Carotenoid synthesis in Streptomyces setonii ISP5395 is induced by the gene crtS, whose product is similar to a sigma factor

In many species of actinomycetes, carotenogenesis can be photoinduced. The capacity to respond to photoinduction is, however unstable and, in various strains of Streptomyces, is lost at a relatively high frequency. In Streptomyces setonii ISP5395, which normally produces no carotenoids, carotenoid-producing mutants can be obtained following protoplast regeneration. We report here the characterization of a gene, crtS, which was isolated from one such mutant and can confer on wild-type S. setonii ISP5395 cells the capacity to synthesize carotenoids. Sequence analysis of crtS reveals an open reading frame, which shows homology to genes that encode alternative sigma factors in Bacillus subtilis. We propose that crtS encodes a sigma factor which is necessary for the expression of a cryptic gene(s) for carotenoid biosynthesis in S. setonii ISP5395.     

Fluctuation of algal alkaline phosphatase activity and the possible mechanisms of hydrolysis of dissolved organic phosphorus in Lake Barato

For freshwater cyanobacteria, the autofluorescence of phycocyanin is quite high while the in vivo fluorescence (IVF) yield of chlorophyll-a is relatively low, apparently because of low chlorophyll concentrations associated with photosystem II. In eucaryotic phytoplankton, even those with phycobili-protein accessory pigments (e.g. some cryptophytes), the opposite is true. Thus, an IVF ratio which relates phycocyanin to chlorophyll-a signals could be a good index of relative cyanobacterial abundance in the field. Spectrofluorometric scans of whole cells from laboratory cultures indicated that the ratio Em₆₆₀ @ Ex₆₃₀/Em₆₈₀ @ Ex₄₃₀ could be a very sensitive cyanobacterial indicator. Tandem flowthrough fluorometers were then fitted with the appropriate interference filters and their discriminatory power was evaluated with mixtures of cyanobacterial and eucaryotic phytoplankton. Although subject to many of the constraints of other IVF assays, tandem fluorometry should be particularly appropriate for real-time mapping of the relative spatial and temporal distributions of broad phytoplankton taxa in continuous vertical of horizontal profiles in lakes.     

In vivo priming of mouse CTL precursors directed to product of a newly defined minor H-42 locus is under a novel control of class II MHC gene

In a previous study, we discovered a new mouse minor histocompatibility antigen encoded by a locus at 8.5 cM apart from the H-2 complex, and we have since named the locus H-42. One allele of H-42, which is named H-42a, had been elucidated, but the other alleles, which we tentatively named H-42b, have not been elucidated. In the present study, we explored MHC control on the anti-H-42a cytotoxic T lymphocyte (CTL) responsiveness in H-42b mice. In vivo immunization (i.v. injection) of H-42b mice with 5 to 30 X 10(6) spleen cells (SC) bearing allogeneic H-42a antigen but carrying H-2 complex (mouse MHC) matched with the H-42b mice failed to prime anti-H-42a CTL but induced stable and specific anti-H-42a CTL unresponsiveness, i.e., tolerance, in the H-42b recipient mice. In contrast, H-2 heterozygous H-42b F1 mice injected with SC bearing H-42a alloantigen on either of the parental H-2 haplotypes were effectively primed to generate anti-H-42a CTL. Exploration of the region or subregion in the H-2 complex of H-42a donor SC that should be compatible with H-42b recipient mice for the induction of their anti-H-42a CTL tolerance demonstrated that the compatibility at I region, most probably I-A subregion, but not at K, S, or D region, determined the induction of the tolerance. MHC class II compatible H-42a skin graft (SG) to H-42b mice, however, consistently primed the anti-H-42a CTL in the H-42b recipients. These results were discussed in several aspects, including uniqueness of MHC class II control on the CTL response to minor H-42a antigen, possibility of inactivation of responding anti-H-42a precursor CTL or helper T cells in H-42b mice by encountering the veto cells present in MHC class II-matched H-42a SC population, and significance of the present observations as a mechanism of CTL tolerance to self-components.     

