Probing for Binding Regions of the FtsZ Protein Surface through Site-Directed Insertions: Discovery of Fully Functional FtsZ-Fluorescent Proteins

Abstract

FtsZ, a bacterial tubulin homologue, is a cytoskeletal protein that assembles into protofilaments that are one subunit thick. These protofilaments assemble further to form a "Z ring" at the center of prokaryotic cells. The Z ring generates a constriction force on the inner membrane and also serves as a scaffold to recruit cell wall remodeling proteins for complete cell division in vivo One model of the Z ring proposes that protofilaments associate via lateral bonds to form ribbons; however, lateral bonds are still only hypothetical. To explore potential lateral bonding sites, we probed the surface of Escherichia coli FtsZ by inserting either small peptides or whole fluorescent proteins (FPs). Among the four lateral surfaces on FtsZ protofilaments, we obtained inserts on the front and back surfaces that were functional for cell division. We concluded that these faces are not sites of essential interactions. Inserts at two sites, G124 and R174, located on the left and right surfaces, completely blocked function, and these sites were identified as possible sites for essential lateral interactions. However, the insert at R174 did not interfere with association of protofilaments into sheets and bundles in vitro Another goal was to find a location within FtsZ that supported insertion of FP reporter proteins while allowing the FtsZ-FPs to function as the sole source of FtsZ. We discovered one internal site, G55-Q56, where several different FPs could be inserted without impairing function. These FtsZ-FPs may provide advances for imaging Z-ring structure by superresolution techniques.

Importance: One model for the Z-ring structure proposes that protofilaments are assembled into ribbons by lateral bonds between FtsZ subunits. Our study excluded the involvement of the front and back faces of the protofilament in essential interactions in vivo but pointed to two potential lateral bond sites, on the right and left sides. We also identified an FtsZ loop where various fluorescent proteins could be inserted without blocking function; these FtsZ-FPs functioned as the sole source of FtsZ. This advance provides improved tools for all fluorescence imaging of the Z ring and may be especially important for superresolution imaging.

Keywords: Venus; green fluorescent protein; mEos; mMaple; superresolution; tubulin.

FIG 1

Negative-stain EM images of FtsZ-FP insertions. (A) FtsZ-55-mVenus-56. (B) FtsZ-55-dVenus-56. FtsZ was present at 1 mg/ml in HMK100 buffer. Bar, 200 nm.

FIG 2

Structural model of FtsZ with YFP inserted between G55 and Q56. The FtsZ structure is based on PDB file 2VAW (Pseudomonas aeruginosa FtsZ [27]). The N-terminal subdomain is shown in dark blue, the C-terminal subdomain is shown in cyan, and GDP is shown by orange spheres. YFP (PDB entry 1YFP) is shown in yellow. The 3- and 11-aa tails of YFP were added as extended peptides, and the 5-aa linkers are shown in magenta and red. The figure was prepared by the PyMOL Molecular Graphics System, version 1.8 (Schrödinger, LLC).

FIG 3

Average cell lengths of select strains grown in LB at 37°C. The left four bars show BW27783 strains with the FtsZ-FP-encoding gene incorporated (“i”) into the genome. The right four bars show JKD7 strains with FtsZ expressed from the pJSB2 plasmid (with 0.2% arabinose). The FtsZ-FP constructs (with the FP inserted at Q55-G56) were mVenus, mCerulean, and mNeonGreen, with wtFtsZ as a control. mCerulean and mNeonGreen functioned in suppressor strains, indicated as S1 and S2. The number of cells measured for each strain was 111.

FIG 4

Ranges of cell length for select strains. (A) BW27783 strains with genomically expressed FtsZ-FPs. (B) JKD7 strains expressing FtsZ from the pJSB2 plasmid (with 0.2% arabinose). Cells were grown in LB at 37°C. Note the different x axes for panels A and B.

FIG 5

Cells expressing FtsZ-G55-mNeonGreen-Q56 during exponential growth. (A and B) JKD7 suppressor strain S2 (with FtsZ-G55-mNeonGreen-Q56 expressed from pJSB2) grown in LB plus 0.2% arabinose at 37°C (A) or in M9 medium plus 0.2% arabinose at 23°C (B). (C and D) BW27783 with genomically expressed FtsZ-G55-mNeonGreen-Q56i, grown in LB at 37°C (C) or in M9 medium at 23°C (D). Note that the light levels were adjusted for individual images to optimize the visibility of the Z ring and the cytoplasm. Panel A was originally much brighter than the others.

FIG 6

(A and D) JKD7 suppressor strain S6 (with FtsZ-G55-mEos3.1-Q56 expressed from pJSB2) grown in LB at 37°C (A) and in M9 medium at 23°C (D). (B and E) JKD7 suppressor strain S1 (with FtsZ-G55-mMaple2-Q56 expressed from pJSB2) grown in LB at 37°C (B) and in M9 medium at 23°C (E). (C and F) JKD7 suppressor strain S2 (with FtsZ-G55-mMaple3-Q56 expressed from pJSB2) grown in LB at 37°C (C) and in M9 medium at 23°C (F). Elongated cells with multiple Z rings were found for growth in LB at 37°C. However, the cells in M9 medium at 23°C were shorter, with single Z rings. Note that the light levels were adjusted for individual images to show the Z ring and the cytoplasm. Growth in LB at 23°C gave improved Z rings similar to those from growth in M9 at 23°C, suggesting that temperature was the important parameter.

FIG 7

Cartoon model showing two subunits of an FtsZ pf (PDB entry 3VOA [58]; Staphylococcus aureus FtsZ). The bottom subunit shows sites where the 10-aa GSTLELEGST peptide was inserted. (A) View of the front of FtsZ, from which the C terminus emanates (orange). (B) View of the back of FtsZ, from which the N terminus emanates (orange). Proteins with insertions at dark green sites provided complementation, those with insertions at light green sites generated suppressors, and those with insertions at red sites failed to function in vivo. GDP is shown in blue. The amino acids preceding inserts are shown as spheres, and the E. coli amino acid numbers are indicated.

FIG 8

Negative-stain EM images of polymerization with the 10-aa insert at R174. (A to D) Wild-type FtsZ. (E to H) FtsZ with the 10-aa insertion after R174. FtsZ was present at 1 mg/ml. (A and E) DEAE-dextran (0.5 mg/ml) and 5 mM EDTA in HMK100 buffer. (B and F) DEAE-dextran (0.5 mg/ml) in HMK100 buffer. (C and G) Ca²⁺ (10 mM) in HMK100 buffer. (D and H) Ten percent polyvinyl alcohol in HMK100 buffer. Bar, 200 nm.