The Transcriptional Repressor CcpN from Bacillus subtilis Uses Different Repression Mechanisms at Different Promoters

Abstract

CcpN, a transcriptional repressor from Bacillus subtilis that is responsible for the carbon catabolite repression of three genes, has been characterized in detail in the past 4 years. However, nothing is known about the actual repression mechanism as yet. Here, we present a detailed study on how CcpN exerts its repression effect at its three known target promoters of the genes sr1, pckA, and gapB. Using gel shift assays under nonrepressive and repressive conditions, we showed that CcpN and RNA polymerase can bind simultaneously and that CcpN does not prevent RNA polymerase (RNAP) binding to the promoter. Furthermore, we investigated the effect of CcpN on open complex formation and demonstrate that CcpN also does not act at this step of transcription initiation at the sr1 and pckA and presumably at the gapB promoter. Investigation of abortive transcript synthesis revealed that CcpN acts differently at the three promoters: At the sr1 and pckA promoter, promoter clearance is impeded by CcpN, whereas synthesis of abortive transcripts is repressed at the gapB promoter. Eventually, we demonstrated with Far Western blots and co-elution experiments that CcpN is able to interact with the RNAP ␣-subunit, which completes the picture of the requirements for the repressive action of CcpN. On the basis of the presented results, we propose a new working model for CcpN action.CcpN, a transcriptional repressor from Bacillus subtilis, mediates CcpA-independent carbon catabolite repression of at least three genes: sr1, encoding a small RNA and pckA and gapB (1, 2), encoding two gluconeogenic enzymes (3, 4). Since its discovery in 2005, CcpN has been thoroughly investigated. Binding properties and binding motifs were examined, revealing that CcpN possesses two asymmetric binding sites that are bound cooperatively and positioned differently at the three regulated promoters (5): At the sr1 promoter, binding sites are located upstream of the Ϫ35 region and between the Ϫ35 and the Ϫ10 region, while binding sites cover the Ϫ35 as well as the Ϫ10 region at the pckA promoter. One operator at the gapB promoter overlaps the Ϫ10 region, the second one is located around ϩ20. ATP and low pH have been identified as signals required for CcpN-mediated repression (6) and the detailed biophysical properties of CcpN-DNA interaction have been reported (7). In addition, it has been shown that CcpN controls central carbon fluxes in the metabolism of B. subtilis and that the growth defect of CcpN knock-out mutants is caused by ATP dissipation via extensive futile cycling (8). It has been demonstrated that a CcpN knock-out is able to increase the industrial production of riboflavin in B. subtilis by a deregulation of the gapB gene (9). However, nothing is known about the actual repression mechanism of CcpN as yet.Initiation of transcription is a stepwise process (10), beginning with binding of RNA polymerase (RNAP) 2 to the promoter and formation of a loose closed complex, which is then rearranged into a tighter closed complex. This is followed by the ...

Dual-Function RNAs

“…The 205-nt sRNA is expressed under gluconeogenic and repressed under glycolytic conditions mainly by CcpN but also to a minor extent by CcpA, both regulators are involved in carbon catabolite repression (Figure 4) (22, 62–64). …”

Characterisation of Bacillus subtilis Transcriptional Regulators Involved in Metabolic Processes

“…The repression mechanism of CcpN has been elucidated using various techniques [91]. EMSAs demonstrated that CcpN and RNA polymerase can bind simultaneously to the sr1, pckA or gapB promoter and that CcpN cannot impede closed complex formation at these promoters.…”

Small regulatory RNAs from low-GC Gram-positive bacteria

“…CcpN binds upstream of and overlapping the sr1 promoter, 33 whereas CcpA binds 250 upstream of the transcription start site (TSS) at a cre site. CcpN represses sr1 transcription in the presence of ATP and slightly acidic pH (6.5) 34 by interacting with the α-subunit of the RNA polymerase, thereby inhibiting promoter escape 35 . Transcriptomics and northern blotting suggested a second target for SR1, the gapA operon.…”