Serotype 1a O-antigen modification: molecular characterization of the genes involved and their novel organization in the Shigella flexneri chromosome

Abstract

The factors responsible for serotype 1a O-antigen modification in Shigella flexneri were localized to a 5.8-kb chromosomal HindIII fragment of serotype 1a strain Y53. The entire 5.8-kb fragment and regions up- and downstream of it (10.6-kb total) were sequenced. A putative three-gene operon, which showed homology with other serotype conversion genes, was identified and shown to confer serotype 1a O-antigen modification. The serotype conversion genes were flanked on either side by phage DNA. Multiple insertion sequence (IS) elements were located within and upstream of the phage DNA in a composite transposon-like structure. Host DNA homologous to the dsdC and the thrW proA genes was located upstream of the IS elements and downstream of the phage DNA, respectively. The sequence analysis indicates that the organization of the 10.6-kb region of the Y53 chromosome is unique and suggests that the serotype conversion genes were originally brought into the host by a bacteriophage. Several features of this region are also characteristic of pathogenicity islands.

FIG. 1

O-antigen structure of S. flexneri serotypes Y and 1a.

FIG. 2

Western immunoblot of LPS preparations. Membranes were probed with MASF-Y5 or MASF-I as the primary antibody, and O antigens were detected by chemiluminescence. SFL124 (serotype Y) and S. flexneri Y53 (serotype 1a) were used as controls. LPS prepared from SFL1243 (SFL124 containing orf3) is recognized by both Y- and 1a-specific monoclonal antibodies, whereas LPS from SFL1244 (SFL124 containing orf1, orf2, and orf3) is recognized only by MASF-I.

FIG. 3

The organization of the O-antigen modification genes in S. flexneri 1a. Arrows represent the direction of each IS. The O-antigen modification gene cluster is shaded.

FIG. 4

Determination of copy number and organization of gtrI in wild-type S. flexneri 1a strains. The chromosomal DNA from the strains indicated was digested with EcoRI and HindIII and subjected to Southern hybridization with ³²P-labelled gtrI as a probe. The molecular weight marker was EcoRI-digested SPP1 bacteriophage DNA (Progen).