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Analysis of Shigella flexneri Wzz (Rol) function by mutagenesis and cross‐linking: Wzz is able to oligomerize
Author(s) -
Daniels Craig,
Morona Renato
Publication year - 1999
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.1999.01591.x
Subject(s) - periplasmic space , shigella flexneri , amino acid , mutagenesis , biology , transmembrane protein , transmembrane domain , mutant , site directed mutagenesis , biochemistry , escherichia coli , gene , receptor
The modal length or degree of polymerization (dp) of the Shigella flexneri O‐antigen is determined in an unknown manner by the Wzz/Rol protein. The Wzz protein is anchored into the cytoplasmic membrane by two transmembrane domains (TM1 amino acids 32–52; TM2 amino acids 295–315) with the central loop of the protein located in the periplasm. Plasmids were constructed encoding hybrid Wzz proteins consisting of regions of S. flexneri Wzz (Wzz SF ) and Salmonella typhimurium Wzz (Wzz ST ). These imparted O‐antigen modal chain lengths that implied that the carboxy‐terminal region of Wzz was involved in chain length determination. Site‐directed mutagenesis was undertaken to investigate the functional significance of highly conserved residues in amino‐/carboxy‐terminal domains of Wzz SF . Some of the Wzz SF variants resulted in O‐antigen modal chain lengths much shorter than those of wild‐type Wzz SF , whereas other mutants inactivated Wzz SF function entirely and a third class had a longer O‐antigen chain length distribution. The data indicate that amino acids throughout the length of the Wzz SF protein are important in determination of O‐antigen modal chain length. In vivo cross‐linking experiments were performed to investigate the interactions between Wzz proteins. The experiments indicated that the Wzz SF protein is able to form dimers and oligomers of at least six Wzz SF proteins. A carboxy‐terminal‐truncated Wzz SF protein having the amino terminal 194 amino acids was able to oligomerize, indicating that the amino‐terminal region is sufficient for the Wzz–Wzz interaction observed. Shortened Wzz SF proteins having internal deletions in the amino‐terminal region were also able to oligomerize, suggesting that residues 59–194 are not essential for oligomerization. Cross‐linking of Wzz SF proteins with mutationally altered residues showed that loss of Wzz SF function may be correlated to a reduced/altered ability to form oligomers, and that mutational alteration of glycine residues in the TM2 segment affects Wzz SF –Wzz SF dimer mobility in SDS polyacrylamide gels. These results provide the first evidence of protein–protein interactions for proteins involved in O‐antigen polysaccharide biosynthesis.