Analysis of the gene of Vibrio hollisae encoding the hemolysin similar to the thermostable direct hemolysin of Vibrio parahaemolyticus

The gene (designated as Vh-tdh) of Vibrio hollisae 9041 encoding a hemolysin similar to the thermostable direct hemolysin (TDH) of V. parahaemolyticus contained a 567-base-pair open reading frame (ORF), which was 93.3-93.5% homologous to those of the tdh genes of V. parahaemolyticus, V. cholerae non-01, and V. mimicus encoding TDH or similar hemolysins. Comparative analysis of the nucleotide sequence containing the Vh-tdh ORF with published nucleotide and amino acid sequences suggested that the Vh-tdh gene and other tdh genes diverged from a common ancestral gene, that the divergence was closely associated with the evolutionary divergence of V. hollisae from other species of genus Vibrio, and that strain-to-strain variation of the Vh-tdh gene exists in V. hollisae.     

Contribution of a Salt Bridge Triad to the Thermostability of a Highly Alkaline Protease from an Alkaliphilic Bacillus Strain*

Summary Crystallographic analysis of the highly alkaline M-protease from an alkaliphilic Bacillus strain shows the occurrence of a unique salt bridge triad Arg19–Glu271–Arg275 (in subtilisin BPN′ numbering), which is not found in less alkaline true subtilisins BPN′ and Carlsberg from Bacillus amyloliquefaciens and Bacillus licheniformis, respectively. Because the corresponding residues are all Gln residue in the subtilisin BPN′, Gln residue was engineered into the position(s) 19, 271 and/or 275 in M-protease by site-directed mutagenesis. Disruptions of the salt bridge caused the reduction of the thermostability of the mutant proteins at alkaline pH with the following decreasing order of thermal inactivation rate; the wild-type > Arg275 → Gln > Glu271 → Gln > Arg19 → Gln/Glu271 → Gln/Arg275 → Gln > Arg19 → Gln. This result provides the evidence that the salt bridge triad contributes to the thermostability and structural rigidity of the highly alkaline M-protease.     

Differences in genotypes of Helicobacter pylori from different human populations

DNA motifs at several informative loci in more than 500 strains of Helicobacter pylori from five continents were studied by PCR and sequencing to gain insights into the evolution of this gastric pathogen. Five types of deletion, insertion, and substitution motifs were found at the right end of the H. pylori cag pathogenicity island. Of the three most common motifs, type I predominated in Spaniards, native Peruvians, and Guatemalan Ladinos (mixed Amerindian-European ancestry) and also in native Africans and U.S. residents; type II predominated among Japanese and Chinese; and type III predominated in Indians from Calcutta. Sequences in the cagA gene and in vacAm1 type alleles of the vacuolating cytotoxin gene (vacA) of strains from native Peruvians were also more like those from Spaniards than those from Asians. These indications of relatedness of Latin American and Spanish strains, despite the closer genetic relatedness of Amerindian and Asian people themselves, lead us to suggest that H. pylori may have been brought to the New World by European conquerors and colonists about 500 years ago. This thinking, in turn, suggests that H. pylori infection might have become widespread in people quite recently in human evolution.     

Structure of Novel Enzyme in Mannan Biodegradation Process 4-O-β-d-Mannosyl-d-Glucose Phosphorylase MGP

The crystal structure of a novel component of the mannan biodegradation system, 4-O-β-d-mannosyl-d-glucose phosphorylase (MGP), was determined to a 1.68-Å resolution. The structure of the enzyme revealed a unique homohexameric structure, which was formed by using two helices attached to the N-terminus and C-terminus as a tab for sticking between subunits. The structures of MGP complexes with genuine substrates, 4-O-β-d-mannosyl-d-glucose and phosphate, and the product d-mannose-1-phosphate were also determined. The complex structures revealed that the invariant residue Asp131, which is supposed to be the general acid/base, did not exist close to the glycosidic Glc-O4 atom, which should be protonated in the catalytic reaction. Also, no solvent molecule that might mediate a proton transfer from Asp131 was observed in the substrate complex structure, suggesting that the catalytic mechanism of MGP is different from those of known disaccharide phosphorylases.     

