Peptide inhibitor of cytokinesis during sporulation in Bacillus subtilis

Abstract

Cytokinesis in bacteria is mediated by the tubulin-like protein FtsZ, which forms a Z-ring at the division site. Using FtsZ as bait in a two-hybrid screen, we discovered a 40-amino-acid peptide, termed MciZ, from Bacillus subtilis that appeared to interact with FtsZ. Cells engineered to produce MciZ during growth formed aseptate filaments that lacked Z-rings. A mutant resistant to the toxic effects of MciZ during growth harboured an amino acid substitution near the GTP binding pocket of FtsZ. Synthetic MciZ inhibited the GTPase activity of FtsZ and its ability to polymerize. MciZ was produced during sporulation under the control of the transcription factor sigma(E). In the absence of MciZ, the mother-cell compartment of the sporangium aberrantly formed a Z-ring at a time in development when cytokinetic events normally have ceased. We conclude that MciZ is a previously unrecognized inhibitor of FtsZ that prevents inappropriate Z-ring formation during sporulation. MciZ showed little sequence similarity to other peptides in the databases, except the mouse antimicrobial peptide CRAMP, which we speculate works in part by inhibiting cytokinesis.

Figure 1. MciZ and FtsZ

(A) Map of the mciZ-containing region of the chromosome. The grey box indicates the position of the inferred, σ^E-controlled promoter. Shown below are the inferred amino acid sequences for MciZ from B. subtilis and from its orthologs B. cereus, B. anthracis, and B. licheniformis. Also shown is the sequence of CRAMP. The four MciZ sequences and the CRAMP sequence exhibit approximately 21% identity and 32% similarity, with black indicating identical residues and grey similar residues. B. licheniformis MciZ peptide possesses a unique C-terminal tail of 14 amino acids that is not shown in this alignment. (B) The crystal structure of B. subtilis FtsZ is shown with a molecule of GDP modeled in using an alignment to the M. jannaschii FtsZ co-crystal structure with GDP (Oliva et al., 2007). The nucleotide binding site is magnified in the right-hand panel and residue R143 is revealed in space-fill form and colored in red.

Figure 2. MciZ and CRAMP inhibit FtsZ ring formation

Z-rings were visualized by fluorescence microscopy using B. subtilis or E. coli cells in which, as indicated, FtsZ itself was tagged with YFP or GFP, or in which the FtsZ-binding protein ZapA was tagged with GFP. Panels A and B show cells of B. subtilis strain AH113, which harbors a xylose-inducible copy of mciZ and an IPTG-inducible copy of yfp-ftsZ. The strain was grown in LB medium in the presence of IPTG (A) alone or with IPTG and xylose (B). Panels C shows cells of B. subtilis strain FG347 (wild type) and D cells of AH178 (mciZΔ::kan), which both harbored gfp-zapA. The cells were examined by fluorescence microscopy 3.5 hours after entry into sporulation, a time that coincided with peak levels of mciZ expression. In panel D, Z-rings adjacent to the engulfing forespore compartment are labeled with yellow arrows and one at the forespore-distal pole of the sporangium with a white arrow. Panels E and F show cells of E. coli strain JOE650, which expressed a copy of ftsZ-gfp. The cells were grown in minimal growth medium without (E) or with added CRAMP peptide (F) and examined by fluorescence microscopy. All cells in (A) through (F) were stained with TMA-DPH membrane dye.

