The gene encoding Clostridium sordellii phospholipase C (Csp) was cloned and expressed as a histidine-tagged (His-tag) protein, and the protein was purified to compare its enzymatic and biological activities with those of Clostridium perfringens phospholipase C (Cpa) and Clostridium bifermentans phospholipase C (Cbp). Csp was found to consist of 371 amino acid residues in the mature form and to be more homologous to Cbp than to Cpa. The egg yolk phospholipid hydrolysis activity of the His-tag Csp was about one-third of that of His-tag Cpa, but the hemolytic activity was less than 1% of that of His-tag Cpa. His-tag Csp was nontoxic to mice. Immunization of mice with His-tag Cbp or His-tag Csp did not provide effective protection against the lethal activity of His-tag Cpa. These results indicate that Csp possesses similar molecular properties to Cbp and suggest that comparative analysis of toxic and nontoxic clostridial phospholipases is helpful for characterization of the toxic properties of clostridial phospholipases.

An enzyme mediating the reductive dechlorination of tetrachloroethylene (PCE) from cell-free extracts of Clostridium bifermentans DPH-1 was purified, cloned, and sequenced. The enzyme catalyzed the reductive dechlorination of PCE to cis-1,2-dichloroethylene via trichloroethylene, at a Vmax and Km of 73 nmol/mg protein and 12 microM, respectively. Maximal activity was recorded at 35 degrees C and pH 7.5. Enzymatic activity was independent of metal ions but was oxygen sensitive. A mixture of propyl iodide and titanium citrate caused a light-reversible inhibition of enzymatic activity suggesting the involvement of a corrinoid cofactor. The molecular mass of the native enzyme was estimated to be approximately 70 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) revealed molecular masses of approximately 35 kDa and 35.7 kDa, respectively. A broad spectrum of chlorinated aliphatic compounds (PCE, trichloroethylene, cis-1,2-dichloroethylene, trans-1,2-dichloroethylene, 1,1-dichloroethylene, 1,2-dichloropropane, and 1,1,2-trichloroethane) was degraded. With degenerate primers designed from the N-terminal sequence (27 amino acid residues), a partial sequence (81 bp) of the encoding gene was amplified by polymerase chain reaction (PCR) and sequenced. Southern analysis of C. bifermentans genomic DNA using the PCR product as a probe revealed restriction fragment bands. A 5.0 kb ClaI fragment, harboring the relevant gene (designated pceC) was cloned (pDEHAL5) and the complete nucleotide sequence of pceC was determined. The gene showed homology mainly with microbial membrane proteins and no homology with any known dehalogenase, suggesting a distinct PCE dehalogenase.

Conservation of the sporulation processes between Bacillus spp. and Clostridium spp. was investigated through evolutionary and complementation analyses of sigma(E). Alignment of partial predicted sigma(E) amino acid sequences from three Bacillus spp., Paenibacillus polymyxa and five Clostridium spp. revealed that amino acid residues previously reported to be involved in promoter utilization (M124, E119 and N120) and strand opening (C117) are conserved among all these species. Phylogenetic analyses of various sigma factor sequences from endospore-forming bacteria revealed that homologues of sigma(E), sigma(K) and sigma(G) clustered together regardless of genus, suggesting a common origin of sporulation sigma factors. The functional equivalence between Clostridium acetobutylicum sigma(E) and Bacillus subtilis sigma(E) was investigated by complementing a non-polar B. subtilis sigma(E) null mutant with the spoIIG operon from either B. subtilis (spoIIG(Bs)) or C. acetobutylicum (spoIIG(Ca)). Single-copy integration of spoIIG(Bs) into the amyE locus of the sigma(E) null mutant completely restored the wild-type sporulation phenotype, while spoIIG(Ca) only partially restored sporulation. Maximal expression of spoIIG(Ca)-lacZ occurred approximately 12 h later than maximal expression of spoIIG(Bs)-lacZ. Differences in temporal expression patterns for spoIIG(Ca) and spoIIG(Bs) in the B. subtilis background may at least partially explain the observed sporulation complementation phenotypes. This study suggests a common phylogenetic ancestor for sigma(E) in Bacillus spp. and Clostridium spp., although regulation of sigma(E) expression may differ in these two genera.

