ComX activity of growing in biofilms

Abstract

In many streptococci, including Streptococcus mutans, genetic competence is regulated by a quorum sensing system mediated by a competence stimulating peptide (CSP) pheromone, encoded by the comC gene. In Streptococcus pneumoniae, a central component of this system is ComX, which acts as an alternative sigma factor to activate competence genes involved in DNA uptake and processing. The quorum sensing system responsible for genetic competence induction in S. mutans has been linked to biofilm formation and the acid tolerance response. To examine the response of comX to CSP in S. mutans, a transcriptional fusion of the comX promoter (pcomX) with lacZ was constructed to generate reporter vector pcomx::pALH122 (replicative vector) and transformed into S. mutans UA159 comC-, which is unable to produce endogenous CSP. CSP was added and pcomX::lacZ relative expression index (REI) examined, revealing a 2-fold increase in maximal beta-gal activity 5 and 10 min after CSP addition. The effect of endogenous CSP on pcomX::lacZ expression was also examined by measuring REI in cells grown as a biofilm; peak pcomX activity was observed at 3 h. To determine the temporal pattern of transformation frequency, pMA2, a Spr shuttle vector, was transformed into biofilm-grown cells, with maximal transformation frequency observed at 3 h. Confocal microscopy was performed to examine pcomX activity using a similarly constructed green fluorescent protein reporter vector, pcomX::gfp, in a 4-h biofilm, revealing active pcomX activity in high cell density areas within the biofilm population. These results demonstrated a positive correlation between pcomX activity, natural transformation and competence development in biofilms.

Up-regulation of competence- but not stress-responsive proteins accompanies an altered metabolic phenotype in Streptococcus mutans biofilms

“…As to whether the maintenance of competence in biofilms allows DNA to be assimilated from the external environment primarily as a nutrient source or to increase genetic diversity by recombination remains to be determined (Jefferson, 2004;Claverys et al, 2000). The fact that only 0?03 % of S. mutans are transformed in biofilms (Aspiras et al, 2004) may be evidence in favour of the former proposal and a reassessment of the true function of competence is required in this and other bacteria, particularly when growing in their natural sessile state as biofilms.…”

Plausible Drug Targets in the Streptococcus mutans Quorum Sensing Pathways to Combat Dental Biofilms and Associated Risks

“…The peptides were designed to have a targeted domain (8-aminoacid region of the CSP) fused with an antimicrobial peptide domain to show a robust species SigX (ComX) comR DNA transport, uptake and Recombination [12,36] ComAB nlmAB (comAB) Mutacin IV (bacteriocins), Maturation and export of CSP outside the cell [16,49] ComD comC/comD/comE (TCSTS) Class II bacteriocins and HK receptor for CSP signal [14,22] immA and immB (VicRK) comC Self-immunity proteins for protection from bacteriocins, acid tolerance [55] Lux S luxS Interspecies communication [39,40] Indian…”

“…ComX, an alternative sigma factor (r x ) activates the late competence genes primarily involved in the processing and recombination of the foreign DNA to the chromosome of Streptococci [12]. In S. pneumoniae, the up-regulation of r x is induced in response to the activation of the competence cascade via., CSP-mediated ComCDE pathway.…”

“…Upon activation with the CSP, the membrane-bound HK receptor integrates the signal to its cognate cytoplasmic response regulator (ComD) to further activate the alternate sigma factor, SigX (ComX) [60]. Also, its recognizing sites are well characterized to be a set of non-canonical consensus region (CIN box) that lie in the promoter regions (TACGAATA) of various ComX dependent genes, ssbB, dalA, ccl, celAB, cAAB, and cinA-recA [12,61]. Similarly, in S. mutans, it is postulated that the CSP-mediated activation of r x , an alternate sigma factor up-regulates the expression of several competence genes that includes comX, comCDE (positive feedback loop) and other early com genes, comW (r x Stabilizing factor).…”

“…and comE encodes a precursor of competence-stimulating peptide (CSP), the HK sensor protein, and a cognate RR, respectively [10,12,13]. The genes comC and comDE are closely located on the same chromosome and the peptide (CSP) is synthesized as a collective result of their gene products [14,15].…”