Photosynthetic directed endosymbiosis to investigate the role of bioenergetics in chloroplast function and evolution

Bacterial strains, compounds, and reagents

Synechococcus elongatus strains were derived from Synechococcus elongatus PCC 7942 (Syn7942). This strain was obtained from Prof. Susan Golden’s lab (University of California San Diego, UCSD). We used S. cerevisiae ρ + (MATa leu2-3,112 lys2 ura3-52 his3ΔHindIII arg8Δ::URA3 [cox2-60])³⁰ as a host for S. elongatus endosymbionts.

Sequence similarity networks analysis of CAE46506 homologs

Sequence similarity networks (SSN) analysis was based on the PSI-BLAST (Position-Specific Iterated BLAST) search using nucleotide transporter (NTT) protein from Candidatus Protochlamydia amoebophila UWE25 (NCBI Sequence ID CAE46506.1) as a query sequence. By using the NCBI database, non-redundant protein sequences (nr) we curated 500 sequences as prelimany data. In addition, organism specific PSI-BLAST was performed, for red algae (taxid:2763) and higher plants (taxid:3193). There are a total of 500 sequences of non-redundant protein sequences (nr) which have identity coverage of 48.44 to 100 % with query coverage of 92 to 100%. Similarly top 500 sequences were selected from higher plants (taxid:3193) which have identity coverage of 48.44 to 100 % and query coverage of 91 to 97%. Finally, red algal protein hits were selected with identity coverage of 53.22 to 57.03% and query coverage of 73 to 96%. The EFI- Enzyme Similarity Tool (EFI-EST, https://efi.igb.illinois.edu/efi-est/) was used to generate sequence similarity networks (SSNs). A data set of 1010 sequences was used to build the SSN. An alignment score of 120 was used for generating the networks described in this study.

Identification of Cyanophora paradoxa ADP/ATP translocase and phylogenetic analysis

Identification of complete glaucophyte Cyanophora paradoxa putative ADP/ATP translocase performed after BLASTp search to whole genome data base (http://cyanophora.rutgers.edu/cyanophora_v2018/) using CAX16542.1 (partial sequence) as query sequence. The identified complete Cyanophora paradoxa putative ADP/ATP translocase TRINITY_DN12309_c0_g1_i1:235-2400 (http://cyanophora.rutgers.edu/cyanophora_v2018/, TransDecoder) was included for further multiple sequence alignment and phylogenetic analysis. In order to get a better view of NTT and ADP/ATP translocase diversity, a small dataset contains total 104 sequences were selected from endoparasite, archaea, protozoa, metazoan, dinoflagellates, euglena, chlorella, red algae, heterokonta, diatoms, green algae, moss, glaucophytes, higher plants (Fig. 2C). All retrieved FASTA sequences were aligned by Clustal Omega online multiple sequence alignment tool (https://www.ebi.ac.uk/Tools/msa/clustalo/). A phylogenetic tree was constructed using MEGA11 software, employing the Maximum Likelihood method and the JTT matrix-based model, with 500 rapid bootstrap replicates. The tree with the highest log likelihood (-9419.02) is shown. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories ( + G, parameter=0.9047)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 11.68% sites). Finally, the phylogenetic tree (Fig. 2C and Supplementary Fig. 1) was visualized by iTOL⁴⁰.

Construction of plasmids

Primers and gBlock gene fragments were purchased from Integrated DNA Technologies (IDT). Primers are listed in Supplementary Data 3. DNA fragments were amplified by Polymerase Chain Rreaction, PCR (Q5 Hot Start High-Fidelity 2X Master mix, NEB catalog # M0494S), and assembled into vectors by Gibson assembly. Gene fragments of ADP/ATP carrier genes are listed in Supplementary Table 1 and Supplementary Data 1. The genes were codon-optimized for S. elongatus expression using IDT codon optimization browser tool (https://www.idtdna.com/CodonOpt). Cyanovectors were obtained from Prof. Susan Golden’s lab (UCSD). Cloning vectors were transformed into One Shot® ccdB Survival™ 2 T1R Chemically Competent Cells (Invitrogen A10460) according to manufacturer’s specifications.

The plasmids pBD1 – pBD6 are derived from the pML17 (Supplementary Data 2). Vector maps are included in Supplementary Fig. 4, and detailed vector map links are provided in Supplementary Table 2.

pML+ XP_005536231.1: pML17 was linearized by PCR using the oligonucleotides pML17_F/ R. A gBlock of the Cyanidioschyzon merolae strain 10D; XP_005536231.1 gene codon optimized for S. elongatus was amplified by PCR using the oligonucleotides BD2F/R. The amplified DNA fragment was inserted into linearized pML17 by Gibson assembly to afford pBD1.

pML+ KAF6002099.1: A codon optimized gBlock of the Cyanidiococcus yangmingshanensis KAF6002099.1 gene was amplified by PCR using the oligonucleotides BD3F/R and inserted into linearized pML17 by Gibson assembly to afford pBD2.

pML+ PXF48205.1: A codon optimized gBlock of the Gracilariopsis chorda PXF48205.1 gene codon-optimized for S. elongatus was amplified by PCR using the oligonucleotides BD4F/R and inserted into linearized pML17 by Gibson assembly to afford pBD3.

pML+ XP_005707402.1: A codon optimized gBlock of the Galdieria sulphuraria XP_005707402.1 gene was amplified by PCR using the oligonucleotides BD5F/R and inserted into linearized pML17 by Gibson assembly to afford pBD4.

pML+ NP_178146.1: A codon optimized gBlock of the Arabidopsis thaliana NP_178146.1 gene was amplified by PCR using the oligonucleotides BD6F/R and inserted into linearized pML17 by Gibson assembly to afford pBD5.

pML+ NC_003076.8 (locus_tag=AT5G01500): A codon optimized gBlock of the Arabidopsis thaliana NC_003076.8 gene was amplified by PCR using the oligonucleotides BD7F/R and inserted into linearized pML17 by Gibson assembly to afford pBD6.

pML+CAX16542.1: A codon optimized gBlock of the Cyanophora paradoxa CAX16542.1gene was amplified by PCR using the oligonucleotides BD11F/R and inserted into linearized pML17 by Gibson assembly to afford pBD7.

pML+PXF48205.1-Flag: A same codon optimized gBlock of the Gracilariopsis chorda PXF48205.1-flag gene codon-optimized for S. elongatus was amplified by PCR using the oligonucleotides BD8F/R. The pML17 back bone was PCR isolated using BD9F/R and inserted into linearized pML17 by Gibson assembly to afford pBD3-flag.

pML+NC_003076.8-Flag: A codon optimized gBlock of the Arabidopsis thaliana NC_003076.8-flag gene was amplified by PCR using the oligonucleotides BD10F/R and inserted into linearized pML17 by Gibson assembly to afford pBD6-flag.

pML_SNARE: The SNARE genes encode Ctr-incA, and CT_813 genes were PCR isolated using oligonucleotides BD13F/1R. The linear vector backbone was PCR amplified using oligonucleotides BD15F/16 R. Performed Gibson assembly to afford pBD8.

pML+TRINITY_DN12309: A codon optimized gBlock of the Cyanophora paradoxa TRINITY_DN12309 gene was amplified by PCR using the oligonucleotides BD7F/R and inserted into linearized pML17 by Gibson assembly to afford pBD9.

Engineering of cyanobacteria strains

Log-phase Syn7942 cultures (15 mL) were centrifuged for 10 min at 3000 × g and 24 °C. The pellet was washed with 10 mL NaCl (10 mM) and resuspended in 0.3 mL BG-11 at room temperature. To this suspension was added the plasmid (1.5 μL), and the mixture was added to a 1.5 mL microcentrifuge tube and shaken in the dark (12–16 h at 70 rpm). Transformed cells were spread on BG-11 agar plates supplemented with appropriate antibiotic(s) and incubated at 37 °C under 30-50 μmol photons·s⁻¹m⁻² for ≤10 days. PCR analysis of recombinant loci was used to evaluate genomic recombination.

SynJEC3 was generated by transformation of wild-type Syn7942 with pML17 to give a recombinant mutant which ectopically expresses Ctr-incA, CT813 and an ADP/ATP translocase from the NSII locus.

SynBD1 was generated by transformation of wild-type Syn7942 with pBD1 to give a recombinant mutant which ectopically expresses Ctr-incA, CT813 and Cyanidioschyzon merolae strain 10D XP_005536231.1 a putative chloroplast ADP/ATP translocase from the NSII locus.

SynBD2 was generated by transformation of wild-type Syn7942 with pBD2 to give a recombinant mutant which ectopically expresses Ctr-incA, CT813 and Cyanidiococcus yangmingshanensis KAF6002099.1 a putative ADP/ATP carrier from the NSII locus.

SynBD3 was generated by transformation of wild-type Syn7942 with pBD3 to give a recombinant mutant which ectopically expresses Ctr-incA, CT813 and Gracilariopsis chorda PXF48205.1 a putative chloroplast ADP/ATP translocase from the NSII locus.

SynBD4 was generated by transformation of wild-type Syn7942 with pBD4 to give a recombinant mutant which ectopically expresses Ctr-incA, CT813 and Galdieria sulphuraria XP_005707402.1 a putative ADP/ATP translocase from the NSII locus.

SynBD5 was generated by transformation of wild-type Syn7942 with pBD5 to give a recombinant mutant which ectopically expresses Ctr-incA, CT813 and Arabidopsis thaliana NP_178146.1 a putative ADP/ATP translocase from the NSII locus.

SynBD6 was generated by transformation of wild-type Syn7942 with pBD6 to give a recombinant mutant which ectopically expresses Ctr-incA, CT813 and Arabidopsis thaliana NC_003076.8 a putative thylakoid an ADP, ATP carrier protein from the NSII locus.

SynBD3-Flag was generated by transformation of wild-type Syn7942 with pBD3-flag to give a recombinant mutant which ectopically expresses Ctr-incA, CT813 and Gracilariopsis chorda PXF48205.1-Flag a putative plastidic ADP/ATP translocase from the NSII locus.

SynBD6-Flag was generated by transformation of wild-type Syn7942 with pBD6-flag to give a recombinant mutant which ectopically expresses Ctr-incA, CT813 and Arabidopsis thaliana NC_003076.8 a putative thylakoid an ADP, ATP carrier protein from the NSII locus.

SynBDsnare strain was generated by transformation of wild-type Syn7942 with pBD8 to give a recombinant mutant which ectopically expresses Ctr-incA, CT813 proteins from the NSII locus.

SynBD7 strain was generated by transformation of wild-type Syn7942 with pBD7 to give a recombinant mutant which ectopically expresses Ctr-incA, CT813 proteins from the NSII locus.

SynBD8 strain was generated by transformation of wild-type Syn7942 with pBD9 to give a recombinant mutant which ectopically expresses Ctr-incA, CT813 proteins from the NSII locus.

Luciferase assay to measure ATP release by engineered cyanobacteria

Trace levels of contaminating ATP was removed from ADP solution (Sigma A2754) through treatment with hexokinase: ADP (80 mM, pH 7.5) was incubated with D-glucose (200 mM), MgCl₂ (2 mM) and hexokinase (Sigma H4502-500UN) (0.04 U/μL) at room temperature for 2 h. The mixture was filtered through an Amicon Ultra 0.5 column (14,000 × g, 15 min) and the flowthrough was stored at -20 °C. Syn7942 (wild-type), SynBD1 to SynBD5 cells were grown for 3 d to reach densities of OD₇₅₀ of 0.6. For each assay, cells were harvested by centrifugation (3000 × g, 5 min, room temperature), and washed with 20 mM Tris-HCl buffer (pH 8.0). The cells were normalized to a density (OD₇₅₀) of 1.5. The normalized cells were then incubated with ATP solution (Sigma G8877) (10 mM, pH 7.5) for 15 min at 37 °C and washed three times with 20 mM Tris-HCl to eliminate extracellular ATP. ADP (80 µM final concentration) was added, and the cells were incubated at 37 °C. The suspensions were centrifuged (10,000 × g, 5 min) and the supernatant ATP concentration was determined by using luciferase assay (ATP determination kit, Life Technologies – #A22066). ATP standards (provided with the kit) were used to obtain calibration curves.

Luciferase assay to measure ATP uptake

Syn7942 (wild-type), SynBD2, SynBD3 to SynBD6 cells were grown for 3 days to reach densities of OD₇₅₀ of 0.6. For each assay, cells were harvested by centrifugation (3000 × g, 5 min, room temperature), and the pellet was washed once with 20 mM Tris-HCl buffer (pH 8.0). The cells were normalized to OD₇₅₀ of 1.5. The cells were kept in dark for overnight prior to incubation with ATP. The normalized cells were then incubated with ATP solution (Sigma G8877) (50 nM, pH 7.5) at 37 °C for 2, 4, 6 minutes. The mixtures were then centrifuged (10,000 × g, 5 min) and the supernatant ATP concentration was determined by using luciferase assay (ATP determination kit, Life Technologies – #A22066). ATP standards (provided with the kit) were used to obtain calibration curves.

