Molecular characterization of clostridium botulinum type B progenitor toxin gene and its toxic component

Abstract

Botulinum neurotoxins(BoNT) are the most potent biological toxins which have been known to block the release of neurotransmitter from presynaptic nerve terminal and produce clinical syndromes of rapidly progressive weakness. The toxin is composed of two subunits containing light chain(responsible for Zn-dependent endopeptidase activity)and heavy chain(responsible for binding to its receptor and channel formation). However, the toxins are associated with nontoxic-nonhemagglutinin component (NTNH; about 130kDa) and hemagglutinin components (Hem; about 33 kDa, 17 kDa and 70 kDa), and a series of complexes raging from 220 kDa to 900 kDa of molecular weight are subsequently formed. Serotype B progenitor toxin gene was cloned and characterized. Nucleotide sequence analysis revealed that the genes for toxin, NTNH, unidentified 22 kDa open reading frame (Orf22), and HEM/33, 17 and 70 were clustered. Northern blot analysis showed that a set of these six genes were organized into three transcription units. Cluster 1 encompasses genes for the toxin and nontoxic-nonhemaggluinin component, forming an operon; the toxin gene can be transcribed alone or co-transcribed with nontoxic-nonhemagglutinin component. Cluster 2 encompasses three genes including Hem33,Hem17,and Hem70 which also form an operon and are transcribed as a tricistronic RNA. The orientation of the three genes was opposite to those of toxin and NTNH gene. The third transcriptional unit existed between NTNH gene and HEM33 gene. The gene encodes Orf22, which is basic protein with 9.0 calculated pI value, presents an helix-turn-helix motif. The primary sequence of Orf22 is turned out to have similarity with a number of many bacterial regulatory proteins such as uvi gene product in Clostridium perfringens and sigma factor in Escherichia coli. These characteristics of Orf22 imply that Orf22 might be a regulatory protein, which controlled the expression of toxin, NTNH and Hem genes. Based on the sequence alignment o...