Efficient delivery of large DNA from Escherichia coli to Synechococcus elongatus PCC7942 by broad-host-range conjugal plasmid pUB307

Efficient delivery of large DNA from Escherichia coli to Synechococcus elongatus PCC7942 by... Abstract Synechococcus elongatus PCC7942, a cyanobacterium that uses light and carbon dioxide to grow, has a high ability to incorporate DNA by transformation. To assess the effective delivery of large DNA in plasmid form, we cloned the endogenous plasmid pANL (46.4 kbp) into a BAC vector of Escherichia coli. The plasmid p38ANL (54.3 kbp) replaced the native plasmid. To assess the delivery of larger DNA into PCC7942, p38ANL was fused to the broad-host-range conjugal transfer plasmid pUB307IP (53.5 kbp). The resulting plasmid pUB307IP501 (107.9 kbp) was transmitted from E. coli to PCC7942 by simple mixing of donor and recipient cultures. PCC7942 transcipients possessed only pUB307IP501, replacing the preexisting pANL. In contrast, the pUB307IP501 plasmid was unable to transform PCC7942, indicating that natural transformation of DNA may be restricted by size limitations. The ability to deliver large DNA by conjugation may lead to genetic engineering in PCC7942. conjugal transfer, cyanobacteria, endogenous plasmid, natural genetic transformation, plasmid stability Cyanobacteria are photosynthetic prokaryotes that are abundant in natural habitats and able to produce oxygen through the conversion of sunlight and carbon dioxide. To improve the potential environmental applications of cyanobacteria in an energy-saving and economical manner requires genetic engineering using a synthetic biology approach (1–3). We evaluated the species Synechocystis sp. PCC6803 and Synechococcus elongatus PCC7942, hereafter called PCC6803 and PCC7942, because they are recognized as unicellular freshwater model organisms (2, 3) and are the targets of our genome synthesis work (4, 5). We have previously reported transfer of the whole genome of PCC6803 into the genome of Bacillus subtilis using the B. subtilis genome (BGM) vector system (4, 5). However, this chimeric genome of PCC6803 and B. subtilis has provided many challenges, most of which remain unresolved (6, 7). By contrast, delivery of large DNA from B. subtilis to cyanobacteria may be less of a challenge. Owing to their natural competency, PCC6803 and PCC7942 offer basic genome engineering tools such as the ability to knock-in and knock-out regions of the genome (1, 2, 4). In addition, the use of a broad-host-range conjugal transfer system based on RP4 is more attractive. However, the conjugal transfer of DNA by a plasmid can only be established in PCC6803, as the lack of a stable plasmid appropriate for gene delivery into PCC7942 is a major obstacle (2). We attempted to explore effective tools for delivering large DNA into PCC7942 because of the advantages of PCC7942 in comparison with PCC6803, such as higher transformation efficiency (2), no restriction modification (8) and faster growth (3). The endogenous plasmid pANL of PCC7942 (9) was cloned and assessed for its suitability as a stable plasmid. We established pANL derivatives in combination with an RP4-based conjugal plasmid (10–12) as engineering tools to deliver large DNA. Materials and Methods Bacterial strains The Escherichia coli, B. subtilis and S. elongatus strains used in this study are listed in Table I. Luria–Bertani (LB) broth was used to grow E. coli and B. subtilis bacteria at 37°C unless specified. Strain PCC7942 was provided by H. Yoshikawa, Tokyo University of Agriculture, Tokyo, Japan. All of the PCC7942 derivatives in this study were grown in BG-11 liquid medium (1, 2) in CELLSTAR 10 containers (50 ml, Greiner Bio-One, Frickenhausen, Germany) at 25°C with continuous illumination (60 μE/m2 s). The containers were shaken at 60 rpm on the Double Shaker NR-30 (TAITEC, Saitama, Japan). Cultures were established from a single colony grown on solid BG11 medium, reaching stationary phase after approximately 7 days with an optical density at 600 nm (OD600) of 1.5–1.7, measured using the GeneQuant pro Spectrophotometer (GE Healthcare Japan, Tokyo, Japan). The BG11 medium was supplemented with chloramphenicol (Cm) at a final concentration of 5 μg/ml to select PCC7942 transformants and transcipients. The preparation and transformation of competent B. subtilis cells were performed as described previously (4, 5). Transformation of PCC7942 by a plasmid carrying the Cm acetyl transferase gene p38ANL and pUB307IP501 (Fig. 1) was conducted using an established method (1, 2). The electroporation of DNA into E. coli was performed using the MicroPulser Electroporator (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer’s instructions. The antibiotics used to detect resistance in B. subtilis were 5 μg/ml Cm, 100 μg/ml spectinomycin and 10 μg/ml tetracycline. The antibiotics used to detect resistant E. coli transformants were 100 μg/ml ampicillin and 20 μg/ml tetracycline. Table I. Bacterial strains and plasmids Bacterial strains Genotype or insert (bp) Antibiotic selection Reference or source E. coli DH5α F- λ- Φ80dlacZΔM15 deoR Laboratory stock Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rK-, mK+) phoA, supE44 thi-1 gyrA96 relA1 deoR recA1 DH10B F- λ- Φ80dlacZΔM15 mcrA Laboratory stock Δ(mrr-hsdRMS-mcrBC)ΔlacX74 deoR recA1 araD139 Δ(ara leu)7697 galU galK rpsL endA1 nupG MIC148 rnhA:: Tn3 of W3110b) ApRa Laboratory stock B. subtilis RM125 leuB8 arg-15 ΔSPß (5) BEST310 RM125 plus proB:: pBR[BAC cI-spc] SpcR (15) pr-neo BUSY31327 BEST310 plus p38 integrated CmR, NmR This study in the proB:: BAC S. elongatus PCC7942 pANL (46,366) Laboratory stock BUSY31173A p38ANL (54,774) transformant CmR This study BUSY31173A p38ANL (54,774) transformant CmR This study BUSY31187 pUB307IP501(107,869) transcpient CmR This study BUSY31188 pUB307IP501 transcipient CmR This study Plasmids E. coli transformants Antibiotic selection References or sources p108BGMC CmR (14) p108BGME CmR (14) p108BGFC MIC8400c (7,960) CmR This study p108BGFE MIC8602d (8,408) CmR This study p38 P38c (54,774) CmR This study p38ANL MEC1633e (54,326) CmR This study pUB307b MEC8515e (53,514) TcR, KmR Laboratory stock pUB307IPb MIC8718e (53,543) TcR This study pUB307IP501b MEC1726e (107,869) TcR, CmR This study pUB307IP501b MEC1729e TcR, CmR This study pUB307IP501b MEC1730e TcR, CmR This study Bacterial strains Genotype or insert (bp) Antibiotic selection Reference or source E. coli DH5α F- λ- Φ80dlacZΔM15 deoR Laboratory stock Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rK-, mK+) phoA, supE44 thi-1 gyrA96 relA1 deoR recA1 DH10B F- λ- Φ80dlacZΔM15 mcrA Laboratory stock Δ(mrr-hsdRMS-mcrBC)ΔlacX74 deoR recA1 araD139 Δ(ara leu)7697 galU galK rpsL endA1 nupG MIC148 rnhA:: Tn3 of W3110b) ApRa Laboratory stock B. subtilis RM125 leuB8 arg-15 ΔSPß (5) BEST310 RM125 plus proB:: pBR[BAC cI-spc] SpcR (15) pr-neo BUSY31327 BEST310 plus p38 integrated CmR, NmR This study in the proB:: BAC S. elongatus PCC7942 pANL (46,366) Laboratory stock BUSY31173A p38ANL (54,774) transformant CmR This study BUSY31173A p38ANL (54,774) transformant CmR This study BUSY31187 pUB307IP501(107,869) transcpient CmR This study BUSY31188 pUB307IP501 transcipient CmR This study Plasmids E. coli transformants Antibiotic selection References or sources p108BGMC CmR (14) p108BGME CmR (14) p108BGFC MIC8400c (7,960) CmR This study p108BGFE MIC8602d (8,408) CmR This study p38 P38c (54,774) CmR This study p38ANL MEC1633e (54,326) CmR This study pUB307b MEC8515e (53,514) TcR, KmR Laboratory stock pUB307IPb MIC8718e (53,543) TcR This study pUB307IP501b MEC1726e (107,869) TcR, CmR This study pUB307IP501b MEC1729e TcR, CmR This study pUB307IP501b MEC1730e TcR, CmR This study Plasmid size indicated by base pairs in parenthesis. aApR, ampicillin resistance; CmR, chloramphenicol resistance; KmR, kanamycin resistance; SpcR, spectinomycin