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Journal of Bacteriology, November 2005, p. 7444-7459, Vol. 187, No. 21
0021-9193/05/$08.00+0     doi:10.1128/JB.187.21.7444-7459.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Regulation of the pspA Virulence Factor and Essential pcsB Murein Biosynthetic Genes by the Phosphorylated VicR (YycF) Response Regulator in Streptococcus pneumoniae{dagger}

Wai-Leung Ng, Ho-Ching Tiffany Tsui, and Malcolm E. Winkler*

Department of Biology, Indiana University, Bloomington, Indiana 47405

Received 19 June 2005/ Accepted 8 August 2005

The VicRK (YycFG) two-component regulatory system (TCS) is required for virulence of the human respiratory pathogen Streptococcus pneumoniae (pneumococcus). The VicR (YycF) response regulator (RR) is essential through its positive regulation of pcsB, which encodes an extracellular protein that mediates murein biosynthesis. To determine other genes that are regulated by VicR, we performed microarray analyses on a unique {Delta}vicR deletion mutant, which was constructed by uncoupling regulation of pcsB. Results from these microarray experiments support the idea that the VicR RR exerts strong positive regulation on the transcription of a set of genes encoding important surface proteins, including the PspA virulence factor, two proteins (Spr0096 and Spr1875) containing LysM peptidoglycan-binding domains, and a putative membrane protein (Spr0709) of unknown function. To demonstrate direct regulation, we performed band shift and footprinting experiments using purified unphosphorylated VicR and phosphorylated VicR-P, which was prepared by reaction with acetyl phosphate. VicR and VicR-P bound to regions upstream of pcsB, pspA, spr0096, spr1875, and spr0709. Phosphorylation of VicR to VicR-P increased the apparent strength and changed the nature of binding to these regions. DNase I footprinting of VicR and VicR-P bound to regions upstream of pcsB, pspA, spr0096, and spr1875 showed protection of extended regions containing a degenerate sequence related to a previously proposed consensus. These combined approaches did not support autoregulation of the vicRKX operon or substantive direct regulation of fatty acid biosynthesis by VicR or VicR-P. However, the {Delta}vicR mutant required fatty acids in some conditions, which supports the notion that the VicRK TCS may mediate membrane integrity as well as murein biosynthesis and virulence factor expression in S. pneumoniae.

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

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

Journal of Bacteriology, November 2005, p. 7444-7459, Vol. 187, No. 21
0021-9193/05/$08.00+0     doi:10.1128/JB.187.21.7444-7459.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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