Experimental Designs

To quantitatively validate the ability of URA_Cm-Cm and URA_Cm-Gs as selection markers for targeted gene insertion, we constructed a pair of gene cassettes, each containing the EGFP transgene and one of the selection markers, which was designed to be targeted into the convergent intergenic region of CMD184C and CMD185C (Figure 1B). This locus was selected as the target site because it was very short and was not expected to contain promoter activities that potentially affect gene expression in its vicinity. Shown in Figure 1B is the basic strategy used for one-step insertion of the EGFP gene and selection markers by homologous recombination. Colony PCRs using the primer set F1/R1 were set up as a primary screen for strains in which the left arm of the introduced fragment had been recombined at the CMD184C locus on the genome. We found that 35% of the colonies screened (38/108) produced a PCR fragment with an expected length in a transformation experiment using URA_Cm-Cm as a selection marker. In contrast, only 10% (11/108) were positive when URA_Cm-Gs was used instead. Among these positive clones, we selected four independent, uracil-prototrophic strains from each marker. The resultant transformants were named CC strains for URA_Cm-Cm (CC14, CC18, CC24, and CC27), and CG strains for URA_Cm-Gs (CG1, CG2, CG3, and CG6). We used specific pairs of primers for PCRs and verified the occurrence of the recombination events at the CMD184C region (Figure 1C, F1/R1) and the CMD185C/CMD186C region (Figure 1C, F2/R2) in both CC and CG strains. However, when PCRs were performed using another pair of primers that was designed to amplify the whole inserted regions, different products were amplified between CC and CG strains (Figure 1C, F1/R2). In all CC strains, a single-sized fragment with an expected length of 8.2 kb was amplified, demonstrating that single-copy insertion events had occurred. In contrast, the expected 8.2-kb fragment was poorly or rarely amplified from CG strains. Moreover, the strains CG1 and CG2 additionally produced a fragment of 3.9 kb, the same length amplified from the wild-type (10 D) and parental (M4) strains. These results suggested that single-copy insertion rarely occurred with the construct containing the chimeric selection marker, yielding a mixture of revertants and undesired transformants.

The Authentic Selection Marker URA_Cm-Cm Guarantees Single-copy Insertion whereas the Chimeric Marker URA_Cm-Gs Causes Multicopy Insertion

We then analyzed the genomic structures of CC and CG strains by Southern blotting analyses. A hybridization probe was designed to recognize both URA_Cm-Cm and URA_Cm-Gs as well as the endogenous URA5.3 gene. The first set of analyses against KpnI-digested genomic fragments identified a 5.8-kb band corresponding to the endogenous URA5.3 locus in all CC and CG strains (Figure 2A, KpnI, double arrowhead). This result demonstrated that neither construct altered the genomic structures of the endogenous URA5.3 locus. In CC strains, an additional ∼12-kb fragment with the same intensity was detected (Figure 2A, KpnI, single arrowhead), indicating that the predicted single-copy insertion via a double-crossover reaction had occurred at the CMD184C locus. In contrast, larger fragments were observed in CG strains, implicating multicopy insertion of the introduced fragments. Moreover, the copy number was apparently variable among these CG strains as judged by their signal intensities. Southern blotting against undigested and KpnI-digested genomic DNA confirmed that the large-sized fragment observed in CG2 was not an artifact derived from incomplete digestion of genomic DNA (Figure 2A, CG2). The second set of analyses against EcoRV-digested genomic fragments further confirmed predicted single-copy insertion in CC strains, and multicopy insertion in CG strains. The size of the positive fragments was variable among different CG strains.

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Figure 2. Characterization of the genome structure of CC and CG strains.

(A) Southern blotting analyses of CC strains (CC14, CC18, CC24 and CC27) and CG strains (CG1, CG2, CG3 and CG6) along with 10 D and M4. Schematic representations include the position of the probe (yellow bars) and restriction enzyme sites used in the Southern blotting analyses. The URA_Cm-Gs gene is a chimera of C. merolae (1 to 515 bp) and G. sulphuraria (516 to 1416 bp) sequences. The hybridization probe recognizes the introduced fragments (both URA_Cm-Gs and URA_Cm-Cm) as well as the endogenous URA5.3 gene. The arrowhead indicates the predicted position of a fragment produced by a single-copy-insertion event. The double arrowhead indicates the predicted position of a fragment derived from the endogenous URA5.3 locus. The predicted sizes of these fragments are as follows: KpnI, 12.4 kb for CC and CG, 5.8 kb for endogenous URA5.3; EcoRV, 8.2 kb for CC and CG, 11.4 kb for endogenous URA5.3. A part of the ethidium bromide (EtBr)-stained gel is shown as a loading control. (B) Quantitative-PCR analyses of CC and CG strains, along with 10 D and M4, to estimate the copy number of CMD184C-end (b) and EGFP (c). The value of (b) and (c) in each strain was normalized against that of CMD184C-mid (a), located outside of the introduced DNA fragment, to estimate their copy number. The bars indicate the SD (n = 3). (C) PCR analysis to detect tandem insertion. The positions of PCR primers (F3 and R3) are shown in (B) and their sequences were shown in Table S1 with No. 29 and No. 30, respectively.

