The “low affinity” c-di-GMP receptor YdaK (K_D 1 μM) does not serve as an effector protein to modulate swarming motility directly (Gao et al., 2013), but instead affects BF formation by positively regulating the production of an unknown EPS synthesized by the products of the ydaJ-N operon (Bedrunka and Graumann, 2017).

We wanted to investigate the potential involvement of DGCs and thus c-di-GMP in EPS production via induction of YdaK. Deletion and overexpression of ydaK alone have not revealed any phenotypic effects so far. Therefore, we initially introduced two expression constructs pSG1164-PB53 (P_xyl-ydaJ-N) and pSG1164-PB55 (P_xyl-ydaK-N), steering the expression of the transcriptional units ydaJ-N and ydaK-N respectively, into the genome of wild type B. subtilis NCIB3610, and into that of a dgc triple mutant strain lacking the native c-di-GMP-synthesizing components (strain DS1809, ΔdgcK ΔdgcW dgcP::tet, Figure 1A).

As shown in an earlier study (Bedrunka and Graumann, 2017), induction of ydaJ-N (strain NCIB3610-PB53, P_xyl-ydaJ-N) and overexpression of ydaK-N (strain NCIB3610-PB55, P_xyl-ydaK-N) respectively, lead to strongly altered and wrinkled colony morphologies and inhibited surface spreading behavior (Figure S1 and Figure 1A, second and third row, respectively) of B. subtilis on solid BF medium (Branda et al., 2001). These changes are indicative of EPS production as demonstrated for different bacterial species (Serra et al., 2013).

Essentially, inactivation of all three dgc genes prevents the synthesis of this unknown EPS in both induction strains (Figure 1A, fifth row: strain DS1809-PB53, ΔdgcK, -P, -W, P_xyl-ydaJ-N; sixth row: strain DS1809-PB55, ΔdgcK, -P, -W, P_xyl-ydaK-N). The morphology of these resembled that of the wild type supporting the c-di-GMP dependence of the ydaJ-N related EPS machinery (Figure 1A, first row).

We proceeded to test the effect of ydaK-N induction (construct pSG1164-PB55, P_xyl-ydaK-N) on BF-promoting medium in single dgc gene mutant backgrounds (Figure 1B). Importantly, in strains in which the endogenous locus dgcK was deleted (strain DS9305-PB55, ΔdgcK, P_xyl-ydaK-N, Figure 1B, third row) production of EPS by YdaK-N was completely abolished despite the presence of xylose, as respective colony architectures resembled wild type appearance (NCIB3610, P_sigB-ydaJ-N, Figure 1B, first row). In contrast, disruption of dgcP did not impair EPS production via YdaK-N, reflected by the altered colony morphology in the case ydaK-N induction (strain DS9537-PB55, ΔdgcP, P_xyl-ydaK-N, Figure 1B, right column, forth row), compared to the dgcK background deletion strain (Figure 1B, right column, third row). Similarly, deletion of dgcW also resulted in altered BF formation upon induction of ydaK-N (strain DS9883-PB55, ΔdgcW, P_xyl-ydaK-N, Figure 1B right column, fifth row) implying that EPS production via YdaK-N is independent of DgcP and DgcW under our experimental conditions, but dependent of DgcK.

