Novel Modulators Controlling Entry into Sporulation in Bacillus subtilis

Novel Modulators Controlling Entry into Sporulation in Bacillus subtilis Sharon Garti-Levi,a Ashlee Eswara,b* Yoav Smith,c Masaya Fujita,b Sigal Ben-Yehudaa Department of Microbiology and Molecular Genetics, Institute for Medical Research Israel-Canada (IMRIC), The Hebrew University-Hadassah Medical School, The Hebrew University of Jerusalem, Jerusalem, Israela; Department of Biology and Biochemistry, University of Houston, Houston, Texas, USAb; Genomic Data Analysis Unit, The Hebrew University-Hadassah Medical School, The Hebrew University of Jerusalem, Jerusalem, Israelc

Upon nutrient deprivation, Bacillus subtilis initiates the developmental process of sporulation by integrating environmental and extracellular signals. These signals are channeled into a phosphorelay ultimately activating the key transcriptional regulator of sporulation, Spo0A. Subsequently, phosphorylated Spo0A regulates the expression of genes required for sporulation to initiate. Here we identified a group of genes whose transcription levels are controlled by Spo0A during exponential growth. Among them, three upregulated genes, termed sivA, sivB (bslA), and sivC, encode factors found to inhibit Spo0A activation. We furthermore show that the Siv factors operate by reducing the activity of histidine kinases located at the top of the sporulation phosphorelay, thereby decreasing Spo0A phosphorylation. Thus, we demonstrate the existence of modulators, positively controlled by Spo0A, which inhibit inappropriate entry into the costly process of sporulation, when conditions are favorable for exponential growth.

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hen nutrients are limited, certain bacteria can execute the developmental process of sporulation, whereby an extremely durable cell called a spore is formed, as exemplified by the bacterium Bacillus subtilis. Spore formation is an energy-consuming process involving the expression of hundreds of genes leading to distinct morphological changes (1, 2). At the onset of sporulation, the cell undergoes asymmetric division, giving rise to a small forespore compartment that becomes a mature spore and a larger mother cell, which nurtures the developing spore. Upon the completion of sporulation, the resilient spore is discharged by lysis of the mother cell, and the liberated spore can survive for long periods of time (1, 3, 4). Sporulation is initiated by the activation of the master transcriptional regulator Spo0A. Spo0A is activated by phosphorylation mediated via a relay comprising three main histidine kinases, KinA, KinB, and KinC, through which a phosphoryl group is passed to Spo0F, then to Spo0B, and finally to Spo0A (5–7). Several phosphatases (RapA, RapB, RapE, RapH, and Spo0E) as well as kinase inhibitors (Sda and KipI) were shown previously to act at various steps in the relay to antagonize Spo0A phosphorylation (8–10). The kinase inhibitor Sda is a small protein coupling DNA replication with the onset of sporulation. When DNA replication is perturbed, Sda is produced and interacts directly with KinA to inhibit its autophosphorylation (9, 11). KinA autophosphorylation was also found to be blocked by overproduction of the regulating factor KipI (10, 12). The phosphorylated form of Spo0A (Spo0A⬃P) directly controls the transcription of more than 120 genes by binding their promoters (13). Subsequently, these target genes lead to global changes in gene expression, modifying the transcription profile of over 500 genes and thus triggering sporulation (13–15). Many of the Spo0A-controlled genes are differentially responsive to low and high doses of Spo0A⬃P. Higher levels of Spo0A⬃P activate genes essential for sporulation, whereas lower levels of Spo0A⬃P are sufficient to maintain a pool of factors, regulating alternative developmental fates such as competence and biofilm formation (16, 17).

