Cloning and sequencing of the central region of the flagellin gene from the Gram-positive bacterium Clostridium tyrobutyricum ATCC 25755

The purpose of this study was to sequence the central part of the coding re gion of the Clostridium tyrobutyricum flagellin gene to improve the immunoe nzymatic counting of cells after milk filtration. The coding region was amp lified by PCR, and the amplified products were cloned. A DNA sequence analy sis of positive clones gave us 1,131 nucleotides with a partial calculated flagellin molecular mass of 40,143 Da, The flagellar filament protein seque nce exhibited high levels of homology to sequences of flagellin protein fro m other bacteria in both N- and C-terminal parts, but little homology in th e central domain, A PCR-restriction fragment length polymorphism analysis o f amplified C, tyrobutyricum flagellin gene products confirmed the variabil ity of the central domain. The flagellin mRNA was determined to be 1.1 kb i n size, which suggests a monocistronic mRNA, Furthermore, the deduced prote in flagellin contains eleven potential N-glycosylation sites and one sequen ce rich in serine, which could be modified by O-glycosylation.