Strains and culture conditions
The wild type Gonium pectorale Müller strains SAG 12.85, NIES-1710, and CCAP 32/14 were obtained from the Culture Collection of Algae at the University of Göttingen (SAG), Germany [36], the Microbial Culture Collection at the National Institute for Environmental Studies (NIES) (Tsukuba, Japan) and the Culture Centre of Algae and Protozoa (CCAP) (Ambleside, Scotland), respectively. Cultures were grown in Jaworski's Medium (JM) [37] at 23°C or 29°C in an 8 h dark/16 h light (~10,000 lux) cycle. Cultures were grown in 10 ml glass tubes with caps that allow for gas exchange or in 50 ml and 300 ml Erlenmeyer flasks, which were aerated via Pasteur pipettes with 40 cm3 and 55 cm3 sterile air/min, respectively. Transgenic strains that express the aphVIII gene were grown in JM in the presence of 1 μg paromomycin/ml (paromomycin sulfate, Sigma-Aldrich, St. Louis, MO).
Transformation vectors
The plasmid pPmr3 contains the 0.8 kb S. rimosus aphVIII gene, which confers resistance to paromomycin, a V. carteri hsp70A-rbcS3 hybrid promoter (0.5 kb and 0.27 kb of upstream sequences), and a 3'-UTR from the V. carteri rbcS3 gene (0.53 kb of downstream sequence), and the total size of plasmid pPmr3 is 5.1 kb, which includes the pBluescript II vector backbone [21]. The plasmid paphG contains the 0.8 kb S. rimosus aphVIII gene, a C. reinhardtii hsp70A-rbcS2 hybrid promoter (0.26 kb and 0.22 kb of upstream sequences), intron 1 (0.15 kb) of the C. reinhardtii rbcS2 gene 42 bp upstream of the translation start codon, and a 3'-UTR of the C. reinhardtii rbcS2 gene (0.22 kb of downstream sequence), and the plasmid paphG contains sixteen repeats of this hybrid gene in the same orientation, which results in a 28.4 kb insert. The total size of plasmid paphG is 31.4 kb, which includes the pBluescript II vector backbone [22]. The plasmid ptubar4 contains the 7.8 kb V. carteri arylsulfatase (ars) gene, a V. carteri β2-tubulin promoter (0.5 kb of upstream sequence), and a V. carteri arylsulfatase 3'-UTR (2.3 kb of downstream sequence), and the total size of plasmid ptubar4 is 13.2 kb, which includes the pUC18 vector backbone [30]. The plasmid pHsp-HA contains the 3.2 kb V. carteri hsp70A gene with its own promoter (2.5 kb of upstream sequence) and its own 3'-UTR (0.75 kb of downstream sequence), and the coding sequence is tagged with a sequence coding for the HA-epitope. The total size of plasmid pHsp-HA is 9.4 kb, which includes the pBluescript II vector backbone [25]. The plasmid pPsaD-GLuc contains the 0.57 kb luciferase (luc) gene from G. princeps, which was engineered to match the codon usage in C. reinhardtii, a C. reinhardtii psaD promoter (0.8 kb of upstream sequence), and a C. reinhardtii psaD 3'-UTR (0.55 kb of downstream sequence). The total size of plasmid pPsaD-GLuc is 5.0 kb, which includes the pBluescript II vector backbone [27]. The plasmid pHsp70A-GLuc contains the 0.57 kb luciferase (luc) gene from G. princeps (codon-optimized for C. reinhardtii) fused to a 0.8 kb DNA fragment that contains the first three exons of the hsp70B gene of C. reinhardtii, and the hybrid gene is driven by the C. reinhardtii hsp70A promoter (0.26 kb of upstream sequence) and the 3'-UTR comes from the C. reinhardtii rbcS2 gene (0.22 kb of downstream sequence). The total size of plasmid pHsp70A-GLuc is 4.9 kb, which includes the pBluescript II vector backbone [27].
Preparation of plasmid DNA
Plasmid DNA was purified routinely using the E.Z.N.A.® Plasmid Mini Kit II (Peqlab, Erlangen, Germany). Large plasmids (paphG) were purified from 50–100 ml E. coli cultures as described [38], but the anion exchange column step was omitted. The obtained plasmid DNA was further purified using the E.Z.N.A.® Cycle Pure Kit (Peqlab).
