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Volume 161, Issue 9

Detection of Wzy/Wzz interaction in Free

Abstract

The O antigen (Oag) component of lipopolysaccharide (LPS) is important for virulence and a protective antigen. It is synthesized by the Wzy-dependent mechanism. Wzy has 12 transmembrane segments and two large periplasmic loops. The modal chain length of the Oag is determined by Wzz. Experimental evidence supports multi-protein interactions in the Wzy-dependent pathway. However, evidence for direct interaction of Wzy with the other proteins of the Wzy-dependent pathway is limited. Initially, we purified Wzy-GFP-His and detected the presence of a dimeric form. cross-linking was then performed in an mutant strain carrying plasmids encoding Wzy-GFP-His and untagged Wzz. Following solubilization with -dodecyl-β--maltopyranoside (DDM) and affinity purification of Wzy-GFP-His, Western immunoblotting with Wzz antibody detected co-purification of Wzz; this was supported by MS analysis. To the best of our knowledge, this is the first reported isolation of a complex between Wzy and Wzz. Wzy mutants (WzyR164A, WzyV92M, WzyY137H, and WzyR250K) whose properties are affected by Wzz were able to form complexes with Wzz. Their mutational alterations did not affect the interaction of Wzy with Wzz. Thus, the interaction may involve many regions of Wzy.

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2015-09-01
2024-04-20
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Detection of Wzy/Wzz interaction in Shigella flexneri
Microbiology 161, 1797 (2015); https://doi.org/10.1099/mic.0.000132
/content/journal/micro/10.1099/mic.0.000132
/content/journal/micro/10.1099/mic.0.000132
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