M9 salt medium supplemented with 5 g L−1 glucose was used for the detection of complementation. Restriction analyses of
the recombinant plasmids and Ca2+-dependent transformation of E. coli cells were performed in accordance with routine experimental protocols (Sambrook & Russell, 2001). Plasmid transformations of P. ananatis were performed according to Katashkina et al. (2009). Commercially available preparations of restrictases, T4 DNA ligase and the Klenow fragment of E. coli DNA selleck polymerase I (Fermentas, Lithuania) were used. The PCR fragments used for cloning were generated using AccuTaq-LA DNA polymerase (Sigma). Sigma products were used for the isolation of plasmid DNA. Primers were purchased from Syntol (Russia). CRIM plasmids were propagated in the CC118λpir+ strain (Herrero et al., 1990). Preparation of crude E. coli membrane fractions and enzymatic determination of PQQ in cultural media were performed according to the procedure developed and circumstantially described by Geiger & Gorisch (1986). PQQ-mGDH activity was assayed as described by Matsushita et al. (1981). The assay mixture contained (in a total volume of 200 μL) 25 mM potassium phosphate buffer, pH 6.5; 0.67 mM phenazine methosulfate; Stem Cell Compound Library 0.1 mM 2,6-dichlorophenolindophenol;
4 mM sodium azide; and 20 μL of the association mixture. The enzymatic reaction was started by adding 44 nmol of glucose. The change in A600 nm was recorded continuously, and the initial velocity is expressed in ΔA600 nm min−1. Spectrophotometric Cell press measurements
were made using a Synergy 2 multidetection microplate reader (BioTek Instruments Inc.). The total protein concentration was determined using the Bio-Rad Protein Assay (Bio-Rad) according to the manufacturer’s instructions. The capillary electrophoresis Quanta 4000E system (Waters) was used for the determination of gluconic acid in fermentation broth (Kenney, 1991). At the start of this work, it was known that P. ananatis SC17(0) cells accumulate gluconic acid when grown on minimal medium with glucose as the sole carbon source. GDH enzymatic activity, measured according to the method of Matsushita et al. (1981), was clearly detected in crude membrane fractions of these cells in reaction in the presence and absence of PQQ, which indicated that GDH was partially extracted in the holoenzyme form (see Table 2). Moreover PQQ, which is usually used as a cofactor for bacterial mGDH, was detected in the cultural medium (Table 3). An ORF (GenBank accession number GU580893) with a potential protein product possessing high homology to the apoenzymes of PQQ-mGDH from E. coli (73%) and P. citrea (63%) was found in the sequenced P. ananatis genome by a computer search. The amino acid residues critically important for interaction with PQQ by E.