controllable expression of proteins using a platform for stable integration of the expression cassette into the genome. The respective gene is translationally fused to the promoter of the petJ gene encoding cytochrome c(553) that is repressed by copper. Maximal expression from this promoter is achieved under copper depletion, whereas normal copper concentrations in standard medium lead to low expression rates. We show here the application of this system for construction of a conditional knockout mutant for the ferrochelatase, which is an essential enzyme in heme biosynthesis. Using different amounts of copper in the medium we were able to control the amount of ferrochelatase in the cell resulting in a varying expression of the phenotype.
Journal of Biotechnology 06/2012; 162(1):75-80. · 3.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Thirteen Lactobacillus strains isolated from the gastrointestinal microbiome of people from the territory of the former Soviet Union have been studied
for resistance to 15 antibiotics of different nature, namely, penicillins, aminoglycosides, macrolides, lincosamides, tetracyclines,
chloramphenicol, and rifampicin. The strains included four strains of L. plantarum, four of L. helveticus, three of L. casei/paracasei, one of L. rhamnosus, and one of L. fermentum. All strains showed relative sensitivity to ampicillin, chloramphenicol, rifampicin, roxithromycin, erythromycin, and azithromycin,
while none of them were sensitive to all tested antibiotics. L. plantarum strains had the broadest resistance spectra: one strain was resistant to tetracycline and three aminoglycosides and three
strains were resistant to tetracycline and five aminoglycosides; one strain demonstrated high resistance to clindamycin and
two strains to lincomycin. At the same time, two L. plantarum strains demonstrated resistance to benzylpenicillin coupled with sensitivity to ampicillin, another β-lactam antibiotic.
Such resistance was clearly not related to the β-lactamase activity and could be explained by a specific mutation in one of
the penicillin-binding proteins of the cell wall. Strains of L. helveticus, L. casei/paracasei, L. rhamnosus, and L. fermentum exhibited cross resistance to two to five different aminoglycosides. A PCR test of the resistance determinants for the widely
clinically used antibiotics, tetracycline, chloramphenicol, and erythromycin revealed the presence of the tetM gene of conjugative transposon in L. casei/paracasei and two L. helveticus strains. Nucleotide sequence analysis of the amplified tetM fragments demonstrated their high homology with the tetM genes of Enterococcus faecalis and Streptococcus pneumoniae. The strains carrying tetM were tested for the genes of replication and conjugative transfer of plasmids in lactic acid bacteria. The results indicated
that these strains contain genes identical or highly homologous to the rep and trsK genes of the plca36 plasmid and rep gene of the pLH1 and pLJ1 plasmids of lactic acid bacteria. The tetM gene is probably not expressed in strains sensitive to the corresponding antibiotic. However, the investigated lactobacilli
cannot be directly used as probiotics, as they may serve as a source of genes for antibiotic resistance in the human microbiome.
–lactic acid bacteria–probiotics–human gastrointestinal microbiome–antimicrobial agents–antibiotic resistance–plasmids–genes tetW/O
[Show abstract][Hide abstract] ABSTRACT: Serine/threonine protein kinases (STPKs) are the major participants in intracellular signal transduction in eukaryotes, such as yeasts, fungi, plants, and animals. Genome sequences indicate that these kinases are also present in prokaryotes, such as cyanobacteria. However, their roles in signal transduction in prokaryotes remain poorly understood. We have attempted to identify the roles of STPKs in response to heat stress in the prokaryotic cyanobacterium Synechocystis sp. PCC 6803, which has 12 genes for STPKs. Each gene was individually inactivated to generate a gene-knockout library of STPKs. We applied in vitro Ser/Thr protein phosphorylation and phosphoproteomics and identified the methionyl-tRNA synthetase, large subunit of RuBisCO, 6-phosphogluconate dehydrogenase, translation elongation factor Tu, heat-shock protein GrpE, and small chaperonin GroES as the putative targets for Ser/Thr phosphorylation. The expressed and purified GroES was used as an external substrate to screen the protein extracts of the individual mutants for their Ser/Thr kinase activities. The mutants that lack one of the three protein kinases, SpkC, SpkF, and SpkK, were unable to phosphorylate GroES in vitro, suggesting possible interactions between them towards their substrate. Complementation of the mutated SpkC, SpkF, and SpkK leads to the restoration of the ability of cells to phosphorylate the GroES. This suggests that these three STPKs are organized in a sequential order or a cascade and they work one after another to finally phosphorylate the GroES.
