A combination of glycerol and manganese promotes biofilm formation in Bacillus subtilis

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1 JB Accepts, published online ahead of print on 5 April 2013 J. Bacteriol. doi: /jb Copyright 2013, American Society for Microbiology. All Rights Reserved. 1 2 A combination of glycerol and manganese promotes biofilm formation in Bacillus subtilis via the histidine kinase KinD signaling Moshe Shemesh 1,2* and Yunrong Chai 1,3, 1 Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA 2 Department of Food Quality and Safety, Institute for Postharvest Technology and Food Sciences, Agricultural Research Organization (ARO), The Volcani Center, POB 6, Beth-Dagan 50250, Israel 3 Department of Biology, Northeastern University, Boston, Massachusetts 02115, USA addresses: MS: moshesh@volcani.agri.gov.il YC: y.chai@neu.edu * Corresponding author These authors contributed equally to this work Running title: KinD signaling governs biofilm formation. Key words: biofilm formation, Bacillus subtilis, histidine kinase, KinD signaling. 1

2 ABSTRACT The spore-forming Bacillus subtilis forms matrix enclosed biofilms in response to environmental cues that to date remain poorly defined. Biofilm formation depends on the synthesis of an extracellular matrix, which is indirectly regulated by the transcriptional regulator Spo0A. The activity of Spo0A depends on its phosphorylation state. The level of phosphorylated Spo0A (Spo0A~P) is controlled by a network of kinases and phosphatases, which respond to environmental and physiological signals. In spite of significant progress in understanding biofilm development, the fundamental question of how cells sense the environmental cues that trigger biofilm formation has largely remained unaddressed. Here, we report that biofilm formation of B. subtilis in LB medium is triggered by a combination of glycerol and manganese (GM). Moreover, LBGM medium significantly stimulates the biofilm-associated sporulation and production of an undefined brown pigment. We further show that transcription of the major operons responsible for matrix production and biofilm formation is dramatically enhanced in response to GM. We also establish that KinD is a principal histidine kinase for sensing the presence of GM, exclusively by its extracellular CACHE domain. Finally, we show that GM has a similar biofilm-promoting effect in two related Bacillus species, B. licheniformis and B. cereus, indicating that the biofilm-promoting effect of GM is conserved in Bacillus species. INTRODUCTION The vast majority of bacteria often grow as elaborate multicellular communities, referred to as biofilms (1, 2). Biofilm formation is an ancient prokaryotic adaptation, which allows bacteria to survive in hostile environments (2, 3). The spore-forming bacterium Bacillus subtilis can form architecturally complex colonies on solid medium and pellicles at the interface of liquid medium 2

3 and air (4). In spite of significant progress in understanding the biofilm development in B. subtilis, the fundamental question concerning specific signals that trigger biofilm formation has largely remained unaddressed. Biofilm formation depends on the synthesis of an extracellular matrix that holds the constituent cells together. The matrix has two main components, an exopolysaccharide synthesized by the products of the epsa-o operon, and amyloid fibers encoded by tasa located in the tapa (formerly yqxm) operon (5-7). Both operons are under the negative control of two repressors, SinR and AbrB, which act synergistically to repress the matrix genes (7, 8). Loss of either one results in formation of an extremely robust biofilm (7, 8). Derepression is triggered in part by the action of SinI (7), whose expression is in turn activated by Spo0A (8, 9). Spo0A also directly represses the gene for AbrB, a transition state regulator, in post-exponential phase cells (10). The activity of Spo0A depends on its phosphorylation state (11). Low and intermediate levels of phosphorylated Spo0A (Spo0A~P) lead to induction of the epsa-o and tapa operons, which results in production of the extracellular matrix and thus biofilm formation (12). At high levels of Spo0A~P, the matrix genes are repressed (12). Simultaneously, sporulation genes are induced, and as a result the matrix-producing cells transform to become spores. The level of Spo0A~P is controlled by a network of histidine kinases that directly or indirectly phosphorylate Spo0A (11). Five distinct sensor kinases (KinA, KinB, KinC, KinD, and KinE) have the capability of transferring a phosphoryl group into the phosphorelay to control the level of Spo0A~P present at any moment in the cell (13-17). The current thinking in the field is that these kinases respond to various environmental and physiological cues, but the nature of these cues and how the kinases respond to them is not known in most cases. 3

4 It was recently proposed that the kinase KinD contains an extracellular domain, so called CACHE domain (18), for sensing small chemical molecules released from plant host during colonization (19). In another recent study (20), the authors suggested that the transmembrane domain of KinD is involved in osmosensing. Based on those recent studies, it seems that KinD is likely bi-functional (or multi-functional) in signal sensing. Here we show that a combination of glycerol and manganese (GM) strongly promotes biofilm formation as well as biofilm-associated sporulation and pigment production in B. subtilis grown in LB medium. We further report that the histidine kinase KinD is a major kinase for sensing the biofilm-promoting effect of GM. We also provide evidence indicating that the biofilm-promoting effect of GM is conserved amongst the Bacillus species. MATERIALS AND METHODS Strains and growth media. Strains used and generated in this study are listed in Supplemental Table 2 and are isogenic unless otherwise indicated. For routine growth all strains were propagated in Lysogeny broth (LB; 10 g of tryptone, 5 g of yeast extract and 5 g of NaCl per liter) or on solid LB medium supplemented with 1.5% agar. The B. subtilis wild strain NCIB3610 and its derivatives were regularly cultured in LB medium. Biofilms were generated at 30 C in the novel biofilm promoting medium LBGM (LB +1% (v/v) glycerol + 0.1mM MnSO 4 ), and were assayed similarly as described previously (21). When appropriate, antibiotics were added at the following concentrations for growth of B. subtilis: 10 μg/ml of tetracycline, 100 μg/ml of spectinomycin, 10 μg/ml of kanamycin, 5 μg/ml of chloramphenicol, and 1 μg/ml of erythromycin. For growth of E. coli, the concentrations of added antibiotics were as indicated previously (21). 4

