A simple and rapid method to prepare efficient electro-competent cells of Xanthomonas campestris pv. campestris was generated, with up to 100-fold transformation efficiencies over the existing procedures. The overnight cultures were treated with sucrose solution and micro-centrifuged at room temperature; the entire electro-competent cells generation process can be completed in 15 minutes. It overcomes the complication and time-consuming shortcomings of the traditional conjugation or electro-transformation methods in this strain. Both the replicative plasmids and non-replicative plasmids could be transformed or integrated efficiently using this method. And the DNA concentration, cells growth stage, field strength and recovery time all had influences on the transformation efficiency. In the optimal conditions, the transformation efficiency for the replicative plasmids was 10 9 transformants per microgram DNA, and for non-replicative plasmids was 150 transformants per microgram DNA. Further with the homology sequences, two chromosomal target genes were deleted efficiently and the knockout strains were obtained easily.
Xanthomonas campestris is a gram-negative, pathogenic bacterium belonging to the γ-subdivision of Proteobacteria. The variant X. campestris pv. campestris (Xcc) generally invades and multiplies in cruciferous plant vascular tissues, resulting in the characteristic “black rot” symptoms of blackened veins and V-shaped necrotic lesions at the foliar margin [
As Xanthomonas species can secret xanthan gum, extracellular polysaccharide (EPS) and many extracellular enzymes, it is not sensitive to chemical treatments that induce cell competence necessary for transformation [
To simplify the DNA transfer and gene replacement procedures in Xanthomonas, we construct a fast method to generate high competent cells. Combining with the optimized electro-transformation conditions, the replicative plasmids DNA and the non-replicative plasmids DNA could be efficiently transformed into Xcc 8004; further with this method, two chromosomal genes were deleted and two knockout mutant strains were generated successfully.
All strains and plasmids used in this work were listed in
The oligonucleotides were synthesized and sequenced by Invitrogen Ltd. (Shanghai, China), and their sequences were listed in
Two non-replicative plasmids were constructed from plasmid pK18mobsacB [
Strains and plasmids | Genotype or description | Source |
---|---|---|
Strains | ||
E. coli DH5α | SupE44 DlacU169 (Φ80lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 | [ |
E. coli S17-1(λpir) | recA, pro, hsdR−M+RP4: 2-Tc:Mu: Km Tn7 λpir (ATCC 47055) | [ |
Xcc 8004 | A rifampicin-resistant strain derived from Xcc NCPPB No. 1145 (isolated from infected cauliflower, B. oleraceavar. botrytis, in Sussex, UK, 1958) | [ |
Plasmids | ||
pDN19 | 7.8 kb, IncP, oriT, Tcr | [ |
pUFR027 | 9.3 kb, IncW, MobP, Apr, Kanr | [ |
pRKaraRed | 10.9 kb, IncP, oriT, Tcr | [ |
pK18mobsacB | 5.7 kb, RP4 oriT, Kanr | [ |
pEX18Gm | Gene replacement vector, oriT, Apr | [ |
pK18mobsacB-Xc1075 | 7.2 kb, pK18mobsacB with the upstream 500 bp fragment of XC1075, accC1 and the downstream 500 bp fragment of XC1075, Kanr | This work |
pK18mobsacB-Xc2659 | 7.2 kb, pK18mobsacB with the upstream 500 bp fragment of XC2659, accC1 and the downstream 500 bp fragment of XC2659, Kanr | This work |
Name | Sequences | Description |
---|---|---|
XC1075-U-F | 5’-GTAGGAATTCatgactgatcaatcctctaaacgcggg-3’ | Amplification primers for the upstream 500 bp fragment of XC1075 |
XC1075-U-R | 5’-ctgaacggcgttgccgcatc-3’ | |
