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Toxigenic Clostridium difficile isolates harbor a 19 kb pathogenicity locus that encodes the genes for toxins A and B. Toxins A and B are among the largest known bacterial toxins expressing potent cytotoxicity and enterotoxicity, and thus the major virulence factors in C. difficile associated diarrhea. Cloning and sequencing of toxin genes is of interest for studies of molecular pathogenesis. We report the amplification and cloning of the complete toxin A gene into an Escherichia coli expression vector. Ten clones analyzed contained the complete toxin A gene. Four of these clones showed cytotoxic activity in cell culture, and were positive for toxin A as determined by ELISA. Toxin A expression was confirmed by Western immunoblot analysis. The presence of cytotoxic activity in cell culture suggests that toxin A activity is independent of other genes in the pathogenicity locus.

The toxin genes of Clostridium difficile have been previously cloned by reconstructing the entire gene in a series of steps in sequence using several cloned fragments. Amplification of a 7.9 kb fragment corresponding to the toxin B gene (tcdB) was obtained with EXPAND Long Template PCR system. The amplified fragment was inserted into the E. coli expression vector pBAD and cloned into competent E. coli TOP 10 cells. tcdB gene sequences representing the complete toxin gene were detected in 3/120 (2.5%) clones analyzed. Culture filtrates of 2/3 clones were found to have cytotoxic activity in human lung fibroblasts. The recombinant protein expressed in E. coli was identified as toxin B by Western immunoblot analysis using C. sordellii antitoxin. This rapid cloning method may be useful in determining the role that individual genes in the pathogenicity locus (PaLoc) play in the virulence of C. difficile. Our results also suggest that the activity of toxin B is independent of other genes in the PaLoc.

Toxigenic Clostridium difficile is the etiologic agent of C. difficile-associated diarrhoea (CDAD), the most common cause of hospital-acquired infectious diarrhoea. The genes tcdA and tcdB, which encode for the toxin A and B proteins, are part of the pathogenicity locus (PaLoc) of toxigenic C. difficile. Genetic and virulence studies at the molecular level in C. difficile have been hindered by the lack of techniques for DNA manipulation in this species. We describe the electroporation of DNA fragments from a toxigenic isolate into a non-toxigenic strain of C. difficile. Using previously described methods of electroporation into Clostridium spp., the complete toxin B gene and polymerase chain reaction (PCR) fragments of the PaLoc were cloned and electroporated into a non-toxigenic strain of C. difficile. The resulting transformed clones were screened for the introduced gene fragments by PCR, which confirmed their presence. This is the first description of introduction of DNA into C. difficile by electroporation., (Copyright 2001 Academic Press.)

The nucleotide sequences of a diabetogenic variant of encephalomyocarditis (EMC) virus (D variant, ifp- phenotype) and a nondiabetogenic variant of EMC virus (B variant, ifp+ phenotype) have been compared. These variants differ at eight sites. The poly(C) tract of EMC-B is three bases shorter than that of EMC-D. Of the seven sites at which nucleotide substitutions were confirmed using RNA templates, four resulted in amino acid changes in three different proteins, the leader peptide, 1B (VP2), and 1D (VP1). The biological significance of these differences can now be investigated.

The RNA of a diabetogenic variant of encephalomyocarditis (EMC) virus (D variant, ifp- phenotype) and a nondiabetogenic variant of EMC virus (B variant, ifp+ phenotype) which were derived from the same virus stock (M strain) have been compared. The size of both genomes is estimated at 7.7 kb. The poly(C) tract of EMC-D is estimated at 144 bases, whereas that of EMC-B is 141 bases in length. The untranslated 5' terminal 103 nucleotides are identical for B and D with preservation of a stable terminal hairpin structure. The entire open reading frame of both variants has been cloned and the restriction maps of 12 different enzymes are identical. These maps were compared to a computer-generated restriction map of another strain of EMC virus which has been cloned and sequenced by Palmenberg et al. (1984, Nucleic Acids Res. 12, 2969-2985). Approximately 50% of the restriction sites of the B and D variants had similar locations in this strain of EMC. We conclude that significant genetic variation exists between the M strain (B and D variants) and the Palmenberg strain of EMC virus. However, the B and D variants are very similar at the molecular level and comparison of their nucleotide sequences will be necessary to reveal the basis for their different biological properties.

Simple and sensitive methods to directly detect the human immunodeficiency virus (HIV) are needed for routine use in the clinical laboratory. In this study, we compared DNA probes prepared by: (1) nick translation with biotinylated dATP; (2) direct covalent biotinylation with photobiotin; (3) direct covalent reaction with 2-acetylaminofluorene (AAF); and (4) a standard radioactive (32P) nick translation procedure. These four DNA probes were hybridized with dilutions of purified target HIV DNA blotted onto nitrocellulose strips. Hybridization was detected using a complex of strepavidin-alkaline phosphatase [for (1) and (2)], alkaline phosphatase-tagged antibodies [for (3)] and by autoradiography [for (4)]. Alkaline phosphatase was detected colorimetrically using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. After 1 h, AAF probes were most sensitive (amount detected less than 5 pg), followed by biotin (10 pg), photobiotinylated probes (20 pg) and the radioactive probe (10 pg). The AAF probes were then used to detect HIV DNA in infected CEM cells. We conclude that non-radioactive DNA labelling methods can be used to directly detect HIV DNA under conditions compatible with present clinical laboratory procedures.