The Division Defect of a Bacillus subtilis minD noc Double Mutant Can Be Suppressed by Spx-Dependent and Spx-Independent Mechanisms

ABSTRACT

During growth, bacteria increase in size and divide. Division is initiated by the formation of the Z-ring, a ring-like cytoskeletal structure formed by treadmilling protofilaments of the tubulin homolog FtsZ. FtsZ localization is thought to be controlled by the Min and Noc systems, and here we explore why cell division fails at high temperature when the Min and Noc systems are simultaneously mutated. Microfluidic analysis of a minD noc double mutant indicated that FtsZ formed proto-Z-rings at periodic interchromosome locations but that the rings failed to mature and become functional. Extragenic suppressor analysis indicated that a variety of mutations restored high temperature growth to the minD noc double mutant, and while many were likely pleiotropic, others implicated the proteolysis of the transcription factor Spx. Further analysis indicated that a Spx-dependent pathway activated the expression of ZapA, a protein that primarily compensates for the absence of Noc. In addition, an Spx-independent pathway reduced the length of the cytokinetic period, perhaps by increasing divisome activity. Finally, we provide evidence of an as-yet-unidentified protein that is activated by Spx and governs the frequency of polar division and minicell formation.

IMPORTANCE Bacteria must properly position the location of the cell division machinery in order to grow, divide, and ensure each daughter cell receives one copy of the chromosome. In Bacillus subtilis, cell division site selection depends on the Min and Noc systems, and while neither is individually essential, cells fail to grow at high temperature when both are mutated. Here, we show that cell division fails in the absence of Min and Noc, due not to a defect in FtsZ localization but rather to a failure in the maturation of the cell division machinery. Suppressor mutations that restored growth were selected, and while some activated the expression of ZapA via the Spx stress response pathway, others appeared to directly enhance divisome activity.

INTRODUCTION

Most bacteria are surrounded by a semielastic cell wall made of peptidoglycan that protects the cell membrane from hyperexpansion due to the osmotic pressure of the cytoplasm. When cells divide, peptidoglycan growth must be redirected from synthesis that expands the cell perimeter to synthesis that bisects the mother cell into two daughters (1). Ultimately, cell division requires two critical events. First, the cell must identify a site for division that falls between replicated chromosomes such that upon completion, each daughter would receive one full copy of the genome (2, 3). Second, a large complex of proteins known as the divisome must be recruited to the site of cell division and, once activated, synthesizes peptidoglycan to constrict the cell envelope and ultimately complete cytokinesis (4–6). Both events are mediated by FtsZ, a protein that marks the site of cell division and serves as a scaffold for the recruitment of the divisome complex (7).

FtsZ is a GTP-binding and hydrolyzing homolog of eukaryotic tubulin that forms protofilament polymers inside the cell (8–12). Early work indicated that FtsZ localized as a ring and was an early component of the divisome that recruited subsequent proteins by direct interaction (13–16). Later work indicated that localization of FtsZ was dynamic as rings formed at midcell, increased in intensity, constricted in diameter, and ultimately disappeared after the completion of cytokinesis (17–21). FtsZ dynamism includes, but is not limited to, the phenomenon of treadmilling where each individual monomer is stationary within a protofilament, but the profilament as a whole appears to travel as new monomers are added to one end and lost from the other (22–25). FtsZ dynamism is critical for function not only to reposition the FtsZ ring for each round of division but may also play a role in cytokinesis as protofilaments are in constant motion circumnavigating the division plane (26–28). Dynamic control of FtsZ localization is thought to be essential in the process of cell division site selection, symmetrical division, and cell shape control (29, 30).

One system that controls the dynamic localization of FtsZ is the Min system (31). Not all bacteria encode Min, but those that do tend to encode the two most central components, MinC and MinD (32). MinD is a membrane-associated protein that binds to and activates MinC (33–36). MinC in turn binds to and antagonizes FtsZ (37–40). Localization of MinC and MinD is topologically restricted by various mechanisms depending on the organism. In Bacillus subtilis, MinC and MinD are restricted to the cell poles by the multipass transmembrane protein MinJ and the scaffolding network of DivIVA (41–46). Polar localization is related to Min function and, in the case of B. subtilis, the FtsZ ring remains after division is completed and requires the Min system for depolymerization such that in the absence of Min, FtsZ rings persist at the polar location indefinitely (47–52). Constitutive maintenance of multiple Z-rings and a defect in FtsZ monomer recycling puts an increased emphasis on de novo FtsZ synthesis for division, and competition between the multiple rings for FtsZ recruitment results in elongated cells (52–55). Finally, recruitment of FtsZ to the persistent polar FtsZ rings eventually gives rise to polar division events and the generation of anucleoid minicells (48, 52, 56). While cells mutated for the Min system do not suffer a growth rate defect, Min is nonetheless important for FtsZ redistribution and cell size control.

Another factor that controls the dynamic localization of FtsZ is the phenomenon of nucleoid occlusion that prevents FtsZ localization in the vicinity of the nucleoid (57–62). In B. subtilis, nucleoid occlusion is mediated, at least in part, by the DNA-binding Noc protein (63, 64). Noc binds to many sites across the chromosome and also encodes an amphipathic helix that targets/anchors Noc to the membrane (64, 65). Consistent with a role in nucleoid occlusion, decondensed spiral-like FtsZ intermediates are observed in close proximity to the chromosome when Noc is mutated (63). Recent microfluidic analysis indicated that the decondensed FtsZ filaments originated as a subpopulation of FtsZ that departed the cytokinetic ring and migrated along the membrane to the future site of cell division (66). Thus, multiple points of Noc recruitment of DNA to the membrane is thought to act as a passive palisade that discourages FtsZ migration over the chromosome and thereby corrals FtsZ between the nucleoids. Cells mutated for the Noc system do not suffer a growth rate defect, but Noc is nonetheless important for restricting FtsZ migration and concentrating FtsZ to improve division efficiency.

While neither the Min system nor the Noc system is essential when disrupted on their own, double mutants defective in both systems simultaneously are synthetically sick (63, 67). Here, we show that a minD noc double mutant has a temperature-sensitive synthetic lethal phenotype, and we explore the conditional essentiality by monitoring double mutants in microfluidic growth channels and by isolating mutants that restore high-temperature growth. We find that when Noc is depleted in a minD mutant, treadmilling FtsZ protofilaments leave the cytokinetic ring and localize to internucleoid spaces, but the FtsZ rings are defective in maturation as they fail to reach sufficient concentration to induce cytokinesis. Suppressor mutations that restored high temperature growth to the minD noc double mutant were found in a variety of genes, and we focused on those genes involved in the proteolytic regulation of the global stress response transcription factor Spx. We found that elevated Spx levels increased the expression of the FtsZ-cross-linking protein ZapA, a condition that primarily masks the absence of Noc. We also found that ClpX inhibited another pathway that is Spx independent and acts downstream of FtsZ localization, likely at the level of the divisome, to increase the rate of cytokinesis.

RESULTS

The FtsZ ring localizes normally but fails to mature in the absence of MinD and Noc.

The Noc protein was serendipitously discovered when mutations in the noc gene were found to be synthetically sick with mutations in the min system incubated under growth conditions of 30°C (63, 67). Previous work on the double mutant was conducted in domesticated laboratory strains, and to confirm that the growth defect was not dependent on genetic background, we attempted to reproduce the phenotype in the ancestral B. subtilis strain NCIB3610. A minD noc double mutant was successfully generated and grew like the wild type on plates and in liquid media incubated at 30°C. Due to the absence of a growth defect under reported conditions, we wondered whether the phenotype was temperature sensitive. To assess temperature sensitivity, the wild type, both single mutants, and the double mutant were serially diluted, spotted on LB agar plates separately, and incubated at four different temperatures: 22, 30, 37, and 42°C (Fig. 1A; see also Fig. S1A in the supplemental material). Whereas the wild type and minD and noc single mutants exhibited similar viability at all temperatures tested, the number of CFU in the minD noc double mutant was dramatically reduced at 37 and 42°C (Fig. 1A; see also Fig. S1A). We conclude that the ancestral strain is more tolerant than laboratory strains for the minD noc double mutant, and we further conclude that the combination of the two mutations is synthetic lethal at high temperatures.

FIG 1.

