Cloning and Sequencing of the Central Region of the Flagellin Gene from the Gram-Positive Bacterium Clostridium tyrobutyricum ATCC 25755

• Published in: MICROBIOLOGY and IMMUNOLOGY 1999, Vol.43(1), pp.1-8
• Main Authors: Arnold, Françoise, Bédouet, Laurent, Batina, Pierre, Robreau, Georges
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacteriology; Base Sequence; Biological and medical sciences; Blotting, Northern; Cloning, Molecular; Clostridium - classification; Clostridium - genetics; Clostridium tyrobutyricum; DNA Primers; DNA sequencing; Flagellin; Flagellin - genetics; Flagellin - metabolism; Fundamental and applied biological sciences. Psychology; Genes, Bacterial; Genetics; Glycosylation; Glycosylation sites; Microbiology; Molecular Sequence Data; PCR amplification; Polymerase Chain Reaction - methods; Polymorphism, Restriction Fragment Length; Sequence Analysis, DNA
• ISSN: 0385-5600; 1348-0421
• DOI: 10.1111/j.1348-0421.1999.tb02365.x

The purpose of this study was to sequence the central part of the coding region of the Clostridium tyrobutyricum flagellin gene to improve the immunoenzymatic counting of cells after milk filtration. The coding region was amplified by PCR, and the amplified products were cloned. A DNA sequence analysis of positive clones gave us 1, 131 nucleotides with a partial calculated flagellin molecular mass of 40, 143 Da. The flagellar filament protein sequence exhibited high levels of homology to sequences of flagellin protein from other bacteria in both N- and C-terminal parts, but little homology in the central domain. A PCR-restriction fragment length polymorphism analysis of amplified C. tyrobutyricum flagellin gene products confirmed the variability of the central domain. The flagellin mRNA was determined to be 1.1kb in size, which suggests a monocistronic mRNA. Furthermore, the deduced protein flagellin contains eleven potential N-glycosylation sites and one sequence rich in serine, which could be modified by O-glycosylation.