EMSA Recombinant K. pneumoniae Fur protein was expressed in E. coli and purified as previously described [22]. DNA fragment of the putative promoter region of ryhB was respectively PCR amplified by using specific primer sets (Table 2). The purified His6-Fur was incubated with 10-ng DNA in a 15 μl solution containing 50 mM Tris–HCl (pH 7.5), 100 mM NaCl, 100 mM dithiothreitol, 200 μM MnCl2,
and 1 μg/μl BSA at room temperature for 20 min. The samples were then loaded onto a native gel of 5% nondenaturing polyacrylamide RG7420 datasheet containing 5% glycerol in 0.5× TB buffer (45 mM Tris–HCl, pH 8.0, 45 mM boric acid). Gels were electrophoresed with a 20-mA current at 4°C and then stained with SABR safe Gel stain (Invitrogen). FURTA FURTA was performed according to the method described by Stojiljkovic et al. [64]. DNA sequences containing a putative Fur box were PCR amplified with specific primer sets and then cloned into pT7-7. The resulting plasmids were introduced into the E. coli strain H1717, and the transformants were plated onto MacConkey-lactose plates containing 100 μg/ml ampicillin and 30 μM Fe(NH4)2(SO4)2. The indicator strain H1717 contained a chromosomal fhuF::lacZ fusion, and a low affinity Fur box A-1210477 order has been demonstrated in the fhuF promoter.
The introduction of pT7-7 derived plasmids carrying Fur-binding sequences could thus cause the removal of Fur from the fhuF Fur box [60]. H1717 harboring pT7-7 was Florfenicol used as a negative control. Colony phenotype was observed after incubation
at 37°C for 10 h. Red colony (Lac+) denoted a FURTA-positive phenotype and indicated the binding of Fur to the DNA sequence cloned into the pT7-7 plasmid. Extraction and quantification of CPS CPS was extracted and quantified as previously described [65]. The glucuronic acid Repotrectinib datasheet content, represents the amount of K. pneumoniae K2 CPS, was determined from a standard curve of glucuronic acid (Sigma-Aldrich) and expressed as micrograms per 109 CFU [46]. qRT-PCR Total RNAs were isolated from early-exponential-phase grown bacteria cells by use of the RNeasy midi-column (QIAGEN) according to the manufacturer’s instructions. RNA was DNase-treated with RNase-free DNase I (MoBioPlus) to eliminate DNA contamination. RNA of 100 ng was reverse-transcribed with the Transcriptor First Strand cDNA Synthesis Kit (Roche) using random primers. qRT-PCR was performed in a Roche LightCycler® 1.5 Instrument using LightCycler TaqMan Master (Roche). Primers and probes were designed for selected target sequences using Universal ProbeLibrary Assay Design Center (Roche-applied science) and listed in Additional file 2: Table S1. Data were analyzed using the real time PCR software of Roche LightCycler® 1.5 Instrument. Relative gene expressions were quantified using the comparative threshold cycle 2-ΔΔCT method with 23S rRNA as the endogenous reference.