Thus, our experiments demonstrate that activation of EPS production via the degenerated GGDEF domain protein YdaK relies on the integrity of dgc genes and furthermore indicate that YdaK is activated via DgcK under BF-promoting conditions, which results in activation of EPS production in a c-di-GMP dependent manner. In order to further support the hypothesis of YdaK activation via c-di-GMP, we performed site directed replacement mutagenesis of conserved I-site (inhibitory site) residues in YdaK proposed to be involved in c-di-GMP binding (Gao et al., 2013, Figure 1C). Based on sequence analysis, YdaK likely has a primary and secondary inhibitory site (Gao et al., 2013). The putative secondary I-site motif RxxR is found at residues R157 to R160, whereas the putative primary I-site motif RxxD (R202 to D205) locates five residues upstream of the degenerated active site motif SDERI (conserved motif GGDEF). To test the activity of the primary I-site variants, YdaK^R202A and YdaK^D205A (pXG003-PB80 & pXG003-PB81, respectively) were introduced at the ectopic amyE locus of strains that harbored a xylose-dependent promoter driving ydaLMN expression (pSG1164-PB56; Figure 1C). Induction of ydaLMN alone (strain NCIB3610-PB56, P_xyl-ydaL-N, Figure 1C, second row) did not result in EPS production as reflected by unaltered colony morphology of the correponding strain compared to ydaK-N overexpression (strain NCIB3610-PB55, P_xyl-ydaK-N; Figure 1C, first row) in agreement with a previous study (Bedrunka and Graumann, 2017). Complementation with a wild type copy of ydaK in trans restored EPS production upon xylose and IPTG addition (strain NCIB3610-PB56-XG003, P_xyl-ydaLMN, amyE::P_IPTG-ydaK, Figure 1C, third row). However, when strain NCIB3610-PB56 (P_xyl-ydaL-N) was complemented with ydaK alleles encoding the R202A (NCIB3610-PB56-PB80, P_xyl-ydaLMN, amyE::P_IPTG-ydaK^R202A, Figure 1C, forth row) or D205A point mutants (NCIB3610-PB56-PB81, P_xyl-ydaLMN, amyE::P_IPTG-ydaK^D205A, Figure 1C, fifth row) respectively, EPS production was abolished. These observations suggest that an intact primary I-site motif of YdaK is required for c-di-GMP binding and thus YdaK activity.

Given that EPS production mediated by YdaK-N is suppressed in the absence of all three dgc genes and specifically upon disruption of dgcK (Figures 1A,B), we were interested whether the loss of the ydaK-N expression phenotype in a dgc triple mutant can be complemented only with dgcK or whether additional dgc genes (dgcP, dgcW) can positively influence the production of the unknown EPS, e.g., when they are overexpressed. Therefore, we introduced each dgc gene individually at the ectopic amyE locus under the control of an IPTG-inducible promoter, into the genome of the dgc triple mutant, which induces ydaK-N upon xylose addition, and tested BF formation in the presence or absence of xylose and IPTG, respectively (Figure 2A). As expected, the overexpression of dgcK restored modified BF formation upon addition of xylose and IPTG (Figure 2A, first column, first row, strain: DS1809-PB55-XG004, ΔdgcK, -P, -W, P_xyl-ydaK-N, amyE::P_IPTG-dgcK) as observed in wild type backgrounds (Figure 1B, second column, second row, strain NCIB3610-PB55, P_xyl-ydaK-N). Interestingly, also an overexpression of dgcP restored hyper-wrinkle formation (Figure 2A, second column, first row, strain: DS1809-PB55-XG002, ΔdgcK, -P, -W, P_xyl-ydaK-N, amyE::P_IPTG-dgcP).

In contrast to dgcK and dgcP (encoding GGDEF domain proteins), reintroduction of full length dgcW (coding for a GGDEF-EAL domain tandem protein) at the amyE site (Figure 2A, third column, first row, strain: DS1809-PB55-XG001, ΔdgcK, -P, -W, P_xyl-ydaK-N, amyE::P_IPTG-dgcW) did not restore altered biofilm morphology of strain NCIB3610-PB55 (P_xyl-ydaK-N). However, colonies grown in the presence of IPTG/xylose (Figure 2A, third column, first row) and IPTG (Figure 2A, third column, third row) respectively, exhibited a distinct phenotype compared to the wild type (Figure 1A, first column, first row). Overexpression of dgcW resulted in a visible reduction of thick wrinkle structures and in a loss of the associated central ring that usually marks the initial inoculation area, in a dgc triple mutant overexpressing ydaK-N (Figure 2A, third column, first row) and also in wild type background (Figure 2B, third column, first row).

