[Dominant phylotypes in the 16S rRNA gene clone libraries from bacterial mats of the Uzon caldera (Kamchatka, Russia) hydrothermal springs].

In situ analysis of the 16S rRNA genes form bacterial mats of five hydrothermal springs (36-58 degrees C) in the Uzon caldera (Kamchatka, Russia) was carried out using clone libraries. Eight clone libraries contained 18 dominant phylotypes (over 4-5%). In most clone libraries, the phylotype of the green sulfur bacterium Chlorobaculum sp. was among the dominant ones. The phylotypes of the green nonsulfur bacteria Chloroflexus and Roseiflexus and of purple nonsulfur bacteria Rhodoblastus, Rhodopseudomonas, and Rhodoferax were also among the dominant ones. Cyanobacteria were represented by one dominant phylotype in a single spring. Among nonphototrophic bacteria, the dominant phylotypes belonged to Sulfyrihydrogenibium sp., Geothrixsp., Acidobacterium sp., Meiothermus sp., Thiomonas sp., Thiofaba sp., and Spirochaeta sp. Three phylotypes were not identified at the genus level. Most genera of phototrophic and nonphototrophic organisms corresponding to the phylotypes from Uzon hydrotherms have been previously revealed in the hydrotherms of volcanically active regions of America, Asia, and Europe. These results indicate predominance of bacterial mats carrying out anaerobic photosynthesis in the hydrotherms of the Uzon caldera.

[Novel site-specific endonucleases F-TflI, F-TflII and F-TflIV encoded by the bacteriophage T5]. / Novye sait-spetsificheskie éndonukleazy F-TfI, F-TfII i F-TfIV, kodiruemye bakteriofagom T5.

Novel site-specific H-N-H endonucleases F-TflI, F-TflII and F-TflIV were identified. These endonucleases are encoded by open reading frames localized in the bacteriophage T5 tRNA gene region. The endonuclease F-TflIV was shown to introduce double-strand break into pseudo palindromic 17 bp DNA sequence yielding 1 bp extensions with 3'-overhangs. In contrast to F-TflIV, endonucleases F-TflI and F-TflII cleave only one strand of their asymmetric divergent DNA substrates. Each of these endonucleases introduces interruptions into only the particular strand (template or coding). Amino acid sequences of the endonucleases under study are highly homologous in the H-N-H motif regions and C-terminal sequences, forming putative catalytic domain. Endonuclease F-TflIV N-terminal region is homologous to the amino acid sequences representing H-T-H recognition domain found in LuxR family transcription regulators. Putative recognition NUMOD4 motif characteristic for a number of H-N-H endonucleases was shown to be also present in the endonuclease F-TflI and F-TflII N-terminal sequences. Two-domain structure was proposed for endonucleases F-TflI, F-TflII and F-TflIV with N-terminal recognition domain and C-terminal catalytic domain. A hypothesis of evolutionary origin of these endonucleases as a result of catalytic and recognition domains recombination was suggested.

[Transduction of plasmid antibiotic resistance determinants with pseudo-T-even bacteriophages]. / Transduktsiia determinant antibiotikorezistentnosti plazmid psevdo-T-chetnymi bakteriofagami.

Transduction of antibiotic resistance determinants of the plasmid pBR322 with pseudoT-even bacteriophages RB42, RB43, and RB49 was studied. It is established that antibiotic resistance determinants of plasmid pBR322 from Escherichia coli recA(+)- and recA(-)-donor strains do not differ significantly in respect to the efficiency of transduction. Amber mutants RB43-21, RB43-33, and a double amber mutant RB43am21am33 were obtained. These mutants facilitated transduction experiments in some cases. Transduction of antibiotic resistance markers of the vector plasmid pBR325 and recombinant plasmid pVT123, containing a DNA fragment with hoc segE uvsW genes of phage T4, was studied. The frequency of appearance of transductants resistant to pseudoT-even bacteriophages used in transduction was determined, and the sensitivity of resistant transductants to 32 RB bacteriophages and also to phages lambda, T2, T4, T5, T6, T7, and BF23 was estimated. The efficiency of plating pseudoT-even bacteriophages RB42 and RB43 on strain E. coli 802 himA hip carrying mutations in genes that encode subunits of the Integration Host Factor (IHF) was shown to be higher than on isogenic strain E. coli 802. The growth of pseudoT-even bacteriophages limited in vivo by modification-restriction systems of chromosomal (EcoKI, EcoBI), phage (EcoP1I), and plasmid (EcoRI, EcoR124I, and EcoR124II) localization was analyzed. It was shown that these phages were only slightly restricted by the type I modification-restriction systems EcoBI, EcoR124I, and EcoR124II. Phage RB42 was restricted by systems EcoKI, EcoP1I, and EcoRI; phage RB43, by systems EcoKI and EcoRI; and phage RB49, by the EcoRI modification-restriction system.

[Terminal oxidases in different genera of the family Microbacteriaceae]. / Terminal'nye oksidazy predstavitelei razlichnykh rodov semeistva Microbacteriaceae.

The nature of terminal oxidases in representatives of four different genera of the family Microbacteriaceae was studied. It was found that the late-logarithmic and early-stationary cells of all of the investigated strains of the genera Plantibacter and Okibacterium contain the aa3-type cytochrome oxidase. Bacteria of the genera Rathayibacter and Agreia synthesize three oxidases, the bb3- and aa3-type cytochrome oxidases and nonheme cyanide-resistant oxidase, in proportions dependent on the cultivation conditions and the growth phase. Oxygen deficiency in the cultivation medium induces the synthesis of the bd-type oxidase in all of the microorganisms studied. The data obtained provide evidence that the type of terminal oxidases, along with the known chemotaxonomic characteristics, may serve to differentiate the genera of the family Microbacteriaceae at the phenotypic level.

