Direct evidence for requirement of phosphatidylglycerol in photosystem II of photosynthesis

Abstract

Phosphatidylglycerol (PG) is considered to play an important role in the ordered assembly and structural maintenance of the photosynthetic apparatus in thylakoid membranes. However, its function in photosynthesis remains poorly understood. In this study we have identified a pgsA gene of Synechocystis sp. PCC6803 that encodes a PG phosphate synthase involved in the biosynthesis of PG. A disruption of the pgsA gene allowed us to manipulate the content of PG in thylakoid membranes and to investigate the function of PG in photosynthesis. The obtained pgsA mutant could grow only in the medium containing PG, and the photosynthetic activity of the pgsA mutant dramatically decreased with a concomitant decrease of PG content in thylakoid membranes when the cells grown in the presence of PG were transferred to the medium without PG. This decrease of photosynthetic activity was attributed to the decrease of photosystem (PS)II activity, but not to the decrease in PSI activity. These findings demonstrate that PG is essential for growth of Synechocystis sp. PCC6803 and provide the first direct evidence that PG plays an important role in PSII.

Figure 1

Comparison between the deduced amino acid sequence of PGP synthase of Synechocystis sp. encoded by the pgsA gene and the deduced amino acid sequence of PGP synthase of E. coli. The amino acid residues conserved in both sequences are indicated by asterisks. Hyphens indicate gaps that were added to maximize the alignment of the sequences.

Figure 2

Insertional mutagenesis of the pgsA gene of Synechocystis sp. A, Structure of the pgsA gene in the wild type and the insertional mutant of Synechocystis sp. The directions of transcriptions of Km^r and the pgsA gene are indicated by arrows. B, PCR analysis of the pgsA gene in the wild type (lane 1) and the pgsA mutant (lane 2). The positions of DNA size markers are indicated on the right. The positions of the primers used for PCR reactions were shown by arrowheads in Figure 2A. C, Southern hybridization analysis of the pgsA gene in the wild type (lane 1) and the pgsA mutant (lane 2). Genomic DNAs extracted from the wild type and the pgsA mutant cells were digested with XbaI. One microgram of DNA was applied to each lane. The membrane was hybridized at 42°C using the pgsA gene of Synechocystis sp. as a DNA probe.

Figure 3

Growth profile of the wild type and the pgsA mutant of Synechocystis sp. A, Growth of the wild type and the pgsA mutant on agar plates. The cells grown in the medium containing 60 μm PG were streaked onto plates containing 20 μg mL⁻¹ kanamycin and 60 μm PG (+PG) or no PG (−PG), and the plates were incubated at 30°C for 3 weeks. B, Wild type (○) and mutant cells (●) were grown in the medium containing 60 μm PG. The values are the averages and sd from three independent experiments. C, Growth profile of the wild type (○) and the pgsA mutant (●) in the medium without PG. The cells grown to stationary phase in the medium containing PG were transferred to the medium not containing PG. The values are the averages and sd from three independent experiments.

Figure 4

Effect of PG concentration on the growth of the pgsA mutant of Synechocystis sp. The pgsA mutant cells grown to logarithmic growth phase in the medium containing PG were transferred to media containing various concentrations of PG. ●, 0 μm; □, 2 μm; ◊, 5 μm; x, 20 μm; ▵, 60 μm. The values are averages from two independent experiments.

Figure 5

Changes in PG content and photosynthetic activity of intact cells of the wild type and the pgsA mutant of Synechocystis sp. after a deprivation of PG from the growth medium. A, Changes in PG content. Wild type (○) and mutant (●) cells grown in the medium containing 20 μm PG were transferred to the medium without PG and incubated for the designated times. The values are averages from two independent experiments. B, Changes in photosynthetic activity. Wild type (●) and mutant (○) cells grown in the medium containing 20 μm PG were transferred to the medium without PG and incubated for the designated times. In the case of the mutant, the culture was divided into two flasks after a deprivation of PG for 6 d. The culture in one flask was further incubated without PG, whereas that in the other was supplemented with 20 μm PG. The arrow indicates the time of addition of PG. The values are averages from two independent experiments.