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. Author manuscript; available in PMC: 2009 May 1.
Published in final edited form as: Mol Microbiol. 2008 Feb 19;68(3):588–599. doi: 10.1111/j.1365-2958.2008.06173.x

PEPTIDE INHIBITOR OF CYTOKINESIS DURING SPORULATION IN BACILLUS SUBTILIS

Aaron A Handler 1, Joo Eun Lim 1, Richard Losick 1
PMCID: PMC2603569  NIHMSID: NIHMS81447  PMID: 18284588

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 harbored 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 σE. 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.

Introduction

Of central importance to cell division in bacteria is the polymerization of the tubulin-like protein FtsZ into a cytokinetic Z-ring (Addinall et al., 1996, Bi & Lutkenhaus, 1991, Levin & Losick, 1996, Ma et al., 1996, Pogliano et al., 1997, Wang & Lutkenhaus, 1993, Wang & Lutkenhaus, 1996, Weart et al., 2007). Assembly of the Z-ring is the focal point of numerous regulatory proteins that govern when, and where in the cell, the division septum will form. Some regulatory proteins promote Z-ring formation whereas others act negatively [reviewed in (Goehring & Beckwith, 2005, Margolin, 2000, Weart et al., 2007)]. This regulation is of particular importance in the spore-forming bacterium Bacillus subtilis, the subject of this investigation.

Like other bacteria that undergo binary fission, B. subtilis normally forms Z-rings at the mid-cell position when it is growing. However, after it enters the pathway to sporulate, B. subtilis switches the position of Z-ring to sites near both poles of the cell (Ben-Yehuda & Losick, 2002, Levin & Losick, 1996, Rudner & Losick, 2001, Piggot & Losick, 2002). Normally, only one of these Z-rings is converted into a division septum, the other one being disassembled. The division septum, which is asymmetrically positioned, partitions the developing cell or sporangium into a small forespore compartment and a large mother-cell compartment (Piggot & Losick, 2002, Rudner & Losick, 2001). The two compartments initially lie side-by-side but in subsequent development the forespore, which is destined to become the spore, is wholly engulfed by the mother cell. Finally, when sporulation is complete, the mature spore is released from the sporangium by lysis of the mother cell.

Here we report the discovery of an additional negative regulator, termed MciZ (for mother cell inhibitor of FtsZ), of cytokinetic ring formation in B. subtilis. MciZ is a 40-amino acid peptide that acts during sporulation to block Z-ring formation in the mother cell and does so by binding to, and inhibiting the polymerization of, FtsZ.

Results and Discussion

A yeast two-hybrid screen reveals a conserved peptide that interacts with FtsZ

To discover additional regulators of Z-ring formation in B. subtilis, we carried out a yeast two-hybrid screen using FtsZ as bait, as described in the Experimental Procedures. The screen was carried out using a B. subtilis genomic library generously provided by H. Yoshikawa (Fukushima et al., 2006). We discovered a small open-reading frame mciZ whose inferred product, a 40-amino acid peptide, appeared to interact strongly with FtsZ. The open-reading frame was not annotated previously, but the coding sequence for MciZ and its location relative to the flanking genes nudF and yqkF are conserved in at least four Bacillus species (Fig. 1A).

Figure 1. MciZ and FtsZ.

Figure 1

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(A) Map of the mciZ-containing region of the chromosome. The grey box indicates the position of the inferred, σE-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.

MciZ inhibits Z-ring formation and does so by direct interaction with FtsZ

To investigate the effect of MciZ on Z-ring formation, Z-rings were visualized using the Yellow Fluorescent Protein (YFP) fused to FtsZ in cells engineered to synthesize the peptide in response to the inducer xylose during growth. Induction caused the formation of aseptate filaments that were deficient in Z-rings (Fig. 2A–B). Consistent with the observed block in cell division, artificially inducing the synthesis of MciZ during growth markedly impaired viability, greatly reducing the number of colony forming units when the engineered cells were plated on medium containing xylose (data not shown).

Figure 2. MciZ and CRAMP inhibit FtsZ ring formation.

Figure 2

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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.

Taking advantage of the toxicity of MciZ, we isolated a suppressor mutant of the engineered cells that survived treatment with xylose. The mutant grew normally but produced minicells, a phenotype observed previously for other FtsZ mutants (Feucht & Errington, 2005). The suppressor mutant harbored a mutation in the gene for FtsZ, causing a substitution of lysine for arginine-143 (R143K). The crystal structure of B. subtilis FtsZ with GDP modeled in is presented in Fig. 1B (Oliva et al., 2007). It can be seen that the site of the R143K substitution is near the binding pocket for guanine nucleotide. The results so far are consistent with the idea that MciZ is an inhibitor of Z-ring formation that targets FtsZ.

Next, we confirmed that MciZ binds to FtsZ by affinity chromatography using His-tagged FtsZ and synthetic MciZ peptide. The results show that His-tagged FtsZ caused the retention of MciZ (Fig. 3A). In a complementary experiment, His-tagged MciZ caused the retention of native FtsZ in an extract of growing cells but not the retention of an unrelated protein (SigA) (Fig. 3B).

Figure 3. MciZ binds to FtsZ.

Figure 3

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(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 σA, and as expected σA was not detected in the elution fraction (lower panel).

