Figure 2

Analysis of the lux strains. A. Schematic depiction of the mutant strains after the first and second rounds of recombination. In lux ^prim, the nptI-sacB cassette interrupts the luxB gene. In lux ^sec, loss of the cassette leads to the reconstitution of the entire luxAB operon under the regulation of the psbA2 promoter. Annealing sites of the primers used for genotyping (P1-6) and of the DS_lux probe used for Southern hybridization as well as the positions of the XmaI restriction sites are indicated. B. Complete segregation of the Synechocystis lux strains generated by the genetic manipulations represented in A. Genotyping PCR was performed on five independent lux ^prim first-round recombinants and, for each of them, two second-round recombinants. Note that lux ^prim#2-5 behaved like lux ^prim#1. Expected sizes of the amplicons generated by the used primer pairs were: P1/P2, 2.7 kbps; P3/P4, 2.3 kbps; P1/P4, 2.3 kbps; P5/P6, 1 kbps. Negative control (n.c.) was included. C. Southern analysis of genomic DNA from Synechocystis WT and lux mutants. The XmaI restriction map of the slr0168 genomic region in the analyzed strains and the probe used for hybridization are shown in A. Five μg of DNA were loaded per lane. Genomic fragments F1 (7.5 kbps) and F2 (8.3 kbps) were detected in lux ^prim, the fragment F3 (11 kbps) in the lux ^sec strains. Note that lux ^prim#2-5 behaved like lux ^prim#1. The lane corresponding to WT is also shown. D. Drop test of lux mutants on selective media. Liquid cultures (OD₇₃₀: 0.4) were washed with BG11 without glucose and spotted (15 μl each) onto BG11 medium containing either 100 μg/ml kanamycin or 5% sucrose or no supplement. Colonies from the non-selective plate were incubated with 1 mM decanal and luminescence was determined to quantify luciferase activity.