April 2013 Volume 195 Number 7

Although Spo0A has been implicated in controlling developmental transitions, it is also expressed during exponential growth from a weak promoter, controlled by the housekeeping sigma factor ␴A (see Fig. S1 in the supplemental material) (18–21). Here we identify a group of genes regulated by Spo0A during growth, termed sivA, sivB, and sivC (siv for sporulation-inhibitory vegetative gene), encoding factors that actively inhibit sporulation when nutrients are available. MATERIALS AND METHODS Strains and plasmids. B. subtilis strains are listed in Table S2 in the supplemental material. Plasmid constructions are described in the supplemental material. Primers are listed in Table S3 in the supplemental material. General methods. Growth and sporulation were carried out at 37°C as described previously (22). For microscopy, cells were grown in hydrolyzed casein (CH) medium and induced to sporulate at an optical density at 600 nm (OD600) of 0.5 by suspension in Sterlini and Mandelstam medium (sporulation salts). Spore formation was assayed by inducing sporulation in Schaeffer’s liquid medium (Difco sporulation medium [DSM]) for 24 h and determining colony formation of heat-treated spores (80°C for 20 min). Cells harboring kinA, kinB, or kinC under the control of an isopropyl-␤-D-thiogalactopyranoside (IPTG)-inducible promoter (17) were grown in CH medium and induced to sporulate at an OD600 of 0.2 by the addition of 0.2 mM IPTG. Strains overexpressing sivA or sivB were induced by the addition of 0.2 mM or 0.5 mM IPTG, respectively.

Received 30 November 2012 Accepted 16 January 2013 Published ahead of print 18 January 2013 Address correspondence to Sigal Ben-Yehuda, [email protected]. * Present address: Ashlee Eswara, U.S. Food and Drug Administration, Center for Biologics Evaluation and Research, Rockville, Maryland, USA. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /JB.02160-12. Copyright © 2013, American Society for Microbiology. All Rights Reserved. doi:10.1128/JB.02160-12

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Fluorescence microscopy. Fluorescence microscopy was carried out as described previously (23). Briefly, 500 ␮l of cells was centrifuged and resuspended in 10 ␮l of 1⫻ phosphate-buffered saline (PBS) supplemented with the membrane stain FM1-43 or FM4-64 (1 ␮g/ml) (Molecular Probes, Invitrogen). For observing green fluorescent protein (GFP) fusions, a chamber slide containing 1% agarose was used (24). System control and image processing were performed by using MetaMorph 7.4 software (Molecular Devices). RNA extraction. Cells grown in CH medium to an OD600 of 0.5 were collected, and mRNA was extracted by using an RNeasy kit (Qiagen) according to the manufacturer’s protocol. RNA concentration was determined by using a NanoDrop 2000C instrument (Thermo Scientific). Sample integrity was evaluated by capillary gel electrophoresis using the Agilent 2100 Bioanalyzer. Microarray analysis. cDNA synthesis, fragmentation, and end-terminal biotin labeling were performed according to the standard Affymetrix prokaryotic GeneChip expression protocol, with the following modifications: the initial amount of RNA was 20 ␮g, cDNA synthesis was performed with SuperScript III (Molecular Probes, Invitrogen), and cDNA reaction mixtures were incubated overnight at 42°C. Finally, labeled cDNA samples were hybridized onto Affymetrix GeneChip B. subtilis arrays. Hybridized arrays were stained and washed by using the Affymetrix 450 fluidic station. After staining, arrays were scanned with a GC3000 scanner. Statistics. Robust multiarray average (RMA) normalization was performed on CELL files by using Partek Genomic Suite 6.5. Additional statistical analysis was carried out by using the Spotfire software package (Somerville, MA) and custom Matlab (R2010B) listed routines. The histogram plot of the log2 ratio resembled a normal shape distribution. Only genes showing significant statistical changes in two independent biological experiments are listed in Tables 1 and 2. Considered downregulated genes exhibited a log2 ratio of less than 0.5, and upregulated genes exceeded a log2 ratio of 2.5. All genes had a z score value lower or higher than ⫾2. Quantitative real-time PCR. Total RNA (4 ␮g) was treated with RQ1 DNase (Promega) and subjected to cDNA synthesis using SuperScript III reverse transcriptase (Molecular Probes, Invitrogen), according to the manufacturer’s protocol. Standard quantitative realtime PCRs (qRT-PCRs) were conducted by using SYBR green mix (LightCycler 480 SYBR Green I Master; Roche), and fluorescence detection was performed by using a Rotor-Gene 3000A instrument (Corbett Life Science). qRT-PCR primers (see Table S3 in the supplemental material) were designed by using Primer3 (v.0.4.0) software (available online). The hbsU gene was used to normalize expression data, as its expression level is constant during vegetative growth. Each assay was performed in duplicates with mRNA templates prepared from two independent biological repeats. Immunoblot analysis. A cell culture (10 ml) was grown in rich medium (CH medium or LB), harvested at an OD600 of 0.7, and centrifuged for 20 min at 4,000 rpm at 4°C. Samples were resuspended in 2 ml 1⫻ PBS with 5 ␮g/ml DNase I (Sigma), 0.2 mg/ml 4-(2-aminoethyl) benzenesulfonylfluoride hydrochloride (AEBSF) (Calbiochem), and 0.02% SDS. Proteins were extracted by using a FastPrep-24 system (MP Biomedicals) (3 cycles of 6.5 m/s for 45 s each time, with 2 min of cooling on ice between cycles). Next, samples were centrifuged for 10 min at 4,000 rpm at 4°C, and the supernatant was collected and supplemented with 4⫻ cold acetone and incubated for 60 min at ⫺20°C for protein precipitation. Samples were then centrifuged for 30 min at 12,000 rpm, the supernatant was discarded, and the acetone was allowed to evaporate for 10 min at 25°C. For protein extraction from the supernatant fraction, the following procedure was carried out: a cell culture (300 ml) was grown in rich medium (CH medium or LB), harvested at an OD600 of 0.7, and centrifuged for 30 min at 4,000 rpm at 4°C. The supernatant was filtered (0.22 ␮m; Corning), and the proteins were precipitated with 30 ml cold trichloroacetic acid (TCA) overnight at 4°C. Next, samples were centrifuged for 30 min at 10,000 rpm at 4°C, and the fluid was discarded. The protein pellet was washed twice with 100 ␮l cold acetone, and samples were centrifuged for