Fig. 4 shows the results of experiments to investigate the effect of MciZ on the polymerization of FtsZ as measured using 90°-angle light scattering (Mukherjee & Lutkenhaus, 1999). It can be seen that MciZ was highly effective in inhibiting polymerization at low and intermediate concentrations of GTP and only partially effective at high concentrations of nucleotide.

Figure 4. MciZ inhibits the polymerization of FtsZ.

Figure 4

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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).

Because MciZ inhibited polymerization and because the GTPase activity of FtsZ depends on inter-subunit interactions (Dai et al., 1994, de Boer et al., 1992, Mukherjee et al., 1993, Mukherjee & Lutkenhaus, 1998, Oliva et al., 2004, Sossong et al., 1999, Wang & Lutkenhaus, 1993), we anticipated that the peptide would also inhibit nucleotide hydrolysis. Indeed, as shown in Fig. 5A and 5B, MciZ inhibited the GTPase activity of FtsZ. The isolation of a resistance mutant with an amino acid substitution near the GTP pocket (above) raises the possibility that MciZ works by occluding GTP from the nucleotide binding pocket of FtsZ.

Figure 5. MciZ inhibits the GTPase activity of FtsZ.

Figure 5

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(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).

MciZ is produced under sporulation control and blocks Z-ring formation in the mother cell

Using a fusion to lacZ as a reporter, we found that mciZ was induced at an intermediate stage of sporulation, although at a low level (Fig. 6B). This low level of transcription was dependent on the mother-cell-specific regulatory protein σE (Fig. 6B). A fusion of lacZ to B. anthracis mciZ was also expressed in B. subtilis in a σE-dependent manner and, interestingly, the level of expression was higher than for the B. subtilis fusion (Fig. 6C). In keeping with the idea that mciZ is under the direct control of σE, all four orthologs are preceded by sequences that conform exactly to the canonical “−10” sequence for promoters recognized by σE-containing RNA polymerase and partially to the canonical “−35” sequence, with the B. anthracis “−35” conforming more closely than the B. subtilis “−35” sequence (Fig. 1A and Fig. 6A).

Figure 6. Expression of mciZ is under control of the σE factor.

Figure 6

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(A) The putative σE 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 σE (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).

Since σE is confined to the mother cell, it seemed possible that MciZ is responsible for blocking Z-ring formation in the large chamber of the sporangium (Piggot & Losick, 2002, Rudner & Losick, 2001). To investigate this possibility, we visualized Z-ring formation during sporulation using Green Fluorescent Protein (GFP) fused to the FtsZ-binding protein ZapA (Gueiros-Filho & Losick, 2002). Indeed, a Z-ring was frequently observed in the mother-cell compartment of mciZ mutant sporangia (Fig. 2D), but rarely in wild type sporangia (Fig. 2C). Specifically, at hour 3.5 of sporulation the frequency of wild type sporangia with Z-rings in the mother cell was 4% (among 428 sporangia scored) whereas the frequency for mciZ mutant sporangia with Z-rings was 62% (out of 486 scored).

Nonetheless, the absence of MciZ was insufficient to allow the mother cell to divide and did not result in a measurable decrease in the efficiency of spore formation (data not shown). Three other proteins (SpoIID, SpoIIM, and SpoIIP) are known to contribute to preventing cytokinesis in the mother cell (Eichenberger et al., 2001). It seems likely that MciZ is redundant in the presence of these other septation-inhibiting proteins. The redundancy may explain why the absence of MciZ did not block spore formation. Nevertheless, the conservation of mciZ in at least four relatives of B. subtilis, including distant members of the genus (B. cereus and B. anthracis; Fig. 1A), indicates that MciZ confers a fitness advantage for sporulation.

In toto, the results so far are consistent with the idea that MciZ acts during sporulation to block Z-ring formation in the mother cell and does so by inhibiting FtsZ. To investigate this idea further, we examined Z-ring formation during sporulation in cells of the FtsZ-R143K mutant, which was resistant to the toxic effects of MciZ during growth (above). Whereas Z-rings were rarely observed in wild-type sporangia that were at the stage of engulfment, greater than 50% of mutant sporangia (of a total of 559 examined) at a similar stage of development exhibited a Z-ring (data not shown).

Nucleoid occlusion restricts Z-ring formation to the poles in sporangia lacking MciZ

Strikingly, the Z-ring in mciZ mutant sporangia exhibited an extreme polar placement, usually at the forespore-proximal end of the mother cell (Fig. 2D and Fig. 7A). A possible explanation for this observation was that the mother-cell chromosome occluded Z-rings from forming at mid-cell positions, as it is known that in vegetative cells the nucleoid contributes to restricting Z-rings to the cell poles (Wu & Errington, 2004). This phenomenon is mediated by the nucleoid occlusion protein Noc (Wu & Errington, 2004). To investigate whether Noc was responsible for the polar localization of Z-rings in mciZ mutant sporangia, we created a strain that was doubly mutant for mciZ and noc. The results of Fig. 7D-F show that in the absence of Noc, Z-rings, and indeed sometimes more than one Z-ring, formed at variety of positions within the mother cell. A strain deleted for noc alone was indistinguishable from wild type and did not form Z-rings at high frequency during sporulation (data not shown).

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

Figure 7

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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.

