Ryuichi Moriyama

A spore-lytic enzyme released from Bacillus cereus spores during germination.

The exudate of fully germinated spores of Bacillus cereus IFO 13597 in 0.25 M sodium phosphate buffer, pH 7.0, was found to contain a spore-lytic enzyme. This enzyme was found to cause loss of absorbance in coat-stripped spore suspensions and phase-darkening of the spores but had minimal activity on isolated peptidoglycan substrates. The enzyme was purified in an active form and identified as a 24 kDa protein which is either an amidase or a peptidase. The amino-terminal 19 residues had the following sequence: FSNQVIQRGASGEKVIELQ. The spore-lytic enzyme retained its activity in a medium of a relatively high ionic strength containing a non-ionic surfactant such as nonaethyleneglycol n-dodecyl ether. This activity was optimum at a salt concentration of about 30 mM in assay buffer at neutral pH. In contrast to the enzyme in a spore-bound form, the enzyme in solution was shown to be heat-sensitive and was readily inactivated by thiol reagents.

Partial Characterization of an Enzyme Fraction with Protease Activity Which Converts the Spore Peptidoglycan Hydrolase (SleC) Precursor to an Active Enzyme during Germination of Clostridium perfringens S40 Spores and Analysis of a Gene Cluster Involved in the Activity

A spore cortex-lytic enzyme of Clostridium perfringens S40 which is encoded by sleC is synthesized at an early stage of sporulation as a precursor consisting of four domains. After cleavage of an N-terminal presequence and a C-terminal prosequence during spore maturation, inactive proenzyme is converted to active enzyme by processing of an N-terminal prosequence with germination-specific protease (GSP) during germination. The present study was undertaken to characterize GSP. In the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), a nondenaturing detergent which was needed for the stabilization of GSP, GSP activity was extracted from germinated spores. The enzyme fraction, which was purified to 668-fold by column chromatography, contained three protein components with molecular masses of 60, 57, and 52 kDa. The protease showed optimum activity at pH 5.8 to 8.5 in the presence of 0.1% CHAPS and retained activity after heat treatment at 55 degrees C for 40 min. GSP specifically cleaved the peptide bond between Val-149 and Val-150 of SleC to generate mature enzyme. Inactivation of GSP by phenylmethylsulfonyl fluoride and HgCl(2) indicated that the protease is a cysteine-dependent serine protease. Several pieces of evidence demonstrated that three protein components of the enzyme fraction are processed forms of products of cspA, cspB, and cspC, which are positioned in a tandem array just upstream of the 5 end of sleC. The amino acid sequences deduced from the nucleotide sequences of the csp genes showed significant similarity and showed a high degree of homology with those of the catalytic domain and the oxyanion binding region of subtilisin-like serine proteases. Immunochemical studies suggested that active GSP likely is localized with major cortex-lytic enzymes on the exterior of the cortex layer in the dormant spore, a location relevant to the pursuit of a cascade of cortex hydrolytic reactions.

A gene (sleC) encoding a spore-cortex-lytic enzyme from Clostridium perfringens S40 spores ; cloning, sequence analysis and molecular characterization

Antiserum was raised against a 31 kDa spore-cortex-lytic enzyme, which is released during germination of Clostridium perfringens S40 spores. Western blotting of dormant spore and vegetative cell fractions separated by SDS-PAGE indicated that the 31 kDa enzyme is spore-specific and that the enzyme in the dormant spore exists as a 36 kDa protein which has no cortex-lytic activity. A gene encoding the 31 kDa enzyme, sleC, was cloned into Escherichia coli using a synthetic oligonucleotide as a hybridization probe and the nucleotide sequence of the entire gene was determined. The N-terminal amino acid sequence of the 36 kDa protein was found in this reading frame, confirming that the 36 kDa protein is a pro-form of the 31 kDa enzyme. The deduced amino acid sequence indicated that the 31 kDa enzyme is produced as a precursor, comprising three portions; an N-terminal prepro-sequence (114 amino acid residues), a pro-sequence (35 amino acid residues) and a mature enzyme (289 amino acid residues). It is suggested that the 36 kDa pro-enzyme is non-covalently attached to the exterior of the cortex layer, and that the proform is processed to release the active enzyme during germination.