Features of two hepatitis B virus (HBV) DNA integrations suggest mechanisms of HBV integration.

Two integrated hepatitis B virus (HBV) DNA molecules were cloned from two primary hepatocellular carcinomas each containing only a single integration. One integration (C3) contained a single linear segment of HBV DNA, and the other integration (C4) contained a large inverted duplication of viral DNA at the site of a chromosome translocation (O. Hino, T.B. Shows, and C.E. Rogler, Proc. Natl. Acad. Sci. USA 83:8338-8342, 1986). Sequence analysis of the virus-cell junctions of C3 placed the left virus-cell junction at nucleotide 1824, which is at the 5' end of the directly repeated DR1 sequence and is 6 base pairs from the 3' end of the long (L) negative strand. The right virus-cell junction was at nucleotide 1762 in a region of viral DNA (within the cohesive overlap) which shared 5-base-pair homology with cellular DNA. Sequence analysis of the normal cellular DNA across the integration site showed that 11 base pairs of cellular DNA were deleted at the site of integration. On the basis of this analysis, we suggest a mechanism for integration of the viral DNA molecule which involves strand invasion of the 3' end of the L negative strand of an open circular or linear HBV DNA molecule (at the DR1 sequence) and base pairing of the opposite end of the molecule with cellular DNA, accompanied by the deletion of 11 base pairs of cellular DNA during the double recombination event. Sequencing across the inverted duplication of HBV DNA in clone C4 located one side of the inversion at nucleotide 1820, which is 2 base pairs from the 3' end of the L negative strand. Both this sequence and the left virus-cell junction of C3 are within the 9-nucleotide terminally redundant region of the HBV L negative strand DNA. We suggest that the terminal redundancy is a preferred topoisomerase I nicking region because of both its base sequence and forked structure. Such nicking would lead to integration and rearrangement of HBV molecules within the terminal redundancy, as we have observed in both our clones.     

Substrate Preference in a Strain of Megasphaera elsdenii, a Ruminal Bacterium, and Its Implications in Propionate Production and Growth Competition

The NIAH 1102 strain of Megasphaera elsdenii utilized lactate in preference to glucose when the two substrates were present. Even when lactate was supplied to cells fermenting glucose, the cells switched substrate utilization from glucose to lactate and did not utilize glucose until lactate decreased to a low concentration (1 to 2 mM). Since substrate utilization was shifted gradually without intermittence, typical diauxic growth was not seen. The cyclic AMP content did not rise markedly with the shift in substrate utilization, suggesting that this nucleotide is not involved in the regulation of the shift. It was unlikely that propionate was produced from glucose, which was explicable by the fact that lactate racemase activity dropped rapidly with the exhaustion of lactate and cells actively fermenting glucose did not possess this enzyme. A coculture experiment indicated that M. elsdenii NIAH 1102 is overcome by Streptococcus bovis JB1 in the competition for glucose, mainly because M. elsdenii NIAH 1102 is obliged to utilize lactate produced by S. bovis JB1; i.e., glucose utilization by M. elsdenii NIAH 1102 is suppressed by the coexistence of S. bovis JB1.     

Fragmentation of re-formation of mitotic Golgi apparatus detected by a centrifugal method

The fragmentation/re-formation process of the Golgi apparatus during mitosis was studied by flotation centrifugation in a stepwise sucrose density gradient. The mitotic Golgi fraction was obtained from Chinese hamster ovary cells synchronized with thymidine and nocodazole. The Golgi apparatus detected by a marker enzyme, galactosyltransferase, was separated into two peaks by the flotation centrifugation. The amount of the Golgi recovered at the lower density peak was less in the mitotic cells than in the interphase cells. The separation profile of the mitotic Golgi returned to that of the interphase Golgi by further incubation of the mitotic cells. The re-formation of the fragmented Golgi was inhibited by nocodazole and vinblastine, but not by actinomycin D and cycloheximide.     