MciZ is similar to the mouse antimicrobial peptide CRAMP

We detected no bacterial peptides or proteins in the databases with sequence similarity to MciZ and its orthologs. Interestingly, however, MciZ was similar to the mouse antimicrobial peptide CRAMP, with a particularly high level of similarity being seen in the central region of the peptides (Fig. 1A). In this regard, we were struck by the report of Rosenberger et al. that CRAMP causes filamentation of the enteric bacterium Salmonella typhimurium (Rosenberger et al., 2004). In extension of these findings, we tested the effects of CRAMP on Z-ring formation using the related enteric bacterium Escherichia coli. The results show that addition of synthetic CRAMP (a gift of R. Hancock) to the growth medium caused the formation of elongated cells, which were deficient in Z-rings (Fig. 2E-F). (No such effect was seen with B. subtilis.) Some filamentation was seen with 5–10 μg/ml of CRAMP but the strongest effect was seen with 15 μg/ml. Specifically, based on measurements of 502 untreated cells and 414 CRAMP-treated cells, the average increase in cell length in response to CRAMP was 56% and the maximum increase was 295%.

We do not know if this effect was due to a direct effect of CRAMP on FtsZ, but were able to rule out the alternative hypothesis that CRAMP was acting indirectly by triggering the SOS system of E. coli. In response to certain stress conditions E. coli synthesizes an inhibitor of FtsZ termed SulA (Huisman & D'Ari, 1981). We found that cells of a sulA mutant of E. coli behaved similarly to the wild type when treated with CRAMP (data not shown).

It is tempting to speculate that inhibition of cytokinesis contributes to the antibacterial effects of CRAMP in innate immunity. If so, then CRAMP would need to penetrate the cell envelope of enteric bacteria and once inside the cell would act to inhibit Z-ring formation. Antimicrobial peptides generally act by impairing membrane function, but precedents for peptides that pass through the bacterial membrane to act on targets inside the cell include insect antimicrobial peptides that inhibit the bacterial heat shock protein DnaK (Kragol et al., 2001) and the E. coli bacteriocin microcin B17, which inhibits DNA gyrase (del Castillo et al., 2001). A variety of mechanisms have been proposed for how peptides cross a membrane [reviewed in (Yeaman & Yount, 2003)], including the formation within the membrane of a pore-like complex (Hara et al., 2001a, Hara et al., 2001b, Uematsu & Matsuzaki, 2000). If our speculation is correct, inhibition of cytokinesis is unlikely to be the only mode of action of CRAMP, as it likely causes membrane damage as well as acting intracellularly. Finally, a recent report indicates that cells of the bacterial pathogen Mycobacterium tuberculosis growing in human macrophages are filamentous and deficient in Z-rings (Chauhan et al., 2006). Conceivably, an antimicrobial peptide produced in human macrophages also acts by blocking Z-ring formation.

Experimental Procedures

General methods

All B. subtilis strains are derivatives of the prototrophic strain PY79 (Youngman et al., 1983) and are listed in Table 1. Relevant strains of E. coli are also provided. Cells of B. subtilis and E. coli were grown in LB medium at 37°C unless indicated otherwise. The cultures were inoculated at an OD600 of 0.05 from an overnight culture grown in the same medium. Sporulation was induced by transferring cells growing in hydrolyzed casein (CH) medium to the resuspension medium of Sterlini and Mandelstam (Sterlini & Mandelstam, 1969, Harwood & Cutting, 1990). Time of sporulation was measured from the time of transfer into the resuspension medium. Preparation of B. subtilis competent cells was carried out by the one-step method (Wilson & Bott, 1968). Transformations were carried out essentially as described in Kunst and Rapoport (Kunst & Rapoport, 1995). Assays of β-galactosidase activity were performed as previously described and are presented in Miller units (Harwood & Cutting, 1990, Miller, 1972). Antibiotics were used at the following concentrations: chloramphenicol, 5 μg/ml; spectinomycin, 100 μg/ml; erythromycin plus lincomycin, 1 μg/ml and 25 μg/ml; kanamycin, 5 μg/ml; ampicillin, 100 μg/ml.

Table 1.

