Mutational analysis of the Shigella flexneri O-antigen polymerase Wzy: identification of Wzz-dependent Wzy mutants.
J Bacteriol
; 197(1): 108-19, 2015 Jan 01.
Article
en En
| MEDLINE
| ID: mdl-25313393
ABSTRACT
The O-antigen (Oag) component of lipopolysaccharide (LPS) is a major virulence determinant of Shigella flexneri and is synthesized by the O-antigen polymerase, WzySf. Oag chain length is regulated by chromosomally encoded WzzSf and pHS-2 plasmid-encoded WzzpHS2. To identify functionally important amino acid residues in WzySf, random mutagenesis was performed on the wzySf gene in a pWaldo-TEV-GFP plasmid, followed by screening with colicin E2. Analysis of the LPS conferred by mutated WzySf proteins in the wzySf-deficient (Δwzy) strain identified 4 different mutant classes, with mutations found in periplasmic loop 1 (PL1), PL2, PL3, and PL6, transmembrane region 2 (TM2), TM4, TM5, TM7, TM8, and TM9, and cytoplasmic loop 1 (CL1) and CL5. The association of WzySf and WzzSf was investigated by transforming these mutated wzySf plasmids into a wzySf- and wzzSf-deficient (Δwzy Δwzz) strain. Comparison of the LPS profiles in the Δwzy and Δwzy Δwzz backgrounds identified WzySf mutants whose polymerization activities were WzzSf dependent. Colicin E2 and bacteriophage Sf6c sensitivities were consistent with the LPS profiles. Analysis of the expression levels of the WzySf-GFP mutants in the Δwzy and Δwzy Δwzz backgrounds identified a role for WzzSf in WzySf stability. Hence, in addition to its role in regulating Oag modal chain length, WzzSf also affects WzySf activity and stability.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Shigella flexneri
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Proteínas Bacterianas
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Regulación Bacteriana de la Expresión Génica
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Regulación Enzimológica de la Expresión Génica
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Glicosiltransferasas
Tipo de estudio:
Diagnostic_studies
Idioma:
En
Revista:
J Bacteriol
Año:
2015
Tipo del documento:
Article
País de afiliación:
Australia