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TABLE 1 Genes negatively controlled by Spo0A during vegetative growtha Gene

Description

Ratio

pstA pstBA pstC pstS ptsBB pyrB pyrC pyrP tcyP cysK ycbA senS fadN gerD katB manA manP yclF yczF ydzE yhdN yobH yqcE yrkG yvsH yxeL yxeM yxeN yxeP yxeQ

Phosphate ABC transporter Phosphate ABC transporter Phosphate ABC transporter Phosphate ABC transporter Phosphate ABC transporter Pyrimidine biosynthesis Pyrimidine biosynthesis Uracil permease Cysteine uptake Cysteine synthetase A Sensor kinase Transcriptional regulator Acetoacetyl coenzyme A Involved in germination Peroxidehydrogen detoxification Mannose utilization Mannose uptake Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown

⫺4.71 ⫺4.01 ⫺4.30 ⫺6.27 ⫺2.63 ⫺2.86 ⫺2.46 ⫺2.73 ⫺3.44 ⫺2.31 ⫺2.61 ⫺2.26 ⫺2.01 ⫺2.27 ⫺2.02 ⫺2.19 ⫺2.46 ⫺2.15 ⫺2.01 ⫺2.12 ⫺2.16 ⫺2.08 ⫺2.02 ⫺2.06 ⫺2.08 ⫺2.23 ⫺2.24 ⫺2.44 ⫺2.22 ⫺2.39

a Microarray analysis was performed by using cDNA derived from transcripts of wildtype (PY79) and ⌬spo0A (RL2242) cells (OD600 of 0.5) grown in rich medium (CH medium). Listed are Spo0A-downregulated genes. Ratio indicates the log2 wild-type/ ⌬spo0A ratio. The entire genomic data set is presented in Table S1 in the supplemental material.

an additional 10 min at 10,000 rpm at 4°C. The remaining acetone was allowed to evaporate for 10 min at 25°C. Mass spectrometry confirmed the extraction of bona fide extracellular proteins (e.g., Hag and TasA) (25) by utilizing this procedure. Protein fractions were separated by using SDS-PAGE (12% or 15% polyacrylamide gel), electroblotted onto a polyvinylidene difluoride (PVDF) transfer membrane (Immobilon-P; Millipore), and blocked with 5% skim milk powder (Difco, Becton Dickson and Company) in 1⫻ TBST (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.1% Tween 20). Protein detection was performed by using polyclonal rabbit anti-SivA antibodies (1:2,500 dilution) (Hylabs, Israel) incubated overnight at 4°C. Peroxidase-conjugated goat anti-rabbit secondary antibodies (1:10,000 dilution) (Jackson ImmunoResearch) were incubated for 60 min at 25°C. A SuperSignal EZ-ECL kit (Thermo Scientific) was used for final detection, according to the manufacturer’s instructions. Protein expression and purification. His-tagged proteins were expressed in Escherichia coli BL21(DE3). Proteins were soluble, and thus, purification steps were carried out at 4°C, as described previously (24, 26, 27). In vitro phosphorylation. Phosphorylation reactions were performed as described previously (27).

