, 2007). LipR has high sequence homology with several members of the bacterial enhancer-binding proteins family, for example, CbrB of P. aeruginosa and NtrC of Pseudomonas putida and E. coli. These proteins comprise a receiver domain, an AAA+ domain with

ATPase activity, and a DNA-binding domain (Ghosh et al., 2010). We have purified LipR from P. alcaligenes to be able to characterize it for ATPase activity and DNA-binding capability. Bacterial enhancer-binding proteins are normally phosphorylated by their cognate sensor kinases, yet many can also be phosphorylated in vitro by low-molecular-weight phosphor donors, such as acetyl phosphate or carbamoyl phosphate (Deretic et al., 1992; Lukat et al., 1992; McCleary & Stock, 1994). LipR was activated by in vitro phosphorylation with carbamoyl phosphate, but not with acetyl phosphate. Indeed, response regulators have variable sensitivity AZD9668 to small molecule phosphor donors (Lukat et al., 1992; Molle & Buttner, 2000; Schar et al., 2005). An ATP hydrolysis assay with LipR and in vitro phosphorylated LipR, LipR-P, demonstrated that both proteins are able to hydrolyze ATP, with LipR-P having a slightly higher ATPase activity (Fig. 3). Incubation with PlipA199 DNA resulted in a stimulation of check details this ATPase activity. In agreement with these results, it has been shown that phosphorylation of NtrC and the presence of DNA, containing specific NtrC

binding sites, stimulated its ATPase activity (Weiss et al., 1991; Austin & Dixon, 1992). The intrinsic instability of the phospho-aspartate impedes characterization of activities and identification. In our hands, surface plasmon resonance (SPR) was a more suitable technique than gel retardation assay (data not shown) to measure DNA binding by purified LipR. For SPR, we immobilized biotinylated fragments of DNA to a streptavidin sensor chip and

injected LipR or LipR-P to measure binding. The sensorgrams demonstrated that LipR-P, but not LipR, is able to bind specifically STK38 and strongly to PlipA199 (Fig. 4). In addition, mutation of three nucleotides in the UAS unequivocally showed that the DNA-binding site is located in this UAS (Fig. 4). This is in accordance with results of others, which show that phosphorylation of response regulators increases their binding ability to DNA (Aiba et al., 1989; Fiedler & Weiss, 1995; Huang et al., 1997). As phosphorylation is important for LipR activity, we set out to determine which aspartate residue is involved in this process. On the basis of homology with regulators such as CheY and NtrC (Sanders et al., 1989), LipR-D52 was expected to be phosphorylated. Mass spectrometric analysis of LysC/trypsin-digested LipR demonstrated the phosphorylation to occur within peptide 41–65 with sequence YSIPTFDLVVSDLRLPGAPGTELIK and containing two aspartate residues: D47 and D52 (in bold).