Characterization of the Synechocystis strain PCC 6803 penicillin-binding proteins and cytokinetic proteins FtsQ and FtsW and their network of interactions with ZipN

Abstract

Because very little is known about cell division in noncylindrical bacteria and cyanobacteria, we investigated 10 putative cytokinetic proteins in the unicellular spherical cyanobacterium Synechocystis strain PCC 6803. Concerning the eight penicillin-binding proteins (PBPs), which define three classes, we found that Synechocystis can survive in the absence of one but not two PBPs of either class A or class C, whereas the unique class B PBP (also termed FtsI) is indispensable. Furthermore, we showed that all three classes of PBPs are required for normal cell size. Similarly, the putative FtsQ and FtsW proteins appeared to be required for viability and normal cell size. We also used a suitable bacterial two-hybrid system to characterize the interaction web among the eight PBPs, FtsQ, and FtsW, as well as ZipN, the crucial FtsZ partner that occurs only in cyanobacteria and plant chloroplasts. We showed that FtsI, FtsQ, and ZipN are self-interacting proteins and that both FtsI and FtsQ interact with class A PBPs, as well as with ZipN. Collectively, these findings indicate that ZipN, in interacting with FtsZ and both FtsI and FtQ, plays a similar role to the Escherichia coli FtsA protein, which is missing in cyanobacteria and chloroplasts.

FIG. 1.

Proportions of various cell types observed in the Synechocystis mutants constructed in this study. In each case, at least 200 randomly chosen cells were classified in the following three categories: single cells, doublets of dividing cells, and cloverleaf-type cell aggregates. Strains are indicated on the x axis, and the percentage of each category is indicated on the y axis (error bars represent the standard deviation). These experiments were performed twice with two independent clones harboring the same mutation.

FIG. 2.

Morphology of Synechocystis mutant cells depleted in the PBPs of either class A or B. Shown are a phase-contrast image (A; scale bar = 1 μm) and flow cytometry analysis (B) of WT or mutant (Δ) cells totally or partially lacking (Table 3) the indicated PBP. For each mutant, the FSC histogram (bold lines) has been overlaid with that of the WT to better visualize the influence of the mutation on cell size. These experiments were performed twice with two independent clones harboring the same mutation.

FIG. 3.

Morphology and size of Synechocystis mutant cells depleted in class C type 4 PBPs. Shown are a phase-contrast image (A; scale bar = 1 μm) and flow cytometry analysis (B) of WT or mutant (Δ) cells totally or partially lacking (Table 3) the indicated PBP. For each mutant, the FSC histogram (bold lines) has been overlaid with that of the WT to better visualize the influence of the mutation on cell size. These experiments were performed twice with two independent clones harboring the same mutation.

FIG. 4.

Morphology and size of Synechocystis mutant cells depleted in class C type AmpH PBPs. Shown are a phase-contrast image (A; scale bar = 1 μm) and flow cytometry analysis (B) of WT or mutant (Δ) cells totally or partially lacking (Table 3) the indicated PBP. For each mutant, the FSC histogram (bold lines) has been overlaid with that of the WT to better visualize the influence of the mutation on cell size. These experiments were performed twice with two independent clones harboring the same mutation.

FIG. 5.

Morphology and size of Synechocystis mutant cells depleted in proteins FtsQ and FtsW. Shown are a phase-contrast image (A; scale bar = 1 μm) and flow cytometry analysis (B) of WT or mutant (Δ) cells lacking the indicated protein. For each mutant, the FSC histogram (bold lines) has been overlaid with that of the WT to better visualize the influence of the mutation on cell size. These experiments were performed twice with two independent clones harboring the same mutation.

FIG. 6.

Two-hybrid analysis of interactions between PBPs and the cytokinetic proteins FtsQ, FtsW, and ZipN. The occurrence of interaction between the tested proteins coproduced in E. coli DHM1 cells of the BACTH system was ascertained by the production of the β-galactosidase reporter enzyme whose activity (i) turned the corresponding cells (indicated by the gray rectangles in panel A) blue on X-Gal-containing medium and (ii) reached a high value (B; beta-gal). Each bar represents the mean value of two measurements performed on the cellular extracts of three reporter clones originating from independent transformations, and the error bars represent the standard deviation. Cells producing the two interacting ZipN-fusion proteins were used as the positive control (5,900 ± 750 β-Gal units), while those producing a single or no reporter protein served as the negative controls (65 ± 50 U of background β-Gal activity).

FIG. 7.

Schematic representation of the web of interactions among the 10 presently studied proteins and ZipN, the cytokinetic factor we previously characterized. The spherical morphology of Synechocystis is represented by the circle. The double arrows indicate the presently reported interactions. Black and white letters stand for the proteins respectively indispensable and dispensable to Synechocystis. Black and white squares indicate that the corresponding mutants display decreased and increased cell sizes, respectively. Gray rectangles indicate that the corresponding mutants do not show any morphological defects. The hatched rectangle reminds us that the ZipN-depleted mutant has an aberrant spiral shape (26).