The Threshold Level of the Sensor Histidine Kinase KinA Governs Entry into Sporulation in Bacillus subtilis

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1 JOURNAL OF BACTERIOLOGY, Aug. 2010, p Vol. 192, No /10/$12.00 doi: /jb Copyright 2010, American Society for Microbiology. All Rights Reserved. The Threshold Level of the Sensor Histidine Kinase KinA Governs Entry into Sporulation in Bacillus subtilis Prahathees Eswaramoorthy, Daniel Duan, Jeffrey Dinh, Ashlee Dravis, Seram Nganbiton Devi, and Masaya Fujita* Department of Biology and Biochemistry, University of Houston, Houston, Texas Received 22 April 2010/Accepted 17 May 2010 Sporulation in Bacillus subtilis is controlled by a complex gene regulatory circuit that is activated upon nutrient deprivation. The initial process is directed by the phosphorelay, involving the major sporulation histidine kinase (KinA) and two additional phosphotransferases (Spo0F and Spo0B), that activates the master transcription factor Spo0A. Little is known about the initial event and mechanisms that trigger sporulation. Using a strain in which the synthesis of KinA is under the control of an IPTG (isopropyl- -D-thiogalactopyranoside)-inducible promoter, here we demonstrate that inducing the synthesis of the KinA beyond a certain level leads to the entry of the irreversible process of sporulation irrespective of nutrient availability. Moreover, the engineered cells expressing KinA under a H -dependent promoter that is similar to but stronger than the endogenous kina promoter induce sporulation during growth. These cells, which we designated COS (constitutive sporulation) cells, exhibit the morphology and properties of sporulating cells and express sporulation marker genes under nutrient-rich conditions. Thus, we created an engineered strain displaying two cell cycles (growth and sporulation) integrated into one cycle irrespective of culture conditions, while in the wild type, the appropriate cell fate decision is made depending on nutrient availability. These results suggest that the threshold level of the major sporulation kinase acts as a molecular switch to determine cell fate and may rule out the possibility that the activity of KinA is regulated in response to the unknown signal(s). Upon nutrient starvation, Bacillus subtilis cells undergo a highly organized differentiation program, utilizing more than 500 of the 4,200 genes in the genome to produce spores as a last-resort survival strategy (27, 34, 41, 56). These dormant progeny, whose metabolic processes are inactive, can survive until conditions become favorable for growth. When cells encounter nutrient deprivation, a multistep phosphotransfer reaction, termed phosphorelay, involving a major sensor kinase, KinA, and two phosphotransferases, Spo0F and Spo0B, is triggered (6, 29, 53). The ultimate goal of the phosphorelay is to activate Spo0A, the master transcriptional regulator of sporulation, through phosphorylation (Spo0A P) (6, 29). The traditional point of view of the sporulation initiation mechanism is that multiple environmental signals that are produced under nutrient starvation conditions might reach a certain level (29, 56). A sufficient quantity of signal(s) might then be recognized at several different entry points in the phosphorelay and processed for Spo0A activation. It was suggested previously that one of the inputs to trigger the phosphorelay requires the activation of KinA (27, 29). Presumably, the mechanism of the activation of the sensor kinase relies on the * Corresponding author. Mailing address: Department of Biology and Biochemistry, University of Houston, Houston, TX Phone: (713) Fax: (713) mfujita@uh.edu. Supplemental material for this article may be found at Present address: Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD Present address: University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX Published ahead of print on 28 May ability of the N-terminal sensor domain of KinA to recognize an as-yet-unknown sporulation signal, which is produced only under starvation conditions, and induce a conformational change to autophosphorylate the catalytic histidine residue (H405) in the C-terminal domain (35, 54, 64). When the phosphorelay becomes active, Spo0A is additionally subjected to control at the level of its synthesis by a positive-feedback loop, and thus, Spo0A P levels increase further as a result of feedback regulation (Fig. 1A) (22, 40, 52, 59). Increased levels of Spo0A P result in the repression of transcription of the abrb gene (encoding a transcription regulator, AbrB) (24), leading to the derepression of transcription of the sigh (spo0h) gene, encoding H, an alternative subunit of RNA polymerase (RNAP) (22, 57). As a result, an elevation in the concentration of the H RNAP leads to a stimulation of the transcription of kina, spo0f, and spo0a (Fig. 1AB) (29, 57). In addition to H RNAP, Spo0A P is required for the induced transcription of spo0f and spo0a (22, 58, 59), thereby setting up a self-reinforcing closed cycle (Fig. 1A). The phosphorelay activity is also modulated by dedicated phosphatases that remove phosphoryl groups from Spo0F P (e.g., RapA) and from Spo0A P (Spo0E) (39). In addition to these regulations, a recent study reported that AbrB is inactivated by the action of an antirepressor, AbbA, which binds to AbrB (4). As a result, the antirepressor prevents the repressor from binding to operator sites but only in a subset of target genes. In fact, the sigh gene is not under AbbA control. Thus, AbbA-mediated repression and antirepression events might be required for competence, cannibalism, and biofilm formation but not for sporulation (4). Upon the activation of this regulatory circuit, the levels of the Spo0A protein and activity increase gradually during the early stages of sporulation (19), and the progressive increase in 3870