https://doi.org/10.1371/journal.pone.0073608.g002

To rigorously estimate the inserted copy number in each strain, we performed quantitative-PCR analyses. The copy number of the CMD184C-end sequence (Figure 2B, b) and the EGFP sequence (Figure 2B, c) in each strain was estimated by being normalized against the CMD184C-mid sequence (Figure 2B, a) that is located outside of the introduced DNA fragment. We found that CC strains contained ∼1 copy each of the CMD184C-end and EGFP sequences (Figure 2B, graphs), a result perfectly consistent with that obtained by Southern blotting (Figure 2A). In striking contrast, the estimated copy numbers of CMD184C-end and EGFP in the CG strains were high and variable among the different strains (from ∼3 up to ∼48 copies). Moreover, the estimated numbers of the CMD184C-end and EGFP sequences sometimes diverge (e. g., in CG3). We then designed and performed a PCR analysis to seek for the possibility of tandemly repeated insertion of the introduced fragments (Figure 2C). If the fragments were inserted into a tandem manner, this analysis would detect amplification of a ∼3-kb fragment. As expected, no such bands were amplified from the control and CC strains. In the CG strains, however, positive bands with different lengths and different strengths were amplified, suggesting that radically rearranged sets of the fragments were inserted into the genome in these strains. The current data do not eliminate the possibility that some of the introduced fragments might be inserted into loci outside of the targeted region (i.e., CMD184C/CMD185C).

The Copy Number of Gene Insertion Closely Reflects Transgene Expression

Having examined the genomic property of the transformants, we next investigated the growth property of each strain. It was found that all transformants displayed growth curves indistinguishable from those of M4 (parental strain) and 10 D (wild type) in MA2 medium containing uracil (Figure 3A, left). In MA2 medium containing no uracil, the uracil-auxotrophic strain M4 failed to grow, but all four CC strains grew almost normally as the wild-type strain (Figure 3A, right). The growth rates of CG strains were variable, with a tendency that strains with low-copy-number insertion grew slowly (e. g., CG6).

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Figure 3. Growth of and EGFP expression from the transformants.

(A) Growth curves of the CC and CG strains in MA2 medium in the presence or absence of uracil. (B) Immunoblotting analysis against total lysates with anti-GFP. The arrowhead indicates the position of EGFP. A CBB-stained part of the gel is shown as a loading control. (C) Fluorescent images showing EGFP signals detected in the CC and CG strains. The CG strains emitted much higher levels of EGFP signals than the CC strains. Exposure times under blue-light excitation are 0.5 sec for 10 D, M4 and CC stains, and 0.2 sec for CG strains. The lower panels show typical G1 and M phase cells (CG1), indicating that the EGFP signals distributed throughout the cytosol. Note that the chloroplasts emitted red autofluorescence. Bars, 5 µm (upper panels) and 1 µm (lower panels). (D) Flow cytometry analysis of EGFP fluorescence. The broken line indicates the mode value calculated from the 10 D data, representing the sum of background and autofluorescence signals.

https://doi.org/10.1371/journal.pone.0073608.g003

We then looked at the expression level of the EGFP transgene in each strain. Immunoblotting analysis demonstrated that a constant level of EGFP expression was detectable in CC strains, whereas much higher levels of expression were observed in CG strains (Figure 3B). The expression levels in CG strains were highly variable, yet largely proportional to the estimated copy numbers inserted into the genome. Consistently, fluorescence microscopy showed a low and constant level of signals was observed among CC strains, whereas higher and variable levels were detectable in CG strains (Figure 3C). The EGFP signals distributed throughout the cytoplasm in both G1 phase and M phase (Figure 3C, bottom). Finally, flow cytometry analysis of the EGFP signals also substantiated the immunoblotting data (Figure 3D). The fluorescent intensities in CG strains were largely correlated with the gene copy numbers estimated, with an exception of CG2. Although the exact reason for this discrepancy is unknown, it is possible, for instance, that scrambled organization of multiple copies of the transgene on the genome affected the level of protein expression from each copy.

