H-045. Competence for Genetic Transformation in Streptococcus pneumoniae: Termination of Activity of the Alternative Sigma Factor, ComX, Is Independent of Proteolysis and of Expression of Early Genes

A. Piotrowski, D. A. Morrison;
Univ. of Illinois, Chicago, IL.

Competence for genetic transformation in S. pneumoniae is a transient physiological state whose appearance and rapid disappearance is coordinated by a peptide pheromone (CSP). The pheromone receptor activates transcription of two downstream genes, comX and comW, and about 15 other “early” genes, some of whose functions are unknown. ComX, a sigma factor that appears transiently during competence induction, drives transcription of “late” genes, many of which are essential for transformation. In vivo, ComW stabilizes ComX against proteolysis by protease ClpEP and stimulates activity of ComX. Interestingly, stabilization of ComX by a clpP mutation does not extend the period of competence. We considered the hypothesis that the rapid decay of competence arises from a rapid loss of ComW, and thus of its ComX stimulating activity, so that ComX might persist but have little transcriptional activity. By Western Analysis we found that ComW is indeed a transient protein and that its lability is dependent on the protease ClpCP. To ask whether stabilizing both ComX and ComW would prolong ComX activity we monitored late gene expression in a clpP mutant by fusing lacZ to the late competence gene, ssbB. Late gene transcription terminated as rapidly in the clpP mutant as in wild type. We conclude that termination of late gene expression is not dependent on proteolysis of ComX or ComW. To test if early genes are required for termination of ComX activity, an artificial comXcomW operon was inserted at the Aga locus so comX and comW could be regulated independently of any other early genes. For a high level of competence, this strain required raffinose, growth at 30C, a high level of aga expression, and a deficiency in clpC. When all of these conditions were met, competence and late gene expression in this strain reached levels similar to those in wild type cells induced with CSP. Competence and late gene expression were also transient in this strain despite continued expression of aga and even when both clpE and clpC are missing. From this, we conclude that termination of late gene expression does not require induction of any early genes.