TY - JOUR
AU - Ikeuchi, Masahiko
AB - Abstract We improved genetic transformation of the thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1, by combining electroporation with a top agar method. Transformation was also improved when a disruptant of a putative type I restriction endonuclease (tll2230) was used as recipient cells. In particular, some constructs, with which wild type has never been transformed, were successfully integrated into the tll2230-disruptant. Single-crossover recombination was detected more frequently than the double-crossover recombination. In accordance with the presence of all the homologs of pil genes in Synechocystis sp. PCC 6803, we found that T. elongatus is naturally transformable with exogenous DNA. (Received September 29, 2003; Accepted December 7, 2003) Introduction Cyanobacteria are oxygenic photosynthetic prokaryotes, from which plant chloroplasts have evolved as endosymbionts. The photosynthetic apparatus of cyanobacteria is practically the same as those of higher plants, including both photosystem I and photosystem II. The genetic analysis of the photosynthetic apparatus has been performed mostly in the cyanobacterium Synechocystis sp. PCC 6803, because this strain possesses a natural DNA uptake system and is able to grow heterotrophically (Grigorieva and Shestakov 1982, Williams 1988). However, the protein complexes such as photosystem I and photosystem II from these strains are not stable enough for biochemical or structural analyses. By contrast, some thermophilic strains have been established as a source of highly stable protein complexes (Jordan et al. 2001, Kamiya and Shen 2003, Zouni et al. 2001). They inhabit various hot springs and exhibit a number of unique properties such as flocculation and motility in addition to the stability of proteins. Thermosynechococcus elongatus BP-1 is a unicellular rod-shaped cyanobacterium, and grows at an optimal temperature 55°C (Yamaoka et al. 1978). Because of the thermostability of proteins and protein complexes and determination of the entire genome sequence (Nakamura et al. 2002), development of genetic engineering techniques of this organism became an urgent issue for analysis of protein complexes. For example, chlorophyll-binding proteins and protein supercomplexes, which are difficult to be express in Escherichia coli, will be the initial target for the genetic manipulation of this organism. Transformation by electroporation of T. elongatus has been developed (Katoh et al. 2001, Muhlenhoff and Chauvat 1996), but the efficiency of transformation was often low and also dependent on target genes. Here, we report an improvement of electrotransformation and competence of natural transformation. Results Top agar We often observed higher efficiency of colony formation when T. elongatus cells were embedded in top agar. We, therefore, examined the top agar during screening on agar plates after electroporation. Typical results are shown in Fig. 1. In this case, over 400 transformants were obtained by single electroporation with psbT::CmR construct. This transformation efficiency with the top agar was about 3.5 times higher than that without top agar. On the other hand, the colony size of transformants was 4- to 5-fold smaller in the top agar. This is partially because motile cells form spreading colonies on the agar surface, while motility is suppressed in the top agar. Further, cells seem to grow slightly slower in the top agar than on the surface especially at the later phase of growth probably due to limitation of CO2. Natural transformation In Synechocystis, natural transformation largely depends on pili structure on the cell surface like some Gram-negative bacteria (Yoshihara et al. 2001, Yoshihara et al. 2002, Okamoto and Ohmori 2002, Mattick et al. 1996, Darzins and Russell 1997). When the entire genome of T. elongatus was determined (Nakamura et al. 2002), we could find all the homologs of pilA, pilB, pilC, pilD, pilG, pilH, pilI, pilJ, pilL, pilM, pilN, pilO, pilQ, pilT, and comA, which are critical for assembly of the pili and/or natural transformation competence in Synechocystis (Okamoto and Ohmori 2002, Yoshihara et al. 2001, Yoshihara et al. 2002). Thus, we examined the natural competence of T. elongatus with various test plasmids. Without any specific pretreatment, cells were successfully transformed with the constructs of double-crossover recombination. It should be noted that efficiency of this natural transformation was much less than that of Synechocystis. The efficiency (2.5×10–6 transformants/recipient cells) was about 1/10 to 1/20 of that of electroporation in T. elongatus. However, it is still very useful to get transformants, because it is much simpler than the electroporation described here. We further examined the effects of growth phase on natural transformation (Fig. 2). The competency was relatively high throughout the exponential growth phase and was greatly reduced in the stationary phase as in the case for some other naturally transformable cyanobacteria (Grigorieva and Shestakov 1982, Stevens and Porter 1980). Disruption of a gene for type I