Antisense-RNA-mediated decreased synthesis of small, acid-soluble spore proteins leads to decreased resistance of clostridium perfringens spores to moist heat and UV radiation

Abstract

Previous work has suggested that a group of alpha/beta-type small, acid-soluble spore proteins (SASP) is involved in the resistance of Clostridium perfringens spores to moist heat. However, this suggestion is based on the analysis of C. perfringens spores lacking only one of the three genes encoding alpha/beta-type SASP in this organism. We have now used antisense RNA to decrease levels of alpha/beta-type SASP in C. perfringens spores by approximately 90%. These spores had significantly reduced resistance to both moist heat and UV radiation but not to dry heat. These results clearly demonstrate the important role of alpha/beta-type SASP in the resistance of C. perfringens spores.

FIG. 1.

Alignments of sequences of Ssp proteins and ssp genes and construction of a plasmid containing ssp2 in the antisense orientation to its promoter. (A) Amino acid sequences of B. subtilis SspC and Ssp1, Ssp2, and Ssp3 of C. perfringens are shown using the one-letter amino acid code. The symbols : and * below the sequences indicate residues that are conserved in all three C. perfringens proteins and residues conserved in the B. subtilis and the C. perfringens proteins, respectively. The dashes in the SspC sequence indicate the position of a gap that was introduced to maximize the sequence alignment with the C. perfringens proteins. (B) Alignment of nucleotide sequences of C. perfringens ssp genes. The symbols * and : below the sequences indicate nucleotides that are identical in all three sequences and nucleotides that are identical in ssp2 and in either ssp1 or ssp3, respectively. The alignment was performed using the ClustalW program and modified with Microsoft PowerPoint. CPP194 and CPP195 indicate forward and reverse primers, respectively, to amplify an internal ssp2 fragment. (C) Construction of plasmid pDR81 containing the ssp2 fragment in the antisense direction to the ssp2 promoter (ssp2 pro). oriCP and oriEC indicate origins of replication for C. perfringens and E. coli, respectively; rep indicates the replication gene from pIP404; ermBP indicates an Em^r gene; and lacZ′ is the gene encoding the β-galactosidase α-peptide. The various genes are transcribed towards the arrowheads.

FIG. 2.

Levels of α/β-type SASP in spores of various C. perfringens strains. (A) α/β-Type SASP was extracted from equal amounts of spores (50 mg [dry weight]) of various C. perfringens strains, lyophilized, and dissolved in 25 μl of 8 M urea, 10-μl aliquots were run on polyacrylamide gels at low pH, and the gel was stained with Coomassie brilliant blue. The spores from which extracts were prepared are SM101(pSG22) (wild-type strain carrying the control plasmid), DR101(pSG22) (an isogenic ssp3 mutant carrying the control plasmid), SM101(pDR81) (wild-type strain carrying the plasmid expressing ssp2 asRNA), and DR101(pDR81) (ssp3 mutant carrying the plasmid expressing ssp2 asRNA). The arrow indicates the major band that was immunoreactive with B. subtilis SspC antiserum. (B) Western blot of samples run on a gel as shown in A. The blot was probed with antiserum against B. subtilis SspC and developed for chemiluminescence detection to identify immunoreactive species. (C) Densitometric analysis of the α/β-type SASP band intensities with extracts from spores of various C. perfringens strains run on polyacrylamide gels at low pH and stained with Coomassie blue. Densitometric analysis was performed as described in Materials and Methods, and the results are averages of three independent experiments.

FIG. 3.

Thermal death curves of spores of various C. perfringens strains. Spores of strains SM101(pSG22) (•) (wild type), DR101(pSG22) (⧫) (ssp3 mutant with the control plasmid), SM101(pDR81) (▪) (wild type expressing ssp2 asRNA), and DR101(pDR81) (▴) (ssp3 mutant expressing ssp2 asRNA) were heated at 100°C for various times, and CFU/ml were determined as described in Materials and Methods.

FIG. 4.

UV survival curves for spores of various C. perfringens strains. Spores of strains SM101(pSG22) (wild type) (•), SM101(pDR81) (wild type expressing ssp2 asRNA) (▪), and DR101(pDR81) (ssp3 mutant expressing ssp2 asRNA) (▴) were exposed to UV radiation for various times, and CFU/ml were determined as described in Materials and Methods.