Integration of a Galdieria plasma membrane sugar transporter enables heterotrophic growth of the obligate photoautotrophic red alga Cynanidioschyzon merolae

Abstract

The unicellular thermoacidophilic red alga Cyanidioschyzon merolae is an emerging model organism of photosynthetic eukaryotes. Its relatively simple genome (16.5 Mbp) with very low-genetic redundancy and its cellular structure possessing one chloroplast, mitochondrion, peroxisome, and other organelles have facilitated studies. In addition, this alga is genetically tractable, and the nuclear and chloroplast genomes can be modified by integration of transgenes via homologous recombination. Recent studies have attempted to clarify the structure and function of the photosystems of this alga. However, it is difficult to obtain photosynthesis-defective mutants for molecular genetic studies because this organism is an obligate autotroph. To overcome this issue in C. merolae, we expressed a plasma membrane sugar transporter, GsSPT1, from Galdieria sulphuraria, which is an evolutionary relative of C. merolae and capable of heterotrophic growth. The heterologously expressed GsSPT1 localized at the plasma membrane. GsSPT1 enabled C. merolae to grow mixotrophically and heterotrophically, in which cells grew in the dark with glucose or in the light with a photosynthetic inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and glucose. When the GsSPT1 transgene multiplied on the C. merolae chromosome via the URA _Cm-Gs selection marker, which can multiply itself and its flanking transgene, GsSPT1 protein level increased and the heterotrophic and mixotrophic growth of the transformant accelerated. We also found that GsSPT1 overexpressing C. merolae efficiently formed colonies on solidified medium under light with glucose and DCMU. Thus, GsSPT1 overexpresser will facilitate single colony isolation and analyses of photosynthesis-deficient mutants produced either by random or site-directed mutagenesis. In addition, our results yielded evidence supporting that the presence or absence of plasma membrane sugar transporters is a major cause of difference in trophic properties between C. merolae and G. sulphuraria.

Keywords: Cyanidioschyzon merolae; Galdieria sulphuraria; heterotroph; mixotroph; plasma membrane sugar transporter.

Figure 1

Production of a Cyanidioschyzon merolae strain expressing HA‐GsSPT1 (Ap‐SPT1 strain). (a) A schematic diagram of the insertion of HA‐SPT1 transgene and URA selection marker into the chromosomal URA locus. The first line indicates introduced linear DNA, the second line indicates the genomic structure of the uracil‐auxotrophic mutant M4 (derived from C. merolae 10D), and the third line indicates genomic structure of Ap‐SPT1. The asterisk shows the position of a frameshift mutation in the URA gene resulting in truncation of the C‐terminal half of the URA protein in M4. To express HA‐GsSPT1,APCC promoter (600‐bp upstream flanking sequence of the APCC ORF) and β‐tubulin terminator (200‐bp downstream flanking sequence of β‐tubulin ORF) were connected to the upstream and downstream regions of the HA‐SPT1 ORF, respectively. (b) Immunoblotting of total cell lysate with an anti‐HA antibody showing expression of HA‐SPT1 in the Ap‐SPT1 strain. The wild‐type (WT) strain was used as a negative control. The arrowhead indicates the HA‐SPT1 protein that was expressed specifically in Ap‐SPT1. (c) Phase‐contrast (PC) and immunofluorescent images of the WT and Ap‐SPT1 cells stained with an anti‐HA antibody. The cell indicated with the arrowheads was enlarged and shown in the images. The red color is autofluorescence of the chloroplast (Chl). Green fluorescence is the signal detecting HA‐SPT1 (HA). Bars = 2 μm

Figure 2

Properties of heterotrophic and mixotrophic growth of Ap‐SPT1 strain. (a) Growth curves of Ap‐SPT1 in the inorganic MA2 medium supplemented with 0, 12, 25, and 50 mM glucose in the dark. As a control, the wild‐type (WT) was also grown in MA2 with 0 and 25 mM glucose in the dark. Cultures in the dark were started by inoculating cells maintained photoautotrophically (in MA2 in the light) into respective media. Error bars indicate the standard deviations of three independent cultures. (b) Growth curves of WT and Ap‐SPT1 in the light (40 μmol m⁻² s⁻¹) or dark with or without 25 mM glucose and/or 10 μM DCMU. Error bars indicate the standard deviations of three independent cultures. (c) Oxygen evolution rates of WT in the light or dark with or without DCMU. Error bars indicate the standard deviations of three independent cultures. *p < 0.05; NS, not significant (t‐test). (d) Growth of Ap‐SPT1 in the light or dark with or without DCMU in the presence of glucose. The left graph shows growth of Ap‐SPT1 when the cells grown autotrophically were inoculated at day 0. The right graph shows growth of Ap‐SPT1 when the cells in the light with glucose and DCMU (day 35 in b) were inoculated at day 0. (e) Image showing the WT and Ap‐SPT1 cultures grown under respective conditions (at day 13 in b)

