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Title
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Mutational analysis of the major periplasmic loops of Shigella flexneri Wzy: identification of the residues affecting O antigen modal chain length control, and Wzz-dependent polymerization activity.
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Authors
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P.Nath,
R.Morona.
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Ref.
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Microbiology, 2015,
161,
774-785.
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PubMed id
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Abstract
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The O antigen (Oag) component of LPS is a major Shigella flexneri virulence
determinant. Oag is polymerized by WzySf, and its modal chain length is
determined by WzzSf and WzzpHS2. Site-directed mutagenesis was performed on
wzySf in pWaldo-wzySf-TEV-GFP to alter Arg residues in WzySf's two large
periplasmic loops (PLs) (PL3 and PL5). Analysis of the LPS profiles conferred by
mutated WzySf proteins in the wzySf deficient (Δwzy) strain identified residues
that affect WzySf activity. The importance of the guanidium group of the Arg
residues was investigated by altering the Arg residues to Lys and Glu, which
generated WzySf mutants conferring altered LPS Oag modal chain lengths. The
dependence of these WzySf mutants on WzzSf was investigated by expressing them
in a wzySf and wzzSf deficient (Δwzy Δwzz) strain. Comparison of the LPS
profiles identified a role for the Arg residues in the association of WzySf and
WzzSf during Oag polymerization. Colicin E2 and bacteriophage Sf6c
susceptibility supported this conclusion. Comparison of the expression levels of
different mutant WzySf-GFPs with the wild-type WzySf-GFP showed that certain Arg
residues affected production levels of WzySf in a WzzSf-dependent manner. To our
knowledge, this is the first report of S. flexneri WzySf mutants having an
effect on LPS Oag modal chain length, and identified functionally significant
Arg residues in WzySf.
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