High-temperature synthetic lethality of a minD noc double mutant can be genetically suppressed. Dilution plating of wild type (DK1042), minD (DK7744), noc (DK443), minD noc (DK7298) (A); minD noc yjbH (DK8811) and minD noc clpX (DK8369) (B); minD noc zapA (DK8800), minD noc yjbH zapA (DK8851), and minD noc clpX zapA (DK8824) (C); and minD noc spx (DK8767), minD noc yjbH spx (DK8849), and minD noc clpX spx (DK8738) (D) strains at 30 and 37°C. Exponential growing liquid cultures were adjusted to an OD₆₀₀ of ∼1 and then serially diluted to 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, 10⁻⁵, and 10⁻⁶. Next, 10-μl portions of cells were spotted for each concentration. Additional temperatures of 22 and 42°C are included in Fig. S1 in the supplemental material.

To explore why cells failed to grow at high temperature in the absence of both the Min and Noc systems, we generated a conditional double mutant in which the noc gene was expressed from an IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible promoter and inserted at an ectopic site in the chromosome (minD noc amyE::P_spank-noc). To determine functionality, the IPTG-inducible noc construct was introduced at low temperature to a minD noc double mutant background containing both a cytoplasmic mRFPmars and an mNeongreen-FtsZ fusion reporter. The IPTG-inducible construct appeared to be functional and capable of complementing a noc mutation as the strain resembled the minD mutant when grown in broth at 37°C with ≥0.1 mM IPTG but showed increased filamentation at lower levels of IPTG induction (see Fig. S2A). Next, the strain was introduced to the microfluidic channels and grown at 37°C with 0.1 mM IPTG induction for 3 h after which the IPTG was washed out and replaced with IPTG-deficient media. B. subtilis divides by septation with subsequent cell separation, and thus, cell division events were conservatively defined as a local 20 % decrease in mRFPmars cell body intensity (52, 66). Prior to IPTG washout, cells of the minD noc double mutant induced for noc grew in a manner similar to that reported for a minD mutant alone such that cells were elongated, and frequent minicells were observed (Fig. 2A) (52). Over the next 5 to 6 h after washout, the cells grew but gradually stopped dividing until the entire population became filamentous and ultimately lysed (Fig. 2B; see also Movie S1 in the supplemental material). We conclude that noc depletion in a minD mutant background resulted in a cell division defect and cell death consistent with synthetic lethality.

FIG 2.

Microfluidic analysis of growth, division, and chromosome segregation in minD noc double mutants. Time-lapse fluorescence imaging of a microfluidic channel was performed for the minD noc (P_spank-noc) mutants (A to F), minD noc clpX mutant (G), and minD noc yjbH mutant (H) growing at steady state. (A and B) Fluorescence microscopy of a min noc (P_spank-noc) mutant (DK6375) growing with 0.1 mM IPTG (A) and after washout with media lacking IPTG (B). Cytoplasmic mRFPmars false colored red (top), mNeongreen-FtsZ false colored green (middle), and an overlay of the two colors (bottom). Graphs are a quantitative analysis of mRFPmars fluorescence intensity (red line) and mNeongreen fluorescence intensity (green line) to match the fluorescence images immediately above. (C and D) Fluorescence images of a minD noc (P_spank-noc) mutant (DK6962) growing with 0.1 mM IPTG (C) and after washout with media lacking IPTG (D). Chromosomes (HBsu-mCherry) false colored purple (top), mNeongreen-FtsZ false colored green (middle), and an overlay of the two colors (bottom). Graphs are a quantitative analysis of mCherry fluorescence intensity (purple) and mNeongreen fluorescence intensity (green) to match the fluorescence images immediately above. (E and F) Fluorescence images of a minD noc (P_spank-noc) mutant (DK8394) growing with 0.1 mM IPTG (E) and after washout with media lacking IPTG (F). Cytoplasmic mRFPmars false colored red (top), ZapA-mNeongreen false colored cyan (middle), and an overlay of the two colors (bottom). Graphs are a quantitative analysis of mRFPmars fluorescence intensity (red) and mNeongreen fluorescence intensity (cyan) to match the fluorescence images immediately above. (G) Fluorescence images of a minD noc clpX mutant (DK8390) expressing cytoplasmic mCherry protein (falsely colored red; top) and mNeongreen-FtsZ (falsely colored green; middle) with an overlay of the two (bottom). Graphs are a quantitative analysis of mCherry fluorescence intensity (red) and mNeongreen fluorescence intensity (green) to match the fluorescence images immediately above. (H) Fluorescence images of a minD noc yjbH mutant (DK8817) expressing cytoplasmic mCherry protein (falsely colored red; top) and mNeongreen-FtsZ (falsely colored green; middle) with an overlay of the two (bottom). Graphs represent a quantitative analysis of mCherry fluorescence intensity (red) and mNeongreen fluorescence intensity (green) to match the fluorescence images immediately above. For all the panels, two different y axes are used. The left axis corresponds to the mRFP mars (red), HBsu-mCherry (purple), or mCherry signals (red), and the right axis corresponds to the mNeongreen-FtsZ (green) or ZapA-mNeongreen signal (cyan). All images are reproduced at the same magnification.

Cells might fail to divide when MinD is mutated and Noc is depleted if FtsZ failed to localize at interchromosome locations. Prior to IPTG washout, periodic foci of mNeongreen-FtsZ fluorescence were observed either at the midcell, to mark the future site of cell division, or at the cell pole as a remnant of cytokinesis (Fig. 2A) (52). After IPTG washout, FtsZ still localized as periodic foci but with a reduced fluorescence intensity (Fig. 2B) (see Movie S2). Moreover, FtsZ peak fluorescence still formed in between regularly spaced chromosomes labeled by the expression of mCherry fused to the nucleoid binding protein HBsu both prior to (Fig. 2C) and after IPTG washout (Fig. 2D) (68, 69). Thus, the minD noc double mutant fails to divide, not because the FtsZ fails to be periodically enriched in interchromosome spaces, but because the Z-rings that nucleate successfully fail to mature to wild-type intensity.

One reason that FtsZ might not mature into an intense ring is if the total amount of FtsZ was diminished in the absence of Min and Noc such that the pool of monomers was insufficient. Quantitative analysis of the fluorescence microscopy data, however, indicated that the fluorescence intensity per unit cell length of the minD noc double mutant prior to IPTG washout was similar to the fluorescence intensity over the same length interval after washout (Fig. 3A, left). Thus, FtsZ levels appeared to be constant. In a parallel approach, Western blot analysis with an antibody raised against FtsZ and a separate antibody against the constitutive control protein SigA was performed on the wild type and on the minD noc (P_spank-noc) double mutant in which the noc construct was induced with various amounts of IPTG. The amount of FtsZ remained constant relative to the amount of SigA in both strains regardless of the level of IPTG induction (Fig. 3B). Based on the results of both the cell biological and biochemical analyses, we conclude that the inability to form Z-rings in the absence of MinD and Noc was not due to a reduction in FtsZ levels.

FIG 3.

ClpX and YjbH regulate zapA expression through Spx. (A, left panel) Bar graph of the average mNeongreen-FtsZ fluorescence intensity in individual cells of wild-type (WT; DK8162), minD noc (P_spank-noc) (DK6375) mutant growth with 0.1 mM IPTG (pre-washout) and without IPTG (post-washout), minD noc clpX (DK8390), minD noc yjbH (DK8817), clpX (DK7320), and yjbH (DK8741). (A, right panel) Bar graph of the average ZapA-mNeongreen fluorescence of wild-type (WT, DK8138) and minD noc (P_spank-noc) (DK8394) mutant growth with 0.1 mM IPTG (pre-washout) and without IPTG (post-washout), minD noc clpX (DK8387), minD noc yjbH (DK8749), clpX (DK8147), and yjbH (DK8742). The fluorescence intensity of each strain was normalized to the wild type. Over 100 cells were measured per condition (except “postwashout”), and error bars represent standard deviations. Cells became filamentous in the “postwashout” condition, and thus fluorescence intensity was measured over a similar length as the “prewashout” condition. (B) Western blot analysis of B. subtilis cell lysates of zapA (DK8174), wild type (DK1042), noc (DK443), minD (DK7744), and minD noc (P_spank-noc) (DK7369) grown in the presence of the amount of IPTG, separately probed with anti-FtsZ primary antibody (top), anti-ZapA primary antibody (middle), and anti-SigA primary antibody (bottom). (C) Western blot analysis of B. subtilis cell lysates of zapA (DK8174), wild type (DK1042), clpX (DK5565), yjbH (DS8875), spx (DK6677), clpX spx (DK8880), yjbH spx (DK8881), minD noc clpX (DK8369), and minD noc yjbH (DK8811), separately probed with anti-FtsZ primary antibody (top), anti-ZapA primary antibody (middle), and anti-SigA primary antibody (bottom). (D) β-Galactosidase assays of P_zapA-lacZ transcriptional fusions for the wild type (DK8591), spx (DK8627), clpX (DK8628), clpX spx (DK8631), yjbH (DK8883), and yjbH spx (DK8890) at 37°C. Error bars represent standard deviations of three replicates. (E) β-Galactosidase assays of P_zapA-lacZ transcriptional fusions for the wild type (DK8591) and spx (DK8627) grown at either 37°C or at room temperature (22°C), as indicated. Error bars represent standard deviations of three replicates.

