The role of the cytoskeletal proteins MreB and FtsZ in multicellular cyanobacteria

Abstract

Multiseriate and true-branching cyanobacteria are at the peak of prokaryotic morphological complexity. However, little is known about the mechanisms governing multiplanar cell division and morphogenesis. Here, we study the function of the prokaryotic cytoskeletal proteins, MreB and FtsZ in Fischerella muscicola PCC 7414 and Chlorogloeopsis fritschii PCC 6912. Vancomycin and HADA labeling revealed a mixed apical, septal, and lateral trichome growth mode in F. muscicola, whereas C. fritschii exhibits septal growth. In all morphotypes from both species, MreB forms either linear filaments or filamentous strings and can interact with FtsZ. Furthermore, multiplanar cell division in F. muscicola likely depends on FtsZ dosage. Our results lay the groundwork for future studies on cytoskeletal proteins in morphologically complex cyanobacteria.

Keywords: Cyanobacteria; FtsZ; MreB; Stigonematales; cytoskeleton; morphogenesis.

Fig. 1

Presence of ftsZ and mreB homologs in cyanobacteria. (A) Illustration depicting the cellular morphologies and life stages ofF. muscicola, C. fritschii, and Anabaena wild‐type trichomes. (B) Depiction of representative cyanobacterial species with different cell shapes and morphologies and from different habitats. The complete dataset is shown in File S1. Species, where homologous genes to ftsZ, ftsQ, mreB, mreC, and mreD were detected, are marked by a green square. Organisms, where a homolog was not found, are marked by a gray square.

Fig. 2

Fluorescent vancomycin labeling reveals different growth modes in multicellular cyanobacteria. (A–G) Bright‐field and merged chlorophyll autofluorescence (red) and BODIPY ^®FL Vancomycin (Van‐FL) fluorescence micrographs of (A–C) F. muscicola, (D–F)C. fritschii, and (G) Anabaena cells stained with 5 µg·mL⁻¹Van‐FL. Micrographs indicate different growth stages of the respective cyanobacterium: (A upper image) hormogonia, (A lower image) hormogonia in the transition phase to young linear trichome. Apical staining was observed in 123 apical cells (39%) while 191 showed septal staining only (61%),n = 314. (B) young branches, (C, F) heterocysts, (D) hormogonium (as indicated by the linear growth mode, which is restricted to the early growth stages (i.e. hormogonia) ofC. fritschii), (E) multiseriate trichome, and (G) mature trichome. White arrows indicate heterocysts. (B) and (B inlay) additionally show Van‐FL staining pattern at the branching points of newly formed lateral branches. Van‐FL staining patterns were consistent among three independent experiments. Scale bars: 10 µm.

Fig. 3

Localization of GFP‐MreB in F. muscicola, C. fritschii, and Anabaena. Bright‐field and merged chlorophyll autofluorescence (red) and GFP fluorescence micrographs of (A–E) F. muscicola, (F–J) C. fritschii, or (K–M) Anabaena expressing gfp‐mreB from (A–C, F–I, K–L) P_petE or from (D, E, J, M) P_mreB. Figures show (A) mature trichome with nascent hormogonium, (B, E–G, J, K–M) mature trichomes and (D, H, I, J inlay) hormogonia. A heterocyst is marked with an orange triangle. Blue triangles indicate longitudinal GFP‐MreB filaments that appear to traverse the cells along the growth axis. Note: Anabaenaexpressing gfp‐mreBfrom P_petEshows several small filaments throughout the cells when grown on BG11 medium, while polar GFP‐MreB plugs are only observed upon transfer to BG11₀(BG11 without combined nitrogen), which we found to seemingly increase P_petE‐driven expression, thus indicating that the GFP spots seen in (L) could be inclusion body‐like aggregates. Scale bars: (A–G, J–M) 10 µm or (H, I) 5 µm.