A new assay of X-prolyl dipeptidyl-aminopeptidase activity in human serum with glycylproline p-phenylazoanilide as substrate

Summary A new assay procedure for X-prolyl dipeptidyl-aminopeptidase activity in human serum was developed with glycylproline p-phenylazoanilide tosylate as substrate. p-Phenylazoaniline liberated by the enzyme reaction was measured photometrically at 493 nm after stopping the reaction with acid. This assay was simple and sensitive, and less than 50 l of human serum was required for the assay. Km value was 2.5 mm and the optimum pH was 8.7. After disc gel electrophoresis of human serum, the enzyme activity could be distinctly observed as a reddish band on the gel when the gel was incubated with this substrate.     

Antigenic determinants of the 70,000 molecular weight glycoprotein of woolly monkey type C RNA virus.

The 70,000 molecular weight glycoprotein (gp70) of a type-C RNA virus originally isolated from a woolly monkey has been partially purified and immunologically characterized. Evidence that this viral protein is viral coded was derived from studies showing its antigenic properties to be unaltered by virus passage in cells of different species. A broadly reactive competition immunoassay was developed utilizing antiserum prepared against feline leukemia virus to precipitate 125I-labeled woolly monkey virus gp70. Gibbon and woolly viruses, as well as feline and several mouse type-C viruses, all reacted with equal efficiency in this assay. In contrast, an endogenous virus of the baboon failed to cross-react, suggesting that viruses of this latter group are less immunologically related to the others. In a homologous competition immunoassay for the woolly viral glycoprotein, the woolly virus was readily distingusihed from otherwise colsely related viruses of gibbon apes. These findings demonstrate the pronounced type-specific antigenic dterminants possessed by this viral protein. The antigenic determinants of gp70 responsible for neutralization have also been investigated.     

Biological safety in virological field with special considerations on class II biological safety cabinets

The most critical point for the biosafety is not sophisticated devices or facilities, but education of workers and their compliance to the regulation. Appropriate devices should be carefully selected in the introduction of new devices, and they should be properly maintained. The class II biosafety cabinet is one of the delicate safety equipments. It should be kept adequately maintained throughout the lifetime of the cabinet to insure safety of the laboratory. For the maintenance, appropriate measuring equipments should be used by trained technicians. The recently enforced law for control of recombinant DNA researches should be applied for the handling of pathogens even in non-recombinant DNA researches after proper modifications.     

Endospores of halophilic bacteria of the family Bacillaceae isolated from non-saline Japanese soil may be transported by Kosa event (Asian dust storm)

Background

Natural microbial communities are extremely complex and dynamic systems in terms of their population structure and functions. However, little is known about the in situ functions of the microbial communities.

Results

This study describes the application of proteomic approaches (metaproteomics) to observe expressed protein profiles of natural microbial communities (metaproteomes). The technique was validated using a constructed community and subsequently used to analyze Chesapeake Bay microbial community (0.2 to 3.0 μm) metaproteomes. Chesapeake Bay metaproteomes contained proteins from pI 4–8 with apparent molecular masses between 10–80 kDa. Replicated middle Bay metaproteomes shared ~92% of all detected spots, but only shared 30% and 70% of common protein spots with upper and lower Bay metaproteomes. MALDI-TOF analysis of highly expressed proteins produced no significant matches to known proteins. Three Chesapeake Bay proteins were tentatively identified by LC-MS/MS sequencing coupled with MS-BLAST searching. The proteins identified were of marine microbial origin and correlated with abundant Chesapeake Bay microbial lineages, Bacteroides and α-proteobacteria.

Conclusion

Our results represent the first metaproteomic study of aquatic microbial assemblages and demonstrate the potential of metaproteomic approaches to link metagenomic data, taxonomic diversity, functional diversity and biological processes in natural environments.