Metagenomic Analysis of the Dynamic Changes in the Gut Microbiome of the Participants of the MARS-500 Experiment, Simulating Long Term Space Flight.

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.

[Cyanobacterial cell division: genetics and comparative genomics of cyanobacterial cell division].

Division of cyanobacteria serves as a model for studying division of plant chloroplasts. Analysis of mutants obtained by methods of "forward" and "reverse" genetics underlies effective vertical strategy for studying genetics of cell division in these photoautotrophic prokaryotes. Comparative genomic analysis indicates that some cyanobacterial genes involved in the control of cell division have homologs among cyanobacteria, green algae, and higher plants, some others, only in bacteria, whereas the remaining genes are specific only for cyanobacteria.

[Inversion of phototaxis in cells of Synechocystis sp. PCC 6803 determined by a mutation in the regulatory gene prqR].

Phototaxis, positive (movement toward the light source) or negative (from the light source) mediates the adaptation of cyanobacteria to varying wave lengths and illumination intensity. The transcription regulator PrqR of the family TetR is known as a repressor of the prqRA operon controlling resistance to the oxidative stress inducer methyl viologen in the cyanobacterium Synechocystis sp. PCC6803. However, it was shown in this work that mutation prqRL17Q affecting the DNA-binding domain of the PrqR protein, which causes derepression of the prqRA operon and enhances cell resistance to methyl viologen, additionally determines negative phototaxis induced with daylight and red light of low intensity. The inactivation of gene prqA did not affect cell motility in mutant PqR carrying mutation prqRL17Q and in the wild-type strain characterized by positive phototaxis appearing in response to the light of low intensity. Moreover, a mutant with deletion prqR did not differ from the wild-type strain with respect to phototaxis type suggesting that the specificity of the regulator protein was changed in cells carrying prqRL17Q mutation. Note that changes in transcription of pilA genes that control biogenesis of pili providing for cell motility were not detected in mutant PqR, and, in agreement with data of atomic force microscopy, the type of pili formation is identical in prqR mutants and the wild-type strain. Meanwhile, mutant PqR manifested a decrease in transcription of gene taxD1 encoding the photoreceptor of red light that is required for the positive phototaxis of cyanobacteria. These data imply that mutation prqRL17Q changes the specificity of the PrqR repressor protein and thereby affects the regulation of phototaxis at the level of photoperception and signal transduction in cells.

[The sll0886 gene, controlling light-activated heterotrophic growth, is involved in regulating phototaxis in cyanobacterium Synechocystis sp. PCC 6893].

The sll0886 gene, controlling light-activated heterotrophic growth (LAHG), was tested for the role in regulating phototaxis in cyanobacterium Synechocystis sp. PCC 6803. Insertional inactivation of the gene in the genome of a wildtype strain did not affect positive (toward light) or negative (away from high light) phototaxis. However, cells lost motility when sll0886 inactivation was combined with the prqRL17Q mutation, which determined negative phototaxis at low light. Immotile cells with the prqRL17Q mutation and the inactivated sll0886 gene did not display any defect in the formation of type IV pili, essential for phototaxis. Hence, the function, rather than biogenesis, of pili was affected. It was concluded that the sll0886 gene, coding for a TPR family protein, is involved in controlling negative phototaxis of cyanobacteria at the level of photoreception and signal transduction and that its role is mediated by the unidentified redundant gene whose function is suppressed by the prqRL17Q mutation.

[Effect of oxidative stress inductors on the photosynthetic apparatus in cyanobacterium Synechocystis sp. PCC 6803 Prq20 mutant resistant to methyl viologen].

The damaging effect of oxidative stress inductors: methyl viologen, benzyl viologen, cumene hydroperoxide, H2O2, menadion, and high irradiance on the photosynthetic apparatus of cyanobacterium Synechocystis sp. PCC 6803 in cells of the wild type strain and the methyl viologen-resistant Prq20 mutant with the disrupted function of the regulatory gene prqR has been investigated by measuring the delayed fluorescence of chlorophyll a and the rate of CO2dependent -O2 gas exchange. It has been shown that the damage to the photosynthetic apparatus in the Prq20 mutant as compared with the wild type was less in the presence of methyl viologen and benzyl viologen. Reasons for the enhanced resistance of the photosynthetic apparatus in the mutant Prq20 to methyl viologen and benzyl viologen are discussed.