DNA Research 01/2011; 18(3):137-51. · 4.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The article reviews the main systems that regulate gene expression in cyanobacteria in response to various treatments: low and high temperatures, salt, hyperosmotic and oxidative stresses. The systems for perception of light are also reviewed. Functional
characteristics are presented for known two-component regulatory systems, eukaryotic-type serine-threonine protein kinases, σ-subunits of RNA-polymerase, DNA-binding transcription factors. Different mechanisms of perception of stress signals are analyzed, including changes in DNA supercoiling under different stress conditions.
Keywordscyanobacteria–gene expression–histidine kinases–sensors–serine-threonine protein kinases–stress–transcription factors
[Show abstract][Hide abstract] ABSTRACT: The species and strain genetic diversity of bacterial cultures belonging to the genus Lactobacillus, which were isolated from the gastrointestinal microbiome of the human population living in the former Soviet Union in the years 1960-1980, was studied. The bacteria demonstrated probiotic characteristics. Phylogenetic analysis of sequences of the gene coding for 16S rRNA detected earlier by us, showed that the gene found in bacteria isolated from the intestinal content of healthy adults and represented by species L. plantarum, L. helveticus, L. casei/paracasei, L. rhamnosus, and L. fermentum has high homology (97-100%) with this gene in type representatives of the species. The genotypic and strain diversity of cultures was studied using RAPD-PCR and nonspecific primers. This method with the use of the ERIC-1 primer gave reliable and reproducible results as compared that using with M13 and MSP primers and allowed the identification of examined bacteria belonging to the genus Lactobacillus at the level of species and certification at the strain level.
[Show abstract][Hide abstract] ABSTRACT: Thirteen strains of industrial bacterial cultures of the genus Lactobacillus (from a collection of Gabrichevsky Research Institute of Epidemiology and Microbiology) were studied. These strains were used for decades in Russian Federation for food and drug production, as ferments for lactic acid products, for production of probiotics, biologically active and veterinary preparations. Complex analysis of data on cultures obtained using microbiological and molecular-genetic methods was conducted for the first time. Biochemical characteristics of these cultures were studied and the sequence of the proximal region of 16S ribosomal RNA gene was determined. The employment of the test system API-50CHL was shown to broaden the opportunities of a more accurate biochemical identification of bacteria belonging to the genus Lactobacillus, in comparison with the set ANAEROTEST-23. According to the results obtained in a comparative analysis of nucleotide sequences of 16S rRNA gene, all strains examined show 97-99% homology of the proximal region of this gene with that of the type representatives of studied species. These data allowed taxonomic reclassification of the species position of cultures with consideration of the more advanced level of systematics. Nucleotide sequences of gene fragments of examined lactobacilli strains were recorded in NCBI database (accession numbers of deposits GU560031, GU560032, GU560033, GU560034, GU560035, GU560036, GU560037, GU560038, GU560039, GU560040, GU560041, GU560042, GU560043).
