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Journal of Bacteriology, January 2003, p. 349-358, Vol. 185, No. 1
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.1.349-358.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Transient Association of an Alternative Sigma Factor, ComX, with RNA Polymerase during the Period of Competence for Genetic Transformation in Streptococcus pneumoniae

Ping Luo and Donald A. Morrison*

Laboratory for Molecular Biology, Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois 60607

Received 13 June 2002/ Accepted 8 August 2002

Natural transformation in Streptococcus pneumoniae is regulated by a quorum-sensing system that acts through accumulation and sensing of a peptide pheromone (competence-stimulating peptide [CSP]) to control many competence-specific genes acting in DNA uptake, processing, and integration. The period of competence induced by CSP lasts only 15 min (quarter-height peak width). The recently identified regulator ComX is required for the CSP-dependent expression of many competence-specific genes that share an unusual consensus sequence (TACGAATA) at their promoter regions. To test the hypothesis that this regulator acts as a transient alternative sigma factor, ComX was purified from an Escherichia coli overexpression strain and core RNA polymerase was purified from a comX-deficient S. pneumoniae strain. The reconstituted ComX-polymerase holoenzyme produced transcripts for the competence-specific genes ssbB, cinA, cglA, celA, and dalA and was inhibited by anti-ComX antibody, but not by anti-{sigma}70 antibody. Western blotting using antibodies specific for ComX, {sigma}70, and poly-His revealed a transient presence of ComX for a period of 15 to 20 min after CSP treatment, while RNA polymerase remained at a constant level and {sigma}A remained between 60 and 125% of its normal level. ComX reached a molar ratio to RNA polymerase of at least 1.5. We conclude that ComX is unstable and acts as a competence-specific sigma factor.

* Corresponding author. Mailing address: University of Illinois at Chicago, Room 4110, 900 S. Ashland Ave., Chicago, IL 60607. Phone: (312) 996-6839. Fax: (312) 413-2691. E-mail: damorris{at}uic.edu.

Journal of Bacteriology, January 2003, p. 349-358, Vol. 185, No. 1
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.1.349-358.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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