RESULTS

Genes controlled by Spo0A during growth. To investigate the putative function of Spo0A during exponential phase, we com-


Novel Modulators Controlling Entry into Sporulation

TABLE 2 Genes positively controlled by Spo0A during vegetative growtha Gene

Description

Ratio

yweA yuaB ylqB wapA wprA dhbA dhbF yknW yueB pspA des bdhA ykwC rapA srfAA srfAB srfAC srfAD phrF phrC comS tasA floT ydjG ydjH yhjA yjcM yqgA yrpD yuaF yueC yukE yvlB yxaL yybN

sivA sivB (bslA), biofilm formation sivC Cell wall-associated protein precursor Cell wall-associated protein precursor Biosynthesis of siderophore bacillibactin Biosynthesis of siderophore bacillibactin SdpC toxin resistance (permease) Receptor for phage SPPI Phage shock protein Membrane phospholipid desaturase Fermentation Putative beta-hydroxyacid dehydrogenase Phosphatase controlling sporulation Surfactin synthetase Surfactin synthetase Surfactin synthetase Surfactin synthetase Competence phosphatase regulator Competence phosphatase regulator Competence signal transduction Biofilm production Similar to flotillin Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown

2.55 2.79 1.7 4.14 3.26 2.99 3.43 2.5 3.09 3.98 2.99 4.31 2.73 2.86 2.55 3.68 2.68 2.69 2.75 2.75 4.13 3.5 2.92 3.1 3.61 4.14 4.88 3.97 3.45 3.48 2.63 9.14 3.3 4.34 4.54

Since sivA is missing a typical Spo0A binding site, we further substantiated that it is indeed controlled by Spo0A during growth. To this end, we performed quantitative real-time PCR (qRT-PCR) analysis, demonstrating that sivA expression decreases dramatically (⬃22-fold) in the absence of Spo0A (Fig. 1A). In line with these results, a strain harboring a sivA-gfp fusion exhibited a diffusible cytoplasmic fluorescence signal during exponential phase that was hardly detectable in the absence of Spo0A (Fig. 1B and C).

a Microarray analysis was performed by using cDNA derived from transcripts of wildtype (PY79) and ⌬spo0A (RL2242) cells (OD600 of 0.5) grown in rich medium (CH medium). Listed are Spo0A-upregulated genes. Ratio indicates the log2 wild-type/⌬spo0A ratio. The siv genes investigated in this study are shown in boldface type. The entire genomic data set is presented in Table S1 in the supplemental material.

pared the transcriptional profiles of wild-type and spo0A mutant cells grown in rich medium. Our analysis revealed a group of approximately 60 genes that are directly or indirectly controlled by Spo0A (Tables 1 and 2; see also Table S1 in the supplemental material), some of which were previously identified as members of the Spo0A regulon (13, 14, 16). The downregulated genes harbor a prominent group involved in phosphate uptake (pst genes), genes involved in various metabolic pathways (e.g., tcyP and cysK), and several genes with unknown function (Table 1). The positively regulated genes include those participating in various differentiating pathways, such as surfactin (srfAA-srfAD) and exopolysaccharide (tasA) syntheses required for biofilm formation as well as genes involved in competence acquisition (phrC, phrF, and comS) (Table 2). In addition, among the upregulated transcripts, a significant group of genes with unknown function was revealed, harboring several genes that were previously shown to be activated by low levels of Spo0A (16). Within this group, yweA (here sivA), encoding a relatively small protein of 154 amino acids, was chosen for further characterization.


FIG 1 sivA is regulated by Spo0A during vegetative growth. (A) qRT-PCR analysis of sivA expression was performed by using cDNA derived from transcripts of wild-type (wt) (PY79) and ⌬spo0A (RL2422) cells (OD600 of 0.5) grown in rich medium (CH medium). Arbitrary units (AU) represent relative mean expression levels of sivA. The standard deviation was calculated from three repeats of two independent biological experiments. (B) Wild-type (SG81), ⌬spo0A (SG131), ⌬abrB (SG233), and ⌬spo0A ⌬abrB (SG245) cells producing SivA-GFP grown in rich medium (CH medium) were collected (OD600 of 0.5) and monitored by fluorescence microscopy. All images were normalized to the same intensity range. The scale bar corresponds to 1 ␮m. (C) The SivA-GFP signal derived from the strains described above for panel B was measured and quantified. Fluorescence values following background subtraction are shown in arbitrary units and represent the average fluorescence signal of at least 300 cells of each strain.

