Spontaneous VLP (virus-like particle) production and VLP-mediated gene transfer into Escherichia coli AB1157 as recipient was demonstrated. Five marine isolates (Alc 096, Alc 233, Alc 252, Agrobacterium kieliense and Flavobacterium sp. 11604) were investigated for their potential to produce VLP as well as for the gene transfer capability of these VLPs to the E. col1 recipient. These strains are classified as ubiquinone-10-possessing marine bacteria (QIOMB) in the 16s-rRNA Superfamily IV VLPs were obtained from 100 h cultured broth of all strains examined. VLP-host ratio after 100 h growth culture was: Alc 233, 1.54; Alc 252, 1.26; Alc 096, 1.06; Flavobacterjum sp. 11604, 0.69; and A. kieliense, 0.06. These ratios were smaller than those found in the marine environment. However, the spontaneously produced VLP number can be considered as high because the reported numbers are relatively low from coliphage h (0.005) and phage Mu (-0.0001). VLP-mediated gene transfer was examined using an auxotrophic mutant of E. coli (AB1157) with 4 amino acid deficiencies (leu, pro, his, arg)as recipient at multiplicity of infection (MOI) of 0.1. Through this treatment, VLPs showed lethal effect on the recipient. The survival rate of control was: Alc 096, 7%; Alc 252, 8 % ; A. kieliense, 17%; Flavobacterium sp. 11604,31%; and Alc 233,40%. At the same time, all the purified VLPs derived from these 5 strains successfully transferred genes to rescue genetlc defects of the recipi.ent. Overall average efficiency of VLP-mediated gene transfer at MO1 of 0.1 was estimated to be between 2.62 X' 10-3 and 3.58 X 10-'per VLP particle. Loci of employed genetic markers were dispersed on the E. coli chromosome with mutual distance of 121, 1154, 1397 and 364 kb between them. Since VLPs from different sources showed similar gene transfer efficiency in respect to the genet~c marker rescued, it is suggested that VLPs from QlOMB transferred genes as generalized transduction. These results indicate that the VLPs produced by certain marine bacteria may be an important element for both non-specific generalzed horizontal gene transfer towards a broad range of bacterial hosts and population control in the marine environment.

The bactericidal effects of "Virus-like particles" ("ST-VLPs") derived from a sulphur-turf microbial mat consisting of the Aquificales bacterium, were examined using Escherichia coli AB1157 and Bacillus subtilis PS9 as recipients. The recipient population was reduced by up to one order of magnitude with transduction frequencies of between 10 -3 and 10 -4 per particle. Broad host range and xenotrophic behaviour imply that "ST-VLPs" do not fit the current concept of a "virus" with a narrow host range. Such novel VLPs would control the bacterial population, and genetically enhance biodiversity.

As one of the mechanisms for horizontal gene transfer among microorganisms in marine environments, the production and significance of Phage-like particles (PLPs) were investigated using Ubiquinone 10 (Q10) possessing marine bacteria. 2, 4-Dichlorophenoxyacetic acid (2, 4-D) utilization was found as a characteristic feature of marine bacteria examined, and was employed as a marker for gene transfer. Their Plasmid-like elements (PLEs) transformed Escherichia coli AB1157 with the efficiency of 106-10? CFU/ugDNA.The PLPs produced from Agrobacterium kieliense and Flavobacterium sp. 11604 showed 70-95% bactericidal activity on E. coli. At the same time, PLPs from A. kieliense transferred 2, 4-D utilization with efficiency of ca. 0.02% and those from F. sp. I1604 cured four amino acid requirements with efficiency of ca. 0.1% for each amino acid. The PLP production was generally shared by the bacteria investigated. These results indicate that the PLPs produced by certain marine bacteria may be an important new element for non-specific generalized transduction in the environment.

Aquificales, a thermophilic bacterium of the deepest-branching lineage of the domain Bacteria produced viruslike particles (VLPs). Mature VLPs resided in 6.5±4.4% (n=341) of bacterial cells. A count of the free particles in water from geothermal hot spring showed high abundance (1.41±1.70´10 7 VLP/ml, n=12). The molecular mass of the VLP-encapsulated DNA was 406.4±10.1 kb (n=13).