Two family G xylanase genes from Chaetomium gracile and their expression in Aspergillus nidulans

With oligonucleotides based on the amino-terminal and internal amino-acid sequences of a xylanase, two xylanase genes, cgxA and cgxB, were isolated and sequenced from Chaetomium gracile wild and mutant strains. Each gene isolated from both strains was essentially the same as far as nucleotide sequences were compared. The mature CgXA and CgXB xylanases comprise 189 and 211 amino acids, respectively, and share 68.5% homology. The CgXA was found to be the major enzyme in the mutant strain. Comparison of these amino-acid sequences with xylanase sequences from other origins showed that they have a high degree of identity to the family G xylanases. The cgxA and cgxB genes were introduced into Aspergillus nidulans and found to be expressed with their own promoters.

A Novel Lipolytic Enzyme, YcsK (LipC), Located in the Spore Coat of Bacillus subtilis, Is Involved in Spore Germination

The predicted amino acid sequence of Bacillus subtilis ycsK exhibits similarity to the GDSL family of lipolytic enzymes. Northern blot analysis showed that ycsK mRNA was first detected from 4 h after the onset of sporulation and that transcription of ycsK was dependent on SigK and GerE. The fluorescence of the YcsK-green fluorescent protein fusion protein produced in sporulating cells was detectable in the mother cell but not in the forespore compartment under fluorescence microscopy, and the fusion protein was localized around the developing spores dependent on CotE, SafA, and SpoVID. Inactivation of the ycsK gene by insertion of an erythromycin resistance gene did not affect vegetative growth or spore resistance to heat, lysozyme, or chloroform. The germination of ycsK spores in a mixture of L-asparagine, D-glucose, D-fructose, and potassium chloride and LB medium was also the same as that of wild-type spores, but the mutant spores were defective in L-alanine-stimulated germination. In addition, zymogram analysis demonstrated that the YcsK protein heterologously expressed in Escherichia coli showed lipolytic activity. We therefore propose that ycsK should be renamed lipC. This is the first study of a bacterial spore germination-related lipase.

Structure of a β-Glucosidase Gene from Ruminococcus albus and Properties of the Translated Product

A gene encoding β -glucosidase from Ruminococcus albus F-40, an anaerobic cellulolytic bacterium dominant in rumen, was cloned into Escherichia coli and sequenced. The structural gene of the β -glucosidase consisted of 2841 bp encoding 947 amino acid residues without a characteristic signal peptide. A typical promoter sequence was located upstream of the initiation ATG codon. Palindromic sequences were found both upstream and downstream of the structural gene. Codon usage of the gene was similar to that of CMCase of R. albus , and the bias of the G+C content in the positions of the codons was negligible. The deduced amino acid sequence of R. albus β -glucosidase showed high homology with β -glucosidases from several microorganisms. There were two homologous regions; A and B. A sequence of the supposed active region containing aspartic acid residue for Aspergillus wentii β -glucosidase A3 was conserved in region B of the R. albus β -glucosidase sequence. The gene product, β -glucosidase, was released from E. coli transformant cells into the culture supernatant, even though the gene did not encode a signal peptide. From the culture supernatant, the enzyme was purified as a homogeneous protein, of which the N-terminal amino acid sequence was identical to that of the enzyme from R. albus cells and that deduced from DNA sequence. These enzymes were also identical immunologically.

The N‐terminal prepeptide is required for the production of spore cortex‐lytic enzyme from its inactive precursor during germination of Clostridium perfringens S40 spores

A spore cortex‐lytic enzyme of Clostridium perfringens S40 is synthesized during sporulation as a precursor consisting of four domains. After cleavage of an N‐terminal preregion and a C‐terminal proregion, inactive proenzyme (termed C35) is converted to active enzyme by processing of an N‐terminal prosequence with germination‐specific protease (GSP) during germination. The present results demonstrated that the cleaved N‐terminal prepeptide remained associated with C35. After the isolated complex was denatured and dissociated in 6u2003M urea solution, removal of urea regenerated a prepeptide–C35 complex which produces active enzyme when incubated with GSP. However, isolated C35 alone could not be activated by GSP. The prepeptide–C35 complex was more heat stable than active enzyme. Thus, non‐covalent attachment of the prepeptide to C35 is required to assist correct folding of C35 and to stabilize its conformation, suggesting that the prepeptide functions as an intramolecular chaperone. Recombinant proteins, which have prepeptide covalently bonded to C35, were processed by GSP as well as the in vivo prepeptide–C35 complex, and the full length of the N‐terminal presequence was needed to fulfil its role. Although the C‐terminal prosequence is present as an independent domain which is not involved in the activation process of the enzyme, it appears that the N‐terminal prosequence contributes to the regulation of enzyme activity as an inhibitor of the enzyme.

