A putative promoter region was searched upstream of the start codon of chrASO using the BPROM Softberry online software (http://www.softberry.com/berry.phtml?topic=bprom&group=programs&subgroup=gfindb ). A DNA fragment corresponding to the 400 bp upstream from the ATG was amplified by PCR using S. oneidensis MR1-R chromosomal DNA, digested with XbaI and SpeI, and ligated upstream of the lacZ gene of vector pACYC184-lacZ, which was previously constructed by cloning the β-galactosidase-encoding gene lacZ from pGE593 into the vector pACYC184 (S1 Table ) [30 (link),31 (link)]. The ligation product was transformed into E. coli strain MC1061. The resulting plasmid called pchrASO::lacZ was introduced into S. oneidensis MR1-R by conjugation. Plasmid construction was checked by DNA sequencing. As a control, another fusion, named pmxd450::lacZ, was similarly constructed using a DNA fragment corresponding to the 450 bp upstream from the ATG of mxdA [32 (link)]. S. oneidensis MR1-R strain containing pchrASO::lacZ or pmxd450::lacZ was grown overnight on LB-agar plate. The overnight culture was diluted to an OD600nm = 0.1 in fresh liquid LB medium containing various concentrations of chromate (0, 0.05, 0.1 and 0.2 mM) and incubated in semi-aerobiosis at 28°C for 16 hours prior to β-galactosidase activity quantification. The β-galactosidase activity was determined by a Miller assay adapted for use with plate reader [33 ]. Briefly, cells were transferred into a microtiter plate and OD600nm was measured using a Tecan Spark 10M microplate reader. Cells were then lysed using lysozyme and PopCulture reagent (Sigma-Aldrich) prior to incubation with Z buffer (62 mM Na2HPO4, 45 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, 50 mM β-mercaptoethanol). O-nitrophenyl-ß-D-galactopyranoside (ONPG) was added to the mix before kinetic quantifications of OD420nm. Slope of the obtained curves were calculated and β-galactosidase activity (arbitrary units) was determined by the following equation:
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