5 Strain construction. All insertion deletion mutants were generated by using long flanking homology PCR (22). The generated constructs were inserted by double crossover recombination into neutral integration sites (amye and laca) in the genome of B. subtilis by inducing natural competence (4). The KinD-DegS hybrid kinase was created with overlapping PCR using the primers listed in Supplementary Table S3. In brief, the first DNA fragment covering the promoter region and the N-terminal sequence of kind was amplified by PCR using primers kind-degs-f1 and kind-degs-mr1. A second DNA fragment covering the C-terminal sequence of degs was amplified by PCR using primers kind-degs-mf1 and kind-degs-r1. These two PCR fragments were joined together after another round of overlapping PCR amplification. The final PCR products were cloned into BamHI and EcoRI restriction sites of the vector pdg1662 (23), which is a vector for amye locus integration and carries the chloramphenicol resistance marker. Expression of the chimeric kinase is therefore under the control of the kind native promoter in the recombinant plasmid. The generated vector was transformed into E. coli DH5α cells. The insert was verified by PCR as well as by DNA sequencing. The purified plasmid DNA was transformed into naturally competent cells of PY79. The constructs were then transferred to NCIB3610 by SPP1 phage transduction as described previously (6, 21). The insertion deletion mutant of ΔglpK (FC96) was constructed using long-flanking PCR mutagenesis by Frances Chu and provided to us as a gift. Construction of the insertion deletion mutant of ΔglpF (YC877) was performed similarly by applying long-flanking PCR mutagenesis (22). The four primers used for generating ΔglpF deletion mutation are delta/glpf-p1, delta/glpf-p2, delta/glpf-p3, and delta/glpf-p4, listed in Supplemental Table 3. KinC and KinD overexpression strains were originally constructed by Masaya Fujita in the PY79 strain 5

6 background [MF1889 and MF2147; (24)]. The overexpression constructs were then introduced into NCIB3610 strain background by SPP1 phage-mediated transduction, generating YC1014 and YC1015, respectively. Assays of biofilm formation. B. subtilis cells were grown in LB broth at 37 C to mid-log phase. For colony formation, 2-µl of the cells was spotted onto the LBGM medium (or other LB-based media) solidified with 1.5% agar. Plates were incubated at 30 C for 72 hours prior to analysis. For pellicle formation, 9-µl of the cells was mixed with 9-ml of LBGM broth (or other LB-based broth) in 6-well plates (VWR). Plates were incubated at 30 C for 48 hours. Images were taken using either a Nikon CoolPix 950 digital camera or using a SPOT camera (Diagnostic Instruments, USA). Assays of β-galactosidase activities. The entire colonies formed on solid medium were collected and resuspended in PBS buffer. Typical long bundled chains of cells in the biofilm colony were disrupted using mild sonication as described previously (5). Optical density of the cell samples was normalized using O.D One milliliter of cell suspensions was collected and assayed as described previously (21). Sporulation assay. Biofilm colonies were formed on LB and LBGM solid media at 30ºC as described above. The entire colonies were collected and suspended in PBS buffer. Chained and bundled cells in the biofilm colony were disrupted by mild sonication. Cells were serialdiluted. Heat kill was performed at 80ºC for 20 min in a water bath. Total cell numbers before and after heat kill were quantified by the plating method. Sporulation efficiency was calculated by dividing the number of total viable spores after heat kill by the number of total cells before heat kill. 6

7 Growth curve experiment. The wild type (3610), ΔglpK (FC96) and ΔglpF (YC877) mutant cells were first grown in LB medium at 23 C overnight. Next morning, cells were diluted 1:100 into M9 minimal medium (25) with either 0.5% glucose or 05% glycerol as the sole carbon source. Cells were grown in shaking culture condition (150 rpm) at 37 C and cell optical density of the samples was measured periodically for a total of 12 hours. Each condition has 3 replicates and the growth curve experiment was repeated twice. Representative results were shown. RNA extraction and reverse transcription real-time PCR. The details of real-time RT-PCR experiments can be found in Supplemental Materials and Methods. RESULTS A combination of glycerol and manganese promotes multicellular development by B. subtilis. The starting point of this study was the finding that a combination of glycerol and manganese (GM) strongly promotes biofilm formation by the B. subtilis strain 3610 (4) in Lysogeny broth (LB) medium (Fig. 1A, hereafter referred to as LBGM). This observation was quite surprising because LB medium is considered not optimal for biofilm formation for B. subtilis 3610 (Fig. 1A; (21)). Both components, glycerol (1% v/v) and manganese (0.1 mm), were required for the robust biofilm phenotype as the presence of glycerol alone (1% v/v, LBG) or manganese alone (0.1 mm, LBM) had little or relatively less profound stimulation on biofilm formation (Fig. 1A). Noteworthy, it was previously shown that addition of glycerol or manganese alone could stimulate biofilm formation by a B. subtilis B1 strain albeit at much higher concentrations: up to 80 gram per liter for glycerol (8% v/v) and up to 1000 mm for manganese (26). Apparently, in our study there was a synergistic activity between glycerol and 7