Interaction of glyceraldehyde-3-phosphate dehydrogenase with the cytoplasmic pole of band 3 from bovine erythrocyte membrane: The mode of association and identification of the binding site of band 3 polypeptide

Four fragments derived from the cytoplasmic pole of bovine band 3 were isolated, and their ability to bind glyceraldehyde-3-phosphate dehydrogenase from bovine erythrocyte and their amino-terminal primary structure were examined. It was suggested that the 50-kDa fragment, an entire cytoplasmic pole of band 3, contained the blocked amino-terminal end of band 3. Three other fragments, 45-, 39-, and 38-kDa fragments, were produced by cleavage at distances of molecular weight 5000, 11,000, and 12,000 respectively, from the amino-terminus of the 50-kDa fragment. Among these, the 50- and 45-kDa fragments complexed with the enzyme to inhibit its catalytic activity under conditions of low ionic strength, in a fashion similar to that in humans. Affinity for the enzyme was not significantly affected by disruption of the higher order structure of the fragments. The enzyme was found to be inactivated by association with synthetic polyanions, accompanied by conformational alteration. This supports participation of electrostatic interactions as the holding force between the enzyme and band 3, as suggested by I-H. Tsai et al. [1982) J. Biol. Chem. 257, 1438-1442). The 45-kDa fragment was just as potent an inhibitor of the enzyme as the parent fragment, and its amino-terminal region displayed a polyanionic character. These results allow us to map the enzyme binding site of bovine band 3 to a distance of molecular weight approximately 5000 from the amino-terminal end of band 3. Furthermore, comparison of sequence data from different species showed that the species-specific region of band 3 polypeptide centers around the amino-terminal portion.

A study on the separation of reconstituted proteoliposomes and unincorporated membrane proteins by use of hydrophobic affinity gels, with special reference to band 3 from bovine erythrocyte membranes.

Alkyl-Sepharose 4B with octyl, decyl, or dodecyl groups as an alkyl chain was a good adsorbent for any type of detergents and a variety of proteins, but not for phospholipids in a vesicle form. When these gels were added to the mixtures of reconstituted proteoliposomes prepared by using bovine band 3 and the protein unincorporated into liposomes, free band 3 in solution was adsorbed onto the gels and the proteoliposomes could be recovered by filtration, suggesting that this procedure, when applicable, permits a rapid isolation of proteoliposomes without loss and dilution of the sample. In addition, the results indicated that Bio-Beads SM-2 resin, which is virtually nonadsorbing for most proteins, can be used in removing any kind of detergents from those protein-detergent mixtures.

Effect of detergent on protein structure. Action of detergents on secondary and oligomeric structures of band 3 from bovine erythrocyte membranes.

With special interest in the mode of action of zwitterionic detergents on proteins, a variety of detergents were examined for their ability to disrupt the secondary and quaternary structures of an anion transport protein, band 3, and its cytoplasmic 38 kDa fragment from bovine erythrocyte membranes and for their effect on the binding of an anion transport inhibitor to band 3. Nonionic detergents and Chaps also acted as a nondenaturant in these instances, as well accepted for other proteins. Though deoxycholate and cholate inhibited the binding of an anion transport inhibitor to band 3, these detergents did not show any effect on the native structure of band 3. Zwitterionic detergents (Zwittergent 3-10, Zwittergent 3-12 and N, N-dimethyl-N-dodecyl glycine) were suggested to denature the water-soluble 38 kDa fragment at concentrations above the critical micelle concentration, but to be weak in disrupting interacting forces between hydrophobic membrane-bound domains of band 3. The results indicated that these zwitterionic detergents are similar in the mode of denaturing action to dodecyltrimethylammonium bromide rather than sodium dodecyl sulfate.