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The xylanolytic bacterium Paenibacillussp. strain W-61 encodes three extracellular xylanase genes, xyn1, xyn3, and xyn5. In this study, we identified a transcriptional activator required for transcription of the xyn3gene in strain W-61. The activator, AxyR, contained the highly homologous AraC-type DNA binding domain and required xylobiose, xylotriose, or xylotetraose as cofactor for binding to the xyn3promoter region.

This paper presents a new approach for analyzing spectroscopic characteristic of metallic surfaces using spectroscopic and image analysis. This method is useful for contactless and non destructive analysis of cultural heritage. Spectral luminance, CIELAB, and CIEXYZ value of more than 80 metallic surfaces were measured with spectrometer and scanned to examine spectroscopic characteristics of foils by using multiband images. This analysis through imaging can improve the method for extracting difference related to types of metallic foils. Firstly, the spectral reflectance of each foil was measured ranging from 220 to 850 nm in steps of 1 nm. The images were captured with color and monochromatic camera using color filter in order to analyze the targets by multispectral approach. Then, principal component analysis (PCA) was conducted with image pixel value of each target. The results have shown that the spectral reflectance whose peak and change rate at a particular wavelength region differed from each foils, and that the multispectral images extracted the difference in spectral characteristics related to different types of metallic foils and Japanese papers. This could be useful in distinguishing among foils. This provides some promise that unknown metallic foils can be identified through the measurement of their spectroscopic features.

A xylanase gene, xyn1, which encodes Paenibacillussp. strain W-61 xylanase 1 (Xyn1), was cloned in Escherichia coli. xyn1encodes 211 amino acid residues, including 28 amino acid residues of a signal peptide. The deduced amino acid sequence of the mature Xyn1 showed 95.7%, 84.0%, and 83.7% identity to family 11 xylanases of Aeromonas caviaeME-1, Paenibacillussp., and Bacillus stearothermophilusrespectively. The physico-chemical properties of recombinant Xyn1 were very similar to those of intact Xyn1, except for the molecular mass. The pattern of xylooligosaccharides generated by rXyn1 was investigated by fluorophore-assisted carbohydrate electrophoresis (FACE). The degradation rate of xylohexaose by rXyn1 increased markedly as compared with that of xylopentaose. Xylohexaose had a single preferential point of cleavage by rXyn1. On the basis of the pattern of action of xylooligosaccharides, the number of subsites was estimated to be six. The catalytic site was located between the third and the fourth subsites from non-reducing end.

Carotovoricin Er (CtvEr), which is produced by a plant soft rot disease causative agent, Erwinia carotovorasubsp. carotovoraEr, is a high-molecular-weight bacteriocin showing Myoviridae phage-tail-like morphology with contractile sheath and plural tail fibers. We determined the complete nucleotide sequences of CtvEr genes on the E. carotovoraEr chromosome and report that CtvEr genes consist of lysis cassette, major and minor structural protein gene clusters. Four promoters were identified. The lysis gene cassette, which is composed of the genes for lysis enzyme and holin, was also identified and characterized. The nucleotide sequences and organization of the genes for CtvCGE, which is produced by E. carotovorastrain CGE234-M403 with the morphology similar to CtvEr, were also determined and compared to that of CtvEr, and it was found that CtvCGE is almost identical to CtvEr except for tail fibers which are involved in the killing spectra of both bacteriocins. We also explain that the gene organization and the deduced amino acid sequences of both carotovoricins are very close to those of prophage, which is lysogenized in the chromosome on Salmonella entericaserovar Typhi CT18. These findings strongly suggest that Ctv evolved as a phage tail-like bacteriocin from a common ancestor with Salmonella typhiprophage.

The D-T neutron skyshine experiments were carried out at the Fusion Neutronics Source (FNS) of JAERI with a port at the roof in March 2002 and March 2003. The concrete thickness of the roof and the wall of a FNS target room are 1.15 and 2 m, respectively. The FNS skyshine port with a size of 0.9 × 0.9 m2was open during the experimental period. The source neutron intensity was -1.7 × 1011n/s in those skyshine experiments. The highest total dose rate measured was about 0.5 μSv/h at a distance of 30 m from the D-T target point and the dose rate was attenuated to about 0.1 μSv/h at a distance of 100 m and 0.02 μSv/h at a distance of 550 m. Those experimental results were analyzed by Monte Carlo code MCNP-4C with the nuclear data library JENDL-3.2, where the FNS building and the measurement field were modeled with a simplified cylindrical geometry, and the pine forest was modeled by homogenized cell with a height of 10m. The MCNP calculation agreed well both neutron and secondary gamma-ray dose rate distributions.