Coating of microprojectiles
For particle gun transformation (most successful combination of parameters as provided in Table 2), gold microprojectiles of 0.6 μm in diameter (Bio-Rad, Hercules, CA) were coated with the required plasmids. To that end, ~3 mg gold microprojectiles in 50 μl H2O were quickly mixed with 5 μg DNA of the circular selectable marker plasmid (concentration > 0.4 μg/μl), 5 μg DNA of the circular co-bombarded plasmid (if applicable), 50 μl 2.5 M CaCl2, and 20 μl 0.1 M spermidine (Sigma-Aldrich). Mixing was sustained for 30 min at 4°C. After the addition of 200 μl EtOH at room temperature, the suspension was centrifuged for 2–3 s at ~5000 g. The pellet was washed three times with 100 μl EtOH (at -20°C) and centrifuged for 2–3 s at ~5000 g. Finally, the DNA-coated particles were resuspended in 60 μl EtOH and kept at 4°C for use within 3 h.
Determination of cell concentration
In G. pectorale the number of cells per colony varies. Therefore, we refer to "cells/ml" rather than "colonies/ml". Cell concentration was determined using a hemacytometer with Neubauer ruling.
Stable nuclear transformation by particle gun
One hundred fifty milliliters of a logarithmically growing G. pectorale culture that contained ~6 × 104 cells/ml was harvested by centrifugation (800 g, 8 min, swing-out rotor) and resuspended in a total volume of 12 ml JM. Two milliliters of the suspension was spread evenly on a cellulose acetate membrane filter with a pore size of 1.2 μm and a diameter of 47 mm (Whatman, London, UK), and the filter was placed on top of a stack of absorbent paper that soaked up all the excess liquid. Stable transformation of Gonium (most successful combination of parameters as provided in Table 2) was performed using a Biolistic® PDS-1000/He (Bio-Rad) particle gun. One-sixth of the DNA-coated microprojectiles were spread on a macrocarrier (Bio-Rad), which was placed in a macrocarrier holder (Bio-Rad). The distance between macrocarrier and stopping screen (Bio-Rad) was set to 8 mm. The helium pressure was defined by rupture disks with a burst pressure of 1100 psi (Bio-Rad). The gap between rupture disk and macrocarrier was adjusted to 7 mm. The membrane filter with its layer of G. pectorale was positioned in the bombardment chamber, the distance between the stopping screen and target cells was adjusted to 6 cm, and the chamber was partly evacuated to 27 inch Hg. After particle bombardment, the colonies were washed off from the membrane filter with JM. The procedure was repeated five times, and the colonies from the six bombardments were pooled and evenly distributed among ten 50 ml Erlenmeyer flasks that contained a final volume of ~35 ml JM each. Bombarded colonies were incubated under standard conditions for 48 h, and then 1 μg paromomycin/ml was added. Within 24 h, non-transformed cells died, which resulted in a clarification of the medium. After another 9–16 days of incubation in the presence of the antibiotic, greening of a flask showed the initial presence of at least one paromomycin-resistant cell that led to a population of transformants. No more than one transformant per flask was computed.
Re-isolation of transformants
For detailed analyses, transformants were re-isolated to ensure uniform genetic condition. For this, a serial dilution of an exponentially growing Gonium culture was performed in a Terasaki plate (Nunc™ MicroWell™ MiniTrays; Thermo Fisher Scientific, Langenselbold, Germany), which was filled with 10 μl JM medium per well. Under microscopic control, a single Gonium colony was finally transferred into a standard glass tube with JM medium containing 1 μg paromomycin/ml and incubated under standard conditions.
Paromomycin-resistance assay
Transformants or wild type strains were transferred into glass tubes with increasing concentrations of paromomycin in JM. At the beginning of the assay, each tube contained ~12,000 healthy cells in a total volume of 10 ml. Incubation under standard conditions continued for eight days. Subsequently, the tubes were analyzed for either viable, green cells/colonies or cell lysis with some white remains of dead cells/colonies.