This rather modest but reproducible BF-inhibiting phenotype became more severe when a DgcW variant was overproduced lacking the C-terminal EAL domain (Figure 2A, forth column, third row; Figure 2B, forth column, first row). Overexpression of dgcW-Δeal caused an entire loss of wrinkle formation in the center of the macro-colony in both genomic backgrounds, in DS1809-PB55 (Figure 2A, forth column, third row, strain DS1809-PB55-XG086, ΔdgcK, -P, -W, P_xyl-ydaK-N, amyE::P_IPTG-dgcWΔeal) and in NCIB3610 (Figure 2B, forth column, first row, strain NCIB3610-XG086, amyE::P_IPTG-dgcWΔeal). Furthermore, we found that induction of ydaK-N accompanied by an overexpression of dgcW-Δeal lead to enhanced wrinkle formation and altered BF formation (Figure 2A, forth column, first row, strain DS1809-PB55-XG086, ΔdgcK, -P, -W, P_xyl-ydaK-N, amyE::P_IPTG-dgcWΔeal). This suggests that a truncated version of DgcW is able to provide c-di-GMP to activate EPS production via YdaK.

In summary, our complementation analysis reveals that both DgcK and DgcP support EPS production when being overproduced, whereas DgcW is only able to do so upon deletion of the GGDEF-adjacent EAL domain. Therefore, we suggest that the activity of the EAL domain (potentially c-di-GMP hydrolysis) of DgcW masks the enzymatic activity of its neighboring (upstream) GGDEF domain (c-di-GMP synthesis). Because the overproduction of DgcW-ΔEAL by itself had a strong effect on BF morphology, our findings suggest that DgcW affects a pathway different from that of the yda operon, and that therefore, at least two independent c-di-GMP processes occur during BF formation in case of ydaK-N and dgcWΔeal overexpression.

Several DGCs in different bacterial species have been reported to occur in complex with their effector proteins/targets in order to maintain signal specificity within certain signaling cascades (Lindenberg et al., 2013; Dahlstrom et al., 2015). Additionally, it has been suggested that differential subcellular localization patterns of DGCs may affect their function and thus the interplay with its corresponding effectors (Merritt et al., 2010; Zhu et al., 2016). This prompted us to perform comparative localization studies of the c-di-GMP receptor YdaK and of the corresponding DGCs (DgcK and DgcP) that were able to restore EPS production mediated by YdaK-N in a dgc triple mutant (Figure 2A).

In a recent study, we investigated the subcellular localization of YdaK and of its potential downstream targets, the putative glycosyltransferases YdaM and YdaN, by fluorescence microscopy (Bedrunka and Graumann, 2017). YdaK-, YdaM-, and YdaN-mV-YFP fusions (mV: monomeric Venus) expressed from their original promoter and at their native site on the chromosome, formed static subcellular clusters (usually one or two single foci per cell) at the membrane, predominantly at the septa and/or at cell poles in exponentially growing B. subtilis cells. YdaM/YdaK and YdaM/YdaN fluorescent fusions co-localized to the same cellular positions supporting the idea that specific protein localization at the cell membrane might be necessary to facilitate protein-protein interactions and EPS production.

Functional C-terminal mV-YFP fusions of full-length YdaK induced from the ectopic amyE site were able to support EPS activation in a mutant of undomesticated B. subtilis that induced ydaL-N upon xylose addition (strain NCIB3610-PB56-PB57, P_xyl-ydaL-N, amyE::P_xyl-ydaK-mV-yfp, Figure S2) as depicted in Figure 3Aii. Ectopically induced YdaK-mV-YFP fusions primarily localized at the peripheries of cells as double foci at mid cell and/ or at the cell poles revealing high signal intensities but also as lateral patches with rather low signal intensities (Figure 3Aiii). As an additional control, we included a C-terminal fluorescent fusion of YdaK lacking its 4 predicted TMHs (transmembrane helices, Figure 3Bi) in this study.