8 manganese in stimulating biofilm phenotype when added at much lower concentrations. We also attempted to replace glycerol in this combination with a variety of different carbon sources at similar concentrations including sugars such as glucose, galactose, mannose, and sucrose, and organic acids such as succinic acid, glutamic acid, and lactic acid. None of the above tested carbon sources, when combined with manganese, had a strong biofilm-promoting activity similar to that of glycerol (data not shown). This indicates that glycerol is unique in stimulating biofilm formation. We further wondered if simple carbohydrates such as glucose, which represses utilization of glycerol via catabolite repression (27), could impair glycerol-dependent biofilm stimulation. We therefore tested the affect of addition of glucose to the LBGM. Surprisingly, our results show that addition of glucose does not impair the biofilm formation in LBGM (Fig. S1). Biofilm formation depends on the synthesis of extracellular matrix, whose production is specified by two major operons: the epsa-o and tapa operons (5-7). The epsa-o operon is responsible for the production of the exopolysaccharide whereas the tapa operon is responsible for the production and assembly of an amyloid-like fiber (21, 28). We hypothesized that the dramatic increase in biofilm formation in the presence of GM could be due to upregulation of those genes involved in matrix synthesis. To test this hypothesis, we analyzed the effect on matrix gene expression by addition of GM using transcriptional fusions of the promoters for epsa-o and tapa to the lacz gene encoding β-galactosidase. The expression of the two transcriptional fusions was enhanced drastically in response to the addition of GM (Fig. 1B), increasing about 20- and 9-fold for tapa (left-hand panel) and epsa-o (right-hand panel), respectively. This result suggests that the strong biofilm-stimulating activity in response to the addition of GM was indeed due to upregulation of the matrix genes. To differentiate the contribution of glycerol alone and manganese alone in upregulation of the matrix genes, we 8

9 similarly measured transcription of epsa-o (by using the above transcriptional fusion) in cells from the biofilms formed on LB, LBG and LBM, respectively. Interestingly, the addition of glycerol alone to LB has a quite significant effect, whereas the addition of manganese alone has a less profound effect on epsa-o transcription (Fig. S2A). This result was rather unexpected since biofilm phenotype in LBM seems more significant than in LBG (Fig. 1A). The presence of GM also significantly stimulated biofilm-associated sporulation; there was a more than 100-fold increase in the sporulation efficiency in a biofilm colony grown on LBGM compared to that of LB (Fig. 1C). In addition, the presence of GM also promoted production of a brown pigment and secretion of the pigment around the biofilm colony (Fig. S3A). Interestingly, the secreted pigment seemed to be biofilm specific since a biofilm-defective strain of B. subtilis (ΔepsH), which bears a null mutation in one of the eps genes whose products are involved in making exopolysaccharides and is thus unable to form biofilms (7), did not show significant pigment secretion (Fig. S3B). Furthermore, the pigment production was not associated with sporulation as the sigf mutant, which is blocked in sporulation due to the mutation in the gene encoding the key sporulation sigma factor SigF (29), was still able to secret the brown pigment (Fig. S3B). It will be interesting to characterize the chemical composition and understand the function of the pigment in future studies. The biofilm-promoting effect of GM is mediated mainly by the histidine kinase KinD. We next wished to elucidate the signaling pathway that enables GM to trigger such robust biofilm formation. Our assumption was that Spo0A must be involved in this process, since it is known to have a pivotal role in regulating biofilm formation (9). The five sensory histidine kinases (from KinA to KinE) that are capable of donating the phosphoryl group to Spo0A are the 9

10 putative candidates for sensing the presence of GM (14, 16). We wondered whether any one of these sensor kinases was involved in sensing the presence of GM and promoting biofilm formation. To answer this question we screened mutants for each of the five sensor kinases for their ability to respond to the addition of GM. As shown in Fig. 2, the kind mutant (and less significantly the kinc mutant) had the most defective phenotype, while all other mutants were able to respond to the addition of GM by forming robust biofilm. Moreover, the elevated expression of matrix genes in response to GM discussed above was found to depend on KinD (Fig. S2B). These results indicate that KinD might be principally responsible for sensing the presence of GM. Interestingly, the biofilm phenotype of the kind mutant was almost as defective as that of the mutants deficient in phosphorelay, namely spo0f, spo0b and spo0a (Fig. 2), which further indicates that the presence of GM was sensed by KinD and then transduced to Spo0A via the phosphorelay. It was previously shown that a mutation in the abrb gene, whose protein product is a transition state regulator and controls both epsa-o and tapa, suppresses the biofilm defective phenotype caused by Δspo0A (30). In agreement with this data, we found that deletion in the abrb gene also suppressed the kind mutation, once again suggesting that KinD is located upstream of Spo0A and AbrB in the pathway (Fig. 2). The biofilm-forming phenotype for many of the above mutants was previously examined in another biofilm-inducing medium, MSgg (17, 31). In comparison to LBGM, MSgg is a minimal medium in which the mutants deficient in phosphorelay ( spo0f, spo0b and spo0a) were also found to be severely defective in biofilm formation (9, 31). On the contrary however, the mutants for each of the individual kinases, especially KinC and KinD, showed little or very mild phenotypic difference from that of the wild type strain in MSgg (Fig. S4 and (17)). Therefore, KinD (and to a lesser degree KinC) does seem to be more important in promoting 10

