Abstract
A recent study detected genes encoding type B botulinum neurotoxin in some type A strains of Clostridium botulinum that exhibit no type B toxin activity. In this study, we investigated the presence, structure, linkage, and organization of genes encoding botulinum neurotoxin (BoNT) and other components of the progenitor complex. Sequence analysis showed that the silent BoNT/B gene is highly related to that from authentic proteolytic type B C. botulinum. However, a stop signal and deletions were found within the sequence. A non-toxin nonhemagglutinin gene (NTNH) was mapped immediately upstream of both the BoNT/A and silent BoNT/B genes. Significantly the NTNH gene adjacent to the defective BoNT/B gene was "chimeric, " the 5'- and 3'-regions of the gene had high homology with corresponding regions of the type B NTNH gene, while the 471-amino acid sequence in the central region was identical to NTNH of type A. Hemagglutinin genes HA-33 and HA-II were not found adjacent to the NTNH/A gene, but instead there was an unidentified open reading frame previously reported in strains of C. botulinum types E and F. By contrast HA-II, HA-33, and NTNH genes were located immediately upstream of the silent BoNT/B gene. Pulsed-field gel electrophoretic analysis of chromosomal DNA digests indicated the distance between type A and B gene clusters to be less than 40 kilobases.
Highlights
A recent study detected genes encoding type B botulinum neurotoxin in some type A strains of Clostridium botulinum that exhibit no type B toxin activity
The results indicate that both strains 588 and 667 contain one copy of the gene for each neurotoxin (BoNT/A and botulinum neurotoxin (BoNT)/B), two copies of nontoxin nonhemagglutinin gene (NTNH) genes, but only one copy of the HA-33 gene (Fig. 1)
In NdeI digests, two fragments hybridized with NN-180 probe. One of these displayed a positive signal with probes Ha-1a and 120b, and the other with probe 334a. These results indicate that single copies of NTNH and HA-33 genes are linked to the BoNT/B gene
Summary
Vol 271, No 18, Issue of May 3, pp. 10786 –10792, 1996 Printed in U.S.A. Genetic Characterization of Clostridium botulinum Type A Containing Silent Type B Neurotoxin Gene Sequences*. Franciosa et al [5], using polymerase chain reaction (PCR) methodologies, reported the detection of genes encoding type B neurotoxin (BoNT/B) in a large number (43 of 79) of strains of C. botulinum type A, only one of which produced any demonstrable type B toxin. The silent type B gene was detected by PCR in three of the type A strains (type A(B)) using four different primer pairs or combinations designed for amplification of 5Ј-terminal, central, and 3Ј-terminal regions, as well as nearly the entire gene [5], sequences within those segments could be altered in major or subtle ways It is not known whether genes encoding other components (e.g. NTNH and HA-33) normally associated with BoNT production are present. The study reveals major alterations in the organization, structure, and expression of the genes encoding proteins of the toxin complexes
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