Another reason that cells might fail to form intense FtsZ rings is a failure in Z-ring condensation. Condensation of FtsZ rings appears to be mediated, at least in part, by the FtsZ-associated protein ZapA that binds to and cross-links FtsZ protofilaments (21, 70, 71). To explore ZapA dynamics, mNeongreen was fused to ZapA and monitored in a minD noc (P_spank-noc) mutant background both before and after IPTG washout at 37°C. Prior to washout, ZapA-mNeongreen fluorescence was observed both at the future midcell and at the poles in a pattern reminiscent of that observed for FtsZ (Fig. 2E). After washout, the ZapA-mNeongreen became more diffuse, like that observed with mNeongreen-FtsZ (Fig. 2F). Also similar to FtsZ, quantitative analysis indicated that ZapA-mNeongreen levels remained constant after Noc depletion in microfluidic channels (Fig. 3B), and Western blot analysis with antibodies raised against ZapA protein (Fig. 3C) indicated that ZapA levels were constant regardless of the level of Noc induction. Unlike mNeongreen-FtsZ, however, ZapA-mNeongreen did not display periodic foci after Noc depletion but rather only formed intense rings at locations where cell division would eventually occur (Fig. 2F; see also Movie S3). We conclude that ZapA and FtsZ are maintained at wild-type levels in the absence of MinD and Noc and that ZapA localization is an indicator of mature Z-rings that promote cytokinesis. We further conclude that cells die in the absence of Min and Noc because Z-rings fail to mature.

An increase in FtsZ ring condensation overrides the absence of Noc in the minD noc double mutant.

To gain insight into FtsZ-ring maturation, a forward genetic screen was performed to identify genes, which when mutated could permit growth of the minD noc double mutant at high temperature. A transposon mutagenesis system was introduced and propagated in the minD noc double mutant background, and the resulting transposants from seven parallel mutagenesis experiments were plated at the nonpermissive 42°C temperature (72). Mutant colonies were isolated from the pool grown at high temperature with a net frequency of approximately 1 × 10⁻³, or 1 in 1,000 transposants. Two candidates were clonally isolated from each mutagenesis pool and backcrossed to a minD noc double mutant parental background at the permissive temperature. In each case the resulting backcrossed transposon conferred the ability to restore growth at 42°C upon subsequent culturing confirming genetic linkage between the insertion and the phenotype. The location of each of the 14 transposon insertions was identified by inverse PCR and sequencing (Table 1).

TABLE 1.

Transposon insertions that restored high-temperature growth to a minD noc double mutant

Location	Annotation of gene product	Sequence taga	Strain
rho	Transcription terminator	TATGGATGA	DK8705
rho	Transcription terminator	TATTTCCTC	DK8716
rho	Transcription terminator	TACAACTTA	DK8707
rho	Transcription terminator	TATGGACTT	DK8708
ggaB	Teichoic acid biosynthesis	TATGGTTAT	DK8710
ggaB	Teichoic acid biosynthesis	TATAAAAAA	DK8714
yaaT/ricT	Regulator of RNase Y/Spo0A	TACAATGTA	DK8711
hemX	Regulator of heme biosynthesis	TAAAATAGG	DK8713
clpX	AAA+ unfoldase	TAAAAGACG	DK8712
Upstream clpP	Polar on clpP, AAA+ protease	TATCATGTA	DK8709
Upstream clpP	Polar on clpP, AAA+ protease	TACCTAAAA	DK8715
yjbH	Proteolytic chaperone for Spx	TACCTCTTC	DK8703
yjbI	Polar on yjbH, hemoglobin-like	TATAAAGAG	DK8704
Upstream yjbI	Polar on yjbH, hemoglobin-like	TACTCCTTT	DK8706

^aSequence tag indicates the 9 bp immediately adjacent to transposon insertion site.

Eight of the transposons disrupted a variety of genes that could give rise to pleiotropic effects. For example, four of the transposons disrupted the rho gene encoding the transcriptional terminator protein Rho, suggesting that RNA antitermination may be important for growth in the absence of MinD and Noc (73, 74). One transposon insertion disrupted the yaaT gene, encoding YaaT (RicT), involved in the regulation of Spo0A phosphorylation and RNase Y (75–78). Two other insertions were found in the ggaB gene, encoding GgaB, a protein involved in teichoic acid biosynthesis (79), and one other insertion disrupted hemX, encoding HemX, a regulator of HemA-dependent reduction of glutamyl-tRNA (80). The six remaining strains, however, were disrupted for genes related to regulatory proteolysis. In particular, three disrupted either the clpX gene, encoding an ATP-dependent unfoldase for the ClpP protease (81, 82), or inserted upstream of the gene encoding ClpP, such that the amount of ClpP might be reduced by polarity downstream of the insertion site. Importantly, three other transposons either directly disrupted, or were upstream of and likely polar on the expression of the proteolytic adaptor protein YjbH, which directs ClpXP-mediated degradation of the global stress response transcription factor Spx (83–87). While one or more mechanisms of suppression may be at work, we focused our attention on the mutants disrupted for the ClpXP/YjbH system as their effects could likely be attributed to the Spx protein.

Mutation of either ClpX or YjbH rescued high temperature growth (Fig. 1B) and efficient cell division to the minD noc double mutant background by restoring the formation of intense rings of FtsZ (Fig. 2G and H). One way in which growth might be improved in the absence of MinD and Noc is if the suppressor mutations in clpX and/or yjbH increased levels of FtsZ protein (63). The fluorescent intensity of mNeongreen-FtsZ did not appear to increase; however, (Fig. 3A, left), and Western blot analysis indicated that FtsZ protein levels were similar to the wild type in either suppressor background (Fig. 3C). We conclude that growth rescue was not correlated with an increase in FtsZ. Another way in which growth might be improved is if the suppressors increased expression of ZapA, a protein which when overexpressed has been shown to compensate for the absence of Noc (66). Fluorescence intensity of ZapA-mNeongreen was 2-fold higher (Fig. 3A, right), and Western blot analysis indicated that transposon insertions in clpX and yjbH both increased ZapA protein levels relative to the wild type (Fig. 3C). Moreover, mutation of zapA exacerbated the temperature-sensitive growth phenotype of the minD noc double mutant and abolished the ability to rescue growth at 37°C when either yjbH or clpX were also mutated (Fig. 1C). We conclude that high-temperature growth rescue in the minD noc double mutant by mutation of either clpX or yjbH was correlated with an increase in ZapA expression and that ZapA was necessary for suppression.

To determine whether an increase in ZapA levels was sufficient to restore high-temperature growth in the absence of MinD and Noc, an IPTG-inducible copy of zapA was introduced to a minD noc double-mutant background that also encoded mNeongreen-FtsZ and constitutive cytoplasmic mRFPmars. The resulting strain grew at both 30 and 37°C even in the absence of IPTG, indicating that the low basal level expression from the IPTG-inducible promoter was sufficient to promote growth (Fig. 4A). At levels up to 0.001 mM IPTG, fluorescence microscopy revealed cells that appeared elongated with diffuse FtsZ fluorescence resembling the strain when no IPTG was added (Fig. 4A). At 0.01 mM IPTG, however, cell length decreased, FtsZ formed multiple rings per cell, and minicells were observed, perhaps suggesting that the noc mutation was being masked, thereby revealing a minD-like phenotype (Fig. 4A). As IPTG levels increased to 0.03 mM, excess ZapA became inhibitory, and cells appeared elongated once more (Fig. 4A). At still higher levels of IPTG, growth was inhibited entirely. Thus, 0.01 mM IPTG was the optimum concentration to bypass the minD noc double mutant, and quantitative microfluidic analysis supported that the cell dimensions, FtsZ localization pattern, and minicell frequency resembled that of cells mutated for minD alone (Fig. 4B). We conclude that titrated overexpression of ZapA was sufficient to rescue growth to the minD noc double mutant and likely did so by compensating for the absence of Noc.

