Stable Expression of Antibiotic-Resistant Gene ble from Streptoalloteichus hindustanus in the Mitochondria of Chlamydomonas reinhardtii

The mitochondrial expression of exogenous antibiotic resistance genes has not been demonstrated successfully to date, which has limited the development of antibiotic resistance genes as selectable markers for mitochondrial site-directed transformation in Chlamydomonas reinhardtii. In this work, the plasmid pBSLPNCB was constructed by inserting the gene ble of Streptoalloteichus hindustanus (Sh ble), encoding a small (14-kilodalton) protective protein into the site between TERMINVREP-Left repeats and the cob gene in a fragment of mitochondrial DNA (mtDNA) of C. reinhardtii. The fusion DNA-construct, which contained TERMINVREP-Left, Sh ble, cob, and partial nd4 sequence, were introduced into the mitochondria of the respiratory deficient dum-1 mutant CC-2654 of C. reinhardtii by biolistic particle delivery system. A large number of transformants were obtained after eight weeks in the dark. Subsequent subculture of the transformants on the selection TAP media containing 3 ìg/mL Zeomycin for 12 months resulted in genetically modified transgenic algae MT-Bs. Sequencing and Southern analyses on the mitochondrial genome of the different MT-B lines revealed that Sh ble gene had been integrated into the mitochondrial genome of C. reinhardtii. Both Western blot, using the anti-BLE monoclonal antibody, and Zeomycin tolerance analysis confirmed the presence of BLE protein in the transgenic algal cells. It indicates that the Sh ble gene can be stably expressed in the mitochondria of C. reinhardtii.


Introduction
Unlike the genetic transformation of the nucleic and chloroplast genomes, genetic modification of the mitochondrial genome is still very limited in higher plants and animals [1,2], with successful stable expression of foreign functional genes in the mitochondria only demonstrated in Saccharomyces cerevisiae [3,4]. Besides the Saccharomyces cerevisiae, Chlamydomonas reinhardtii is a good choice for mitochondrial studies, as it can undergo mitochondrial transformation with homologous genes [1,5,6] or heterologous protein [7]. It has distinct advantages in comparison to higher plants as it is unicellular, haploid, is amenable to tetrad analysis, and its' three genomes are theoretically subject to specific transformation [8]. However, the stable expression of exogenous antibiotic resistance genes in C. reinhardtii mitochondria have not been routinely performed to date, and an effective selectable marker for screening the successful mitochondrial transformation has not yet been found.
To construct a transformation and screening system, we have developed a recombinant plasmid containing Sh ble gene, which was isolated from Streptoalloteichus hindustanus and confers Zeomycin resistance. Zeomycin is a copper-chelated glycopeptide antibiotic, structurally related to the group of bleomycin and phleomycin type antibiotics, and it is toxic to both prokaryotic and eukaryotic cells. The antibiotic is effective on most aerobic cells and is therefore useful for selection of cells that express the Sh ble gene in bacteria, eukaryotic microorganisms, plants and animal cells. As there is no cross resistance with other currently used animal cells markers, this antibiotic can also be used to isolate clones resistant to other selecting agents. In some cases, the Sh ble protein can be fused to other proteins (such as the green fluorescent protein) for visual screening and drug selection of transfected eukaryotic cells [9]. The Sh ble gene displays interesting characteristics which make it a useful marker for the nuclear transformation in C. reinhardtii [10,11,12].
In this study, a Sh ble gene was integrated into the mitochondrial genome of C. reinhardtii cc-2654 by homologous recombination. Subsequently, Zeomycin resistant protein was expressed in the mitochondria of C. reinhardtii. To our knowledge, this is the first report of stable expression of an exogenous antibiotic-resistant gene in the mitochondria of photosynthetic organism. It provides the opportunity to develop the Sh ble gene as a selectable marker for mitochondrial site-directed transformation in C. reinhardtii.

Construction of mitochondrial expression vector containing Sh ble gene
The expression vector containing a resistance marker was constructed by inserting the Sh ble gene into pBsLPNC. The left arm and PNC sequences were amplified from the mitochondrial genome of C. reinhardtii CC-124 and inserted into pBluescript II SK to create pBsLPNC. The Sh ble gene was amplified and cloned into plasmid pBsLPNC. In the resulting plasmid pBsLPNCB, the Sh ble gene was flanked by the left arm and PNC fragments. The DNA fragment PNC contains the cob gene and a part of the nd4 gene. The fragment containing the Sh ble gene was integrated to the mitochondrial genome of C. reinhardtii cc-2654 by homologous recombination mediated by this part of nd4 gene ( Figure 1).