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Additionally, the signal was increased in cells lacking the global transcriptional repressor AbrB, indicating that its expression level is largely repressed by this regulator (Fig. 1B and C). The abrB gene is negatively controlled by Spo0A; however, when expressed, the protein inhibits spo0A transcription in a feedback loop and thus prevents expression of transition-stage-specific genes during growth (26, 28). Importantly, monitoring the expression of sivAgfp in a ⌬spo0A ⌬abrB double mutant strain revealed the expression level to be lower than that observed for the wild-type strain (Fig. 1B and C). Taken together, the activation of sivA by vegetatively produced Spo0A is more prominent than its repression by AbrB. SivA inhibits entry into sporulation through the KinA pathway. Since Spo0A is essential for entry into sporulation and for activation of sivA during growth, we assessed whether SivA influences sporulation. Initially, we compared the sporulation efficiencies of cells lacking sivA and wild-type cells. No apparent difference in efficiency of polar septum formation was displayed by the mutant cells; however, the level of production of mature spores was slightly increased (Fig. 2A; see also Fig. S2 in the supplemental material). Furthermore, overexpression of sivA resulted in a decrease in the formation of polar septa and mature spores (Fig. 2B and C), implying that SivA plays a role in inhibiting entry into sporulation. To further dissect how SivA exerts its effect on sporulation, we tested its influence on the histidine kinases KinA, KinB, and KinC, which activate Spo0A and are present during growth (29, 30). We utilized a previously developed system whereby cells growing under nutrient-rich conditions are induced to sporulate by overexpressing each of the kinases separately (17). Sporulation driven by KinA was enhanced in the absence of SivA, as indicated by polar septum formation (Fig. 3A). However, lack of SivA had no measurable effect when sporulation was triggered through KinB or KinC (Fig. 3B). Thus, we conclude that SivA inhibits entry into sporulation specifically through the KinA pathway. BslA (SivB), a SivA homolog, inhibits entry into sporulation. A search of the B. subtilis genome database revealed the existence of a small protein, YuaB/BslA (here called SivB for simplicity), harboring significant homology to SivA (43% identity). sivB was shown previously to be responsive to low levels of Spo0A and encodes an extracellular protein implicated in biofilm formation (16, 31–33). Similarly to sivA, microarray and qRT-PCR analyses indicated that sivB is positively regulated by Spo0A during growth (Table 2 and Fig. 4A). Fusion of the sivB promoter to GFP revealed the signal to be dependent on Spo0A and to increase in the absence of the repressor AbrB (Fig. 4B). Investigation of the effect of sivB on sporulation uncovered that, as for sivA, deletion of sivB had no apparent influence on the induction of polar septa, yet the production of spores was enhanced (Fig. 2A; see also Fig. S2 in the supplemental material). Consistently, its overexpression resulted in decreased sporulation efficiency (Fig. 2B and C). Furthermore, when sivB-deleted cells were triggered to sporulate in rich medium by activating the sporulation kinases, increased sporulation levels were specifically displayed upon KinA induction (Fig. 3A and C). This increase was even more pronounced than that detected in the absence of sivA (Fig. 3A). However, deletion of both sivA and sivB had no added effect on sporulation (Fig. 3A), suggesting that the encoded proteins impinge on the same target and that the ⌬sivB mutant appears to be epistatic to the ⌬sivA mutant. We surmise that sivA and sivB are controlled by Spo0A during growth, and

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FIG 2 sivA and sivB affect sporulation. (A) Wild-type (wt) (PY79), ⌬sivA (SG77), and ⌬sivB (SG113) cells were induced to sporulate in DSM medium, and spore formation was detected before and after heat treatment by CFU (see Materials and Methods). The histogram shows percent sporulation that was calculated by percent CFU after heat kill/total counts. The standard deviation was calculated from the means of four experiments from two independent biological repeats. (B) Wild-type (PY79) cells and cells overexpressing (o/ex) sivA (SG168) or sivB (SG310) were induced to sporulate in sporulation salt medium supplemented with IPTG, and sporulation efficiency was measured (2 h). (Top) Representative images of sporulating cells stained with FM1-43. (Bottom) Percentage of sporulation measured by the appearance of polar septa. At least 400 cells from each strain were counted. The standard deviation was calculated from the means of several fields of cells obtained from three independent biological repeats. The scale bar corresponds to 1 ␮m. (C) Wildtype (PY79) cells and cells overexpressing sivA (SG168) or sivB (SG310) were induced to sporulate in DSM medium supplemented with IPTG, and spore formation was detected as percent heat-resistant CFU/total counts (see Materials and Methods). The standard deviation was calculated from the means of three independent biological repeats.

