Localization of germination-specific spore-lytic enzymes in Clostridium perfringens S40 spores detected by immunoelectron microscopy

The localization of germination-specific spore-lytic enzymes, an amidase and a muramidase, in Clostridium perfringens S40 spores was examined by immunoelectron microscopy with respective antisera raised against the enzymes and a colloidal gold-immunoglobulin G complex. For both antisera, immunogold particles were visualized on the outside of the cortex of dormant spores, and they were not detected in germinated spores and decoated spores.     

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.     

Mode of action of a germination-specific cortex-lytic enzyme, SleC, of Clostridium perfringens S40

The hydrolysis of the bacterial spore peptidoglycan (cortex) is a crucial event in spore germination. It has been suggested that SleC and SleM, which are conserved among clostridia, are to be considered putative cortex-lytic enzymes in Clostridium perfringens. However, little is known about the details of the hydrolytic process by these enzymes during germination, except that SleM functions as a muramidase. Muropeptides derived from SleC-digested decoated spores of a Bacillus subtilis mutant that lacks the enzymes, SleB, YaaH and CwlJ, related to cortex hydrolysis were identified by amino acid analysis and mass spectrometry. The results suggest that SleC is most likely a bifunctional enzyme possessing lytic transglycosylase activity and N-acetylmuramoyl-L-alanine amidase activity confined to cross-linked tetrapeptide-tetrapeptide moieties of the cortex structure. Furthermore, it appears that during germination of Clostridium perfringens spores, SleC causes merely small and local changes in the cortex structure, which are necessary before SleM can function.     

Phylogenetic analysis of the pathogenic anaerobe Clostridium perfringens using the 16S rRNA nucleotide sequence.

Clostridium perfringens, the first pathogenic clostridium examined, was placed in the nonmycoplasma subgroup of the low-dG+dC-content gram-positive cluster on the basis of the results of a phylogenetic analysis in which we used 16S rRNA comparisons. The closest relative that has been identified to date is Clostridium pasteurianum.     

Extraction of Clostridium perfringens spores from bottom sediment samples.

Two extraction-separation procedures were developed and evaluated for use in conjunction with the mCP membrane filter method for the enumeration of Clostridium perfringens spores in bottom sediments. In the more facile of the two procedures, a distilled-water suspension of the sediment sample is pulse sonicated for 10 s and allowed to settle. Portions of the supernatant are then removed for membrane filtration. This procedure is recommended for general use. The more complicated procedure is recommended for situations in which the presence of high levels of toxic materials is suspected or in which relatively low spore densities are present in fine silts. In this procedure, sonication is followed by a distilled water wash. The centrifuged sediment is resuspended in distilled water and mixed with the components of a two-phase separation system (50% polyethylene glycol in distilled water and 25% sucrose in 3 M phosphate buffer [pH 7.1]). After equilibration of the system and low-speed centrifugation, the top phase and interphase are removed, mixed, and membrane filtered. The recoveries of C. perfringens spores by the two procedures, when used in conjunction with the mCP method, were comparable to each other and significantly greater than those by the British most-probable-number method. It was estimated that more than 85% of the spores were recovered by the procedures. The precision of the sonicate-and-settle-mCP procedure was markedly better than that obtained theoretically by the most-probable-number method and approached that theoretically attributable to counting an average of 85 colonies on each of two plates.     

Cloning and characterization of a conjugated bile acid hydrolase gene from Clostridium perfringens.

The gene encoding a conjugated bile acid hydrolase (CBAH) from Clostridium perfringens 13 has been cloned and expressed in Escherichia coli, and its nucleotide sequence has been determined. Nucleotide and predicted amino acid sequence analyses indicated that the gene product is related to two previously characterized amidases, a CBAH from Lactobacillus plantarum (40% identity) and a penicillin V amidase from Bacillus sphaericus (34% identity). The product is apparently unrelated to a CBAH from C. perfringens for which N-terminal sequence information was determined. The gene product was purified from recombinant E. coli and used to raise antibody in rabbits. The presence of the protein in C. perfringens was then confirmed by immunoblot analysis. The protein was shown to have a native molecular weight of 147,000 and a subunit molecular weight of 36,100, indicating its probable existence as a tetramer. Disruption of the chromosomal C. perfringens CBAH gene with a chloramphenicol resistance cartridge resulted in a mutant strain which retained partial CBAH activity. Polyacrylamide gel electrophoresis followed by enzymatic activity staining and immunoblotting indicated that the mutant strain no longer expressed the cloned CBAH (CBAH-1) but did express at least one additional CBAH (CBAH-2). CBAH-2 was immunologically distinct from CBAH-1, and its mobility on native polyacrylamide gels was different from that of CBAH-1. Furthermore, comparisons of pH optima and substrate specificities of CBAH activities from recombinant E. coli and wild-type and mutant C. perfringens provided further evidence for the presence of multiple CBAH activities in C. perfringens.     