[Show abstract][Hide abstract] ABSTRACT: In living cells, the perception of environmental stress and the subsequent transduction of stress signals are primary events in the acclimation to changes in the environment. Some molecular sensors and transducers of environmental stress cannot be identified by traditional and conventional methods. Based on genomic information, a systematic approach has been applied to the solution of this problem in cyanobacteria, involving mutagenesis of potential sensors and signal transducers in combination with DNA microarray analyses for the genome-wide expression of genes. Forty-five genes for the histidine kinases (Hiks), 12 genes for serine-threonine protein kinases (Spks), 42 genes for response regulators (Rres), seven genes for RNA polymerase sigma factors, and nearly 70 genes for transcription factors have been successfully inactivated by targeted mutagenesis in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Screening of mutant libraries by genome-wide DNA microarray analysis under various stress and non-stress conditions has allowed identification of proteins that perceive and transduce signals of environmental stress. Here we summarize recent progress in the identification of sensory and regulatory systems, including Hiks, Rres, Spks, sigma factors, transcription factors, and the role of genomic DNA supercoiling in the regulation of the responses of cyanobacterial cells to various types of stress.
[Show abstract][Hide abstract] ABSTRACT: To study strains of bacteria from Lactobacillus genus using combination of microbiological and molecular biological methods in order to define more accurately their systematic position and biochemical characteristics.
Thirteen cultures of Lactobacillus bacteria isolated from stool of healthy persons were studied: L. plantarum CS 396, L. plantarum 8-PA-3, L. plantarum 421-2, L. fermentum 90-TC-4, L. delbrueckii gKNM 101, L. delbrueckii gKNM 526, L. acidophilus Er 317/402 NARINE, L. acidophilus 100 ash, L. acidophilus NK-1, L. acidophilus NNIE, L. acidophilus K3sh24, L. brevis gKNM 23 11, L. casei gKNM 577. Their enzymatic activity relative to 50 sugars was studied using API-50 system. Structure of proximal region of 16S rRNA gene was studied also.
According to results of 16S rRNA gene sequence analysis strains were divided on 2 groups: 1) L. casei gKNM 577, L. plantarum 8-PA-3, L. plantarum CS 396, which species belonging corresponded to stated description. Comparison of nucleotide sequence of 16S rRNA gene of group 2 strains with nucleotide sequences database revealed that cultures NK-1, Er315/402 NARINE, 100 ash, NNIE identified early as L. acidophilus belong to species L. helveticus; L. brevis gKNM 23 and L. acidophilus K3sh24--to group L. casei/paracasei, L. delbrueckii gKNM 101 and L. fermentum 90-TC-4--to L. plantarum, L. delbrueckii gKNM 526--to L. fermentum, and L. plantarum 421-2--to L. rhamnosus.
Obtained data allowed to perform taxonomic reclassification of species belonging of studied probiotic cultures of lactobacilli according to modem level of systematic of bacteria.
Zhurnal mikrobiologii, epidemiologii, i immunobiologii 01/2010;
[Show abstract][Hide abstract] ABSTRACT: Plants and other photosynthetic organisms experience frequent changes in environment. Their ability to survive depends on
their capacity to acclimate to such changes. In particular, fluctuations in temperature and/or osmolarity affect the fluidity
of cytoplasmic and thylakoid membranes. The molecular mechanisms responsible for the perception of changes in membrane fluidity
have not been fully characterized. However, the analysis of genome-wide gene expression with DNA microarrays has provided
a powerful new approach to studies of the contribution of membrane fluidity to gene expression and to the identification of
environmental sensors. In this chapter, we summarize the knowledge on the mechanisms that regulate membrane fluidity, on putative
sensors that perceive changes in membrane fluidity, and on the subsequent expression of genes that ensures acclimation to
a new set of environmental conditions.
