Abstract
CbrAB is a high ranked global regulatory system exclusive of the Pseudomonads that responds to carbon limiting conditions. It has become necessary to define the particular regulon of CbrB and discriminate it from the downstream cascades through other regulatory components. We have performed in vivo binding analysis of CbrB in P. putida and determined that it directly controls the expression of at least 61 genes; 20% involved in regulatory functions, including the previously identified CrcZ and CrcY small regulatory RNAs. The remaining are porines or transporters (20%), metabolic enzymes (16%), activities related to protein translation (5%) and orfs of uncharacterised function (38%). Amongst the later, we have selected the operon PP2810-13 to make an exhaustive analysis of the CbrB binding sequences, together with those of crcZ and crcY. We describe the implication of three independent non-palindromic subsites with a variable spacing in three different targets; CrcZ, CrcY and operon PP2810-13 in the CbrAB activation. CbrB is a quite peculiar σN—dependent activator since it is barely dependent on phosphorylation for transcriptional activation. With the depiction of the precise contacts of CbrB with the DNA, the analysis of the multimerisation status and its dependence on other factors such as RpoN o IHF, we propose a model of transcriptional activation.
Highlights
The ability to recognize and convert external environmental stimuli into appropriate physiological responses is of fundamental importance for all organisms
Our previous work using standard molecular methods such as Electrophoretic Mobility Shift Assays (EMSA) and DNA footprinting analysis showed that CbrB binds to Mechanism of transcriptional activation of CbrB in Pseudomonas putida regions upstream crcZ and crcY, and identified the precise binding contacts of CbrB in these promoter regions [7], we performed a genome-wide screen for CbrB binding sequences throughout the complete genome in order to better define the CbrB binding site and its primary regulon
The complete list of the candidate CbrB regulated genes is presented in Table 1, and some captures of the genomic regions that were further analysed are shown in S1 Fig. As an internal quality control for experiment, we detected a 4.3 and 2.6 -fold enrichment after inmunoprecipitation of the two previously characterised CbrB targets CrcZ and CrcY, respectively
Summary
The ability to recognize and convert external environmental stimuli into appropriate physiological responses is of fundamental importance for all organisms. Several distinct global regulatory networks can participate in co-ordinating gene expression programmes under different situations. Signal transduction is predominantly mediated by two-component regulatory systems (TCSs) consisting of a sensor kinase (SK) and a cognate response regulator. Mechanism of transcriptional activation of CbrB in Pseudomonas putida
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