The production of pectin lyase (Pnl) and carotovoricin (Ctv), as well as cell lysis in the plant-pathogenic bacterium Erwinia carotovorasubsp. carotovoraEr are induced by mitomycin C. Here, Pnl and Ctv production and cell lysis were found to be temperature-dependent. The optimal temperature for Pnl production was 30°C. However, the optimal temperature for both Ctv production and cell lysis was 23°C, at which Pnl production was reduced to 47% of the maximum. These data suggest the possible existence of novel regulation system(s) for the production of Pnl and Ctv, and cell lysis, in addition to the well-documented regulation system of recA, rdgA, and rdgBgenes.

Staphylococcus aureusP83 has Panton-Valentine leukocidin (PVL)-like genes, lukMand lukF-PV. Here, lukMand lukF-PVgenes were found on the genome of a prophage, which was designated as φPV83-pro. The precise genome size was 45,636 bp with attcore sequences of 10 base pairs. Sixty-four ORFs were identified on the φPV83-pro genome, including two extra operons, lukM-lukF-PVand orfs63-64. The lukM-lukF-PVcluster was located 2.1 kb upstream of the attLsite. The most striking feature of the φPV83-pro genome was a constituent of at least 4 regions from φ11, φPVL, and other phages, i.e., (i) attsites identical with those of φ11, (ii) a cossequence and the genes encoding packaging and head proteins of φPVL (occupied half region of φPV83-pro), and (iii) the other two regions which showed no significant similarity with known phages (occupied about 40% of φPV83-pro). Furthermore, two insertion sequences, ISSA1and ISSA2were integrated into attLsite and orf44, respectively. φPV83-pro was not induced as phage particles from S. aureusP83 regardless of its treatment with mitomycin C. The insertion of ISSA1into the attLsite was one of the reasons of the failure of the induction of the phage particles by mitomycin C treatment of the strain P83.

Staphylococcal γ-hemolysin and leukocidin are bi-component cytolysins, consisting of LukF (or Hlg1)/Hlg2 and LukF/LukS, respectively. Here, we purified serum inhibitors of γ-hemolysin and leukocidin from human plasma. Protein sequencing showed that the purified inhibitors of 62, 57, 50 and 38 kDa were the vitronectin fragments with truncation(s) of the C-terminal or both N- and C-terminal regions. The purified vitronectin fragments specifically bound to the Hlg2 component of γ-hemolysin and the LukS component of leukocidin to form high-molecular-weight complexes with them, leading to inhibition of the toxin-induced lysis of human erythrocytes and human polymorphonuclear leukocytes, respectively. Intact vitronectin also showed inhibitory activity to the toxins. The ability of γ-hemolysin and leukocidin to bind vitronectin and its fragments is a novel function of the pore-forming cytolysins.

Staphylococcus aureusP83 (ATCC 31890) produces five components, I to V for synergistic toxins, leukocidin and γ-hemolysin [Sudo et al., Biosci. Biotech. Biochem., 56, 1786–1789 (1995)]. We report here the identification of component II, which is designated LukF-PV(P83) and its gene (lukF-PV(P83)). The lukF-PV(P83) gene was found to be one base downstream of the stop codon of the lukMgene. The deduced amino acid sequence of LukF-PV(P83) showed 78.4% identity with that of LukF-PV. The lukMand lukF-PV(P83) genes were encoded as one operon like that of Panton-Valentine leukocidin.