Mitochondrial transformation and screening of transgenic algae MT-Bs
The respiratory deficient dum-1 mt 2 CC-2654 mutant of C. reinhardtii was used as a recipient strain. The CC-2654 mutant can not grow in dark, because the left fragment containing TERMINVREP-Left and cob gene of its mtDNA was truncated. The Sh ble gene was sandwiched between the TERMINVREP-Left arm sequence and cob gene from the mitochondrial genome of wild-type strain C. reinhardtii CC-124. The fusion DNA-construct was introduced into the mtDNA of C. reinhardtii CC-2654 by biolistic particle delivery system. The transformants were obtained in the dark after 8 weeks. Subsequent subculture of the transfromants on the selection TAP media containing 3 ìg/mL Zeocin for 12 months resulted in genetically modified transgenic algae named C. reinhardtii MT-B ( Figure 2A). Actually, one week of subculturing on selective medium was enough to obtain homozygous transformants. 12 months was used to reveal whether long period would influence transformants. We found that the 12 months' transformants and one week' transformants had no obvious genetic differentiation. The presence of transgenes in transformants was further investigated. For comparison, DNA samples isolated from wild-type CC-124, respiratory-deficient mutant CC-2654 and transgenic MT-B were used for PCR amplification of Sh ble (about 380 bp) using primers B1/B2. The expected PCR bands were found in the MT-B transformants only ( Figure 2B).

Analysis of the ble integrated site in the mitochondrial genome of MT-B
To locate the ble gene, a 4 kb fragment of mtDNA was analyzed by DNA sequencing. Results showed that left arm sequence was from 1-504 bp, Sh ble was 557-931 bp and the PNC fragment containing cob and nd4 was 931-2737 bp. The sequence from 2738-3491 bp was the other part of nd4 gene in the mtDNA of cc-2654. This demonstrated that the construct fragment was integrated to the mitochondrial genome of C. reinhardtii cc-2654 by homologous recombination mediated by part of the nd4 gene.
Total DNA was digested with Bam HI, Nde I and Sac II, respectively, and blotted with the ble-probe. A single band of approximately 0.8 kb was present in Bam HI digested DNA. Nde I and Sac II digests also showed a single band of approximately 3.3 kb (Figure 3). The result matched the restriction map of mitochondrial genome of MT-B. Only one southern hybridized band with three restriction enzymes demonstrated that there was Northern blot analysis was used to detect the steady-state level of the transcript of Sh ble ( Figure 4B). Total RNA was probed with a probe specific for the Sh ble transcript. A significant signal band of 0.38 kb was detected, corresponding to the predicted size of the ble transcript. This proved a good indicator for the transcriptional activity of Sh ble in transgenic MT-B.  Expression of the BLE protein in the mitochondria of C. reinhardtii Western blot analysis was performed to detect the Sh ble gene product at a protein level using monoclonal anti-BLE antibody. Total soluble protein of the transgenic algae was used for the analysis and a protein band with 13.7 kDa was detected in transgenic algae as expected ( Figure 5). This indicated that the transgene Sh ble was stably expressed in mitochondria of the transformant MT-B.
Resistance and Sensitivity of transgenic algae MT-Bs to Zeomycin in C. reinhardtii Sensitivity of C. reinhardtii transformants to Zeomycin was observed using different concentrations of Zeomycin added to the solid TAP plate. 2 mg/ml Zeomycin was found to completely inhibit the growth of C. reinhardtii CC-124 and CC-2654. However, the transgenic strain MT-B contained ble gene grew well on TAP agar plate containing 3 mg/ml Zeomycin. 5 mg/ml Zeomycin inhibited visible the growth of C. reinhardtii MT-B after incubation ( Figure 6). The data suggested that mitochondrial expression of the Sh ble gene rendered the transformants certain levels of resistance against Zeomycin.

The expression levels of the exogenous Sh ble and the mitochondrial genes in transgenic MT-B
The semi-quantitative RT-PCR methods were used to investigate the expression differences of Sh ble, cob, nd4, nd5, cox1 and nd2 among transgenic MT-B, cc-2654 and wild type cc-124. The results showed: (1) the insertion of Sh ble gene did not significantly affect other mitochondrial genes expression. (2) Theoretically, the Sh ble and other mitochondrial genes should belong to constitutive co-expression, which was initiated by the bi-directional promoter between nd5 and cox1 [13]. However, our results showed that the expression level of exogenous Sh ble was less than other mitochondrial genes in transgenic MT-B (Figure 7). The possible factors that effected the expression of Sh ble gene included the codon bias, promoter and intron [14,15].