FIG 4.

Artificial overexpression of ZapA suppresses the absence of Noc. (A) Fluorescence images of a minD noc double mutant (DK8480) containing an IPTG-inducible copy of ZapA grown in broth culture containing the indicated amount of IPTG at 37°C. A minD mutant (DK8352) is provided for comparison. Constitutively expressed cytoplasmic mRFPmars is false colored red, and mNeongreen-FtsZ is false colored green. The scale bar is 5 μm and applies to all panels. (B) Histogram of individual cell lengths for minD mutant (DK5155, red) and a minD noc double mutant (DK8480, purple) containing an IPTG-inducible copy of ZapA grown in 0.01 mM IPTG (zapA⁺) and measured on the microfluidic device. (C) Bar graph of the percentage of cell division events that occurred on the cell pole for the minD mutant (DK5155, red) and a minD noc double mutant (DK8480, purple) containing an IPTG-inducible copy of ZapA grown in 0.01 mM IPTG (zapA⁺). Manual counting of >500 cell division events was performed to ensure minicells were accurately recorded.

ZapA expression might increase in the absence of ClpX and YjbH if the transcription factor Spx, normally degraded by the complex, instead accumulated and activated the expression of the zapA gene. To measure transcriptional regulation, the promoter region of zapA was cloned upstream of the lacZ gene encoding the LacZ β-galactosidase, and P_zapA-lacZ expression increased 2-fold when either yjbH or clpX was mutated (Fig. 3D). We note that a 2-fold increase in zapA transcription was consistent with the 2-fold increase in ZapA protein level observed in Western blotting (Fig. 3C) and the 2-fold increase in fluorescence intensity of ZapA-mNeongreen observed in either mutant background (Fig. 3A, right). Moreover, the increase in transcription by reporter gene assay and increase in ZapA protein accumulation by Western blotting was abolished in both clpX spx and yjbH spx double mutants (Fig. 3C and D). Finally, suppression by the yjbH mutation was Spx-dependent as a minD noc yjbH spx quadruple mutant was as temperature sensitive as the minD noc double mutant alone (Fig. 1D). We note, however, that mutation of spx did not restore temperature sensitivity to the minD noc clpX triple mutant (Fig. 1D). Nonetheless, we conclude that mutation of either yjbH or clpX overrides the absence of Noc by an Spx-dependent increase in ZapA expression. Furthermore, we infer that mutation of clpX confers an additional mechanism of growth rescue that is Spx-independent.

An acceleration of cytokinesis overrides the absence of MinD in the minD noc double mutant.

We wondered whether the Spx-dependent mechanism, as revealed by the yjbH mutation, also had an effect in the absence of MinD alone. One consequence of the minD mutation is that cell division events occur at the cell pole generating anucleoid minicells (Fig. 5A) (53, 56, 88). Mutation of yjbH reduced the polar division frequency of the minD mutant 2-fold (Fig. 5A), an effect likely mediated by elevated levels of Spx. Conversely, mutation of Spx exacerbated the minD minicell phenotype, by increasing the frequency of polar division 2-fold (Fig. 5A), such that nearly half of all divisions now occurred at the cell pole. Moreover, the mutation of spx was epistatic to yjbH as the polar division frequency of the minD yjbH spx triple mutant was elevated to levels that resembled the minD spx double mutant alone (Fig. 5A). Thus, the frequency of minicell formation can be altered, and Spx appears to control an inhibitor of minicell formation. Finally, artificial overexpression of zapA in the minD mutant background also elevated minicell frequency (Fig. 5A), suggesting that ZapA was likely not the inhibitor of minicell formation under Spx control. We infer that Spx regulates cell division not only by increasing expression of ZapA but also by controlling the expression or activity of another cell division regulator in parallel.

FIG 5.

Minicell formation is inhibited by an Spx-dependent mechanism. (A) Bar graph of the percentage of cell division events that occurred at the cell pole in the following mutants: minD (DK5155, red), minD clpX (DK7332, blue), minD yjbH (DK8747, cyan), minD spx (DK8918, orange), minD spx clpX (DK8922, olive), minD spx yjbH (DK8958, green), and minD (P_hyspank-zapA) mutants grown with 0.01 mM IPTG (DK8353, magenta). (B to E) Histogram of individual cell lengths for indicated mutants labeled with the same color scheme as described in panel A. For panels B to E, manual counting of >400 cell division events for each strain was performed to ensure minicells were accurately recorded. Panel B also includes data for the wild type (gray, DK5133).

To determine whether mutation of clpX had the same effect as mutation of yjbH in the absence of minD, we examined division in the minD clpX double mutant. Consistent with the ClpX-dependent proteolysis of Spx, mutation of clpX phenocopied mutation of yjbH in decreasing the minD polar division frequency (Fig. 5A). Mutation of Spx, however, was only partially epistatic to mutation of ClpX as the polar division frequency of the minD clpX spx triple mutant was only partially elevated (Fig. 5A). Mutation of minD also causes nucleoid-containing mother cells to become elongated (Fig. 5B) (53, 54), but mutation of clpX decreased minD mother cell length to that resembling the wild type (Fig. 5B), whereas simultaneous mutation of minD and yjbH (Fig. 5B) or minD and zapA overexpression did not (Fig. 5C). We conclude that the phenotypes of mother cell elongation and polar division frequency are separable, such that changes to one do not necessarily result in changes to the other. Nonetheless, mutation of spx was fully epistatic for cell length in both the minD clpX spx and minD yjbH spx triple mutants (Fig. 5D and E), suggesting that the effect of ClpX on cell length control can be overridden by the absence of Spx. Regardless, we sought to use the cell length differential between the minD clpX and minD yjbH double mutants to explore the Spx-independent mechanism of minD noc suppression.

Mutation of ClpX was shown to suppress the cell division defect that occurs when MinC and MinD are overexpressed, and the mechanism was attributed to ClpX acting as a direct inhibitor of FtsZ polymerization (89, 90). To determine the effect of ClpX on FtsZ dynamics, minD yjbH and minD clpX double mutants containing mNeongreen-FtsZ and cytoplasmic red fluorescence were grown and compared in microfluidic channels. One hundred cells from each strain were chosen at random through the course of growth, and four events were sequentially measured: the appearance of an FtsZ ring, accumulation of FtsZ to peak intensity, cell division, and FtsZ-ring disappearance (Fig. 6A and B). Data from tracking the four events were then used to calculate various parameters of cell division (19, 52, 66). Each of the cell division parameters in the minD yjbH mutant were similar to that reported for minD (Fig. 6C), and thus any differences in the minD clpX double mutant would likely reflect the action of the Spx-independent mechanism of minD noc suppression. Mutation of clpX did not appear to alter FtsZ dynamics in the minD mutant since polar FtsZ rings exhibited indefinite persistence (Fig. 6C), and the FtsZ-ring maturation time was similar to that of minD yjbH (Fig. 6C). Instead, mutation of ClpX resulted in a statistically significant reduction in cytokinesis (defined as the time between peak FtsZ-ring fluorescence and cell division) likely mediated at the level of divisome components downstream of FtsZ in the septation pathway. Moreover, faster cytokinesis produced shorter cells in the minD clpX double mutant which may also have caused a positive value in the medial Z-ring delay time, indicating that, as in the wild type, the new FtsZ ring formed in the same generation as the division event (Fig. 6C). We conclude that the Spx-independent mechanism of minD suppression does not change the rate of Z-ring maturation but instead likely accelerates the activation and/or function of the divisome.

FIG 6.