[Show abstract][Hide abstract] ABSTRACT: The charged quaternary ammonium compounds--methyl, ethyl and benzyl viologens--generate reactive oxygen species in photosynthetic cells. Three independent methyl viologen-resistant spontaneous mutants of Synechocystis sp. PCC 6803 were identified, in which the conserved R115 residue of the Slr1174 protein was replaced with G115, L115 and C115. The Slr1174 protein of the DUF990 family is related to the permease subunit of an ABC-2-type transporter and its R115 mutation was found to be solely responsible for the observed methyl viologen resistance. Bioinformatic analysis showed that in various bacterial genomes, two genes encoding another permease subunit and the ATPase component of an ATP-binding cassette transporter form putative operons with slr1174 orthologs, suggesting that the protein products of these genes may form functional transporters. The corresponding genes in Synechocystis sp. PCC 6803, i.e. slr0610 for the permease and slr1901 for the ATPase, did not form such an operon. However, insertional inactivation of any slr1174, slr0610 or slr1901 genes in both the wild-type and the R115-resistant mutant resulted in increased sensitivity to methyl, ethyl and benzyl viologens; moreover, single- and double-insertion mutants did not differ in their viologen sensitivity. Our data suggest that Slr1901, Slr1174 and Slr0610 form a heteromeric ATP-binding cassette-type viologen exporter, in which each component is critical for viologen extrusion. Because the greatest increase in mutant sensitivity was observed in the case of ethyl viologen, the three proteins have been named EvrA (Slr1901), EvrB (Slr1174) and EvrC (Slr0610). This is the first report of a function for a DUF990 family protein.
[Show abstract][Hide abstract] ABSTRACT: Genome-wide investigations of gene expression at the transcriptional level in cyanobacteria, using DNA microarrays, have allowed identification of genes whose expression is induced or repressed by various types of environmental stress and also of previously uncharacterized genes that appear to be involved in stress responses. Acclimation to stress begins with perception of stress and transduction of the stress signal. A combination of the systematic mutation of potential sensors and transducers and DNA microarray analysis has led to significant progress in understanding the mechanisms of perception of and reaction to environmental stress in cyanobacteria. In this chapter, we summarize recent progress in the identification of stress-inducible genes and of systems that regulate responses to stress that has been made using DNA microarrays in cyanobacteria and, in particular, in Synechocystis sp. PCC 6803.
[Show abstract][Hide abstract] ABSTRACT: In previous studies, we characterized five histidine kinases (Hiks) and the cognate response regulators (Rres) that control the expression of approximately 70% of the hyperosmotic stress-inducible genes in the cyanobacterium Synechocystis sp. PCC 6803. In the present study, we screened a gene knock-out library of Rres by RNA slot-blot hybridization and with a genome-wide DNA microarray and identified three Hik-Rre systems, namely, Hik33-Rre31, Hik10-Rre3, and Hik16-Hik41-Rre17, as well as another system that included Rre1, that were involved in perception of salt stress and transduction of the signal. We found that these Hik-Rre systems were identical to those that were involved in perception and transduction of the hyperosmotic stress signal. We compared the induction factors of the salt stress- and hyperosmotic stress-inducible genes that are located downstream of each system and found that these genes responded to the two kinds of stress to different respective extents. In addition, the Hik33-Rre31 system regulated the expression of genes that were specifically induced by hyperosmotic stress, whereas the system that included Rre1 regulated the expression of one or two genes that were specifically induced either by salt stress or by hyperosmotic stress. Our observations suggest that the perception of salt and hyperosmotic stress by the Hik-Rre systems is complex and that salt stress and hyperosmotic stress are perceived as distinct signals by the Hik-Rre systems.
Journal of Biological Chemistry 07/2005; 280(22):21531-8. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To establish the role of the two putative type I leader peptidases (LepB1 and LepB2) encoded in the genome of the cyanobacterium Synechocystis sp. strain PCC 6803, we generated independent knockout mutants for both genes by introducing kanamycin resistance cassettes into the two open reading frames (sll0716 [lepB1] and slr1377 [lepB2], respectively). Although the insertion was successful in both instances, it was not possible to select homozygous mutant cells for lepB2, suggesting that the function of this gene is essential for cell viability. In contrast, LepB1 is apparently essential only for photoautotrophic growth, because homozygous lepB1::Km(r) cells could be propagated under heterotrophic conditions. They were even capable to some extent of photosynthetic oxygen evolution. However, the photosynthetic activity decreased gradually with extended incubation in the light and was particularly affected by high light intensities. Both features were indicative of photooxidative damage, which was probably caused by inefficient replacement of damaged components of the photosynthetic machinery due to the lack of a leader peptidase removing the signal peptides from photosynthetic precursor proteins. Indeed, processing of the PsbO precursor polypeptide to the corresponding mature protein was significantly affected in the mutant, and reduced amounts of other proteins that are synthesized as precursors with signal peptides accumulated in the cells. These results strongly suggest that LepB1 is important for removal of the signal peptides after membrane transport of the components of the photosynthetic machinery, which in turn is a prerequisite for the biogenesis of a functional photosynthetic electron transport chain.
