Allosteric activation of exopolysaccharide synthesis through cyclic di-GMP-stimulated protein–protein interaction
2012; Springer Nature; Volume: 32; Issue: 3 Linguagem: Inglês
10.1038/emboj.2012.315
ISSN1460-2075
AutoresSamuel Steiner, Christian Lori, Alex Boehm, Urs Jenal,
Tópico(s)Infections and bacterial resistance
ResumoArticle30 November 2012free access Allosteric activation of exopolysaccharide synthesis through cyclic di-GMP-stimulated protein–protein interaction Samuel Steiner Samuel Steiner Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Christian Lori Christian Lori Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Alex Boehm Alex Boehm LOEWE-Zentrum für Synthetische Mikrobiologie, Philipps-Universität Marburg, Marburg, Germany Search for more papers by this author Urs Jenal Corresponding Author Urs Jenal Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Samuel Steiner Samuel Steiner Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Christian Lori Christian Lori Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Alex Boehm Alex Boehm LOEWE-Zentrum für Synthetische Mikrobiologie, Philipps-Universität Marburg, Marburg, Germany Search for more papers by this author Urs Jenal Corresponding Author Urs Jenal Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Author Information Samuel Steiner1, Christian Lori1, Alex Boehm2 and Urs Jenal 1 1Biozentrum, University of Basel, Basel, Switzerland 2LOEWE-Zentrum für Synthetische Mikrobiologie, Philipps-Universität Marburg, Marburg, Germany *Corresponding author. Biozentrum, University of Basel, Klingelbergstrasse 50/70, 4056 Basel, Switzerland. Tel.:+41 61 267 2135; Fax:+41 61 267 2118; E-mail: [email protected] The EMBO Journal (2013)32:354-368https://doi.org/10.1038/emboj.2012.315 There is a Have you seen? (February 2013) associated with this Article. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In many bacterial pathogens, the second messenger c-di-GMP stimulates the production of an exopolysaccharide (EPS) matrix to shield bacteria from assaults of the immune system. How c-di-GMP induces EPS biogenesis is largely unknown. Here, we show that c-di-GMP allosterically activates the synthesis of poly-β-1,6-N-acetylglucosamine (poly-GlcNAc), a major extracellular matrix component of Escherichia coli biofilms. C-di-GMP binds directly to both PgaC and PgaD, the two inner membrane components of the poly-GlcNAc synthesis machinery to stimulate their glycosyltransferase activity. We demonstrate that the PgaCD machinery is a novel type c-di-GMP receptor, where ligand binding to two proteins stabilizes their interaction and promotes enzyme activity. This is the first example of a c-di-GMP-mediated process that relies on protein–protein interaction. At low c-di-GMP concentrations, PgaD fails to interact with PgaC and is rapidly degraded. Thus, when cells experience a c-di-GMP trough, PgaD turnover facilitates the irreversible inactivation of the Pga machinery, thereby temporarily uncoupling it from c-di-GMP signalling. These data uncover a mechanism of c-di-GMP-mediated EPS control and provide a frame for c-di-GMP signalling specificity in pathogenic bacteria. Introduction Most bacteria are able to switch from a motile planktonic 'lifestyle' to growth in surface-associated multicellular communities known as biofilms. Within these structures, cells are encased in a self-produced extracellular polymeric matrix that is typically composed of proteinaceous adhesin factors, DNA and exopolysaccharides (EPS) (Branda et al, 2005; Flemming and Wingender, 2010). This complex biofilm structure is known to protect bacteria from antimicrobials, physical stresses and the predation by the host immune system. Bacterial biofilms are often associated with chronic infections and infection relapses causing health problems of growing importance (Costerton et al, 1999; Mah and O#x2027;Toole, 2001; Davies, 2003; Hall-Stoodley et al, 2004; Fux et al, 2005). The second messenger bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) plays a central role in integrating environmental and cellular cues to control this major bacterial 'lifestyle' transition by disfavouring single cell behaviour and by promoting biofilm formation. C-di-GMP is synthesized from GTP by diguanylate cyclases (DGCs) that harbour a conserved GGDEF domain (Paul et al, 2004) and is degraded to the linear dinucleotide pGpG by specific phosphodiesterases (PDEs) that harbour either a conserved EAL (Christen et al, 2005) or HD-GYP domain (Ryan et al, 2006; Hengge, 2009; Schirmer and Jenal, 2009). While DGCs and PDEs have been analysed in detail, both structurally and functionally, little is known about how c-di-GMP acts on downstream targets. Only a few c-di-GMP-specific receptor protein families have been described up to now, for most of which mechanistic details are lacking (Lee et al, 2007; Merighi et al, 2007; Christen et al, 2007; Duerig et al, 2009; Newell et al, 2011; Sondermann et al, 2011). In Escherichia coli, c-di-GMP regulates several cellular processes including EPS production, the biogenesis of fimbriae, flagellar-based motility and RNA degradation (Pesavento et al, 2008; Monteiro et al, 2009; Boehm et al, 2009; Tagliabue et al, 2010; Boehm et al, 2010; Paul et al, 2010; Fang and Gomelsky, 2010; Tuckerman et al, 2011; Povolotsky and Hengge, 2012). To colonize surfaces, E. coli produces the EPS poly-β-1,6-N-acetylglucosamine (poly-GlcNAc) (Wang et al, 2004). This linear homopolymer was implicated in biofilm formation in a wide variety of pathogenic bacteria