Primer design
Oligonucleotide primers were designed using the primer analysis software Oligo 6 (Molecular Biology Insights, Cascade, CO), DNASIS™ (version 7.00; Hitachi Software Engineering, San Francisco, CA), and Primer Express® (Applied Biosystems, Foster City, CA).
Isolation of genomic DNA
Thirty-five milliliters of a logarithmically growing culture was harvested by centrifugation (3500 g, 10 min). The pellet, which had a wet weight of ~80 mg, was washed twice with H2O, centrifuged 2×, resuspended in H2O, and frozen in liquid nitrogen. Frozen samples were pulverized in a mortar. After homogenization, the sample was warmed to 65°C, and lysis buffer (Qiagen, Hilden, Germany) that contained RNase A1 was added. Genomic DNA was isolated using the spin columns of the DNeasy® Plant Mini Kit (Qiagen).
Larger amounts of genomic DNA were prepared by conventional methods [39], using tris-saturated phenol (Roti®-phenol; Roth, Karlsruhe, Germany).
Genomic PCR
Genomic PCR was carried out in a total volume of 50 μl, which contained ~100 ng of genomic DNA, 300 nM of each primer, 0.2 mM dNTP mix, 1.5 mM MgCl2, and 2.6 units of Expand High Fidelity enzyme mix in 1× Expand High Fidelity buffer (Roche Applied Science, Basel, Switzerland). PCR was performed on a T3 Thermocycler PCR system (Biometra, Göttingen, Germany) using the following conditions: 40 cycles of 94°C for 20 s, 55°C for 30 s, and 72°C for 45 s and a final extension was at 72°C for 10 min. The PCR products were cloned and sequenced.
Southern blotting
After restriction enzyme digest, genomic DNA fragments were separated on 1% agarose gels, vacuum transferred to nylon membranes (Hybond-N®; Amersham Biosciences, Little Chalfont, UK), and fixed to the membrane by baking for 30 min at 120°C using standard protocols [39]. A 282 bp fragment of the aphVIII coding region was amplified by PCR (Expand High Fidelity Plus PCR System; Roche Applied Science) and simultaneously labeled using a digoxigenin DNA labeling mix (Roche Applied Science). A 343 bp fragment of the coding region of the luciferase (luc) gene from G. princeps (codon-optimized for C. reinhardtii) was amplified in the same way. Pre-hybridization at 52°C, hybridization at 52°C, and washing steps were carried out in standard solutions (Roche Applied Science). Detection of the hybridizing bands was done by using an anti-digoxigenin-alkaline phosphatase conjugate (1:7500 dilution) and the chemiluminescent substrate CDP Star®, in accordance with the instructions of the supplier of the chemiluminescence reagent (Roche Applied Science). Chemiluminescence-sensitive films (Retina XBA; Fotochemische Werke, Berlin, Germany) were subsequently exposed to the membranes for 2–15 min.
Isolation of total RNA
Total RNA was isolated from ~9 × 106 Gonium cells using the membrane-based SV Total RNA Isolation System (Promega, Madison, WI). RNA quantification and purity checks were done by agarose gel electrophoresis and by measuring absorption at 260 and 280 nm with an Ultrospec™ 2100 pro UV/Visible Spectrophotometer (GE Healthcare, Uppsala, Sweden).
Reverse Transcription (RT)-PCR
First strand cDNA synthesis was performed using 1 μg total RNA and Moloney murine leukemia virus (MMLV) reverse transcriptase lacking ribonuclease H activity (H minus), according to the manufacturer's instructions (Promega). Subsequent PCR was carried out using the Mid Range PCR system, according to the instructions provided by the vendor (Peqlab). PCR was performed on a T3 Thermocycler PCR system (Biometra) using the following cycling conditions: 40 cycles of 94°C for 20 s, 55°C for 30 s, and 68°C for 45 s and a final extension at 68°C for 10 min. The RT-PCR products were cloned and sequenced.