The corresponding construct was introduced into the genome of NCIB3610-PB56 (resulting strain: NCIB3610-PB56-PB100, P_xyl-ydaL-N, amyE::P_xyl-ydaKΔ4tmh-mV-yfp; upper panel in Figure 3Bii, Figure S2). In contrast to YdaK-mV-YFP full length fusions (Figure 3A) and wild type YdaK (strain NCIB3610-PB55-PB16, P_xyl-ydaL-N, amyE::P_xyl-ydaK; lower panel in Figure 3Bii), the truncated variants YdaKΔ4TMH-mV-YFP failed to restore altered BF formation upon overproduction resulting in unaffected colony morphologies. This is most likely due to the fact that removal of TMHs results in a cytoplasmic distribution of these fusion proteins (Figure 3Biii), which is not due to degradation of the fusions as verified by Western blots of cell extracts using an antibody against GFP (Figure S2). Thus, YdaK must localize in a complex with its downstream effector proteins YdaLMN at its native membrane position in order to activate EPS production at specific sites of the bacterial cell membrane, which we have already hypothesized earlier.

In view of these finding, we wondered whether DgcK, the specific c-di-GMP delivering DGC for YdaK (Figure 1B), would resemble YdaK localization (Figure 3C). Initially, we have examined the subcellular localization of C-terminal DgcK-mV-YFP produced from the original locus in B. subtilis NCIB3610 cells (strain NCIB3610-PB01, P_dgcK-dgcK-mV-yfp). Fusion proteins were hardly detectable suggesting low expression levels of dgcK-mV-yfp under our experimental conditions. Therefore, we visualized the subcellular localization of N- and C-terminal mV-YFP fusions of DgcK originated from the ectopic amyE locus upon xylose addition (strain NCIB3610-PB90, amyE::P_xyl-dgcK-mV-yfp). Only the translational C-terminal DgcK-mV-YFP proved to be functional (Figure 3Cii, Figure S3). The phenotypes of the dgc triple mutant strains, inducing ydaK-N from the original locus and dgcK-mV-yfp from the amyE locus (DS1809-PB55-PB90, ΔdgcK, -P, -W, P_xyl-ydaK-N, amyE::P_xyl-dgcK-mV-yfp), were similar to those of the ΔdgcK, -P, -W strains carrying the P_xyl-ydaK-N construct and the wild-type dgcK allele (DS1809-PB55-PB90, ΔdgcK, -P, -W, P_xyl-ydaK-N, amyE::P_IPTG-dgcK) in that both strains had altered BF morphologies and were comprised in colony spreading behavior (compare Figure 2A, first column and Figure 3Cii, first row). In contrast to this, overproduction of the fluorescent protein mV-YFP alone from the endogenous locus and induction of ydaK-N in a triple DGC mutant did not result in EPS production (DS1809-PB55-1193NLMV, ΔdgcK, -P, -W, P_xyl-ydaK-N, amyE::P_xyl-mV-yfp).

Similarly to our observation of YdaK-mV-YFP clustering (Figure 3Aiii), DgcK-mV-YFP fusion proteins also formed assemblies that retained a preference of localization to the cell poles and septa in NCIB3610 (Figure 3Ciii). This observation implies that DgcK and YdaK might be in spatial proximity at these cellular positions and might establish a spatially linked signal-effector cluster at the membrane. In addition to the comparable localization patterns of YdaK-mV-YFP and DgcK-mV-YFP (Figure 3), both fusion proteins exhibited a similar movement behavior when overproduced. We performed time-lapse experiments with both fusion proteins produced from the amyE locus upon xylose addition in exponentially growing B. subtilis NCIB3610 wild type cells. Upon continuous illumination (515 nm) with 100 ms intervals, foci detected at the septa were predominantly static, displaying negligible movement within a time span of several 100 ms (Figure 4A).

Polar foci were especially observed in case of YdaK-mV-YFP fusions and dynamic movement of both protein foci with lower “resting times” at the lateral cell periphery (Movies S1, S2). Although we observed events of co-localization in only 30% of total signals counted between DgcK-CFP (amyE locus) and YdaK-m-YFP (Figure 4B, strain NCIB3610-PB37-PB10, amyE::P_xyl-dgcK-cfp, P_ydaK-ydaK-mV-yfp), our data strongly suggest that c-di-GMP signaling and YdaK activation by DgcK occurs at the cell membrane and employs close spatial proximity of the players involved.

