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Journal of Bacteriology, May 2007, p. 3369-3381, Vol. 189, No. 9
0021-9193/07/$08.00+0     doi:10.1128/JB.01938-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Mutations Blocking Side Chain Assembly, Polymerization, or Transport of a Wzy-Dependent Streptococcus pneumoniae Capsule Are Lethal in the Absence of Suppressor Mutations and Can Affect Polymer Transfer to the Cell Wall

Bobbi Xayarath and Janet Yother^*

Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294

Received 21 December 2006/ Accepted 13 February 2007

Extracellular polysaccharides of many bacteria are synthesized by the Wzy polymerase-dependent mechanism, where long-chain polymers are assembled from undecaprenyl-phosphate-linked repeat units on the outer face of the cytoplasmic membrane. In gram-positive bacteria, Wzy-dependent capsules remain largely cell associated via membrane and peptidoglycan linkages. Like many Wzy-dependent capsules, the Streptococcus pneumoniae serotype 2 capsule is branched. In this study, we found that deletions of cps2K, cps2J, or cps2H, which encode a UDP-glucose dehydrogenase necessary for side chain synthesis, the putative Wzx transporter (flippase), and the putative Wzy polymerase, respectively, were obtained only in the presence of suppressor mutations. Most of the suppressor mutations were in cps2E, which encodes the initiating glycosyltransferase for capsule synthesis. The cps2K mutants containing the suppressor mutations produced low levels of high-molecular-weight polymer that was detected only in membrane fractions. cps2K-repaired mutants exhibited only modest increases in capsule production due to the effect of the secondary mutation, but capsule was detectable in both membrane and cell wall fractions. Lethality of the cps2K, cps2J, and cps2H mutations was likely due to sequestration of undecaprenyl-phosphate in the capsule pathway and either preclusion of its turnover for utilization in essential pathways or destabilization of the membrane due to an accumulation of lipid-linked intermediates. The results demonstrate that proper polymer assembly requires not only a functional transporter and polymerase but also complete repeat units. A central role for the initiating glycosyltransferase in controlling capsule synthesis is also suggested.

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* Corresponding author. Mailing address: Department of Microbiology, 845 19th Street South, BBRB 661/12, Birmingham, AL 35294-2170. Phone: (205) 934-9531. Fax: (205) 975-6715. E-mail: jyother{at}uab.edu

Published ahead of print on 23 February 2007.

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Journal of Bacteriology, May 2007, p. 3369-3381, Vol. 189, No. 9
0021-9193/07/$08.00+0     doi:10.1128/JB.01938-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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