B. subtilis
Strain Genotype/relevant features Source/construction/reference
PY79 prototroph Youngman et al., 1983
RL1061 sigE Δ::mls Kenney & Moran, 1987
FG347 amyE ::Pxyl -gfp -zapA (cat) Gueiros-Filho & Losick, 2002
AH75 amyE ::PmciZ(B.sub)-lacZ (spc) pAH86 →PY79
AH93 amyE ::Pxyl-mciZ (cat) pAH103 →PY79
AH95 mciZ Δ::kan sequence interrupted: codons 4–37
AH113 amyE ::Pxyl-mciZ (cat), thrC ::Pspac-yfp -ftsZ (erm) AH175 →AH93
AH162 amyE ::PmciZ(B.anth) -lacZ (spc) pAH120 →PY79
AH171 sigE Δ::mls, amyE ::PmciZ(B.anth)-lacZ (spc) AH162 →RL1061
AH173 sigE Δ::mls, amyE ::PmciZ(B.sub) -lacZ (spc) AH75 →RL1061
AH175 thrC ::Pspac -yfp -ftsZ (erm) pAH135 →PY79
AH178 aahZ Δ::kan, amyE ::Pxyl-gfp-zapA (cat) FG347 →AH95
AH183 noc Δ::spc sequence interrupted: codons 3–227
AH187 aahZ Δ::kan, noc Δ::spc AH183 →AH95
AH188 aahZ Δ::kan, noc Δ::spc, amyE ::Pxyl -gfp -zapA (cat) FG347 →AH187
AH217 noc Δ::spc, amyE ::Pxyl -gfp -zapA (cat) FG347 →AH183
E. coli
Strain Genotype/relevant features Source/construction/reference
JOE650 MC4100 araD+ att φ 80::pJC114(P204-ftsZ-gfp) Goehring et al, 2005
JL3 B. subtilis FtsZ-His6 expression strain pJL1 →BL21 CodonPlus RIL
AH190 His6-MciZ expression strain pAH124 →BL21 CodonPlus RIL
W3110 B. subtilis FtsZ expression strain Wang & Lutkenhaus, 1993
(pBS58)
(pCXZ)
Plasmid Genotype/relevant features Source/construction/reference
pAH86 amyE ::PmciZ(B.sub)-lacZ (spc) integration plasmid See Methods
pAH103 amyE ::Pxyl-mciZ (cat) integration plasmid See Methods
pAH120 amyE ::PmciZ(B.anth)-lacZ (spc) integration plasmid See Methods
pAH124 PT7-His6-MciZ (kan) expression plasmid See Methods
pAH134 thrC ::Pspac -yfp -ftsZ (erm) integration plasmid See Methods
pJL1 PT7-FtsZB.sub -His6 (amp) expression plasmid See Methods
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DNA methods

DNA manipulations and cloning were carried out according to standard methods (Sambrook & Russel, 2001). Genomic DNA was prepared using the Wizard® Genomic DNA Purification Kit (Promega). Plasmid preparation from E. coli strain DH5α was performed using the QIAprep Spin Miniprep Kit (Qiagen). The long-flanking homology polymerase chain reaction (LFH-PCR) technique (Wach, 1996) was used for constructing B. subtilis deletion strains. PCR reactions were performed using either Pfu DNA polymerase (Stratagene) or the Expand High Fidelity PCR System (Roche). DNA sequencing was carried out either in the Molecular and Cellular Biology departmental facility at Harvard University, Cambridge, MA, USA or in the Molecular Biology Core Facility at the Dana-Farber Cancer Institute, Boston, MA, USA.

Plasmid construction

To construct pAH86 (amyE::PmciZ(B.sub)-lacZ spc), a 218 bp DNA fragment containing the putative mciZ promoter, ribosome binding site, and first 5 codons of the mciZ open reading frame was amplified using primers oAH146 (5′-GGAATTCAGGGTGTTTCACAAT-3′) and oAH144 (5′-ACGCGTCGACGCGGTGCACTTTCAC-3′). The PCR product was digested with EcoRI and SalI (sites underlined in primer sequences above) and cloned into pDG1728 (Guerout-Fleury et al., 1996), cut with the same enzymes, upstream of the lacZ coding sequence.

To construct pAH103 (amyE::Pxyl-mciZ cat), a 150 bp DNA fragment containing the mciZ ribosome binding site and open reading frame was amplified using primers oAH143 (5′-ACGCGTCGACCATACTAGAGCAAAAGG-3′) and oAH111 (5′-CGGGATCCTTATGGCTTTGAGATCC-3′). The PCR product was digested with SalI and BamHI (sites underlined in primer sequences above) and cloned into pOR277ΔSal (Gueiros-Filho & Losick, 2002), cut with the same enzymes, downstream of the xyl promoter.

To construct pAH120 (amyE::PmciZ(B.anth)-lacZ spc), a 219 bp fragment containing the putative B. anthracis mciZ promoter, ribosome binding site, and first 6 codons of the B. anthracis mciZ open reading frame was amplified using primers oAH177 (5′-GGAATTCGGTGATTTACAATTTCACGT-3′) and oAH179 (5′-ACGCGTCGACTAAAATATAAACTTTCAT-3′). The PCR product was digested with EcoRI and SalI (sites underlined in primer sequences above) and cloned into pDG1728 (Guerout-Fleury et al., 1996), cut with the same enzymes, upstream of the lacZ coding sequence. B. anthracis genomic DNA, which served as a template for amplification of the PCR fragment, was a gift of J. Dworkin, Columbia University.

To construct pAH124 (PT7-His6-MciZ kan), a 120 bp fragment containing the mciZ open reading frame was amplified using primers oAH175 (5′-GGAATTCCATATGAAAGTGCACCGCATG-3′) and oAH176 (5′-CCGCTCGAGTTATGGCTTTGAGATCCAAT-3′). The PCR product was digested with NdeI and XhoI (sites underlined in primer sequences above) and cloned into pET28a (Novagen), cut with the same enzymes, such that the mciZ ORF was in frame with the upstream hexahistadine tag coding sequence. A stop codon in primer oAH176 ensured that the encoded protein would not additionally possess a C-terminal hexahistadine tag.

