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.

[Application of robotic technology to the needs in the medical service of the Armed Forces].

Application of robotic technology to the needs in the medical service of the Armed Forces. Further development of the medical service is inseparably associated with the implementation of robot technology into the practice of medical support of the Armed Forces of the Russian federation. For this purpose it is necessary to create a clinical scientific research centre of robot technology and interdepartmental scientific research simulation training center on the basis of the Kirov Military Medical Academy. It is also necessary to provide development of medical robotic complexes of tactical level of the medical service.

[Evolution aspects of intraspecific polymorphysm of Arabidopsis thaliana genes coding subunit I of Mg-chelatase complex].

Intraspecific polymorphism in the CHLI 2 gene, coding subunit I of Mg-chelatase complex that forms Mg-protoporphyrin IX was investigated in 19 ecotypes of Arabidopsis thaliana. Sequence divergence by 35 nucleotides was found; 12 of them result into amino acid change in third exon where functional domains of the protein are located. In one of two found divergent sequence haplogroups, Col haplogroup an excess of low-frequency polymorphism was revealed, showing the action of purifying selection and indicating a functional significance of CHLI 2 gene. Paths of evolutionary dynamics for CHLI 1 and CHLI 2 genes determining different forms of subunit I of Mg-chelatase complex of Arabidopsis thaliana are proposed.

[Determination of type and spatial pattern formation of flower organs: dynamic model of development].

The mathematical model imitating floral organ spatial pattern formation (positioning) was developed. Computer experiments performed on its basis demonstrated that organ spatial pattern formation in typical crucifer flower occurred in successive order: medial sepals, carpels, lateral sepals, long stamens, petals and short stamens. The positioning was advanced in two directions, acropetally in the perianth and basipetally in the stamens and carpels. The organ type specifying and positioning take place non-simultaneously in different floral areas. The organ type specifying passed ahead of organ primordial spatial pattern formation. The modeling of flower development of several mutants demonstrated that arabidopsis genes AP2 and AG in addition to specifying floral organ types also determine the particular zones in the floral meristem for futur organ development. The AG gene controls the formation of basipetal patterning zones where the reproductive organs develop, AP2 maintains the proliferative activity in the floral meristem that form acropetal patterning zones where perianth organ develop.

[Genetic control of hydrogen metabolism in cyanobacteria].

The development of methods for the use of phototrophic cyanobacteria as producers of molecular hydrogen via bioconversion of solar energy is a promising filed of hydrogen energetics. Artificial optimization of hydrogen formation and release is based on studying the genetic control of hydrogen metabolism and the use of genetic approaches for obtaining efficient producer strains. Data on genes coding for the hydrogenases that are responsible for hydrogen uptake and production in cyanobacteria are summarized. Bioinformatic methods have been used to construct the scheme of the hydrogen metabolism gene network of nitrogen-fixing heterocyst cyanobacteria. The possible approaches to constructing the cyanobacterium strains producing molecular hydrogen that would be promising for photobiotechnology by mutagenesis and genetic engineering methods are discussed in terms of this model and analysis of the data on hydrogen-producing mutants.

[Interspecies polymorphism of peroxidase genes, localized on chromosome 5 of Arabidopsis thaliana].

Comparative analysis of nucleotide sequences in five peroxidase genes AtPrx52-AtPrx56 located in the left arm of chromosome 5 was performed by using six Arabidopsis thaliana ecotypes and lines (Columbia, Dijon-M, Blanes-M, Enkheim-M, Ler, K-156). Significal differences (up to 20 times) in the levels of nucleotide variation between these genes were detected: tandem duplicated genes AtPrx53 and AtPrx54 have the highest and the AtPrx56 gene has the lowest level of nucleotide diversity. The genes AtPrx53 and AtPrx54 were characterized by allelic dimorphism; the nonrandom association between nucleotide polymorphic sites within the AtPrx54 was shown. The connection between gaplotype of these genes and the mobility of anionic peroxidase izoforms was detected. Since two gaplotypes of AtPrx53 were coding proteins, which differed by two significant amino acid substitutions, we supposed that differences in mobility of anionic peroxidase izoforms caused by the diallelic polymorphism in amino acid sequence of AtPrx53 protein.

Involvement of the SppA1 peptidase in acclimation to saturating light intensities in Synechocystis sp. strain PCC 6803.

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.

