NUCLEOTIDE-SEQUENCE OF THE RHAMNOSE BIOSYNTHETIC OPERON OF SHIGELLA-FLEXNERI 2A AND ROLE OF LIPOPOLYSACCHARIDE IN VIRULENCE

N1308, a chromosomal Tn5 mutant of Shigella flexneri 2a, was described previously as a lipopolysaccharide (LPS) mutant with a short O side c hain. N1308 formed foci, but not plaques, in LLC-MK2 cell monolayers a nd was negative in the Sereny test. In this study, the wild-type locus inactivated in N1308 was cloned and further defined by means of compl ementation analysis. A 4.3-kb BstEII-XhoI fragment of S. flexneri 2a Y SH6200 DNA was sufficient to restore both normal LPS and virulence phe notype to the mutant. DNA sequencing of this region revealed four gene s, rfbA, rfbB, rfbC, and rfbD, encoding the enzymes required for the b iosynthesis of activated rhamnose. The four genes were expressed in Es cherichia coli, and the expected protein products were visualized by s odium dodecyl sulfate-polyacrylamide gel electrophoresis. N1308 was sh own to have normal levels of surface IpaC and IpaD, while a Western bl ot (immunoblot) of whole-cell lysates or outer membrane fractions indi cated an elevated level of appropriately localized VirG. An in vitro i nvasion assay revealed that N1308 had normal primary invasive capacity and was able to multiply and move normally within the initial infecte d cell. However, it exhibited a significant reduction in its ability t o spread from cell to cell in the monolayer. A double immunofluorescen ce assay revealed differences between LLC-MK2 cells infected with the wild-type YSH6000 and those infected with N1308. The wild-type bacteri a elicited the formation of the characteristic F actin tails, whereas N1308 failed to do so. However, N1308 was capable of inducing depositi on of F-actin, which accumulated in a peribacterial fashion with only slight, if any, unipolar accumulation of the cytoskeletal protein.