To construct pAH134 (thrC::Pspac-yfp-ftsZ erm), a 1149 bp fragment containing the ftsZ open reading frame was amplified using primers oFG12 (5′-AAGCTAGCGGCCGCATGTTGGAGTTCGAAACAAAC-3′) and oAH217 (5′-ACATGCATGCTTAGCCGCGTTTATTACGGTT-3′). The PCR product was digested with NotI and SphI (sites underlined in primer sequences above) and cloned into pFG23 (thrC::Pspac-yfp-minD erm), cut with the same enzymes, such that the ftsZ ORF replaced that of minD and was in frame with the upstream yfp coding sequence. Plasmid pFG23 was a gift of F. Gueiros-Filho, Universidade de Sao Paulo.

To construct pJL1 (PT7-ftsZB.sub-His6 amp), a 1146 bp fragment containing the B. subtilis ftsZ open reading frame was amplified using primers oAH211 (5′-GGAATTCCATATGTTGGAGTTCGAAACAAA-3′) and oAH212 (5′-CCGCTCGAGGCCGCGTTTATTTACGGTTTC-3′). The PCR product was digested with NdeI and XhoI (sites underlined in primer sequences above) and cloned into pET21b (Novagen), cut with the same enzymes, such that the ftsZ ORF was in frame with the downstream hexahistidine tag coding sequence.

Transformation of yeast

Yeast transformations were performed essentially as described in Gietz and Woods (Gietz & Woods, 2002). The yeast strain to be transformed was grown overnight in 5 ml of YPAD medium at 30°C. OD600 was determined in order to estimate the culture titer, and the cells were recovered by centrifugation at 3000 x g for 5 min. Approximately 2.5 x 108 cells were inoculated into 50 ml of pre-warmed (30°C) YPAD medium, and the culture was shaken at 30°C for 3–4 hours until the cell titer reached approximately 2 x 107 cells/ml. Cells were harvested by centrifugation at 3000 x g for 5 min, washed in ½ volume of sterile distilled water, and recovered by centrifugation as described above. The cell pellet was washed a second time in 1 ml of sterile distilled water, the cells recovered by centrifugation at maximum speed in a microfuge, and finally resuspended in 1 ml of sterile distilled water. For each transformation a 100 μl aliquot of the cell suspension was removed, subjected to centrifugation at maximum speed for 1 min in a microfuge, and the cell pellet set aside. Meanwhile, a transformation mix was prepared from the following components (added in the order listed): 240 μl of 50% PEG, 36 μl of 1.0 M lithium acetate, 50 μl of 2 mg/ml single stranded carrier DNA, and 34 μl of plasmid DNA dissolved in water. The transformation mix was added to the cell pellet and the cells resuspended using a vortex mixer for 1 min. The transformation mixture was then incubated at 42°C for 40 min in a water bath. The cells were collected by centrifugation at maximum speed for 1 min in a microfuge and the pellet gently resuspended in 1 ml of sterile distilled water. Cells were plated on appropriate SC omission medium as required to select for the plasmid.

Preparation of plasmid DNA from yeast

The yeast strain containing the plasmid of interest was grown overnight at 30°C in 5 ml of YPAD. Cells were recovered by centrifugation at 3000 x g for 5 min and the supernatant removed. 0.3 g of fine glass beads, 0.2 ml of Yeast Lysis Buffer, and 0.2 ml of 1:1 phenol:chloroform were added to the cell pellet, and the cells resuspended using a vortex mixer for 2 min. Following addition of 0.2 ml 1X TE, the mixture was subjected to centrifugation at maximum speed for 5 min in a microfuge. Two volumes of ethanol were added to the aqueous phase, and the solution was mixed and centrifuged at maximum speed for 3 min in a microfuge to recover the precipitated DNA. The pellet was rinsed with 0.5 ml of cold 70% ethanol, air-dried for 15 min at room temperature, and resuspended in 20 μl of 1X TE. The plasmid was transformed into E. coli strain DH5α and the final preparation was obtained using a standard mini-prep procedure, as described above.

Yeast two-hybrid screen

Yeast two-hybrid screening was carried out using yeast strains PJ69-4a and PJ69-4α (James et al., 1996). A B. subtilis genomic library was generously provided by H. Yoshikawa (Yoshimura, 2001) and was re-amplified in the Losick laboratory by Q. Pan (Pan, 2003). Briefly, the library was constructed by cloning of genomic fragments approximately 0.5 kb-2.0 kb in length, generated by sonication of chromosomal DNA from B. subtilis strain 168, into plasmid pGADT7 (Clontech). The library consisted of an estimated 5.6 x 106 independent clones. Yeast strain PJ69-4a was transformed with library DNA as described above, except that the protocol was scaled up by 10-fold. The resulting cells were plated onto SC-Leu omission medium and the plates incubated at 30°C until colonies appeared. Approximately 4 x 106 transformants were collected and pooled by scraping the colonies from the plates and resuspending the cells to a final volume of 10 ml in 1X PBS containing 10% glycerol. The resuspension was divided into 1 ml aliquots and frozen at −80°C.

