The plasmid DNA electrophoretic mobility shift assay signaling pathway (PD.EMSA) and genomic DNA electrophoretic mobility shift assay (GD.EMSA) methods involve incubation of purified DNA-binding protein with fractionated DNA, followed by electrophoresis through a native polyacrylamide gel using sodium boric acid (SB) buffer. In this study, the restriction endonuclease Bsp143I was used for DNA fragmentation. The use of SB buffer, a low conductivity medium, and a 14-cm gel as well as running the gel for 3–6 hours at low voltage, allowed unbound DNA fragments to migrate far from the top of the gel while ChvI-bound fragments remained near the wells

(see Additional file 1). Inclusion of EDTA in the buffer resulted in no retardation of electrophoretic mobility suggesting an involvement of the putative Mg2+ site for ChvI-DNA interaction (see Additional file 2). The slower migrating bands were excised from the gel, purified, and cloned into pUC18 vector from which the insert DNA could be sequenced from each end to determine the extent of each www.selleckchem.com/products/prt062607-p505-15-hcl.html fragment. Bsp143I-digested pTC198 plasmid DNA was used to perform PD.EMSA (see Additional file 1). This pUC19 clone contains a 5-kb KpnI-fragment from S. meliloti Rm1021 spanning across the entire chvI-hprK genomic sequence including the intergenic region between pckA and chvI[10]. This plasmid

was employed to optimize the method with a smaller number of fragments than with genomic DNA, thus providing a better resolution on the gel but also increasing the chances of binding to areas surrounding chvI and exoS to test for Thymidine kinase possible autoregulation of ExoS/ChvI. Regulation of the adjacent gene pckA by chvG-chvI has been previously shown for A. tumefaciens using reporter gene fusion assays [19], therefore this experiment was also aimed at testing if S. meliloti ChvI could bind upstream of pckA. Following the excision of electrophoretic bands from PD.EMSA of pTC198, DNA fragments were cloned into BamHI-linearized pUC18 and sequenced from both ends. Out of four

inserts sequenced, three represent a 176-bp fragment (genomic origin from 48523 to 48699) coding for the region upstream of SMc02753, including its start codon. A single clone contained a 395-bp region spanning the upstream sequence of chvI and past the translational start site (genomic origin from 51887 to 52281). These results suggest that ChvI might autoregulate its transcription but most importantly, it shows a direct binding affinity between the ChvI and the upstream sequence of manXhpr operon part of the PTS system. The ChvI binding to the 176-bp fragment was also confirmed by performing a gel shift assay using a PCR-amplified DNA fragment from pLB102 and the purified ChvI protein (data not shown). Further delineation of this binding was not performed. After GD.