XC1075-D-F | 5’-gcagcaagaccaagggctacatg-3’ | Amplification primers for the downstream 500 bp fragment of XC1075 |
XC1075-D-R | 5’-GTAGAAGCTTttatttggcgctggcggcc-3’ | |
XC2659-U-F | 5’-GTAGGAATTCatgtcgacattgcttcgtccctccc-3’ | Amplification primers for the upstream 500 bp fragment of XC2659 |
XC2659-U-R | 5’-gggcgattgaccacacctgcg-3’ | |
XC2659-D-F | 5’-gtggcgatgccggtggtgc-3’ | Amplification primers for the downstream 500 bp fragment of XC2659 |
XC2659-D-R | 5’-GTAGAAGCTTttaccgctgcggcaacgcgtag-3’ | |
accC1-1075-F | 5’-GATGCGGCAACGCCGTTCAGatgttacgcagcagcaacgatgt-3’ | Amplification primers for the accC1 fragment for fusion with two flanking fragments of XC1075 |
accC1-1075-R | 5’-GTAGCCCTTGGTCTTGCTGCttaggtggcggtacttgggt-3’ | |
accC1-2659-F | 5’-GCAGGTGTGGTCAATCGCCCatgttacgcagcagcaacgatgt-3’ | Amplification primers for the accC1 fragment for fusion with two flanking fragments of XC2659 |
accC1-2659-R | 5’-GTGGCGATGCCGGTGGTGCAttaggtggcggtacttgggt-3’ |
The oligonucleotides were named according to gene name and were listed from 5’ to 3’. F and R primers represented forward and reverse primers, respectively. Lowercase bases matched the template; the capitalized bases in primers accC1-1075-F/R and accC1-2659-F/R indicated sequences for overlap PCR; and the capitalized and lined bases in the other primers were sequences of restriction enzyme sites.
producing plasmids pK18mobsacB-Xc1075 and pK18mobsacB-Xc2659 (
The Xcc 8004 electro-competent cells were made based on a micro-centrifugation procedure. Single colony of Xcc 8004 was inoculated into 10 ml LB medium (in 50 ml bottle) and cultured overnight at 28˚C with 200 rpm shaking. The overnight culture was equally distributed into four 5 ml centrifuge tubes, and cells were harvested by centrifugation at 8000 rpm for 5 min at room temperature. The cell pellets in each tube were re-suspended with 1 ml of 250 mM sucrose, and transferred into 1.5 ml Eppendorf tubes. Centrifugations and re-suspensions with 1 ml of 250 mM sucrose were repeated three times at 12,000 rpm for 2 min at room temperature. Finally, cells in four tubes were re-suspended with total 100 μl of 250 mM sucrose, and the final cell concentration was about 109 ~ 1010 colony forming units (CFU)/ml. 50 μl aliquot of the competent cells was used for one electroporation experiment.
Cells cultured at different stages (OD600 = 0.4 ~ 1.2) were used to prepare the electro-competent cells as described above. Electroporation was carried out with 50 μl of competent cells and no more than 10 μl of plasmids DNA. Electro-transformation was performed in a 0.1 cm ice-cold electroporation cuvette, on a Bio-Rad GenePulser II. The electric field strength varied from 10 KV/cm to 20 KV/cm. The concentrations of plasmids DNA ranged from 50 ng to 1 μg. Cells without plasmids DNA were used as negative controls. After the pulse, immediately added 1.0 ml LB broth into the electroporation cuvette, and transferred into a 17 × 100 mm round-bottom, sterile glass tube. The electroporated cells were incubated at 28˚C for 0 ~ 3 hours. Either transformation mixture dilutions (for replicative plasmids) or the entire transformation mixtures (for non-replicative plasmids) was screened on antibiotic-imbued plates and incubated at 28˚C until colonies appeared (usually 48 ~ 72 h). Counted the number of colonies and extracted the plasmids or chromosomal DNA for analysis. For the transformation groups of non-replicative plasmids, the colonies were screened on LB plates with Gm and LB plates with Kan to distinguish double cross-over events from single cross-over events.