In addition to the localization of YdaK and DgcK, we also monitored mV-YFP labeled DgcP fusions in B. subtilis NCIB3610 (Figure 5, Figure S4). DgcP is a c-di-GMP synthesizing protein containing N-terminal GAF domains (putative sensor domain) and a C-terminal GGDEF domain, hinting that it is most likely a soluble protein in contrast to YdaK, DgcK, and DgcW. To test for the functionality of N- and C-terminal fusions, we applied the same complementation assay as described for DgcK (compare to Figure 3). Overexpression of mV-yfp-dgcP and of dgcP-mV-yfp respectively, in a DGC triple mutant inducing YdaK-N (strains: DS1809-PB55-PB85, ΔdgcK, -P, -W, P_xyl-ydaK-N, amyE::P_xyl-mV-yfp-dgcP; DS1809-PB55-PB86, ΔdgcK, -P, -W, P_xyl-ydaK-N, amyE::P_xyl-dgcP-mV-yfp) caused altered BF colony morphologies (Figures 5A,B, “MSgg panel”) in the same manner as seen for overexpression of dgcK-mV-yfp (Figure 3Bii).

Interestingly, stable mV-YFP-DgcP (Figure 5A, left panel, Figure S4) and DgcP-mV-YFP (Figure 5A, right panel, Figure S4) both assembled to subcellular clusters at the periphery of exponentially growing cells (produced from the ectopic amyE locus, strains NCIB3610-PB85, amyE::P_xyl-mV-yfp-dgcP; NCIB3610-PB86, amyE::P_xyl-dgcP-mV-yfp). Furthermore, both fusion proteins moved dynamically through the cell, but frequently arrested at the cell membrane for several 100 ms intervals (Figure 5B, Movie S3, Movie S4). A similar behavior was observed for DgcP-mV-YFP whose synthesis was initiated by its native promoter at its original locus resulting in low amounts of fusion proteins (Figure 5C, Figure S4). Thus, we conclude that DgcP would be able to deliver c-di-GMP to YdaK potentially through spatial proximity at the cell membrane. However, it is equally possible that YdaK is activated simply by elevated cytosolic c-di-GMP levels following the overproduction of DgcP (see deltaEAL-DgcW).

DNA manipulation and Escherichia coli DH5α transformations were carried out using standard techniques (Mamiatis et al., 1985; Gibson et al., 2009). E. coli strains were routinely cultivated at 37°C in Lysogeny Broth (LB) medium supplemented with 100 μg ml⁻¹ ampicillin. Lists of utilized plasmids and oligonucleotides are provided in Tables S2, S3 respectively. All constructs were verified by DNA sequencing.

Bacillus subtilis strains used in this study derived from the non-domesticated strain NCIB3610 (BGSC) or its transformable derivative DK1042 (Gift from D. Kearns). For transformation of both classes of derivatives, B. subtilis overnight cultures were grown in liquid LB at 30°C and were diluted to OD₆₀₀ 0.08 in 10 ml of a modified competence medium (Zafra et al., 2012). Inoculated cells were further incubated at 37°C and 200 rpm. Upon entry into stationary phase (OD₆₀₀ 1.4–1.6), 300–500 ng of purified genomic DNA were added to 1 ml culture of NCIB3610 derivatives and 0.5-1 μg of plasmid DNA to 1 ml culture of DK1042 derivatives respectively. Cells were further incubated at 37°C and 200 rpm for 2 h followed by selection on solid medium with the appropriate antibiotic. Final antibiotic concentrations were: 5 μg ml⁻¹ chloramphenicol, 50–100 μg ml⁻¹ spectinomycin and 10 μg ml⁻¹ tetracycline. Table S1 provides a detailed description of strains and whether they were generated by plasmid- or by chromosomal DNA-transformation.