Yeast strain PJ69-4α was transformed as described above with a Gal4BD-FtsZ fusion plasmid, encoding a fusion of the Gal4 DNA binding domain to the N-terminus of FtsZ, to create strain AH306. The Gal4BD-FtsZ fusion plasmid was derived from pGBDU, which has been described previously (James et al., 1996). Strain AH306 was grown in 25 ml SC-Ura omission medium for 5 hours at 30°C. At the same time, a 50 ml volume of SC-Leu omission medium was inoculated with a single 1ml aliquot of the PJ69-4a pGADT7 library strain, and was also grown for 5 hours at 30°C. A 50 ml volume of YPAD was inoculated with 25 OD600 units of the AH306 culture and 37.5 OD600 units of the library culture, and mating was allowed to proceed by overnight incubation at 30°C. 250 μl aliquots of the mating culture were plated onto SC-Ade omission medium to select for Ade+ two-hybrid positives. Dilutions of the mating culture were plated onto SC-Leu, SC-Ura, and SC-Leu-Ura omission media to determine the mating efficiency, which was calculated to be 16.8%. Of approximately 1.4 x 107 diploids tested, 438 ADE+ positives were recovered.

To eliminate false positive interactions, the 438 ADE+ positive clones were replica plated onto SC-Leu-Ura and SC-His media. Only clones that could successfully grow on both were chosen for further analysis, of which there were 179. These 179 ADE+LEU+URA+HIS+ clones were then patched onto SC-Ade omission medium containing 5-fluoroorotic acid in a second step of false positive elimination. Only one clone failed to grow, while the remaining178 grew successfully and were recovered for further analysis. Plasmid DNA was isolated from these 178 clones as described above. B. subtilis genomic insert DNA was amplified from the pGADT7 plasmids using primers oQP284 (5′-TAATACGACTCACTATAGGGC-3′) and oQP285 (5′-AGATGGTGCACGATGCACAG-3′), which are complementary to sequences flanking the multiple cloning site. The PCR products were sequenced to determine the identity of the genomic insert DNA as described above.

Microscopy

Samples (0.25–1.0 ml) were withdrawn from cultures of LB, resuspension medium, or N-minimal medium, depending on the experiment. The sample was subjected to centrifugation at 10,000 x g for 1 min in a microfuge and the supernatant was removed. The cell pellet was washed with 1X PBS, centrifuged as just described, and resuspended in 15 μl of 1X PBS containing 1 μM TMA-DPH membrane dye. In experiments where DNA was to be examined by DAPI staining, the cells were instead resuspended in 15 μl of 1X PBS containing 1 μg/ml of FM4-64 membrane dye and 2 μg/ml of DAPI. 3 μl of this cell suspension was transferred onto a glass cover slip, and the cover slip was placed onto a microscope slide covered with a 1% agarose pad. The agarose pad served to immobilize the cells and was necessary for maintenance of Z-rings during the course of the experiment. The equipment used and the capture and analysis of the images were as previously described (Fujita & Losick, 2002).

The 1% agarose pads were prepared as follows. For experiments in which the cells were harvested from LB culture, a 1% agarose solution was made in 1X minimal medium (0.1 M MOPS, pH 7.0, 5 mM K2HPO4, 5 mM KH2PO4, 2 mM MgCl2, 50 mM MnCl2, 1 μM ZnCl2, 2 μM thiamine, 0.05 mg/ml phenylalanine, 0.05 mg/ml tryptophan, 0.05 mg/ml threonine, 0.5% glycerol, 0.5% glutamate). For experiments in which the cells were harvested from cultures grown in A+B medium or N-minimal medium, 1% agarose solutions were instead made in A+B or N-minimal medium, respectively. 800 μl of the molten 1% agarose solution was applied to a microscope slide, and a second slide was placed on top in order to flatten the agarose. After cooling for 1 min, one slide was gently removed, leaving the agarose pad which remained adhered to the other slide.

For the experiment depicted in Fig. 2A–B, the growth medium was supplemented with 150 μM IPTG and 0.05% xylose to induce expression of yfp-ftsZ and mciZ, respectively. For the experiments shown in Fig. 2C–D and Fig. 6, gfp-zapA expression was switched on by addition of 0.25% xylose to the sporulation medium 30 minutes prior to examination. For the experiment shown in Fig. 2E–F, cells were grown for 16 hours in N-minimal medium (Nelson & Kennedy, 1971) containing 0.3% v/v glycerol, 0.1% casamino acids, and 8 μM MgCl2. 2 μM IPTG was added as a supplement to induce expression of gfp-ftsZ. If desired, synthetic CRAMP peptide was added to a final concentration of 15 μM.

Isolation of MciZ-resistant mutants

Five independent cultures of B. subtilis strain AH93 were grown in 5 ml of LB at 37°C to OD600 = 0.8–1.0. Cells from each culture were concentrated to OD600 = 4.0–5.0, which corresponded to a titer of approximately 2.2 x 109 cells/ml, and 100 μl aliquots were plated onto LB supplemented with 0.5% xylose to select for spontaneously arising MciZ-resistant mutants. The plates were incubated for 16 hours at 37°C, and a total of 30 colonies were picked from among the plates for further analysis. Genomic DNA was prepared from each of the 30 MciZ-resistant strains as described above, and was used in PCR reactions for amplification of ftsZ. The resulting PCR products were sequenced as described above. A single clone was identified that harbored a G-to-A transition at position 428 within the ftsZ open reading frame, corresponding to an R143K substitution in the FtsZ protein.

