Fructose-1,6-bisphosphate (FBP) aldolase (EC 126.96.36.199) was purified 97-fold from a halophilic archaebacterium Haloferax mediterranei, with a specific activity of 2.8. The enzyme was characterized as a Class II aldolase on the basis of its inhibition by EDTA and other metal chelators. The enzyme had a specific requirement for divalent metal Fe2+ for activity. Sulfhydryl compounds enhanced aldolase activity.
The effect of low doses of ionizing and nonionizing radiation on the radiation response of yeast Saccharomyces cerevisiae toward ionizing and nonionizing radiation was studied. The wild-type strain D273-10B on exposure to 54 Gy gamma radiation (resulting in about 10% cell killing) showed enhanced resistance to subsequent exposure to UV radiation. This induced UV resistance increased with the incubation time between the initial gamma radiation stress and the UV irradiation. Exposure to low doses of UV light on the other hand showed no change in gamma or UV radiation response of this strain. The strains carrying a mutation at rad52 behaved in a way similar to the wild type, but with slightly reduced induced response. In contrast to this, the rad3 mutants, defective in excision repair, showed no induced UV resistance. Removal of UV-induced pyrimidine dimers in wild-type yeast DNA after UV irradiation was examined by analyzing the sites recognized by UV endonuclease from Micrococcus luteus. The samples that were exposed to low doses of gamma radiation before UV irradiation were able to repair the pyrimidine dimers more efficiently than the samples in which low gamma irradiation was omitted. The nature of enhanced repair was studied by scoring the frequency of induced gene conversion and reverse mutation at trp and ilv loci respectively in strain D7, which showed similar enhanced UV resistance induced by low-dose gamma irradiation. The induced repair was found to be essentially error-free. These results suggest that irradiation of strain D273-10B with low doses of gamma radiation enhances its capability for excision repair of UV-induced pyrimidine dimers.
The Brn1 reductase melanin biosynthesis gene in the fungal genus Bipolaris was sequenced in 74 strains of 22 species. The Brn1 region was highly conserved among the species examined at the nucleotide and the amino acid levels. To elucidate the phylogenetic relationships among Bipolaris species, trees were inferred from nucleotide sequences of this region. Species in these trees formed exclusive clusters clearly separated from one another, except for B. panici-miliacei and B. setariae, and B. victoriae and B. zeicola. When unidentified strains were added to this tree, they fell within known species or formed independent clusters. These data indicated that the Brn1 gene region was suitable for species-level systematics within the genus. The results also suggest that Bipolaris consists of two or more clades that may reflect teleomorphic connections.
Studies on the interaction of the insect pathogenic bacterium, Xenorhabdus nematophilus (Enterobacteriaceae), with its nematode and insect hosts would be greatly assisted if a luminescent phenotype were generated that would allow the detection of viable bacteria in vivo without the necessity for disruption of the cellular interactions. The plasmid, pMGM221, containing the luminescence gene (luxCDABE) of Vibrio harveyi was introduced into different strains (DD136 and 19061) and phases (one and two) of X. nematophilus by triparental mating. For reproducible and efficient conjugation, it was necessary to use older cultures (96–160 h) in the stationary phase of X. nematophilus for mating with relatively small differences (<2-fold) in transconjugant yield for the different strains and phases of X. nematophilus. All transconjugants emitted high levels of light with optimum bioluminescence at 27°C in Luria broth at pH 8.0 containing 20 g/L NaCl; pH, osmolarity, and temperature conditions were similar to those encountered by the bacteria in the hemolymph of the larvae of Galleria mellonella. Plasmids were detected in the transconjugants after 6 months of subculturing the bacteria without antibiotic selection. Aside from light emission, luminescent transconjugants had the same physiological properties as the nonluminescent parental strains, including identical rates of growth, production of exoenzymes, removal from and subsequent emergence into the insect's hemolymph, bacterial-induced hemocyte damage, suppression of prophenoloxidase activation, and the ability to kill G. mellonella larvae. Light-emitting larvae could readily be detected by eye in a dark room, and all bacteria reisolated from dead larvae were luminescent. These properties validate the use of luminescent X. nematophilus not only as a means of following bacterial host interactions, but also as a potential agent to follow the infection and death of the insect population.