Spore lytic enzyme released from Clostridium perfringens spores during germination.

The exudate of fully germinated spores of Clostridium perfringens was found to contain a large amount of a spore lytic enzyme which acted directly on alkali-treated spores of the organism to cause germination. Although no detectable amount of the enzyme was found in dormant spores during germination in a KCl medium, the enzyme was produced rapidly and released into the medium. The optimal conditions for enzyme activity were pH 6.0 and 45 degrees C. Maximum activity occurred in the presence of various univalent cations at a concentration of 50 mM. The enzyme was readily inactivated by several sulfhydryl reagents. A strong reducing condition was generated in the ionic germination of the spores, a minimum Eh level of -350 mV being reached 30 min after initiation of germination. Furthermore, adenosine triphosphate-dependent pyruvate:ferredoxin oxidoreductase (EC 1.2.7.1) was identified in both dorman and germinated spores. The relationship between the release of active enzyme and the generation of reducing conditions during germination is discussed.     

Germination-specific cortex-lytic enzymes from Clostridium perfringens S40 spores: time of synthesis, precursor structure and regulation of enzymatic activity

Germination-specific enzymes, an amidase and a muramidase, of Clostridium perfringens S40 were synthesized at the time of forespore formation during sporulation. The amidase had a unique precursor structure consisting of four domains: the N-terminal pre-sequence, the N-terminal pro-sequence, mature enzyme and the C-terminal pro-sequence. The N-terminal pre-sequence and the C-terminal pro-sequence were sequentially processed at the time of development of phase-bright spores, and the resulting inactive pro-enzyme was activated by cleavage of the N-terminal pro-sequence with a specific protease during germination. A possible mechanism for the regulation of activity of muramidase, which is produced as a mature form and does not need processing for activation, is presented.     

Purification and characterization of neuraminidase from Clostridium perfringens.

Clostridium perfringens cells were cultivated on a large scale using an automatic system. Neuraminidase secreted by the cells into the culture medium was purified 380 000-fold by: precipitation with ammonium sulfate between 50 and 85% saturation, filtration on Sephadex G-75, electrophoresis on polyacrylamide gel, and by isoelectric focusing. Three enzyme fractions with different migration rates were obtained by preparative disc electrophoresis in polyacrylamide gel, and five fractions with isoelectric points between pH 4.7 and 5.4 were observed after isoelectric focusing. This microheterogeneity disappeared after denaturation of the enzyme in 0.1% sodium dodecylsulfate or 8M urea. The isoelectric point of the denatured enzyme corresponded to pH 4.3. All enzyme fractions were identical with regard to their immunological and kinetic properties; they had the same molecular weights. The origin of the different "conformers" of neuraminidase is discussed. The existence of genuine isoenzymes could largely be excluded. The yield of neuraminidase was 65%, which corresponded to about 10 mg of pure enzyme from 100 l of culture medium. The enzyme was free of protease and various other glycosidase activities. The neuraminidase preparation appeared not to be contaminated by other proteins as judged by electrophoretic analysis using either the native enzyme or the enzyme denatured by sodium dodecylsulfate or urea; ultracentrifugation; chromatography on Sephadex G-200; and immunological methods. The molecular weights of the native or denatured enzyme were found to be in the range between 60 000 and 69 000 (on an average 63 750) using four independent methods. The existence of subunits of neuraminidase was excluded. The neuraminidase exhibited a spec. act. of 580 or 615 U/mg protein with glycopeptides from edible birds' nests or sialyllactose, respectively, as substrates. Additional kinetic properties and the UV-absorption spectrum of the enzyme are described.     

Molecular cloning and nucleotide sequence of the gene encoding the major peptidoglycan hydrolase of Lactococcus lactis, a muramidase needed for cell separation.