For routine growth, B. subtilis cells were streaked from frozen stocks onto LB agar plates and incubated overnight at 37°C. Overnight cultures were grown at 30°C and 200 rpm in LB under antibiotic selective pressure and at 37°C previous to phenotypical tests and fluorescence microscopy. Prior to microscopy cells were washed twice in S7₅₀ minimal medium containing 1.0% (v/v) fructose, 0.1% (v/v) glutamate, 0.004% (v/v) casamino acids (Jaacks et al., 1989). For induction of the xylose promoter, xylose was added up to 0.1% (v/v). For induction of the hyperspank promoter, the culture medium was supplemented with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG).

Undomesticated B. subtilis NCIB3610 strains were cultured in LB containing appropriate antibiotics at 30°C for 14 h. Daily cultures were grown in LB at 37°C to an OD₆₀₀ of 1.0 without antibiotics. For biofilm growth, bacteria from a liquid LB culture were collected and transferred to liquid MSgg medium [5 mM potassium phosphate (pH 7.0), 100 mM 3-(N-morpholino) propane-sulfonic acid (pH 7.0), 2 mM MgCl₂, 700 μM CaCl₂, 50 μM MnCl₂, 100 μM FeCl₃, 1 μM ZnCl₂, 2 μM thiamine, 0.5% glycerol, 0.5% glutamate]. Cells were incubated for additional 30 min at 37°C and 200 rpm before inoculation (2 μl) on MSgg plates (MSgg medium fortified with 1.5% Bacto agar, 6- well plates, dried overnight) supplemented with Congo Red (40 μg ml⁻¹) and Coomassie Brilliant Blue (20 μg ml⁻¹) and with or without 0.1% (v/v) xylose and or 1 mM IPTG (Branda et al., 2001; Asally et al., 2012). Plates were sealed and incubated up to 72 h at 25°C. Colony morphology was documented over time using the ChemiDocTM MP System (BIO-RAD). For each strain, we analyzed 3 biological replicates in at least two independent experiments.

Cells were grown in LB rich medium under selective pressure to the exponential growth phase at 37°C. D-xylose was added in different concentrations [0.001, 0.01, and 0.1% (v/v)] to the growth media to induce expression of genes downstream of the encoded fusion protein at original locus or the encoded fusion protein itself at the amyE locus for 45 min at 37°C. For microscopy, 2 μl of washed cells were spotted on a coverslip and immobilized by a thin agarose pad [1% (w/v) agarose in S7₅₀ minimal medium]. Fluorescence microscopy was performed using a Zeiss Axio Observer A1 equipped with a 100 × TIRF objective (numerical aperture NA of 1.45) using the setup from Visitron Systems (Munich, Germany). YFP fluorophores were excited by exposure to a 515 nm laser beam and CFP fluorophores to 445 nm. Images were acquired with an Evolve EM-CCD camera (Photometrix) and were processed with ImageJ (National Institutes of Health, Bethesda, MD).

To validate expression levels of fusion genes and stability of fusion proteins respectively, cells were grown in LB medium at 37°C until exponential phase and gene expression was artificially induced with different xylose concentrations in case of amyE encoded gene fusions. 45 min after induction and incubation at 37°C and 200 rpm, equal amounts of cells were resuspended in lysis buffer (50 mM EDTA, 100 mM NaCl, 2.5 mg ml⁻¹ lysozyme, 0.1 mg ml⁻¹ RNase, 0.01 mg ml⁻¹ DNase, pH 7.5) and incubated for 20 min at 37°C. SDS sample buffer (final concentration, 1 X) was added to the cell lysate and boiled at 95°C for 10 min, except for lysates which derived from strain NCIB3610-PB90 (amyE::P_xyl-dgcK-mV-yfp). These samples were incubated at RT for 1 h prior to SDS-PAGE on 4–20% Tris/ Glycin gradient gels which have been also used for separation of YdaK fusion proteins. DgcP fusion proteins were separated via SDS-PAGE on 12% gels. The proteins were transferred to a nitrocellulose membrane, applying the semidry Western blotting method for 1 h and 45 mA and YFP-fused proteins were visualized by a primary polyclonal α-GFP antiserum (dilution, 1:500) and a secondary goat α-rabbit antiserum coupled to a horseradish peroxidase.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.