11 robust biofilm formation in LBGM than in MSgg. Since MSgg also contains glycerol (0.5% v/v) and manganese (0.1 mm), one possible explanation for the difference in the two media is that in MSgg, the roles of the sensory kinases (especially KinC and KinD) in stimulating biofilm formation are redundant whereas in LBGM, their roles on that are much more complementary. As further evidence, the ΔkinCΔkinD double mutant is severely defective in both LBGM and MSgg (Fig. 2 and (17)). If KinD and/or KinC are indeed that important in stimulating biofilm formation in LBGM, overexpression of KinD or KinC may lead to increased levels of biofilm production. Moreover, the presence of GM may further enhance the robustness of the biofilms. To test this, we constructed two strains in which the native kind (or kinc) gene is now under the control of the hyperspank promoter (see Materials and Methods). These two strains overproduce either KinD (YC1078) or KinC (YC1077) upon addition of the inducer IPTG. As shown in Fig. 3A, overexpression of KinD alone (+IPTG, but in the absence of GM) in LB medium has resulted in slightly elevated levels of biofilm formation when compared to the very thin pellicles formed in the absence of IPTG. In LBGM medium, over-expression of KinD (+IPTG) resulted in formation of thick and structured floating pellicles, compared to those of the same strain not treated with the inducer (-IPTG, Fig. 3A). The results from KinC overexpression (Fig. 3B) were unexpected because overexpression of KinC in the presence of GM had a strong negative impact on growth especially under static culture conditions and no pellicle formation was observed (Fig. 3B). Our results further strengthened the hypothesis that the sensory kinase KinD mediates the biofilmstimulating effect of GM and that both the protein levels of KinD and the presence of GM are important for biofilm stimulation. 11

12 To further support the above hypothesis that KinD mediates the biofilm-inducing effect of GM, we compared the transcription of epsa-o (by using the same transcriptional fusion described above) in the ΔkinD mutant in LB, LBG and LBM, respectively. As seen in Fig. S2B, there is no significant change in epsa-o transcription when glycerol or manganese alone are added to LB, indicating that KinD mediates the biofilm-stimulating effect of GM. The presence of GM is sensed by the extracellular sensor domain of KinD. We further asked: what is the unique characteristic of KinD distinguishing it from other sensor kinases? According to bioinformatics analysis (see Supplemental Materials and Methods), KinD bears an extracellular CACHE domain (19). CACHE is present in many bacterial sensory histidine kinases and is capable of sensing small molecules often in the presence of cofactors - metal ions (Table S1) (18). This finding is consistent with the hypothesis that KinD can directly sense glycerol or its derivative through the CACHE domain, while the Mn 2+ could be a possible cofactor of the interaction. A KinD CACHE mutant was previously constructed in order to test the role of this domain in KinD in sensing plant-released chemical signals during colonization and rootassociated biofilm formation of B. subtilis on plant host (19). The CACHE mutant of KinD contains amino acid substitutions in the putative signal recognition motif ( 131 RSFF 134 > 131 LLDS 134 ) and may have lost the ability to bind to its cognate signal molecule (19). We took advantage of that and tested whether the KinD CACHE mutant is also defective in stimulating biofilm formation in response to the addition of GM. Indeed, and as shown in Fig. 4, the strain expressing the CACHE mutant of KinD (ΔkinD, amye::kind mut ) was unable to stimulate biofilm formation even in the presence of GM, similar to what was observed for the ΔkinD mutant, 12

13 whereas the mutant strain with the wild type kind complementation (ΔkinD, amye::kind WT ) formed robust biofilms comparable to those of the wild type cells (Fig. 4). This result strongly indicates that the biofilm-promoting activity of GM is mediated by the CACHE domain of KinD. To further prove the involvement of the CACHE domain in sensing the presence of GM we constructed a hybrid histidine kinase, which harbors the CACHE domain from KinD and the kinase and the ATPase domains from DegS (Fig. 5A). DegS is a sensory histidine kinase that is able to phosphorylate DegU, a DNA-binding response regulator (32). The DegS-DegU system controls many genes (both positively and negatively) including the fla/che operon that encodes dozens of genes involved in motility and chemotaxis (33). Following the introduction of the hybrid kinase construct to a degs kind mutant, the expression of fla/che operon is now controlled by the chimeric kinase. We applied real-time RT-PCR to examine the expression of the fla/che operon in the hybrid kinase background in the presence or absence of glycerol (see Materials and Methods). We specifically measured the expression of the flgb gene, the first gene in the fla/che operon. It was previously shown that highly phosphorylated DegU represses expression of the fla/che operon (33). We therefore hypothesized that if glycerol or its derivative activates DegU phosphorylation via the hybrid kinase the flgb gene should be down-regulated. The results from the real-time RT-PCR analysis showed that the addition of glycerol downregulated transcription of flgb very strongly. There was about 10-fold reduction in the abundance of mrna encoding flgb in response to glycerol (Fig. 5B). The similar experiment in the kind degs double mutant strain background without the hybrid kinase showed no significant changes in flgb expression in response to glycerol (Fig. S5). This result provides further evidence that the CACHE domain of KinD can independently sense glycerol. Interestingly, the 13

14 hybrid kinase was not activated in response to the Mn 2+ (data not shown), which probably indicates that manganese does not act directly on KinD in the native situation Glycerol uptake or metabolism mutants are partially capable of promoting biofilm formation in response to GM. We wanted to further test whether glycerol itself or metabolic derivatives of glycerol exerted the biofilm-promoting effect. To do so, we constructed two deletion mutants (ΔglpF and ΔglpK) that are defective in either glycerol uptake or metabolism. Among the deleted genes, glpf encodes a glycerol uptake facilitator and the mutation blocks the uptake of glycerol into the cells (34). The glpk gene encodes a glycerol kinase that is required in the first step of glycerol metabolism (34). We then assayed the mutants for their ability to respond to the addition of glycerol and to promote biofilm formation. As shown in Fig. 6A, both glpk and ΔglpF are only partially capable of responding to the addition of GM by promoting relatively less profound biofilm formation (Fig. 6A). This result indicates that the biofilmpromoting effect of glycerol may not be attributed solely to glycerol itself; rather, an intermediate metabolite of glycerol (catabolized by GlpK) may also be important in biofilm stimulation. We picked the ΔglpK and ΔglpF mutants for further assays of growth in a minimal medium with glycerol as the sole carbon source. As shown in Fig. 6B, both mutants failed to grow in the minimal medium with glycerol as the sole carbon source (although the ΔglpF mutant grew slightly in late time points) but grew well in the minimal medium with glucose as the sole carbon source (Fig. 6B). The result confirms that the glpk-encoded glycerol kinase and glpfencoded glycerol transport facilitator are critical in utilization of glycerol as a carbon source. Next we wanted to address how would products of glycerol metabolism be involved in biofilm formation if the glpk and glpf deletions are still capable of responding to GM? One 14