Conclusions and Perspectives

In the current study, we have firmly established a technique of targeted gene insertion in C. merolae, demonstrating that the use of the authentic URA_Cm-Cm selection marker guarantees single-copy insertion into the genome. It is important to emphasize that modified versions of this technique have now been successfully applied to targeted disruption and targeted replacement (tagging) of condensin genes [16]. On the other hand, the chimeric URA_Cm-Gs gene, a selection marker used in the previous studies, is found to result in multicopy insertion at high frequencies. We reason that the activity of G. sulphuraria OMP-decarboxylase is substantially lower than that of the C. merolae counterpart in C. merolae cells, and that multicopy insertion of URA_Cm-Gs is positively selected for rescuing the mutant allele of M4. It is anticipated that the current results will substantially simplify and strongly encourage future genetic analyses in C. merolae, thereby facilitating our understanding of the fundamental mechanisms of cell proliferation, organelle inheritance and adaptation to stressful environments. Moreover, the technique established here will open an avenue to regulated overexpression of transgenes and may help create cellular metabolites potentially useful for the development of next-generation biofuels that utilize algal ability for carbon fixation.

Cell Cultures

The wild-type C. merolae strain 10D-14 [17] and the uracil-auxotrophic mutant M4 [14] were used in the current study. They were maintained by gyratory culture (130 rpm), in either 2× Allen’s medium [18] or MA2 medium [11] containing uracil (0.5 mg/mL), at pH 2.3 and 42°C under continuous light. Cell cultures were diluted to an OD₇₅₀ of 0.4, incubated under continuous light with aeration for 24 h, and then used for downstream analyses.

Targeted Gene Insertion

Primers used for plasmid construction are listed in Table S1. To make the URA_Cm-Cm selection marker, a DNA fragment containing the URA5.3 gene (including an 897-bp upstream sequence and a 471-bp downstream sequence) was amplified by PCR using the primer set No. 1/2 and C. merolae 10 D genomic DNA as a template. A CMD184C fragment (containing the last 1,961 bp of the CMC184C ORF and its 1.9-kb downstream sequence) and a pQE80 vector were amplified by PCR with the primer sets No. 3/4 and No. 5/6, respectively. The CMD184C fragment was subcloned into pQE80 vector (QIAGEN) using the In-Fusion HD Cloning Kit (Clontech). The resultant pD184 vector was amplified by PCR with the primer set No. 7/8. An upstream sequence of CMO250C (−600 to −1), the EGFP ORF (Takara Bio Inc.), and a downstream sequence of β-tubulin (+1 to +200) were amplified by PCR with the primer sets No. 9/10, No. 11/12, and No. 13/14, respectively, and subcloned into pQE vector using the In-Fusion HD Cloning Kit. Using the resultant pO250-EGFP vector as the template DNA, the assembled fragment (the upstream region of CMO250C [−600 to −1], EGFP ORF, the downstream of β-tubulin [+1 to +200]) was amplified by PCR with the primer sets No. 15/16, and subcloned into amplified pD184 vector. The resultant pD184-O250-EGFP vector was amplified with the primer sets No. 17/18. The URA_Cm-Cm selection marker was subcloned into the amplified pD184-O250-EGFP vector. The resultant pD184-O250-EGFP-URA_Cm-Cm vector was used as a template to amplify the assembled set of fragments (containing a part of the CMD184C ORF and 3′-UTR [1270 to +25], CMO250C 5′-UTR, EGFP, CMO250C 5′-UTR, EGFP, β-tubulin 3′-UTR, the URA_Cm-Cm marker, and the CMD184C downstream sequence [+28 to +1,448]) with the primer set No. 19/20. The amplified 8.1-kb DNA fragment was used for transformation.

To remove the sequence containing the OMP-decarboxylase domain of URA_Cm-Cm from the pD184-O250-EGFP-URA_Cm-Cm vector, the vector was amplified by PCR with the primer set No. 21 and 22. The sequence containing the OMP-decarboxylase domain of G. sulphuraria URA5.3 was amplified by PCR using the primer set No. 23/24 and URA_Cm-Gs selection marker as the template DNA [15], and then the sequence was subcloned into the amplified pD184-O250-EGFP-URA_Cm-Cm vector. The resultant pD184-O250-EGFP-URA_Cm-Gs was used as a template to amplify the assembled fragment (containing a part of the CMD184C ORF and 3′-UTR [1270 to +25], CMO250C 5′-UTR, EGFP, CMO250C 5′-UTR, EGFP, β-tubulin 3′-UTR, the URA_Cm-Gs marker, and the CMD184C downstream sequence [+28 to +1,448]) by PCR with the primer set No. 19/20 and the extremely high-fidelity PCR polymerase PrimeSTAR Max (Takara). The amplified 8.1-kb DNA fragment was used for transformation.