A Spx-independent mechanism reduces cytokinetic period for minD mutant. (A and B) Kymograph analysis of the minD clpX mutant (DK7332, A) and minD yjbH mutant (DK8747, B) grown in a microfluidic channel and imaged every 2 min. Cytoplasmic mCherry signal is false colored red (left) and overlaid with mNeongreen-FtsZ that is false colored green (right). Events necessary for defining division parameters are indicated and labeled as follows: a, septation; b, appearance of a nascent Z-ring; c, disappearance of a Z-ring; d, FtsZ peak intensity achieved. Note that “c” events are absent since FtsZ rings do not disappear in the absence of MinD and instead persist indefinitely. Each event designation is given a number as follows: 0 for the preceding generation and 1 for the current generation. Thin white lines indicate cell tracking and lineage analysis. (C) Graphs of 100 manually tracked cell division cycles for the wild type (DK5133, gray), minD mutant (DK5155, red), minD clpX double mutant (DK7332, blue), and min yjbH double mutant (DK8747, cyan) are presented as bars of average values and whiskers of standard deviations for the following parameters: “cell cycle” is the time between septation events (between consecutive “a” events); “Z-ring medial delay” is the time between a septation event and the appearance of a Z-ring that will eventually give rise to the next medial division event (between an “a” event and a “b” event that will give rise to the next round of septation); “Z-ring maturation period” is the time between Z-ring appearance and when that Z-ring achieves peak local intensity (between consecutive “b” and “d” events); “Z-ring persistence period” is the time between the appearance of a Z-ring and the disappearance of that Z-ring (between consecutive “b” and “c” events); “Z-ring polar duration” is the time between a septation event and the disappearance of the Z-ring resulting from that septation event (between consecutive “a” and “c” events); and “cytokinesis period” is the time between a matured Z-ring to cell septation events (between a “d” event and the “a” event that is caused by that particular Z-ring). Columns marked with a single asterisk are not significantly different (P > 0.05) from each other but are statistically different from columns marked with two asterisks (P < 0.05) by Student t test analysis. Data used for statistical analysis are included as Table S3 in the supplemental material. Note that the Z-ring medial delay of the minD and minD yjbH double mutants were negative on average because the medial Z-ring that would eventually promote cell division was formed in the preceding generation. The Z-ring persistence period and Z-ring polar duration in the minD, minD clpX, and minD yjbH are indefinite because FtsZ persists at the division site after cell division and never disappears during the course of the experiments. (D) Timelines of the various events, indicated in the bar graph, are color coded to match the indicated parameter of like color above and annotated with relevant events marked by the defining letters.

DISCUSSION

Min and Noc are thought to be two systems that help determine cell division site selection by controlling FtsZ localization. While neither system is essential in B. subtilis, cell division fails for unknown reasons when both are mutated simultaneously. Here, we show that in the absence of both Min and Noc, FtsZ becomes periodically enriched in interchromosomal spaces but fails to mature into an intense ring, such that cells subsequently elongate and eventually lyse. We interpret the double mutant phenotype in the context of models that suggest Noc sterically prevents FtsZ protofilaments from migrating away from the division plane, and Min depolymerizes FtsZ protofilaments to recycle monomers (52, 66). In the absence of Min, FtsZ monomers recycling is impaired, and FtsZ-ring maturation depends primarily on spatial restriction of de novo synthesized FtsZ to interchromosomal locations by Noc-mediated corralling. In the absence of Noc, a subpopulation of protofilaments migrates from one division plane to the next, and the nascent Z-ring matures by Min-mediated monomer recycling. In the absence of both, the FtsZ protofilaments wander indefinitely and are neither stabilized by spatial restriction nor by sufficient monomer availability. Thus, neither system directs the localization of FtsZ to the interchromosome location per se, but both enable FtsZ to reach a sufficient local concentration needed to activate the divisome.

To better understand Z-ring maturation and activation of the divisome, we selected for second site suppressor mutations that restored high temperature growth in the absence of MinD and Noc. One class of suppressors disrupted components important for the regulatory proteolysis of the global stress response transcription factor Spx (86, 87). Spx is proteolyzed by the ClpXP machinery, and mutants that disrupted or impaired production of the Spx-specific adaptor protein YjbH restored growth (83–85). Consistent with the proteolytic model and suppression due to elevated levels of Spx, growth rescue by the absence of YjbH was abolished when Spx was mutated. While Spx is an RNA polymerase-interacting protein with a large regulon (91–93), ZapA was predicted as a potentially important target because ZapA, but not FtsZ, protein levels were elevated in mutants enhanced for Spx. Moreover, Spx activated expression from the promoter of zapA, and previously published ChIP-seq analysis indicated that Spx interacts upstream of the P_zapA promoter (91). Thus, when YjbH is mutated, Spx levels increase in the cell, zapA transcription is increased, and growth is restored to the minD noc double mutant by increased levels of the ZapA protein.

Genetically, ZapA was both necessary and sufficient for growth rescue, since its mutation abolished suppression in the absence of YjbH, and its overexpression rescued growth to the minD noc double mutant background. ZapA is a small FtsZ-binding protein thought to condense and bundle protofilaments, and its artificial expression has been shown to suppress FtsZ condensation defects in the absence of Noc (24, 66, 70, 71, 94). Consistent with Noc suppression, titrated induction of ZapA gave rise to cell dimensions and FtsZ localization patterns in the minD noc double mutant that were indistinguishable from a minD mutant alone (Fig. 7). The importance of ZapA however extends beyond the specific yjbH suppressor background as, unlike the absence of Spx, the absence of ZapA further lowered the permissive growth temperature for the minD noc double mutant from 30°C to 22°C. We note that transcription from the P_zapA promoter increased to levels sufficient for suppression simply by growing cells at low temperature, but the elevated expression was only partly dependent on Spx (Fig. 3F). Thus, we suggest that elevated zapA expression also helps explain the low temperature growth of minD noc double mutant. We infer that because Spx is not required for low temperature growth, other mechanisms of zapA activation are likely at work.

FIG 7.

Model for the Spx-dependent and Spx-independent mechanisms of minD noc suppression. Arrows indicate activation, and T bars indicate repression. Written in red are the interpretations of how the various pathways reported here override either the absence of Min or Noc.

We also found evidence of an Spx-independent mechanism that restored high-temperature growth to the minD noc double mutant, as mutation of ClpX did not precisely phenocopy mutation of YjbH. Specifically, whereas mutation of Spx abolished growth rescue in the absence of YjbH but not in the absence of ClpX (Fig. 1D). Furthermore, mutation of ClpX also improved the temperature tolerance of the minD noc zapA triple mutant (Fig. 1C). Thus, mutation of ClpX increases Spx and ZapA levels but also engages an Spx-independent mechanism of suppression in parallel. Previous work indicated that ClpX interacts directly with FtsZ to inhibit FtsZ polymerization (89, 90), but our microfluidic analysis indicated that the Spx-independent suppression was not primarily mediated by changes in FtsZ dynamics. ClpX instead appeared to act downstream of FtsZ maturation. Indeed, the primary effect on division of the minD clpX double mutant was an acceleration of cytokinesis, likely by activating other components of the divisome (Fig. 7). Our results may be consistent with the model of direct interaction between the two proteins, but whereas ClpX antagonizes FtsZ polymerization in vitro, the in vivo effect may primarily manifest at higher-order FtsZ-divisome interactions. How ClpX antagonizes the divisome is unknown, but the mechanism is likely conserved as similar effects have been observed in the related bacterium Staphylococcus aureus (95).

Here, we report further evidence that the activity of the divisome is subject to regulation after FtsZ maturation. Specifically, the frequency of minicell formation could be increased or decreased 2-fold when either Spx or YjbH was mutated, respectively. We infer that the frequency at which FtsZ rings become activated for cytokinesis is regulated, either across the entire cell, or specifically governed at the poles. A specific effect on polar divisome activation is implicated by the observation that the frequency of minicell formation is separable from cell length effects suggesting that medial and polar Z-rings are differentially regulated. If true, Z-rings may differ based on their location, an idea previously raised in E. coli based on differential sensitivity to inhibition by MinCD (96). While the identity of the putative polar division inhibitor is unknown, our data suggest that inhibition is likely encoded as part of the Spx regulon, and this product may also be directly proteolyzed by ClpX (Fig. 7). Why B. subtilis would encode a regulator of polar Z-ring activation during vegetative growth is unclear, but this could be related to the process of sporulation in which polar septa give rise to the forespore under starvation conditions (97, 98).

The results presented here indicate that cells mutated for both MinD and Noc fail to grow at a high temperature due to a failure of Z-ring maturation and that a variety of extragenic suppressor mutations restored growth by more than one mechanism. Two mechanisms were implicated by the study of ClpX and the stress response transcription factor Spx, but we note that mutations outside the system were also obtained. In particular, mutation of GgaB restored growth, perhaps implicating an intersection between FtsZ maturation and the synthesis of teichoic acid (95). Furthermore, mutation of the transcriptional terminator Rho also restored growth, and strong phenotypes for rho mutations in B. subtilis have been notoriously difficult to identify (99, 100). Thus, in addition to the Spx-dependent and Spx-independent mechanisms of minD noc suppression reported here, there may be additional pathways revealed through the future study of the remaining suppressor classes. We note that different mutations were found to suppress synthetic lethality of E. coli doubly mutated for MinD and the Noc-analog SlmA, but these suppressors were found to act through the ZapB protein that is absent in B. subtilis (101). Whatever the case, the relative ease with which the conditional essentiality of the Min and Noc system can be bypassed suggest that there are likely multiple pathways that aid Z-ring maturation and may help explain why many bacteria lack either one or both systems. Thus, Min and Noc are simply two ways to help the Z-ring to mature rather than being integral parts of cell division site selection per se.