A gene of Lactococcus lactis subsp. cremoris MG1363 encoding a peptidoglycan hydrolase was identified in a genomic library of the strain in pUC19 by screening Escherichia coli transformants for cell wall lysis activity on a medium containing autoclaved, lyophilized Micrococcus lysodeikticus cells. In cell extracts of L. lactis MG1363 and several halo-producing E. coli transformants, lytic bands of similar sizes were identified by denaturing sodium dodecyl sulfate (SDS)-polyacrylamide gels containing L. lactis or M. lysodeikticus cell walls. Of these clearing bands, corresponding to the presence of lytic enzymes with sizes of 46 and 41 kDa, the 41-kDa band was also present in the supernatant of an L. lactis culture. Deletion analysis of one of the recombinant plasmids showed that the information specifying lytic activity was contained within a 2,428-bp EcoRV-Sau3A fragment. Sequencing of part of this fragment revealed a gene (acmA) that could encode a polypeptide of 437 amino acid residues. The calculated molecular mass of AcmA (46,564 Da) corresponded to that of one of the lytic activities detected. Presumably, the enzyme is synthesized as a precursor protein which is processed by cleavage after the Ala at position 57, thus producing a mature protein with a size of 40,264 Da, which would correspond to the size of the enzyme whose lytic activity was present in culture supernatants of L. lactis. The N-terminal region of the mature protein showed 60% identity with the N-terminal region of the mature muramidase-2 of Enterococcus hirae and the autolysin of Streptococcus faecalis. Like the latter two enzymes, AcmA contains C-terminal repeated regions. In AcmA, these three repeats are separated by nonhomologous intervening sequences highly enriched in serine, threonine, and asparagine. Genes specifying identical activities were detected in various strains of L. lactis subsp. lactis and L. lactis subsp. cremoris by the SDS-polyacrylamide gel electrophoresis detection assay and PCR experiments. By replacement recombination, an acmA deletion mutant which grew as long chains was constructed, indicating that AcmA is required for cell separation.     

Activation and injury of Clostridium perfringens spores by alcohols.

The activation properties of Clostridium perfringens NCTC 8679 spores were demonstrated by increases in CFU after heating in water or aqueous alcohols. The temperature range for maximum activation, which was 70 to 80 degrees C in water, was lowered by the addition of alcohols. The response at a given temperature was dependent on the time of exposure and the alcohol concentration. The monohydric alcohols and some, but not all, of the polyhydric alcohols could activate spores at 37 degrees C. The concentration of a monohydric alcohol that produced optimal spore activation was inversely related to its lipophilic character. Spore injury, which was manifested as a dependence on lysozyme for germination and colony formation, occurred under some conditions of alcohol treatment that exceeded those for optimal spore activation. Treatment with aqueous solutions of monohydric alcohols effectively activated C. perfringens spores and suggests a hydrophobic site for spore activation.     

Molecular cloning and nucleotide sequence of the gramicidin S synthetase 1 gene

The entire gene for gramicidin S synthetase 1 (GS 1) was cloned into the plasmid vector pUC18, and the nucleotide sequences of the GS 1 gene and its flanking region were determined. The full-length clone was 4,539 base pairs long and had an open reading frame of 3,294 nucleotides coding for 1,098 amino acids. The calculated molecular weight of 123,474 agreed with the apparent molecular weight of 120,000 found in SDS-PAGE of GS 1 from B. brevis. The nucleotide sequence of GS 1 gene was highly homologous to that of tyrocidine synthetase 1. The overall similarity between the deduced amino acid sequences of the two genes was 57.5%. The gene product of clone GS309 was easily purified to an essentially homogeneous state by ammonium sulfate fractionation followed by DEAE-Sepharose CL-6B, Ultrogel AcA-34, and second DEAE-Sepharose CL-6B column chromatography. The purified protein catalyzed the D-phenylalanine-dependent ATP-32PPi exchange reaction which is specific for GS 1 activity, and the specific activity of the purified product was nearly the same as the purified GS 1 from B. brevis. The product also showed a weak phenylalanine racemase activity.     

Purification and characterization of N-acetylneuraminate lyase from Clostridium perfringens.

Clostridium perfringens cells were cultivated on a large scale using an automatic system. 2) N-Acetylneuraminate lyase, which is a cytosolic enzyme, was liberated from the bacteria by cell lysis using lysozyme in hypotonic solution. The enzyme was purified 770-fold by precepitation with ammonium sulfate, filtration on Sephadex A-50 and final preparative electrophoresis in a 7.5% polyacrylamide gel. Yield: 12 mg from 1 kg wet cell paste; specific activity: 167 nkat/mg protein. 3) The enzyme preparation appeared homogeneous in analytical disc electrophoresis, in gel electrophroesis in 0.1% sodium dodecylsulfate or 8m urea and in immunoelectrophoresis. Contaminating enzyme activities were not detected. 4) The isoelectric point of pH 4.7 was found for the enzyme. At 278 nm a molar extinction coefficient of 6.4 x 10(4)M-1 Xcm-1 was determined. The enzyme exhibited a Km value for N-acetylneuraminic acid of 2.8mM at its pH optimum of pH 7.2. The pH dependence of the Km value gives evidence that an ionizing guoup in the active center of the enzyme with a pKe value of 6.4 may be involved in the catalytic reaction. Pyruvate inhibited the cleavage reaction of N-acetylneuraminic acid competitively; Ki = 2.9mM. 5) An average molecular weight of 99200 was determined for the native enzyme using different methods. After denaturation in sokium dodecylsulfate or urea, a mean molecular weight of only 50000 could be demonstrated, indicating the existence of two enzyme subunits. The lyase molecule was shown by electron microscopy, using a negative staining technique, to consist of two hemispherical parts. 6) Two active sites per native enzyme molecule, probably corresponding to one active site per subunit, were found by incubation of the enzyme with radioactive pyruvate followed by borohydride reduction. The results obtained from chemical modification of the lyase with 5-diazonium-1H-tetrazole and iodocaetamide under various conditionsare interpreted as evidence for the presence of two reactive histidine residues in the enzyme molecule. It is probable that one residue per subunit forms the nucleophilic group participating in enzyme catalysis. A model suggesting the mechanism of reversible cleavage of N-acylneuraminic acids by the lyase is presented.     

