Generation of pcsR2 Mutant, Complementation, and Overexpression

A derivative of 30–84WT containing a pcsR2-deletion mutation was generated using strategies and methods described previously (Hmelo et al., 2015) with minor modifications. Briefly, fragments containing the upstream and downstream coding sequences (459 base pair each) flanking pcsR2 were amplified by PCR using the primer pairs 4807KO-UP-F-EcoRI (4807KO-1) and 4807K0-UP-R-PstI (4807KO-2), and 4807KO-DN-F-PstI (4807KO-3) and 4807KO-DN-R-HindIII (4807KO-4), respectively. The two amplicons each have 24 nt overlap at 3′ and 5′ end, respectively, due to the overlap of 4807KO-2 and 4807KO-3. Using the primers 4807KO-1 and 4807KO-4 and the products of the previous PCR as a template, overlap PCR was performed. The new product was a DNA fragment that fused the upstream and downstream coding sequence of pcsR2 with a PstI site at their junction. This fragment was ligated into the EcoRI and HindIII restriction enzyme sites in the multiple-cloning region of the suicide vector pEX18Ap. The modified plasmid was then transformed into E. coli and screened via blue-white color on LB with ampicillin and Xgal. A Km resistance cassette with its promoter (916 bp) was amplified from pUC4K using primer pairs KmPstI-F and KmPstI-R. Following PstI digestion, the resistance cartridge was inserted into the PstI site of the modified pEX18Ap construct via T4 ligation. The final construct was conjugated into 30–84WT, and positive transformants were selected on LB (amended with Km and Rif). A double-crossover mutant (30–84ΔpcsR2) was obtained by counter-selection with LB amended with Km and 6% sucrose and checked by PCR primers 4807KO-UP-UP-F(4807Check1), 4807-DN-DN-R(4807Check2), KmPstI-F, 4807qPCR-R PCR was performed using FideliTaq^TM DNA Polymerase (Affymetrix, Santa Clara, CA) or GoTaq^® Green Master Mix (Promega, Madison, WI) according to manufacturer instructions. Vector constructions were verified via Sanger sequencing using an ABI 3130xl Genetic Analyzer (Laboratory for Genome Technology, Texas A&M University). E. coli transformation and P. chlororaphis conjugation were performed as described previously (Pierson and Thomashow, 1992; Wang et al., 2012).

For complementation of 30–84ΔpcsR2, a plasmid constitutively expressing pcsR2 via the Ptac promoter was created. This was done by replacing the coding sequence of lacZ with pcsR2 in the expression vector pGT2Ptac:lacZ. The coding sequence of pcsR2 was PCR amplified with the primers 4807-F-BamH1 and 4807-R-HindIII. The resulting fragment was digested with BamHI and HindIII and cloned into the GT2Ptac:lacZ expression vector creating a Ptac:pcsR2 fusion, and the plasmid (pGT2PcsR2) was introduced into E. coli. The sequence was confirmed by Sanger sequencing. The expression vector was transformed to 30–84ΔpcsR2 by conjugation to generate 30–84ΔpcsR2(pGT2PcsR2), the complemented version of the mutant. A pcsR2 overexpression derivative also was obtained by conjugating the plasmid into 30–84WT to generate 30–84WT(pGT2PcsR2). For all comparisons utilizing the complement strain, 30–84WT and 30–84ΔpcsR2 containing the plasmid with no insert (NI), e.g., 30–84WT(NI) and 30–84ΔpcsR2(NI) were used. Standard growth curves were obtained for strains grown in AB + CAA at 28°C, with agitation.