2 VOL. 192, 2010 THRESHOLD OF SPORULATION KINASE IN B. SUBTILIS 3871 FIG. 1. Schematic diagram of the sporulation network for the wildtype and artificial strains used in this study. Arrows and T-shaped bars indicate positive and negative actions, respectively. (A) Phosphorelay regulatory network in the wild-type strain. Upon nutrient starvation, a major sensor kinase (KinA) undergoes autophosphorylation and provides phosphate input to the master transcriptional regulator Spo0A (0A) via two additional regulators, Spo0F (0F) and Spo0B (0B). Phosphorylated Spo0A (0A P) becomes a positive or negative regulator for sporulation genes, including those for Spo0A itself, Spo0F, and the transition state transcription regulator AbrB. AbrB represses the transcription of a gene for H, which is also essential for sporulation. Thus, phosphorylated Spo0A represses abrb, thereby stimulating H expression. As a result, the transcription of genes for KinA, Spo0F, and Spo0A are activated in the closed-loop system. (B) Transcriptional regulation of kina in the wild-type (wt) strain. The wild-type strain, where the native promoter of kina is transcribed by H, is able to sporulate in response to nutrient starvation. (C) The ASI (artificial sporulation induction) strain, where the native promoter of kina was replaced by the hyper-spank promoter (P hy-spank ), produces various levels of kina mrna in proportion to the concentration of IPTG, and thus, varied KinA protein levels are depicted with the shades of gray and dark arrows. Therefore, the ASI strain is able to sporulate irrespective of nutrient availability in the presence of IPTG (14, 19). (D) Transcriptional regulation of kina expression in the COS (constitutive sporulation) strain (P citg -kina). A strain where the native promoter of kina was replaced by the citg promoter produces a higher level of kina mrna and, thus, the KinA protein. Therefore, the engineered strain is able to sporulate under nutrient-rich conditions in a constitutive manner. the level of activated Spo0A (Spo0A P) explains the temporal expression pattern of the low- and high-threshold Spo0A-regulated genes (18). A set of 121 genes is known to be under the direct control of Spo0A P (37). Among them, genes involved in auxiliary and nonsporulating processes, such as cannibalism (25), are turned on at an earlier phase with a low dose of Spo0A P, while genes involved directly in sporulation are activated at a later phase with a high dose of Spo0A P (18). Recently, it was reported that a culture population of genetically identical B. subtilis cells under identical sporulation conditions shows phenotypic variations, which may arise from cell-to-cell variability of Spo0A activity: cells in a certain subpopulation possessing a sufficient level of Spo0A activity sporulate, while others with an insufficient level of Spo0A activity do not (8, 12, 14a, 61). This type of phenotypic bifurcation, known as bistability, heterogeneity, or heterochronicity, may help a bacterium to survive under unexpected environmental circumstances (62). Thus, growth (nonsporulating state) and sporulation are mutually exclusive and controlled by the interplay of signaling pathways that execute the intrinsic genetic program (27, 29, 52). In the scheme of the closed circuit (Fig. 1A), it is expected that in an abrb mutant, sporulation can be induced with an increase in H activity followed by an increase in Spo0A activity in a constitutive manner. However, the most puzzling point in this circuit is that a strain harboring the abrb gene knockout shows no significant enhancement of the timing and temporal pattern of sporulation gene expression to cause a massive initiation of sporulation during growth (47, 57). Therefore, the key unanswered question regarding the feedback regulation of the phosphorelay is what is the first component to be activated upon starvation to trigger phosphorelay? Currently, GTP has been known as the only metabolite whose intracellular level is monitored by a GTP-sensing repressor, CodY (44, 50). When cells are growing under nutrient-rich conditions, cellular GTP levels are elevated, and genes under the control of CodY are repressed. Conversely, when cells are limited for nutrients, GTP levels are low, resulting in the derepression of CodYregulated genes. In fact, spo0a is among the targets of CodY (38). However, CodY cannot be the primary factor to initiate sporulation because a mutant lacking the GTP sensor does not trigger massive sporulation during growth (44). Thus, in the KinA pathway demonstrated in this study, there is no direct evidence to support the traditional view of multiple entry points sensing various signals to trigger the phosphorelay. Most importantly, sporulation signals that would directly activate KinA have never been identified (13, 14). One of the difficulties in elucidating the problem is that there are many possible pathways that affect the activity of the sporulation initiation network in wild-type cells (52). To overcome these limitations, we take a simplified approach in which the network is decoupled into subsystems. Thus, we commenced our analysis with an artificially constructed strain in which the synthesis of KinA is under the control of an IPTG (isopropyl- -D-thiogalactopyranoside)-inducible hyper-spank promoter (P hy-spank ) (14, 19). Using this strain, we recently demonstrated that cells in the presence of an inducer sporulate efficiently under both nutrient-rich and starvation conditions in a manner entirely dependent on IPTG (13, 14). To gain a better understanding of the initial event that triggers phosphorelay activation, in this report, we first show that inducing the synthesis of KinA beyond a certain level results in the threshold activity of Spo0A, thereby allowing sporulation to proceed efficiently. Second, in contrast, sporulation is not induced efficiently in the strain expressing H under the IPTG-inducible promoter during the exponential phase of growth. Moreover, neither phosphorelay nor sporulation can be triggered by inducing the synthesis of Spo0F, Spo0B, or Spo0A. Third, we construct a strain expressing KinA under a strong H -dependent promoter, which is not involved in sporulation, and demonstrate that sporulation is induced efficiently and constitutively in the engineered strain during the exponential phase of growth under nutrient-rich conditions and also under starvation conditions. These results suggest that the major sporulation kinase acts as a switch to determine cell fate.

