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Journal of Bacteriology, May 2009, p. 3024-3040, Vol. 191, No. 9
0021-9193/09/$08.00+0     doi:10.1128/JB.01505-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Influences of Capsule on Cell Shape and Chain Formation of Wild-Type and pcsB Mutants of Serotype 2 Streptococcus pneumoniae {triangledown} ,{dagger}

Skye M. Barendt,1 Adrian D. Land,1 Lok-To Sham,1 Wai-Leung Ng,1,{ddagger} Ho-Ching T. Tsui,1 Randy J. Arnold,2 and Malcolm E. Winkler1*

Department of Biology,1 Department of Chemistry, Indiana University Bloomington, Bloomington, Indiana 474052

Received 24 October 2008/ Accepted 20 February 2009

PcsB is a protein of unknown function that plays a critical role in cell division in Streptococcus pneumoniae and other ovococcus species of Streptococcus. We constructed isogenic sets of mutants expressing different amounts of PcsB in laboratory strain R6 and virulent serotype 2 strain D39 to evaluate its cellular roles. Insertion mutagenesis in parent and pcsB+ merodiploid strains indicated that pcsB is essential in serotype 2 S. pneumoniae. Quantitative Western blotting of wild-type and epitope-tagged PcsB showed that all PcsB was processed into cell-associated and secreted forms of the same molecular mass and that cell-associated PcsB was moderately abundant and present at {approx}4,900 monomers per cell. Controlled expression and complementation experiments indicated that there was a causative relationship between the severity of defects in cell division and decreasing PcsB amount. These experiments also showed that perturbations of expression of the upstream mreCD genes did not contribute to the cell division defects of pcsB mutants and that mreCD could be deleted. Unexpectedly, capsule influenced the cell shape and chain formation phenotypes of the wild-type D39 strain and mutants underexpressing PcsB or deleted for other genes involved in peptidoglycan biosynthesis, such as dacA. Underexpression of PcsB did not result in changes in the amounts or composition of lactoyl-peptides, which were markedly different in the R6 and D39 strains, and there was no correlation between decreased PcsB amount and sensitivity to penicillin. Finally, microarray analyses indicated that underexpression of PcsB may generate a signal that increases expression of the VicRK regulon, which includes pcsB.

* Corresponding author. Mailing address: Department of Biology, Indiana University Bloomington, Jordan Hall, Room 142, Bloomington, IN 47405. Phone: (812) 856-1318. Fax: (812) 855-6705. E-mail: mwinkler{at}bio.indiana.edu

{triangledown} Published ahead of print on 6 March 2009.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

{ddagger} Present address: 322 Lewis Thomas Lab, Princeton University, Washington Rd., Princeton, NJ 08544.

Journal of Bacteriology, May 2009, p. 3024-3040, Vol. 191, No. 9
0021-9193/09/$08.00+0     doi:10.1128/JB.01505-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



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