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The hemgene cluster, which consists of hemA, cysGB, hemC, hemD, hemB, and hemLgenes, and encodes enzymes involved in the biosynthetic pathway from glutamyl‐tRNA to uroporphyrinogen III, has been identified by the cloning and sequencing of two overlapping DNA fragments from Clostridium perfringensNCTC8237. The deduced amino acid sequence of the N‐terminal region of C. perfringensHemD is homologous to those reported for the C‐terminal region of Salmonella typhimuriumCysG and Clostridium josuiHemD. C, perfringensCysGBis a predicted 220‐residue protein which shows homology to the N‐terminal region of S. typhimuriumCysG. Disruption of the cysGBgene in C. perfringensstrain 13 by homologous recombination reduced cobalamin (vitamin B12) levels by a factor of 200. When grown in vitamin B12‐deficient medium, the mutant strain showed a four‐fold increase in its doubling time compared with that of the wild‐type strain, and this effect was counteracted by supplementing the medium with vitamin B12. These results suggest that C. perfringensCysGBis involved in the chelation of cobalt to precorrin II as suggested for the CysGBdomain of S. typhimuriumCysG, enabling the synthesis of cobalamin.

The colHgene encoding 116‐kDa collagenase of Clostridium histolyticum(cColH) was cloned into an Escherichia coli‐Bacillus subtilisshuttle vector to develop a method for purification of recombinant collagenase (rColH). When plasmid pJCM310 containing the colHgene was introduced into B. subtilisDB104 and the transformant was grown in LB broth at 37 C, stability of the plasmid was not maintained. However, stability was partly improved by growing the transformant in a modified LB broth containing 0.5 Msodium succinate with gentle shaking at 35 C. When the transformant was grown to an optical density of 0.4 at 600 nm in this medium, pJCM310 was stable and rColH was produced in sufficient amounts. rColH was purified to homogeneity by ammonium sulfate precipitation, gel filtration and ion‐exchange chromatography. The yield of rColH from an 800‐ml culture was 0.53 mg and its specific activity was estimated to be 1,210 U per mg of protein. The purified rColH was capable of degrading native type‐I collagen fibril from bovine achilles tendon, as was demonstrated by zymography. A comparison of the N‐terminal amino acid sequence between cColH and rColH revealed that rColH has 10 extra N‐terminal amino acid residues. However, the peptide mapping of rColH with V8 protease was virtually identical to that of cColH. Furthermore, the molecular mass of rColH was estimated to be 112,999 Da by mass spectrometry, coinciding with the value of 112,977 Da, which was predicted from the nucleotide sequence of the colHgene. Therefore, the recombinant B. subtilisculture is capable of serving as a useful source for enzyme purification.

Clostridium perfringensKZ1340 isolated from Antarctic soil was first classified as Clostridium plagarumand later as a lecithinase‐negative variant of C. perfringens. Although the strain produced no detectable lecithinase (phospholipase C, PLC) activity in the culture supernatant, it was shown by Southern blot hybridization to possess a PLC‐encoding gene (plc). To determine the cause of the PLC deficiency, we cloned and sequenced the plcgene from KZ1340. The deduced amino acid sequence consists of 398 amino acid residues, coinciding with those of the plcgenes previously determined. Tyrosine was substituted for histidine at amino acid position 148, which is thought to bind a zinc ion essential for PLC activity. Northern blot analysis revealed that KZ1340 expressed the plcgene at an extremely low level. Furthermore, the plcgene cloned from C. perfringensstrain 13 into a plasmid was expressed weakly in KZ1340, compared to that in strain 13. This indicates that the former strain represses plcgene expression in trans. When a phylogenetic tree of plcgenes was constructed, the KZ1340 plcgene formed a monophyletic branch along with those of various other C. perfringensstrains, supporting the classification of the strain as a variant of C. perfringens.

The effect of γ‐toxin, a thermolysin‐like metalloprotease of Clostridium perfringens, on the inactive ε‐prototoxin produced by the same organism was examined. When the purified ε‐prototoxin was incubated with the purified γ‐toxin at 37 C for 2 hr, the 32.5‐kDa ε‐prototoxin was processed into a 30.5‐kDa polypeptide, as determined by SDS‐polyacrylamide gel electrophoresis. A mouse lethality test showed that the treatment activated the prototoxin: the 50% lethal doses (LD50) of the prototoxin with and without γ‐toxin treatment were 110 and 70,000 ng/kg of body weight, respectively. The lethal activity of the prototoxin activated by γ‐toxin was comparable to that with trypsin plus chymotrypsin and higher than that with trypsin alone: LD50of the prototoxin treated with trypsin and trypsin plus chymotrypsin were 320 and 65 ng/kg of body weight, respectively. The ε‐toxin gene was cloned and sequenced. Determination of the N‐terminal amino acid sequence of each activated ε‐prototoxin revealed that γ‐toxin cleaved between the 10th and 11th amino acid residues from the N‐terminus of the prototoxin, while trypsin and trypsin plus chymotrypsin cleaved between the 13th and 14th amino acid residues. The molecular weight of each activated ε‐prototoxin was also determined by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. The C‐terminus deduced from the molecular weight is located at the 23rd or 30th amino acid residue from the C‐terminus of the prototoxin, suggesting that removal of not only N‐terminal but also C‐terminal peptide is responsible for activation of the prototoxin.

Recently, certain plans have been made to replace existing motors with the objective of achieving controlled rolling in Japanese plate mills. Application of ac drive systems, which are making rapid progress, can be considered for these plans. A comparison of conventional dc motors, brushless dc motors, and vector-controlled induction motors fed by cycloconverters (cycloconverter IM's) is introduced from the viewpoint of their characteristics such as power-torque curve, efficiency, and influence of harmonic current on the power system. As a result of comparison it is found that the cycloconverter IM's can be satisfactorily applied to the plate mills.