their corresponding proteins operate in similar fashions to inhibit sporulation specifically through the KinA pathway. SivA and SivB directly inhibit the sporulation phosphorelay through KinA. Having established that SivA and SivB inhibit entry into sporulation through the KinA pathway, we explored whether they interfere with KinA activity. To this end, purified KinA was incubated with increasing amounts (0, 0.2, 2, and 5 ␮M) of either purified SivA or SivB and radiolabeled ATP ([␥32 P]ATP). To improve solubility, purified SivA and SivB were lacking their N-terminal signal sequence (see the supplemental



FIG 4 sivB is regulated by Spo0A during vegetative growth. (A) qRT-PCR analysis

FIG 3 Siv factors inhibit entry into sporulation during growth through the phosphorelay. (A) Wild-type (wt) (SG45), ⌬sivA (SG152), ⌬sivB (SG170), and ⌬sivA ⌬sivB (SG236) cells grown in rich medium (CH medium) were induced to sporulate by overexpressing (o/ex) kinA. Cells were collected 1.5 h after induction, stained with FM1-43, and examined by fluorescence microscopy. Sporulation efficiency was scored by the appearance of polar septa. At least 300 cells from each strain were counted. The standard deviation was calculated from the means of several fields of cells obtained from three independent biological repeats. (B) Wild-type (SG47 and SG49) and ⌬sivA (SG153 and SG154) cells grown in rich medium (CH medium) were induced to sporulate by overexpressing kinB or kinC, as indicated. Cells were processed as described above for panel A. (C) Wild-type (SG47 and SG49) and ⌬sivB (SG180 and SG181) cells grown in rich medium (CH medium) were induced to sporulate by overexpressing kinB or kinC, as indicated. Cells were processed as described above for panel A. (D) Wild-type (SG47 and SG49) and ⌬sivC (SG153 and SG154) cells grown in rich medium (CH medium) were induced to sporulate by overexpressing kinA, kinB, or kinC, as indicated. Cells were processed as described above for panel A.

material). An inverse correlation between the levels of the phosphorylated KinA and those of SivA or SivB was detected (Fig. 5A). Consistent with our in vivo observations (Fig. 3A), SivB exhibited a stronger inhibitory effect than SivA (Fig. 5A). As a control, the phosphorylation levels of the distantly related histidine kinase YycG (34, 35) were hardly affected when incubated with the highest concentration of purified SivA or SivB (Fig. 5B). Thus, SivA and SivB interfere directly with the kinase activity of KinA. Next, we examined the influence of SivA and SivB on the phos-


of sivB expression was performed by using cDNA synthesized from transcripts extracted from wild-type (wt) (PY79) and ⌬spo0A (RL2422) cells (OD600 of 0.5) grown in rich medium (CH medium). Arbitrary units (AU) represent relative mean expression levels of sivB (see Materials and Methods). The standard deviation was calculated from three repeats of two independent biological experiments. (B) Wild-type (SG258), ⌬spo0A (SG268), and ⌬abrB (SG266) cells harboring PsivB-gfp grown in rich medium (CH medium) were collected (OD600 of 0.5) and monitored by fluorescence microscopy. All images were normalized to the same intensity range. The scale bar corresponds to 1 ␮m.

