Ken Kurokawa

Genome sequence of Vibrio parahaemolyticus : a pathogenic mechanism distinct from that of V. cholerae

BACKGROUND Vibrio parahaemolyticus, a gram-negative marine bacterium, is a worldwide cause of food-borne gastroenteritis. V parahaemolyticus strains of a few specific serotypes, probably derived from a common clonal ancestor, have lately caused a pandemic of gastroenteritis. The organism is phylogenetically close to V cholerae, the causative agent of cholera. METHODS The whole genome sequence of a clinical V parahaemolyticus strain RIMD2210633 was established by shotgun sequencing. The coding sequences were identified by use of Gambler and Glimmer programs. Comparative analysis with the V cholerae genome was undertaken with MUMmer. FINDINGS The genome consisted of two circular chromosomes of 3288558 bp and 1877212 bp; it contained 4832 genes. Comparison of the V parahaemolyticus genome with that of V cholerae showed many rearrangements within and between the two chromosomes. Genes for the type III secretion system (TTSS) were identified in the genome of V parahaemolyticus; V cholerae does not have these genes. INTERPRETATION The TTSS is a central virulence factor of diarrhoea-causing bacteria such as shigella, salmonella, and enteropathogenic Escherichia coli, which cause gastroenteritis by invading or intimately interacting with intestinal epithelial cells. Our results suggest that V parahaemolyticus and V cholerae use distinct mechanisms to establish infection. This finding explains clinical features of V parahaemolyticus infections, which commonly include inflammatory diarrhoea and in some cases systemic manifestations such as septicaemia, distinct from those of V cholerae infections, which are generally associated with non-inflammatory diarrhoea.

Identification of PGC7, a new gene expressed specifically in preimplantation embryos and germ cells.

The gene expression patterns of primordial germ cells (PGCs) and embryonic stem cells were analyzed by a modified serial analysis of gene expression. During the process, we cloned a novel gene, PGC7, which was preferentially expressed in PGCs. Immunohistochemical analysis revealed that PGC7 was specifically expressed in early pre-implantation embryos, PGCs and oocytes. These results suggest that PGC7 might play an important role in the development of PGCs and oocytes.

Complete nucleotide sequence of the prophage VT1-Sakai carrying the Shiga toxin 1 genes of the enterohemorrhagic Escherichia coli O157: H7 strain derived from the Sakai outbreak.

Shiga toxins 1 and 2 (Stx1 and Stx2) are encoded by prophages lysogenized in enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains. Lytic growth of the phage particles carrying the stx1 genes (stx1A and stx1B) of the EHEC O157:H7 strain RIMD 0509952, which was derived from the Sakai outbreak in 1996 in Japan, was induced after treatment with mitomycin C, but the plaque formation of the phage was not detected. We have determined the complete nucleotide sequence of the prophage VT1-Sakai. The integration site of the prophage was identified within the yehV gene at 47.7 min on the chromosome. The stx1 genes were downstream of the Q gene in the prophage genome, suggesting that their expression was regulated by the Q protein, the regulator of the late gene expression of the phage, which is similar to that of the stx1 or stx2 genes carried by the lambdoid phages reported previously. The sequences of the N gene and its recognition sites, nutL and nutR, were not homologous to those of the phages carrying the stx genes thus far reported, but they were very similar to those of bacteriophage phi21. The sequences of the repressor proteins, CI and Cro, that regulate expression of the early genes had low similarities with those of the known repressors of other phages, and their operator sequences were different from any sequence reported. These data suggest that multiple genetic recombination among bacteriophages with different immunities took place to generate the prophage VT1-Sakai. Comparison between the sequences of VT1-Sakai and lambda suggests that the ancestor of VT1-Sakai was produced by illegitimate excision, like lambda gal and bio phages.

Plasmodium falciparum Phospholipase C Hydrolyzing Sphingomyelin and Lysocholinephospholipids Is a Possible Target for Malaria Chemotherapy

Sphingomyelinase (SMase) is one of the principal enzymes in sphingomyelin (SM) metabolism. Here, we identified a Plasmodium falciparum gene (PfNSM) encoding a 46-kD protein, the amino acid sequence of which is ∼25% identical to that of bacteria SMases. Biochemical analyses of the recombinant protein GST-PfNSM, a fusion protein of the PfNSM product with glutathione-S-transferase, reveal that this enzyme retained similar characteristics in various aspects to SMase detected in P. falciparum–infected erythrocytes and isolated parasites. In addition, the recombinant protein retains hydrolyzing activity not only of SM but also of lysocholinephospholipids (LCPL) including lysophosphatidylcholine and lysoplatelet-activating factor, indicating that PfNSM encodes SM/LCPL-phospholipase C (PLC). Scyphostatin inhibited SM/LCPL-PLC activities of the PfNSM product as well as the intraerythrocytic proliferation of P. falciparum in a dose-dependent manner with ID50 values for SM/LCPL-PLC activities and the parasite growth at 3–5 μM and ∼7 μM, respectively. Morphological analysis demonstrated most severe impairment in the intraerythrocytic development with the addition of scyphostatin at trophozoite stage than at ring or schizont stages, suggesting its effect specifically on the stage progression from trophozoite to schizont, coinciding with the active transcription of PfNSM gene.

Micelle enhanced oxidation of amines by coenzyme PQQ

Abstract Nonezymatic oxidation of amines with the coenzyme PQQ is enhanced by a cationic surfactant to give the corresponding carbonyl compounds, suggesting that the micelle presents a good environment for oxidation with PQQ. The similar reaction is applicable to alcohols though an efficiency is not so high.

Comparative analysis of the whole set of rRNA operons between an enterohemorrhagic Escherichia coli O157:H7 Sakai strain and an Escherichia coli K-12 strain MG1655.

Two primer sets for direct sequence determination of all seven rRNA operons (rrn) of Escherichia coli have been developed; one is for specific-amplification of each rrn operon and the other is for direct sequencing of the amplified operons. Using these primer sets, we determined the nucleotide sequences of seven rrn operons, including promoter and terminator regions, of an enterohemorrhagic E. coli (EHEC) O157:H7 Sakai strain. To elucidate the intercistronic or intraspecific variation of rrn operons, their sequences were compared with those for the K-12 rrn operons. The rrn genes and the internal transcribed spacer regions showed a higher similarity to each other in each strain than between the corresponding operons of the two strains. However, the degree of intercistronic homogeneity was much higher in the EHEC strain than in K-12. In contrast, promoter and terminator regions in each operons were conserved between the corresponding operons of the two strains, which exceeded intercistronic similarity.

Determination of the breakpoints of a chromosomal translocation found in a patient with paroxysmal nocturnal hemoglobinuria

Determination of the breakpoints of a chromosomal translocation found in a patient with paroxysmal nocturnal hemoglobinuria