High-level expression of his-tagged clostridial collagenase in Clostridium perfringens

Clostridium histolyticum collagenase is used to isolate cells from various organs and tissues for tissue engineering, and also to treat destructive fibrosis; thus, the demand for high-grade enzyme preparations is increasing. In this study, we constructed a plasmid encoding C. histolyticum type II collagenase (ColH) with a C-terminal hexahistidine tag (ColH-his) to facilitate the purification of the enzyme through immobilized metal affinity chromatography (IMAC). When ColH-his was expressed in a protease-deficient mutant of Clostridium perfringens, it was produced in the culture supernatant more efficiently than the untagged ColH. ColH-his exhibited the same hydrolytic activity as ColH against 4-phenylazobenzyloxy-carbonyl-Pro-Leu-Gly-Pro-D: -Arg (Pz peptide), a synthetic collagenase substrate. From 100 ml of the culture supernatant, approximately 1 mg of ColH-his was purified by ammonium sulfate precipitation, IMAC, and high-performance liquid chromatography on a MonoQ column. When IMAC was performed on chelating Sepharose charged with Zn(2+) instead of Ni(2+), a potential carcinogenic metal, the specific activities against Pz peptide and type I collagen decreased slightly. However, they were comparable to those reported for other recombinant ColHs and a commercial C. histolyticum collagenase preparation, suggesting that this expression system is useful for large-scale preparation of high-grade clostridial collagenases.     

Clostridium spiroforme toxin is a binary toxin which ADP-ribosylates cellular actin

We have purified from Clostridium spiroforme strain 246 an heterogeneous population of proteins (Sa) ranging from 43 to 47 kilodaltons exhibiting ADP-ribosyl transferase activity as do C. botulinum C2 toxin component I or the ia chain of C. perfringens E iota toxin. C. spiriforme Sa had alone no activity upon injection in mice or inoculated to Vero cells. When spiroforme ADP ribosyl transferase were mixed with a trypsin activated protein (Sb) separated from C. spiroforme bacterial supernatant, a lethal effect in mice and cytotoxicity on Vero cells were recorded. The Sa cross-reacted immunologically with either the light chain of C. perfringens E iota toxin or the ADP-ribosyl transferase from C. difficile 196 strain. No immunological relatedness was observed between Sa and C2 toxin component I. C. spiroforme toxin is thus another binary toxin close to iota.     

Lyophilized Clostridium perfringens 3 alpha- and Clostridium bifermentans 7 alpha-hydroxysteroid dehydrogenases: two new stable enzyme preparations for routine bile acid analysis

Preparations of 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) from Clostridium perfringens were successfully lyophilized into a stable powder form. Purification of the enzyme was achieved using triazine dye affinity chromatography. C. perfringens 3 alpha-hydroxysteroid dehydrogenase was purified 24-fold using Reactive Red 120 (Procion Red) -cross-linked agarose (70% yield). Quantitative measurement of bile acids with the purified enzymes, 3 alpha-hydroxysteroid dehydrogenase and 7 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.159) from Clostridium bifermentans (strain F-6), was achieved spectrophotometrically. Standard curves with chenodeoxycholic acid (CDC) and cholic acid were linear within a concentration range of 20-100 microM. Analysis of mixtures of ursodeoxycholic acid and CDC showed the additive nature of the 3 alpha-hydroxysteroid dehydrogenase and showed also that 7 alpha-hydroxyl groups were independently quantified by the 7 alpha-hydroxysteroid dehydrogenase. Bile acids in Folch extracts of human bile samples were measured using purified preparations of Pseudomonas testosteroni 3 alpha-hydroxysteroid dehydrogenase, C. perfringens 3 alpha-hydroxysteroid dehydrogenase, Escherichia coli 7 alpha-hydroxysteroid dehydrogenase and C. bifermentans (strain F-6) 7 alpha-hydroxysteroid dehydrogenase. Statistical comparison validated the use of C. perfringens 3 alpha- and C. bifermentans 7 alpha-hydroxysteroid dehydrogenases for the quantification of bile acids in bile.     

Response of White Mice to Cells and Culture Constituents of Clostridium perfringens

Broth cultures of Clostridium perfringens (ATCC 10543) were fractionated by ammonium sulfate precipitation and Sephadex G-150 chromatography. Components isolated, as well as some enzymes present in the culture, were assayed for toxicity by feeding to white mice. Early work indicated that when a meat-fat-starch slurry, infected with C. perfringens, was fed to mice, the intestinal passage time was reduced. By using large numbers of mice as test animals and analyzing the data statistically, we found that C. perfringens and several fractions from the culture supernatant significantly affected the mice. A toxic material present in the supernatant was not identifiable as phospholipase C. Phospholipase C and physphorylcholine affected the intestinal passage time of the mice only when large amounts were given. The enzyme, neuraminidase, and another unidentified compound present in the supernatant affected the passage time when very small amounts were fed to mice.     