restriction endonuclease Generally, restriction of foreign DNA is somehow critical in bacterial transformation. In fact, we often observed that transformation efficiency strongly depends on target genes, suggesting that sequence-specific processes such as restriction may underline it. Here, we disrupted a gene of putative type I restriction endonuclease, tll2230. It is the only candidate for the restriction endonuclease in the genome based on sequence analysis (Nakamura et al. 2002). A mutant of tll2230-disruption was successfully segregated without any detectable phenotype. When cells were transformed with the test plasmid of psbT::CmR by electroporation, efficiency was approximately five times higher in the tll2230-disruptant than in wild type (Table 1). Natural transformation efficiency in the disruptant was also significantly higher than in the wild type, although the overall efficiency was much lower than electroporation. It is of note that some constructs such as ndhE-His and ndhL-His, with which wild type has never been transformed in our hands, were successfully integrated into the tll2230-disruptant. Single-crossover and double-crossover recombination In general, both single- and double-crossover events can take place for integration of homologous DNA fragments into cyanobacterial chromosomes. To examine this, we constructed two recombinant plasmids by inserting the same screening cassette outside (for single-crossover) or inside the same genomic fragment (for double-crossover) (Fig. 3). When introduced by electroporation, both constructs gave a number of transformants (Table 2). Notably, the transformation efficiency was about 10 times higher for the single-crossover than for the double-crossover. Genetic recombination with both single-crossover and double-crossover was confirmed by Southern hybridization (Fig. 4). The hybridized bands of the single-crossover recombinants were larger than that of the double-crossover recombinants by the size of vector portion. On the other hand, natural transformation with the single-crossover construct gave no transformants, but some with the double-crossover construct (Table 3). This seems to indicate that the natural transformation involves fragmentation of the DNA during incorporation in contrast with uptake of the circular DNA by electroporation. The higher efficiency of single-crossover than the double-crossover in electroporation could be accounted for by assuming that length of the uninterrupted homologous DNA is critical for efficient recombination. Discussion The improved transformation of T. elongatus by electroporation was achieved by combination of the top agar method, disruption of the restriction endonuclease gene (tll2230), and the single-crossover recombination. For the first time, natural transformation was observed in thermophilic cyanobacteria. In spite of the low efficiency, it would be the first choice for genetic manipulation, because no laborious procedures are required. In the previous report (Muhlenhoff and Chauvat 1996), the transformants were selected in liquid culture by gradually raising the concentrations of antibiotics. The frequency of electrotransformation could not be estimated accurately since independent transformants were not recognized separately. In this study, we could evaluate transformation procedures by using top agar. It also improved efficiency of colony formation of T. elongatus. The top agar was also reported to be essential for colony formation of marine Synechococcus species (Brahamsha 1996). This may be due to sensitivity of certain cyanobacterial species to desiccation. Disruption of the putative type I endonuclease gene (tll2230) remarkably increased the efficiency of transformation. In addition, some constructs, with which wild type was not transformable, were successfully integrated into the tll2230-disruptant. Type I system is known to carry out both methylation and cleavage reactions of DNA within a protein complex. Subunits are encoded by hsdRMS genes in many bacteria (Murray 2000, Bickle and Kruger 1993). HsdM subunit methylates the own DNA with sequence specificity, which is determined by HsdS subunit. The third HsdR subunit is essential for restriction of unmethylated DNA. Improved transformation efficiency in the tll2230-disruptant strongly suggested that it works as a functional type I endonuclease in T. elongatus. Two accompanying genes, tll2228 and tll2229, are homologous to hsdM and hsdS, respectively. However, another restriction barrier seems to be still active in the tll2230 mutant, since a considerable difference in transformation efficiency was observed even in this mutant depending on target DNA fragments. In the literature, a type II restriction endonuclease activity (SelI) has been reported from T.elongatus to degrade DNA at CGCG (Miyake et al. 1992). Sequence-specific methylation of the genomic DNA was observed at RGATCY (T. Kinoshita and M. Ikeuchi, unpublished results). These observations suggest the presence of more restriction barriers in this organism. In the T. elongatus genome, we detected two ORFs (tlr1578 and tlr1640) as homologs of