So-called sulfur-turf microbial mats, which are macroscopic white filaments or bundles consisting of large sausage-shaped bacteria and elemental sulfur particles, occur in sulfide-containing hot springs in Japan. However, no thermophiles from sulfur-turf mats have yet been isolated as cultivable strains. This study was undertaken to determine the phylogenetic positions of the sausage-shaped bacteria in sulfur-turf mats by direct cloning and sequencing of 16S rRNA genes amplified from the bulk DNAs of the mats. Common clones with 16S rDNA sequences with similarity levels of 94.8 to 99% were isolated from sulfur-turf mat samples from two geographically remote hot springs. Phylogenetic analysis showed that the phylotypes of the common clones formed a major cluster with members of theAquifex-Hydrogenobacter complex, which represents the most deeply branching lineage of the domain bacteria. Furthermore, the bacteria of the sulfur-turf mat phylotypes formed a clade distinguishable from that of other members of theAquifex-Hydrogenobacter complex at the order or subclass level. In situ hybridization with clone-specific probes for 16S rRNA revealed that the common phylotype of sulfur-turf mat bacteria is that of the predominant sausage-shaped bacteria.

Incubation of the amino acid-deficient strain Escherichia coli AB1157 with particles harvested from an oligotrophic environment revealed evidence of horizontal gene transfer (HGT) with restoration of all deficiencies in revertant cells with frequencies up to 1.94 × 10(-5). None of the markers were preferentially transferred, indicating that the DNA transfer is performed by generalized transduction. The highest gene transfer frequencies were obtained for single markers, with values up to 1.04 × 10(-2). All revertants were able to produce particles of comparable size, appearing at the beginning of the stationary phase. Examination of the revertants using electron microscopy showed bud-like structures with electron-dense bodies. The particles that display the structural features of membrane vesicles were again infectious to E. coli AB1157, producing new infectious particles able to transduce genetic information, a phenomenon termed serial transduction. Thus, the <0.2-μm particle fraction from seawater contains a particle size fraction with high potential for gene transfer. Biased sinusoidal field gel electrophoresis indicated a DNA content for the particles of 370 kbp, which was higher than that of known membrane vesicles. These findings provide evidence of a new method of HGT, in which mobilizable DNA is trafficked from donor to recipient cells via particles.

, were separately purified and counted using a qNano particle analyzer. These EVs, showing different buoyant densities, were identically spherical in shape, and their sizes varied from 80 to 210 nm in diameter, with 120-and 190-nm sizes predominant. The average size of DNA packaged into EVs was about 14 kb. The DNA of the EVs in band C was sequenced and assembled. Mapping of the T. onnurineus NA1 T EV (ToEV) DNA sequences onto the reference genome of the parent archaeon revealed that most genes of T. onnurineus NA1 T were packaged into EVs, except for an ϳ9.4-kb region from TON_0536 to TON_0544. The absence of this specific region of the genome in the EVs was confirmed from band B of the same culture and from bands B and C purified from a different batch culture. The presence of the 3=-terminal sequence and the absence of the 5=-terminal sequence of TON_0536 were repeatedly confirmed. On the basis of these results, we hypothesize that the unpackaged part of the T. onnurineus NA1 T genome might be related to the process that delivers DNA into ToEVs and/or the mechanism generating the ToEVs themselves.

Virus-like particles (VLPs) were collected from geothermal vent water samples in the drift-way at Toyoha Mine, Hokkaido, Japan (-500 m level, 63.5°C) whose VLP and bacterial abundance was (No/ml±SD, n: 500), VLP: 9.60±0.29´10 8 and bacteria: 3.61±0.14´106 . VLPs ranged in diameter from 30 to 320 nm, and the major size distribution (ca 62%) was 83.3±3.3 nm (n: 843). Ultrafiltration followed by CsCl density equilibrium ultracentrifugation gave purified TY-VLPs: 6.64´10 13 . Regardless of UV treatment, TY-VLP reduced the efficiency of plating to 68.6-83.4% at a multiplicity of infection of ca 0.3 on Escherichia coli AB1157. Generalised transduction was observed on E. coli AB1157 with a frequency between 10 -4 and 10 -5 cells/particle using TY-VLPs without UV-treatment. The growth of generated E. coli transductants (TY-E-trans) was compared to that of an E. coli transductant (ST-E-trans) generated by Aquificales originating VLP (Chiura, 2002). The extent of the maximum growth of both transductants was ca 40% of the parental E. coli used as a recipient. TY-E-trans acquired "budding-like" particle productivity, which has been demonstrated for ST-E-trans. ST-E-trans produced five different size particles, whose DNA content ranged between 291.6 and 382.0 kb, and TY-E-trans produced ten different size particles between 68.5 and 190.2 kb, respectively.