AN ALTERNATIVE SIGMA-FACTOR CONTROLS TRANSCRIPTION OF FLAGELLAR CLASS-III OPERONS IN ESCHERICHIA-COLI - GENE SEQUENCE, OVERPRODUCTION, PURIFICATION AND CHARACTERIZATION

Based on the studies of the FLiA protein in Bacillus subtilis (Bs) and Salmonella typhimurium (St), the Escherichia coli (Ec) fliA gene has been proposed to encode a flagellar-specific sigma factor, sigma(28). In this study, the complete nucleotide (nt) sequence of Ec fliA was determined. The fliA coding region consists of 717 nt starting with a GTG start codon and ending with a TAA stop codon. The gene product is predicted to be 239 amino acids (26435 Da). Sequence comparison between Ec FliA and the sigma(28) Of St revealed 93.7% identity. Gene fliA was amplified by the polymerase chain reaction, subcloned into expression vector pT7-7, and overexpressed. The overproduced 28-kDa FliA protein, recognized by the St anti-sigma(28) antibody, was purified to homogeneity. The purified protein was able to initiate transcription from the tar promoter in the presence of RNP core enzyme. We conclude that FliA functions as an alternative sigma factor sigma(28) which is specific for flagellar operons in Ec.