A range of clostridial species produce phospholipases C. The zinc metallo phospholipases C have related sequences but different properties. All of these enzymes may be arranged, like alpha-toxin as two-domain proteins. Differences in enzymatic, haemolytic and toxic properties may be explained by differences in amino acids at key positions.

The phospholipases C of C. perfringens (alpha-toxin) and C. bifermentans (Cbp) show >50% amino acid homology but differ in their hemolytic and toxic properties. We report here the purification and characterisation of alpha-toxin and Cbp. The phospholipase C activity of alpha-toxin and Cbp was similar when tested with phosphatidylcholine in egg yolk or in liposomes. However, the hemolytic activity of alpha-toxin was more than 100-fold that of Cbp. To investigate whether differences in the carboxy-terminal domains of these proteins were responsible for differences in the hemolytic and toxic properties, a hybrid protein (NbiCalpha) was constructed comprising the N domain of Cbp and the C domain of alpha-toxin. The hemolytic activity of NbiCalpha was 10-fold that of Cbp, and the hybrid enzyme was toxic. These results confirm that the C-terminal domain of these proteins confers different properties on the enzymatically active N-terminal domain of these proteins.

Clostridium bifermentans spores contain two major small, acid-soluble, proteins (SASP) termed SASP-alpha and beta. The amino acid sequences of SASP-alpha and beta are almost identical, and are very similar to those of alpha/beta-type SASP from spores of C. perfringens and various Bacillus species. However, the C. bifermentans proteins contain an extra five amino acids in the middle of their sequence. Surprisingly, no gamma-type SASP were found in C. bifermentans or C. perfringens spores, although these are the most prominent SASP in spores of Bacillus species.

The phospholipase C gene from Clostridium perfringens was isolated, and its sequence was determined. It was found that the structural gene codes for a protein of 399 amino acid residues. The NH2-terminal residues have the typical features of a signal peptide and are probably cleaved after secretion. Escherichia coli cells harboring the phospholipase C gene-containing plasmid expressed high levels of this protein in the periplasmic space. Phospholipase C purified from E. coli transformants was enzymatically active, hemolytic to erythrocytes, and toxic to animals when injected intravenously. The phospholipase C gene from a related organism, Clostridium bifermentans, was also isolated. The two phospholipase C genes were found to be 64% homologous in coding sequence. The C. bifermentans protein, however, was 50-fold less active enzymatically than the C. perfringens enzyme.

A gene (cbm71) encoding a 71,128-Da mosquitocidal protein (Cbm71) was obtained by screening a size-fractionated XbaI digest of total genomic DNA from Clostridium bifermentans subsp. malaysia CH18 with two gene-specific oligonucleotide probes. The sequence of the Cbm71 protein, as deduced from the sequence of cbm71, corresponds to that of the 66-kDa protein previously described as one of the mosquitocidal components of C. bifermentans subsp. malaysia. Cbm71 shows limited similarities with Bacillus thuringiensis delta-endotoxins, especially in the four first conserved blocks. However, Cbm71 was not immunologically related to any of the Cry toxins and thus belongs to a novel class of mosquitocidal protein. The cbm71 gene was expressed in a nontoxic strain of B. thuringiensis, and Cbm71 was produced during sporulation and secreted to the supernatant of culture. Trichloroacetic-precipitated supernatant preparations were toxic for mosquito larvae of the species Aedes aegypti, Culex pipiens, and Anopheles stephensi.

Three new open reading frames were found downstream from cbm71, a toxin gene from Clostridium bifermentans malaysia (Cbm) strain CH18. The first one (91bp downstream) called cbm72, is 1857bp long and encodes a 71727-Da protein (Cbm72) with a sequence similar to that of Bacillus thuringiensis delta-endotoxins. This protein shows no significant toxicity to mosquito larvae. The two others, cbm17.1 (462bp) and cbm17.2 (459bp), are copies of the same gene encoding Cbm P18 and P16 polypeptides and located 426bp and 1022bp downstream from cbm72, respectively. They encode 17189-Da and 17451-Da proteins with sequences 44.6% similar to that of Aspergillus fumigatus hemolysin; however, they were not hemolytic in the conditions tested.