Cloning, sequencing, and characterization of the gene encoding flagellin, flaC, and the post-translational modification of flagellin, FlaC, from Clostridium acetobutylicum ATCC824

Clostridium acetobutylicum is an anaerobic, motile, industrially important organism. In order to investigate the relationship between motility and solvent production, the gene which encodes the flagellin was cloned, sequenced and the corresponding amino acid sequence deduced. The gene was designated flaC, based upon significant amino acid sequence similarity with flagellin proteins from other organisms. The flaC gene was found to be 825 bp in length which could potentially encode a 275-amino acid protein, with a calculated molecular weight of 29 505Da. Primer extension analysis revealed the presence of a single transcriptional start site 72 bp upstream of the flaC translation start codon, with an inverted repeat present downstream of the structural gene that has the characteristics of a stem-loop structure and thus may act as a rho-independent transcription terminator. The putative promoter that was identified shared strong similarity to sigma(28)-type promoters that have been identified upstream of flagellin genes in other species. Previous work using western immunoblots had identified that the flagellin protein was a ca. 42-kDa protein, yet this did not agree with the size expected based on nucleotide sequence analysis. PCR analysis revealed that there was no re-arrangement of the flaC structural gene. To further characterize the difference between the two observations, FlaC was investigated for a post-transnational modification such as glycosylation. Protein analysis revealed that the FlaC protein was glycosylated, with a terminal sialyl residue as demonstrated by treatment with neuraminidase. (C) 2000 Academic Press.