A single vector-based strategy for marker-less gene replacement in Synechocystis sp. PCC 6803

Abstract

Background: The cyanobacterium Synechocystis sp. PCC 6803 is widely used for research on photosynthesis and circadian rhythms, and also finds application in sustainable biotechnologies. Synechocystis is naturally transformable and undergoes homologous recombination, which enables the development of a variety of tools for genetic and genomic manipulations. To generate multiple gene deletions and/or replacements, marker-less manipulation methods based on counter-selection are generally employed. Currently available methods require two transformation steps with different DNA plasmids.

Results: In this study, we present a marker-less gene deletion and replacement strategy in Synechocystis sp. PCC 6803 which needs only a single transformation step. The method utilizes an nptI-sacB double selection cassette and exploits the ability of the cyanobacterium to undergo two successive genomic recombination events via double and single crossing-over upon application of appropriate selective procedures.

Conclusions: By reducing the number of cloning steps, this strategy will facilitate gene manipulation, gain-of-function studies, and automated screening of mutants.

Figure 1

Schematic depiction of the single-step double recombination strategy. The first recombination step (upper panel), involving a double crossover between the homologous regions HR1 and HR2 of the vector and the genomic target sequence, leads to genomic integration of the construct. Note that the integrated gene of interest (GOI) is split into two parts, 5’ and 3’, the sequences of which partially overlap (shaded box). The 5’ and 3’ GOI segments are separated from each other by the nptI-sacB selection cassette, which renders the first recombinant mutants resistant to kanamycin and sensitive to sucrose. After complete segregation of the replacement under positive selection in the presence of kanamycin, which ensures the total elimination of the endogenous target gene function, release of the selective pressure allows the second recombination to take place (lower panel). In this step, a crossover involving the overlapping regions of the split GOI leads to the excision of the nptI-sacB cassette. Negative selection on sucrose yields colonies that have lost the entire sacB marker and carry the intact, functional GOI in place of the endogenous target gene.

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.

Figure 3

Analysis of the psaA strains. A. Schematic depiction of mutant strains after the first and second rounds of recombination following transformation with the psaA construct. The psaA construct is integrated in the Synechocystis psaA gene. In psaA^prim the nptI-sacB cassette interrupts the At psaA gene. In psaA^sec, loss of the cassette leads to the reconstitution of the entire At psaA under the control of the native cyanobacterial promoter. Positions of the primers used for genotyping (P2, P7-10) are indicated. B.In vivo absorption spectra of WT, psaA^prim and psaA^secSynechocystis strains. The peaks at 438 and 681 nm correspond to the maxima of Chl a absorption, the peak at 628 nm corresponds to the absorption maximum of PC. The spectra were normalized to the absorbance at 730 nm. C. Complete segregation of the generated Synechocystis psaA strains, as demonstrated by PCR analysis. Primer positions are given in A. Expected sizes of the amplicons generated by the used primer pairs were: P7/P8, 2 kbps; P7/P2, 3 kbps; P9/P10, 2.3 kbps. Negative control (n.c.) was included. D. Drop test of WT, psaA^prim and psaA^secSynechocystis strains on selective media and under different light conditions. Liquid cultures at OD₇₃₀ of 0.4 were washed with BG11 without glucose and spotted (15 μl each) on non-selective BG11 medium or on BG11 containing 100 μg/ml kanamycin or 5% sucrose. When tested for autotrophic growth, cells were grown in continuous light at 30 μmol photons m^-2 s^-1 on BG11 without glucose. When grown in Light Activated Heterotrophic Growth (LAHG) conditions, the cells were incubated in the dark on BG11 supplemented with 5 mM glucose, and exposed to light for 5 min every 24 hours.