3 3872 ESWARAMOORTHY ET AL. J. BACTERIOL. MATERIALS AND METHODS Strain construction. The parental strain for all experiments was B. subtilis strain PY79 (68). Details of strains used in this study are presented in Table S1 in the supplemental material. Details of the constructions are available upon request. Plasmid construction. All recombinant DNA and plasmid constructions were performed with Escherichia coli DH5 according to standard procedures. Details of plasmid construction and oligonucleotide DNA primers used for PCR are described in Tables S2 and S3, respectively, in the supplemental material. Media. For sporulation induction conditions, B. subtilis cells were grown in hydrolyzed casein (CH) growth medium (55) at 37 C and induced to sporulate by the resuspension method described previously by Sterlini and Mandelstam (SM medium) (55). L-Threonine supplement (40 g/ml) was added to the culture for strains harboring reporter genes at the thrc locus in SM medium. LB (Luria- Bertani) medium was used for DNA manipulations in E. coli and also for B. subtilis as the rich medium when indicated. Sporulation and -galactosidase assays. Assays for sporulation and -galactosidase activity were performed as described previously (20). Immunoblot analysis. Immunoblot analysis was performed with polyclonal anti-green fluorescent protein (GFP) (45), anti-spo0a (17), and anti- A (17) antibodies. Alkaline phosphatase-conjugated anti-igg antibodies were used as the secondary antibodies. The antigen-antibody complex was detected by 5-bromo-4-chloro-3-indolyl phosphate (BCIP) nitro blue tetrazolium chloride (NBT) color development substrate (Promega). The intensities of each band were quantified with an image analyzer (FluorChem; Alpha Innotech). A,a constitutively expressed protein, was used as a loading control. The protein levels were normalized to both the levels of A and then the levels of each of the corresponding proteins in the wild-type strain. Fluorescence microscopy. Fluorescence microscopy was performed as described previously (14). Cells expressing fluorescent proteins were captured by a microscope. To randomize the sampling, all cells within a field were analyzed. Intensities of fluorescent proteins (arbitrary units/pixel) were analyzed electronically by use of Slidebook software (Intelligent Imaging Innovations, Inc.). RESULTS A subtle change in the KinA level exerts a potent effect to trigger efficient sporulation. While the signal(s) that triggers the initiation of sporulation remains unknown, our previous study has demonstrated that the artificial induction of the synthesis of KinA leads to a gradual increase in the levels and activity of Spo0A and the subsequent triggering of sporulation with wild-type efficiency in cells, even under conditions of nutrient excess. To extend the data from prior studies (14, 18, 19), we quantified the KinA and Spo0A levels and sporulation efficiency in response to various concentrations of IPTG (isopropyl- -d-thiogalactopyranoside) in the artificial induction (artificial sporulation initiation [ASI]) strain (Fig. 1C). We note that the effects exerted by KinA induction showed a phosphorelay dependency (see Fig. S1 in the supplemental material) (19). The engineered cells were suspended in sporulation (SM) medium in the presence of various concentrations of IPTG and then examined for protein expression and sporulation efficiency. Quantitative immunoblot analysis showed that KinA levels were related linearly to the concentration of IPTG (Fig. 2A and B). Thus, the level of KinA increased approximately in proportion to the concentration of IPTG over the range of 0 to 10 M, followed by a saturation of the levels from 10 to 20 M. Similarly, the levels of Spo0A increased gradually according to the KinA protein level as a result of the feedback regulation of the phosphorelay (Fig. 1A and 2A and C) (22, 29). In contrast, the efficiency of sporulation, measured by the production of heat-resistant CFU, steeply increased over a narrow range between 4 and 8 M (Fig. 2D). Importantly, when the KinA protein level in the ASI became a level FIG. 2. Necessary and sufficient level of KinA to trigger sporulation. The top panels show immunoblots of extracts from cells of a strain (MF3352) in which kina-gfp was under the control of the IPTG-inducible promoter P hy-spank. The synthesis of KinA was induced in the strain by the addition of the indicated concentrations of IPTG ( M) just after the cells were suspended in SM medium (A) for normal sporulation conditions and at mid-log phase (OD 600 of 0.5) in LB medium for nutrient-rich conditions (E). Extracts were prepared from cells collected at 2 h after the addition of IPTG, at which time all the phosphorelay proteins reached steady-state levels. Protein samples were subjected to SDS-PAGE followed by immunoblot analysis with anti-gfp, anti-spo0a, and anti- A antibodies. The anti- A immunoblot served as a control for loading (17). Shown are representative blot images from one of three independent experiments. The bottom shows the corresponding levels of KinA (B and F) and Spo0A (C and G) at the indicated concentrations of IPTG ( M). The results shown in the bar graph represent the averages of data from three independent experiments with standard deviations. Cells of the wild-type strain harboring the kina-gfp gene construct (MF3593) were suspended in SM medium to induce sporulation. Protein extracts from the sporulating wild-type strain were prepared from cells collected 2 h after suspension in SM medium and served as a control (wt). The intensities of each band were quantified with an image analyzer (FluorChem; Alpha Innotech) as described in Materials and Methods. The number of spores per milliliter of culture was measured 16 h after suspension in SM medium for the wild-type strain or after treatment with inducer for the strains harboring IPTG-inducible constructs cultured in SM or LB medium (D and H). similar to that observed for sporulating wild-type cells at an IPTG concentration of approximately 8 M, sporulation with wild-type efficiency was obtained. Although in vivo, the correlation between the activity (level of phosphorylation) and the protein level cannot be measured directly due to technical limitations, it was demonstrated by indirect measurements that these two parameters are well correlated: levels of KinA and Spo0A are proportional to their activities (14, 18). In vitro, it was demonstrated previously that the protein level and the phosphorylation level are proportional (26). To examine the possibility that not only levels but also the activity of KinA are regulated by some unknown starvationinduced signal, we repeated the same experiment under nutrient-rich culture conditions. As shown in Fig. 2E to H, even

