The objective of this study was to determine how ComE co-ordinates the temporal gene expression of both nlmC and comC. Both luciferase gene fusion and real-time RT-PCR were used to characterize promoter activities of nlmC and comC in different mutant backgrounds. EMSAs were used to analyse ComE binding to the wild-type and DR deletion mutants of the nlmC–comC intergenic region. We demonstrated that ComE is required for both activation of nlmC transcription and repression of comC gene expression. Furthermore, we showed that the two 11 bp direct repeats in the upstream regions of nlmC and comC are required for ComE binding to the promoter region, suggesting that the two direct repeats may serve as the binding sites for ComE.
The strongest evidence supporting the bifunctionality of ComE in regulating nlmC and comC gene expression came from the mutagenesis studies of comE. In a comE null mutant background, nlmC gene expression was nearly abolished, whilst comC gene expression was increased to the same level as in CSP-induced wild-type cells (). Further support for ComE bifunctionality came from the expression pattern analyses of nlmC and comC. In overnight cultures, when ComE was presumably absent, comC gene expression displayed its strongest induction; in contrast, nlmC gene expression was reduced to below detectable levels. In addition, the differential response of nlmC and comC to CSP induction could also add further support to the opposing regulatory mechanisms for nlmC and comC gene expression via ComE. For example, the maximal induction for nlmC is usually between 50- and 100-fold (), whereas the maximal induction for comC is usually between 3- and 5-fold (). In addition to the potentially dissimilar promoter strengths, an additional factor in the different levels of CSP induction could result from the positive feedback loop regulation of nlmC gene expression vs the derepressive regulation of comC gene expression. Unlike nlmC induction, the derepression of comC in response to CSP could not be further enhanced by autoinduction of the comED operon.
As a further support for the direct role of ComE in nlmC and comC gene regulation, purified ComE was shown to bind to the nlmC–comC intergenic region. This binding was also dependent upon the presence of two direct repeats, which are reminiscent of the S. pneumoniae ComE binding site. In the presence of both direct repeats, there are two shifted bands, presumably reflecting binding of ComE to either one or both direct repeats (). Deletion of one direct repeat resulted in only one shifted band, whilst deletion of both direct repeats abolished ComE binding completely. These results suggest that ComE can bind to either direct repeat; however, whether the presence of both direct repeats promotes cooperative binding is not known.
The direct involvement of the two direct repeats in gene expression of nlmC
was tested by deletion mutagenesis. Deletion of the two direct repeats diminished nlmC
gene expression (). This is consistent with previous findings (van der Ploeg, 2005
), which showed that point mutations made in either of the direct repeats severely impaired nlmA
gene expression [nlmA
has a near-identical promoter region to nlmC
(Kreth et al., 2006
)]. Unexpectedly, the same direct repeat deletion also abolished comC
gene expression. This result is rather puzzling as the two direct repeats exist only on the DNA strand encoding the promoter of nlmC
, not that encoding the comC
promoter. Two explanations may be entertained at this point. (1) The promoter region of comC
may overlap the direct repeats region; thus deletion of the direct repeats may have abolished RNA polymerase binding to the comC
promoter. (2) The comC
transcript may start further upstream of the direct repeat region and the direct repeat region is required for RNA stability of the comC
transcript. Since real-time RT-PCR measures RNA at steady state, rapid degradation of the comC
transcript as a result of the DR deletion may have resulted in the nearly undetectable levels of comC
gene expression as observed in . Although the detailed mechanisms of how ComE regulates comC
gene expression await further research, our data appear to suggest that the mechanism of comC
transcription is more complicated than is currently accepted.
Taken together, this study demonstrates that ComE plays a very different regulatory role in S. mutans
from that of its homologue in S. pneumoniae
. Interestingly, both of the C-termini from the ComE proteins of S. pneumoniae
and S. mutans
share high homology to the DNA binding domains of AlgR/AgrA/LytR family members. In addition, both proteins bind to direct repeat sequences that match extremely well to the predicted binding sites for the AlgR/AgrA/LytR family members (Nikolskaya & Galperin, 2002
). However, despite this similarity, S. mutans
has evolved a repressive mode of regulation for comC
and a delayed competence response to the addition of CSP. This suggests that the mechanism of competence regulation in S. mutans
may be quite distinct from that in S. pneumoniae