Discussion
C. reinhardtii is the only photosynthetic organism whose mitochondria can be genetically modified with homologous mtDNA [13]. Early attempts to develop a mitochondrial transformation system for the green alga C.reinhardtii relied on the complementation of respiratory deficient mutant cc-2654 with the cob gene encoding apocytochrome b from wild type cc-124. After integration with the transgene cob, genetically modified respiratory deficient mutants can grow in the dark and the transformants can consequently be easily screened. The transformant selection could not be applied to higher multicellular organisms, because these organisms can not survive due to respiratory defect. It was reported that two plasmids were cotransformed by biolistic bombardment, one plasmid carried the nuclear selectable marker, and the other contained a mitochondrial transgene. At first, transformants were screened by the selectable marker of nuclear transformation under light illumination, then they were transferred into dark condition for further screening of mitochondrial transformants, but this approach did not work well [16]. So far, no effective antibiotic resistance genes are stably expressed in mitochondria of photosynthetic organisms,    which limits the development of mitochondrial genetic transformation.
Zeomycin is an antibiotic that is sensitive in yeast, green algae, plant and animal cells. The ble gene from Streptoalloteichus hindustanus can be stably expressed in the nuclear genomes of mammalian cells [17], plants [18], yeast [19] and green alga C. reinhardtii [20], so that transformants of these organisms become Zeomycin resistant and can be screened using Zeomycincontaining selection media. However, although stable expression of foreign genes has been achieved [21], and many marker genes for transformant screening have been found in the nuclear or chloroplast genomes of C. reinhardtii [22,23], the mitochondrial expression of exogenous marker genes has not been successfully expressed to date. In this study, a fusion DNA-construct, which contained TERMINVREP-Left, Sh ble, cob, and partial nd4 sequence, was introduced into the mitochondria of respiratory deficient dum-1 mutant CC-2654 of C. reinhardtii. Proof that the DNA-construct had been inserted into the mtDNA of respiratory deficient mutant cc-2654 was demonstrated as the transformant MT-B was able to grow on the TAP selection media containing 3 ìg/mL Zeomycin under dark conditions. The sequence analysis of left 4 kb fragment of the MT-B mtDNA furtherly confirmed that the Sh ble gene was integrated into the mtDNA by homologous recombination. Previous work has found mitochondrial pseudogenes in the nucleus of eukaryotes, with many copies of nuclear counterparts of mtDNA were found in nuclear DNA [24]. In this study the southern blot detection of RFLPs with three restriction enzymes clearly demonstrated that there was only one copy of Sh ble in the transformant MT-B (Figure 3). It indicated that Sh ble gene was only integrated to the mitochondrial genome of transformant MT-B and none of the Sh ble gene had been inserted into the nuclear mtDNA pseudogenes of C. reinhardtii by homologous recombination.
The transgenic algae MT-B kept the Zeomycin resistance after it was cultured in TAP media for two years ( Figure 6). We never observed a loss of Zeomycin resistance in transgenic algae maintained on non-selective condition. It showed that the expression of the Sh ble gene was stable in the mitochondria of transgenic algae. To our knowledge, this is the first report of stable expression of an exogenous antibiotic resistance gene in the mitochondria of C.reinhardtii.
It is unclear how algal cells acquire the Zeomycin resistance by the expression of BLE protein inside the mitochondria of transformants MT-B. Zeomycin is a DNA intercalating agent that can destroy all three cellular genomes of algal cells. Theoretically then, the expression of the BLE protein in the mitochondria would protect only the mitochondrial DNA, with no protection provided to the nuclear and the chloroplastic DNA. However, our experimental results showed that the transgenic MT-Bs could grow on TAP agar plate containing 3-5 mg/ml Zeomycin ( Figure 6). This indicates that the BLE protein is exported from the mitochondria in the transgenic MT-B. The putative explanations are that: within the mitochondria exists a specific protein export mechanism [25,26], BLE is a small molecule of the heterologous protein (13 kD), it can be delivered outside mitochondria or out of the algae by the protein export mechanism to create Zeomycin resistance. Further detailed pathway studies will be required.
A cell of C. reinhardtii contains 30-50 copies of the mitochondrial genome [16]. In a transformation event, not every mitochondrial genome is integrated with the foreign gene fragment, and heterogeneous cells containing both transformed and non transformed mitochondria exist. As a transformant selection process proceeds, separations of heterogeneous mitochondrial genome occured and homogenous transformant cells were finally obtained. In the C. reinhardtii chloroplasts, this homogenization process of transformant cells is quite fast [27], but the process of mitochondrial homogenization is very slow in the respiratory deficient mutant of C. reinhardtii; heterogeneous transformant clones still remained after incubation in a dark environment for 2-3 months [1]. However, TAP plates containing Zeomycin were used to enhance the pressure of transformant selection and homogenization in this study. When the transgenic clones were subcultured on selection media containing 3 mg/ml Zeomycin for one week, our results showed that all transformant lines were homoplasmic (Figure 2). This suggests that Zeomycin resistance selection was able to accelerate the homogenization process of mitochondrial transformants.
The resistance to Zeomycin was much weaker in the transgenic strains MT-B than that previously reported in algae [11,12], mammalian cells [17], plants [18], yeast [19] or bacteria [28]. Sh ble gene expression allowed the growth of transgenic bacteria at 25-50 mg/mL Zeomycin on a low-salt growth medium. Transgenic yeast, plants and mammalian cells can grow at 100 mg/mL of Zeomycin. The transgenic MT-B that contained Sh ble gene in mitochondrial genome could only grow on TAP agar plate containing 3-5 mg/ml Zeomycin. However, the lower mitochondrial expression of Sh ble gene was enough to be used to generate stable mitochondrial transformants by selection against Zeomycin. The expression of the Sh ble gene based on the mitochondria could be developed as a resistance marker of expression system for transgenic research and expression of other kinds of recombinant proteins in mitochondria of C. reinhardtii.
Our results showed that the transgenic alga MT-B could produce BLE proteins after subculturing for 24 months. It indicates that the gene Sh ble can be stably expressed in the mitochondria of C. reinhardtii. As the full sequence of Sh ble contains only 375 bp, it is suitable and feasible to be used as a selectable marker for the mitochondrial genetic transformation of C. reinhardtii. In particular it makes it possible to integrate a transgene into different sites within the C. reinhardtii mitochrodrial genome by site-specific recombination, not just limited to the left site [6]. Our results shed light on mitochondrial reverse genetics studies and genetic engineering in the photosynthetic organisms.