4 VOL. 192, 2010 THRESHOLD OF SPORULATION KINASE IN B. SUBTILIS 3873 under nutrient-rich conditions, inducing the synthesis of KinA produced essentially the same results as those obtained under nutrient-limited culture conditions. Furthermore, we found that the KinA level in the wild-type strain is lower under nutrient-rich conditions than under starvation conditions (see Fig. 6 and 7) (14). Again, the induction of KinA to the level corresponding to that of the wild type under conditions of starvation was necessary and sufficient to reach wild-type sporulation efficiency. These results supported the notion that the increase in the level of KinA beyond a certain level is a crucial event to trigger phosphorelay activation and sporulation. Once the KinA level reaches the critical level, the developmental process of sporulation becomes irreversible. We note that protein factors (such as Sda, KipI, and YheH) were reported previously to inhibit KinA activity during growth in rich medium (7, 23, 65). All those protein factors show inhibitory effects on sporulation only when artificially overexpressed (7, 23, 65). Furthermore, the disruption of these genes had little or no effect on the sporulation efficiency (7, 23, 65). Alternatively, the effects of these negative factors on sporulation initiation might be cumulative, in which phenotypic effects become evident only when a combination of these mutant alleles is examined. Thus, we suggest that these modulators might inhibit KinA activity to fine-tune to some extent in rich medium but possibly not act as primary regulators of KinA activity. Induction of the synthesis of H induces the expression of H -dependent genes but fails to trigger sporulation in nutrient-rich medium. During sporulation, H RNAP stimulates the transcription of the genes for Spo0A, Spo0F, and KinA from a H -dependent promoter (Fig. 1A and B). Transcription from H -dependent promoters in these phosphorelay component genes is essential for efficient sporulation, and thus, H contributes to gene expression during sporulation. Our results indicate that inducing the synthesis of KinA during growth under nutrient-rich conditions confers the phosphorylation of Spo0A through the phosphorelay and then induces sporulation efficiently (Fig. 2) (14, 19). It is known that the gene encoding KinA is under the direct control of the H RNAP (Fig. 1B) (42). Accordingly, we wondered whether inducing the synthesis of H would induce sporulation during growth, as was observed in the case of KinA (Fig. 2) (14, 19). First, we confirmed that inducing the synthesis of the H protein is sufficient to induce H activity in rich medium (LB) as reported previously (5). For this demonstration, we used a fusion of the gene for H to the IPTG-inducible, hyper-spank promoter (5). It was reported previously that citg, the structural gene for fumarase, is transcribed from two promoters, one of which (citg P2) is transcribed by H (15, 60). Thus, we fused the citg P2 promoter to the lacz structural gene (P citg - lacz) and introduced it into a strain that carries the IPTGinducible H to examine -galactosidase activity as a transcriptional reporter of the H -dependent promoter. For simplicity, here, the citg P2 promoter is referred to as the citg promoter or P citg. Using this strain, we determined the optimum IPTG concentrations to induce active H. As shown in Fig. 3A, the level of transcription from the P citg promoter reached the saturation point at approximately 50 M. The maximum level was similar to that for the wild-type strain under normal sporulation conditions in SM medium. In the absence of an inducer, significantly low activity was detected. Thus, we determined the FIG. 3. Induction of the synthesis of the H protein induces transcription from the H -dependent promoter but not sporulation. (A) - Galactosidase activities were measured for strains harboring the P hy-spank -sigh construct and lacz fused to the citg (MF2256) and spo0a Ps (MF2265) promoters. Cells of the P hy-spank -sigh construct were treated with the indicated concentrations of IPTG at the midexponential phase of growth (OD 600 of 0.5) in LB medium. Samples were collected at hour 2 of induction and assayed for -galactosidase activity. The wild-type strain (that is, without P hy-spank -sigh) harboring the same reporter gene was induced to sporulation in SM medium, and samples were assayed at hour 2 of induction as controls. (B) Cells of the wild-type (wt) strain (PY79) (a) and the strains harboring the P hy-spank -kina (MF1887) (b and c), P hy-spank -sigh (EG232) (d), and P hy-spank -spo0a (MF2008) (e) constructs were treated with the vital membrane stain FM4-64 at hour 3 after suspension in SM medium for the wild-type strain or after treatment with the inducer for the strains harboring IPTG-inducible constructs cultured in LB medium. Cells were observed by fluorescence microscopy. IPTG concentrations used were 10 M for P hy-spank -kina and 50 M for P hy-spank -sigh and P hyspank-spo0a as determined from previous studies (18, 19) and this study. Bar, 2 m. optimum IPTG concentration to induce H -dependent citg expression to the wild-type level under normal sporulation conditions. Second, accordingly, we expected that inducing the synthesis of the H protein could induce the transcription of kina and, thus, KinA activity and thereby Spo0A activity through phosphorelay and then finally trigger sporulation. As shown in Fig. 3A, however, transcription from the spo0a Ps promoter, which is known to be dependent on both H and Spo0A P, was not significantly detectable in the presence of various amounts of the inducer. Furthermore, we observed that only a small fraction of the cells proceeded to sporulation under these conditions (Fig. 3Bd), while inducing the synthesis of KinA triggered sporulation efficiently (Fig. 3Bc), comparable to that for the wild type under normal sporulation conditions (Fig. 3Ba and Table 1). As a negative control, the induction of the synthesis of only Spo0A (without inducing the synthesis of other components) showed little or no effect on sporulation (Fig. 3Be and Table 1), indicating that the phosphorylation of Spo0A with the upstream components of the phosphorelay is essential

5 3874 ESWARAMOORTHY ET AL. J. BACTERIOL. TABLE 1. Induction of the synthesis of Spo0F, Spo0B, Spo0A, or H is not sufficient to trigger sporulation KinA Spo0F Spo0B Spo0A H CFU/ml CFU/ml CFU/ml CFU/ml CFU/ml Efficiency Efficiency Efficiency Efficiency Efficiency IPTG concn ( M) Spores Viable cells Spores Viable cells Spores Viable cells Spores Viable cells Spores Viable cells FIG. 4. Time course of H and Spo0A activities in strains harboring P hy-spank -sigh and P hy-spank -kina. -Galactosidase activities were measured in the strains harboring P hy-spank -sigh or P hy-spank -kina with lacz reporters to the following promoters: spo0f in P hy-spank -sigh (MF2264) (A) and P hy-spank -kina (MF2113) (B), spo0a Ps in P hy-spank -sigh (MF2265) (C) and P hy-spank -kina (MF2117) (D), kina in P hy-spank -sigh (MF2273) (E) and P hy-spank -kina (MF2111) (F), and citg in P hy-spank - sigh (MF2256) (G) and P hy-spank -kina (MF2257) (H). Cells of the IPTG-inducible constructs were treated with and without IPTG at the mid-exponential phase of growth (OD 600 of 0.5) in LB medium. Symbols for each IPTG expression system are indicated at the top. Samples were collected at the indicated times after the addition of IPTG and assayed for -galactosidase activity. IPTG concentrations used were 10 M for P hy-spank -kina and 50 M for P hy-spank -sigh. All experiments were performed at least three times independently. Representative data are shown. to trigger sporulation, as demonstrated previously (19). Little or no sporulation was observed for the strain harboring the IPTG-inducible kina without an inducer (Fig. 3Bb). To investigate details of these results further, we examined transcriptional activities from promoters for kina ( H regulated), spo0a Ps ( H and Spo0A P regulated), and spo0f ( H and Spo0A P regulated) in a time course experiment after the addition of IPTG under rich-medium (LB) conditions. As shown in Fig. 4A and C, transcription from both the spo0a Ps and spo0f promoters was detected partially but not fully even at later time points from inducing the synthesis of H. We note that a higher concentration of IPTG was not effective to induce transcription from these promoters, while transcription from the citg promoter was induced efficiently even at lower concentrations of inducer (Fig. 3A and data not shown for the spo0f promoter). We further note that the activation of transcription from the spo0a Ps promoter is essential to induce sporulation (49). We then examined whether the transcription level from the kina promoter was increased after the synthesis

