The carbon catabolite repressor CcpA mediates optimal competence development in Streptococcus oligofermentans through post‐transcriptional regulation

Abstract

Natural transformation increases the genetic diversity of bacteria, but is costly and must be strictly controlled. We previously found that deletion of ccpA, a key regulator of carbon catabolite repression (CCR), reduced transformation efficiency of Streptococcus oligofermentans, the current work further investigated the regulatory mechanisms of CcpA. The competence operon comCDE is subjected to basal and autoregulatory transcription. A luciferase reporter detected a transcriptional readthrough (TRT) from the upstream tRNA Arg into the comCDE operon, which was induced by L -arginine. Insertion of the Escherichia coli T1T2 terminator downstream of tRNA Arg abolished TRT, and reduced the basal com-CDE transcription by 77% and also the transformation efficiency. Deletion of ccpA increased tRNA Arg TRT and tRNA Arg -comCDE polycistronic transcript by twofold. An in vitro transcription assay determined that CcpA promoted the transcription termination of tRNA Arg TRT, and RNA EMSA and SPR assays detected equal binding affinity of CcpA to both the RNA and DNA of tRNA Arg . These results indicate that CcpA controls the basal comCDE transcription by post-transcriptional actions. Overexpression of comDE or its phospho-mimicking mutant comDE D58E reduced transformation efficiency, indicating that excessive ComE impairs competence development. CCR-regulated competence was further confirmed by higher tRNA Arg TRT but lower transformation efficiency in galactose than in glucose.

The RNA-bound proteome of MRSA reveals post-transcriptional roles for helix-turn-helix DNA-binding and Rossmann-fold proteins

“…This suggests that CcpA could have a role in transcription termination or regulates the stability of these RNAs. Consistent with the former, Streptococcus oligofermentans CcpA binds tRNA Arg in vitro and deletion of ccpA resulted in modest transcriptional readthrough of tRNA Arg 45 . This points towards a role for CcpA in transcription termination.…”

Investigating the Streptococcus sinensis competence regulon through a combination of transcriptome analysis and phenotypic evaluation

“…Another example is catabolite control protein A (ccpA), a regulatory metabolic protein that directs selective metabolism of available sugars and has been found to play a role in competence in two Mitis group streptococci: S. cristatus and S. gordonii [61,62]. Transcriptome analysis of S. gordonii during competence revealed that ccpA was downregulated during the competence state, while a similar observation was made with S. sinensis Forsyth1A (Table 5) [23].…”

“…Researchers had previously found that CcpA indirectly aids in the regulation of early competence genes, including comCDE, in Mitis group streptococci [62,76]. For example, it was found that an S. oligofermentans ΔccpA mutant had higher expression levels of phosphorylated ComE, which is known to bear great influence on other competence genes, such as comX [61].…”

“…For example, it was found that an S. oligofermentans Δ ccpA mutant had higher expression levels of phosphorylated ComE, which is known to bear great influence on other competence genes, such as comX [61]. As such, ccpA is potentially downregulated in early competence to allow for the adequate expression of early competence genes that are needed for successful transformation.…”

RNase Z Oxidative Degradation Impedes tRNA Maturation and is Involved in Streptococcal Translation Regulation in Response to Oxidative Stress

“…5′ and 3′ RACEs were performed as described previously ( 57 ) with slight modifications. Briefly, So-RNaseZ-digested products of tRNA precursor transcripts were purified on 10% urea-PAGE gels using a ZR small-RNA PAGE recovery kit (Zymo Research, Irvine, CA, USA), and then the 5′ and 3′ ends were ligated with 50 pmol of universal microRNA (miRNA)-cloning linker 5′-CAGACUGGAUCCGUCCUC-App-3′ and 5′-AppCUGUAGGCACCAUCAAU-ddC-3′ (synthesized by Integrated DNA, Coralville, IA, USA), respectively, with the addition of 20 U of T4 RNA ligase (Ambion, Austin, TX, USA) in the presence of 40 U of RNasin-Plus.…”