resistance; NmR, neomycin resistance. bF- λ- IN(rrnD-rrnE)1 rph-1. cλ lysogeny DH5α. dλ lysogeny DH10B. eDH5α. Table I. Bacterial strains and plasmids Bacterial strains Genotype or insert (bp) Antibiotic selection Reference or source E. coli DH5α F- λ- Φ80dlacZΔM15 deoR Laboratory stock Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rK-, mK+) phoA, supE44 thi-1 gyrA96 relA1 deoR recA1 DH10B F- λ- Φ80dlacZΔM15 mcrA Laboratory stock Δ(mrr-hsdRMS-mcrBC)ΔlacX74 deoR recA1 araD139 Δ(ara leu)7697 galU galK rpsL endA1 nupG MIC148 rnhA:: Tn3 of W3110b) ApRa Laboratory stock B. subtilis RM125 leuB8 arg-15 ΔSPß (5) BEST310 RM125 plus proB:: pBR[BAC cI-spc] SpcR (15) pr-neo BUSY31327 BEST310 plus p38 integrated CmR, NmR This study in the proB:: BAC S. elongatus PCC7942 pANL (46,366) Laboratory stock BUSY31173A p38ANL (54,774) transformant CmR This study BUSY31173A p38ANL (54,774) transformant CmR This study BUSY31187 pUB307IP501(107,869) transcpient CmR This study BUSY31188 pUB307IP501 transcipient CmR This study Plasmids E. coli transformants Antibiotic selection References or sources p108BGMC CmR (14) p108BGME CmR (14) p108BGFC MIC8400c (7,960) CmR This study p108BGFE MIC8602d (8,408) CmR This study p38 P38c (54,774) CmR This study p38ANL MEC1633e (54,326) CmR This study pUB307b MEC8515e (53,514) TcR, KmR Laboratory stock pUB307IPb MIC8718e (53,543) TcR This study pUB307IP501b MEC1726e (107,869) TcR, CmR This study pUB307IP501b MEC1729e TcR, CmR This study pUB307IP501b MEC1730e TcR, CmR This study Bacterial strains Genotype or insert (bp) Antibiotic selection Reference or source E. coli DH5α F- λ- Φ80dlacZΔM15 deoR Laboratory stock Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rK-, mK+) phoA, supE44 thi-1 gyrA96 relA1 deoR recA1 DH10B F- λ- Φ80dlacZΔM15 mcrA Laboratory stock Δ(mrr-hsdRMS-mcrBC)ΔlacX74 deoR recA1 araD139 Δ(ara leu)7697 galU galK rpsL endA1 nupG MIC148 rnhA:: Tn3 of W3110b) ApRa Laboratory stock B. subtilis RM125 leuB8 arg-15 ΔSPß (5) BEST310 RM125 plus proB:: pBR[BAC cI-spc] SpcR (15) pr-neo BUSY31327 BEST310 plus p38 integrated CmR, NmR This study in the proB:: BAC S. elongatus PCC7942 pANL (46,366) Laboratory stock BUSY31173A p38ANL (54,774) transformant CmR This study BUSY31173A p38ANL (54,774) transformant CmR This study BUSY31187 pUB307IP501(107,869) transcpient CmR This study BUSY31188 pUB307IP501 transcipient CmR This study Plasmids E. coli transformants Antibiotic selection References or sources p108BGMC CmR (14) p108BGME CmR (14) p108BGFC MIC8400c (7,960) CmR This study p108BGFE MIC8602d (8,408) CmR This study p38 P38c (54,774) CmR This study p38ANL MEC1633e (54,326) CmR This study pUB307b MEC8515e (53,514) TcR, KmR Laboratory stock pUB307IPb MIC8718e (53,543) TcR This study pUB307IP501b MEC1726e (107,869) TcR, CmR This study pUB307IP501b MEC1729e TcR, CmR This study pUB307IP501b MEC1730e TcR, CmR This study Plasmid size indicated by base pairs in parenthesis. aApR, ampicillin resistance; CmR, chloramphenicol resistance; KmR, kanamycin resistance; SpcR, spectinomycin resistance; NmR, neomycin resistance. bF- λ- IN(rrnD-rrnE)1 rph-1. cλ lysogeny DH5α. dλ lysogeny DH10B. eDH5α. Fig. 1 View largeDownload slide Construction of pANL-based plasmids for PCC7942. (A) The BAC plasmid from p108BGMC (14) was converted to p108BGFC by inserting a Pr-mazF gene cassette provided by I-PpoI in the I-PpoI site of p108BGMC. (B) Library construction of the PCC7942 genome using p108BGFC after BamHI partial digestion (Sato, M. and Itaya M., to be published elsewhere). The BamHI site in the anL16 gene located in Synpcc7942_B2642 (9). (C and D) Conversion from p38 to p38ANL by the two-step method via BEST310 (15) to BUSY31327 described in the Materials and Methods. Only genome structures of these B. subtilis with a BAC integration locus and neomycin resistance gene (neo) are illustrated. Homologous recombination with the BAC sequence of the BEST310 genome is indicated by X. (E) Fusion of p38ANL to pUB370IP at the I-PpoI site. pUB307IP is described in the Materials and Methods. Location of the oriV of pUB307 is shown by circle. Plasmid sizes are indicated in parentheses (bp). Plasmid structures are drawn to an arbitrary size. Two cat genes present in the BAC, one for B. subtilis selection (bold arrow) and the other for E. coli selection (thin arrow). The latter might confer PCC7942 resistance to Cm (our unpublished observations). Fig. 1 View largeDownload slide Construction of pANL-based plasmids for PCC7942. (A) The BAC plasmid from p108BGMC (14) was converted to p108BGFC by inserting a Pr-mazF gene cassette provided by I-PpoI in the I-PpoI site of p108BGMC. (B) Library construction of the PCC7942 genome using p108BGFC after BamHI partial digestion (Sato, M. and Itaya M., to be published elsewhere). The BamHI site in the anL16 gene located in Synpcc7942_B2642 (9). (C and D) Conversion from p38 to p38ANL by the two-step method via BEST310 (15) to BUSY31327 described in the Materials and Methods. Only genome structures of these B. subtilis with a BAC integration locus and neomycin resistance gene (neo) are illustrated. Homologous recombination with the BAC sequence of the BEST310 genome is indicated by X. (E) Fusion of p38ANL to pUB370IP at the I-PpoI site. pUB307IP is described in the Materials and Methods. Location of the oriV of pUB307 is shown by circle. Plasmid sizes are indicated in parentheses (bp). Plasmid structures are drawn to an arbitrary size. Two cat genes present in the BAC, one for B. subtilis selection (bold arrow) and the other for E. coli selection (thin arrow). The latter might confer PCC7942 resistance to Cm (our unpublished observations). Plasmids The PCC7942 strain used in this study contains only the large plasmid pANL, as the small plasmid pANS reported in the original strain was lost (9). pUB307 was a gift from M. Tsuda, Tohoku University, Sendai, Japan. The conjugal transfer of RP4 derivatives to broad hosts has been reported in certain cyanobacteria (1, 2) and in cells (10, 11) in relation to the type IV secretion system (12). In vitro DNA manipulation Plasmid preparation from E. coli was performed as described previously (13). Type II restriction enzymes and T4 DNA ligase were obtained from Toyobo (Tokyo, Japan), except for I-PpoI, which was from Promega (Madison, WI, USA). The PCR primers were prepared by DATE Concept Co. (Sapporo, Japan). PCR-mediated amplification was carried out using Ex Taq Hot Start (TaKaRa, Kusatsu, Shiga, Japan). Conjugal transfer of pUB307 and pUB307IP between E. coli cells pUB307 (tetracycline resistance, kanamycin resistance) self-transmits between E. coli cells and can also be used in the transmission of mobile plasmids carrying the oriT sequence to a variety of cells. pUB307 was converted to a starter plasmid appropriate for universal use in broad-host investigations. pUB307 has unique restriction enzyme sites for BglII, EcoRI, HindIII and MluI. An adapter HindIII–I-PpoI–HindIII was inserted into the HindIII site in the kanamycin gene of pUB307 using two oligos, 5′-agcttATGACTCTCTTAAGGTAGCCAAAa-3′ and 5′-agcttTTTGGCTACCTTAAGAGAGTCATa-3′, to obtain pUB307IP in E. coli (Table I). The structure of pUB307IP is illustrated in Fig. 1. Conjugation activities for pUB307 derivatives were measured by our protocol indicated in Table II. Briefly, 50 μl of the donor MIC8515, selectable by Tc, and the recipient MIC148, selectable by ampicillin, were mixed in 2 ml fresh LB in a 15-ml tube. Rotation was performed at 60 rpm on a tilting-type rotator (Titec Co. Ltd, Saitama, Japan), tilted at 30° from horizontal, at 30°C. The culture was mixed for 4 h followed by sequential dilution, and then 10–25 μl were spotted onto solid LB medium containing ampicillin and tetracycline. Incubation for 17 h at a constant temperature of 30°C yielded 106–107 transcipients/ml. pUB307IP displayed similar conjugal