1887

Abstract

Regulation of the length of the O-antigen (Oag) chain attached to LPS in is important for virulence and is dependent on the inner-membrane protein Wzz. A lack of high-resolution structural data for Wzz has hampered efforts so far to correlate mutations affecting function of Wzz with structure and describe a mechanism for chain length regulation. Here we have used secondary structure prediction to show that the periplasmic domain of the Wzz protein has three regions of significant coiled-coil (CC) potential, two of which lie within an extended helical region. We describe here the first site-directed mutagenesis study to investigate the role of individual predicted CC regions (CCRs) in Wzz function and oligomerization. We found that CCRs 2 and 3 are necessary for wild-type Oag chain length regulation by Wzz. The cross-linking profile of mutants affected in the three CCRs was not altered, indicating that individually each CCR is not required for oligomerization. Interestingly, the CCR3 mutation resulted in a temperature-sensitive phenotype and an inhibitory effect on Oag polymerization. Analysis of Wzz and the mutant constructs in a mutant showed that DegP did not affect the function of wild-type Wzz but its presence influenced the phenotype of the Wzz CCR3 mutant. Additionally, the phenotype of the Wzz CCR3 mutant was suppressed by a mutation near the putative cytoplasmic C-terminus of Wzz.

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2008-04-01
2019-10-21
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