To analyze the feasibility of this method in DNA transfer and gene modification, this method was compared with other two transformation methods, one electroporation method and one conjugation method using E. coli S17-1 (λpir) [
Three plasmids, pUFR027, pDN19 and pRKaraRed was selected as target plasmids to test the efficiency of this method, because the broad host-range vectors with the RK-2 origin or RK-6 origin could replicate in Xanthomons (
Plasmids | Replicative | Size (kb) | Amount (ng) | Selection | Transformation efficiency (transformants/μg DNA) | |||
---|---|---|---|---|---|---|---|---|
Recovery time (h) | ||||||||
0 | 1 | 2 | 3 | |||||
pDN19 | Yesa | 7.8 kb | 200 | Tcr | 2.3 × 103 | 1.7 × 107 | 9.6 × 107 | 6.3 × 107 |
pRKaraRed | Yesa | 10.9 kb | 200 | Tcr | 1.4 × 103 | 4.0 × 106 | 6.8 × 107 | 3.4 × 107 |
pUFR027 | Yesa | 9.3 kb | 200 | Kanr | 1.9 × 103 | 5.6 × 106 | 3.5 × 107 | 4.9 × 107 |
pK18mobsacB-Xc0751 | Nob | 7.2 kb | 1000 | Kanr | 6 | 64 | 151 | 136 |
pK18mobsacB-Xc2728 | Nob | 7.2 kb | 1000 | Kanr | 8 | 79 | 138 | 142 |
In all cases, 50 μl aliquots of electro-competent cells were transformed with the indicated amounts of DNA. 1 ml LB medium was added and the cells were either immediately plated on selective plates (0 h) or after 1 - 3 h shaking at 28˚C. Selective plates consisted of LB with 10 μg/ml Tc, 10 μg/ml Kan or 10 μg/ml Gm. Transformants were counted after about 48 ~ 72 h incubation at 28˚C. aAfter incubation for the indicated amount of time, cells were diluted up to 105-fold in LB medium and then plated on selective plates to yield single colonies. The numbers shown are the averages from three separate experiments; bSince these pK18mobsacB-based plasmids do not replicate in Xanthonomas, antibiotic resistant colonies after transformation were the result of either plasmid-integration (merodiploid formation) into the chromosome via a single or double cross-over event. After incubation for the indicated amount of time, the entire mixture was screened on a single LB plate with 10 μg/ml Gm. Colonies were re-selected on LB added 10 μg/ml Gm and LB added 10 μg/ml Kan plates to distinguish double from single cross-over events. Data shown are the total numbers of Gmr colonies including two cross-over events.
First we analyzed whether the three plasmids could be transformed into Xcc 8004. According to previous work, we collected cells of Xcc 8004 at OD600 about 0.8 and treated cells with sucrose solution and micro-cen- trifugation to generate the competent cells. Then we electro-transformed 200 ng of plasmids DNA into these cells with following electrical parameters: electrodes of 0.1 cm gap, 14 KV/cm, 2 h recovery time. Results showed that all three plasmids could be transformed into Xcc 8004 and the transformants were around 109 CFU per microgram DNA (
Then we analyzed whether the effect of DNA concentration on the transformation efficiency. We found increasing DNA concentration could enhance the transformation efficiency from 1.5 × 106 to 2.7 × 108, when the concentration of plasmid DNA was changed from 50 ng to 1 μg per 50 μl competent cells (
Also the influence of cell growth stage to the transformation efficiency was detected and 200 ng of plasmid pUFR027 was used. The highest transformation efficiency was observed when cells were at the late exponential phase to the early stationary phase (OD600 = 0.6 ~ 1.0), about 16 ~ 20 hours (
Further we analyzed the effect of electric intensity and 14 ~ 18 KV/cm field strengths were found to be optimal (
In addition, the recovery time also had great impact on the transformation efficiency. When cells were screened on the selective plate immediately after electroporation, the transformation efficiency was only about 2 × 103 per microgram input DNA; however, one- or two-hour recover could improve it significantly by three to four logs (
In a word, this method was feasible to generate efficient electro-competent cells; combining with optimized electroporation parameters, the plasmids DNA could be transformed into Xcc 8004 efficiently.
