Toshio Shida

Identification and characterization of novel cell wall hydrolase CwlT: a two-domain autolysin exhibiting n-acetylmuramidase and DL-endopeptidase activities.

A cell wall hydrolase homologue, Bacillus subtilis YddH (renamed CwlT), was determined to be a novel cell wall lytic enzyme. The cwlT gene is located in the region of an integrative and conjugative element (ICEBs1), and a cwlT-lacZ fusion experiment revealed the significant expression when mitomycin C was added to the culture. Judging from the Pfam data base, CwlT (cell wall lytic enzyme T (Two-catalytic domains)) has two hydrolase domains that exhibit high amino acid sequence similarity to dl-endopeptidases and relatively low similarity to lytic transglycosylases at the C and N termini, respectively. The purified C-terminal domain of CwlT (CwlT-C-His) could hydrolyze the linkage of d-γ-glutamyl-meso-diaminopimelic acid in B. subtilis peptidoglycan, suggesting that the C-terminal domain acts as a dl-endopeptidase. On the other hand, the purified N-terminal domain (CwlT-N-His) could also hydrolyze the peptidoglycan of B. subtilis. However, on reverse-phase HPLC and mass spectrometry (MS) and MS-MS analyses of the reaction products by CwlT-N-His, this domain was determined to act as an N-acetylmuramidase and not a lytic transglycosylase. Moreover, the site-directed mutagenesis analysis revealed that Glu-87 and Asp-94 are sites related with the cell wall lytic activity. Because the amino acid sequence of the N-terminal domain of CwlT exhibits low similarity compared with those of the soluble lytic transglycosylase and muramidase (goose lysozyme), this domain represents “a new category of cell wall hydrolases.”

CD81 and CD9 work independently as extracellular components upon fusion of sperm and oocyte

Summary When a sperm and oocyte unite into one cell upon fertilization, membranous fusion between the sperm and oocyte occurs. In mice, Izumo1 and a tetraspanin molecule CD9 are required for sperm-oocyte fusion as one of the oocyte factors, and another tetraspanin molecule CD81 is also thought to involve in this process. Since these two tetraspanins often form a complex upon cell-cell interaction, it is probable that such a complex is also formed in sperm-oocyte interaction; however, this possibility is still under debate among researchers. Here we assessed this problem using mouse oocytes. Immunocytochemical analysis demonstrated that both CD9 and CD81 were widely distributed outside the oocyte cell membrane, but these molecules were separate, forming bilayers, confirmed by immunobiochemical analysis. Electron-microscopic analysis revealed the presence of CD9- or CD81-incorporated extracellular structures in those bilayers. Finally, microinjection of in vitro-synthesized RNA showed that CD9 reversed a fusion defect in CD81-deficient oocytes in addition to CD9-deficient oocytes, but CD81 failed in both oocytes. These results suggest that both CD9 and CD81 independently work upon sperm-oocyte fusion as extracellular components.

DRUG BINDING TO HIGHER ORDERED DNA STRUCTURES : ETHIDIUM BROMIDE COMPLEXATION WITH PARALLEL QUADRUPLE-STRANDED DNA

Abstract The interaction between ethidium bromide and a parallel G4-DNA, which is a quadruplex composed from four oligonucleotides containing a dG cluster, has been investigated. [d(TTGGGGTT)]4 formed a complex with ethidium bromide, which was assumed to be intercalated between the adjacent guanine tetrads of the quadruplex. #This paper is dedicated to Prof. Yoshihisa Mizuno on the occasion of his 75th birthday.

Role of the tryptophan residue in the vicinity of the catalytic center of exonuclease III family AP endonucleases: AP site recognition mechanism

The mechanisms by which AP endonucleases recognize AP sites have not yet been determined. Based on our previous study with Escherichia coli exonuclease III (ExoIII), the ExoIII family AP endonucleases probably recognize the DNA-pocket formed at an AP site. The indole ring of a conserved tryptophan residue in the vicinity of the catalytic site presumably intercalates into this pocket. To test this hypothesis, we constructed a series of mutants of ExoIII and human APE1. Trp-212 of ExoIII and Trp-280 of APE1 were critical to the AP endonuclease activity and binding to DNA containing an AP site. To confirm the ability of the tryptophan residue to intercalate with the AP site, we examined the interaction between an oligopeptide containing a tryptophan residue and an oligonucleotide containing AP sites, using spectrofluorimetry and surface plasmon resonance (SPR) technology. The tryptophan residue of the oligopeptide specifically intercalated into an AP site of DNA. The tryptophan residue in the vicinity of the catalytic site of the ExoIII family AP endonucleases plays a key role in the recognition of AP sites.

Production of long-chain levan by a sacC insertional mutant from Bacillus subtilis 327UH.