phorylation levels of the downstream relay components. Indeed, phosphorylation levels of Spo0F and Spo0B were reduced when either SivA or SivB was supplemented (Fig. 5C). Consequently, the final acceptor of the relay, Spo0A, displayed a comparable decreasing trend of phosphorylation (Fig. 5C). This decrease could be even more pronounced in vivo, whereby KinA levels are induced by Spo0A⬃P via phosphorelay feedback regulation (26, 36, 37), while in vitro, the levels of KinA, as well as of other phosphorelay components, remained constant. We conclude that SivA and SivB directly inhibit KinA activity. This reduced activity decreases the phosphorylation levels of the intermediate components, inhibiting Spo0A activation and delaying entry into sporulation during growth. Of note, SivA and SivB were identified as extracellular proteins (25, 33, 38), while KinA is a cytoplasmic kinase (39, 40). Therefore, the interaction between the kinase and its inhibitors is likely to occur in the cytoplasm. Consistent with this idea, at least some of the SivA and SivB protein molecules are found to be located in the intracellular fraction (see Fig. S3 in the supplemental material) (31), thus enabling such interactions. It is possible that SivA and SivB are dual-function proteins displaying different activities according to their localization. SivC inhibits sporulation through the KinB and KinC pathways. Investigation of the array of Spo0A-upregulated genes dur-

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FIG 5 KinA autophosphorylation is inhibited directly by SivA and SivB. (A) Purified KinA (0.2 ␮M) was incubated (30°C for 20 min) with increasing amounts (0, 0.2, 2, and 5 ␮M) of purified SivA (amino acids 27 to 154) or purified SivB (amino acids 26 to 181) in the presence of radiolabeled ATP ([␥-32P]ATP). (Top) Autoradiogram of phosphorylated KinA in the absence (⫺) and presence of SivA or SivB. (Bottom) The phosphorylation levels of KinA are indicated in arbitrary units (AU) as values relative to those for the control (⫺). (B) As a control, a 1 ␮M concentration of the distantly related histidine kinase YycG (amino acids 218 to 611) was incubated with 5 ␮M purified SivA (A) or SivB (B). (Left) Autoradiogram of phosphorylated YycG in the absence (⫺) and presence of SivA or SivB. (Right) The phosphorylation levels of YycG are indicated in arbitrary units as values relative to those for the control (⫺). (C) Purified phosphorelay components were incubated (30°C for 20 min) with radiolabeled ATP ([␥-32P]ATP) in the absence (no addition) or presence of 5 ␮M purified SivA or SivB. (Top) Autoradiograms of phosphorylation reaction mixtures including KinA (0.2 ␮M) (lane 1); KinA and Spo0F (0.05 ␮M) (lane 2); KinA, Spo0F, and Spo0B (0.5 ␮M) (lane 3); and KinA, Spo0F, Spo0B, and Spo0A (10 ␮M) (lane 4) (of note, when all the components of the relay are present, only the final acceptor Spo0A⬃P is detectable). (Bottom) The phosphorylation levels of each component (KinA, Spo0F, Spo0B, or Spo0A) are indicated in arbitrary units as values relative to those for the control (⫺). The standard deviation was calculated from three repeats of at least two independent experiments.

ing growth uncovered an additional factor, YlqB, here termed SivC (Table 2). sivC was also shown to be a low-Spo0A-threshold gene (16). Consistently, we found that the vegetative expression of SivC-GFP was dependent on Spo0A (see Fig. S4A in the supplemental material). Deletion of sivC resulted in a greater sporulation efficiency than that of the wild-type strain (see Fig. S4B in the

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supplemental material), implying that the encoded protein acts to inhibit entry into sporulation. We then monitored sporulation efficiency induced in rich medium by KinA, KinB, or KinC in the absence and presence of SivC. Unlike SivA and SivB, which specifically influence sporulation through KinA, SivC inhibits sporulation mediated by KinB or KinC (Fig. 3D). These results reinforce



FIG 6 Model for inhibition of sporulation during growth. During vegetative growth, sporulation is inhibited by a low threshold level of Spo0A, which upregulates the expression of factors such as SivA, SivB, and SivC that in turn inhibit its own activation. SivA and SivB inhibit the KinA pathway, while SivC inhibits the KinB and KinC pathways.

the view that during growth, sporulation is blocked by an array of proteins controlled by Spo0A that target the most upstream components of the phosphorelay. DISCUSSION

