The Pseudomonas putida HskA hybrid sensor kinase controls the composition of the electron transport chain Emma Sevilla, 1 Hortencia Silva-Jiménez, 2 Estrella Duque, 2 Tino Krell 2 and Fernando Rojo 1 * 1 Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain. 2 Department of Environmental Protection, Estación Experimental del Zaidín, CSIC, 18008 Granada, Spain. Summary Sensor kinases play a key role in sensing and responding to environmental and physiological signals in bacteria. In this study we characterized a previously unknown orphan hybrid sensor kinase from Pseudomonas putida, which is conserved in several Pseudomonads. Inactivation of the gene coding for this sensor kinase, which we have named HskA, modified the expression of at least 85 genes in cells growing in a complete medium. HskA showed a strong influence on the composition of the electron transport chain. In cells growing exponentially in a complete medium, the absence of HskA led to a sig- nificant reduction in the expression of the genes coding for the bc1 complex and for the CIO and Cbb3-1 terminal oxidases. In stationary phase cells, however, lack of HskA caused a higher expression of the Cyo terminal oxidase and a lower expression of the Aa3 terminal oxidase. The HskA polypeptide shows two PAS (signal-sensing) domains, a transmit- ter domain containing the invariant phosphorylatable histidine and an ATP binding site, and a receiver domain containing the conserved aspartate capable of transphosphorylation, but lacks an Hpt module. It is therefore a hybrid sensor kinase. Phosphorylation assays showed that purified HskA undergoes auto- phosphorylation in the presence of ATP. Introduction Bacterial cells have diverse mechanisms to sense and respond to environmental and physiological changes. One of the most widespread relies on the so-called two- component regulatory systems (TCSs). The prototypical TCS includes two proteins, a sensor histidine kinase (HK) and a response regulator (RR). The HK typically contains a variable input domain that senses the stimuli, and a conserved transmitter domain with an invariant histidine residue that is phosphorylated in response to the stimuli. The response regulator (RR) has a conserved receiver domain linked to a variable output domain (Stock et al., 2000; Gao and Stock, 2009; see Fig. 1). Upon autophos- phorylation of the HK at the conserved histidine, the phos- phoryl group is transferred to a conserved aspartate of the RR, which in turn elicits an appropriate response, typically a gene regulation or chemotaxis process (Casino et al., 2010; Galperin, 2010; Krell et al., 2010; 2011). Some TCSs are more complex and have extra modules in the HK or in the RR, or can also incorporate auxiliary proteins called connector proteins (Rodrigue et al., 2000; Gao and Stock, 2009). In the so-called unorthodox systems, the HK possesses a receiver domain adjacent to the trans- mitter domain (Fig. 1); this receiver domain is similar to that of response regulators and is linked to a phospho- transfer module (Hpt). The phosphoryl group is sequen- tially transferred from the autophosphorylated histidine to an aspartate of the receiver domain, and then to a histi- dine in the Hpt module. This module can then transfer the phosphoryl group to the response regulator, thereby establishing a His–Asp–His–Asp phosphorelay. The Hpt module can also exist as an independent Hpt protein; in this case, the HK is called a hybrid HK (Fig. 1). Histidine kinases are often membrane-bound proteins, but can also be cytosolic. PAS domains are probably the most frequent sensor domains found in HKs. They can monitor a wide variety of stimuli including light, oxygen concentration, changes in the redox status of the cell or of the electron transport chain, and the presence of various small molecules (Taylor and Zhulin, 1999). PAS domains show limited similarity at the amino acid sequence level, but appear to have a conserved 3D fold (Hefti et al., 2004). Many of them bind cofactors or ligands, which are required for the detection of the input signals. The number of two-component systems and HKs present in a given bacterium is related both to its genome size and to its metabolic and ecological versatility (Ulrich Received 27 June, 2012; accepted 13 November, 2012. *For corre- spondence. E-mail frojo@cnb.csic.es; Tel. (+34) 91 585 45 39; Fax (+34) 91 585 45 06. Environmental Microbiology Reports (2013) 5(2), 291–300 doi:10.1111/1758-2229.12017 © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd