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.2015 Apr 21:6:6925.
doi: 10.1038/ncomms7925.

Designer diatom episomes delivered by bacterial conjugation

Affiliations

Designer diatom episomes delivered by bacterial conjugation

Bogumil J Karas et al. Nat Commun..

Abstract

Eukaryotic microalgae hold great promise for the bioproduction of fuels and higher value chemicals. However, compared with model genetic organisms such as Escherichia coli and Saccharomyces cerevisiae, characterization of the complex biology and biochemistry of algae and strain improvement has been hampered by the inefficient genetic tools. To date, many algal species are transformable only via particle bombardment, and the introduced DNA is integrated randomly into the nuclear genome. Here we describe the first nuclear episomal vector for diatoms and a plasmid delivery method via conjugation from Escherichia coli to the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. We identify a yeast-derived sequence that enables stable episome replication in these diatoms even in the absence of antibiotic selection and show that episomes are maintained as closed circles at copy number equivalent to native chromosomes. This highly efficient genetic system facilitates high-throughput functional characterization of algal genes and accelerates molecular phytoplankton research.

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Conflict of interest statement

J.C.V. is executive chairman and co-chief scientific officer of Synthetic Genomics Inc., H.O.S. is co-chief scientific officer and a member of the board of directors and C.A.H. is chairman of the scientific advisory board. All three of these authors and the J. Craig Venter Institute hold shares of Synthetic Genomics Inc. The remaining authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Conjugative transfer of plasmids fromE. coli toP. tricornutum.
(a) Map of the plasmids p0521 and p0521s and their derivation fromP. tricornutum scaffold 25.OriT, origin of transfer;URA3, gene encoding orotidine 5′-phosphate decarboxylase fromS. cerevisiae, and ShBle, phleomycin-resistance cassette withP. tricornutum FcpF promoter andFcpA terminator. (b) Average number ofP. tricornutum colonies obtained per conjugation for different ‘cargo' plasmid variants of p0521s. Deletions of features indicated by Δ, pRL443 is the RP4 conjugative plasmid. (c) Maps of plasmids used to test the importance of theP. tricornutum- and yeast-derived regions. Plasmids pPtPuc1-4 differ in whether they have theP. tricornutum-derived region from p0521s (Pt R1R2, green) or the yeast-derivedCEN6-ARSH4-HIS3 (yellow). Other plasmid features present in all four plasmids are described on the pPtPuc1 map. (d) Number ofP. tricornutum colonies obtained after conjugation with the pPtPuc plasmids (‘cargo' plasmids). Features of each plasmid are noted in the table under the figure (for example, theP. tricornutum-derived region from p0521S, Pt R1R2, or the yeast elements,CEN6-ARSH4-HIS3). Error bars denote one s.d. of the mean from at least three biological replicates per experiment.
Figure 2
Figure 2. Demonstration thatP. tricornutum episomes replicate as stable, circular, low-copy plasmids (a–e), and expression and localization of proteins encoded on theP. tricornutum episome p0521s (F-I).
(a) Cultures ofP. tricornutum containing p0521s (clone 9, Supplementary Fig. 3), were subcultured in seawater medium for 28 days with or without antibiotic selection and plated (Supplementary Fig. 5). DNA from five antibiotic-resistant colonies from each culture (Supplementary Fig. 5D) was recovered inE. coli and isolated plasmids were separated by agarose gel electrophoresis. Shown are rescued plasmids derived from separateP. tricornutum colonies that were initially subcultured for 28 days without (lanes 1–5) or with (lanes 6–10) antibiotic selection. ‘M' designates supercoiled marker and ‘C' designates the original plasmid (isolated from clone 9) introduced intoP. tricornutum. Arrow denotes supercoiled plasmid band. (b) Stability of p0521-Se containing a 49-kbS. elongatus fragment. Ten independently transformedP. tricornutum lines containing p0521-Se were subcultured in liquid media with selection for 60 days, followed by episome rescue and separation of plasmids by agarose gel electrophoresis. Arrow denotes supercoiled plasmid band. (c) Plasmids extracted fromP. tricornutum were untreated, treated with exonuclease, ClaI endonuclease or a combination of exonuclease and ClaI. Treated plasmids were transformed intoE. coli and the number of transformed colonies was plotted (error bars indicate one s.d. of the mean from three biological replicates). (d) Agarose gel electrophoresis of plasmids extracted fromP. tricornutum and treated with nucleases. Lanes from left to right: (1) 1 kb+ ladder (NEB), (2) p0521s control (fromE. coli), (3) p0521s exonuclease-treated (extracted fromP. tricornutum), (4) p0521s untreated (extracted fromP. tricornutum), (5) 1 kb+ ladder (NEB), (e) Copy number of p0521s inP. tricornutum determined by qPCR. Cm (Cat gene) and His (HIS3 gene) are loci found on the episome backbone; Ure (urease, protein ID 29702) and NR (nitrate reductase, protein ID 54983) are loci encoded onP. tricornutum nuclear chromosomes 18 and 20, respectively; Rbc (RuBisCO small subunit) and CytB (Cytochromoe B) are loci found on theP. tricornutum chloroplast and mitochondrial chromosomes, respectively. Error bars denote one s.d. of the mean from three biological replicates. (f) Wild-typeP. tricornutum, fluorescence measured with GFP settings. (g)P. tricornutum expressing CFP translationally fused to beta-carbonic anhydrase (Protein ID 51305) localized to the chloroplast pyrenoid encoded on plasmid p0521s. (h)P. tricornutum expressing YFP translationally fused to mitochondrial urea transporter (Protein ID 39772) encoded on plasmid p0521s. (i)P. tricornutum expressing GFP localized to the cytoplasm encoded on plasmid p0521s. Scale bar forf–i indicates 5 μm.
Figure 3
Figure 3. Episome replication inT. pseudonana.
(a) Conjugation fromE. coli toT. pseudonana results in increased conjugation efficiency when the yeastCEN6-ARSH4-HIS3 region is included on the plasmid (pTpPuc3) compared with control plasmid lacking this region (pTpPuc4). Error bars denote one s.d. of the mean from at least three biological replicates. (b,c) Images ofT. pseudonana wild type (b) and exconjugants expressing YFP translationally fused to PEPCK (Protein ID_5186) encoded on a p0521s-derived episome (c). Scale bar, 2.5 μm.
Figure 4
Figure 4. Suggested strategies to clone sequences of interest into diatom episomes.
(a) Genes or pathways of interest can be assembled into p0521s replacing theURA3 gene. Incoming DNA should be prepared with sequence overlaps to the plasmid regions flankingURA3 and assembled in yeast spheroplasts with counter-selection on 5FOA. (b) Smaller sequences interest (equivalent in size to single expression cassettes) can be assembled into pPtPuc3 using Gibson assembly or other cloning strategy. Sequences can be introduced anywhere in the plasmid, but we typically insert at the 3′ region of theCEN6-ARSH4-HIS3 sequence. (c) Any existing plasmid can be modified with the yeastCEN6-ARSH4-HIS3 sequence amplified from p0521s, pPtPuc3 or other source to enable episomal replication inP. tricornutum orT. pseudonana. TheoriT sequence should also be included (if it is not already present in the vector sequence) if conjugation fromE. coli is chosen as the method of plasmid introduction.
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