6 VOL. 192, 2010 THRESHOLD OF SPORULATION KINASE IN B. SUBTILIS 3875 of H was induced. The results indicate that transcription from the kina and citg promoters was activated to a different fold by inducing the synthesis of H, while no increase in the reporter activity was detected in the absence of an inducer (Fig. 4E), as observed in the case of the citg promoter (Fig. 4G). These results indicate that inducing the synthesis of H during growth is sufficient to induce the transcription of H -dependent genes, including kina and citg, but it is not sufficient to induce the expression of the phosphorelay components, including Spo0F and Spo0A, nor is it sufficient to trigger sporulation. Finally, we determined the level of KinA under the induction of H synthesis in the P hyspank -sigh strain by immunoblot analysis. As shown in Fig. 7, we found that the level of KinA under H induction was slightly lower ( 60 to 70% of that in the sporulating wild-type strain) (see Fig. 7, lane 3) than that for the wild-type strain under sporulation conditions (see Fig. 7, lane 1) and comparable to that for the wild-type strain growing under nutrient-rich conditions (see Fig. 7, lane 2). In contrast, however, when the synthesis of KinA was induced, the level of transcription from the spo0f and spo0a Ps promoters was significantly increased (Fig. 4B and D), and sporulation was also induced efficiently (Fig. 3Bc), as described previously (19). Interestingly, the kinetic profile of transcription from the promoter of kina or citg was indistinguishable from that both with and without the induction of the synthesis of KinA (Fig. 4F and H). Taken together, these results suggest that transcription from the intrinsic kina promoter is not induced sufficiently, even with an excess amount of active H during growth, to increase the level of the KinA protein to trigger Spo0A activation via phosphorelay. Therefore, we conclude that an increase in the KinA protein level above a threshold is essential and sufficient to induce phosphorelay and thereby Spo0A phosphorylation and, finally, sporulation, as suggested previously (19), although the regulatory mechanism is not known. After submission of the manuscript, we learned that de Jong et al. (10) independently obtained results indicating that the Spo0A P level is not increased when H expression is artificially induced, which is consistent with our results presented here. Induction of the synthesis of Spo0F, Spo0B, or Spo0A is not sufficient to trigger a massive induction of sporulation under nutrient-rich conditions. We further examined the effect of inducing the synthesis of Spo0F or Spo0B under the control of the IPTG-inducible P hy-spank promoter on triggering sporulation under nutrient-rich conditions. Inducing the synthesis of Spo0A was not sufficient to induce sporulation efficiently, as reported previously (19), and was used as a control in this study (Fig. 3Be). Similarly, sporulation was not induced efficiently by inducing the synthesis of Spo0F and Spo0B with various concentrations of IPTG under rich-medium (LB) conditions (Table 1). We confirmed that the sporulation-defective phenotype caused by the endogenous spo0f and spo0b knockouts was complemented by the expression of the proteins from the IPTG-inducible constructs under sporulation conditions (SM) (data not shown). Thus, we conclude that the artificial induction of the functional Spo0F and Spo0B proteins is not sufficient to trigger a massive initiation of sporulation under nutrient-rich conditions, while a gradual increase in the level of all four components, including Spo0F and Spo0B, was observed FIG. 5. Transcription from promoters for sporulation genes in the COS strain. -Galactosidase activities were measured for the strains harboring lacz fused to the kina (MF2107) (A), citg (MF312) (B), spoiig (MF750 and MF3136) (C and E), and spoiiq (MF199 and MF2997) (D and F) promoters. The wild-type strain (PY79) harboring each reporter gene was used as a control. Cells were cultured in SM medium for normal sporulation conditions and in LB medium for nutrient-rich conditions. Samples were collected at the indicated times and assayed for -galactosidase activity. Time zero is defined as the time of suspension in SM medium or the time of the mid-exponential phase of growth (OD 600 of 0.5) in LB. Symbols are indicated at the top. Note that the scales in the y axis for C and E and D and F are different. All experiments were performed at least three times independently. Representative data are shown. during an early stage of sporulation in the wild type under normal sporulation conditions (see Fig. 6) (14a). Increase in KinA synthesis under the control of a strong H -dependent promoter induces sporulation constitutively. It was reported previously that the induction of kina gene expression requires the sigh gene but not the spo0a, spo0b, and spo0f genes at the onset of sporulation (2). It is also evident that the levels of transcriptional activities from the kina promoter are relatively low ( 10 Miller units as -galactosidase activities for the reporter assay) (1, 2, 22). Also, our data confirmed those previously reported results (Fig. 4E and F and 5A) and indicated that inducing the synthesis of H was not sufficient to trigger sporulation (Fig. 3). Thus, we wondered whether kina gene regulation could contribute to cell fate decisions. To test this idea, we constructed a strain that expresses kina under the control of a strong H -dependent promoter for citg (15, 60), which is independent of and unrelated to phosphorelay feedback regulation. We then examined the sporulation process in this strain under both nutrient-rich (LB) and normal sporulation (SM) conditions. We already confirmed that the promoter for citg is dependent on H in vivo (Fig. 3 and 4G). We then examined the intrinsic promoter activities of citg and kina in the wild-type background during growth under nutrient-rich (LB) and normal sporulation (SM) conditions. These

