CHARACTERIZATION OF THE RFC REGION OF SHIGELLA-FLEXNERI

The O antigen of the Shigella flexneri lipopolysaccharide (LPS) is an important virulence determinant and immunogen. We have isolated S. fle xneri mutants which produce a semi-rough LPS by using an O-antigen-spe cific phage, Sf6c. Western immunoblotting was used to show that the LP S produced by the semi-rough mutants contained only one O-antigen repe at unit. Thus, the mutants are deficient in production of the O-antige n polymerase and were termed rfc mutants. Complementation experiments were used to locate the rfc adjacent to the rfb genes on plasmid clone s previously isolated and containing this region (D. F. Macpherson, p. Morona, D. W. Beger, K.-C. Cheah, and P. A. Manning, Mel. Microbiol 5 :1491-1499, 1991). A combination of deletions and subcloning analysis located the rfc gene as spanning a 2-kb region. Insertion of a kanamyc in resistance cartridge into a SalI site in this region inactivated th e rfc gene. The DNA sequence of the rfc region was determined. An open reading frame spanning the SalI site was identified and encodes a pro tein with a predicted molecular mass of 43.7 kDa. The predicted protei n is highly hydrophobic and showed little sequence homology with any o ther protein. Comparison of its hydropathy plot with that of other Rfc proteins from Salmonella enterica (typhimurium) and Salmonella enteri ca (muenchen) revealed that the profiles were similar and that the pro teins have 12 or more potential membrane-spanning segments. A comparis on of the S. flexneri rfc gene and protein product with other rfc and rfc-like proteins revealed that they have a similarly low percentage o f G+C content and have similar codon usage, and all have a high percen tage of rare codons. An attempt to identify the S. flexneri Rfc protei n was unsuccessful, although proteins encoded upstream and downstream of the rife gene could be identified. Examination of the distribution of rare or minor codons in the rfc gene revealed that it has several m inor codons within the first 25 amino acids. This is in contrast to th e upstream gene rfbG, which also has a high percentage of rare codons but whose gene product could be detected. The positioning of the rare codons in the rfc gene may restrict translation and suggests that mino r isoaccepting tRNA species may be involved in translational regulatio n of rfc expression. The low percentage of G+C content of rfc genes ma y be a consequence of the selection pressure to maintain this form of control.