glnK gene mutagenesis. A - Schematic diagram depicting the mutagenesis procedure (modified from Clerico et al., 2007 ). The vector pKΔK (pK19MOBSACB derivative) harbors the flanking regions of the glnK gene (red). This suicide plasmid was delivered by conjugation to A. amazonense and integrated in the target site (orange) by homologous recombination, generating a merodiploid strain (containing both, wild-type and mutant alleles) that was selected by kanamycin since there is a resistance marker (white) present in the vector. The black box represents the region deleted. Subsequently, the merodiploid strain was cultivated and the cells that underwent a second recombination event were selected by sucrose, since the sacB marker present in the vector is lethal in the presence of this substance. The kanamycin-sensitive/sucrose resistant colonies were evaluated by PCR. B - Identification of the mutant strains by PCR using primers that flank the deletion site. The primers glnK_NdeI_up and glnK_BamHI_do utilized in this procedure are represented by the small green arrows in Figure 3A. NC - negative control, WT - wild type, MER - merodiploid, numbers - strains tested. C - Verification of the mutant strains by PCR using primers that flank the recombination sites. The primers conf_glnK_up and conf_glnK_do are represented by the small black arrows in Figure 3A. NC - negative control, WT - wild type, numbers - strains tested.
Sant'Anna et al. BMC Microbiology 2011 11:107 doi:10.1186/1471-2180-11-107