Purification and characterization of a recombinant alpha-N-acetylgalactosaminidase from Clostridium perfringens

Clostridium perfringens alpha-N-acetylgalactosaminidase (alphaNAG) hydrolyzed the terminal N-acetyl-alpha-d-galactosamine from the blood type A(2) antigen producing H antigen, blood type O. Blood type O is universally compatible in the ABO system. Purification of the native enzyme is difficult with very low yields. To obtain the enzyme in satisfactory yield, the gene encoding the clostridial enzyme was cloned in an Escherichia coli T7 expression system. A highly purified preparation of recombinant alphaNAG was obtained from cell lysates by ion-exchange chromatography and high-pressure liquid chromatography. The final preparation was homogeneous by SDS-PAGE with a molecular mass of 71.96kDa and the native molecular weight of 72.42kDa. The enzyme was highly selective for terminal N-acetylgalactosamine residues. No other significant exoglycosidase activities, particularly neuraminidase, were detected. The pH optimum of the enzyme was between 6.5 and 7.0 and activity was relatively unaffected by ionic strength. ELISA experiments demonstrated activity against blood type A(2) epitope. These characteristics were similar to those of native alphaNAG from C. perfringens. With adequate expression in E. coli, sufficient recombinant alphaNAG enzyme mass can be obtained for potential use in enzymatic conversion of human blood type A(2) red blood cells to universally transfusable type O red blood cells.     

Reversal of radiation-dependent heat sensitization of Clostridium perfringens spores.

The effect of solute concentration on the sensitization of Clostridium perfringens spores to heat by ionizing radiation was investigated. As we have shown previously, spores of C. perfringens treated with gamma radiation are now sensitive to subsequent heat treatments than are spores that receive no radiation treatment. When gamma-irradiated spores were heated in the presence of increasing concentrations of glycerol or sucrose, the heat sensitivity induced by irradiation was progressively decreased. The magnitude of the increase in heat resistance induced by extracellular solutes was greater in gamma-irradiated spores than in nonirradiated spores. Based on these observations, it is proposed that the induction of heat sensitivity in spores by radiation is related to the loss of osmoregulatory or dehydrating mechanisms in irradiated spores.     

Molecular cloning and nucleotide sequence of the streptavidin gene.

Using synthetic oligonucleotides as probes we have cloned the streptavidin gene from a genomic library of Streptomyces avidinii. Nucleotide sequence analysis indicated that a 2 Kb DNA-fragment contained the entire coding region, a signal peptide region and the 3' and 5' flanking regions of the gene. The deduced amino acid sequence shows several interrupted blocks of homology with the amino acid sequence of chicken egg-white avidin. Analysis of the secondary structure suggests a high content of beta-structure in both proteins and considerable overall structural similarity between them.     

Complete nucleotide sequence and genetic organization of the bacteriocinogenic plasmid, pIP404, from Clostridium perfringens

The complete nucleotide sequence of the bacteriocinogenic plasmid, pIP404, from Clostridium perfringens has been determined. The plasmid genome comprises 10,207 bp and has a dA + dT content of 75%. Functions have been tentatively assigned to 6 of the 10 open reading frames and an origin-like region of repeated sequence identified. The codon usage of this extremely dA + dT rich plasmid is highly unusual and displays a pronounced preference for codons with the lowest dG + dC content. Only one of the genes from pIP404 was expressed at a significant level in Escherichia coli, suggesting that the atypical codon usage could represent a major obstacle to heterologous gene expression.     

A germination-specific spore cortex-lytic enzyme from Bacillus cereus spores: cloning and sequencing of the gene and molecular characterization of the enzyme.

A gene (sleB) encoding a 24-kDa germination-specific spore cortex-lytic enzyme, probably an N-acetylmuramyl-L-alanine amidase, was cloned from Bacillus cereus, and its nucleotide sequence was determined. It was indicated that the enzyme is produced as a 259-residue protein with a signal sequence of 32 residues and is present in dormant spores in its active form. Sulfhydryl reagents inactivated the enzyme, but mutation of a single cysteine of the protein, Cys-258, to Gly did not cause complete inactivation of the enzyme, suggesting that the residue does not function as the catalytic center of enzyme.