Mitochondrial Transformation Procedures
A biolistic PDS-1000 He particle delivery system (Bio-Rad, USA) was used for mitochondrial transformation. Gold particles (30 mg, 0.6 mM) were precipitated in a 1.5 ml Eppendorf tube. The pellet was resuspended in 1 ml of 70% ethanol and 1 ml of sterile water. The water was removed after gold particles were precipitated by centrifugation. Particles were resuspended with 5 ml of DNA (1 mg/ml). Subsequently, 50 ml of freshly prepared 2.5 M CaCl 2 and 20 ml of 0.1 M spermidine was added to the DNA-gold particle mixtures. It was vortex mixed and centrifuged. The pellet was washed with 70% ethanol and the particles were suspended in 48 ml of 100% ethanol. C. reinhardtii strain dum-1 mt 2 CC-2654 was grown in liquid TAP medium up to the exponential phase (5,6610 6 cells/ml). Cells were then collected and spread on TAP agar plates at 22uC in the light for 2 days. 8 ml of DNA particle suspension was loaded into the biolistic apparatus, and each plate was bombarded at 1,350 psi of helium. After bombardment, the cells were maintained on 1% TAP agar plate containing 100 mg/ml ampicillin and 3 mg/ml Zeocin in the light at 22uC for one day and then in darkness at 22uC for 7-8 weeks [1,5,6].

Southern Blot and RT-PCR Analysis
Genomic DNA was isolated by using the DNeasy kit (Takara, Japan). The purified DNA was digested with restriction enzymes and the digested DNA fragments were separated on a 0.8% agarose gel. The DNA in the gel was blotted onto Hybond N+ (Amersham Biosciences) and probed with a random-primed ble probe using DIG high prime DNA labeling and detection starter kit I (Roche) according to the manufacturer's instructions.

Western blot analysis
For detection of the BLE protein [13], total soluble protein was isolated from 20 ml cultures of C. reinhardtii grown to 5610 6 cells/ ml. Cells were then collected by centrifugation at 5,000 g for 10 minutes and lysed by sonication in buffer containing 20 mM Tris-HCl, 10 mM EDTA, 50 mM NaCl, pH 7.4, and 1.0 mM phenylmethylsulfonyl fluoride. The cell extract was centrifuged at 19,000 g for 30 minutes. Protein samples were boiled for 5 minutes, separated by 10% SDS-PAGE gel and transferred electrophoretically onto polyvinylidene difluoride membranes (Millipore, Bedford, MA). The blots were blocked with 5% bovine serum albumin and treated with rabbit anti-sh ble antibodies (Cayla,France) at a dilution of 1:5000. After washing, the blot was incubated with alkaline phosphatase conjugated goat anti-rabbit IgG secondary antibody (Proteintech Group, USA) and developed with BCIP/NBT alkaline phosphatase substrate solution (Promega, USA).