activity. Table II. DNA transmission from E. coli to PCC7942 Donor: E. coli or Plasmid Growth stage Recipient PCC7942 Recipient E. coli (MIC148) Stational Rapid growing Stational Rapid growing MEC1726 (pUB307IP501) Stational 1,826 or 2,214 nd >106 nd Rapid growing nd 19 or 128 MEC1633 (p38ANl) Stational 0 nd Rapid growing nd 0 MIC8718 (pUB307IP) Stational 0 nd >106 nd Rapid growing nd 0 pUB307IP501 TF N/A 204 p38ANL TF N/A >104 Donor: E. coli or Plasmid Growth stage Recipient PCC7942 Recipient E. coli (MIC148) Stational Rapid growing Stational Rapid growing MEC1726 (pUB307IP501) Stational 1,826 or 2,214 nd >106 nd Rapid growing nd 19 or 128 MEC1633 (p38ANl) Stational 0 nd Rapid growing nd 0 MIC8718 (pUB307IP) Stational 0 nd >106 nd Rapid growing nd 0 pUB307IP501 TF N/A 204 p38ANL TF N/A >104 Conditions are detailed in the text. Number of colonies from PCC7942 on BG11 plate supplemented by Cm. TF: transformation, nd: not done, N/A not applicable. Table II. DNA transmission from E. coli to PCC7942 Donor: E. coli or Plasmid Growth stage Recipient PCC7942 Recipient E. coli (MIC148) Stational Rapid growing Stational Rapid growing MEC1726 (pUB307IP501) Stational 1,826 or 2,214 nd >106 nd Rapid growing nd 19 or 128 MEC1633 (p38ANl) Stational 0 nd Rapid growing nd 0 MIC8718 (pUB307IP) Stational 0 nd >106 nd Rapid growing nd 0 pUB307IP501 TF N/A 204 p38ANL TF N/A >104 Donor: E. coli or Plasmid Growth stage Recipient PCC7942 Recipient E. coli (MIC148) Stational Rapid growing Stational Rapid growing MEC1726 (pUB307IP501) Stational 1,826 or 2,214 nd >106 nd Rapid growing nd 19 or 128 MEC1633 (p38ANl) Stational 0 nd Rapid growing nd 0 MIC8718 (pUB307IP) Stational 0 nd >106 nd Rapid growing nd 0 pUB307IP501 TF N/A 204 p38ANL TF N/A >104 Conditions are detailed in the text. Number of colonies from PCC7942 on BG11 plate supplemented by Cm. TF: transformation, nd: not done, N/A not applicable. Conversion of p38 to p38ANL p38 was integrated into the BAC sequence already inserted into the BEST310 genome by homologous recombination (15). The resulting B. subtilis BUSY31327 indicated in Fig. 1, selected by 5 μg/ml Cm, carried the whole pANL sequence in the genome BAC locus and had lost the Pr-mazF gene. The BUSY31327 genome DNA prepared in an agarose block by complete I-PpoI digestion produced a linear fragment of approximately 50 kbp by pulsed-field gel electrophoresis (PFGE), corresponding to the integrated p38 lacking the Pr-mazF gene (data not shown). The fragment recovered from the gel was circularized by T4DNA ligase and transferred by electroporation into E. coli DH5α to produce 78 transformants after selection using 15 μg/ml Cm at 37°C. Seven transformants carried the same plasmid structure. A representative plasmid, named p38ANL, is shown in Fig. 1 and listed as MEC1633 in Table I. Results Cloning of the whole pANL plasmid from PCC7942 A BAC library for the whole genome of PCC7942 was constructed by BamHI partial digestion as described in Fig. 1. The whole pANL plasmid fused to the BAC plasmid p108BGFC (Fig. 1) was detected and named p38. The structure of p38 is shown in Fig. 1. The BAC vector was inserted into the BamHI site of the anL16 gene of pANL. The Pr-mazF gene encoding sequence-nonspecific RNase in the BAC was converted to an I-PpoI recognition sequence. The conversion, performed as described in Materials and Methods, yielded p38ANL in E. coli (Table I). A stable shuttle plasmid p38ANL between E. coli and PCC7942 Purified p38ANL was used to transform PCC7942. Rapidly growing PCC7942 (OD600 = 0.47) yielded transformants (>104/μg) after selection on BG11 medium supplemented with 5 μg/ml Cm as summarized in Table II. Plasmids from two of the transformants, named BUSY31173A and BUSY31173B, were analysed. Agarose gel plugs constructed using 5 ml of the stationary-phase cultures were subjected to PFGE and compared with the native plasmid pANL. As shown in Fig. 2, the two transformants were confirmed to possess p38ANL, as the sole plasmid. Endogenous pANL seemed to be replaced by the cloned plasmid. pANL is reported to be essential for PCC7942 because of genes located in replication and maintenance regions (9). The anL16 gene inactivated by BAC insertion is located outside these regions and was consistent with stable replication and maintenance of p38ANL. Replacement via a potential incompatibility mechanism remains to be investigated. Fig. 2 View largeDownload slide PCC7942 transformants by p38ANL. The endogenous pANL, indicated by a white asterisk (*) in lane PCC7942, was replaced by p38ANL in BUSY31173A and BUSY31173B, shown by a horizontal arrow. Supercoiled plasmids migrate relative to size, but slower than linear DNA of the same size. Size marker, concatemeric lambda (48.5 kbp × n) indicated by horizontal thin lines, is shown on the left. Lambda HindIII digests, with their sizes, are indicated by thick horizontal lines. The pulsed-field gel electrophoresis (PFGE) was run at 14°C, 5 V/cm, with a pulse time of 120 s and running time of 18 h. Fig. 2 View largeDownload slide PCC7942 transformants by p38ANL. The endogenous pANL, indicated by a white asterisk (*) in lane PCC7942, was replaced by p38ANL in BUSY31173A and BUSY31173B, shown by a horizontal arrow. Supercoiled plasmids migrate relative to size, but slower than linear DNA of the same size. Size marker, concatemeric lambda (48.5 kbp × n) indicated by horizontal thin lines, is shown on the left. Lambda HindIII digests, with their sizes, are indicated by thick horizontal lines. The pulsed-field gel electrophoresis (PFGE) was run at 14°C, 5 V/cm, with a pulse time of 120 s and running time of 18 h. Insertion of the conjugal plasmid pUB307 into the p38ANL plasmid To examine whether pANL can carry larger DNA than the BAC (7,960 bp), the plasmid pUB307IP (54,543 bp) was inserted into the I-PpoI site of p38ANL, as shown in Fig. 1. p38ANL, purified from preparative PFGE, and pUB307IP were both linearized by I-PpoI digestion and ligated. The ligation product transferred by electroporation to E. coli DH5α yielded two colonies on an LB plate supplemented with Cm and tetracycline. One colony possessed the plasmid pUB307IP501 (MEC1726), as listed in Table I. Conjugal transfer of pUB307IP501 to PCC7942 Transmission of pUB307IP501 from E. coli to PCC7942 was examined first by simple mixing of freshly made stationary phase cultures. As indicated in Table II, in a 2.0-ml tube, 500 μl donor E. coli (MEC1726) from a stationary phase culture grown in LB for 17 h at 30°C were mixed with 500 μl PCC7942 from BG11 after growing for 1 week at 25°C (OD600 = 1.5). DNase I was added (final concentration, 0.34 ng/ml) to the donor and recipient separately at 5 min before mixing. The mixing tube was shaken mildly with 60 rpm rotation at 30°C. Half (500 μl) of the mix was transferred after 2 h, and the other half after 5 h, to fresh 2.0 ml tubes and washed with 500 μl BG11 twice and plated on two BG11 plates supplemented with Cm. A week later, the 2-h mix produced 1,826 colonies, and the 5-h mix produced 2,214 colonies. The E. coli donor harbouring pUB307IP (MIC8718) or p38ANL (MEC1633) produced no colonies (Table II). As the simple mix of stationary phase cultures resulted in a number of colonies, 500 μl rapidly growing donor and recipient cultures were analysed and summarized in Table II. MEC1726, derived from inoculation of a 50 μl stationary phase culture in 2 mL LB and grown at 30°C for 2 h, and 500 μl rapidly growing recipient PCC7942 at OD600 0.27 and 0.47, respectively were mixed in a 2-ml tube. After gentle shaking for 4 h and two-time washes with 1 ml BG11, all cells were spread onto selection plates. After 7 days, the OD600 0.27 and 0.47 cultures yielded 19 and 128 transcipients of PCC7942, respectively. A higher number of transcipients seemed to be produced from stationary phase cultures. We did not assess the impact of