Purification and characterization of Clostridium perfringens 120-kilodalton collagenase and nucleotide sequence of the corresponding gene.

Clostridium perfringens type C NCIB 10662 produced various gelatinolytic enzymes with molecular masses ranging from approximately 120 to approximately 80 kDa. A 120-kDa gelatinolytic enzyme was present in the largest quantity in the culture supernatant, and this enzyme was purified to homogeneity on the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme was identified as the major collagenase of the organism, and it cleaved typical collagenase substrates such as azocoll, a synthetic substrate (4-phenylazobenzyloxy-carbonyl-Pro-Leu-Gly-Pro-D-Arg [Pz peptide]), and a type I collagen fibril. In addition, a gene (colA) encoding a 120-kDa collagenase was cloned in Escherichia coli. Nested deletions were used to define the coding region of colA, and this region was sequenced; from the nucleotide sequence, this gene encodes a protein of 1,104 amino acids (M(r), 125,966). Furthermore, from the N-terminal amino acid sequence of the purified enzyme which was found in this reading frame, the molecular mass of the mature enzyme was calculated to be 116,339 Da. Analysis of the primary structure of the gene product showed that the enzyme was produced with a stretch of 86 amino acids containing a putative signal sequence. Within this stretch was found PLGP, the amino acid sequence constituting the Pz peptide. This sequence may be implicated in self-processing of the collagenase. A consensus zinc-binding sequence (HEXXH) suggested for vertebrate Zn collagenases is present in this bacterial collagenase. Vibrio alginolyticus collagenase and Achromobacter lyticus protease I showed significant homology with the 120-kDa collagenase of C. perfringens, suggesting that these three enzymes are evolutionarily related.     

Clostridium perfringens Type A Infection of Ligated Intestinal Loops in Lambs

Clostridium perfringens type A suspended in fresh medium was injected into ligated intestinal loops of lambs. Within 7 hr after inoculation, the fluid volume of the loops increased up to seven times. No significant accumulation of fluid occurred in loops receiving grown culture, culture supernatant fluid, or medium alone. alpha-Antitoxin injected along with C. perfringens in fresh medium into intestinal loops did not prevent the accumulation of fluid. It is concluded that alpha-toxin plays no major role in C. perfringens type A enteritis.     

Genomic map of Clostridium perfringens strain 13

A physical and genetic map of Clostridium perfringens strain 13 was constructed. C. perfringens strain 13 was found to have a 3.1-Mb chromosome and a large 50-kb plasmid, indicating that strain 13 has a relatively small genome among C. perfringens strains. A total of 313 genetic markers were mapped on the chromosome of strain 13. Compared with the physical and genetic map of C. perfringens CPN50, strain 13 had a quite similar genome organization, but with a large deletion (approximately 400 kb) in a particular segment of the chromosome. Among several toxin genes, a beta2 toxin gene that is a novel virulence gene in C. perfringens was found to be located on the 50-kb plasmid.     

Genes encoding homologues of three consecutive enzymes in the butyrate/butanol-producing pathway of Clostridium acetobutylicum are clustered on the Clostridium difficile chromosome

Abstract Screening of a Clostridium difficile ψEMBL3 gene library with antisera raised against C. difficile culture supernatant identified several clones expressing a 31-kDa protein. A 1.8-kb Hin dIII fragment subcloned from one of the clones was sufficient for expression of the 31-kDa polypeptide. Southern blot analysis showed a region homologous to this fragment to be present in all of 13 different C. difficile strains tested. Sequence analysis of the 1.8-kb fragment revealed three adjacent open reading frames. A database search showed that these three open reading frames appeared to encode homologues of three consecutive enzymes in the butanol/butyrate-producing pathway of Clostridium acetobutylicum (crotonase, β-hydroxybutyryl coenzyme A dehydrogenase and thiolase).     

Partial characterization of a 36-kDa protein from Clostridium botulinum type E that inhibits trypsin and chymotrypsin

A 36-kDa trypsin inhibitor was purified from Clostridium botulinum type E culture supernatant by multiple molecular sieve and ion exchange chromatographic steps. The sequence of the amino-terminal 13 amino acid residues of this single-chain protein is Asn.Gln.Glu.Val.Phe.Asn.Met.Pro.Lys.Phe.Ser.Thr.Ala-. This novel protein that also inhibits chymotrypsin is produced by an organism that does not appear to produce any protease.     