A hyper extracellular protein producer, Bacillus subtilis 327UH, produced large amounts of levan in a medium containing 20% sucrose, and the yield of levan after 10 hours was more than 60%, when based on the fructose amount of sucrose. After transformation of 327UH with a levanase-deficient 168SC (sacC::Cmr) chromosomal DNA, a Cmr transformant 327UHSC (sacC::CmrdegSU(Hy)) produced 3 times longer levan than that of the wild type.

Characterization and comparison of mitochondrial DNAs and rRNAs from Penicillium urticae and P. chrysogenum

Mitochondrial DNA (mt DNA) from a patulin producer, Penicillium urticae (synonym P. griseofulvum), was 27.8 kb +/- 0.6 kb in size by electron microscopy and 27.2 kb by agarose gel electrophoresis. Restriction endonuclease maps for nine restriction enzymes were constructed, and eleven fragments which covered the total range of the mt DNA were cloned into the Escherichia coli plasmid vector pUC19. Southern analysis of the native genomes of P. urticae and P. chrysogenum with six of the cloned fragments as probes indicated similar genome arrangements as well as similar restriction maps. Both the large and small rRNA genes of P. urticae and P. chrysogenum were located on these restriction maps using Southern hybridization, and the result also supported the similar arrangement. Agarose/formaldehyde gel electrophoresis indicated that the small rRNA was 1.5 kb in size in both species; but, surprisingly, the large rRNA was 4.2 kb in size for P. urticae and 3.5 kb for P. chrysogenum. These sizes were, respectively, 1.1 kb and 0.4 kb larger than those from the very closely related Aspergillus nidulans.

Properties of DNA Duplexes Containing an Abasic Site: Effect of Nucleotide Residue of the Opposite Part of a 2-Deoxyribosylformamide Residue

Abstract To elucidate the conformational properties of a DNA duplex with a 2-deoxyribosylformamide residue (dF), oligomers containing this abasic residue and a propanediol residue in the center of the helix were synthesized and characterized by UV absorption and CD. By a comparison of the Tms of the duplexes containing a dF residue and a propanediol residue (P), the stability of the duplex containing the abasic damage was judged to depend on the kind of nucleotide residue on the opposite side of the abasic residue rather than the kind of abasic residue.

Characterization of the AP endonucleases from Thermoplasma volcanium and Lactobacillus plantarum: Contributions of two important tryptophan residues to AP site recognition.

Escherichia coli AP endonuclease (ExoIII) and its human homolog (APE1) have the sole tryptophan residue for AP site recognition (AP site recognizer) but these residues are at different positions near the catalytic sites. On the other hand, many bacterial AP endonucleases have two tryptophan residues at the same positions of both ExoIII and APE1. To elucidate whether these residues are involved in AP site recognition, the ExoIII homologs of Thermoplasma volcanium and Lactobacillus plantarum were characterized. These proteins showed AP endonuclease and 3′-5′exonculease activities. In each enzyme, the mutations of the tryptophan residues corresponding to Trp-280 of APE1 caused more significant reductions in activities and binding abilities to the oligonucleotide containing an AP site (AP-DNA) than those corresponding to Trp-212 of ExoIII. These results suggest that the tryptophan residue corresponding to Trp-280 of APE1 is the predominant AP site recognizer, and that corresponding to Trp-212 of ExoIII is the auxiliary recognizer.

Accumulation of a recombinant Aspergillus oryzae lipase artificially localized on the Bacillus subtilis cell surface

cutL cDNA encoding an extracellular lipase, L1, from Aspergillus oryzae was fused to the cell wall-binding domain (CWB) region of a plasmid, pHCB3R. SDS-polyacrylamide gel electrophoresis (PAGE) and zymography of proteins extracted from the cell surface of Bacillus subtilis 168 harboring a fused lipase plasmid (pHCB3RCL) revealed that the fused gene product, CWB-CutL, was localized in the B. subtilis cell wall and retained lipase activity. B. subtilis WASD (wprA sigD), recently used for the accumulation of CWB-LipB (the CWB protein fused with B. subtilis lipase B), was also a suitable host for the accumulation of CWB-CutL, the amount being 10% of the total proteins extracted from the cell surface.

Incipient complex formation between AP endonucleases and DNA containing AP site: A vital role of the tryptophan residue

To elucidate whether the tryptophan residues in the vicinity of the catalytic site are involved in AP site recognition and are critical for AP endonuclease activity, the AP endonucleases of the four subtypes in the ExoIII AP endonuclease family were characterized and compared the positions of the tryptophan residues. The positions of the catalytic amino acid residues, corresponding to Glu-34, Asp-229, and His-259 of ExoIII, are strictly conserved. On the other hand, the positions of the tryptophan residues, which are critical to the incipient complex formation, do not exist at a fixed position. There are four patterns at the position of the essential tryptophan residue.