7 3876 ESWARAMOORTHY ET AL. J. BACTERIOL. TABLE 2. Sporulation is initiated constitutively in the COS strain irrespective of nutrient availability Medium Strain Viable cells CFU/ml Spores Efficiency LB Wild type COS SM Wild type COS results indicated that the promoter activity of citg, measured by a lacz transcriptional fusion assay, was approximately 10 times higher than that of kina under rich-medium conditions (Fig. 5A and B). Interestingly, under sporulation conditions in SM medium, the promoter activity of citg increased further to approximately 2-fold of that under rich-medium (LB) conditions, while the promoter activity of kina showed similar levels under both conditions (Fig. 5AB). Finally, we constructed a strain that has a kina gene under the control of the citg promoter (P citg -kina) (Fig. 1D) and examined various parameters of the sporulation process as described below. First, we examined the number of spores (spores per milliliter of culture) in the wild-type strain and the engineered strain (P citg -kina) under rich (LB) and sporulation (SM) conditions. As shown in Table 2, the number of spores in the engineered strain was essentially of a similar degree ( 10 8 spores) under both conditions, comparable to that of the wild type under sporulation conditions. The numbers of viable cells and spores dropped slightly but showed a similar degree ( 10 8 spores) in the engineered strain under sporulation conditions compared to those for the wild type. Under rich conditions in the wild-type strain, the number of spores was significantly lower ( 10 4 spores), while the number of viable cells was slightly increased ( 10 9 spores). The COS strain produced fewer viable cells than the wild-type strain in LB medium (approximately 50%), perhaps because a large fraction of the COS population chooses sporulation instead of continued growth. Nevertheless, these results indicated that sporulation is initiated constitutively in this engineered strain (P citg -kina) irrespective of culture conditions. Thus, here, we call this engineered strain COS (constitutive sporulation). Next, reporter gene expression was examined to monitor Spo0A activity and polar septum formation. For Spo0A activity, the Spo0A-dependent spoiig promoter was fused to the lacz gene. To monitor polar septation, the forespore-specific F -dependent spoiiq promoter was fused to the lacz gene. These reporter constructs were introduced into the strains, and -galactosidase activities were examined. Under sporulation conditions, for the wild-type as a control experiment, transcription from spoiig and spoiiq was induced well, while it was not detected under nutrient-rich conditions (Fig. 5C and D). In contrast, for the COS strain, these reporter activities were significantly higher than those for the wild type under both nutrient-rich and sporulation conditions (Fig. 5E and F). We note that the increased activity from P spoiig in the COS strain declined from hour 2 under sporulation conditions, perhaps due to the fact that transcriptional repression might occur at later times, when E, one of the products of the spoiig operon, is produced enough, while it was further increased under nutrient-rich conditions (Fig. 5E). Under these conditions, however, the reporter activity from P spoiiq increased earlier under sporulation conditions than under nutrient-rich conditions (Fig. 5F). Nevertheless, these two sporulation genes were expressed efficiently in the COS strain irrespective of culture conditions. To confirm expression levels for the key regulator proteins KinA and Spo0A, cell extracts were prepared from wild-type and COS strains and analyzed by immunoblotting. Under growing conditions for the wild-type cells in nutrient-rich medium, levels of KinA and Spo0A are known to be less than those under sporulation conditions (14, 19). These results were confirmed, as shown in Fig. 6 and 7. In contrast, in the COS strain under the same conditions, levels of KinA and Spo0A were significantly higher at an early stage of growth (optical density at 600 nm [OD 600 ] of 0.5) than those in the wild-type strain. These protein levels in the COS strain were approximately two times higher than those in the sporulating wild-type strain under both nutrient-rich and sporulating conditions (Fig. 6), resulting in efficient spore formation in the COS strain, as shown in Table 2. These results indicated that a strain expressing KinA under the strong H promoter induces sufficient levels of the Spo0A protein and activity to permit sporulation and displays a constitutive-sporulation phenotype even under nutrient-rich conditions. Finally, to visualize the sporulation process in individual cells, we constructed wild-type and COS strains that express yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), and red fluorescent protein (mcherry) from three different promoters for each strain. To monitor growth states, the A -dependent abrb promoter was fused to the gene for YFP. To monitor Spo0A activity, the Spo0A-dependent spoiia promoter was fused to the gene for CFP. To monitor forespore formation, the F -dependent spoiiq promoter was fused to the gene for mcherry. All three reporter genes were introduced into each strain as a single-copy transgene. As can be seen for the wild-type strain in rich medium, YFP from the abrb promoter was expressed predominantly in a culture population during the exponential to early stationary phases (1 to 2 h) (Fig. 8A to C). In contrast, expression of CFP from spoiia promoter was not detectable, and thus, no polar septum formation was detected as mcherry expression from the spoiiq promoter (Fig. 8A to C). Under sporulation conditions in SM medium, however, both CFP and mcherry signals were detected in the mother cell and forespore, respectively, in a compartment-specific manner after hour 2. On the other hand, the expression of YFP from the abrb promoter was significantly decreased over the time of sporulation (Fig. 8M to O), indicating that sporulation was induced efficiently in the wild type. In the COS strain, the compartment-specific expression of CFP and mcherry was detected in the cells not only under sporulation conditions (Fig. 8S to U) but also under nutrientrich conditions (Fig. 8G to I). Efficiencies of sporulation in these two strains were examined by counting mcherry signals from the P spoiiq promoter at hour 3 (Fig. 8C, I, O, and U). Results were essentially consistent with those for the viable and heat-resistant spore assays shown in Table 2. These results confirmed that the gene expression program of sporulation is induced successfully and that the sporulation process is com-

