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Two Distinct Functions of ComW in Stabilization and Activation of the Alternative Sigma Factor ComX in Streptococcus pneumoniae

Chang Kyoo Sung and Donald A. Morrison^*

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

Received 21 October 2004/ Accepted 27 January 2005

Natural genetic transformation in Streptococcus pneumoniae is controlled by a quorum-sensing system, which acts through the competence-stimulating peptide (CSP) for transient activation of genes required for competence. More than 100 genes have been identified as CSP regulated by use of DNA microarray analysis. One of the CSP-induced genes required for genetic competence is comW. As the expression of this gene depended on the regulator ComE, but not on the competence sigma factor ComX (^X), and as expression of several genes required for DNA processing was affected in a comW mutant, comW appears to be a new regulatory gene. Immunoblotting analysis showed that the amount of the ^X protein is dependent on ComW, suggesting that ComW may be directly or indirectly involved in the accumulation of ^X. As ^X is stabilized in clpP mutants, a comW mutation was introduced into the clpP background to ask whether the synthesis of ^X depends on ComW. The clpP comW double mutant accumulated an amount of ^X higher (threefold) than that seen in the wild type but was not transformable, suggesting that while comW is not needed for ^X synthesis, it acts both in stabilization of ^X and in its activation. Modification of ComW with a histidine tag at its C or N terminus revealed that both amino and carboxyl termini are important for increasing the stability of ^X, but only the N terminus is important for stimulating its activity.

Present address: Dept. of Pathology NRB-0939, Harvard Medical School, Boston, MA 02115.

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