Vipp1 deletion mutant of Synechocystis: a connection between bacterial phage shock and thylakoid biogenesis?

Abstract

Plant chloroplasts originated from an endosymbiotic event by which an ancestor of contemporary cyanobacteria was engulfed by an early eukaryotic cell and then transformed into an organelle. Oxygenic photosynthesis is the specific feature of cyanobacteria and chloroplasts, and the photosynthetic machinery resides in an internal membrane system, the thylakoids. The origin and genesis of thylakoid membranes, which are essential for oxygenic photosynthesis, are still an enigma. Vipp1 (vesicle-inducing protein in plastids 1) is a protein located in both the inner envelope and the thylakoids of Pisum sativum and Arabidopsis thaliana. In Arabidopsis disruption of the VIPP1 gene severely affects the plant's ability to form properly structured thylakoids and as a consequence to carry out photosynthesis. In contrast, Vipp1 in Synechocystis appears to be located exclusively in the plasma membrane. Yet, as in higher plants, disruption of the VIPP1 gene locus leads to the complete loss of thylakoid formation. So far VIPP1 genes are found only in organisms carrying out oxygenic photosynthesis. They share sequence homology with a subunit encoded by the bacterial phage shock operon (PspA) but differ from PspA by a C-terminal extension of about 30 amino acids. In two cyanobacteria, Synechocystis and Anabaena, both a VIPP1 and a pspA gene are present, and phylogenetic analysis indicates that VIPP1 originated from a gene duplication of the latter and thereafter acquired its new function. It also appears that the C-terminal extension that discriminates VIPP1 proteins from PspA is important for its function in thylakoid formation.

Figure 1

Alignment of PspA and VIPP1 protein sequences from bacteria and plants. Amino acid residues that are conserved in at least half of the sequences are boxed in black, and conserved amino acid residue changes are marked by gray boxes. The start of the mature VIPP1 protein from pea after removal of the transit sequence is indicated by an asterisk.

Figure 2

Phylogeny of bacterial and plant VIPP1 and PspA proteins supports the close relationship between the plant and cyanobacterial proteins. Maximum likelihood analysis of PspA and VIPP1 proteins from various organisms was performed with puzzle (17) and protml (18).

Figure 3

sll0617 encodes a cyanobacterial VIPP1 homologue, and it seems to be located exclusively in the plasma membrane. (a) Immunoblotting with α-VIPP1 and α-PspA antisera identifies VIPP1 in Synechocystis. Lanes 1–3, immunoblot performed with α-VIPP1; lanes 4–6, immunoblot performed with α-PspA; lanes 1 and 4, total leaf extract from A. thaliana; lanes 2 and 5, total protein extract from Synechocystis; lanes 3 and 6, PspA, heterologously expressed in and purified from E. coli. (b) Synechocystis wild-type cells were separated into outer membrane (OM), plasma membrane (PM), cytoplasm (SOL), and thylakoids (THY). Each fraction was analyzed by immunoblotting for the presence of VIPP1. Antisera against the 75-kDa outer membrane protein (α-syn-Toc75), large subunit of Rubisco (α-RbcL), ATP synthase (α-AtpA/B), and nitrate reductase subunit A (α-NrtA) were used as markers for the purity of the different subfractions. (c) sll0617 was disrupted by the insertion of a kanamycin-resistance cassette at position 454 of the coding region. (d) Southern blot analysis was carried out with wild-type and Δsynvipp1 genomic DNA to confirm the disruption of sll0617. (e) Immunoblotting with α-VIPP1 protein establishes an extreme reduction of the level of VIPP1 protein in the Δsynvipp1 mutant compared with wild-type and rescue cells.

Figure 4

Ultrathin sections reveal that Δsynvipp1 mutant cells contain no orderly structured thylakoids but instead have very few membrane-like structures dispersed throughout the cytoplasm. They are unable to carry out photosynthesis. The phenotype can be rescued by plasmidal expression of VIPP1. (a–c) Ultrathin sections of wild-type, Δsynvipp1 mutant, and rescue cells. (a′–c′) Analysis of oxygen evolution and consumption in wild-type, Δsynvipp1, and rescue cells.