Abstract
The CbrA/CbrB system is a two-component signal transduction system known to participate in the regulation of the cellular carbon/nitrogen balance and to play a central role in carbon catabolite repression in Pseudomonas species. CbrA is composed of a domain with similarity to proteins of the solute/sodium symporter family (SLC5) and domains typically found in bacterial sensor kinases. Here, the functional properties of the sensor kinase CbrA and its domains are analyzed at the molecular level using the system of the soil bacterium P. putida KT2440 as a model. It is demonstrated that CbrA can bind and transport L-histidine. Transport is specific for L-histidine and probably driven by an electrochemical proton gradient. The kinase domain is not required for L-histidine uptake by the SLC5 domain of CbrA, and has no significant impact on transport kinetics. Furthermore, it is shown that the histidine kinase can autophosphorylate and transfer the phosphoryl group to the response regulator CbrB. The SLC5 domain is not essential for these activities but appears to modulate the autokinase activity. A phosphatase activity of CbrA is not detected. None of the activities is significantly affected by L-histidine. The results demonstrate that CbrA functions as a L-histidine transporter and sensor kinase.
Highlights
Transporters integral to cytoplasmic membranes usually catalyze the selective uptake of nutrients or the extrusion of metabolic end products and toxic solutes
Besides sodium-motive force-dependent transporters for proline (PutP of archaea and bacteria7), monosaccharides (SGLT of bacteria and higher eukaryotes8) and others[9,10,11], the family contains bacterial proteins in which a complete SLC5 domain is connected via a STAC (SLC5 and two-component systems (TCSs) Associated Component) domain to domains found in histidine kinases or diguanylate cyclase[5,12,13]
The resulting mutant P. putida LW1 was transformed with pUCP-Tc-cbrA or pUCP-Tc, cbrA expression from the lac promoter was induced by IPTG, and uptake of 3H-L-histidine was analyzed (Fig. 2a)
Summary
Transporters integral to cytoplasmic membranes usually catalyze the selective uptake of nutrients or the extrusion of metabolic end products and toxic solutes. A mutant of Pseudomonas fluorescens SBW25 devoid of known histidine uptake systems was previously shown to grow on L-histidine, and CbrA was identified as being responsible for L-histidine uptake in that strain[26] These results suggest that CbrA responds to extracellular L-histidine, and that transport and signal transduction are coupled[26]. We set out to further explore the functional properties and interactions of the individual domains of CbrA in the soil bacterium Pseudomonas putida KT2440 For this purpose, we deleted individual domains of CbrA and analyzed the impact of the deletion on the expression of known target genes in P. putida KT2440. CbrA can transport L-histidine, autokinase, phosphotransfer and phosphatase activities are not influenced by the amino acid
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