8 VOL. 192, 2010 THRESHOLD OF SPORULATION KINASE IN B. SUBTILIS 3877 FIG. 6. Immunoblot analysis to measure levels of the KinA and Spo0A proteins during the initiation of sporulation. (A) Cell extracts were prepared from the wild-type strain (MF929) or the COS strain (MF2969) harboring a gene for GFP-tagged KinA at the indicated times after suspension in SM medium or after the mid-exponential phase of growth (OD 600 of 0.5) in LB medium. Protein samples were subjected to SDS-PAGE followed by immunoblot analysis with anti-gfp antibodies. Also, the accumulation of Spo0A in each strain was analyzed by immunoblotting with anti-spo0a antibodies. (B) Kinetics of protein synthesis from the immunoblot analysis shown in A, normalized to A levels and then to the maximum level of each protein. The intensities of each band were quantified with an image analyzer (FluorChem; Alpha Innotech) as described in Materials and Methods. Quantification of the relative level of each protein is shown in Fig. S2 in the supplemental material. All experiments were performed at least three times independently. Representative data are shown. pleted efficiently in the COS strain even under nutrient-rich conditions. Finally, we visualized H activity using the P citg -gfp reporter in the wild type and COS strains at the single-cell level under both nutrient-rich and sporulation conditions. In the wild-type strain under nutrient-rich conditions, fluorescence from P citg - gfp was detected heterogeneously throughout the cell population, but the intensities were relatively low (Fig. 9A to C). However, under sporulation conditions in the wild-type strain, the intensity and distribution of fluorescent signals became higher and broader, respectively (Fig. 9G to I). In the COS strain under nutrient-rich conditions, levels of intensity and distribution similar to those in the sporulating wild-type strain were observed (compare Fig. 9D to F to G to I). Under sporulation conditions in the COS strain, the intensities of signals increased further, consistent with the results of the lacz reporter assay (Fig. 5B), and the fluorescent signals were detected in a more heterogeneous pattern (Fig. 9J to L). Forespore formation was also detected as CFP signals from the P spoiiq -cfp reporter in the same cell (Fig. 9). These results support the notion (22, 58) that the phosphorelay amplifies not only the Spo0A activity but also H activity under feedback regulation in both the wild-type and COS strains. FIG. 7. Comparison of levels of KinA in cells of the sporulating wildtype strain, cells of the wild-type strain grown under nutrient-rich conditions, and cells of the strain harboring P hy-spank -sigh grown under nutrientrich conditions in the presence of IPTG. Sporulating wild-type cells (strain MF929) were harvested at hour 2 after suspension in SM medium (lane 1). Wild-type cells grown in rich medium were harvested at hour 2 after the mid-exponential phase of growth (OD 600 of 0.5) (lane 2). Cells of the strain harboring P hy-spank -sigh (MF3976) were harvested at hour 2 after the addition of IPTG (50 M final concentration) at the mid-exponential phase of growth (OD 600 of 0.5) (lane 3). Protein samples were subjected to SDS-PAGE followed by immunoblot analysis with anti-gfp antibodies to detect the GFP-tagged KinA protein. Relative levels were normalized to A levels and then to the maximum level of the protein in sporulating wild-type cells (lane 1). Average values standard deviations from three experiments are shown. *, P 0.01 compared to the level of the sporulating wild type. DISCUSSION In this study, we show that by using an IPTG-inducible KinA strain, the phosphorelay is activated and leads to sporulation, irrespective of nutrient availability, when the KinA protein level increases beyond a certain level (Fig. 2 to 4). Furthermore, we demonstrate that the engineered strain expressing KinA under a H -dependent promoter that is similar to, but stronger than, the native kina promoter induces sporulation in a constitutive manner (Fig. 5 to 9). Based on these results, we suggest that the threshold level of the kinase acts as a molecular switch to determine the initiation of sporulation. Our

9 3878 ESWARAMOORTHY ET AL. J. BACTERIOL. FIG. 8. Fluorescence microscopy analysis of the sporulation process. Cells of the wild-type strain (MF3756) or the COS strain (MF3757) harboring P abrb -yfp, P spoiia -cfp, and P spoiiq -mcherry were collected at the indicated times of incubation as defined in the legend of Fig. 4. Two different culture conditions (LB and SM media) were examined. Fluorescence microscopy was carried out as indicated in Materials and Methods. The fluorescence images were falsely colored yellow for YFP, cyan for CFP, and magenta for mcherry. The corresponding phase-contrast images are shown in black and white. The pixel intensities are shown as an intensity plot on the right for each color image. Also shown are percentages of cells with mcherry expressed in the forespore, indicating the sporulation efficiencies for the strains. Bar, 2 m. FIG. 9. Comparison of H activities between sporulating and nonsporulating cells in a culture population. Cells of the wild-type strain (MF3022) or the COS strain (MF3011) harboring P citg -gfp and P spoiiq - cfp were collected at hour 3 of incubation as defined in the legend of Fig. 4. Fluorescence microscopy was carried out as indicated in Materials and Methods. The fluorescence images were falsely colored green for GFP and yellow for CFP. The corresponding phase-contrast images are shown in black and white. The pixel intensities are shown as an intensity plot on the right for each color image. Shown are the histograms indicating the relative numbers of cells with GFP expressed under the indicated culture conditions. The y axes indicate the relative cell numbers in each plot (total cell counts are defined as 1). GFP intensities in the microscope images were measured by Slidebook software (Intelligent Imaging Innovations, Inc.) and are shown as arbitrary units on the x axes. Over 200 cells were counted randomly. The shaded area in the graphs corresponds to a population of nonsporulation cells detected in A ( 95%). Bar, 2 m. results may rule out the possibility that the autophosphorylation activity of KinA is regulated in response to the unknown signal(s), which was originally believed to be essential for signal sensing under starvation conditions. Regulation of expression of phosphorelay components upon starvation. Very little is known about the regulation of kina gene expression except that the promoter for kina has a H RNAP recognition sequence. Genome-wide analysis of the H regulon brings the total regulon to 49 promoters controlling the expression of at least 87 genes. Among them, promoters for kina and citg are absolutely dependent on H without any additional transcription factors and show increased transcription with the overexpression of H during growth (5). In contrast, transcription from the spo0a Ps and spo0f promoters is not activated by inducing the synthesis of H during growth but is successfully upregulated by the induction of KinA (Fig. 3 and 4) (5). The phosphorylated form of Spo0A is absolutely required, together with H, for the activation of these two phosphorelay component promoters and, thus, for the initiation of sporulation (22). Asai et al. reported previously that the expression of kina is induced in the mutants of the phosphorelay components although at lower levels than those for the wild-type strain (2). Those results (2) suggest that (i) the induction of kina transcription is dependent on H but is not mediated exclusively by any component of the phosphorelay pathway, (ii) levels of H accumulated in the cell without any functional phosphorelay components are sufficient for the transcription of kina, and (iii) an additional unknown factor(s) that regulates the levels of H in response to nutritional conditions might exist. It was reported previously that H activity is controlled at the posttranscriptional level and responds to a variety of external con-