MATERIALS AND METHODS

Strains and growth conditions.

Bacillus subtilis strains (Table 2) were grown in lysogeny broth (LB; 10 g tryptone, 5 g yeast extract, and 10 g NaCl per liter) or on LB plates fortified with 1.5 % Bacto agar at 37°C. Antibiotics were supplemented when appropriate at the following concentrations: 100 μg/ml spectinomycin, 5 μg/ml kanamycin, 10 μg/ml tetracycline, 5 μg/ml chloramphenicol, and macrolide-lincosamide-streptogramin B (MLS;1 μg/ml erythromycin, 25 μg/ml lincomycin). For P_hyspank promoter-dependent gene expression, 1 mM IPTG was added to the medium, if not otherwise indicated.

TABLE 2.

Strains

Strain	Genotype
DK443	noc::kan
DK1042	Wild type
DK3391	amyE::Physpank-mRFPmars spec
DK5133	mNeongreen-ftsZ ΔepsH amyE::Physpank-mcherry spec
DK5155	mNeongreen-ftsZ ΔepsH amyE::Physpank-mcherry spec minD::TnYLB kan
DK5565	clpX::kan
DK5605	pTnFLX spec mls
DK6330	amyE::Pspank-noc spec
DK6339	noc::mls
DK6375	mNeongreen-ftsZ ΔepsH ycgO::PsigA-mRFP mars mls noc::kan amyE::Pspank-noc spec
DK6677	spx::spec
DK6962	mNeongreen-ftsZ ΔepsH sacA::hbsu-mCherry cat noc::mls amyE::Pspank-noc spec minD::TnYLB kan
DK7279	minD::cat
DK7298	noc::kan minD::cat
DK7320	mNeongreen-ftsZ ΔepsH amyE::Physpank-mcherry spec clpX::kan
DK7332	mNeongreen-ftsZ ΔepsH amyE::Physpank-mcherry spec clpX::kan minD::cat
DK7369	noc::kan amyE::Pspank-noc spec minD::cat
DK7744	minD::cat
DK8132	amyE::Physpank-zapA spec
DK8138	ΔepsH zapA-mNeongreen amyE::Physpank-mcherry spec
DK8140	noc::kan minD::cat amyE::Physpank-zapA spec
DK8147	ΔepsH zapA-mNeongreen amyE::Physpank-mcherry spec clpX::kan
DK8162	mNeongreen-ftsZ ΔepsH ycgO::PsigA-mRFP mars mls
DK8174	ΔzapA
DK8352	mNeongreen-ftsZ ΔepsH ycgO::PsigA-mRFPmars mls minD::cat
DK8353	mNeongreen-ftsZ ΔepsH amyE::Physpank-zapA spec ycgO::PsigA-mRFPmars mls minD::cat
DK8369	noc::mls clpX::kan minD::cat
DK8387	ΔepsH zapA-mNeongreen amyE::Physpank-mCherry spec minD::cat clpX::kan noc::mls
DK8390	mNeongreen-ftsZ ΔepsH amyE::Physpank-mCherry spec clpX::kan minD::cat noc::mls
DK8394	ΔepsH zapA-mNeonGreen ycgO::PsigA-mRFPmars mls amyE::Pspank-noc spec minD::cat
DK8480	mNeongreen-ftsZ ΔepsH amyE::Physpank-zapA spec ycgO::PsigA-mRFPmars mls minD::cat noc::kan
DK8524	mNeongreen-ftsZ amyE::Pspank-noc spec ycgO::PsigA-mRFP mars mls
DK8591	amyE::PzapA-lacZ cat
DK8627	spx::spec amyE::PzapA-lacZ cat
DK8628	clpX::TnYLB kan amyE::PzapA-lacZ cat
DK8631	spx::spec clpX::TnYLB kan amyE::PzapA-lacZ cat
DK8703	noc::kan minD::cat yjbH::TnFLX spec
DK8704	noc::kan minD::cat yjbI::TnFLX spec
DK8705	noc::kan minD::cat rho::TnFLX spec
DK8706	noc::kan minD::cat upstream of yjbJ::TnFLX spec
DK8707	noc::kan minD::cat rho::TnFLX spec
DK8708	noc::kan minD::cat rho::TnFLX spec
DK8709	noc::kan minD::cat upstream of clpP::TnFLX spec
DK8710	noc::kan minD::cat ggaB::TnFLX spec
DK8711	noc::kan minD::cat yaaT::TnFLX spec
DK8712	noc::kan minD::cat clpX::TnFLX spec
DK8713	noc::kan minD::cat hemX::TnFLX spec
DK8714	noc::kan minD::cat ggaB::TnFLX spec
DK8715	noc::kan minD::cat upstream of clpP::TnFLX spec
DK8716	noc::kan minD::cat rho::TnFLX spec
DK8738	noc::mls clpX::kan minD::cat spx::spec
DK8741	mNeongreen-ftsZ ΔepsH amyE::Physpank-mcherry spec yjbH::TnKRM kan
DK8742	ΔepsH zapA-mNeongreen amyE::Physpank-mcherry spec yjbH::TnKRM kan
DK8747	mNeongreen-ftsZ ΔepsH amyE::Physpank-mcherry spec yjbH::TnKRM kan minD::cat
DK8749	ΔepsH zapA-mNeongreen amyE::Physpank-mcherry spec noc::mls yjbH::TnKRM kan
DK8767	spx::spec minD::cat noc::kan
DK8800	ΔzapA minD::cat noc::kan
DK8811	minD::cat yjbH::TnKRM kan noc::mls
DK8817	mNeongreen-ftsZ ΔepsH amyE::Physpank-mcherry spec noc::mls yjbH::TnKRM kan
DK8824	ΔzapA noc::mls clpX::kan minD::cat
DK8849	minD::cat yjbH::TnKRM kan noc::mls spx::spec
DK8851	ΔzapA noc::mls yjbH::TnKRM kan minD::cat
DK8880	clpX::kan spx::spec
DK8881	yjbH::TnKRM kan spx::spec
DK8883	yjbH::TnKRM kan amyE::PzapA-lacZ cat
DK8890	spx::spec yjbH::TnKRM kan amyE::PzapA-lacZ cat
DK8918	mNeongreen-ftsZ ΔepsH ycgO::PsigA-mRFPmars mls minD::cat spx::spec
DK8922	mNeongreen-ftsZ ΔepsH ycgO::PsigA-mRFPmars mls minD::cat clpX::kan spx::spec
DK8958	mNeongreen-ftsZ ΔepsH ycgO::PsigA-mRFPmars mls minD::cat spx::spec yjbH::TnKRM kan
DS2231	clpX::TnYLB kan
DS3187	ΔminJ minD::TnYLB kan amyE::Phag-hag(T209C) spec
DS4522	spx::spec
DS8875	yjbH::TnKRM kan

Strain construction.

All strains were generated by direct transformation of DK1042, a derivative of B. subtilis ancestral strain 3610 with enhanced frequency of natural competence for DNA uptake or were transduced into DK1042 by SPP1-mediated transduction (Table 2) (102, 103). All primers used for strain construction are listed in Table S1 in the supplemental material. Cells were mutated for the production of extracellular polysaccharide (EPS) to prevent biofilm formation within the microfluidic device by in-frame markerless deletion of epsH genes, encoding enzymes essential for EPS biosynthesis (104, 105). The mNeongreen fluorescent fusion to FtsZ or ZapA, a generous gift from Ethan Garner (Harvard University), was crossed into the indicated genetic background by SPP1 phage-mediated transduction, and the antibiotic resistance cassette was eliminated by cre-lox recombination (22, 106). The HBsu-mCherry fusion (68, 69) was transduced into the indicated genetic backgrounds by SPP1 phage-mediated transduction. The P_hyspank-mcherry inducible construct was introduced by integrating pEV6 at the amyE locus and selection for spectinomycin resistance (107).

(i) noc::kan and noc::mls.