[Mutants of cyanobacterium Synechocystis sp. PCC6803 with insertion of the sodB gene encoding Fe-superoxide dismutase]. / Mutanty tsianobakterii Synechocystis sp. PCC6803 s insertsiiami v gen sodB, kodiruiushchii Fe-superoksiddismutazu.

The sodB gene encoding the only superoxide dismutase (Fe-SOD) in cells of the cyanobacterium Synechocystis sp. PCC6803 was inactivated with gentamycin resistance aacC1 marker insertions located in the direct or inverted order toward the sodB gene. The corresponding delta sodB12 and delta sodB22 mutants are characterized by the complete absence of superoxide dismutase activity and the loss of viability upon standard photoautotrophic cultivation. Mutant cells can grow under conditions of a decreased illumination intensity and upon addition of NaHCO3 with catalase or bovine serum albumin in the growth medium. The delta sodB22 mutant is auxotrophic for leucine due to the polar effect of insertion into the sodB gene on the downstream leuB gene controlling leucine biosynthesis. These data suggest that Fe-SOD is very important for providing tolerance of Synechocystis cells to oxidative stress and that sodB and leuB genes are organized into a single operon.

[The prqA and mvrA genes encoding carrier proteins control resistance to methyl viologen in the cyanobacterium Synechocystis sp. PCC6803]. / Geny prqA i mvrA, kodiruiushchie belki-transportery, kontroliruiut ustoichivost' k metilviologenu u tsianobakterii Synechocystis sp. PCC6803.

Derivatives with insertional inactivation of prqA and mvrA genes were obtained and studied in the Synechocystis sp. PCC6803 wild-type strain and in the mutant Prq20 resistant to methyl viologen (MV). It was shown that the formation of resistance to MV is associated with the operation of two systems: constitutive and inducible. The prqA gene encoding drug efflux proteins controls the constitutive system of cell resistance to MV. Derepression of the prqA gene is the main reason for an enhanced MV resistance in the Prq20 mutant with impaired repressor function of the PrqR protein. The mvrA gene encoding the transmembrane protein from the family of transporters of sugar and other compounds controls the inducible MV resistance. It is assumed that the MvrA protein is required for efficient elimination from cells of toxic substances formed upon oxidative stress or participates in the repair of membranes destroyed by oxidants. The data obtained demonstrated for the first time that transport systems are involved in the formation of MV resistance in photosynthetic organisms.

[On the involvement of the regulatory gene prqR in the development of resistance to methyl viologen in cyanobacterium Synechocystis sp. PCC6803]. / Ob uchastii reguliartornogo gena prqR v razvitii ustoichivosti k metilviologenu u tsianobakterii Synechocystis sp. PCC6803.

The role of the prqR gene in the regulation of the adaptive response of the cyanobacterium Synechocystis sp. PCC6803 to the oxidative stress induced with methyl viologen (MV) was studied. For this, transcription activity of prqR and the genes, which may be involved in the control of resistance to MV, was determined by means of Northern blot hybridization in wild-type cells and in the MV-resistant Prq20 mutant with a mutation located in the DNA-binding domain of the PrqR protein. It was ascertained that the prqR gene is a component of the prqR-prqA operon and down regulates its transcription. In cells of the wild-type strain containing MV, the autorepressor activity of the PrqR protein enhances and transcription of mvrA and sodB genes encoding an respectively assumed transporter protein and iron-containing superoxide dismutase increases. The prqR gene may be involved in the negative, indirect control of transcription of these genes. The Prq20 mutant is characterized by an MV-independent derepression of the prqR-prqA operon and by a slightly increased transcription of mvrA and sodB genes not stimulated by MV. Nevertheless, the expression of mvrA and sodB genes was lower than in wild-type cells after the MV treatment. On the strength of this evidence, it is assumed that the main mechanism underlying for the resistance to MV in the Prq20 mutant is derepression of the prqA gene, the product of which is homologous to multidrug transporters, drug efflux proteins.

