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J. Bacteriol., 02 1995, 1059-1068, Vol 177, No. 4
R Morona, L van den Bosch and PA Manning
The rfb region of Shigella flexneri encodes the proteins required to
synthesize the O-antigen component of its cell surface lipopolysaccharides
(LPS). We have previously reported that a region adjacent to rfb was
involved in regulating the length distribution of the O-antigen
polysaccharide chains (D. F. Macpherson et al., Mol. Microbiol.
5:1491-1499, 1991). The gene responsible has been identified in Escherichia
coli O75 (called rol [R. A. Batchelor et al., J. Bacteriol. 173:5699-5704,
1991]) and in E. coli O111 and Salmonella enterica serovar typhimurium
strain LT2 (called cld [D. A. Bastin et al., Mol. Microbiol. 5:2223-2231,
1991]). Through a combination of subcloning, deletion, and transposon
insertion analysis, we have identified a gene adjacent to the S. flexneri
rfb region which encodes a protein of 36 kDa responsible for the length
distribution of O- antigen chains in LPS as seen on silver-stained sodium
dodecyl sulfate- polyacrylamide gels. DNA sequence analysis identified an
open reading frame (ORF) corresponding to the rol gene. The corresponding
protein was almost identical in sequence to the Rol protein of E. coli O75
and was highly homologous to the functionally identical Cld proteins of E.
coli O111 and S. enterica serovar typhimurium LT2. These proteins, together
with ORF o349 adjacent to rfe, had almost identical hydropathy plots which
predict membrane-spanning segments at the amino- and carboxy-terminal ends
and a hydrophilic central region. We isolated a number of TnphoA insertions
which inactivated the rol gene, and the fusion end points were determined.
The PhoA+ Rol::PhoA fusion proteins had PhoA fused within the large
hydrophilic central domain of Rol. These proteins were located in the
whole-membrane fraction, and extraction with Triton X-100 indicated a
cytoplasmic membrane location. This finding was supported by sucrose
density gradient fractionation of the whole-cell membranes and of E. coli
maxicells expressing L- [35S]methionine-labelled Rol protein. Hence, we
interpret these data to indicate that the Rol protein is anchored into the
cytoplasmic membrane via its amino- and carboxy-terminal ends but that the
majority of the protein is located in the periplasmic space. To confirm
that rol is responsible for the effects on O-antigen chain length observed
with the cloned rfb genes in E. coli K-12, it was mutated in S. flexneri by
insertion of a kanamycin resistance cartridge. The resulting strains
produced LPS with O antigens of nonmodal chain length, thereby confirming
the function of the rol gene product.(ABSTRACT TRUNCATED AT 400 WORDS)
Copyright © 1995, American Society for Microbiology
Molecular, genetic, and topological characterization of O-antigen chain length regulation in Shigella flexneri
Department of Microbiology and Immunology, University of Adelaide, Australia.
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