The noc::kan and the noc::mls insertion deletion alleles were generated with a modified “Gibson” isothermal assembly protocol (108). Briefly, the region upstream of noc was PCR amplified with the primer pair 3399/3400, and the region downstream of noc was PCR amplified with the primer pair 3401/3402. DNA containing a kanamycin resistance gene (pDG780) (109) or erythromycin resistance gene (pAH52) (110) was amplified with the universal primers 3250 and 3251. The three DNA fragments were combined at equimolar amounts to a total volume of 5 μl and added to a 15-μl aliquot of prepared isothermal assembly master mix (see below). The reaction mixture was incubated for 60 min at 50°C. The completed reaction was then PCR amplified with the primer pair 3399/3402 to amplify the assembled product. The amplified product was transformed into competent cells of PY79 and then transferred to the 3610 background with SPP1-mediated generalized transduction. Insertions were verified by PCR amplification with primers 3399/3402.

(ii) amyE::P_hyspank-mRFPmars.

To generate the IPTG-inducible construct for mRFPmars expression, the gene encoding mRFPmars was PCR amplified from pmRFPmars (Addgene) with the primer pair 4572/4573. The PCR product was purified, digested with NheI and SphI, and cloned into the NheI and SphI sites of pDR111 containing the P_hyspank promoter, the gene conducing the LacI repressor protein, and an antibiotic resistance cassette between the arms of the amyE gene (a generous gift from David Rudner, Harvard Medical School) to generate pDP430. The pDP430 plasmid was transformed into DK1042 to create DK3391.

(iii) ycgO::P_sigA-mRFPmars.

The constitutive mRFPmars construct was generated by isothermal assembly and direct transformation of the linear fragment into B. subtilis. The fragments upstream and downstream of ycgO were PCR amplified with the primer pairs 5270/5271 and 5272/5273, respectively, with chromosomal DNA from strain DK1042 as a template. The fragment containing the P_sigA promoter was PCR amplified with the primer pair 5268/5269 and chromosomal DNA from strain DK1042 as a template. The fragment containing mRFPmars and the spectinomycin resistance cassette was PCR amplified with the primer pair 5266/5267 and chromosomal DNA from strain DK3391 as a template. The four PCR products were purified and mixed in an isothermal assembly reaction that was subsequently amplified by PCR with primers 5270/5273.

(iv) amyE::P_hyspank-zapA spec.

To generate the IPTG-inducible construct for ZapA expression, the gene encoding ZapA was PCR amplified with the primer pair 7278/7279 and chromosomal DNA from strain 3610 as a template. The PCR product was purified, digested with HindIII and SalI, and cloned into the HindIII and SalI sites of pDR111 to generate pYY8. The pYY8 plasmid was transformed into PY79 to create DK8132.

(v) minD::TnYLB kan.

The minD gene was mutated by SPP1 phage-mediated transduction of a transposon mutant allele from DS3187 (minD::TnYLB-1 kan transposon insertion in the middle of the minD gene at sequence tag TAAATAGAG) reported previously (41).

(vi) clpX::kan.

The clpX::kan insertion deletion allele was generated with a modified “Gibson” isothermal assembly protocol. Briefly, the region upstream of clpX was PCR amplified with the primer pair 6048/6049, and the region downstream of clpX was PCR amplified with the primer pair 6050/6051. DNA containing the kanamycin resistance cassette (pDG780) (109) was PCR amplified with the universal primers 3250 and 3251. The three DNA fragments were combined following the “Gibson” isothermal assembly protocol above (see noc::kan). The assembled product was transformed into competent cells of PY79 to generate DK5565 and then transferred to the appropriate strain backgrounds by SPP1-mediated generalized transduction.

(vii) amyE::P_spank-noc spec.

To generate the IPTG-inducible construct for Noc expression, the noc gene was PCR amplified with the primer pair 6409/6410 and chromosomal DNA from strain 3610 as a template. The PCR product was purified, digested with NheI and SalI, and cloned into the NheI and SalI sites of pDR110 containing the P_spank promoter, the gene conducing the LacI repressor protein, and an antibiotic resistance cassette between the arms of the amyE gene (generous gift from David Rudner, Harvard Medical School) to generate pYY3. The pYY3 plasmid was transformed into PY79 to create DK6330.

(viii) minD::cat.

The minD::cat insertion deletion allele was generated with a modified “Gibson” isothermal assembly protocol. Briefly, the region upstream of minD was PCR amplified with the primer pair 6739/6740, and the region downstream of minD was PCR amplified with the primer pair 6741/6742. DNA containing the chloramphenicol resistance cassette (pAC225) (110) was PCR amplified with the universal primers 3250 and 3251. The three DNA fragments were combined following the “Gibson” isothermal assembly protocol above (see noc::kan). The assembled product was transformed into competent cells of PY79 to generate DK7279 and then transferred to the appropriate strain backgrounds by SPP1-mediated generalized transduction.

(ix) ΔzapA.

The in-frame deletion of zapA was generated by transducing the zapA::kan allele (BKK28610) obtained from the Bacillus Genetic Stock Center (The Ohio State University) into a strain of interest. The kanamycin cassette is flanked on either site by lox sequences and was evicted by transformation with the Cre-recombinase-expressing plasmid pDR244 at 30°C and selection for spectinomycin resistance (106). Plasmid pDR244 was subsequently evicted by growth at 37°C in the absence of selection. Eviction of the kanamycin resistance cassette was confirmed by kanamycin sensitivity.

(x) spx::spec.

The spx::spec allele was a generous gift from John Helmann (Cornell University).

(xi) yjbH::TnKRM kan.

The yjbH::TnKRM kan allele was previously reported (111).

(xii) amyE::P_zapA-lacZ cat.

To generate the P_zapA-lacZ transcriptional reporter plasmid pDP537, the promoter region of zapA (P_zapA) was amplified from DK1042 chromosomal DNA with the primer pair 7408/7049, digested with EcoRI and BamHI and cloned into the EcoRI/BamHI sites of pDG268 containing a chloramphenicol antibiotic resistance cassette and a polylinker upstream of the lacZ gene between two arms of amyE (112).

Isothermal assembly reaction.

First, a 5× ITA stock mixture was generated (500 mM Tris-HCl [pH 7.5], 50 mM MgCl₂, 50 mM dithiothreitol [DTT; Bio-Rad], 31.25 mM PEG-8000 [Fisher Scientific], 5.02 mM NAD [Sigma-Aldrich], and a 1 mM concentration of each deoxynucleoside triphosphate [dNTP; New England BioLabs]), aliquoted, and stored at −80°C. An assembly master mixture was made by combining prepared 5× isothermal assembly reaction buffer (131 mM Tris-HCl, 13.1 mM MgCl₂, 13.1 mM DTT, 8.21 mM PEG-8000, 1.32 mM NAD, and a 0.26 mM concentration of each dNTP) with Phusion DNA polymerase (New England BioLabs) (0.033 U/μl), T5 exonuclease diluted 1:5 with 5× reaction buffer (New England BioLabs) (0.01 U/μl), Taq DNA ligase (New England BioLabs) (5,328 U/μl), and additional dNTPs (267 μM). The master mix was aliquoted as 15 μl and stored at –80°C. DNA fragments were combined at equimolar amounts to a total volume of 5 μl and added to a 15-μl aliquot of prepared master mix. The reaction mixture was incubated for 60 min at 50°C.

SPP1 phage transduction.

To 0.1 ml of dense culture grown in TY broth (LB broth supplemented after autoclaving with 10 mM MgSO₄ and 100 μM MnSO₄), serial dilutions of SPP1 phage stock were added and statically incubated for 15 min at 37°C. To each mixture, 3 ml of TYSA (molten TY supplemented with 0.5 % agar) was added, poured atop fresh TY plates, followed by incubation at 37°C overnight. Top agar from the plate containing near confluent plaques was harvested by scraping into a 50-ml conical tube, vortexed, and centrifuged at 5,000 × g for 10 min. The supernatant was treated with a 25-μg/ml DNase final concentration before being passed through a 0.45-μm syringe filter and stored at 4°C. Recipient cells were grown to stationary phase in 2 ml of TY broth at 37°C. The, 0.9-ml portions of cells were mixed with 5 μl of SPP1 donor phage stock; 9 ml of TY broth was added to the mixture and allowed to stand at 37°C for 30 min. The transduction mixture was centrifuged at 5,000 × g for 10 min, the supernatant was discarded, and the pellet was resuspended in the remaining volume. Next, 100 μl of cell suspension was plated on TY fortified with 1.5 % agar, the appropriate antibiotic, and 10 mM sodium citrate.