[Trans- and cis-acting autorepressors of the prqR gene in Synechocystis cyanobacteria sp. PCC6803]. / Trans- i tsis-deistvuiushchaia avtorepressiia gena prqR u tsianobakterii Synechocystis sp. PCC 6803.

Genetic analysis of the allele interactions was carried out with the use of recombinant plasmids and reporter genes to study the autorepressor function of prqR, which negatively regulates the prqR-prqA operon and the response to oxidative stress inductor methyl viologen (MV) in cyanobacterium Synechocystis sp. PCC 6803. The wild-type (WT) prqR showed negative autoregulation and suppressed in trans the derepressed mutant alleles. Frameshift mutation C134fs, which was introduced in prqR by site-directed mutagenesis, impaired the autoregulation, implicating the C-terminal domain in transcriptional repression by PrqR. Missense mutation C134S altering the only redox-sensitive Cys of PrqR, had no effect on prqR expression, indicating that oxidation and consequent disulfide bridging of two PrqR molecules was not responsible for MV-induced autorepression of prqR. Analysis of the prqR-prqA deletion derivatives lacking the promoter and most of prqR revealed weak uncontrollable expression of reporter cat, testifying to the existence of a constitutive promoter in prqA responsible for MV resistance. The interaction of the WT and mutant prqR alleles in Synechocystis cells revealed a cis-dominant character of the alteration of prqR autoregulation. Stimulation of in cis autorepression of prqR was assumed to contribute to the induction of systems protecting cyanobacteria from oxidative stress.

[Mutation in the glmS gene responsible for the cell wall synthesis increases resistance to the herbicide amitrole in the cyanobacterium Synechocystis sp. PCC6803]. / Mutatsiia v gene glmS, kontroliruiushchem sintez kletochnoi stenki, povyshaet ustoichivost' k gerbitsidu amitrolu u tsianobakterii Synechocystis sp. PCC6803.

A DNA fragment transforming the cells of the cyanobacterium Synechocystis sp. PCC 6803 to amitrole (3-amino-1,2,4-triazole) resistance was cloned from the Atr2 mutant resistant to this herbicide. The transforming activity of the cloned fragment was shown to be related to the missence-mutation "Val250-->Leu250" in the glmS gene encoding glucosamine-6-phosphate synthase, a key enzyme of cell wall synthesis. The amino acid substitution is localized in the central nonconservative part of the GlmS protein, far from two reaction centers positioned at the ends of a polypeptide. It is suggested that the mutant protein has lost sensitivity to amitrole. In the wild type strain, this herbicide causes conditional glucosamine auxotrophy (exogenous glucosamine restores ability of the cells to row in the presence of the lethal amitrole concentrations). Val250 is proposed to be allosteric binding site of AM in the GlmS protein of cyanobacterium.

Mutation in ntrC gene leading to the derepression of nitrogenase synthesis in Rhodobacter sphaeroides.

The Rhodobacter sphaeroides mutants Drn12 and Drn21 derepressed for nitrogenase synthesis in the presence of ammonia and impaired in utilization of certain nitrogen sources have been analyzed. Both mutants show a low level of expression of the glnBA operon. The DNA fragment restoring the wild-type phenotype to these mutants contains the 3'-portion of ntrB gene and the entire ntrC gene. Sequence analysis showed that Drn12 bears a missense mutation in the ntrC gene. The mutation results in the replacement of a glycine residue by aspartate within the N-terminal domain of the NtrC protein. Pleiotropic phenotypes of Drn12 and Drn21 appear to be associated with an alteration in the regulation of glnBA expression.

[Cloning and identification of the gene controlling nitrogen metabolism in the photosynthetic purple bacteria Rhodobacter sphaeroides]. / Klonirovanie i identifikatsiia gena, kontroliruiushchego azotistyi metabolizm u fotosinteziruiushchei purpurnoi bakterii Rhodobacter sphaeroides.

