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 sol vent 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 len gth which could potentially encode a 275-amino acid protein, with a calcula ted molecular weight of 29 505Da. Primer extension analysis revealed the pr esence of a single transcriptional start site 72 bp upstream of the flaC tr anslation start codon, with an inverted repeat present downstream of the st ructural gene that has the characteristics of a stem-loop structure and thu s may act as a rho-independent transcription terminator. The putative promo ter that was identified shared strong similarity to sigma(28)-type promoter s that have been identified upstream of flagellin genes in other species. P revious work using western immunoblots had identified that the flagellin pr otein was a ca. 42-kDa protein, yet this did not agree with the size expect ed 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 po st-transnational modification such as glycosylation. Protein analysis revea led that the FlaC protein was glycosylated, with a terminal sialyl residue as demonstrated by treatment with neuraminidase. (C) 2000 Academic Press.