AnabaenadmtA (putative DNA methylase gene of unknown type II system) (Matveyev et al. 2001). Disruption of their flanking genes would give clues for the type II restriction enzymes. In Synechocystis, disruption of recJ encoding 5′-3′ exonuclease to degrade single-stranded DNA gave rise to marked increase in the natural transformation (Kufryk et al. 2002). If this is also the case in T. elongatus, disruption of tlr0174 (recJ) would improve the transformation. We found that the circular DNA can be integrated in the chromosome by single-crossover homologous recombination at high frequency, when introduced by electroporation. This would allow us quick genetic manipulation without designing complicated plasmids. For example, fusion constructs with a strong promoter, a regulatory promoter or expression tags such as His-tag can be easily generated by PCR cloning of a gene of interest into appropriate vectors. Another advantage is plasmid rescue that allows quick cloning of tagged genes, when mutants are generated by single-crossover recombination with plasmids carrying genomic DNA inserts. We demonstrated that T. elongatus is also competent for natural transformation with exogenous DNA in a manner of double-crossover recombination. The lack of single-crossover recombination may be due to digestion of the exogenous DNA prior to incorporation as suggested in several uptake systems (Solomon and Grossman 1996, Dreiseikelmann 1994). We detected the ORF (tll2339) as a homolog of comA (slr0197), which encodes putative DNA-binding and -processing protein and is essential for natural transformation in Synechocystis (Yura et al. 1999, Yoshihara et al. 2001). Judging from the wide distribution of a number of pil genes, we assume that the natural DNA uptake system strongly depends on a pilus structure. On the other hand, the natural transformation could not be observed in a closely related culture strain of T. elongatus (Muhlenhoff and Chauvat 1996). The difference may have arisen from differences in the screening procedures. In conclusion, transformation efficiency of T. elongatus was greatly improved but still lower than that of Synechocystis. Further studies are needed to develop better procedure of transformation but the procedures presented here seem to be sufficient for manipulation of most genes including photosynthesis genes. However, establishment of heterotrophic strain will be crucial for molecular studies of the essential photosynthesis genes. To end this, we are also trying to integrate a glucose transporter gene into the genome of T. elongatus. We believe that the present report should encourage the post-genome analysis in T. elongatus. Materials and Methods Strain and culturing conditions The thermophilic cyanobacterium Thermosynechococcus (formerly Synechococcus) elongatus strain BP-1 derived from a hot spring in Beppu, Japan was used in this study (Yamaoka et al. 1978). Wild type and mutants were grown at 45°C in BG11 medium (Stanier et al. 1971) supplemented with 20 mM TES-KOH (pH 8.2) under continuous illumination with white fluorescence lamps (20–50 µE m–2 s–1) bubbled with air containing 1.0% (v/v) CO2. The tll2230-disrupted mutant was maintained with 10 µg ml–1 streptomycin but propagated in the absence of antibiotics for analytical experiments. Growth of cells in liquid medium was monitored as light scattering of cells at 730 nm. The strains were usually propagated on 1.5% agar plate containing BG11 medium. Transformation The usual transformation experiments were performed with cells at mid-exponential phase. The plasmid DNA was introduced into cells by electroporation basically according to Muhlenhoff and Chauvat (1996) and Katoh et al. (2001). A 40 µl of cell suspension (A730 = 20) was mixed with 2 or 4 µl of 3 µg µl–1 DNA solution and electoroporated at a field strength of 10 kV cm–1. Pulsed cells were mixed with 1 ml BG11 medium followed by shaking for 24 h at 45°C under low light conditions (10–20 µE m–2 s–1). For natural transformation, cells were centrifuged at room temperature and resuspended in BG11 medium at A730 of 20. A 40 µl aliquot of suspension was mixed with 2 or 4 µl of 3 µg µl–1 DNA solution and then incubated with 1 ml BG11 medium by shaking for a day at 45°C under low light conditions. Then, cells were mixed with three volumes of BG11 medium containing 0.35% (w/v) melted agar (Difco, U.S.A.) and spread on an antibiotic-containing BG11 plate. Antibiotics-resistant transformants emerged as green colonies after incubation under continuous illumination (ca. 50 µE m–2 s–1) for about 1 week at 45°C. The concentration of antibiotics in BG11 plates for selection of transformants was 3.4 µg ml–1 chloramphenicol, 10 µg ml–1 streptomycin or 40 µg ml–1 kanamycin. Transformation was performed either with the single-crossover recombination construct or with the double-crossover recombination construct (Fig. 3). Construction of the tll2230-disruptant The DNA fragment harboring tll2230 was amplified by PCR with oligonucleotide primers, 5′-AAATTCGAGGCCCTCAATC-3′ and 5′-TCAGGCATGGTCATACAC-3′. The PCR fragment (2583 bp) was cloned into pT7Blue-T vector (Novagen, U.S.A.). The spectinomycin-resistant cassette, which was derived from the coding region of streptomycin / spectinomycin adenylyltransferase from pRL453 (Elhai and Wolk 1988), was inserted into the fragment at an MscI site to interrupt tll2230. Construction of the test DNA for single- or double-crossover recombination A HincII fragment of 2.6 kb harboring psbB (tlr1530), psbT (tsr1531) and gpmB (tlr1532), which was amplified and cloned into pT7Blue-T vector (Novagen) as described previously (Iwai et al. 2001), was used for further construction of test plasmids. A screening cassette to confer resistance against chloramphenicol was inserted in the middle of the fragment (BanII) or outside the fragment (HincII) (Fig. 3). The former construct allows double-crossover recombination to yield interruption of non-essential gene, psbT (Iwai et al. 2001). The latter allows single-crossover recombination to yield partial duplication of gpmB. Acknowledgments We wish to thank Dr. Teruo Ogawa for the offering of ndhE-His and ndhL-His constructs. This work was supported by Grants-in-Aid for Scientific Research (to M.I.) from the Ministry of Education, Science and Culture, Japan and by the Program for Promotion of Basic Research Activities for Innovative Biosciences of Japan (to M.I.). 1 Corresponding author: E-mail, mikeuchi@bio.c.u-tokyo.ac.jp; Fax, +81-3-5454-4337. View largeDownload slide Fig. 1 Transformants on BG11 (pH 8.2) plates with and without top agar. Cells were incubated for 14 d. View largeDownload slide Fig. 1 Transformants on BG11 (pH 8.2) plates with and without top agar. Cells were incubated for 14 d. View largeDownload slide Fig. 2 Growth phase-dependent competency of the natural transformation. (A) Transformation efficiency is shown for the growth phase (A730) of recipient cells. (B) Growth curve of the recipient cells. Recipient cells were grown at 45°C, at 20–30 µE m–2 s–1, with bubbling of 1% (v/v) CO2. View largeDownload slide Fig. 2 Growth phase-dependent competency of the natural transformation. (A) Transformation efficiency is shown for the growth phase (A730) of recipient cells. (B) Growth curve of the recipient cells. Recipient cells were grown at 45°C, at 20–30 µE m–2 s–1, with bubbling of 1% (v/v) CO2. View largeDownload slide Fig. 3 Test plasmids designed for single (A) or double (B) recombination. A HincII fragment of 2.6 kb cloned into pT7Blue-T vector. The chloramphenicol-resistant cassette (CmR) inserted into the outside the fragment (A) or middle of the fragment (B). View largeDownload slide Fig. 3 Test plasmids designed for single (A) or double (B) recombination. A HincII fragment of 2.6 kb cloned into pT7Blue-T vector. The chloramphenicol-resistant cassette (CmR) inserted into the outside the fragment (A) or middle of the fragment (B). View largeDownload slide Fig. 4 Southern blot analysis of electrotransformants of T. elongatus. Lane 1, DNA of wild type; lane 2, DNA of the double-crossover recombinant; lane 3–7, DNA of the single-crossover recombinants. All DNAs were digested by BstEII and were probed with the antibiotic resistance gene. View largeDownload slide Fig. 4 Southern blot analysis of electrotransformants of T. elongatus. Lane 1, DNA of wild type; lane 2, DNA of the double-crossover recombinant; lane 3–7, DNA of the single-crossover recombinants. All DNAs were digested by BstEII and were probed with the antibiotic resistance gene. Table 1 Effect of tll2230 disruption on electrotransformation   Transformants    psbT::CmR (6 µg)  psbT::CmR (12 µg)  ndhE-His*  ndhL-His*  Wild type  45  186  0  0  tll2230-disruptant  264  786  35  5    Transformants    psbT::CmR (6 µg)  psbT::CmR (12 µg)  ndhE-His*  ndhL-His*  Wild type  45  186  0  0  tll2230-disruptant  264  786  35  5  * C-terminal fusion construct with His-tag and CmR cassette. View Large Table 2 Electrotransformation of wild type with single- or double-crossover construct   Transformants    psbT::CmR (6 µg)  psbT::CmR (12 µg)  Single-crossover  578  3,080  Double-crossover  45  470    Transformants    psbT::CmR (6 µg)  psbT::CmR (12 µg)  Single-crossover  578  3,080  Double-crossover  45  470  View Large Table 3 Growth phase-dependent natural transformation of wild type with single- or double-crossover construct Growth phase [A730]  Transformants  Single-crossover  Double-crossover  0.39  0  10  0.74  0  32  0.83  0  65  0.88  0  80  Growth phase [A730]  Transformants  Single-crossover  Double-crossover  0.39  0  10  0.74  0  32  0.83  0  65  0.88  0  80  View Large Abbreviations !CmR chloramphenicol-resistant cassette. References Bickle, T.A. and Kruger, D.H. ( 1993) Biology of DNA restriction. Microbiol. Rev.  57: 434–450. Google Scholar Brahamsha, B. 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TI - Improved Genetic Transformation of the Thermophilic Cyanobacterium, Thermosynechococcus elongatus BP-1
JO - Plant and Cell Physiology
DO - 10.1093/pcp/pch015
DA - 2004-02-15
UR - https://www.deepdyve.com/lp/oxford-university-press/improved-genetic-transformation-of-the-thermophilic-cyanobacterium-sxoEMoCKE9
SP - 171
EP - 175
VL - 45
IS - 2
DP - DeepDyve
ER -