Microfluidic system.

The microfluidic device was fabricated through a combination of electron beam lithography, contact photolithography, and polymer casting (113). Briefly, the microfluidic device is comprised of fluid and control layers both cast in poly(dimethylsiloxane) (PDMS) and a glass coverslip. The fluid layer lies between the control layer and glass coverslip and contains the microchannel array to trap the bacteria. Media and cells are pumped through the microfluidic channels by on-chip valves and peristaltic pumps that are controlled pneumatically through the top control layer. Each pneumatic valve is controlled by software to apply either vacuum (3 × 10⁴ Pa) or pressure (1.3 × 10⁵ Pa) to open or close individual valves, respectively.

On-device cell culture.

Before cells were loaded into the microfluidic device, the microchannels were coated with 1 % bovine serum albumin (BSA) in LB medium for 1 h to act as a passivation layer. Next, all of the channels were filled with LB medium containing 0.1 % BSA. A saturated culture of cells (∼10 μl) was added through the cell reservoir and pumped into the cell-trapping region. During cell loading, vacuum was applied to the control layer to lift up the microchannel array. After a sufficient number of cells were pumped underneath the channel array, positive pressure was applied to trap individual cells in those channels. Medium was pumped through the microchannels to flush away excess cells and maintain steady-state cell growth in the channel array.

Time-lapse image acquisition.

After inoculation in the microfluidic channels, a period of roughly 3 h elapsed during which cells adjusted to the growth conditions, and growth was monitored over the next 10 h. Fluorescence microscopy was performed either on a Nikon Eclipse Ti-E microscope or an Olympus IX83 microscope. The Nikon Eclipse Ti-E microscope was equipped with a 100× Plan Apo lambda, phase-contrast, 1.45 numerical aperture (NA) oil immersion objective and a Photometrics Prime95B sCMOS camera with Nikon Elements software (Nikon, Inc.). Fluorescence signals from mCherry, mRFPmars, and mNeongreen were captured from a Lumencor SpectraX light engine with matched mCherry, mCherry, and yellow fluorescent protein filter sets, respectively, from Chroma. The Olympus IX83 microscope was equipped with an Olympus UApo N 100×/1.49 oil objective and a Hamamatsu EM-CCD digital camera operated with MetaMorph Advanced software. Fluorescence signals from mCherry, mRFPmars, and mNeongreen were excited with an Olympus U-HGLGPS fluorescence light source with matched tetramethyl rhodamine isocyanate (TRITC), TRITC, and fluorescein isothiocyanate (FITC) filters, respectively, from Semrock. Images were captured from at least eight fields of view at 2-min intervals, if not otherwise indicated. The channel array was maintained at 37°C with a TC-1-100s temperature controller (Bioscience Tools). For all direct comparisons, the same microscope and settings were used.

Data analysis.

An adaptation period following exposure to illumination was observed; thus, data analysis was restricted to periods of steady state. Cell identification and tracking were analyzed by MATLAB programs (The MathWorks, Inc.) (113). The program extracted fluorescence intensity along a line profile down the longitudinal center of each microchannel in the array. The cytoplasmic mCherry or mRFPmars line profiles showed a flat-topped peak on the line where a cell was located, and a local 20 % decrease in fluorescence intensity was used to identify cell boundaries after division. Division events were conservatively measured as the time at which one cell became two according to the decrease in fluorescence intensity. Moreover, cell bodies were tracked from frame to frame in order to construct lineages of cell division, and cell body intensity was determined by the integration of the cytoplasmic mCherry or mRFPmars signal intensities within the confines of the cell. Signals from HBsu-mCherry, mNeongreen-FtsZ, and ZapA-mNeongreen were similarly tracked and measured along the length of the cell. FtsZ and ZapA line profiles were normalized by cell body intensity in order to minimize intensity differences among frames and across different fields of view.

Microscopic image acquisition on agarose pads.

Cells were grown to mid-log phase in liquid culture at 37°C and then imaged on 1 % agarose pads in 1× phosphate-buffered saline. Fluorescence microscopy was performed with a Nikon 80i microscope with a Nikon Plan Apo 100× phase-contrast objective and an Excite 120 metal halide lamp. Cytoplasmic mRFPmars was visualized with a C-FL HYQ Texas Red filter cube, and mNeongreen-FtsZ and ZapA-mNeongreen were visualized with a C-FL HYQ FITC filter cube. Images were captured with a Photometrics Coolsnap HQ2 camera in black and white, false colored, and superimposed with Metamorph image software.

Western blotting.

B. subtilis strains were grown in LB medium to an optical density at 600 nm (OD₆₀₀) of ∼1.0, and 1 ml was harvested by centrifugation, resuspended to 10 OD₆₀₀ in lysis buffer (20 mM Tris [pH 7.0], 10 mM EDTA, 1 mg/ml lysozyme, 10 μg/ml DNase I, 100 μg/ml RNase I, 1 mM phenylmethylsulfonyl fluoride [PMSF]), and incubated 30 min at 37°C. Then, 10 μl of lysate was mixed with 2 μl of 6× SDS loading dye. Samples were separated by 15 % SDS-PAGE. The proteins were electroblotted onto nitrocellulose and developed with a 1:80,000 dilution of anti-SigA primary antibody (a generous gift from Masaya Fujita, University of Houston), a 1:80,000 dilution of anti-FtsZ primary antibody (a generous gift from Petra Levin, Washington University), a 1:1,000 dilution of anti-ZapA primary antibody (a generous gift from David Rudner, Harvard Medical School), and a 1:1,000 dilution secondary antibody (horseradish peroxidase [HRP]-conjugated goat anti-rabbit immunoglobulin G). The immunoblot was developed using an Immun-Star HRP developer kit (Bio-Rad).

Transposon mutagenesis.

The transposon plasmid was introduced into the min noc double mutant (DK7298) of B. subtilis by SPP1 phage transduction at room temperature, selecting for MLS resistance, and incubated at room temperature for 72 h. Subsequently, cells were incubated for 16 h at room temperature in LB medium. To obtain individual colonies, the cell culture was diluted 1:10 with LB medium, and 100 μl of the dilution was plated on a LB plate fortified with 1.5 % agar, supplemented with spectinomycin, and grown at the nonpermissive temperature (42°C) overnight. To confirm that the transposon was linked to the suppressor mutation, a lysate was generated on the suppressor mutant, and the transposon was transduced to the parent min noc double mutant strain (DK7298) lacking the suppressor selecting for spectinomycin resistance and grown at 42°C.

Transposon insertion site identification by IPCR.

Cells were grown in LB medium at 37°C for 6 h. DNA was isolated, and 6 μg was digested with 2 U/μg DNA of the restriction endonuclease Sau3AI (NEB). Ligation with 400 U of T4 DNA ligase (NEB) was performed at room temperature for 2 h with 1.5 μg of the digested DNA. Inverse PCR was performed with 0.5 μg of ligated DNA with oligonucleotides 6212 and 6420 in a standard PCR program with a 3-min extension time on a Mastercycler thermocycler (Eppendorf). Phusion DNA polymerase (NEB) was used for PCR amplification. The amplified fragment was analyzed by Sanger sequencing (IDT) with the universal sequencing oligonucleotide 6224.

β-Galactosidase assay.

A 1-ml portion of the cells was harvested from a mid-log-phase (OD₆₀₀ ∼0.5) culture grown in LB broth shaken at 37°C and resuspended in an equal volume of Z buffer (40 mM NaH₂PO₄, 60 mM Na₂HPO₄, 1 mM MgSO₄, 10 mM KCl, and 38 mM β-mercaptoethanol). To each sample, lysozyme was added to a final concentration of 0.2 mg/ml, followed by incubation for 15 min at 30°C. Each sample was diluted appropriately to 500 μl in Z buffer, and the reaction was started with 100 μl of 4 mg/ml O-nitrophenyl β-d-galactopyranoside (in Z buffer) and stopped with 250 μl of 1 M Na₂CO₃. The OD₄₂₀ of the reaction mixtures was recorded, and the β-galactosidase-specific activity was calculated according to the following equation: [OD₄₂₀/(time×OD₆₀₀)] × dilution factor × 1,000.

Dilution plating.

B. subtilis cells were grown in LB medium at room temperature to mid-log phase and then adjusted to an OD₆₀₀ of 1.0. The diluted cultures were serially diluted (10^–1 to 10^–6); 10 μl of each dilution was spotted onto an LB plate, followed by growth overnight at either 22, 30, 37, or 42°C.