Purification and characterisation of a serine peptidase from the marine psychrophile strain PA-43

An extracellular serine peptidase, purified from the culture supernatant of the sub-Arctic psychrophilic bacterium strain PA-43, is monomeric, with a relative molecular mass of 76000, and an unusually low pI of 3.8. The peptidase is active towards N-succinyl AAPF p-nitroanilide and N-succinyl AAPL p-nitroanilide, indicating a chymotrypsin-like substrate specificity. It is inhibited by the serine peptidase inactivator phenylmethylsulfonyl fluoride, but not by EDTA or EGTA, suggesting that added metal ions are not necessary for activity. The enzyme is most active at pH 8.3 and at 55-60 degrees C, although it is unstable at 60 degrees C. It is nevertheless remarkably stable for an enzyme from a psychrophilic microorganism, remaining active after 1 week at 20 degrees C and after five freeze-thaw cycles. Comparison of the N-terminal 40 amino acid residues with other archived sequences revealed highest similarity to the alkaline serine protease (aprx) from Bacillus subtilis.     

Procedures for large-scale production and purification of Clostridium botulinum C1 toxin for preparation of toxoid

Abstract Fortified cooked meat medium containing calcium carbonate (CaCO₃-FCM) supported toxin production of a strain of Clostridium botulinum type C to a level of 2 × 10⁶ mouse i.p. LD₅₀/ml. C1 toxin was purified by sequential steps of acid precipitation from 5-fold diluted culture supernatant in the presence of RNA, 2nd acid precipitation by dialysis, removal of RNA by protamine treatment, removal of excess protamine and bufferisation by ultrafiltration through Amicon PM-30 membrane, sulphopropyl-Sephadex chromatography, and Sephadex G-200 gel filtration. By these procedures, 25 mg or more of highly purified C1 toxin was constantly obtained from a lot of 600-ml culture.     

Complex transcriptional regulation of citrate metabolism in Clostridium perfringens

A Gram-positive, spore-forming bacterium, Clostridium perfringens, possesses genes for citrate metabolism, which might play an important role in the utilization of citrate as a sole carbon source. In this study, we identified a chromosomal citCDEFX-mae-citS operon in C. perfringens strain 13, which is transcribed on three mRNAs of different sizes. Expression of the cit operon was significantly induced when 5 mM extracellular citrate was added to the growth medium. Most interestingly, three regulatory systems were found to be involved in the regulation of the expression of cit genes: 1) the two upstream divergent genes citG and citI; 2) two different two-component regulatory systems, CitA/CitB (TCS6 consisted of CPE0531/CPE0532) and TCS5 (CPE0518/CPE0519); and 3) the global two-component VirR/VirS-VR-RNA regulatory system known to regulate various genes for toxins and degradative enzymes. Our results suggest that in C. perfringens the citrate metabolism might be strictly controlled by a complex regulatory system.     

EtfA catalyses the formation of dipicolinic acid in Clostridium perfringens

Dipicolinic acid (DPA) is a major component of bacterial endospores, comprising 5–15% of the spore dry weight, and is important for spore stability and resistance properties. The biosynthetic precursor to DPA, dihydro-dipicolinic acid (DHDPA), is produced by DHDPA synthase within the lysine biosynthesis pathway. In Bacillus subtilis , and most other bacilli and clostridia, DHDPA is oxidized to DPA by the products of the spoVF operon. Analysis of the genomes of the clostridia in Cluster I, including the pathogens Clostridium perfringens , Clostridium botulinum and Clostridium tetani , has shown that no spoVF orthologues exist in these organisms. DPA synthase was purified from extracts of sporulating C. perfringens cells. Peptide sequencing identified an electron transfer flavoprotein, EtfA, in this purified protein fraction. A C. perfringens strain with etfA inactivated is blocked in late stage sporulation and produces ≤ 11% of wild-type DPA levels. C. perfringens EtfA was expressed in and purified from Escherichia coli , and this protein catalysed DPA formation in vitro . The sequential production of DHDPA and DPA in C. perfringens appears to be catalysed by DHDPA synthase followed by EtfA. Genome sequence data and the taxonomy of spore-forming species suggest that this may be the ancestral mechanism for DPA synthesis.     

酪酸梭菌与婴儿型双歧杆菌对产气荚膜梭菌试管内生物拮抗作用的研究

用酪酸权菌（Clostridium butyricum）和婴儿型双歧杆菌（Bifidobacterium Infantis）对产气荚膜梭菌（Clostriduim perfringens）进行试管内的生物拮抗试验。将酪酸梭菌、婴儿型双歧杆菌及酪酸梭菌和婴儿型双歧杆菌分别对产气荚膜酸菌以一定的比例等量混合接种于GAM液体培养基中进行厌氧培养。实验证明酪酸梭菌和婴儿型双歧能明显抑制产气荚膜梭菌的生长,并且比各自单独培养时显示了较强的生物拮抗作用。