mating on solid plates. Two isolates, named BUSY31187 and BUSY31188, from the 19 colonies described above were grown in liquid BG11 containing chrloramphenicol. An agarose gel plug prepared from a 5-ml culture OD600 > 1.5 was subjected to PFGE, as shown in Fig. 3. A single band corresponding to pUB307IP501 larger than the sizes of the original pANL and p38ANL was the sole replicon in the PCC7942 transcipients. Fig. 3 View largeDownload slide PCC7942 transcipients by pUB307IP501. pUB307IP501 in BUSY31187 and BUSY31188 are indicated by a horizontal arrow on the right. The endogenous pANL is indicated by a white asterisk (*) in the PCC7942 lane and p38ANL in the BUSY31173A by a white arrow on the left. Size marker, concatemeric lambda (48.5 kbp × n) indicated by horizontal thin lines, is shown on the left. The PFGE was run at 14°C, 5 V/cm, with a pulse time of 120 s and running time of 20 h. Fig. 3 View largeDownload slide PCC7942 transcipients by pUB307IP501. pUB307IP501 in BUSY31187 and BUSY31188 are indicated by a horizontal arrow on the right. The endogenous pANL is indicated by a white asterisk (*) in the PCC7942 lane and p38ANL in the BUSY31173A by a white arrow on the left. Size marker, concatemeric lambda (48.5 kbp × n) indicated by horizontal thin lines, is shown on the left. The PFGE was run at 14°C, 5 V/cm, with a pulse time of 120 s and running time of 20 h. Reproducible conjugal transfer of pUB307IP501 from the PCC7942 transcipients To confirm the structure and reproducible conjugal transfer activities, the plasmid isolated from the BUSY31187 was delivered to E. coli DH5α by electroporation. Two E. coli transformants, named MEC1729 and MEC1730, among the colonies on selection plates possessed a plasmid with the same structure as the original pUB307IP501, analysed by restriction digest using several enzymes (data not shown). Both E. coli transformants exhibited conjugal transfer to PCC7942 at a similar rate as that of the original MEC1726. These observations indicate that the pUB307IP501 from E. coli transferred to PCC7942 underwent replication and replaced the original pANL. Transformation PCC7942 by pUB307IP501 Transformation of PCC7942 by pUB307IP501 yielded 204 colonies/μg, a dramatic reduction compared with >104 colonies/μg resulting from the parental p38ANL. Ten random isolates examined by PFGE analyses exhibited plasmids of variable sizes but smaller than pUB307IP501 (data not shown). We did not examine these altered plasmid structures in detail. This observation together with the reduced frequency may indicate difficulties in delivering DNA > 100 kbp using the natural transformation protocol for PCC7942. Discussion We isolated an endogenous plasmid pANL from PCC7942 and demonstrated that a derivative p38ANL possessed a shuttling nature between E. coli and PCC7942. In terms of transformation efficiency and plasmid replacement in PCC7942, p38ANL may be useful as a stable plasmid to deliver engineered DNA. A larger plasmid, pUB307IP501 (107.9 kbp), was constructed (Fig. 1), and its shuttling nature was examined by transformation and conjugation, as this plasmid carries the full-length broad-host-range conjugal transfer plasmid pUB307. It was found that the plasmid transmits efficiently by conjugal processes but not by transformation. Plasmid replacement by incompatibility or homologous recombination remained to be examined. Use of recA mutant of PCC7942 should distinguish the two possibilities. Our results may have unveiled a size limit associated with delivery to PCC7942 by the present transformation protocol and should be verified by p38ANL carrying various lengths of DNA. As pUB307IP501 transfers by conjugation, in particular by simple mixing of donor and recipient cells, could encourage design and construction of pANL-based stable mobile plasmids from E. coli to PCC7942 using pUB307 as a helper plasmid. The pANL-based vector is attractive not only in basic research as a genetics tool but also in technological applications as a DNA delivery method. De novo synthesis of a complete new vector as large as 50 kbp can be prepared accurately using the method called ordered gene assembly in B. subtilis developed by our group (16). Finally, pUB307IP may be a valuable tool to evaluate conjugal transfer from E. coli to a variety of recipients. pUB307IP possesses a single I-PpoI site, a unique 23-bp sequence, to enable insertion of any DNA, if prepared as an I-PpoI fragment. Acknowledgement We thank Dr Naoto Ohtani for technical help with the BAC construction. Funding Institute for Advanced Biosciences. Conflict of Interest None declared. References 1 Heidorn T. , Camsund D. , Huang H.H. , Lindberg P. , Oliveira P. , Stensjö K. , Lindblad P. ( 2011 ) Synthetic biology in cyanobacteria engineering and analyzing novel functions in Methods of Enzymology (Voigt, C., ed.), Vol. 497 , pp. 539 – 579 , Academic Press , New York 2 Taton A. , Unglau F. , Wright N.E. , Wei Y.Z. , Paz-Yepes J. , Brahamsha B. , Palenik B. , Peterson T.C. , Haerizadeh F. , Golden S.S. , Golden J.W. ( 2014 ) Broad-host-range vector system for synthetic biology and biotechnology in cyanobacteria . Nucleic Acids Res . 42 , e136 Google Scholar CrossRef Search ADS PubMed 3 Yu J. , Liberton M. , Cliften P.F. , Head R.D. , Jacobs J.M. , Smith R.D. , Koppenaal D.W. , Brand J.J. , Pakrasi H.B. ( 2015 ) Synechococcus elongatus UTEX 2973, a fast growing cyanobacterial chassis for biosynthesis using light and CO2 . Sci. Rep . 5 , 8132 Google Scholar CrossRef Search ADS PubMed 4 Itaya M. , Fujita K. , Koizumi M. , Ikeuchi M. , Tsuge K. ( 2003 ) Stable positional cloning of long continuous DNA in the Bacillus subtilis genome vector . J. Biochem . 134 , 513 – 519 Google Scholar CrossRef Search ADS PubMed 5 Itaya M. , Tsuge K. , Koizumi M. , Fujita K. ( 2005 ) Combining two genomes in one cell: stable cloning of the Synechocystis PCC6803 genome in the Bacillus subtilis 168 genome . Proc. Natl. Acad. Sci. U.S.A. 102 , 15971 – 15976 Google Scholar CrossRef Search ADS PubMed 6 Itaya M. ( 2009 ) Recombinant genomes in Systems Biology and Synthetic Biology ( Fu P. , Panke S. , eds.) pp. 155 – 194 , John Wiley & Brothers, Inc ., Hoboken, NJ Google Scholar CrossRef Search ADS 7 Kaneko S. , Itaya M. ( 2015 ) Production of mitochondrial genome and chromosomal DNA segments highly engineered for use in mouse genetics by a Bacillus subtilis-based BGM vector system in Advances in Molecular Biology and Medicine: Synthetic Biology ( Meyers R.A. , ed.) pp. 490 – 500 , Wiley-VCH Verlag GmbH and Co ., Weinheim, Germany 8 Wang B. , Yu J. , Zhang W. , Meldrum D.R. ( 2015 ) Premethylation of foreign DNA improves integrative transformation efficiency in Synechocystis sp. strain PCC6803 . Appl. Environ. Microbiol. 81 , 8500 – 8506 . Google Scholar CrossRef Search ADS PubMed 9 Chen Y. , Holtman C.K. , Magnuson R.D. , Youderian P.A. , Golden S.S. ( 2008 ) The complete sequence and functional analysis of pANL, the large plasmid of the unicellular freshwater cyanobacterium Synechococcus elongatus PCC7942 . Plasmid 59 , 176 – 192 Google Scholar CrossRef Search ADS PubMed 10 Heinemann J.A. , Sprague G.F. Jr. ( 1989 ) Bacterial conjugative plasmids mobilize DNA transfer between bacteria and yeast . Nature 340 , 205 – 209 Google Scholar CrossRef Search ADS PubMed 11 Suzuki H. , Yoshida K. ( 2012 ) Genetic transformation of Geobacillus kaustophilus HTA426 by conjugative transfer of host-mimicking plasmids . J. Microbiol. Biotechnol. 22 , 1279 – 1287 Google Scholar CrossRef Search ADS PubMed 12 Low H.H. , Gubellini F. , Rivera-Calzada A. , Braun N. , Connery S. , Dujeancourt A. , Lu F. , Redzej A. , Fronzes R. , Orlova E.V. , Waksman G. ( 2014 ) Structure of a type IV secretion system . Nature 508 , 550 – 553 Google Scholar CrossRef Search ADS PubMed 13 Sambrook J. , Russell D. ( 2001 ) Molecular Cloning: A Laboratory Manual . 3rd edn . Cold Spring Harbor Laboratory Press, Cold Spring Harbor , NY 14 Ohtani N. , Hasegawa M. , Sato M. , Tomita M. , Kaneko S. , Itaya M. ( 2012 ) Serial assembly of Thermus megaplasmid DNA in the genome of Bacillus subtilis 168: a BAC-based domino method applied to DNA with a high GC content . Biotechnol. J . 7 , 867 – 876 Google Scholar CrossRef Search ADS PubMed 15 Kaneko S. , Akioka M. , Tsuge K. , Itaya M. ( 2005 ) DNA shuttling between plasmid vectors and a genome vector: systematic conversion and preservation of DNA libraries using the Bacillus subtilis genome (BGM) vector . J. Mol. Biol . 349 , 1036 – 1044 Google Scholar CrossRef Search ADS PubMed 16 Tsuge K. , Sato Y. , Kobayashi Y. , Gondo M. , Hasebe M. , Togashi T. , Tomita M. , Itaya M. ( 2015 ) Method of preparing an equimolar DNA mixture for one-step DNA assembly of over 50 fragments . Sci. Rep . 5 , 10655 Google Scholar CrossRef Search ADS PubMed Abbreviations Abbreviations BAC bacterial artificial chromosome PFGE pulsed-field gel electrophoresis © The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Biochemistry Oxford University Press