Purification of native FtsZ

The E. coli strain W3110(pBS58)(pCXZ), described previously in Wang and Lutkenhaus (Wang & Lutkenhaus, 1993), was used to overexpress native B. subtilis FtsZ. A 50 ml starter culture was grown overnight at 37°C in LB medium supplemented with ampicillin (100 μg/ml) and chloramphenicol (25 μg/ml), and cells from this culture were used to inoculate a 1 L volume of the same medium. The 1 L culture was grown at 37°C to OD600 = 0.8–1.0, induced with 1 mM IPTG, and harvested after 3 hours. The cell pellet was resuspended in 20 ml of TKEG buffer (50 mM Tris-HCl, pH 8.0, 50 mM KCl, 1 mM EDTA, 10% glycerol). After addition of 1 mM PMSF, 10 mM MgCl2, 2 μg/ml DNaseI, and 100 μg/ml lysozyme, the cell suspension was incubated at 37°C for 15 min, chilled to 4°C on ice, and the cells lysed by sonication. The resulting lysate was centrifuged at 5,000 x g for 10 min, and the supernatant was then subjected to an additional centrifugation step at 100,000 x g for 30 min in an ultrafuge. FtsZ was subsequently purified from the supernatant in two steps. First, ammonium sulfate was added to a final concentration of 40% by dropwise addition of a saturated solution. The mixture was incubated on ice for 20 min, the precipitate was recovered by ultracentrifugation as described above, and the pellet was resuspended in 3 ml of TKEG. Next, polymerization of FtsZ was initiated by addition of 20 mM CaCl2 and 10 mM GTP to the resuspension. The mixture was incubated at 30°C for 5 min and the precipitated FtsZ was recovered by ultracentrifugation at 100,000 x g for 20 min at 20°C. The resulting pellet was resuspended in 2.5 ml TKEG and the solution applied to a PD-10 desalting column (GE Healthcare Life Sciences) equilibrated with TKEG. The protein was eluted in a final volume of 3.5 ml in TKEG, divided into aliquots, and snap-frozen in liquid nitrogen.

Purification of FtsZ-His6

B. subtilis FtsZ-His6 protein was overproduced in E. coli strain BL21 CodonPlus RIL (Stratagene) transformed with a PT7-FtsZ-His6 (amp) expression plasmid. A 50 ml starter culture was grown overnight at 37°C in LB medium supplemented with ampicillin (100 μg/ml) and chloramphenicol (50 μg/ml), and cells from this culture were used to inoculate a 1 L volume of the same medium. The 1 L culture was grown at 37°C to OD600 = 0.6–0.8, induced with 1 mM IPTG, and harvested after 3 hours at 37°C. The cell pellet was resuspended in 20 ml of Lysis buffer (50 mM Tris, pH 8.0, 300 mM NaCl, 10 mM Imidazole), and PMSF was added to a final concentration of 1 mM. Lysis was achieved by sonication and the resulting lysate was subjected to two sequential centrifugation steps. In the first step, centrifugation was carried out at 5,000 x g for 10 min. In the second step, the resulting supernatant was subjected to ultracentrifugation at 100,000 x g for 30 min at 4ºC. The supernatant was recovered and loaded onto a 1 ml column of Ni-NTA agarose (Qiagen) equilibrated with Lysis buffer. The column was washed with 50 ml of Wash buffer (50 mM Tris, pH 8.0, 300 mM NaCl, 20 mM Imidazole), and FtsZ-His6 protein was eluted with 3 x 5 ml aliquots of Elution buffer (50 mM Tris, pH 8.0, 300 mM NaCl, 500 mM Imidazole). The first 5 ml elution, which typically contained the bulk of the protein, was split into two aliquots of 2.5 ml. Buffer exchange into TKEG was accomplished using PD-10 columns (described above). Following concentration, the protein sample was divided into aliquots and snap-frozen in liquid nitrogen.

Purification of His6-MciZ

His6-MciZ was overproduced in E. coli strain BL21 CodonPlus RIL (Stratagene) transformed with a PT7-His6-MciZ (kan) expression plasmid. A 50 ml starter culture was grown overnight at 37°C in LB medium supplemented with kanamycin (30 μg/ml) and chloramphenicol (50 μg/ml), and cells from this culture were used to inoculate a 1 L volume of the same medium. The 1 L culture was grown at 37°C to OD600 = 0.6–0.7, induced with 1 mM IPTG, and harvested after 2 hours. The cell pellet was resuspended in 20 ml of Buffer B (10 mM Tris-HCl, pH 8.0, 0.1 M NaH2PO4, 8 M Urea). Cells were lysed by sonication, chilled to 4°C on ice for 30 min, and subjected to centrifugation at 9,000 x g for 15 min. The supernatant was recovered and loaded onto a 1 ml column of Ni-NTA agarose (Qiagen) equilibrated with Buffer B. The column was washed first with 10 ml of Buffer B, followed by 10 ml of Buffer TN (50 mM Tris-HCl, pH 8.0, 150 mM NaCl). Renaturation of His6-MciZ on the column was accomplished by performing a 50 ml wash with Buffer TN. The protein was eluted with 5 ml of Buffer TN containing 250 mM imidazole, and 2.5 ml of the eluate was applied to a PD-10 desalting column (see above) equilibrated with Buffer TN + 10% glycerol. The protein was eluted in a final volume of 3.5 ml in Buffer TN + 10% glycerol, divided into aliquots, and snap-frozen in liquid nitrogen.

Protein concentration assays

Protein concentration was determined using the Coomassie Plus reagent method (Pierce) or the BCA protein assay reagent method (Pierce) with BSA as the standard. When necessary, proteins were concentrated further using Centricon or Amicon Ultra centrifugal filter units (Millipore).

