SummaryAntisense-RNAs have been investigated in detail over the past 20 years as the principal regulators in accessory DNA elements such as plasmids, phages and transposons. However, only a few examples of chromosomally encoded bacterial antisense RNAs were known. Meanwhile, ª ª ª ª 70 small non-coding RNAs from the Escherichia coli genome have been found, the functions of the majority of which remain to be elucidated. Only one systematic search has been performed for Gram-positive bacteria, so far. Here, we report the identification of a novel small (205 nt) nontranslated RNA -SR1 -encoded in the Bacillus subtilis genome. SR1 was predicted by a computational approach and verified by Northern blotting. Knockout or overexpression of SR1 did not affect growth. SR1 was derepressed under conditions of gluconeogenesis, but repressed under glycolytic conditions. Two regulatory levels could be identified, one involving CcpA, the second, more important, involving the recently identified regulator CcpN.

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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 ...

Two independent cervimycin C (CmC)-resistant clones of Bacillus subtilis were identified, each carrying two mutations in the intergenic region preceding the ABC transporter gene bmrA. In the double mutant, real-time PCR revealed an increased amount of bmrA mRNA with increased stability. Accordingly, isolation of membrane proteins yielded a strong band at 64 kDa corresponding to BmrA. Analyses showed that one mutation optimized the -35 box sequence conferring resistance to 3 μM CmC, while the +6 mutation alone had no effect, but increased the potential of the strain harboring the -35 mutation to grow at 5 μM CmC. Transcriptional fusions revealed an elevated bmrA promoter activity for the double mutant. Electrophoretic mobility shift assays (EMSAs) confirmed a 30-fold higher binding affinity of RNA polymerase for this mutant compared with the wild type, and the effect was due to the -35 box alteration of the bmrA promoter. In vitro transcription experiments substantiated the results of the EMSA. EMSAs in the presence of heparin indicated that the mutations did not influence the formation and/or the stability of open complexes. Half-life measurements demonstrated that the +6 mutation stabilized bmrA mRNA ≈ 2-fold. Overall, we found that an ABC transporter confers antibiotic resistance by the cumulative effects of two mutations in the promoter region.