Figure 3

Production of a Cyanidioschyzon merolae strain overexpressing HA‐GsSPT1 (Cp‐SPT1 strain) by multi‐copy integration of the transgene. (a) Immunoblotting with an anti‐GFP antibody to compare activities of APCC and CPCC promoters. The CBB‐stained PVDF membrane is shown as a loading control. GFP was expressed by APCC promoter (APCCp; 600‐bp upstream flanking sequence of the APCC ORF) or CPCC promoter (CPCCp; 500‐bp upstream flanking sequence of the CPCC ORF) in C. merolae. Two independent transformed clones (#1 and #2) for APCCp and CPCCp were analyzed, respectively. (b) A schematic diagram of the insertion of the HA‐SPT1 transgene and URA_{C m‐Gs} selection marker into the chromosomal region between CMD184C and CMD185C loci. The first line indicates the introduced linear DNA, and the second line indicates the genomic structure of C. merolae M4. The third and fourth lines indicate the genomic structure of the Cp‐SPT1 strain in which a single‐copy and multi‐copy of the HA‐SPT1 and URA_{C m‐Gs} is integrated into the chromosome, respectively. To express HA‐GsSPT1,CPCC promoter and β‐tubulin terminator were connected to the upstream and downstream regions of the HA‐ SPT1 ORF (codon‐optimized to C. merolae), respectively. (c) Quantitative PCR analyses showing the copy number of transgenes in Ap‐SPT1 and Cp‐SPT1 strains. The wild‐type (WT) strain was used as a control. The positions of the amplified sequences were indicated in b (purple arrowhead, β‐tubulin terminator; gray arrowhead; URA promoter). The values of purple and gray were normalized against values of a chromosomal region outside the introduced DNA. The primers used in the qPCR assay are shown in Supporting Information Table S1. The sequences which correspond to purple and gray arrowheads and the region outside the introduced DNA were amplified using the primer set No. 19/20, 21/22, and 23/24. The wild‐type genome possesses one copy of the endogenous β‐tubulin terminator and URA promoter. Besides these endogenous sequences, Ap‐SPT1 genome possesses another copy of β‐tubulin terminator of the transgene (total two copies) but one copy of URA promoter because the transgene with URA marker was integrated into chromosomal URA locus (Figure 1a). Besides the endogenous sequences, Cp‐SPT1 genomes possess additional (~20) copies of β‐tubulin terminator and URA promoter of the transgene. Error bars indicate the standard deviations of three technical replicates. (d) Immunoblotting of total cell lysate with an anti‐HA antibody showing expression of HA‐SPT1 in Cp‐SPT1. The wild‐type (WT) and Ap‐SPT1 strains were used as controls. The CBB‐stained PVDF membrane is shown as a loading control. (e) Phase‐contrast (PC) and immunofluorescent images of the WT and Cp‐SPT1 cells stained with an anti‐HA antibody. The red color is autofluorescence of the chloroplast (Chl). Green fluorescence is the signal detecting HA‐SPT1 (HA). Bar = 2 μm

Figure 4

Heterotrophic and mixotrophic growth properties of Cp‐SPT1 strain. (a) Growth curves of Cp‐SPT1 in the light (40 μmol m⁻² s⁻¹) or dark with or without 25 mM glucose and/or 10 μM DCMU. Error bars indicate the standard deviations of three independent cultures. (b) Immunoblotting of total cell lysate with an anti‐HA antibody showing expression of HA‐SPT1 in Cp‐SPT1 in the light with 25 mM glucose and 10 μM DCMU or in the dark with 25 mM glucose. The CBB‐stained PVDF membrane is shown as a loading control. (c) Colony formation of Ap‐SPT1 and Cp‐SPT1 cells on the starch bed (white) on the gellan gum‐solidified medium. The wild‐type (WT) strain was used as a control. WT, the original Ap‐SPT1 and Cp‐SPT1 cells (grown photoautotrophically in MA2 in the light;) and the acclimated Ap‐SPT1 cells (grown in MA2 with glucose and DCMU in the light) were inoculated onto the starch bed (~5 mm in diameter) on solidified MA2 medium with 10 μM DCMU and 25 mM glucose in the light (40 μmol m⁻² s⁻¹) or with glucose in the dark. 1.6 × 10⁶, 3.2 × 10⁴, 6.4 × 10³, 1.3 × 10³, 2.6 × 10², 5.1 × 10, and 1 × 10 cells of each strain were inoculated onto starch beds as shown in the schema. The starch beds indicated with the arrowheads were enlarged and shown on the right of each original image. The number above enlarged starch bed images indicates days after inoculation of cells when the photographs were taken. Bar = 2 mm