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In Pseudomonas aeruginosa , the CbrA/CbrB two-component system is instrumental in the maintenance of the carbon-nitrogen balance and for growth on carbon sources that are energetically less favorable than the preferred dicarboxylate substrates. The CbrA/CbrB system drives the expression of the small RNA CrcZ, which antagonizes the repressing effects of the catabolite repression control protein Crc, an RNA-binding protein. Dicarboxylates appear to cause carbon catabolite repression by inhibiting the activity of the CbrA/CbrB system, resulting in reduced crcZ expression. Here we have identified a conserved palindromic nucleotide sequence that is present in upstream activating sequences (UASs) of promoters under positive control by CbrB and σ 54 RNA polymerase, especially in the UAS of the crcZ promoter. Evidence for recognition of this palindromic sequence by CbrB was obtained in vivo from mutational analysis of the crcZ promoter and in vitro from electrophoretic mobility shift assays using crcZ promoter fragments and purified CbrB protein truncated at the N terminus. Integration host factor (IHF) was required for crcZ expression. CbrB also activated the lipA (lipase) promoter, albeit less effectively, apparently by interacting with a similar but less conserved palindromic sequence in the UAS of lipA . As expected, succinate caused CbrB-dependent catabolite repression of the lipA promoter. Based on these results and previously published data, a consensus CbrB recognition sequence is proposed. This sequence has similarity to the consensus NtrC recognition sequence, which is relevant for nitrogen control.

The cip-cel cluster of genes plays an important role in the catabolism of the substrate cellulose by Clostridium cellulolyticum . It encodes several key components of the cellulosomes, including the scaffolding protein CipC and the major cellulase Cel48F. All the genes of this cluster display linked transcription, focusing attention on the promoter upstream from the first gene, cipC . We analyzed the regulation of the cipC promoter using a transcriptional fusion approach. A single promoter is located between nucleotides −671 and −643 with respect to the ATG start codon, and the large mRNA leader sequence is processed at position −194. A catabolite-responsive element (CRE) 414 nucleotides downstream from the transcriptional start site has been shown to be involved in regulating this operon by a carbon catabolite repression mechanism. This CRE is thought to bind a CcpA-like regulator complexed with a P-Ser-Crh-like protein. Sequences surrounding the promoter sequence may also be involved in direct (sequence-dependent DNA curvature) or indirect (unknown regulator binding) regulation.

Twelve genes encoding key components of Clostridium cellulolyticum cellulosomes are clustered. Among them, the first, second, and fifth genes encode the assembly factor CipC and the two major cellulases Cel48F and Cel9E, respectively. Cellulolytic clones were selected from the noncellulolytic cipC insertional mutant trans -complemented with a cipC expression vector, in which one homologous recombination event between the 3′ end of the chromosomal cipC gene and the plasmidic cipC gene has restored the cluster continuity. The absence of the enzymes encoded by the cluster in the cipC mutant was thus only due to a strong polar effect, indicating that all genes were transcriptionally linked. Two large transcripts were detected in cellulose-grown cells by Northern hybridization: a 14-kb messenger which carries the cipC-cel48F-cel8C-cel9G-cel9E coding sequences and, in a smaller amount, a 12-kb messenger which carries the genes located in the 3′ part of the cluster. Four smaller transcripts were found in large amounts: a cipC-cel48F bicistronic one and three monocistronic ones, cipC , cel48F , and cel9E . The cipC-cel48F and cel48F messengers were shown to be stable. Analysis by reverse transcription-PCR suggested transcriptional linkage of all of the open reading frames. The production of a primary very large transcript covering the entire cluster was hypothesized. Primer extension analysis has identified two putative transcriptional start sites located 638/637 and 194 nucleotides upstream of the cipC translational start. The processing of the primary transcript would lead to the production of several secondary messengers displaying different stabilities, contributing to fine tuning of expression of individual genes of the operon.

Clostridium cellulolyticum secretes large multienzymatic complexes with plant cell wall-degrading activities named cellulosomes. Most of the genes encoding cellulosomal components are located in a large gene cluster: cipC - cel 48 F - cel 8 C-cel 9 G-cel 9 E - orfX - cel 9 H-cel 9 J-man 5 K-cel 9 M . Downstream of the cel 9 M gene, a new open reading frame was discovered and named rgl 11 Y . Amino acid sequence analysis indicates that this gene encodes a multidomain pectinase, Rgl11Y, containing an N-terminal signal sequence, a catalytic domain belonging to family 11 of the polysaccharide lyases, and a C-terminal dockerin domain. The present report describes the biochemical characterization of a recombinant form of Rgl11Y. Rgl11Y cleaves the α- l -Rha p -(1→4)-α- d -Gal p A glycosidic bond in the backbone of rhamnogalacturonan I (RGI) via a β-elimination mechanism. Its specific activity on potato pectic galactan and rhamnogalacturonan was found to be 28 and 3.6 IU/mg, respectively, indicating that Rgl11Y requires galactan decoration of the RGI backbone. The optimal pH of Rgl11Y is 8.5 and calcium is required for its activity. Rgl11Y was shown to be incorporated in the C. cellulolyticum cellulosome through a typical cohesin-dockerin interaction. Rgl11Y from C. cellulolyticum is the first cellulosomal rhamnogalacturonase characterized.

International audience; Transcription and expression regulation of some individual cel genes (cel5A, cel5I, cel5D and cel44O) of Clostridium cellulolyticum were investigated. Unlike the cip-cel operon, these genes are transcribed as monocistronic units of transcription, except cel5D. The location of the transcription initiation sites was determined using RT-PCR and the mRNA 59-end extremities were detected using primer extension experiments. Similarly to the cip-cel operon, cel5A and cel5I expressions are regulated by a carbon catabolite repression mechanism, whereas cel44O and cel5D expressions do not seem to be submitted to this regulation. The role of the putative transcriptional regulator GlyR2 in the regulation of cel5D expression was investigated. The recombinant protein GlyR2 was produced and was shown to bind in vitro to the cel5D and glyR2 promoter regions, suggesting that besides regulating its own expression, GlyR2 may regulate cel5D expression. To test this hypothesis in vivo, an insertional glyR2 mutant was generated and the effect of this disruption on cel5D expression was evaluated. Levels of cel5D mRNAs in the mutant were 16 fold lower than that of the wild-type strain suggesting that GlyR2 acts as an activator of cel5D expression.