[Insertional inactivation of genes encoding eukaryotic type serine/threonine protein kinases in cyanobacterium Synechocystis sp. PCC 6803]. / Insertsionnaia inaktivatsiaa genov, kodiruiushchikh serin/treoninovye proteinkinazy éukarioticheskogo tipa u tsianobakterii Synechocystis sp. PCC 6803.

Synechocystis sp. PCC 6803 mutants, in which one of the eukaryotic-type serine/threonine protein kinase genes pknD, pknE, pknG, and pknH was inactivated, were obtained by insertion mutagenesis. None of these mutants differed phenotypically from the wild-type strain, indicating that the pknD, pknE, pknG, and pknH genes are not of crucial importance for the photoautotrophically grown cyanobacterium. Mutant with the inactivated pknE gene was resistant to L-methionine-D,L-sulfoximine and especially to methylamine. The resistance was neither due to the impaired transport of these compounds nor to the inhibition of the production of toxic gamma-glutamylmethylamide from methylamine. The data presented suggest that resistance to methylamine may be associated with alterations in the regulation of the glutamine synthetase system and that the PknE protein kinase may be involved in the regulation of nitrogen metabolism in the cyanobacterium studied.

[PXD gene controls synthesis of the three anionic peroxidase isoforms in Arabidopsis thaliana]. / Gen PXD kontroliruet obrazovanie trekh izoform anionnykh peroksidaz Arabidopsis thaliana.

The pxd mutant of Arabidopsis thaliana had a changed spectrum of anionic peroxidases: three anionic isoforms in the pxd mutant plant had the same enzymatic activity but relatively high electrophoretic mobility as compared to the analogous isoforms in the wild type plants. These isoforms were the most active anionic peroxidases and could be found in most plant organs. Genetic analysis showed that all three isoforms were controlled by the PXD gene. The activity of one isoform was affected by indolyl-3-acetic acid and other stress factors. Expressed sequence tags (EST) analysis of all putative peroxidase genes of A. thaliana revealed the group of the most actively and nonspecifically expressed genes. The promoter sequences of these genes were screened to find the cis-elements. We propose that the PXD gene encodes one of the nonspecific anionic peroxidases or a protein involved in posttranscriptional or posttranslational modification of the peroxidases.

[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.

[Study of the functional role of Ctp-family proteins in Synechocystis sp. PCC 6803 cyanobacteria]. / Izuchenie funktsional'noi roli belkov Ctp-semeistva u tsianobakterii Synechocystis sp. PCC 6803.

To understand the functional role of CtpB and CtpC proteins, which are similar to the C-terminal processing CtpA peptidase, the effect of the insertional inactivation of the ctpB and ctpC genes on the phenotypic characteristics of Synechocystis sp. PCC 6803 was studied. The inactivation of the ctpC gene was found to be lethal to the cyanobacterium, which indicates a vital role of the CtpC protein. The mutant with the inactivated ctpB gene had the same photosynthetic characteristics as the wild-type strain. The double mutant@[delta]ctpA delta ctpB with the two deleted genes was identical, in the phenotypic characteristics, to the mutant with a knock-out mutation in the ctpA gene, which was unable to grow photoautotrophically. The data obtained suggest that, in spite of the high similarity of the Ctp proteins, they serve different functions in Synechocystis sp. PCC 6803 cells and cannot compensate for each other.

Deregulation of electron flow within photosystem II in the absence of the PsbJ protein.

The photosystem II (PSII) complex of photosynthetic oxygen evolving membranes comprises a number of small proteins whose functions remain unknown. Here we report that the low molecular weight protein encoded by the psbJ gene is an intrinsic component of the PSII complex. Fluorescence kinetics, oxygen flash yield, and thermoluminescence measurements indicate that inactivation of the psbJ gene in Synechocystis 6803 cells and tobacco chloroplasts lowers PSII-mediated oxygen evolution activity and increases the lifetime of the reduced primary acceptor Q(A)(-) (more than a 100-fold in the tobacco DeltapsbJ mutant). The decay of the oxidized S(2,3) states of the oxygen-evolving complex is considerably accelerated, and the oscillations of the Q(B)(-)/S(2,3) recombination with the number of exciting flashes are damped. Thus, PSII can be assembled in the absence of PsbJ. However, the forward electron flow from Q(A)(-) to plastoquinone and back electron flow to the oxidized Mn cluster of the donor side are deregulated in the absence of PsbJ, thereby affecting the efficiency of PSII electron flow following the charge separation process.