Non-replicative plasmids are widely used in gene deletion and modification by single or double recombination events. In Xanthomonas, gene replacement was performed based on the conjugation with E. coli S17-1 (λpir) [
To test the feasibility of this method for non-replicative plasmid integration and gene deletion, two glucose dehydrogenase genes XC1075 and XC2659 were selected and we constructed two plasmids pK18mobsacB-Xc1075
Plasmids CFU | pDN19 | pRKaraRed | pUFR027 | pK18mobsacB-Xc0751 | pK18mobsacB-Xc2728 |
---|---|---|---|---|---|
This Methoda | 1.1 × 108 | 7.8 × 107 | 3.9 × 107 | 147 | 133 |
Method 1b | 3.7 × 106 | 8.6 × 105 | 1.6 × 106 | ND | ND |
Method 2c | ND | ND | ND | 27 | 19 |
In all cases, 50 μl aliquots of electro-competent cells were transformed with 200 ng of replicative DNA or 1000 ng of non-replicative DNA. ND was not detected. aThe electroporation parameters were cells of OD600 = 0.8, electrodes of 0.1 cm gap, 14 KV/cm, and 2 h recovery time. Transformants were screen on the selective plates and counted after about 48 ~ 72 h incubation at 28˚C. The numbers shown are the averages from three separate experiments; bMethod 1 was the electroporation performed as described previously [
and pK18mobsacB-Xc2659, containing gentamycin encoding gene, the upstream 500 bp and the downstream 500 bp chromosomal flanking regions. Since pK18mobsacB-based plasmids cannot replicate in Xanthonomas, antibiotic resistant colonies after transformation were the result of either plasmid-integration (merodiploid formation) into the chromosome via single or double cross-over event. Results indicated that in the optimal electroporation conditions, each plasmid yielded about 130 - 150 transformants per microgram of input DNA (
integration and recombination; increased amounts of plasmids DNA could improve the efficiency (data not shown).
In brief, it is possible to using this method to transform non-replicative plasmids into Xanthomonas directly to induce plasmid integration; and homologous recombination between chromosome and plasmids could generate knockout mutants.
Electroporation is widely used in many organisms because of its high efficiency. In Xanthomonas, electro- transformation is the major plasmid DNA transfer method as Xanthomonas is insensitive to the chemical treatment [
The main advantages of this method for preparation of electro-competent Xcc 8004 cells described here are its simplicity and speed, without compromising efficiency. The entire procedure can be performed at room temperature with overnight cultures in a microcentrifuge, simply using 250 mM aqueous sucrose solution and taking less than 15 minutes to complete the steps. It is in contrast to traditional procedures which require time-con- suming centrifugation steps, vessels and sometimes complex buffers that need to be refrigerated, and take hours to complete. We also tried to use wide-used 10% glycerol to generate competent cells, and found higher efficiencies can be obtained with sucrose solution, probably because of higher cells sensitivities in the latter treatment (data not shown). The entire procedure, from start (preparation of plasmid DNA) to finish (screening on selective media), takes about 3 hours, and colonies are ready for test in about 2 - 3 days. Despite its speed and simplicity, the transformation efficiencies of this method are comparable to or exceed those of other two methods, sometimes up to 100-fold (
Therefore, this method can accelerate the speed in routine plasmid DNA transfer and gene replacement experiments in Xcc 8004; and the observed transformation efficiencies are sufficient for further analysis. It can be more powerful if conjunction with other marker excision methods (Flp/FRT), which allows recycling of the same selection marker in the same strain for construction of mutants to contain multiple lesions in the same chromosome.
This work was supported in part by National Science Foundation of China (Grant No. 31370152), the Shanghai Pujiang Program (14PJD020) and the Chen Xing Grant of Shanghai Jiao Tong University.
All authors have no conflict of interest to declare.
XiuliWang,DaningZheng,RubingLiang, (2016) An Efficient Electro-Competent Cells Generation Method of Xanthomonas campestris pv. campestris: Its Application for Plasmid Transformation and Gene Replacement. Advances in Microbiology,06,79-87. doi: 10.4236/aim.2016.62008