Showing papers by "Hideo Hayashi published in 1994"

The virR gene, a member of a class of two-component response regulators, regulates the production of perfringolysin O, collagenase, and hemagglutinin in Clostridium perfringens.

Abstract: The perfringolysin O (theta-toxin) gene (pfoA) of Clostridium perfringens was cloned into an Escherichia coli-C. perfringens shuttle vector, and the pfoA gene was expressed in mutants of C. perfringens 13 which lacked the production of perfringolysin O. One group (SI117) could express the pfoA gene, and the other (SI112) could not. A mutation in the regulatory system for pfoA gene expression was suspected in SI112. A chromosomal DNA library constructed from strain 13 was transformed into strain SI112 to identify the regulatory gene(s) for the pfoA gene. Five strains of 10,000 transformants restored perfringolysin O production. All contained a 2.5-kb DNA fragment. This fragment activated the transcription of the pfoA gene and also restored the production of collagenase (kappa-toxin) and hemagglutinin in strain SI112. Deletion analysis showed that a 1.25-kb region was sufficient for the trans activity, and sequence analysis disclosed that open reading frame 2 (ORF2) was located in this region. A homology search for the deduced amino acid sequence revealed that ORF2 was homologous to a response regulator in a two-component signal transduction system. ORF2 was designated virR, and it is suggested that the virR gene plays an important role in the pathogenicity of C. perfringens.

Molecular cloning of two new heat shock genes related to the hsp70 genes in Staphylococcus aureus.

Abstract: We have identified two new heat shock protein genes, orf37 and orf35, in Staphylococcus aureus, located upstream and downstream of grpE(hsp20), dnaK(hsp70), and dnaJ(hsp40) homologous genes in the order orf37-hsp20-hsp70-hsp40-orf35. The transcripts of both orf37 and orf35 were increased by thermal upshift of the culture from 37 to 46 degrees C. The heat shock promoters were located upstream of orf37 and upstream of hsp40. The deduced peptide of orf37 showed similarity with those of orfA in Clostridium acetobutylicum and orf39 in Bacillus subtilis. orf35 was unique in S. aureus and has not yet been described in other bacteria.

The Rice Culture Filtrate of Bacillus cereus Isolated from Emetic-Type Food Poisoning Causes Mitochondrial Swelling in a HEp-2 Cell

Abstract: Rice culture filtrates of Bacillus cereus SA-50, an emetic-type strain, produced a toxin which caused cytoplasmic vacuole formation in HEp-2 and HeLa cells. Electron microscopic observation revealed that the apparent vacuoles in HEp-2 seen under a light microscope were actually swollen mitochondria. The oxygen consumption of HEp-2 cells was accelerated by the addition of the rice culture filtrate as was measured with a polarographic oxymeter; a respiratory control ratio was 1.0 for control cells, while 1.4 for ones with the filtrates. The culture filtrates showed a similar effect on the isolated mouse liver mitochondria; respiratory control ratios for the mitochondria with and without the filtrates were 3.6 and 1.0, respectively. The affecting manner of the culture filtrates on the oxygen consumption of mitochondria was similar to that of 2,4-dinitrophenol, suggesting that the culture filtrate contains a toxin acting as an uncoupler of oxidative phosphorylation in mitochondria. It is likely that the culture filtrates containing the emetic toxin of B. cereus causes mitochondrial swelling with a close relationship to the uncoupling of the oxidative phosphorylation of mitochondria.

Over-expression of the peptide containing the ATP-binding site in Na/K-ATPase α-subunit

Abstract: The deduced primary structure of Na/K-ATPase from the cloned nucleotide sequence revealed that the structure of ATP-hydrolysing active site was highly conserved among P-type ATPases (1,2,4). However, the minute conformation and molecular mechanism of ion transport have not been made clear yet. X-ray crystallography may be an efficient method to reveal the conformation of the protein, but such a hydrophobic membrane-protein has been unable to be purifed without using detergents. We made an assumption that out-of-membrane domain of Na/K-ATPase α-subunit facing to hydrophilic side with catalytic activity could be crystallized like regular soluble proteins (3). We report here that the hydrophilic, catalytic domain (50kDa) of α-subunit has been over-expressed in E. coli and purified without using any detergent, and discuss on the characterization for the peptide.