Efficient delivery of large DNA from Escherichia coli to Synechococcus elongatus PCC7942 by broad-host-range conjugal plasmid pUB307

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved
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0021-924X
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1756-2651
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10.1093/jb/mvy026
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Abstract

Abstract Synechococcus elongatus PCC7942, a cyanobacterium that uses light and carbon dioxide to grow, has a high ability to incorporate DNA by transformation. To assess the effective delivery of large DNA in plasmid form, we cloned the endogenous plasmid pANL (46.4 kbp) into a BAC vector of Escherichia coli. The plasmid p38ANL (54.3 kbp) replaced the native plasmid. To assess the delivery of larger DNA into PCC7942, p38ANL was fused to the broad-host-range conjugal transfer plasmid pUB307IP (53.5 kbp). The resulting plasmid pUB307IP501 (107.9 kbp) was transmitted from E. coli to PCC7942 by simple mixing of donor and recipient cultures. PCC7942 transcipients possessed only pUB307IP501, replacing the preexisting pANL. In contrast, the pUB307IP501 plasmid was unable to transform PCC7942, indicating that natural transformation of DNA may be restricted by size limitations. The ability to deliver large DNA by conjugation may lead to genetic engineering in PCC7942. conjugal transfer, cyanobacteria, endogenous plasmid, natural genetic transformation, plasmid stability Cyanobacteria are photosynthetic prokaryotes that are abundant in natural habitats and able to produce oxygen through the conversion of sunlight and carbon dioxide. To improve the potential environmental applications of cyanobacteria in an energy-saving and economical manner requires genetic engineering using a synthetic biology approach (1–3). We evaluated the species Synechocystis sp. PCC6803 and Synechococcus elongatus PCC7942, hereafter called PCC6803 and PCC7942, because they are recognized as unicellular freshwater model organisms (2, 3) and are the targets of our genome synthesis work (4, 5). We have previously reported transfer of the whole genome of PCC6803 into the genome of Bacillus subtilis using the B. subtilis genome (BGM) vector system (4, 5). However, this chimeric genome of PCC6803 and B. subtilis has provided many challenges, most of which remain unresolved (6, 7). By contrast, delivery of large DNA from B. subtilis to cyanobacteria may be less of a challenge. Owing to their natural competency, PCC6803 and PCC7942 offer basic genome engineering tools such as the ability to knock-in and knock-out regions of the genome (1, 2, 4). In addition, the use of a broad-host-range conjugal transfer system based on RP4 is more attractive. However, the conjugal transfer of DNA by a plasmid can only be established in PCC6803, as the lack of a stable plasmid appropriate for gene delivery into PCC7942 is a major obstacle (2). We attempted to explore effective tools for delivering large DNA into PCC7942 because of the advantages of PCC7942 in comparison with PCC6803, such as higher transformation efficiency (2), no restriction modification (8) and faster growth (3). The endogenous plasmid pANL of PCC7942 (9) was cloned and assessed for its suitability as a stable plasmid. We established pANL derivatives in combination with an RP4-based conjugal plasmid (10–12) as engineering tools to deliver large DNA. Materials and Methods Bacterial strains The Escherichia coli, B. subtilis and S. elongatus strains used in this study are listed in Table I. Luria–Bertani (LB) broth was used to grow E. coli and B. subtilis bacteria at 37°C unless specified. Strain PCC7942 was provided by H. Yoshikawa, Tokyo University of Agriculture, Tokyo, Japan. All of the PCC7942 derivatives in this study were grown in BG-11 liquid medium (1, 2) in CELLSTAR 10 containers (50 ml, Greiner Bio-One, Frickenhausen, Germany) at 25°C with continuous illumination (60 μE/m2 s). The containers were shaken at 60 rpm on the Double Shaker NR-30 (TAITEC, Saitama, Japan). Cultures were established from a single colony grown on solid BG11 medium, reaching stationary phase after approximately 7 days with an optical density at 600 nm (OD600) of 1.5–1.7, measured using the GeneQuant pro Spectrophotometer (GE Healthcare Japan, Tokyo, Japan). The BG11 medium was supplemented with chloramphenicol (Cm) at a final concentration of 5 μg/ml to select PCC7942 transformants and transcipients. The preparation and transformation of competent B. subtilis cells were performed as described previously (4, 5). Transformation of PCC7942 by a plasmid carrying the Cm acetyl transferase gene p38ANL and pUB307IP501 (Fig. 1) was conducted using an established method (1, 2). The electroporation of DNA into E. coli was performed using the MicroPulser Electroporator (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer’s instructions. The antibiotics used to detect resistance in B. subtilis were 5 μg/ml Cm, 100 μg/ml spectinomycin and 10 μg/ml tetracycline. The antibiotics used to detect resistant E. coli transformants were 100 μg/ml ampicillin and 20 μg/ml tetracycline. Table I. Bacterial strains and plasmids Bacterial strains Genotype or insert (bp) Antibiotic selection Reference or source E. coli DH5α F- λ- Φ80dlacZΔM15 deoR Laboratory stock Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rK-, mK+) phoA, supE44 thi-1 gyrA96 relA1 deoR recA1 DH10B F- λ- Φ80dlacZΔM15 mcrA Laboratory stock Δ(mrr-hsdRMS-mcrBC)ΔlacX74 deoR recA1 araD139 Δ(ara leu)7697 galU galK rpsL endA1 nupG MIC148 rnhA:: Tn3 of W3110b) ApRa Laboratory stock B. subtilis RM125 leuB8 arg-15 ΔSPß (5) BEST310 RM125 plus proB:: pBR[BAC cI-spc] SpcR (15) pr-neo BUSY31327 BEST310 plus p38 integrated CmR, NmR This study in the proB:: BAC S. elongatus PCC7942 pANL (46,366) Laboratory stock BUSY31173A p38ANL (54,774) transformant CmR This study BUSY31173A p38ANL (54,774) transformant CmR This study BUSY31187 pUB307IP501(107,869) transcpient CmR This study BUSY31188 pUB307IP501 transcipient CmR This study Plasmids E. coli transformants Antibiotic selection References or sources p108BGMC CmR (14) p108BGME CmR (14) p108BGFC MIC8400c (7,960) CmR This study p108BGFE MIC8602d (8,408) CmR This study p38 P38c (54,774) CmR This study p38ANL MEC1633e (54,326) CmR This study pUB307b MEC8515e (53,514) TcR, KmR Laboratory stock pUB307IPb MIC8718e (53,543) TcR This study pUB307IP501b MEC1726e (107,869) TcR, CmR This study pUB307IP501b MEC1729e TcR, CmR This study pUB307IP501b MEC1730e TcR, CmR This study Bacterial strains Genotype or insert (bp) Antibiotic selection Reference or source E. coli DH5α F- λ- Φ80dlacZΔM15 deoR Laboratory