10 VOL. 192, 2010 THRESHOLD OF SPORULATION KINASE IN B. SUBTILIS 3879 ditions, including ph and nutrient availability (9, 11, 16, 28, 36). However, the precise mechanisms of the regulation of H activity are not completely understood. In support of those previous reports, our data presented here indicate that during growth under nutrient-rich conditions, the overproduction of the H protein shows no further increased enhancement of transcription from the kina promoter and no induction of sporulation (Fig. 3Bd), but instead, levels of transcription activity similar to that for the sporulating wild-type strain are detected (up to approximately 10 Miller units in a lacz reporter assay) (Fig. 4E and F). Most importantly, by using a quantitative immunoblot assay, it was demonstrated that the KinA protein level increased slightly (about 30%) when cells were grown under starvation conditions compared to nutrient-rich or H -overproducing conditions (Fig. 6 and 7). A massive entry into sporulation is reproduced when a subtle increase in the level of the KinA protein is artificially induced by using an IPTG-inducible KinA strain under both nutrient-rich and starvation conditions (Fig. 2). These results suggest that H is required but not sufficient for the increased level of KinA protein synthesis to trigger sporulation efficiently under nutrient-rich conditions. Taken together from all those points, we summarize that in the wild type, (i) there is no difference in the mrna levels of kina between sporulating cells under starvation conditions and nonsporulating cells under nutrient-rich conditions, (ii) the KinA protein level increases slightly when cells are grown under starvation conditions compared to nutrient-rich conditions, and (iii) an increase in the KinA protein level to a certain level (e.g., threshold) is essential and sufficient to trigger phosphorelay and sporulation. Thus, the threshold level of KinA is sufficient to induce the production of phosphorylated Spo0A beyond a critical level to direct the expression of sporulation genes (18). In support of these results, our recent studies indicate that the role of the N-terminal sensor domain of KinA is to stabilize tetramer formation of the kinase but possibly not for signal sensing to activate the C-terminal catalytic domain of the enzyme (13, 14). Furthermore, in in vitro experiments, the purified form of KinA, which is heterologously overexpressed in E. coli, is active (6, 21, 26). These biochemical results indicate that the KinA protein by itself is constitutively active without the involvement of any signal(s). Thus, based on all these facts, it appears that the threshold level of KinA acts as the master molecular switch for the initiation of sporulation. Currently, the control mechanism(s) of the KinA protein level during sporulation is unclear but may be involved in posttranscriptional level control(s), such as regulated proteolytic degradation or modification of the protein. Establishment of a strain that exhibits a constitutive-sporulation (COS) phenotype. Sporulation in the wild-type B. subtilis strain is normally induced under nutrient starvation conditions through complex phosphorelay signal transduction pathways. In this report, a strain exhibiting a constitutive-sporulation phenotype, even in the presence of excess nutrients, is established (Fig. 5, 6, and 8 to 10). When the kina gene is placed under the control of the strong H -dependent citg promoter, which is unrelated to the phosphorelay, sporulation is initiated constitutively, even under rich-medium conditions. The key difference between native (kina) and ectopic (citg) promoters is the strength of transcription activity. Based on the lacz reporter assay, the activity from P citg is 10 times higher than that from P kina (Fig. 4 and 5). In the COS strain, protein levels of KinA and Spo0A are significantly higher at an early time of culture, irrespective of nutrient availability, than those in the wild-type strain under nutrient-rich conditions (Fig. 6). Thus, in the COS strain, even during growth under nutrient-rich conditions, KinA levels continue to increase beyond the threshold level, Spo0A synthesis thereby increases via the feedback regulation of phosphorelay genes, and, finally, Spo0A activity (phosphorylation) increases to trigger sporulation gene expression. These results, derived from experiments carried out with a COS strain, suggest that the intrinsic promoter strength, i.e., the ability to recruit the H RNAP, and not receiving a starvation signal(s) by the sensor domain of KinA as previously believed, is an important factor to determine the cell fate. There is a long history that should remind us that excess nutrients, including carbon and nitrogen, inhibit sporulation (46). Several mutants capable of high sporulation efficiency in the presence of excess glucose have been isolated as cataboliteresistant sporulation mutants. These mutations have been identified in the siga operon (known as crs, for catabolite resistance for sporulation) (32), in spo0a (known as rvt and sof mutations) (30, 31), in tnra (48), in pts (16), and in hpr (scoc) (3). However, the molecular mechanisms controlling the catabolite repression of sporulation with these mutations are largely unknown. Currently, CodY, in a GTP-bound form, is the only regulator that prevents growing cells from initiating sporulation when cellular GTP levels are especially high in the presence of excess glucose (51). However, the GTP-binding protein cannot be a primary nutrient sensor, because a massive induction of sporulation could not be observed for a null mutant of CodY under nutrient-rich conditions (44). Cell cycle and population dynamics. Recently, we demonstrated a single-cell measurement to determine concentrations of each phosphorelay component per cell using a combination of fluorescence microscopy and computational analysis in an isogenic cell population (14a). In that study, phosphorelay components, the KinA, Spo0F, and Spo0B proteins, show a heterogeneous expression pattern in a cell population, and a gradual increase in the protein levels was observed during the initiation process of sporulation. Furthermore, in support of our data presented here, a recent study using a computational modeling simulation reported by Chastanet et al. indicates that a significant increase in the level of all four phosphorelay proteins at early times of sporulation is required for entry into sporulation (8). Thus, we speculate that the successful entry into the sporulation state of the sporulating wild-type strain might be manifested by a threshold of each phosphorelay component. As a result, only a fraction of the cells acquires a sufficient intracellular quorum of the phosphorelay components and proceeds successfully to the completion of the sporulation process. We note that inducing the synthesis of only Spo0F, Spo0B, or Spo0A during growth is not sufficient to trigger a massive entry into sporulation (Table 1). These results suggest that a threshold level of Spo0F, Spo0B, or Spo0A is required but not sufficient for entry into sporulation. In support of and to further extend these notions, we find that the heterogeneity of sporulation in a cell population is still maintained in the COS strain (Fig. 7 and 8). We first expected that