15 possibility was that the phenotypes of the glpk and glpf deletions are actually due to manganese. Indeed, our results indicate that the mutant of glpk or glpf may still respond to manganese (Fig. S6A), as the phenotypes of ΔglpK, ΔglpF and WT strains in LBM are fairly similar. To quantitatively examine the effect of the glpk or glpf deletion on the response of the cells to the addition of manganese, we constructed two reporter strains in which the P espa -lacz reporter fusion was introduced into the glpk and glpf deletion mutants (generating YC1250 and YC1251, respectively). The activities of the P espa -lacz reporter from the above two deletion mutants were compared to that of the wild type strain (YC110) by using cells from biofilm colonies on LBM (Fig. S6A). No significant difference was observed among the three tested strains (Fig. S6B). Taken together, these results suggest that the biofilm-promoting effect of glycerol is likely associated to its metabolism, while manganese has an independent mechanism for activation of Spo0A which is not related to the glycerol metabolism. The biofilm-promoting effect of GM is conserved in Bacillus species. Finally, we wondered whether the biofilm-promoting effect of GM is conserved in other Bacillus species. We tested this hypothesis in two related Bacillus species: Bacillus licheniformis and Bacillus cereus. B. licheniformis is involved mainly in food spoilage while some B. cereus strains are considered as highly pathogenic to human (35, 36). Interestingly, the similar biofilm-promoting effect caused by addition of GM was very dramatic in both bacteria as judged by pigment production as well as formation of wrinkled colonies (Fig. 7A). Addition of GM also strongly stimulated formation of floating pellicles in B. licheniformis, very similar to what was seen in B. subtilis, whereas in B. cereus, only weak floating pellicles were seen in the presence of GM (Fig. 7B). Possible explanation of observed phenotypes might be in high similarity of the CACHE domain of the B. subtilis KinD to the CACHE domain of B. licheniformis KinD (59% sequence identity), but 15

16 relatively lower similarity to CACHE domain of B. cereus KinD (39% sequence identity). Overall, our finding indicates that the biofilm-stimulating effect in the presence of GM could be highly conserved in Bacillus species (Fig. 7C) DISCUSSION A key question that remains unclear in the field of biofilm development is how the cells sense the environmental cues that trigger biofilm formation. B. subtilis provides a facile system to address this question, as the molecular pathways involved in regulation of cellular differentiation are well characterized. B. subtilis cells use the levels of Spo0A~P, controlled by a network of five histidine kinases, as a sign to initiate biofilm formation. Recent studies showed that four histidine kinases, KinA, KinB, KinC, and KinD are predominantly involved in controlling the Spo0A~P level for biofilm formation (17, 37-39). In the present study, we establish that KinD is the most important sensor kinase for biofilm development in LBGM medium. Along with this finding, we note that the KinC also has some contribution to the biofilm phenotype in LBGM medium as a kinc kind double mutant showed very severe - null phenotype, which was very similar to that of the mutants deficient in phosphorelay (Fig. 2). The important role of KinD in biofilm formation and biofilm-associated sporulation has been discussed in several recent studies (19, 20, 40). For example, Aguilar et al. (40) described KinD as a bi-functional kinase/phosphatase likely sensing the presence of exopolysaccharides and proposed that its chief role is to act as a check-point protein linking spore formation to extracellular matrix production during B. subtilis biofilm formation. In another study (20), KinD was proposed to be capable of functioning as an osmosensor, possibly mediated by the transmembrane domain of KinD. KinD also seems to be important in B. subtilis colonization on 16

17 plant host by sensing plant-released signals and promoting biofilm formation on the root surface of the plant (19). It was also recently suggested that the activity of KinD depends on its partner, a lipoprotein termed Med (41). Based on the above studies as well as the present study, we suppose that the signal-sensing ability of KinD is multifaceted. Up to now, we don t fully understand why a single kinase such as KinD possesses the ability to sense multiple distinct signals. It is possible that multiple domains of KinD may play different roles in signal-sensing and signal transduction especially under different inducing conditions. It has been well established that wild type cells of B. subtilis form robust biofilms mainly in specialized media such as MSgg, E-medium and 2X SGG (4, 7, 42). It is not known why those media favor biofilm formation of B. subtilis. LB medium, on the other hand, is least favorable for biofilm formation by B. subtilis. In this study, we found that addition of glycerol and manganese can transform LB medium to a robust biofilm-promoting medium - LBGM. Importantly, both components (glycerol and manganese) were required for robust biofilm phenotype (Fig. 1A), whereas the phenotypic effect of addition of glycerol or manganese alone to LB medium were relatively minor. This result is different from findings of a recent study which suggested that raising either Mn 2+ or glycerol concentration alone in specialized E-medium (minimal medium) by B-1 strain of B. subtilis results in an increase in biofilm formation (26). In addition to biofilm phenotype, the LBGM medium also promoted robust sporulation and production of a brown pigment associated with biofilms. It was previously shown that manganese dependent brown pigmentation of B. subtilis strain 168 was independent of sporulation (43). Our data indicate that the secreted pigment in LBGM medium is biofilm specific (Fig. S3). Although we do not know the function of the secreted pigment, we presume that the pigment production is strongly 17