stock Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rK-, mK+) phoA, supE44 thi-1 gyrA96 relA1 deoR recA1 DH10B F- λ- Φ80dlacZΔM15 mcrA Laboratory stock Δ(mrr-hsdRMS-mcrBC)ΔlacX74 deoR recA1 araD139 Δ(ara leu)7697 galU galK rpsL endA1 nupG MIC148 rnhA:: Tn3 of W3110b) ApRa Laboratory stock B. subtilis RM125 leuB8 arg-15 ΔSPß (5) BEST310 RM125 plus proB:: pBR[BAC cI-spc] SpcR (15) pr-neo BUSY31327 BEST310 plus p38 integrated CmR, NmR This study in the proB:: BAC S. elongatus PCC7942 pANL (46,366) Laboratory stock BUSY31173A p38ANL (54,774) transformant CmR This study BUSY31173A p38ANL (54,774) transformant CmR This study BUSY31187 pUB307IP501(107,869) transcpient CmR This study BUSY31188 pUB307IP501 transcipient CmR This study Plasmids E. coli transformants Antibiotic selection References or sources p108BGMC CmR (14) p108BGME CmR (14) p108BGFC MIC8400c (7,960) CmR This study p108BGFE MIC8602d (8,408) CmR This study p38 P38c (54,774) CmR This study p38ANL MEC1633e (54,326) CmR This study pUB307b MEC8515e (53,514) TcR, KmR Laboratory stock pUB307IPb MIC8718e (53,543) TcR This study pUB307IP501b MEC1726e (107,869) TcR, CmR This study pUB307IP501b MEC1729e TcR, CmR This study pUB307IP501b MEC1730e TcR, CmR This study Plasmid size indicated by base pairs in parenthesis. aApR, ampicillin resistance; CmR, chloramphenicol resistance; KmR, kanamycin resistance; SpcR, spectinomycin resistance; NmR, neomycin resistance. bF- λ- IN(rrnD-rrnE)1 rph-1. cλ lysogeny DH5α. dλ lysogeny DH10B. eDH5α. Table I. Bacterial strains and plasmids Bacterial strains Genotype or insert (bp) Antibiotic selection Reference or source E. coli DH5α F- λ- Φ80dlacZΔM15 deoR Laboratory stock Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rK-, mK+) phoA, supE44 thi-1 gyrA96 relA1 deoR recA1 DH10B F- λ- Φ80dlacZΔM15 mcrA Laboratory stock Δ(mrr-hsdRMS-mcrBC)ΔlacX74 deoR recA1 araD139 Δ(ara leu)7697 galU galK rpsL endA1 nupG MIC148 rnhA:: Tn3 of W3110b) ApRa Laboratory stock B. subtilis RM125 leuB8 arg-15 ΔSPß (5) BEST310 RM125 plus proB:: pBR[BAC cI-spc] SpcR (15) pr-neo BUSY31327 BEST310 plus p38 integrated CmR, NmR This study in the proB:: BAC S. elongatus PCC7942 pANL (46,366) Laboratory stock BUSY31173A p38ANL (54,774) transformant CmR This study BUSY31173A p38ANL (54,774) transformant CmR This study BUSY31187 pUB307IP501(107,869) transcpient CmR This study BUSY31188 pUB307IP501 transcipient CmR This study Plasmids E. coli transformants Antibiotic selection References or sources p108BGMC CmR (14) p108BGME CmR (14) p108BGFC MIC8400c (7,960) CmR This study p108BGFE MIC8602d (8,408) CmR This study p38 P38c (54,774) CmR This study p38ANL MEC1633e (54,326) CmR This study pUB307b MEC8515e (53,514) TcR, KmR Laboratory stock pUB307IPb MIC8718e (53,543) TcR This study pUB307IP501b MEC1726e (107,869) TcR, CmR This study pUB307IP501b MEC1729e TcR, CmR This study pUB307IP501b MEC1730e TcR, CmR This study Bacterial strains Genotype or insert (bp) Antibiotic selection Reference or source E. coli DH5α F- λ- Φ80dlacZΔM15 deoR Laboratory stock Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rK-, mK+) phoA, supE44 thi-1 gyrA96 relA1 deoR recA1 DH10B F- λ- Φ80dlacZΔM15 mcrA Laboratory stock Δ(mrr-hsdRMS-mcrBC)ΔlacX74 deoR recA1 araD139 Δ(ara leu)7697 galU galK rpsL endA1 nupG MIC148 rnhA:: Tn3 of W3110b) ApRa Laboratory stock B. subtilis RM125 leuB8 arg-15 ΔSPß (5) BEST310 RM125 plus proB:: pBR[BAC cI-spc] SpcR (15) pr-neo BUSY31327 BEST310 plus p38 integrated CmR, NmR This study in the proB:: BAC S. elongatus PCC7942 pANL (46,366) Laboratory stock BUSY31173A p38ANL (54,774) transformant CmR This study BUSY31173A p38ANL (54,774) transformant CmR This study BUSY31187 pUB307IP501(107,869) transcpient CmR This study BUSY31188 pUB307IP501 transcipient CmR This study Plasmids E. coli transformants Antibiotic selection References or sources p108BGMC CmR (14) p108BGME CmR (14) p108BGFC MIC8400c (7,960) CmR This study p108BGFE MIC8602d (8,408) CmR This study p38 P38c (54,774) CmR This study p38ANL MEC1633e (54,326) CmR This study pUB307b MEC8515e (53,514) TcR, KmR Laboratory stock pUB307IPb MIC8718e (53,543) TcR This study pUB307IP501b MEC1726e (107,869) TcR, CmR This study pUB307IP501b MEC1729e TcR, CmR This study pUB307IP501b MEC1730e TcR, CmR This study Plasmid size indicated by base pairs in parenthesis. aApR, ampicillin resistance; CmR, chloramphenicol resistance; KmR, kanamycin resistance; SpcR, spectinomycin resistance; NmR, neomycin resistance. bF- λ- IN(rrnD-rrnE)1 rph-1. cλ lysogeny DH5α. dλ lysogeny DH10B. eDH5α. Fig. 1 View largeDownload slide Construction of pANL-based plasmids for PCC7942. (A) The BAC plasmid from p108BGMC (14) was converted to p108BGFC by inserting a Pr-mazF gene cassette provided by I-PpoI in the I-PpoI site of p108BGMC. (B) Library construction of the PCC7942 genome using p108BGFC after BamHI partial digestion (Sato, M. and Itaya M., to be published elsewhere). The BamHI site in the anL16 gene located in Synpcc7942_B2642 (9). (C and D) Conversion from p38 to p38ANL by the two-step method via BEST310 (15) to BUSY31327 described in the Materials and Methods. Only genome structures of these B. subtilis with a BAC integration locus and neomycin resistance gene (neo) are illustrated. Homologous recombination with the BAC sequence of the BEST310 genome is indicated by X. (E) Fusion of p38ANL to pUB370IP at the I-PpoI site. pUB307IP is described in the Materials and Methods. Location of the oriV of pUB307 is shown by circle. Plasmid sizes are indicated in parentheses (bp). Plasmid structures are drawn to an arbitrary size. Two cat genes present in the BAC, one for B. subtilis selection (bold arrow) and the other for E. coli selection (thin arrow). The latter might confer PCC7942 resistance to Cm (our unpublished observations). Fig. 1 View largeDownload slide Construction of pANL-based plasmids for PCC7942. (A) The BAC plasmid from p108BGMC (14) was converted to p108BGFC by inserting a Pr-mazF gene cassette provided by I-PpoI in the I-PpoI site of p108BGMC. (B) Library construction of the PCC7942 genome using p108BGFC after BamHI partial digestion (Sato, M. and Itaya M., to be published elsewhere). The BamHI site in the anL16 gene located in Synpcc7942_B2642 (9). (C and D) Conversion from p38 to p38ANL by the two-step method via BEST310 (15) to BUSY31327 described in the Materials and Methods. Only genome structures of these B. subtilis with a BAC integration locus and neomycin resistance gene (neo) are illustrated. Homologous recombination with the BAC sequence of the BEST310 genome is indicated by X. (E) Fusion of p38ANL to pUB370IP at the I-PpoI site. pUB307IP is described in the Materials and Methods. Location of the oriV of pUB307 is shown by circle. Plasmid sizes are indicated in parentheses (bp). Plasmid structures are drawn to an arbitrary size. Two cat genes present in the BAC, one for B. subtilis selection (bold arrow) and the other for E. coli selection (thin arrow). The latter might confer PCC7942 resistance to Cm (our unpublished observations). Plasmids The PCC7942 strain used in this study contains only the large plasmid pANL, as the small plasmid pANS reported in the original strain was lost (9). pUB307 was a gift from M. Tsuda, Tohoku University, Sendai, Japan. The conjugal transfer of RP4 derivatives to broad hosts has been reported in certain cyanobacteria (1, 2) and in cells (10, 11) in relation to the type IV secretion system (12). In vitro DNA manipulation Plasmid preparation from E. coli was performed as described previously (13). Type II restriction enzymes and T4 DNA ligase were obtained from Toyobo (Tokyo, Japan), except for I-PpoI, which was from Promega (Madison, WI, USA). The PCR primers were prepared by DATE Concept Co. (Sapporo, Japan). PCR-mediated amplification was carried out using Ex Taq Hot Start (TaKaRa, Kusatsu, Shiga, Japan). Conjugal transfer of pUB307 and pUB307IP