The C. merolae strain M4 [14] was used for targeted gene insertion as a parental strain. Cells were grown in MA2 medium [11] containing uracil (0.5 mg/ml) and 5-fluoroorotic acid (FOA) (0.8 mg/ml) in a Erlenmeyer flask with shaking at 130 rpm under continuous white light (80 µmol/s⋅m²)at 40°C. MA2 medium was solidified with 0.5% (w/v) gellan gum (Wako, Japan). To adjust pH to ∼2.3, H₂SO₄ was added at a final concentration of 0.05% (v/v). Transformation was performed as described previously [11], [15]. A 4-µg aliquot of the PCR fragment was used for transformation, and cells were incubated under continuous white light at 40°C until colonies were formed. The colonies were then transferred to starch on solidified MA2 medium containing uracil (0.5 mg/ml).

Southern Blotting Analyses

Cells (10 ml, OD₇₅₀ = 1.0) were harvested, resuspended in 4 ml of nucleic acid extraction buffer (100 mM Tris-HCl, pH 7.5, 50 mM EDTA, and 1% SDS) containing 25 µg/ml proteinase K, and incubated at 37°C for 12 hr. The lysate was extracted with PCI (phenol: chloroform: isoamylalcohol = 25∶24∶1), and total nucleic acids were precipitated with isopropanol in the presence of 20 µg of glycogen. The pellet was dissolved in TE containing RNase A (QIAGEN) at a final concentration of 0.5 mg/ml and incubated at 37°C for 45 min. The genomic DNA was then purified by PCI extraction, followed by isopropanol precipitation. 2 µg of genomic DNA were digested with KpnI or EcoRV, and then separated by 0.8% agarose gel electrophoresis. Transferring DNA from the agarose gel to a nitrocellulose membrane was performed by a conventional alkaline method using 20×SSC buffer. DIG-labeled DNA fragments were prepared by PCR using DIG Probe Synthesis Kit (Roche) with specific primers (Table S1) and used as hybridization probes. The DIG was detected with alkaline phosphatase (AP)-conjugated anti-DIG antibody (Roche) and CDP-Star (Roche), and the signals were visualized with the luminescent image analyzer LAS-3000 (FUJIFILM).

Quantitative PCRs

Real-time PCRs were performed using a CFX96 Real-Time PCR Detection System and C1000 Thermal Cycler (Bio-Rad) in a 20- µl reaction mixture containing 1 µl template DNA, 0.5 nM primers (Table S1) and 10 µl SsoFast EvaGreen Super Mix (Bio-Rad). Standard curves were constructed using serially diluted solutions of DNA isolated from C. merolae cells and the relevant sets of primers. The copy number of the (b) or (c) region in each strain was normalized with the data of the (a) region that is located outside of the introduced DNA fragment (Figure 2B).

Growth Curves

To draw growth curves of C. merolae strains, they were diluted to an OD₇₅₀ of 0.4 in MA2 medium containing uracil, and were grown for a week in order to equalize their growth conditions. The cells were then inoculated in the medium at an OD₇₅₀ of 0.2, and their growth was periodically monitored. Alternatively, the cells were washed twice with a uracil-free MA2 medium, and then inoculated in the uracil-free medium at an OD₇₅₀ of 0.2, and their growth was periodically monitored.

Immunoblotting Analysis

A whole cell lysate prepared from 2.5×10⁶ cells was separated by SDS-PAGE, and then transferred to a PVDF membrane. The membrane was probed with an anti-GFP antibody (Abcam) at a final concentration of 1 µg/ml, followed by goat anti-rabbit HRP-conjugated IgG (Vector Laboratories). The chemiluminescence signals were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore) and LAS-3000 (FUJI FILM).

Fluorescent Microscopy and Flow Cytometry

Images were captured using a fluorescence microscope (BX51; Olympus) with a wide bandpass filter set (U-MWIB2; Olympus) and a three charge-coupled device (CCD) camera system (C7780-10; Hamamatsu Photonics) as described previously [19]. Fluorescent intensities of GFP were measured using JSAN cell sorter (Bay bioscience) with 488-nm laser.