18 associated with biofilm formation. The challenge for our future studies will be to elucidate the exact nature and functionality of the pigment and its connection to biofilm formation. Cells of B. subtilis use a complex phosphoryl transfer system to control the activity of Spo0A, a transcriptional regulator of many cellular processes, including extracellular-matrix production and sporulation. Our findings show that the phosphoryl transfer system and biofilm formation is activated by a combination of glycerol and manganese in LB medium. Our analysis indicated that the histidine kinase KinD, bearing the CACHE extracellular sensing domain, is capable of sensing glycerol or its derivative. Aside from being a signal molecule for the sensor kinase, we speculate that glycerol may play other roles in promoting biofilm formation by B. subtilis. This assumption is in part supported by the observation that the ΔglpK and ΔglpF mutants, which are defective in glycerol uptake or metabolism, lost a portion of their ability to promote robust biofilm formation in response to the addition of GM (Fig. 6). This observation indicates that possibly, yet unknown molecules derived from glycerol metabolism may also contribute to the biofilm activation. Moreover, as we discussed earlier, when KinD senses glycerol, Mn 2+ might act as a cofactor. Alternatively, it is also possible that Mn 2+ act as a cofactor further downstream, by promoting the efficiency of phosphoryl transfer in relay. For example, Mn 2+ was previously shown to be complexed with Spo0F in a crystal structure (44). Spo0F is a critical component of the phosphorelay. Therefore, Mn 2+ might be required for the activity of Spo0F and promote efficiency of the relay; another possibility could be the regulation of Sda activity by Mn 2+ (45) which in turn influences the efficiency of phosphoryl transfer in the relay. Furthermore, as discussed above, the hybrid kinase was not activated in response to the Mn 2+, which also supports the notion that Mn 2+ acts downstream of KinD (in the phosphorelay) in the native condition. 18

19 The ability of forming robust biofilms is conserved in the members of Bacillus genus, which are highly ubiquitous in natural environments. In keeping with the idea that the KinD protein and proteins in the phosphorelay are also conserved in different Bacillus species, we found that the biofilm-promoting effect of GM is conserved in both B. licheniformis and B. cereus. These findings led us to propose that the signaling pathway for biofilm formation (Fig. 7C) is highly conserved in Bacillus species. ACKNOWLEDGEMENTS We would like to thank Prof. Richard Losick for helpful suggestions. We are grateful to Dr. Frances Chu and Dr. Yun Chen for strains. We thank Dr. Ronit Pasvolsky-Gutman from ARO for the helpful discussions. REFERENCES 1. Kolter R, Greenberg EP Microbial sciences: the superficial life of microbes. Nature 441: Hall-Stoodley L, Costerton JW, Stoodley P Bacterial biofilms: from the natural environment to infectious diseases. Nat Rev Microbiol 2: Stewart PS, Costerton JW Antibiotic resistance of bacteria in biofilms. Lancet 358: Branda SS, Gonzalez-Pastor JE, Ben-Yehuda S, Losick R, Kolter R Fruiting body formation by Bacillus subtilis. Proc Natl Acad Sci U S A 98: Branda SS, Chu F, Kearns DB, Losick R, Kolter R A major protein component of the Bacillus subtilis biofilm matrix. Mol Microbiol 59:

20 Chu F, Kearns DB, Branda SS, Kolter R, Losick R Targets of the master regulator of biofilm formation in Bacillus subtilis. Mol Microbiol 59: Kearns DB, Chu F, Branda SS, Kolter R, Losick R A master regulator for biofilm formation by Bacillus subtilis. Mol Microbiol 55: Chu F, Kearns DB, McLoon A, Chai Y, Kolter R, Losick R A novel regulatory protein governing biofilm formation in Bacillus subtilis. Mol Microbiol 68: Hamon MA, Lazazzera BA The sporulation transcription factor Spo0A is required for biofilm development in Bacillus subtilis. Mol Microbiol 42: Strauch M, Webb V, Spiegelman G, Hoch JA The SpoOA protein of Bacillus subtilis is a repressor of the abrb gene. Proceedings of the National Academy of Sciences 87: Burbulys D, Trach KA, Hoch JA Initiation of sporulation in B. subtilis is controlled by a multicomponent phosphorelay. Cell 64: Chai Y, Norman T, Kolter R, Losick R. Evidence that metabolism and chromosome copy number control mutually exclusive cell fates in Bacillus subtilis. EMBO J 30: Grossman AD Genetic networks controlling the initiation of sporulation and the development of genetic competence in Bacillus subtilis. Annu Rev Genet 29: LeDeaux JR, Yu N, Grossman AD Different roles for KinA, KinB, and KinC in the initiation of sporulation in Bacillus subtilis. J Bacteriol 177: Piggot PJ, Hilbert DW Sporulation of Bacillus subtilis. Curr Opin Microbiol 7: Jiang M, Shao W, Perego M, Hoch JA Multiple histidine kinases regulate entry into stationary phase and sporulation in Bacillus subtilis. Mol. Microbiol. 38:

