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.

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.

In Gram-negative bacteria, transport of ferric siderophores through outer membrane is a complex process that requires specific outer membrane transporters and energy-transducing TonB-ExbB-ExbD system in the cytoplasmic membrane. The genome of the non-siderophore-producing cyanobacterium Synechocystis sp. PCC 6803 encodes all putative components of the siderophore-mediated iron uptake system. So far, there has been no experimental evidence for the existence of such a pathway in this organism. On the contrary, its reductive iron uptake pathway has been studied in detail. We demonstrate that Synechocystis sp. PCC 6803 is capable of using dihydroxamate xenosiderophores, either ferric schizokinen (FeSK) or a siderophore of the filamentous cyanobacterium Anabaena variabilis ATCC 29413 (SAV), as the sole source of iron. Inactivation of the tonB gene or the exbB1-exbD1 gene cluster resulted in an inability to utilize these siderophores. At the same time, the inactivation of the feoB gene encoding FeoB plasma membrane ferrous iron transporter, or one of the futB or futC genes encoding permease and ATPase subunit of FutABC ferric iron transporter, did not impair the ability of cells to utilize FeSK or SAV as the sole source of iron for growth. Our data suggest that cyanobacterium Synechocystis sp. PCC 6803 is capable of acquiring iron-siderophore complexes in a TonB-dependent manner without iron reduction in the periplasm.

Mutants of Rhodobacter cupsulafus deficient in growth on nitrogen sources other than NH,+ were compared with mutants of a similar phenotype isolated from Rhodobacter sphueroides. In addition to N2 and some amino acids (glutamate, alanine, proline, arginine), mutants of R. sphmroides and R. cupsulafus strain AD2 were unable to utilize NO? as sole nitrogen source for growth. Under conditions of nitrogen starvation, mutants of both species lacked the methylammonium (ammonium) uptake system, which was found in the wild-type strains under these conditions. The wild-type (adgA) genes complementing these mutants were isolated from gene banks of the respective species and localized to a 2.9 kb BmHI-SdI fragment in R. sphaeroides and to a 1.7 kb SmuI fragment in R. cupsulutus. These two fragments hybridized strongly with each other, showing that they contain homologous sequences. Furthermore, the adgA gene from R. cupsulutus fully restored the wild-type phenotype to Adg-mutants of R. sphueroides and vice versa. Inactivation of the chromosomal adgA gene by insertion of an antibiotic-resistance cassette resulted in a severe inhibition of growth in rich medium and in minimal medium containing NHJ. This suggests that the adgA gene is required for normal growth under all growth conditions.