between E. coli cells pUB307 (tetracycline resistance, kanamycin resistance) self-transmits between E. coli cells and can also be used in the transmission of mobile plasmids carrying the oriT sequence to a variety of cells. pUB307 was converted to a starter plasmid appropriate for universal use in broad-host investigations. pUB307 has unique restriction enzyme sites for BglII, EcoRI, HindIII and MluI. An adapter HindIII–I-PpoI–HindIII was inserted into the HindIII site in the kanamycin gene of pUB307 using two oligos, 5′-agcttATGACTCTCTTAAGGTAGCCAAAa-3′ and 5′-agcttTTTGGCTACCTTAAGAGAGTCATa-3′, to obtain pUB307IP in E. coli (Table I). The structure of pUB307IP is illustrated in Fig. 1. Conjugation activities for pUB307 derivatives were measured by our protocol indicated in Table II. Briefly, 50 μl of the donor MIC8515, selectable by Tc, and the recipient MIC148, selectable by ampicillin, were mixed in 2 ml fresh LB in a 15-ml tube. Rotation was performed at 60 rpm on a tilting-type rotator (Titec Co. Ltd, Saitama, Japan), tilted at 30° from horizontal, at 30°C. The culture was mixed for 4 h followed by sequential dilution, and then 10–25 μl were spotted onto solid LB medium containing ampicillin and tetracycline. Incubation for 17 h at a constant temperature of 30°C yielded 106–107 transcipients/ml. pUB307IP displayed similar conjugal activity. Table II. DNA transmission from E. coli to PCC7942 Donor: E. coli or Plasmid Growth stage Recipient PCC7942 Recipient E. coli (MIC148) Stational Rapid growing Stational Rapid growing MEC1726 (pUB307IP501) Stational 1,826 or 2,214 nd >106 nd Rapid growing nd 19 or 128 MEC1633 (p38ANl) Stational 0 nd Rapid growing nd 0 MIC8718 (pUB307IP) Stational 0 nd >106 nd Rapid growing nd 0 pUB307IP501 TF N/A 204 p38ANL TF N/A >104 Donor: E. coli or Plasmid Growth stage Recipient PCC7942 Recipient E. coli (MIC148) Stational Rapid growing Stational Rapid growing MEC1726 (pUB307IP501) Stational 1,826 or 2,214 nd >106 nd Rapid growing nd 19 or 128 MEC1633 (p38ANl) Stational 0 nd Rapid growing nd 0 MIC8718 (pUB307IP) Stational 0 nd >106 nd Rapid growing nd 0 pUB307IP501 TF N/A 204 p38ANL TF N/A >104 Conditions are detailed in the text. Number of colonies from PCC7942 on BG11 plate supplemented by Cm. TF: transformation, nd: not done, N/A not applicable. Table II. DNA transmission from E. coli to PCC7942 Donor: E. coli or Plasmid Growth stage Recipient PCC7942 Recipient E. coli (MIC148) Stational Rapid growing Stational Rapid growing MEC1726 (pUB307IP501) Stational 1,826 or 2,214 nd >106 nd Rapid growing nd 19 or 128 MEC1633 (p38ANl) Stational 0 nd Rapid growing nd 0 MIC8718 (pUB307IP) Stational 0 nd >106 nd Rapid growing nd 0 pUB307IP501 TF N/A 204 p38ANL TF N/A >104 Donor: E. coli or Plasmid Growth stage Recipient PCC7942 Recipient E. coli (MIC148) Stational Rapid growing Stational Rapid growing MEC1726 (pUB307IP501) Stational 1,826 or 2,214 nd >106 nd Rapid growing nd 19 or 128 MEC1633 (p38ANl) Stational 0 nd Rapid growing nd 0 MIC8718 (pUB307IP) Stational 0 nd >106 nd Rapid growing nd 0 pUB307IP501 TF N/A 204 p38ANL TF N/A >104 Conditions are detailed in the text. Number of colonies from PCC7942 on BG11 plate supplemented by Cm. TF: transformation, nd: not done, N/A not applicable. Conversion of p38 to p38ANL p38 was integrated into the BAC sequence already inserted into the BEST310 genome by homologous recombination (15). The resulting B. subtilis BUSY31327 indicated in Fig. 1, selected by 5 μg/ml Cm, carried the whole pANL sequence in the genome BAC locus and had lost the Pr-mazF gene. The BUSY31327 genome DNA prepared in an agarose block by complete I-PpoI digestion produced a linear fragment of approximately 50 kbp by pulsed-field gel electrophoresis (PFGE), corresponding to the integrated p38 lacking the Pr-mazF gene (data not shown). The fragment recovered from the gel was circularized by T4DNA ligase and transferred by electroporation into E. coli DH5α to produce 78 transformants after selection using 15 μg/ml Cm at 37°C. Seven transformants carried the same plasmid structure. A representative plasmid, named p38ANL, is shown in Fig. 1 and listed as MEC1633 in Table I. Results Cloning of the whole pANL plasmid from PCC7942 A BAC library for the whole genome of PCC7942 was constructed by BamHI partial digestion as described in Fig. 1. The whole pANL plasmid fused to the BAC plasmid p108BGFC (Fig. 1) was detected and named p38. The structure of p38 is shown in Fig. 1. The BAC vector was inserted into the BamHI site of the anL16 gene of pANL. The Pr-mazF gene encoding sequence-nonspecific RNase in the BAC was converted to an I-PpoI recognition sequence. The conversion, performed as described in Materials and Methods, yielded p38ANL in E. coli (Table I). A stable shuttle plasmid p38ANL between E. coli and PCC7942 Purified p38ANL was used to transform PCC7942. Rapidly growing PCC7942 (OD600 = 0.47) yielded transformants (>104/μg) after selection on BG11 medium supplemented with 5 μg/ml Cm as summarized in Table II. Plasmids from two of the transformants, named BUSY31173A and BUSY31173B, were analysed. Agarose gel plugs constructed using 5 ml of the stationary-phase cultures were subjected to PFGE and compared with the native plasmid pANL. As shown in Fig. 2, the two transformants were confirmed to possess p38ANL, as the sole plasmid. Endogenous pANL seemed to be replaced by the cloned plasmid. pANL is reported to be essential for PCC7942 because of genes located in replication and maintenance regions (9). The anL16 gene inactivated by BAC insertion is located outside these regions and was consistent with stable replication and maintenance of p38ANL. Replacement via a potential incompatibility mechanism remains to be investigated. Fig. 2 View largeDownload slide PCC7942 transformants by p38ANL. The endogenous pANL, indicated by a white asterisk (*) in lane PCC7942, was replaced by p38ANL in BUSY31173A and BUSY31173B, shown by a horizontal arrow. Supercoiled plasmids migrate relative to size, but slower than linear DNA of the same size. Size marker, concatemeric lambda (48.5 kbp × n) indicated by horizontal thin lines, is shown on the left. Lambda HindIII digests, with their sizes, are indicated by thick horizontal lines. The pulsed-field gel electrophoresis (PFGE) was run at 14°C, 5 V/cm, with a pulse time of 120 s and running time of 18 h. Fig. 2 View largeDownload slide PCC7942 transformants by p38ANL. The endogenous pANL, indicated by a white asterisk (*) in lane PCC7942, was replaced by p38ANL in BUSY31173A and BUSY31173B, shown by a horizontal arrow. Supercoiled plasmids migrate relative to size, but slower than linear DNA of the same size. Size marker, concatemeric lambda (48.5 kbp × n) indicated by horizontal thin lines, is shown on the left. Lambda HindIII digests, with their sizes, are indicated by thick horizontal lines. The pulsed-field gel electrophoresis (PFGE) was run at 14°C, 5 V/cm, with a pulse time of 120 s and running time of 18 h. Insertion of the conjugal plasmid pUB307 into the p38ANL plasmid To examine whether pANL can carry larger DNA than the BAC (7,960 bp), the plasmid pUB307IP (54,543 bp) was inserted into the I-PpoI site of p38ANL, as shown in Fig. 1. p38ANL, purified from preparative PFGE, and pUB307IP were both linearized by I-PpoI digestion and ligated. The ligation product transferred by electroporation to E. coli DH5α yielded two colonies on an LB plate supplemented with Cm and tetracycline. One colony possessed the plasmid pUB307IP501 (MEC1726), as listed in Table I. Conjugal transfer of pUB307IP501 to PCC7942 Transmission of pUB307IP501 from E. coli to PCC7942 was examined first by simple mixing of freshly made stationary phase cultures. As indicated in Table II, in a 2.0-ml tube, 500 μl donor E. coli (MEC1726) from a stationary phase culture grown in LB for 17 h at 30°C were mixed with 500 