Cloning of the rfb gene region of Shigella dysenteriae 1 and construction of an rfb- rfp gene cassette for the development of lipopolysaccharide-based live anti-dysentery vaccines

Abstract

Recent studies have shown that determinants for the production of O antigen lipopolysaccharide in Shigella dysenteriae 1 are distributed over two distinct genetic elements, the chromosome and a 9 kb plasmid designated pHW400. 1,2 In this communication, we describe the cloning of all determinants necessary for S. dysenteriae 1 O antigen production in E. coli K-12 and their combination in a single plasmid. An RP4::miniMu R-prime plasmid, R-prime 40, containing the his- rfb (histidine biosynthesis-lipopolysaccharide biosynthesis) gene region of the Shigella dysenteriae 1 chromosome was generated. E. coli K-12 bacteria containing R-prime 40 and pSS8, a transposon Tn 5-tagged derivative of pHW400, produced lipopolysaccharide indistinguishable from that of S. dysenteriae 1. Small DNA fragments containing the rfb gene cluster and the rfp gene were subcloned from R-prime 40 and pSS8 and subsequently combined in vector pACYC184 to produce pSS37. This latter plasmid when introduced by transformation into E. coli K-12 provoked the formation of S. dysenteriae 1 O-specific lipopolysaccharide, a feature that suggests it may be useful in the construction of LPS-based live vaccines against the Shiga bacillus.