Intraspecies host specificity of a single-stranded RNA virus infecting a marine photosynthetic protist is determined at the early steps of infection

Viruses are extremely abundant in seawater and are believed to be significant pathogens to photosynthetic protists (microalgae). Recently, several novel RNA viruses were found to infect marine photosynthetic protists; one of them is HcRNAV, which infects Heterocapsa circularisquama (Dinophyceae). There are two distinct ecotypes of HcRNAV with complementary intraspecies host ranges. Nucleotide sequence comparison between them revealed remarkable differences in the coat protein coding gene resulting in a high frequency of amino acid substitutions. However, the detailed mechanism supporting this intraspecies host specificity is still unknown. In this study, virus inoculation experiments were conducted with compatible and incompatible host-virus combinations to investigate the mechanism determining intraspecies host specificity. Cells were infected by adding a virus suspension directly to a host culture or by transfecting viral RNA into host cells by particle bombardment. Virus propagation was monitored by Northern blot analysis with a negative-strand-specific RNA probe, transmission electron microscopy, and a cell lysis assay. With compatible host-virus combinations, propagation of infectious progeny occurred regardless of the inoculation method used. When incompatible combinations were used, direct addition of a virus suspension did not even result in viral RNA replication, while in host cells transfected with viral RNA, infective progeny virus particles with a host range encoded by the imported viral RNA were propagated. This indicates that the intraspecies host specificity of HcRNAV is determined by the upstream events of virus infection. This is the first report describing the reproductive steps of an RNA virus infecting a photosynthetic protist at the molecular level.     

Higher susceptibility to osmolality of the medaka (Oryzias latipes) mutants in orthologue genes of mammalian skin transglutaminases

Transglutaminase (TG) is an essential enzyme to catalyze cross-linking reactions of epidermal proteins. Recently, we biochemically characterized human skin TG orthologues for medaka (Oryzias latipes), a model fish. By genome editing, gene-modified fishes for the two orthologues were obtained, both of which lack the ordinal enzymes. These fish appeared to exhibit higher susceptibility to osmolality at the period of larvae.     

Synthesis and Hybridization Properties of Oligonucleotides Containing (2 S ,3 R )-9-(2,3,4-Trihydroxybutyl)Adenine

The synthesis and properties of oligonucleotides (ONs) containing 9-(2,3,4-trihydroxybutyl)adenine, A ^C2 and A ^C3, are described. The ON containing A ^C2 involves the 3′ → 4′ and 3′ → 5′ phosphodiester linkages in the strand, whereas that containing A ^C3 possesses the 3′ → 4′ and 2′ → 5′ phosphodiester linkages. It was found that incorporation of the analogs, A ^C2 or A ^C3, into ONs significantly reduces the thermal and thermodynamic stabilities of the ON/DNA duplexes, but does not largely decrease the thermal and thermodynamic stabilities of the ON/RNA duplexes as compared with the case of the ON/DNA duplexes. It was revealed that the base recognition ability of A ^C2 is greater than that of A ^C3 in the ON/RNA duplexes.     

Experimental Infection of the Cotton Rat Sigmodon hispidus with Rickettsia rickettsii

Studies of experimental infection of the cotton rat, Sigmodon hispidus, with the virulent Sheila Smith (R type) and the avirulent Si 7 (U type) strains of Rickettsia rickettsii were undertaken to evaluate the role of this native wild mammal in the ecology of Rocky Mountain spotted fever. The Sheila Smith strain, which was highly lethal for guinea pigs, was nonpathogenic for cotton rats. Serial passage of the R-type strain in the cotton rat did not alter the virulence of the agent for cotton rats or guinea pigs. The U-type strain, which was originally recovered from a wild cotton rat, could not be maintained beyond the first passage in this animal host. Rickettsemia in the cotton rat occurred over a 24-hr period after inoculation of the virulent strain but was detected only 1 hr after inoculation of the avirulent strain. The short period of rickettsemia suggests that the cotton rat probably is not an important reservoir of R. rickettsii. Specific complement-fixing antibodies developed rapidly after infection with either strain, but the antibodies evoked by the R strain attained higher titers and persisted longer. Cotton rats previously infected with the Sheila Smith strain developed rickettsemia after reinfection with the same strain, even though relatively high levels of antibody were still present.     

Characteristics of microbial fermentation and potential digestibility of fiber in the hindgut of dugongs (Dugong dugon)

The number of microorganisms in the hindgut of dugongs (Dugong dugon) were estimated and their in vitro volatile fatty acid (VFA) production and degradation of eelgrass measured. Scanning electron microscopy showed that some rod bacteria attached to the surface of plant tissue degraded and eroded the cell walls. Number of starch-, lactate-, cellobiose-, pectin-, xylan- and cellulose-utilizing bacteria, sulfate-reducing bacteria and methane-producing bacteria were estimated at 10⁹ ～ 10¹⁰ colony forming units g^?1. Microorganisms degraded the cellulose and noncellulolytic components of the eelgrass, and about 47.3% of dry matter was degraded after 36?h in vitro incubation. The total VFA concentration was 10.5?mmol?dL^?1 at 36?h incubation, which included 55.7?mol% acetate, 18.0?mol% n-butyrate and 15.1?mol% propionate. The gas composition of in vitro fermentation was 68.4% carbon dioxide, 22.2% methane and 9.4% hydrogen.