[Features of the structure of transfer RNA coded by bacteriophage T5]. / Osobennosti struktury transportnykh RNK, kodiruemykh bakteriofagom T5.

Nucleotide sequence of 24 genes for the bacteriophage T5 tRNAs specific for all amino acids involved in protein synthesis was determined. All of them, except tRNA(Pro), were shown to differ significantly from the generalized "clover-leaf" structure consisting in the displacement of invariant and semi-invariant residues, the absence of pairing in the stems and some other deviations from the canonical parameters of the model. Basing on the available information on the functional activity of the phage-specific tRNAs, one can put forward a suggestion that, at least for some of them, the above anomalies do not essentially influence their activity. A comparison of phage T5 tRNAs with the Escherichia coli and phage T4 counterparts was carried out. The majority of the known elements determining the specificity of E. coli tRNA aminoacylation was also found in all phage T5 tRNAs, except tRNAPhe).

[Biogenesis and secretion of alkaline phosphatase and its mutants in Escherichia coli. III. Substitution of N-terminal amino acids of alkaline phosphatase affect its biogenesis]. / Izuchenie biogeneza i sekretsii shchelochnoi fosfatazy i ee mutantnykhform u Escherichia coli. III. Zameny N-kontsevoi aminokisloty shchelochnoi fosfatazy vliiaiut na ee biogenez.

The effect of the N-terminal amino acid substitution on E. coli alkaline phosphatase biogenesis has been studied. The substitutions of Ser, Gln, Tyr, Leu, Gly, Ala, Glu, Phe, His, Cys, Lys and Pro for Arg(+1) were obtained by creating amber mutation at the corresponding position within phoA gene and expressing this mutated gene in E. coli strains that produce the amber-suppressor tRNAs. All mutant proteins were shown to translocate across the cytoplasmic membrane and possess enzyme activity. The introduction of Pro in +1 position disturbs the cleavage of signal peptide whereas the insertion of the other amino acids does not change the rates of processing in comparison with wild-type protein. All amino acid substitutions affect alkaline phosphatase isoenzyme composition. Some experimental evidence were also obtained on the specificity of protease, which split off N-terminal Arg during alkaline phosphatase maturation.

[Study of the biogenesis and secretion of alkaline phosphatase and its mutant forms in Escherichia coli. I. Introduction of directed mutations into the alkaline phosphatase gene]. / Izuchenie biogeneza i sekretsii shchelochnoi fosfatazy i ee mutantnykhform u Excherichia coli. I. Vvedenie napravlennykh mutatsii v gen shchelochnoi fosfatazy.

Various mutations in E. coli alkaline phosphatase gene were obtained by oligonucleotide-directed mutagenesis. They result in amino acid substitutions in the signal peptide cleavage site [Val for Ala(-1)] and in the N terminus of mature polypeptide chain: Ala for Arg(+1) and Gln for Glu(+4); Gln for Glu(+4). Enzyme activity was observed in all E. coli strains transformed by plasmids with cloned mutant genes. In addition, an amber mutation was introduced into the Arg(+1) position, and the synthesis of mutant alkaline phosphatase was shown in E. coli strains containing suppressor tRNAs specific for Ser, Gln, Tyr, Leu, Ala, Glu, Phe, Gly, His, Pro, and Cys.

[Nucleotide sequence at the site of single-strand DNA breaks in Pseudomonas aeruginosa bacteriophage phi kF77]. / Nukleotidnaia posledovatel'nost' v raionakh kanonicheskikh odnonitevykh razryvov DNK bakteriofaga phi kF77 Pseudomonas aeruginosa.

It is known that DNA molecules from the phage group phi k specific to Pseudomonas aeruginosa possess single-strand breaks (nicks). The sequences around the nicks in the bacteriophage phi kF77 DNA have been determined by various methods. In addition, an EcoRV-HindIII fragment, containing a nick, was cloned into the plasmid pUC9 and sequenced by Maxam-Gilbert technique. The sequence common for all nicks was CCTAohpCTCCGG.

[Primary structure of tRNAAsn of bacteriophage T5]. / Pervichnaia struktura tRNKAsn bakteriofaga T5.

Unformly 32P-labelled phage-specific tRNAAsn was isolated from bacteriophage T5-infected E. coli cells its oligonucleotide fragments were fractionated by thin-layer chromatography on cellulose and the tRNA's primary structure was determined as follows: (formula; see text). Main features of the structure are: displacement of the constant residue A14 by U; absence of G27 X A43 and U30 X psi40 pairing in the anticodon stem; possibility of additional pairing in D-loop. Comparison of T5 tRNAAsn with other asparagine tRNAs is presented.

[Primary structure of proline tRNA of bacteriophage T5]. / Pervichnaia struktura prolinovoi tRNK bakteriofaga T5.

The uniformly 32P-labeled bacteriophage T5 proline tRNA has been isolated from phage-infected E. coli cells by two-dimensional PAGE. Its nucleotide sequence has been determined by conventional techniques (using TLC on cellulose for oligonucleotide fractionation) as follows: (Formula: see text). The tRNA has the anticodon sequence UGG, which can presumably recognize the four proline-specific codons (CCN). It has 70% homology with phage T4 tRNA(Pro).