The reaction was stopped by acidification with formic acid
to 1%. Peptides were separated on a C18 column (Zorbax 300SB-C18) using a nano LC system (Agilent 1200) that was coupled Palbociclib cost to a quadrupole-time-of-flight mass spectrometer (Agilent 6520) with a liquid chromatography-chip electrospray ionization interface. The raw LCMS data were preprocessed using the Agilent MassHunter Qualitative Analysis software (Agilent). For the search in the LipR sequence, a user-defined residue modification has been introduced for Asp phosphorylation and set as variable amino acid modification. SPR measurements were performed on a Biacore 3000 (GE Healthcare) using a streptavidin-coated SA sensor chip Decitabine mouse (GE Healthcare). Chips were conditioned and equilibrated with HBS-P buffer (GE Healthcare; 10 mM HEPES, pH 7.4, 150 mM
NaCl, and 0.005% (v/v) P20 surfactant). A volume of 260 μL of 0.6 μg mL−1 biotinylated DNA fragments were injected at a flow rate of 5 μL min−1 across one of the flow cells of a streptavidin sensor chip resulting in 500–1000 resonance units (RUs). Protein binding experiments were performed at 25 °C at a flow rate of 70 μL min−1. LipR-P and LipR were diluted in HBS-P buffer prior to injections. The sensor surface was regenerated after each cycle with 3 M MgCl2 (30 s contact time). Pseudomonas alcaligenes strains were grown overnight in 2× TY liquid medium. A small volume of each culture (~5 μL), corrected for differences in cell density, was spotted on 1% (v/v) tributyrin as described (Braun et al., 1999). To investigate the involvement of the RpoN protein in the regulation of
lipase expression, we created P. alcaligenes mutant strain Ps1101 C59 purchase by insertional inactivation of rpoN. The effect of rpoN inactivation on lipolytic activity was studied with the indicator assay plates containing tributyrin. As shown in Fig. 1, Ps1101 displayed a remarkably reduced clearance zone, similar to the lipR-inactivated strain Ps1100, as observed earlier (Krzeslak et al., 2008). This finding supports the hypothesis that lipase expression is governed by LipR and RpoN. As a further proof of the involvement of LipR and RpoN in lipA promoter activity, the pTZlipA vector bearing the lipA-lacZ transcriptional fusion was introduced into the strains Ps93, Ps1100, and Ps1101. The level of lipA-lacZ expression in the parental strain Ps93 was higher than of strains Ps1100 and Ps1101 (Fig. 2). This is in agreement with the observation of impaired lipase production on tributyrin plates for the Ps1100 mutant (Krzeslak et al., 2008) and the Ps1101 mutant (Fig. 1) strains. The sequence of LipR and its similarity to other DNA-binding regulators such as CbrB (Abdou et al., 2011) and NtrC (Weiss et al.