A β-1,3-xylanase-producing bacterium, Alcaligenes sp. XY-234, was isolated from the marine environment. The organism produced endo-1,3-β-xylanase at a high level in the culture fluid. The enzyme was purified 292-fold by ammonium sulfate precipitation and several column chromatographies. The final enzyme preparation appeared to be homogeneous on disc gel electrophoresis and SDS-PAGE with a molecular mass of 59 kDa, and the pl was 4.0. The enzyme hydrolyzed β-1,3-xylan and larger xylooligosaccharides than xylobiose to give several xylooligosaccharides, but it could not hydrolyze xylobiose, p-nitrophenyl-β-D-xyloside, and β-1,4-xylan. The Km of the enzyme was 4.0 mg/ml. Optimal pH and temperature were 7.5 and 40°C, respectively. It was stable from pH 6.0 to 10 and at a temperature of less than 40°C. The enzyme was strongly inhibited by 1 mM HgCl2, AlCl3, CuCl2, FeCl3, HgCl2, Pb(CH3COO)2, and N-bromosuccinimide.
A single copy of a reporter gene cassette, such as PGKP-lacZ-LEU2 (promoter-reporter-marker gene) cassette, was inserted into one of 32 positions along chromosome III in Saccharomyces cerevisiae with an interval of approximately 10 kb. The amounts of translational gene product (β-galactosidase) synthesized by the cassette-transformed cells were then determined. The region specificity in chromosome III could be demonstrated in gene expression: two higher-expressed regions (hot regions), 133 and 199 (MAT) regions, and seven lower-expressed regions (cold regions). For the steady and high production of polypeptide, foreign gene products, by yeast, we would like to state that we hope for an insertion of the artificially prepared gene cassette [(promoter)-(foreign gene)-(marker gene)] into a hot region, such as 199 (MAT) region of chromosome III.
The effect of the antibiotics gentamicin, streptomycin, kanamycin, tetracycline, and ampicillin on planktonic cultures of Enterobacter aerogenes, Serratia marcescens, Salmonella derby, Streptococcus mitis, and Staphylococcus epidermidis with and without an application of 70 kHz ultrasound was studied. The ultrasound was applied at levels that had no inhibitory effect on planktonic cultures of bacteria. Measurements of viability at, above, and below the minimum inhibitory concentration of the above antibiotics on the planktonic cultures of these bacteria showed that a simultaneous application of 70 kHz ultrasound and antibiotic significantly increased the effectiveness of selected antibiotics. Bacterial viability was reduced several orders of magnitude when harmless levels of ultrasound were combined with some antibiotics, especially the aminoglycosides. Similar synergistic effects of combined ultrasound and antibiotic treatment were seen in both Gram-positive and Gram-negative bacteria with several classes of antibiotics. These results may have application in the treatment of bacterial infections normally resistant to some antibiotics.
Strain DSK1 is a novel moderately barophilic bacterium isolated from the Japan Trench at a depth of 6,356m. Phylogenetic analysis based on 16S ribosomal DNA sequences showed that strain DSK1 represents a separate lineage with the Shewanella barophiles branch and is closely related to Moritella marina. Comparisons of the temperature and pressure range for growth and some biochemical characteristics indicate that strain DSK1 differs from M. marina and Shewanella barophilicspecies. Furthermore, strain DSK1 displays a low level of DNA similarity to the Moritella and Shewanella type strains; however this isolate characteristically produces DHA (22:6) as a membrane fatty acids, and the fatty acid profile of this strain is similar to that of M. marina. Because of these differences, strain DSK1 appears to represent a novel deep-sea Moritella species. The name Moritella Japonica is proposed. The type strain is JCM 10249.
A methanogenic and sulfate-reducing consortium, which was enriched on medium containing tetrachloroethylene (PCE), had the ability to dechlorinate high concentrations of PCE. Dehalogenation was due to the direct activity of methanogens. However, interactions between methanogenic and sulfate-reducing bacteria involved modification of the dechlorination process according to culture conditions. In the absence of sulfate, the relative percentage of electrons used in PCE dehalogenation increased after an addition of lactate in batch conditions. The sulfate reducers would produce further reductant from lactate catabolism. This reductant might be used by methanogenic bacteria in PCE dechlorination. A mutualistic interaction was observed in the absence of sulfate. However in the presence of sulfate, methanogenesis and dechlorination decreased because of interspecific competition, probably between the H2-oxydizing methanogenic and sulfate-reducing bacteria in batch conditions. In the semicontinuous fixed-bed reactor, the presence of sulfate did not affect dechlorination and methanogenesis. The sulfate-reducing bacteria may not be competitors of H2-consuming methanogens in the reactor because of the existence of microbial biofilm. The presence of the fixed film may be an advantage for bioremediation and industrial treatment of effluent charged in sulfate and PCE. This is the first report on the microbial ecology of a methanogenic and sulfate-reducing PCE-enrichment consortium.