21 McLoon AL, Kolodkin-Gal I, Rubinstein SM, Kolter R, Losick R Spatial regulation of histidine kinases governing biofilm formation in Bacillus subtilis. J Bacteriol 193: Anantharaman V, Aravind L CACHE - a signaling domain common to animal Ca2+-channel subunits and a class of prokaryotic chemotaxis receptors. Trends Biochem. Sci. 25: Chen Y, Cao S, Chai Y, Clardy J, Kolter R, Guo J-h, Losick R A Bacillus subtilis sensor kinase involved in triggering biofilm formation on the roots of tomato plants. Molecular Microbiology 85: Rubinstein SM, Kolodkin-Gal I, McLoon A, Chai L, Kolter R, Losick R, Weitz DA Osmotic pressure can regulate matrix gene expression in Bacillus subtilis. Molecular Microbiology 86: Chai Y, Chu F, Kolter R, Losick R Bistability and biofilm formation in Bacillus subtilis. Mol Microbiol 67: Wach A PCR-synthesis of marker cassettes with long flanking homology regions for gene disruptions in S. cerevisiae. Yeast 12: Guérout-Fleury AM, Frandsen N, Stragier P Plasmids for ectopic integration in Bacillus subtilis. Gene 180: Fujita M, Losick R Evidence that entry into sporulation in Bacillus subtilis is governed by a gradual increase in the level and activity of the master regulator Spo0A. Genes Dev. 19: Sambrook J, Russell DW Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA. 21

22 Morikawa M, Kagihiro S, Haruki M, Takano K, Branda S, Kolter R, Kanaya S Biofilm formation by a Bacillus subtilis strain that produces gamma-polyglutamate. Microbiology 152: Moreno MS, Schneider BL, Maile RR, Weyler W, Saier MH Catabolite repression mediated by the CcpA protein in Bacillus subtilis: novel modes of regulation revealed by whole-genome analyses. Mol. Microbiol. 39: Romero D, Aguilar C, Losick R, Kolter R. Amyloid fibers provide structural integrity to Bacillus subtilis biofilms. Proc Natl Acad Sci U S A 107: Min K-T, Hilditch CM, Diederich B, Errington J, Yudkin MD Sigma F, the first compartment-specific transcription factor of B. subtilis, is regulated by an anti-sigmaf factor that is also a protein kinase. Cell 74: Hamon M, A., Stanley NR, Britton RA, Grossman AD, Lazazzera BA Identification of AbrB-regulated genes involved in biofilm formation by Bacillus subtilis. Mol. Microbiol. 52: Branda SS, Gonzalez-Pastor JE, Dervyn E, Ehrlich SD, Losick R, Kolter R Genes involved in formation of structured multicellular communities by Bacillus subtilis. J. Bacteriol. 186: Mukai K, Kawata M, Tanaka T Isolation and phosphorylation of the Bacillus subtilis degs and degu gene products. Journal of Biological Chemistry 265: Amati G, Bisicchia P, Galizzi A DegU-P Represses Expression of the Motility fla-che Operon in Bacillus subtilis. Journal of Bacteriology 186: Kunst F, Ogasawara N, Moszer I, Albertini AM, Alloni G, Azevedo V, Bertero MG, Bessieres P, Bolotin A, Borchert S, Borriss R, Boursier L, Brans A, Braun M, Brignell SC, 22

23 Bron S, Brouillet S, Bruschi CV, Caldwell B, Capuano V, Carter NM, Choi SK, Codani JJ, Connerton IF, Cummings NJ, Daniel RA, Denizot F, Devine KM, Dusterhoft A, Ehrlich SD, Emmerson PT, Entian KD, Errington J, Fabret C, Ferrari E, Foulger D, Fritz C, Fujita M, Fujita Y, Fuma S, Galizzi A, Galleron N, Ghim SY, Glaser P, Goffeau A, Golightly EJ, Grandi G, Guiseppi G, Guy BJ, Haga K, Haiech J, Harwood CR, Henaut A, Hilbert H, Holsappel S, Hosono S, Hullo MF, Itaya M, Jones L, Joris B, Karamata D, Kasahara Y, Klaerr-Blanchard M, Klein C, Kobayashi Y, Koetter P, Koningstein G, Krogh S, Kumano M, Kurita K, Lapidus A, Lardinois S, Lauber J, Lazarevic V, Lee SM, Levine A, Liu H, Masuda S, Mauel C, Medigue C, Medina N, Mellado RP, Mizuno M, Moestl D, Nakai S, Noback M, Noone D, O'Reilly M, Ogawa K, Ogiwara A, Oudega B, Park SH, Parro V, Pohl TM, Portetelle D, Porwollik S, Prescott AM, Presecan E, Pujic P, Purnelle B, Rapoport G, Rey M, Reynolds S, Rieger M, Rivolta C, Rocha E, Roche B, Rose M, Sadaie Y, Sato T, Scanlan E, Schleich S, Schroeter R, Scoffone F, Sekiguchi J, Sekowska A, Seror SJ, Serror P, Shin BS, Soldo B, Sorokin A, Tacconi E, Takagi T, Takahashi H, Takemaru K, Takeuchi M, Tamakoshi A, Tanaka T, Terpstra P, Tognoni A, Tosato V, Uchiyama S, Vandenbol M, Vannier F, Vassarotti A, Viari A, Wambutt R, Wedler E, Wedler H, Weitzenegger T, Winters P, Wipat A, Yamamoto H, Yamane K, Yasumoto K, Yata K, Yoshida K, Yoshikawa HF, Zumstein E, Yoshikawa H, Danchin A The complete genome sequence of the Gram-positive bacterium Bacillus subtilis. Nature 390: Kotiranta A, Lounatmaa K, Haapasalo M Epidemiology and pathogenesis of Bacillus cereus infections. Microbes Infect 2: Pirttijarvi TS, Graeffe TH, Salkinoja-Salonen MS Bacterial contaminants in liquid packaging boards: assessment of potential for food spoilage. J Appl Bacteriol 81:

