ClpC acts as a negative regulator of competence in Streptococcus thermophilus

Abstract

The alternative sigma factor ComX is a key regulator of natural transformation in members of the genus Streptococcus. ComX controls expression of the late competence genes, which are essential for DNA binding, uptake and recombination. In Streptococcus pneumoniae, it has been demonstrated that ComX is degraded by ClpEP at the end of the competence period. In the present study we show that a different Clp protease complex, ClpCP, contributes to ComX degradation in Streptococcus thermophilus. Mutant strains lacking the ClpC chaperone displayed significantly increased transformability compared with the wild-type strain under conditions where ComX was expressed at relatively low levels. At higher expression levels, ClpCP appears to become saturated and unable to prevent the accumulation of ComX. Together, our results suggest that the role of ClpC is to mediate degradation of ComX when the sigma factor is produced in low amounts, i.e. when the environmental stimulus promoting competence development is weak. This would prevent S. thermophilus from developing the competent state at an inappropriate time and/or place.

Adaptor Protein MecA Is a Negative Regulator of the Expression of Late Competence Genes in Streptococcus thermophilus

“…The implication of ClpC in the posttranslational control of ComX was clearly demonstrated, while the role of ClpE in competence development was not further investigated (1). Concerning the role of ClpE, our results based on reporter strains, Western blotting experiments, and B2H assays show that ClpE does not play a significant role in the control of competence development in strain LMD-9 of S. thermophilus.…”

“…However, the inactivation of mecA is insufficient to activate natural transformation under these conditions (data not shown), while the overexpression of comX led to a low but detectable transformation rate (16). Similarly, no improvement in the transformation rate of a ClpC-deficient strain of LMG18311 grown under nonpermissive THBG conditions was reported (1). Various hypotheses could explain this apparent discrepancy, such as the absence of an induction of one or more key late com genes that could be required for natural transformation or an indirect effect of the inactivation of MecA that could negatively alter the cell status for natural transformation.…”

“…For S. thermophilus LMG18311, both ClpC and ClpE were recently shown to repress the activity of a late com promoter (1). Furthermore, the depletion of ClpC in this strain had no impact on the activity of P comX , but the abundance of ComX was shown previously to be increased in the clpC mutant (1). In order to compare the effects of the deprivation of MecA, ClpC, and ClpE on the activity of P comGA in LMD-9, a range of reporter strains (CB007 derivatives) were constructed, and their Lux activities were compared under conditions not permissive for competence (THBG).…”

“…LMD-9 derivative strains AW001 (ClpC Ϫ ) and AW003 (ClpE Ϫ ), CB007 derivative reporter strains AW002 (ClpC Ϫ ) and AW004 (ClpE Ϫ ), and CB0072 derivative reporter strains AW005 (MecA Ϫ ClpC Ϫ ) and AW006 (MecA Ϫ ClpE Ϫ ) were constructed by replacing the coding sequence of clpC or clpE with a spectinomycin resistance cassette (spc). This was achieved by natural competence (16) using PCR fragments amplified from chromosomal DNA extracted from the corresponding mutant strains of S. thermophilus LMG 18311 (a generous gift from L. S. Håvarstein) (1). The primers used are listed in Table S1 in the supplemental material.…”

“…In Streptococcus pyogenes, the mechanism of ComX stabilization/ degradation is still unknown, except for the implication of the ClpP protease (38). For S. thermophilus, previous extensive in silico analyses did not allow the identification of a homolog of ComW, but during the course of that study, the ClpC ATPase subunit was shown to act as a negative regulator of competence development in this species (1). For Bacillus subtilis, the posttranslational control of the master regulator of competence development, ComK, has also been highlighted (25-27, 30, 36, 37, 40, 43, 45, 47-49).…”