Genomic DNA fragments flanking the Tn5-insertion site in the mutant were amplified by PCR-walking [14]. Tn5-insertion mutant DNA was digested by EcoR V (TaKaRa) and ligated with CHIR99021 the designed adaptor [11]. The adaptor specific primers AP1, AP2, and Tn5-specific primers TnFP1, TnRP1, TnFP2 and TnRP2 were designed for isolating the forward and reverse flanking sequences ( Fig. 3-a). PCR products were

retrieved and purified for sequencing. By aligning both the forward and reverse flanking sequences with the whole genome sequences of Xoo strains PXO99A, KACC10331 and MAFF311018 through NCBI BLAST (http://www.ncbi.nlm.nih.gov/BLAST), the Tn5-insertion site in the mutant was determined. Marker exchange was performed by splice overlap PCR. A fragment containing a kanamycin-encoding gene cassette (KM) was amplified from pKD13 plasmid DNA using primers KD13F and KD13R Selleckchem Trametinib (Table 1). The hrcQ forward flanking fragment (hrcQF1R1) and reverse flanking fragment (hrcQF2R2) were amplified from PXO99A genomic DNA using the primer pairs P69F1/P69R1 and P69F2/P69R2, respectively. Primer P69R1 contains the forward flanking sequence of KM, and P69F2 contains the reverse flanking sequence. The hrcQF1R1 and KM fragments were mixed as template, and primers P69F1 and KD13R were used

to amplify the forward fragment hrcQF-KM. Similarly, the reverse fragment KM-hrcQR was amplified with primers KD13F and P69R2. The hrcQF-KM and KM-hrcQR fragments were individually ligated into the pBluescript II SK (−) vector at an EcoR V restriction enzyme site. The EcoR I site (in the SK vector) and Nco I site (in the kanamycin-encoding gene cassette) were used to construct the plasmid SK-hrcQ. After confirming the insertion by DNA sequencing, the SK-hrcQ plasmid was transferred into a wild-type strain PXO99A by electroporation. The cell

cultures were spread on TSA medium plates containing G protein-coupled receptor kinase kanamycin (Km) at 50 μg mL− 1, incubated at 28 °C for 3 to 4 days. Clones were picked out and cultured in TSA medium plates containing ampicillin (Amp) at 100 μg mL− 1 for the second selection. We picked clones that grew on the kanamycin-containing plates but not on the ampicillin-containing plates. According to the Tn5-insertion site and genome sequence of PXO99A, the wild-type hrcQ gene with its promoter was amplified by PCR using primers PXM69F7 and PXM69R5 ( Table 1). The PCR product, with Hind III and EcoR I restriction sites introduced at the two ends, respectively, was cloned into the pEASY-B (TransGen) vector. After the DNA insert was confirmed by sequencing, the hrcQ-containing fragment was cut out by Hind III and EcoR I digestion and cloned into the broad host range vector pHM1, resulting in the complementary plasmid pHhrcQ, which was then transferred into the mutant strain PXM69 by electroporation using a Gene Pulser Xcell (Bio-Rad) electroporator at 1.8 kV mm− 1. After electro-pulsing cells were incubated in 500-μL PSA medium in a 200 r min− 1 rotary shaker at 28 °C for 1.5 h.