24 Lopez D, Fischbach MA, Chu F, Losick R, Kolter R Structurally diverse natural products that cause potassium leakage trigger multicellularity in Bacillus subtilis. Proc Natl Acad Sci U S A 106: Shemesh M, Kolter R, Losick R. The biocide chlorine dioxide stimulates biofilm formation in Bacillus subtilis by activation of the histidine kinase KinC. J Bacteriol 192: Shank EA, Klepac-Ceraj V, Collado-Torres L, Powers GE, Losick R, Kolter R. Interspecies interactions that result in Bacillus subtilis forming biofilms are mediated mainly by members of its own genus. Proc Natl Acad Sci U S A 108:E Aguilar C, Vlamakis H, Guzman A, Losick R, Kolter R KinD Is a Checkpoint Protein Linking Spore Formation to Extracellular-Matrix Production in Bacillus subtilis Biofilms. mbio Banse AV, Hobbs EC, Losick R Phosphorylation of Spo0A by the histidine kinase KinD requires the lipoprotein med in Bacillus subtilis. J Bacteriol 193: Kobayashi K SlrR/SlrA controls the initiation of biofilm formation in Bacillus subtilis. Mol Microbiol 69: Hullo MF, Moszer I, Danchin A, Martin-Verstraete I CotA of Bacillus subtilis is a copper-dependent laccase. J Bacteriol 183: Mukhopadhyay D, Sen U, Zapf J, Varughese KI Metals in the sporulation phosphorelay: manganese binding by the response regulator Spo0F. Acta Crystallogr D Biol Crystallogr 60:

25 Hoover SE, Xu W, Xiao W, Burkholder WF Changes in DnaA-dependent gene expression contribute to the transcriptional and developmental response of Bacillus subtilis to manganese limitation in Luria-Bertani medium. J Bacteriol 192: Downloaded from on September 22, 2018 by guest 25

26 FIGURE LEGENDS FIG. 1. Effect of glycerol and manganese on B. subtilis 3610 multicellular development. (A) A combination of glycerol (1% v/v) and manganese (0.1 mm) added to LB medium (LBGM) promotes robust biofilm formation. Effects of addition of glycerol (LBG) or manganese (LBM) alone are also shown. An undomesticated wild strain of B. subtilis 3610 was used here. (B) Transcription of the operons responsible for the matrix production is upregulated in response to GM. Results from RL4582 cells that bear the P tapa -lacz transcriptional fusion are presented in the left-hand panel whereas results from RL4548 cells that bear the P eps -lacz transcriptional fusion are presented in the right-hand panel. (C) Addition of GM to LB medium also greatly stimulates the biofilm-associated sporulation as the total spore accounts increased about 100-fold in a biofilm colony formed on LBGM agar plate than that on LB agar plate. FIG. 2. Histidine kinase KinD is a major kinase in sensing the presence of GM. Colony development and pellicle formation on LBGM by the wild type and various mutant strains were compared. The strains used here were as follows: wild type (3610), ΔkinA (RL4566), ΔkinB (RL4563), ΔkinC (RL4565), ΔkinD (RL4569), ΔkinE (RL4570), ΔkinAB (RL4573), ΔkinCD (RL4577), Δspo0F (RL4567), Δspo0B (RL4568), Δspo0A (RL4620), ΔkinDΔabrB (YC863) and ΔabrB (YC668). The ΔkinD mutant showed most defective phenotypes in biofilm formation, fairly similar to that of the mutants deficient in phosphorelay: Δspo0F, Δspo0B and Δspo0A. FIG. 3. Overexpression of KinD stimulates biofilm formation. (A) Overexpression of KinD in the strain YC1078 stimulated floating pellicle formation both in the absence and presence of GM. (B) The effect of KinC overexpression on biofilm formation was similarly accessed in the 26

27 strain YC1077: to overexpress KinC or KinD, IPTG was added to the medium at a final concentration of 100 μm. Cells were incubated at 30 C. Images were taken after 48 hours of incubation. Note that overexpression of KinC in the presence of GM had a strong negative impact on cell growth. FIG. 4. Cells expressing CACHE mutant of KinD fail to form robust biofilms in LBGM. Colony development and floating pellicle formation in LBGM by strains of wild type (3610), ΔkinD mutant complemented with either wild type (CY189) or CACHE mutant of kind (CY190), and ΔkinD mutant (RL4569) are compared. FIG. 5. Sensing of GM is mediated by CACHE, the extracellular sensor domain of KinD. (A) A schematic demonstration of the construction of the hybrid kinase harboring the extracellular sensor domain from KinD and the kinase and the ATPase domains from DegS. (B) Analysis by real-time RT-PCR of the expression of flgb, the first gene in the fla/che operon, which is now under the control of the KinD-DegS chimeric kinase (MS178), in response to glycerol. The results represent the mean ± standard deviation (SD) of two independent experiments. FIG. 6. Mutants defective in glycerol uptake or metabolism are partially capable of promoting biofilm formation in response to GM. (A) The glycerol uptake or metabolism mutants, ΔglpF (YC877) and ΔglpK (FC96) are only partially capable of promoting biofilm formation in response to GM when compared to that of the wild type. (B) Growth curves of the ΔglpF and ΔglpK strains in minimal medium supplemented with either 0.5% glucose (left-hand 27

28 598 panel) or 0.5% glycerol (right-hand panel) as the sole carbon source FIG. 7. The biofilm-promoting effect of GM is conserved in Bacillus species. (A) GM has a strong effect on colony morphology and pigment production in B. licheniformis ATCC8480 and B. cereus ATCC (B) GM promotes formation of floating pellicles in B. licheniformis, but not B. cereus. (B) Schematic representation of signal transduction pathway responsible for the activation of a master regulator of matrix production and biofilm formation in B. subtilis. Downloaded from on September 22, 2018 by guest 28

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