Staphylococcal γ-hemolysin consists of two protein components, F (or HγI) and HγII. To elucidate the mode of action of γ-hemolysin, we studied the binding order of F and HγII to human erythrocytes and the cell-bound state of the two components. The binding of F to human erythrocytes preceded the binding of HγII to the cells, and thereafter hemolysis occurred. Western immunoblot analysis of the cell-bound γ-hemolysin indicated that F and HγII components form high-molecular-mass (150–250 kDa) complexes on the erythrocytes. The toxin complexes were recovered in a Triton X-100-insoluble fraction of the erythrocytes, which contains cytoskeleton proteins. Neither the formation of the toxin complex(es) nor hemolysis occurred when the erythrocytes were treated with proteinase K. Abortion of the complex formation on the proteinase K-treated erythrocytes may be due to the failure of the binding of HγII to the cells, because F bound to the proteinase K-treated erythrocytes to the same extent as to the non-treated erythrocytes.

Aeromonas caviaeW-61 produces five species of xylanases, xylanases 1, 2, 3, 4, and 5 [Nguyen, V.D. et al., Biosci. Biotechnol. Biochem., 56, 1708-1712 (1993) and Appl. Environ. Microbiol., 57, 445-449 (1991)]. While preserving a purified xylanase 3 preparation from A. caviaein solution at 4°C, the xylanase 3 was found to be proteolyzed to give a truncated form with a smaller molecular mass than that of the intact one. It appears likely that the truncated form of xylanase 3 was produced in this particular purification experiment by the action of a contaminating protease. We isolated the truncated form of xylanase 3 (Xyn3tr), of which the C-terminal 102-residue segment is missing. By the chemical analysis of the N- and C-terminal amino acid residues of Xyn3tr and the DNA sequencing analysis of the xylanase 3 gene (xyn3), the N-terminal 398th proline residue of xylanase 3 was found to be the C-terminus of Xyn3tr. Xyn3tr had the activity to form xylotriose (X3), xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6) as main final products from oat spelt xylan. In contrast, intact xylanase 3 released X6 and higher xylo-oligosaccharides as main products. Xylanase 3 hydrolysed X4 through X6. However, Xyn3tr had no activity towards X4 and X5. The recombinant Xyn3tr (XYN3tr) and recombinant xylanase 3 (XYN3) were purified homogeneously from the periplasmic space of E. coliharboring the plasmids pXYN3 and pXYN3tr, which include xyn3and xyn3trgenes, respectively, and their enzymatic activities were measured. The cleavage patterns of oat spelt and xylo-oligosaccharides by XYN3tr were identical with that by intact Xyn3tr. Thus, we conclude that the C-terminal region comprising a 102-residue segment in xylanase 3 is involved in governing the molecular size of xylo-oligosaccharides cleaved from β-1,4-xylan by the enzyme and in the hydrolytic activity towards X4 and X5.

The staphylococcal Panton–Valentine leukocidin (PVL) genes [lukS-PV-lukF-PV]existed in a hexagonal phage-like particle (ϕPVL) isolated from mitomycin C-induced Staphylococcus aureusV8 (ATCC 49775). The genome packed in ϕPVL was a linear double-stranded 40-kb DNA with single-stranded cohesive ends (cos). The [lukS-PV-lukS-PV], attP, and int(integrase géne) of ϕPVL were all located very close to one another within a 4.0 kb-segment on the genome in the order given, and the segment is located at the center from the left and the right cossites. In addition, the [lukS-PV-lukF-PV]-attP-intregion contains 5 direct repeat sequences that show high similarity with the recombinase-binding sites of bacteriophages of S. aureus.

The two staphylococcal bi-component toxins, leukocidin and γ-hemolysin share LukF [Kamio et al, FEBS Lett., 321, 15-18 (1993)]. This report identifies the pivotal amino acid residues in the N-terminal region of LukF for the leukocytolytic and hemolytic activities in the presence of LukS and Hlg2, respectively, measuring the toxin activiy of a series of LukF mutants with truncated N-terminals. The data obtained showed that the LukF mutant TF21, lacking 20 amino acid residues at the N-terminus of LukF, failed to have any hemolytic activity and had less 10% leukocytolytic activity than that of the intact LukF, while 16-residue truncations retained both toxin activities without loss. The LukF mutants lacking 18- through 19-residue segments from the N-terminus showed low toxin activity on both target cells. All mutants having no toxin activity were also not capable of binding to the human erythrocytes. It can thus be concluded that the 3-residue segment, L18Y19K20of LukF is crucial for the biological activity of the toxin.