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Microbiology 154 (2008), 1104-1116; DOI  10.1099/mic.0.2007/014225-0
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Microbiology 154 (2008), 1104-1116; DOI  10.1099/mic.0.2007/014225-0
© 2008 Society for General Microbiology

Coiled-coil regions play a role in the function of the Shigella flexneri O-antigen chain length regulator WzzpHS2

Leanne Purins, Luisa Van Den Bosch, Vanessa Richardson and Renato Morona

Australian Bacterial Pathogenesis Program, Discipline of Microbiology and Immunology, School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA 5005, Australia

Correspondence
Renato Morona
renato.morona{at}adelaide.edu.au

Regulation of the length of the O-antigen (Oag) chain attached to LPS in Shigella flexneri 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 WzzpHS2 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 WzzpHS2. The in vivo 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 WzzpHS2 and the mutant constructs in a S. flexneri degP mutant showed that DegP did not affect the function of wild-type WzzpHS2 but its presence influenced the phenotype of the WzzpHS2 CCR3 mutant. Additionally, the phenotype of the WzzpHS2 CCR3 mutant was suppressed by a cis mutation near the putative cytoplasmic C-terminus of WzzpHS2.


Abbreviations: CC, coiled-coil; CCR, coiled-coil region; IM, inner membrane; Oag, O-antigen; PCP, polysaccharide copolymerase; RU, repeat unit; S-type, short-type; TM, transmembrane; ts, temperature sensitive; VL-type, very long type

The GenBank accession number for the sequence reported in this paper is EU220028.




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