Journal of Bacteriology 06/2005; 187(9):3071-8. · 3.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Geranylgeranyl reductase catalyses the reduction of geranylgeranyl pyrophosphate to phytyl pyrophosphate required for synthesis of chlorophylls, phylloquinone and tocopherols. The gene chlP (ORF sll1091) encoding the enzyme has been inactivated in the cyanobacterium Synechocystis sp. PCC 6803. The resulting DeltachlP mutant accumulates exclusively geranylgeranylated chlorophyll a instead of its phytylated analogue as well as low amounts of alpha-tocotrienol instead of alpha-tocopherol. Whereas the contents of chlorophyll and total carotenoids are decreased, abundance of phycobilisomes is increased in DeltachlP cells. The mutant assembles functional photosystems I and II as judged from 77 K fluorescence and electron transport measurements. However, the mutant is unable to grow photoautotrophically due to instability and rapid degradation of the photosystems in the absence of added glucose. We suggest that instability of the photosystems in DeltachlP is directly related to accumulation of geranylgeranylated chlorophyll a. Increased rigidity of the chlorophyll isoprenoid tail moiety due to three additional CC bonds is the likely cause of photooxidative stress and reduced stability of photosynthetic pigment-protein complexes assembled with geranylgeranylated chlorophyll a in the DeltachlP mutant.
Biochimica et Biophysica Acta 03/2005; 1706(3):195-203. · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The sll1703 gene, encoding an Arabidopsis homologue of the thylakoid membrane-associated SppA peptidase, was inactivated by interposon mutagenesis in Synechocystis sp. strain PCC 6803. Upon acclimation from a light intensity of 50 to 150 microE m(-2) s(-1), the mutant preserved most of its phycobilisome content, whereas the wild-type strain developed a bleaching phenotype due to the loss of about 40% of its phycobiliproteins. Using in vivo and in vitro experiments, we demonstrate that the DeltasppA1 strain does not undergo the cleavage of the L(R)(33) and L(CM)(99) linker proteins that develops in the wild type exposed to increasing light intensities. We conclude that a major contribution to light acclimation under a moderate light regime in cyanobacteria originates from an SppA1-mediated cleavage of phycobilisome linker proteins. Together with changes in gene expression of the major phycobiliproteins, it contributes an additional mechanism aimed at reducing the content in phycobilisome antennae upon acclimation to a higher light intensity.
Journal of Bacteriology 07/2004; 186(12):3991-9. · 3.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Microorganisms respond to hyperosmotic stress via changes in the levels of expression of large numbers of genes. Such responses are essential for acclimation to a new osmotic environment. To identify factors involved in the perception and transduction of signals caused by hyperosmotic stress, we examined the response of Synechocystis sp. PCC 6803, which has proven to be a particularly useful microorganism in similar analyses. We screened knockout libraries of histidine kinases (Hiks) and response regulators (Rres) in Synechocystis by DNA microarray and slot-blot hybridization analyses, and we identified several two-component systems, which we designated Hik-Rre systems, namely, Hik33-Rre31, Hik34-Rre1, and Hik10-Rre3, as well as Hik16-Hik41-Rre17, as the transducers of hyperosmotic stress. We also identified Hik2-Rre1 as a putative additional two-component system. Each individual two-component system regulated the transcription of a specific group of genes that were responsive to hyperosmotic stress.
Journal of Biological Chemistry 01/2004; 279(51):53078-86. · 4.65 Impact Factor