A recombinant plasmid has been selected from the genomic library of Rhodobacter sphaeroides that restores the properties of the wild type strain in the mutant Drn121. The latter possesses the derepressed synthesis of nitrogenase when grown in the light, inability of nitrogen fixation in the dark and growth on potassium nitrate as a single source of nitrogen, disruption of ammonium ions and methylamine transportation, decreased activity of glutamine synthetase. The gene complementing the drn121 mutation is localized within the EcoRI-HindIII fragment of Rhodobacter sphaeroides chromosome 2.25 kb in size. Analysis of the fragment nucleotide sequence has revealed the fragments with a high level of homology to regulatory genes ntrB (the 3'-end) and ntrC of Rhodobacter capsulatus. The plasmid pRCN102, containing the nifR3-ntrB-ntrC operon of Rhodobacter capsulatus, is able to complement the drn121 mutation while its derivatives having inactivated ntrN or ntrC genes are not. Hence, in Rhodobacter sphaeroides mutant Drn121 the mutation is localized in ntrC gene the product of which is involved not only in nitrogen fixation but also in nitrogen metabolism on the whole.

[Use of the plasmid R89S replicon for constructing integration vectors]. / Ispol'zovanie replikona plazmidy R89S dlia konstruirovaniia vektorov integratsii.

It has been shown that the plasmid R89S derivatives can be used as integrative vectors for bacteria in which the plasmid is unable to replicate autonomously. The chromosomal and plasmid fragments of phototrophic bacterium Rhodobacter sphaeroides have been cloned in plasmid pVZ365, a SmRKmR-derivative of R89S. The obtained recombinant plasmids were mobilized into R. sphaeroides cells by the I pcP-group conjugative plasmid R751. The frequencies of the SmR-transconjugants formation are 3.7.10(-5) to 5.6.10(-3) per recipient cell. The formation of the SmR-transconjugants has not been revealed in case of the plasmid pVZ365 mobilization. The recombinant molecules containing R. sphaeroides plasmid fragments have been shown to integrate into endogenous plasmids and form cointegrates with them.

Rapid separation of DNAs by buoyant density in three-layer CsCl gradients.

The formation of three-layer CsCl gradients with a narrow middle layer containing the analyzed material allows the separation of macrospecies according to their buoyant densities during the first hours of centrifugation before equilibrium is attained. The method can be used for the isolation and purification of various DNA molecules, such as mitochondrial and plasmid DNAs.

[X-ray-induced DNA degradation in Aspergillus nidulans cells. Comparative analysis of UV- and Xray-induced DNa degradation]. / Degradatsiia DNK, indutsirovannaia rentgenovskim oblucheniem v kletkakh Aspergillus nidulans. Sravnitel'nyi analiz UF- i RL-indutsirovannoi degradatsii DNK.

X-ray irradiation of Aspergillus nidulans wild strain cells from logarithmic phase of growth lead to delay of DNA synthesis. Insignificant enzymatic DNA degradation accomplished with its repair takes place. There is no direct dependence between doses of irradiation and levels of DNA degradation. Investigation of X-ray induced DNA degradation in the number of uvs-mutants has demonstrated the existence of two branches of DNA degradation: dependent and independent on external source of energy. Dependence of DNA degradation on the protein synthesis before and after irradiation was observed. It is proposed that the level of X-ray induced DNA degradation is determined by two protein systems: initiating and terminating DNA degradation. The comparative analysis of UV- and X-ray induced DNA degradation is carried out.

[UV-induced DNA degradation in Aspergillus nidulans cells]. / UF-indutsirovannaia degradatsiia DNK v kletkakh Aspergillus nidulans.

UV-induced DNA degradation was studied in mycellial cells of Aspergillus nidulans wild type and several uvs mutants. It was shown to be an enzymatic specific process which possibly reflects the excision of pyrimidine dimers from UV-damaged DNA. Inhibition of DNA degradation by caffeine and 2,4-dinitrophenol shows the connection between degradation and repair of DNA. Two ways of DNA degradation were found in A. nidulans cells, one of them being glucose dependent and the other--glucose independent. The dependence of DNA degradation on protein synthesis before and after UV-irradiation was demonstrated. The scheme of ways of DNA degradation and its genetic control were suggested on the basis of uvs mutations effect on UV-induced DNA degradation.