Western blot analysis
Eight hundred milliliters of a logarithmically growing culture containing ~5 × 107 cells was harvested by centrifugation (3500 g, 8 min, swing-out rotor), washed with 20 mM phosphate buffer (pH 7.4), and disrupted using a Sonopuls™ HD2070 sonicator (Bandelin Electronic, Berlin, Germany). The lysate was cleared by centrifugation (86,000 g, 90 min), passed through a Centricon® 100 column (Millipore, Bedford, MA), concentrated on a Centricon® 10 column (Millipore), and used for western blot analysis. Samples were separated on a 10% standard SDS-polyacrylamide gel, electroblotted to a polyvinylidene fluoride membrane (0.45 μm; Millipore), and probed using a purified polyclonal rabbit anti-AphVIII antibody at 1:100 dilution [20, 21]. The secondary antibody was a horseradish peroxidase-linked anti-rabbit-IgG at 1:10,000 dilution (Bio-Rad). Signals were visualized by using the luminol-based chemiluminescent substrate Lumiglo® (Cell Signaling Technology, Danvers, MA) and Hyperfilm™ ECL films (Amersham Biosciences).
Luciferase assays
For assays on light-sensitive films, a Gonium culture (50 ml) with 3–6 × 106 cells/ml was centrifuged, resuspended in 850 μl assay buffer [0.1 M K2HPO4 (pH 7.6), 0.5 M NaCl, 1 mM EDTA] and cells were disrupted using a Sonopuls™ HD2070 sonicator (Bandelin Electronic) and the lysate was transferred to a 24-well plate. After addition of 150 μl 0.05 mM coelenterazine (Fluka, Neu-Ulm, Germany) in assay buffer, the 24-well plate was exposed to a chemiluminescence-sensitive film (Retina XBA; Fotochemische Werke) for 30 s at 20°C [40].
Quantitation of bioluminescence was performed as described by Shao and Bock [27]. For it, 5 ml of a Gonium culture, which has been grown at 23°C to a density of 3–6 × 106 cells/ml, was centrifuged, resuspended in 300 μl sample buffer [1.5 mM Tris-HCl (pH 7.8), 1 mM EDTA], and frozen at -20°C for at least 20 min. After thawing, 20 μl samples were added to 125 μl of the assay buffer [0.1 M K2HPO4 (pH 7.6), 0.5 M NaCl, 1 mM EDTA]. Following incubation for 15 min at 20°C in the dark, samples were transferred to clear polystyrene vials (Sarstedt, Nümbrecht, Germany), 50 μl 0.01 mM coelenterazine was added, and bioluminescence was assayed at 20°C using a MiniLumat LB9506 luminometer (Berthold, Bad Wildbad, Germany). The luminescence was recorded as relative light units.
For analysis of induction of luciferase activity in heat-shocked transformants, organisms were subject to a temperature shift from 23 to 36°C for 1 h, because in preliminary tests, shifts to 36° resulted in the strongest induction in comparison to lower or higher temperatures (data not shown). After a 1 h recovery phase at 23°C, cells were lysed by freezing and thawing and luciferase activity was assayed at 20°C as described above [27]. As a reference, non-heat-shocked transformants were analyzed in the same way. The induction factors were calculated by comparison of heat-shocked with non-heat-shocked samples.
Phylogenetic analysis
Alignment of sequences was done using the MUltiple Sequence Comparison by Log-Expectation program (MUSCLE) [41]. Minor manual optimization of alignments, trimming, and management of multi-aligned data was done with BioEdit v7.0.9 [42]. Alignments were illustrated using GeneDoc 2.6 [43]. The Needleman-Wunsch global alignment algorithm [44] from the European Molecular Biology Open Software Suite (EMBOSS) was used for the comparison of two sequences [45]. Unrooted consensus trees were calculated using the PHYLogeny Inference Package (PHYLIP) [46]. For each consensus tree, 10000 bootstrap resamplings of multi-aligned sequences were generated with Seqboot, distance matrices were computed with Dnadist, trees were constructed using the neighbor-joining method [47] as implemented in Neighbor, and finally a consensus tree was built using Consense. Phylogenetic trees were drawn with TreeView [48].
GenBank accession numbers
The novel sequences that are described in this study have been deposited under the following accession numbers:
Gonium pectorale SAG 12.85: rbcL [GenBank: FJ793553], psaA [GenBank: FJ793556], psaB [GenBank: FJ793559], ITS [GenBank: FJ793562]; Gonium pectorale CCAP 32/14: rbcL [GenBank: FJ793554], psaA [GenBank: FJ793557], psaB [GenBank: FJ793560], ITS [GenBank: FJ793563]; Gonium pectorale NIES-1710: rbcL [GenBank: FJ793555], psaA [GenBank: FJ793558], psaB [GenBank: FJ793561], ITS [GenBank: FJ793564].
