In order to understand the structure and biological significance of integrons and associated gene cassettes in marine polluted sediments, metagenomic DNAs were extracted from sites at Suez and Tokyo Bays. PCR amplicons containing new integrase genes, intI, linked with novel gene cassettes, were recovered and had sizes from 1.8 to 2.5 kb. This approach uncovered, for the first time, the structure and diversity of both marine integron attachment site, attI, and the first gene cassette, the most efficiently expressed integron-associated gene cassette. The recovered 13 and 20 intI phylotypes, from Suez and Tokyo Bay samples, respectively, showed a highly divergence, suggesting a difference in integron composition between the sampling sites. Some intI phylotypes showed similarity with that from Geobacter metallireducens, belonging to Deltaproteobacteria, the dominant class in both sampling sites, as determined by 16S rRNA gene analysis. Thirty distinct families of putative attI site, as determined by the presence of an attI-like simple site, were recovered. A total of 146 and 68 gene cassettes represented Suez and Tokyo Bay unsaturated cassette pools, respectively. Gene cassettes, including a first cassette, from both sampling sites encoded two novel families of glyoxalase/bleomycin antibiotic-resistance protein. Gene cassettes from Suez Bay encoded proteins similar to haloacid dehalogenases, protein disulfide isomerases and death-on-curing and plasmid maintenance system killer proteins. First gene cassettes from Tokyo Bay encoded a xenobiotic-degrading protein, cardiolipin synthetase, esterase and WD40-like β propeller protein. Many of the first gene cassettes encoded proteins with no ascribable function but some of them were duplicated and possessed signal functional sites, suggesting efficient adaptive functions to their bacterial sources. Thus, each sampling site had a specific profile of integrons and cassette types consistent with the hypothesis that the environment shapes the genome.

No abstract

After excavation using a portable submarine driller near deep-sea hydrothermal vents in the Suiyo Seamount, Izu-Bonin Arc, microbial diversity was examined in samples collected from inside the boreholes using an in situ growth chamber called a vent catheter. This instrument, which we devised for this study, consists of a heat-tolerant pipe tipped with a titanium mesh entrapment capsule that is packed with sterilized inorganic porous grains, which serve as an adhesion substrate. After this instrument was deployed inside each of the boreholes, as well as a natural vent, for 3^10 days in the vicinity of hot vent fluids (maxima : 156^305 ‡C), DNA was extracted from the adhesion grains, 16S rDNA was amplified, and randomly selected clones were sequenced. In phylogenetic analysis of more than 120 clones, several novel phylotypes were detected within the O-Proteobacteria, photosynthetic bacteria (PSB)-related K-Proteobacteria, and Euryarchaeota clusters. Members of O-Proteobacteria were frequently encountered. Half of these were classified between two known groups, Corre's B and D. The other half of the clones were assigned to new groups, SSSV-BE1 and SSSV-BE2 (Suiyo Seamount sub-vent origin, Bacteria domain, O-Proteobacteria, groups 1 and 2). From this hydrothermal vent field, we detected a novel lineage within the PSB cluster, SSNV-BA1 (Suiyo Seamount natural vent origin, Bacteria domain, K-Proteobacteria, group 1), which is closely related to Rhodopila globiformis isolated from a hot spring. A number of archaeal clones were also detected from the borehole samples. These clones formed a novel monophyletic clade, SSSV-AE1 (Suiyo Seamount sub-vent origin, Archaea domain, Euryarchaeota, group 1), approximately between methanogenic hyperthermophilic members of Methanococcales and environmental clone members of DHVE Group II. Thus, this hydrothermal vent environment appears to be a noteworthy microbial and genetic resource. It is also noteworthy that some of the findings presented here were made possible by the application of the in situ growth chamber into the hot fluids deep inside the boreholes.

SummaryThe location of the cis-acting regulatory region for histidine-dependent antitermination of the Bacillus subtilis hut operon was determined. A secondary structure, whose sequences partially overlap with the downstream terminator, was found in the regulatory region of the hut transcript. Mutational analysis of the regulatory region showed that the secondary structure was required for histidine-dependent antitermination. An electrophoretic mobility-shift assay demonstrated that, in response to the presence of histidine and Mg 21 , purified HutP bound hut RNA bearing putative secondary structure but not RNA lacking the potential to form putative secondary structure. Native gel electrophoresis showed that HutP existed as a hexamer. A filterbinding assay revealed that the concentration of histidine required for half-maximal binding of HutP to RNA was 3.1 mM and that the K d for binding of HutP to RNA was < 0.56 mM in the presence of histidine. These results suggested that putative secondary structure in the regulatory region of hut mRNA could function as an antiterminator to inhibit the formation of the terminator structure and that HutP causes expression of the hut structural genes by binding to the putative antiterminator structure in response to the presence of histidine.

The Corynebacterium glutamicum insertion sequence IS31831 was used to construct two artificial transposons: Tn31831 and miniTn31831. The transposition vectors were based on a gram-negative replication origin and do not replicate in coryneform bacteria. Strain Brevibacterium flavum MJ233C was mutagenized by miniTn31831 at an efficiency of 4.3 x 10(4) mutants per microgram DNA. Transposon insertions occurred at different locations on the chromosome and produced a variety of mutants. Auxotrophs could be recovered at a frequency of approximately 0.2%. Transposition of IS31831 derivatives led not only to simple insertion, but also to cointegrate formation (5%). No multiple insertions were observed. Chromosomal loci of B. flavum corresponding to auxotrophic and pigmentation mutants could be rescued in Escherichia coli, demonstrating that these transposable elements are useful genetic tools for studying the biology of coryneform bacteria.