Mutagenesis and chemical cross-linking suggest that Wzz dimer stability and oligomerization affect lipopolysaccharide O-antigen modal chain length control.
In Shigella flexneri, the polysaccharide copolymerase (PCP) protein Wzz(SF) confers a modal length of 10 to 17 repeat units (RUs) to the O-antigen (Oag) component of lipopolysaccharide (LPS). PCPs form oligomeric structures believed to be related to their function. To identify functionally important regions within Wzz(SF), random in-frame linker mutagenesis was used to create mutants with 5-amino-acid insertions (termed Wzz(i) proteins), and DNA sequencing was used to locate the insertions. Analysis of the resulting LPS conferred by Wzz(i) proteins identified five mutant classes. The class I mutants were inactive, resulting in nonregulated LPS Oag chains, while classes II and III conferred shorter LPS Oag chains of 2 to 10 and 8 to 14 RUs, respectively. Class IV mutants retained near-wild-type function, and class V mutants increased the LPS Oag chain length to 16 to 25 RUs. In vivo formaldehyde cross-linking indicated class V mutants readily formed high-molecular-mass oligomers; however, class II and III Wzz(i) mutants were not effectively cross-linked. Wzz dimer stability was also investigated by heating cross-linked oligomers at 100 degrees C in the presence of SDS. Unlike the Wzz(SF) wild type and class IV and V Wzz(i) mutants, the class II and III mutant dimers were not detectable. The location of each insertion was mapped onto available PCP three-dimensional (3D) structures, revealing that class V mutations were most likely located within the inner cavity of the PCP oligomer. These data suggest that the ability to produce stable dimers may be important in determining Oag modal chain length.
Papadopoulos
Magdalene
M
Discipline of Microbiology and Immunology, School of Molecular and Biomedical Science, University of Adelaide, Adelaide, South Australia, Australia 5005.
Morona
Renato
R
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics / Q000502:physiology; D016296:Mutagenesis / Q000235:genetics*; D019081:O Antigens / Q000378:metabolism*; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D012763:Shigella flexneri / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D019081:O Antigens; C075969:rol protein, Bacteria
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