The accession numbers of other cited sequences are:
Gonium pectorale NIES-569: rbcL [GenBank: D63437], psaA [GenBank: AB044242], psaB [GenBank: AB044463]; Gonium pectorale UTEX 2570: ITS [GenBank: AF054425]; Gonium pectorale AWCAf2–3: ITS [GenBank: AF054431]; Gonium pectorale AWC-Laos: ITS [GenBank: AF182429]; Gonium pectorale Coleman 16-1: ITS [GenBank: U66969]; Gonium pectorale UTEX 2075: ITS [GenBank: AF054434]; Gonium pectorale UTEX 2581: ITS [GenBank: AF054433]; Gonium octonarium GO-LC-1+: rbcL [GenBank: D63436], psaA [GenBank: AB044241], psaB [GenBank: AB044462]; Gonium octonarium UTEX 842: ITS [GenBank: U66968]; Gonium quadratum NIES-653: rbcL [GenBank: D63438], psaA [GenBank: AB044243], psaB [GenBank: AB044464]; Gonium quadratum AWC-Cal3-3: ITS [GenBank: AF182430]; Gonium quadratum AWC-Cat: ITS [GenBank: AF182431]; Gonium multicoccum UTEX 2580: rbcL [GenBank: D63435], psaA [GenBank: AB044240], psaB [GenBank: AB044461]; Gonium multicoccum UTEX 783: ITS [GenBank: U66967]; Gonium viridistellatum UTEX 2519: rbcL [GenBank: D86831], psaA [GenBank: AB044244], psaB [GenBank: AB044465]; Gonium viridistellatum UTEX 2520: ITS [GenBank: AF182432]; Tetrabaena socialis (= Gonium sociale) NIES-571: rbcL [GenBank: D63443], psaA [GenBank: AB044415], psaB [GenBank: AB044466]; Tetrabaena socialis (= Gonium sociale) UTEX 14: ITS [GenBank: U66976]; Basichlamys sacculifera (= Gonium sacculiferum) NIES-566: rbcL [GenBank: D63430], psaA [GenBank: AB044416], psaB [GenBank: AB044467, AB044468]; Basichlamys sacculifera (= Gonium sacculiferum) UTEX 822: ITS [GenBank: U66972]; Astrephomene gubernaculifera NIES-418: rbcL [GenBank: D63428], psaA [GenBank: AB044234], psaB [GenBank: AB044458]; Astrephomene gubernaculifera UTEX 1393: ITS [GenBank: AF054422]; Astrephomene perforata NIES-564: rbcL [GenBank: D63429], psaA [GenBank: AB044238], psaB [GenBank: AB044460]; Astrephomene perforata UTEX 2475: ITS [GenBank: U66939]; Pandorina morum NIES-574: rbcL [GenBank: D63442], psaA [GenBank: AB044226], psaB [GenBank: AB044452]; Pandorina morum Poona: ITS [GenBank: AF182433]; Eudorina unicocca UTEX 1215: rbcL [GenBank: D63434], psaA [GenBank: AB044209], psaB [GenBank: AB044440]; Eudorina elegans NIES-456: rbcL [GenBank: D63432], psaA [GenBank: AB044199], psaB [GenBank: AB044435]; Pleodorina californica UTEX 809: rbcL [GenBank: D63439], psaA [GenBank: AB044192], psaB [GenBank: AB044430]; Volvox aureus NIES-541: rbcL [GenBank: D63445], psaA [GenBank: AB044182], psaB [GenBank: AB044424]; Volvox carteri NIES-732: rbcL [GenBank: D63446], psaA [GenBank: AB044185], psaB [GenBank: AB044425]; Volvox globator UTEX 955: rbcL [GenBank: D86836], psaA [GenBank: AB044187], psaB [GenBank: AB044428]; Chlamydomonas reinhardtii 137C: rbcL [GenBank: J01399], psaA [GenBank: AB044419], psaB [GenBank: AB044470]; plasmid pPmr3 [GenBank: AY429514].