Sporulation is generally considered a process that is actively induced upon nutrient limitation. Here we show that sporulation is actively inhibited during growth when nutrients are available. This inhibition is controlled by the key sporulation regulator Spo0A when produced vegetatively. We identified an array of genes that are directly or indirectly regulated by Spo0A during growth. At least three Spo0A-upregulated genes, sivA, sivB, and sivC, encode factors that operate to inhibit Spo0A activation, impeding sporulation in the presence of nutrients. In vivo assays as well as in vitro biochemical examinations revealed that these factors inhibit the action of the key kinases located at the top of the sporulation phosphorelay. SivA and SivB inhibit the activity of KinA, whereas SivC affects sporulation through both KinB and KinC pathways (Fig. 6). It was previously shown that the level of the sporulation kinases is important to initiate sporulation and that during growth, it is below the threshold required for entering the process (16, 40, 41). Consistently, deletion of all three siv genes did not lead to massive entry into sporulation during vegetative growth (data not shown). This observation suggests that the Siv factors could act as a finetuning device determining the proper activity of the kinases. Up until now, only two modulators, KipI and Sda, were shown to interact directly with KinA, inhibiting its autokinase activity (9, 10). However, the Siv factors described here share no obvious homology with these modulators, suggesting that they may operate in a different manner. Taken together, we propose that the primary role of the Siv factors, together with additional known and unknown kinase inhibitors, is to reinforce inhibition of kinase activity under nutrient-rich conditions, ensuring entry into the complex process of sporulation only under conditions of starvation. Sporulation is therefore induced by as-yet-unidentified factors that overcome this inhibitory effect and activate the phosphorelay. Interestingly, the levels of the Siv proteins increase at the


onset of sporulation (see Fig. S5 in the supplemental material), suggesting that they might be involved in regulating the activity of Spo0A at a later stage of the process. Indeed, it has been shown that production of an activated form of Spo0A (Spo0A-Sad67) impaired sporulation, signifying the importance of downregulating Spo0A for a successful completion of the process (17). All three Siv proteins harbor a predicted N-terminal signal peptide motif and were previously identified to be extracellular (25, 33, 38), implying that part of their role may be executed in their secreted form. The membrane-associated KinB and KinC (29, 42–44) are affected by SivC, suggesting that this factor may sense an extracellular cue and consequently interact with its cognate kinases to inhibit sporulation. SivA and SivB were found previously to interact with KinA, which is cytoplasmic (39, 40). Therefore, we hypothesize that these proteins exhibit a dual activity: when residing within the cell, the factors directly inhibit KinA autophosphorylation, while in their extracellular form, they monitor the environment and accordingly transduce signals into the phosphorelay to coordinate entry into sporulation, by an as-yetunknown mechanism. B. subtilis can differentiate into a variety of cell types that can undergo competence, biofilm formation, and sporulation by modifying the cellular levels of Spo0A (16, 17). Furthermore, the vegetative expression of spo0A was shown previously to vary dramatically, exhibiting bursts in transcription (45–47). We observed a significant variation in the levels of the Siv factors among vegetatively growing cells (see Fig. S6 in the supplemental material) that may reflect differences in Spo0A activity within a given population. Therefore, it is possible that the heterogeneity in Siv expression contributes to modulate the total amount of active Spo0A in individual cells and thus impacts cellular fate. Notably, SivB, recently termed BslA, was shown to affect extracellular matrix production during biofilm formation and to contribute to the surface repellency of the biofilm in a B. subtilis undomesticated strain (31–33, 48). During this phase, bslA (sivB) was found to be indirectly controlled by the multicellular response regulator DegU (48). The dual function observed for BslA/SivB, as a matrix protein as well as a kinase inhibitor, could be a consequence of the temporal expression and localization of the protein, which differ between exponential growth and the biofilm phase. On the other hand, the activity of the histidine kinase KinD was found to be linked to the presence of extracellular matrix in biofilms (49), suggesting that matrix components, such as BslA/SivB, may have the capacity to modulate kinase activity. Interestingly, sivC was originally found to be a member of the ␴D regulon, which includes genes required for flagellar assembly, motility, and chemotaxis (50), raising the possibility that SivC functions to inhibit sporulation in cells destined to become motile. Altogether, it is possible that the Siv proteins, and factors alike, display different cellular roles during diverse developmental pathways and that their level and mode of activity determine cellular fate. ACKNOWLEDGMENTS We thank L. Mizrachi (Hadassah, Israel) for technical support. We are grateful to members of the Ben-Yehuda laboratory for technical help and discussions. This work was supported by a European Research Council (ERC) starting grant (grant 209130) to S.B.-Y. and by a Norman Hackerman Advanced Research Program grant (grant 003652-0072-2007), a Welch

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Foundation grant (grant E-1627), and a National Science Foundation grant (grant MCB-0920463) to M.F.

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