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

Abstract

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.

FIG. 1

SDS gel electrophoretic patterns of the purified active enzyme fraction (A) as well as the prepeptide-proSCLE complex and α-, β-, and κ-caseins treated or not treated with GSP (B). (A) An aliquot (1 ml) of the main peak fraction containing GSP activity which eluted from a DEAE-5PW column was dialyzed against H₂O, dried by using of rotary evaporator, dissolved in 40 μl of 10 mM Tris-HCl (pH 8.0) containing 1% SDS and 1% 2-mercaptoethanol, and electrophoresed on a 0.1% SDS–10% polyacrylamide gel. (B) The prepeptide-proSCLE complex (lanes 1 and 2), α-casein (lanes 3 and 4), β-casein (lanes 5 and 6), and κ-casein (lanes 7 and 8) (5 to 10 μg each) were incubated with (odd-numbered lanes) or without (even-numbered lanes) GSP (1.5 U) in 40 mM potassium phosphate (pH 7.0) at 30°C for 4 h and electrophoresed on a 0.1% SDS–13.3% polyacrylamide gel. In lanes 1 and 2, 35- and 18-kDa bands are proSCLE and prepeptide, respectively. The standard proteins run were rabbit muscle phosphorylase b (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), bovine carbonic anhydrase (31 kDa), soybean trypsin inhibitor (22 kDa), and lysozyme (14 kDa).

FIG. 2

Physical map of a csp gene cluster of C. perfringens. The ORFs and direction of transcription are indicated by the arrows. The solid lines below the ORF arrows represent the inserts of recombinant plasmids pCP22H, from which the sleC probe was isolated, and pCP70E, from which the nucleotide sequence was derived. Restriction sites are indicated as A (AccI), E (EcoRI), Hc (HincII), Hd (HindIII), Nc (NcoI), Nd (NdeI), P (PvuII), and S (SacI). FPCR is a fragment of DNA cloned by reverse PCR. Sequence data for the HindIII-HindIII region marked by asterisks are given in Fig. 3.

FIG. 3

Nucleotide sequence and deduced amino acid sequence of a 4,648-bp HindIII-HindIII fragment containing cspA, cspB, and partial cspC genes. The deduced amino acid sequences of cspA (nucleotides 171 to 1,907), cspB (nucleotides 1,923 to 3,620), and part of cspC (nucleotides 3,623 to 4,648) are given below the nucleotide sequences (See reference for the 3′-end sequence of cspC and the whole sequence of sleC). The numbers for the nucleotides and amino acids are shown on the right. An inverted repeat is indicated by arrows. Putative ribosome binding sites (rbs) with moderate complementarity to the 3′ end of the 16S rRNA of C. perfringens (11) are underlined. The asterisks indicate termination codons. The amino acid sequences of CspA, CspB, and CspC which agreed with the N-terminal sequences of GSP-57, GSP-52, and GSP-60, respectively, are underlined in bold.

FIG. 3

Nucleotide sequence and deduced amino acid sequence of a 4,648-bp HindIII-HindIII fragment containing cspA, cspB, and partial cspC genes. The deduced amino acid sequences of cspA (nucleotides 171 to 1,907), cspB (nucleotides 1,923 to 3,620), and part of cspC (nucleotides 3,623 to 4,648) are given below the nucleotide sequences (See reference for the 3′-end sequence of cspC and the whole sequence of sleC). The numbers for the nucleotides and amino acids are shown on the right. An inverted repeat is indicated by arrows. Putative ribosome binding sites (rbs) with moderate complementarity to the 3′ end of the 16S rRNA of C. perfringens (11) are underlined. The asterisks indicate termination codons. The amino acid sequences of CspA, CspB, and CspC which agreed with the N-terminal sequences of GSP-57, GSP-52, and GSP-60, respectively, are underlined in bold.

FIG. 4

Sequence alignment (A) and dendrogram analysis (B) of Csp proteins and several subtilisin-like serine proteases. (A) Only the sequences around three canonical residues (asterisks) and an oxyanion binding site (number sign) of the proteases are aligned. Identical residues in more than five proteins are indicated by black boxes. Bold underlining indicates the N terminus of mature or putative mature enzymes. Amino acid residue numbers given in parentheses indicate the first and last residues of amino acid alignments represented. The proteases are as follows: Apy, hyperthermostable protease from Aquifex pyrophilus (7); Aqua, mesothermophile alkaline protease from Thermus aquaticus YT-1 (18); Carls, subtilisin Carlsberg from Bacillus licheniformis (15); ISP, intracellular alkaline protease from Thermoactinomyces sp. strain HS682 (39); PrtP, cell envelope-located protease from Lactobacillus paracasei (14); and Pyro, hyperthermostable protease pyrolysin from Pyrococcus furiosus (41). (B) Dendrogram of Csp proteins and proteases listed above, in which branch lengths are in inverse proportion to the degree of sequence similarity. The tree was constructed using the programs ClustalX and NJPLOT (38).

FIG. 5

Immunological detection of CspC-related proteins in dormant spores. The spore coat fraction was separated from decoated spores as described in Materials and Methods. The decoated spores (1 g of wet weight) were disrupted with a bead beater in a 20-ml tube containing 10 ml H₂O and 10 g of glass beads (diameter, 0.1 mm). The supernatant obtained by centrifugation (15,000 × g for 5 min at 4°C) was used as an extract of disrupted decoated spores. GSP obtained by DEAE-5PW column chromatography, the extract obtained from germinated spores by incubation in CHAPS solution, the spore coat fraction, and the extract of disrupted decoated spores were subjected to 0.1% SDS–10% PAGE followed by immunoblot analysis with anti-rΔ_1–78CspC antiserum. Lanes 1, GSP obtained by DEAE-5PW column chromatography (∼5 μg of protein); 2 and 4, spore coat fraction (∼100 μg of protein for lane 2 and ∼70 μg of protein for lane 4); 3, extract from germinated spores (∼100 μg of protein); 5, extract of disrupted decoated spores (∼100 μg of protein). Samples for lanes 1 and 2 were electrophoresed on a separate gel different from that used for lanes 3 to 5. The standard proteins were the same as those used for Fig. 1.