Western blot analysis for ADP/ATP carrier translocase protein expression in Syn7942 study

Syn7942 control strain and SynBD3-Flag strain were grown in appropriate culture medium describe above at 37 °C, 3000 lux till OD730 ~ 1.2. The cells were resuspended in lysis buffer (50 mM Tris-HCl, pH 8, 5 % SDS, 1 mM β-mercaptoethanol) and lysed by heating them at 90 °C for 10 min. The cell lysate was mixed with 1x Laemmli sample buffer (BIO-RAD, USA). SDS-PAGE was performed after loading 200 µg protein in 12 % acrylamide protein gels. Gel was then stained for total protein using SYPRO Ruby (Invitrogen), according to the manufacturer’s instructions, or used for western blot. For western blot analysis, gels were transferred to an Millipore Immobilon-P 0.45 µm polyvinyl fluoride (PVDF) membrane in NuPAGE transfer buffer (Invitrogen) for 180 min at 60 V in a XCell blot module (Invitrogen) at cold room and the membrane was then incubated in blocking solution (TBS-T; 20 mM Tris-Cl, 150 mM NaCl, 0.02% (v/v) Tween-20, pH 7.6 supplemented with 5% (w/v) non-fat dry milk) for 1 h. All incubation steps were carried under shaking conditions on a rocking table. Blocking agent was discarded and the membrane was incubated with primary antibody–Anti-FLAG Rabbit mAb (Cell Signaling Technology, Catalog # 14793S) used 1: 1000 dilution and 1:12000 secondary antibody–Anti-Rabbit IgG (Sigma, Catalog # A0545). Membranes were washed for 3 × 5 min in TBS-T then incubated for 5 min in ECL substrate consisting of 0.5 mL each of SuperSignal West Dura reagent A and B (Thermo Scientific) and imaged using an iBright 750 (Invitrogen) imaging system.

Fusion of engineered cyanobacteria to S. cerevisiae cox2-60 cells

The standard cell fusion protocol was followed.^23,25,30,38 Briefly, SynJEC3, SynBD2, SynBD3 and SynBD6 engineered mutants were grown under constant light (30-50 μmol photons·s⁻¹m⁻²), with shaking, for 4 days. The cells (30 mL) were then harvested (3000 × g, 10 min, 24 °C), washed twice with BG-11 and resuspended in BG-11 (500 µL). S. cerevisiae cox2-60 was grown aerobically in YPD medium (500 mL) in a 1-liter conical flask for 20 h with shaking at 200 rpm at 30 °C. The yeast cells were harvested (2500 × g, 10 min, 30 °C), washed twice with sterile water, twice with SCEM (1 M sorbitol, 13 mM β-mercaptoethanol) and resuspended in filtered SCEM solution (10 mL) containing Zymolyase 100 T (15 mg total). The suspension was incubated for 1 h at 37 °C resulting in complete spheroplasting of the culture. The suspension was centrifuged for 10 min at 1500 × g at rt. The pellet was washed twice with SCEM solution at room temperature and resuspended in 1 mL SCEM. The spheroplast suspension (5 × 750 µL) was mixed with TSC buffer (10 mM Tris-HCl, 10 mM CaCl₂, 1 M sorbitol, pH 8) (750 µL) and incubated for 10 min at 30 °C in a microcentrifuge tube. The mixture was centrifuged (1500 × g, 10 min) and the supernatant was carefully discarded. The spheroplasts were resuspended in room temperature TSC buffer (5 ×120 µL) and sorbitol (4 M, 60 µL). The prepared S. elongatus cell suspensions SynJEC3, SynBD2, SynBD3 and SynBD6 (120 µL) were added quickly to the spheroplast suspensions, mixed by tube inversion and incubated statically for 30 min at 30 °C. This mixture was then decanted into a round-bottom polypropylene tube containing PEG buffer (20% PEG 8000, 10 mM Tris-HCl, 2.5 mM MgCl₂, 10 mM CaCl₂, pH 8) (2 mL) and incubated statically at 30 °C for 45 min. The cells were centrifuged (1500 × g, 10 min, 24 °C), the supernatant was discarded and 1 mL of YPDS (YPD with 1 M sorbitol added) was added on top of the plating. The cells were incubated under light without shaking for 2 h at 30 °C under 30 rpm. The pellet was then dislodged by flicking the tube and the mixture incubated for 3 h more with gentle shaking (70 rpm), after which the cells were harvested (1500 × g, 10 min, 24 °C), resuspended in 1 M sorbitol (300 μL) and spread on Selection-I medium. After drying for 5 min, a top agar layer of Selection-I medium was overlaid on the cells. The plates were incubated at 30 °C in a 12 h light-dark cycle for four days until colonies appeared between the agar layers. The colonies were extracted from the agar, suspended in 1 M sorbitol, and spotted on Selection-II medium. For subsequent rounds of propagation, cells were scraped from the surface of the agar, resuspended in 1 M sorbitol, and spotted on Selection-III medium. The yeast/cyanobacteria chimeras are sensitive to high constant light and are propagated under 12 h light-dark cycle light dark cycles with light intensity of 50 µE/m² · s.