μl PCC7942 from BG11 after growing for 1 week at 25°C (OD600 = 1.5). DNase I was added (final concentration, 0.34 ng/ml) to the donor and recipient separately at 5 min before mixing. The mixing tube was shaken mildly with 60 rpm rotation at 30°C. Half (500 μl) of the mix was transferred after 2 h, and the other half after 5 h, to fresh 2.0 ml tubes and washed with 500 μl BG11 twice and plated on two BG11 plates supplemented with Cm. A week later, the 2-h mix produced 1,826 colonies, and the 5-h mix produced 2,214 colonies. The E. coli donor harbouring pUB307IP (MIC8718) or p38ANL (MEC1633) produced no colonies (Table II). As the simple mix of stationary phase cultures resulted in a number of colonies, 500 μl rapidly growing donor and recipient cultures were analysed and summarized in Table II. MEC1726, derived from inoculation of a 50 μl stationary phase culture in 2 mL LB and grown at 30°C for 2 h, and 500 μl rapidly growing recipient PCC7942 at OD600 0.27 and 0.47, respectively were mixed in a 2-ml tube. After gentle shaking for 4 h and two-time washes with 1 ml BG11, all cells were spread onto selection plates. After 7 days, the OD600 0.27 and 0.47 cultures yielded 19 and 128 transcipients of PCC7942, respectively. A higher number of transcipients seemed to be produced from stationary phase cultures. We did not assess the impact of mating on solid plates. Two isolates, named BUSY31187 and BUSY31188, from the 19 colonies described above were grown in liquid BG11 containing chrloramphenicol. An agarose gel plug prepared from a 5-ml culture OD600 > 1.5 was subjected to PFGE, as shown in Fig. 3. A single band corresponding to pUB307IP501 larger than the sizes of the original pANL and p38ANL was the sole replicon in the PCC7942 transcipients. Fig. 3 View largeDownload slide PCC7942 transcipients by pUB307IP501. pUB307IP501 in BUSY31187 and BUSY31188 are indicated by a horizontal arrow on the right. The endogenous pANL is indicated by a white asterisk (*) in the PCC7942 lane and p38ANL in the BUSY31173A by a white arrow on the left. Size marker, concatemeric lambda (48.5 kbp × n) indicated by horizontal thin lines, is shown on the left. The PFGE was run at 14°C, 5 V/cm, with a pulse time of 120 s and running time of 20 h. Fig. 3 View largeDownload slide PCC7942 transcipients by pUB307IP501. pUB307IP501 in BUSY31187 and BUSY31188 are indicated by a horizontal arrow on the right. The endogenous pANL is indicated by a white asterisk (*) in the PCC7942 lane and p38ANL in the BUSY31173A by a white arrow on the left. Size marker, concatemeric lambda (48.5 kbp × n) indicated by horizontal thin lines, is shown on the left. The PFGE was run at 14°C, 5 V/cm, with a pulse time of 120 s and running time of 20 h. Reproducible conjugal transfer of pUB307IP501 from the PCC7942 transcipients To confirm the structure and reproducible conjugal transfer activities, the plasmid isolated from the BUSY31187 was delivered to E. coli DH5α by electroporation. Two E. coli transformants, named MEC1729 and MEC1730, among the colonies on selection plates possessed a plasmid with the same structure as the original pUB307IP501, analysed by restriction digest using several enzymes (data not shown). Both E. coli transformants exhibited conjugal transfer to PCC7942 at a similar rate as that of the original MEC1726. These observations indicate that the pUB307IP501 from E. coli transferred to PCC7942 underwent replication and replaced the original pANL. Transformation PCC7942 by pUB307IP501 Transformation of PCC7942 by pUB307IP501 yielded 204 colonies/μg, a dramatic reduction compared with >104 colonies/μg resulting from the parental p38ANL. Ten random isolates examined by PFGE analyses exhibited plasmids of variable sizes but smaller than pUB307IP501 (data not shown). We did not examine these altered plasmid structures in detail. This observation together with the reduced frequency may indicate difficulties in delivering DNA > 100 kbp using the natural transformation protocol for PCC7942. Discussion We isolated an endogenous plasmid pANL from PCC7942 and demonstrated that a derivative p38ANL possessed a shuttling nature between E. coli and PCC7942. In terms of transformation efficiency and plasmid replacement in PCC7942, p38ANL may be useful as a stable plasmid to deliver engineered DNA. A larger plasmid, pUB307IP501 (107.9 kbp), was constructed (Fig. 1), and its shuttling nature was examined by transformation and conjugation, as this plasmid carries the full-length broad-host-range conjugal transfer plasmid pUB307. It was found that the plasmid transmits efficiently by conjugal processes but not by transformation. Plasmid replacement by incompatibility or homologous recombination remained to be examined. Use of recA mutant of PCC7942 should distinguish the two possibilities. Our results may have unveiled a size limit associated with delivery to PCC7942 by the present transformation protocol and should be verified by p38ANL carrying various lengths of DNA. As pUB307IP501 transfers by conjugation, in particular by simple mixing of donor and recipient cells, could encourage design and construction of pANL-based stable mobile plasmids from E. coli to PCC7942 using pUB307 as a helper plasmid. The pANL-based vector is attractive not only in basic research as a genetics tool but also in technological applications as a DNA delivery method. De novo synthesis of a complete new vector as large as 50 kbp can be prepared accurately using the method called ordered gene assembly in B. subtilis developed by our group (16). Finally, pUB307IP may be a valuable tool to evaluate conjugal transfer from E. coli to a variety of recipients. pUB307IP possesses a single I-PpoI site, a unique 23-bp sequence, to enable insertion of any DNA, if prepared as an I-PpoI fragment. Acknowledgement We thank Dr Naoto Ohtani for technical help with the BAC construction. Funding Institute for Advanced Biosciences. Conflict of Interest None declared. References 1 Heidorn T. , Camsund D. , Huang H.H. , Lindberg P. , Oliveira P. , Stensjö K. , Lindblad P. ( 2011 ) Synthetic biology in cyanobacteria engineering and analyzing novel functions in Methods of Enzymology (Voigt, C., ed.), Vol. 497 , pp. 539 – 579 , Academic Press , New York 2 Taton A. , Unglau F. , Wright N.E. , Wei Y.Z. , Paz-Yepes J. , Brahamsha B. , Palenik B. , Peterson T.C. , Haerizadeh F. , Golden S.S. , Golden J.W. ( 2014 ) Broad-host-range vector system for synthetic biology and biotechnology in cyanobacteria . 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( 2001 ) Molecular Cloning: A Laboratory Manual . 3rd edn . Cold Spring Harbor Laboratory Press, Cold Spring Harbor , NY 14 Ohtani N. , Hasegawa M. , Sato M. , Tomita M. , Kaneko S. , Itaya M. ( 2012 ) Serial assembly of Thermus megaplasmid DNA in the genome of Bacillus subtilis 168: a BAC-based domino method applied to DNA with a high GC content . Biotechnol. J . 7 , 867 – 876 Google Scholar CrossRef Search ADS PubMed 15 Kaneko S. , Akioka M. , Tsuge K. , Itaya M. ( 2005 ) DNA shuttling between plasmid vectors and a genome vector: systematic conversion and preservation of DNA libraries using the Bacillus subtilis genome (BGM) vector . J. Mol. Biol . 349 , 1036 – 1044 Google Scholar CrossRef Search ADS PubMed 16 Tsuge K. , Sato Y. , Kobayashi Y. , Gondo M. , Hasebe M. , Togashi T. , Tomita M. , Itaya M. ( 2015 ) Method of preparing an equimolar DNA mixture for one-step DNA assembly of over 50 fragments . Sci. Rep . 5 , 10655 Google Scholar CrossRef Search ADS PubMed Abbreviations Abbreviations BAC bacterial artificial chromosome PFGE pulsed-field gel electrophoresis © The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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The Journal of BiochemistryOxford University Press

Published: Feb 6, 2018

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