Fig. 4

Interaction of FtsZ and MreB in multicellular cyanobacteria. (A) Beta‐galactosidase assays ofEscherichia colicells co‐expressing indicated translational fusion constructs of all possible pairwise combinations offtsZwithmreB. Quantity values are given in Miller units per milligram LacZ of the mean results from three independent colonies. Error bars indicate standard deviations (n = 3). Neg: pKNT25 plasmid carryingftsZco‐transformed with empty pUT18C. Pos: Zip/Zip control. The value indicated with **** is significantly different from the negative control (****P < 0.0001; Dunnett's multiple comparison test and one‐way ANOVA). (B) Anti‐FtsZ_SubsVand anti‐GFP western blots from anti‐GFP co‐IP's from two individual biological samples ofF. muscicola,C. fritschii, andAnabaenacell‐free extracts expressinggfp‐mreBfrom P_mreBor WT control cells. Experiments were performed twice, each time using two independent biological replicates for each sample type (i.e. WT and GFP‐tagged MreB expressing strain). Input samples were taken before addition of anti‐GFP antibodies.

Fig. 5

Assessment of new Anti‐FtsZ_SubsVantibody. (A) Western blot analysis of cell‐free lysates fromF. muscicola,C. fritschii,Anabaena,Synechocystis,Synechococcus, andEscherichia coliusing anti‐FtsZ_SubsVantibody (raised againstF. muscicolaandC. fritschiiFtsZ). As a control, 1 µg purified FtsZ_Fm‐His was included. (B) Immunolocalization of FtsZ inF. muscicola,C. fritschii, andAnabaenausing an anti‐FtsZ_SubsVantibody. White triangles show Z‐rings in branching cells. The orange arrow indicates a heterocyst. The blue triangle points to a matureC. fritschiicell which was not permeabilized. Red triangles indicate septal localization of FtsZ inAnabaena. Results shown in here are representative figures from three independent experiments. Scale bars: 10 µm.

Fig. 6

Fischerella muscicolaandAnabaenaare sensitive to excess FtsZ levels. (A) Micrographs of mating filters showing growth ofF. muscicolaWT colonies andF. muscicolacolonies after transformation with pRL25C plasmids carrying P_petE::ftsZ‐gfp, P_glnA::gfp‐ftsZor P_ftsZ::ftsZ‐gfp. (B) Bright‐field micrograph showing morphological alterations (i.e. swelling) ofF. muscicolacells expressingftsZ‐gfpfrom P_ftsZ. (C–E) Merged chlorophyll autofluorescence (red) and GFP fluorescence and bright‐field micrographs of (C)F. muscicola, (D)Anabaenaor (E)C. fritschiiexpressingftsZ‐gfporgfp‐ftsZfrom P_ftsZ, P_glnAor P_petE. P_petE‐driven expression offtsZ‐gfpinF. muscicolaandAnabaenawas additionally increased using 0.2 µmCuSO₄. White arrows indicate trichomes with multiseriate trichome growth. Blue arrows mark dim Z‐ring formations. Scale bars: (C–E) 10 µm.

Fig. 7

High rate of proteolytic degradation of FtsZ in subsection V cyanobacteria. (A) Anti‐FtsZ_SubsVwestern blots of cell‐free extracts ofF. muscicola,C. fritschii, andAnabaenaincubated at 37 °C (F. muscicolaandC. fritschii) or 30 °C (Anabaena) for 0, 60, 120, or 180 min supplemented or not (w/o) with protease inhibitor cocktails (PIC) and EDTA/EGTA. Cells were either lysed in cyanobacterial lysis buffer (CLB) or in CLB supplemented with 10 mmMgCl₂. EDTA, and EGTA were supplemented as 5 mmeach, except in cases of presence of 10 mmMgCl₂, where 7.5 mmEDTA and EGTA were included. (B) Purified FtsZ_Fm‐His (1 µg per sampling time point) was incubated for the indicated time points in cell‐free extracts ofF. muscicola,C. fritschii, orAnabaenasupplemented or not (w/o) with PIC and EDTA/EGTA. Degradation of FtsZ_Fm‐His was detected using an anti‐His antibody. (A, B) As a loading and specific‐degradation control, levels of RbcL were detected using an anti‐RbcL antibody. Images are representative western blots from duplicate experiments.