Cell count of S. cerevisiae cox2-60/cyanobacteria chimeras

Agar disks containing cells on Selection III medium were extracted manually from the agar plate and placed inside a microcentrifuge tube. Sorbitol (1 M, 200 μL) was added to the surface of the agar and pipetted to make a cell suspension. The tube was centrifuged for 5 s to remove the cell suspension from the agar. The suspension was added to a reusable glass slide and counted in triplicate from a brightfield image using the Countess II FL Automated Cell Counter (Fisher cat. # AMQAF1000) per the manufacturer’s instructions.

Microscopic sample preparation of yeast/cyanobacteria chimeras

Chimeric cells are washed and resuspended in 1 M sorbitol to a high density. These cells were analyzed using total internal reflection fluorescence microscopy (TIRF) on a custom-built microscope with a Zeiss Axiovert 200 M stand²³. A Cobolt diode-pumped 561 nm laser was used to excite phycobilins and normal bright field images were always captured chimera cells. The pTIRF images were acquired with a Photometric 512 Evolve EMCCD camera. Samples were viewed and imaged using a 100x oil immersion objective lens with NA = 1.4. All images were processed with ImageJ 1.53cto overlay qTIRF image and a brightfield image acquired from the same sample position. For analysis of yeast/cyanobacteria chimeras using fluorescence confocal microscopy, the chimeric cells were washed from plate spots using Hank’s Buffered Salt Solution (HBSS; NaCl (140 mM), KCl (5 mM), CaCl₂ (1 mM), MgSO₄ heptahydrate (0.4 mM), MgCl₂ hexahydrate (0.5 mM), Na₂PO₄ dihydrate (0.3 mM), KH₂PO₄ (0.4 mM), D-glucose (6 mM), NaHCO₃ (4 mM)). The cells were resuspended in 50 µL HBSS with 1X ConA (40X Concanavalin A Stock solutions (40X); Thermo Fisher C827 was dissolved in sodium bicarbonate 0.1 M and HBSS). The cells were then incubated 37 °C for 10 min in the dark. The cells are centrifuged at 1500 × g and washed twice with HBSS. The ConA-stained cells were incubated with 50 µL HBSS with 37% paraformaldehyde for 1 hour in a nutator in dark. Samples were analyzed with a commercial Leica SP8 fluorescence confocal microscope. Samples were viewed and imaged through a 63X/1.40 HC PL APO Oil CS2 lens and excited with 488 nm and 561 nm laser. Emission wavelengths in the 510-530 nm range were detected with photomultiplier tube (PMT) detector, and emission wavelengths in the 616-650 range were detected using a high-sensitivity GaAsP HyD detector. Leica Application Suite (LASX) was used to collect raw data. All images were processed with ImageJ 1.53c to overlay the two channels.

Scanning transmission electron microscope sample preparation

The control and chimera cells were pelleted by centrifugation (6000 × g, 5 min). The supernatant was resuspended in fixative (2.5% EM-grade glutaraldehyde and 2.0% EM-grade formaldehyde in 0.1 M sodium cacodylate buffer, pH 7.4) for overnight at 4 °C. The fixative was then removed, replaced briefly with buffer, and then replaced with 1% osmium tetroxide in buffer for 90 min in dark. Each sample was then subjected to 10 min buffer rinse, after which it was placed in 1% aqueous uranyl acetate and left overnight. The next day, samples were dehydrated via a graded ethanol series, culminating in propylene oxide. Following a graded propylene oxide; Epon812 series, the nuclear pellets were embedded in Epon812 prior to cutting. Ultrathin (ca. 90 nm) Epon sections on grids were stained with 1% aqueous uranyl acetate and Reynold’s Lead Citrate solution. After the grids dried, areas of interest were imaged at 160 kV, spot 3 using a Philips/FEI (now Thermo Fisher FEI) Tecnai G2 F20 S-TWIN transmission electron microscope in the Microscopy Suite at the Beckman Institute of Advanced Science and Technology (University of Illinois at Urbana-Champaign).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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• Source: https://www.nature.com/articles/s41467-024-54051-1