CtsR Regulation in mcsAB -Deficient Gram-Positive Bacteria

Abstract

CtsR is an important repressor that modulates the transcription of class III stress genes in Gram-positive bacteria. In Bacillus subtilis, a model Gram-positive organism, the DNA binding activity of CtsR is regulated by McsAB-mediated phosphorylation of the protein where phosphorylated CtsR is a substrate for degradation by the ClpCP complex. Surprisingly, the mcsAB genes are absent from many Gram-positive bacteria, including streptococci; therefore, how CtsR activity is modulated in those bacteria remains unknown. Here we show that the posttranslational modulation of CtsR activity is different in Streptococcus mutans, a dental pathogen. We observed that of all of the Clp-related proteins, only ClpL is involved in the degradation of CtsR. Neither ClpP nor ClpC had any effect on the degradation of CtsR. We also found that phosphorylation of CtsR on a conserved arginine residue within the winged helix-turn-helix domain is necessary for modulation of the repressor activity of CtsR, as demonstrated by both in vitro and in vivo assays. We speculate that CtsR is regulated posttranslationally by a different mechanism in S. mutans and possibly in other streptococci.

ClpL Is Required for Folding of CtsR in Streptococcus mutans

“…CtsR, a major repressor for the Clp ATPases (34,35), mainly regulates the expression of stress response genes by recognizing a tandemly repeated heptanucleotidic sequence known as the CtsR box (35). We recently demonstrated that CtsR is accumulated in larger amounts in cells that do not have a functional ClpL protein (36). This finding was interesting since in most Gram-positive bacteria, CtsR is degraded by ClpCP (37).…”

“…Our previous study showed that the CtsR protein accumulated in large amounts in a clpL-deficient S. mutans strain (36). Further analyses suggested that the accumulated CtsR (HisCtsR) is present predominantly in the pellet fraction and not in the soluble fraction of the cell lysate from the ⌬clpL strain (IBSJ3/pIBJ1).…”

“…The efficiency of renaturation was determined by measuring the intensity of protein bands with ImageQuantTL software on a Coomassie blue-stained gel. The electrophoretic mobility shift assay (EMSA) used to verify the DNA-binding activity of refolded CtsR-Stag was performed as previously described (36). To test the potential for the folding of CtsR aggregates by ClpL, insoluble CtsR-Stag aggregates were incubated with His-ClpL in refolding buffer (20 mM Tris-Cl, 50 mM NaCl, 1 mM DTT, 10% glycerol, 4 mM ATP [pH 7.4]) for 4 h at 37°C.…”

“…Plasmid pIB521 was linearized with BglI and transferred into S. mutans IBSJ3 cells via natural transformation, using a protocol described previously (39), to generate strain IBSJ9. A ␤-glucuronidase assay was performed as previously described (36). Briefly, the OD 600 of an exponential-phase culture was recorded before cell harvesting.…”

Degradation of SsrA-tagged proteins in streptococci

“…A markerless gene replacement method that utilized the Cre-loxP-based recombination system was used for deleting clpC, clpE and clpX genes as described previously (Banerjee & Biswas, 2008;Biswas et al, 2007;Tao et al, 2012). The mutant constructs were verified by PCR and sequencing.…”