Protein binding assays

For the binding experiment depicted in Fig. 3A, the protein binding reaction (200 μl) was assembled in PEM buffer (50 mM PIPES, pH 6.5, 1 mM EDTA, 5 mM MgCl2) containing 0.5 mg/ml BSA, 5 μM FtsZ- His6 protein, and 5 μM synthetic MciZ peptide. The mixture was incubated for 15 min at room temperature, and a 10 μl aliquot was set aside for analysis (input fraction). 40 μl of Ni-NTA agarose (50% slurry equilibrated in PEM buffer) was added to the remaining 190 μl sample, and the mixture was incubated for 5 min at room temperature. The sample was subjected to centrifugation at 2,500 x g for 2 min and the supernatant was set aside for analysis (flow-through fraction). The Ni-NTA agarose beads were washed twice with 100 μl of PEM buffer containing 0.5 mg/ml BSA (wash fractions), and elution of the bound protein was accomplished by washing the beads two additional times with 20 μl of PEM buffer containing 250 mM imidazole (elution fractions). Fractions were subjected to SDS-PAGE using10–20% Tris-Tricine precast gradient gels (Bio-Rad). Protein bands were visualized using Colloidal Blue stain (Invitrogen). Synthetic MciZ peptide was purchased from SynPep Corporation (www.synpep.com).

For the FtsZ pull-down experiment shown in Fig. 3B, B. subtilis strain PY79 was grown in 200 ml LB at 37°C to OD600 = 0.8–1.0. The cells were recovered by centrifugation at 5,000 x g for 15 min, and the cell pellet was resuspended in 10 ml of Lysis Buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 mM imidazole). After addition of 100 μl of 20 mg/ml lysozyme, the cell suspension was incubated at 37°C for 15 min, chilled to 4°C on ice, and the cells were lysed by sonication. The lysate was subjected to centrifugation at 9,000 x g for 15 min, and the cleared lysate was split into two halves. 0.2 mg of His6-MciZ was added to one half, and an equivalent volume of Buffer TN (see above) was added to the second half. The lysate mixtures were rotated for 1 hour at 4°C, after which each was loaded onto a 0.5 ml column of Ni-NTA agarose (Qiagen) equilibrated with Lysis Buffer. The columns were washed twice with 25 ml of Wash Buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 20 mM imidazole), and proteins were eluted with 1 ml of Elution Buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 250 mM imidazole). 500 μl aliquots of each elution were mixed with 500 μl of 10% TCA, and the mixtures were chilled to 4°C on ice for 30 min. The precipitate was recovered by centrifugation at maximum speed for 15 min in a microfuge, and the supernatant was carefully removed. The pellets were each resuspended in 1 ml of acetone, vigorously mixed, and incubated on ice for 15 min. The samples were subjected to centrifugation at maximum speed for 5 min in a microfuge, the supernatants were discarded, and the pellets were air-dried for 15 min. Each pellet was resuspended in SDS-PAGE loading buffer (Sambrook & Russel, 2001). After electroblotting, proteins were detected by incubation of the membrane in either a 1:20,000 dilution of α-FtsZ or a 1:15,000 dilution of α-σA antibodies, followed by incubation in a 1:10,000 dilution of goat anti-rabbit IgG (H+L)-HRP conjugate (Bio-Rad).

FtsZ polymerization assays

90° light scattering measurements of FtsZ polymerization were performed essentially as described (Romberg et al., 2001) using a SPEX FluoroMax fluorometer. 120 μl reactions were assembled in PKM (50 mM PIPES, pH 6.5, 50 mM KCl, 5 mM MgCl2) buffer plus 5 μM native FtsZ protein. Pure synthetic MciZ peptide was added to a final concentration of 5 μM, and polymerization was initiated by the addition of GTP at various concentrations ranging from 25 μM to 500 μM. Measurements were recorded within 2–3 seconds following the addition of GTP.

FtsZ GTPase assays

GTPase assays were performed using the Malachite Green Phosphate Assay Kit (BioAssay Systems). 300 μl reactions were assembled on ice in PKM buffer plus 2.5 μM native FtsZ, various amount of GTP (ranging from 0.075 to 1 mM), and various amounts of synthetic MciZ peptide (ranging from 0 to 1 μM). Polymerization was initiated by shifting the samples to 37°C and the reactions were allowed to proceed for a period of 8 min. 30 μl aliquots were withdrawn each minute into wells of a 96-well plate and were mixed with 50 μl of 80 mM EDTA to quench the reaction; an initial aliquot was also taken immediately upon transfer of the 300 μl reaction mix to 37°C. 20 μl of the Malachite Green Reagent was mixed with each of the quenched reaction samples, and the plate was incubated for 10 min at room temperature. The absorbance at 650 nm was read using a SpectraMax Gemini XPS microplate fluorometer (Molecular Devices). A phosphate standard curve was prepared using a set of phosphate standards ranging in concentration from 0.018 μM to 40.0 μM.

Acknowledgments

We thank H. Yoshikawa for the gift of the B. subtilis genomic library and R. Hancock for the gift of CRAMP. We thank B. Finlay, R. Hancock, and D. Raychaudhuri for helpful discussions. This work was supported by NIH grant GM 18568 to R.L.

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