Figure 3. MciZ binds to FtsZ

(A) His₆-tagged B. subtilis FtsZ binds to synthetic MciZ. Binding reactions were assembled, and following a brief incubation period, the samples were subjected to Ni⁺⁺-NTA affinity chromatography. Aliquots from the load (L), flow through (FT), washes (W1 and W2), and elutions (E1 and E 2) fractions were subjected to SDS-polyacrylamide gel electrophoresis. Bands corresponding to His₆-FtsZ and synthetic MciZ were visualized by staining with Colloidal Blue. The wash fractions were 2-fold more concentrated than the input and flow through, and the elution fractions were 10-fold more concentrated than the input and flow through. In control experiments lacking His₆-FtsZ protein, MciZ was absent from the elution fractions demonstrating that it cannot bind Ni⁺⁺-NTA agarose directly (data not shown). (B) FtsZ co-purifies with His₆-MciZ protein. A cell extract prepared from a vegetative culture of wild type B. subtilis strain PY79 provided a source of FtsZ. Purified His₆-MciZ (on the left) or, as a control, protein buffer (on the right) were mixed with the cell extract, the mixture applied to a nickel-NTA agarose column, and the fractions collected subjected to western blot analysis using α-FtsZ antibodies. Fractions are labeled as in panel A. FtsZ was detected in the elution fraction only when His₆-MciZ was present. A cross-reactive species present in the load and flow through fractions was not bound by MciZ. As a negative control, the blot was probed using antibody against the housekeeping sigma factor σ^A, and as expected σ^A was not detected in the elution fraction (lower panel).

Figure 4. MciZ inhibits the polymerization of FtsZ

Polymerization of FtsZ (5 μM) was initiated by addition of 25 μM, 100 μM, or 500 μM GTP, and the resulting 90° light scattering signal was recorded immediately thereafter. Reactions were performed without MciZ (white bars) or in the presence of pure synthetic MciZ (5 μM) (black bars).

Figure 5. MciZ inhibits the GTPase activity of FtsZ

(A) FtsZ GTPase activity (nmol GTP hydrolyzed/mg FtsZ/min) as a function of MciZ concentration (μM). Polymerization of 2.5 μM B. subtilis FtsZ was carried out in the presence of 1 mM GTP and varying amounts of synthetic MciZ peptide, ranging from 0 μM to 1 μM. Phosphate release was measured using the malachite green assay (27). FtsZ GTPase activity was almost completely abolished at a 1:2.5 molar ratio of MciZ:FtsZ. (B) Polymerization of 2.5 μM B. subtilis FtsZ was performed in the presence of varying concentrations of GTP and in the absence (solid line) or presence of 0.1 μM MciZ (dashed line), 0.2 μM MciZ (dotted line), or 0.5 μM MciZ (dash-and-dot line). Phosphate release was measured as in A. The data are presented in a double-reciprocal (Lineweaver-Burk) plot, wherein the x-axis denotes the inverse of the substrate (GTP) concentration (1/[S]) and the y-axis the inverse of the reaction velocity (1/v).

Figure 6. Expression of mciZ is under control of the σ^E factor

(A) The putative σ^E promoters of B. subtilis mciZ and its three orthologs. Each is composed of −10 and −35 elements that share similarity to the canonical recognition sequences for the mother-cell-specific alternative sigma factor σ^E (Eichenberger et al., 2003). Nucleotide residues that match the canonical promoter sequence are colored black. Within the canonical sequence, capital letters mark the most highly conserved positions and lowercase letters indicate positions that are less conserved. Promoter activity was tested during sporulation with cells harboring translational fusions of lacZ to DNA lying 200 bp immediately upstream of the (B) B. subtilis mciZ (AH75 and AH173) or (C) B. anthracis mciZ (AH162 and AH171) open-reading frames. Strains AH75 and AH162 were wild type for the sigE gene (black squares) whereas strains AH173 and AH171 were mutant for sigE (white squares) (Kenney & Moran, 1987).

Figure 7. Noc excludes Z-rings nonpolar positions the mother cell

Panels A-C show cells of strain AH178 (mciZΔ::kan), which were examined by fluorescence microscopy 3.5 hours after entry into sporulation. This strain produces a fusion of the FtsZ-binding protein ZapA to GFP (green). Cells were stained with DAPI (blue) to reveal the position of the nucleoid and with FM4-64 (red) to reveal membranes. Shown is a sporangium with an aberrant Z-ring in the nucleoid-free zone immediately adjacent to the forespore. From left to right the panels show (A) FM4-64 and GFP, (B) DAPI, and (C) a merged image. Panels D-F show three fields of cells of strain AH188 (mciZΔ::kan, nocΔ::spc), which were examined as described above. Microscopic images (FM4-64 and GFP) are shown on top and are accompanied by interpretative cartoons beneath.