including Staphylococcus spp. and Yersinia pestis, where it can promote virulence and contribute to survival in the animal host (Maira-Litrán et al, 2005; O'Gara, 2007; Cerca et al, 2007; Izano et al, 2007, 2008; Bobrov et al, 2008; Choi et al, 2009; Becker et al, 2009; Conover et al, 2010; Pérez-Mendoza et al, 2011; Yakandawala et al, 2011; Bentancor et al, 2012; Skurnik et al, 2012). In E. coli, poly-GlcNAc is synthesized and secreted by the envelope-spanning Pga machinery (Figure 1A), which is encoded by the pgaABCD operon (Wang et al, 2004). While PgaA and PgaB are required for poly-GlcNAc export, PgaC and PgaD are necessary for poly-GlcNAc synthesis (Figure 1A; Itoh et al, 2008). PgaA is an outer membrane porin that serves to translocate growing poly-GlcNAc chains to the cell surface (Itoh et al, 2008). PgaB is a putative outer membrane lipoprotein that deacetylates about 3% of the GlcNAc residues during poly-GlcNAc export (Wang et al, 2004; Itoh et al, 2008). PgaC is a processive β-glycosyltransferase (GT) of the GT-2 family that is located in the inner membrane and polymerizes poly-GlcNAc from activated UDP-GlcNAc precursor (Saxena and Brown, 1997; Wang et al, 2004; Itoh et al, 2008). The catalytic domain of GT-2 family members is exposed to the cytoplasm (Heldermon et al, 2001; Ciocchini et al, 2006; Bobrov et al, 2008) with sugar transfer through the cytoplasmic membrane being independent of an undecaprenyl phosphate lipid carrier (Gerke et al, 1998). Finally, PgaD is a small protein with two predicted N-terminal transmembrane helices. Its function is unknown and it does not show any obvious similarity to other protein families or domains. However, because PgaD is essential for poly-GlcNAc synthesis (Wang et al, 2004), it was suggested to assist the GT in polymerizing poly-GlcNAc (Itoh et al, 2008). Figure 1.C-di-GMP controls PgaD stability in a PgaC-dependent manner. (A) Schematic representation of the E. coli Pga machinery. See text for details. IM, inner membrane; PP, periplasm; OM, outer membrane. (B) Immunoblot analysis of 3 × Flag-tagged Pga proteins in the E. coli control strain and ΔydeH mutant. The native pga promoter (left panel) was replaced with the Para promoter (right panel). Expression of the araB-pgaA translational fusion was induced with 0.0002% L-arabinose. (C) PgaD levels depend on PgaC and c-di-GMP. Immunoblots of PgaD–3 × Flag are shown for the indicated mutant strains. Expression of pgaC was induced with 0.0002% L-arabinose (left panel) and with 0, 0.0002 and 0.2% L-arabinose (right panel). (D) Graph showing relative PgaD levels upon blocking protein biosynthesis in exponentially growing cells as an average of two independent experiments with standard deviations. Expression of the heterologous DGC dgcA and its active site mutant dgcAmut (D164N) was not induced (leaky expression). (E) Biofilm formation of strains carrying multiple deletions in genes predicted to encode DGCs. The Δ7 strain carries a total of seven deletions (ΔydeH, ΔycdT, ΔyegE, ΔyfiN, ΔyhjK, ΔydaM, ΔyneF). Error bars are standard deviations of the mean of 3–6 replicas. A representative data set (n=2) of the relative cellular c-di-GMP concentrations of the strains is indicated. n/a, not available; bld, below limit of detection. Inset: Immunoblot of PgaD–3 × Flag in the control strain and the Δ7 mutant. Figure source data can be found in Supplementary data. Download figure Download PowerPoint The expression of the E. coli pgaABCD operon is tightly regulated on multiple levels. Most importantly, pgaABCD translation is repressed by the action of the RNA binding protein CsrA (carbon storage regulator A) (Wang et al, 2005). This global regulator, whose activity is firmly regulated via a complex signal transduction cascade, antagonistically controls numerous cellular pathways. For example, it promotes motility, glycolysis and virulence, while repressing EPS production and gluconeogenesis (Romeo et al, 1993; Suzuki et al, 2006; Timmermans and Van Melderen, 2010; Romeo et al, 2012). In addition, CsrA inhibits the expression of ydeH and ycdT, two genes encoding DGCs (Jonas et al, 2008). The observation that YdeH stimulates poly-GlcNAc-dependent biofilm formation (Boehm et al, 2009) argued that the expression of this DGC and its target, the Pga machinery, is coupled via CsrA. YdeH and c-di-GMP were shown to control poly-GlcNAc biogenesis on a post-transcriptional level (Boehm et al, 2009), but the mechanism responsible for this induction is unknown. In this paper, we unravel a novel allosteric mechanism through which c-di-GMP stimulates poly-GlcNAc-dependent biofilm formation in E. coli. We show that c-di-GMP allosterically activates the PgaCD GT complex. We present genetic and biochemical evidence arguing that c-di-GMP binds to both inner membrane components of the Pga machinery, thereby mediating their productive interaction and the formation of an active GT complex. Finally, we demonstrate that in the absence of c-di-GMP PgaD is rapidly degraded, offering the means to shut-off the Pga machinery in response to a c-di-GMP trough and to temporarily uncouple it from c-di-GMP signalling in the absence of de novo synthesis of Pga components. These studies offer a molecular frame for the widespread c-di-GMP-based activation of bacterial EPS systems and provide the basis for signalling specificity of c-di-GMP-controlled systems. Results PgaD in vivo stability depends on c-di-GMP We have previously shown that PgaD steady-state protein levels are positively controlled by c-di-GMP on a post-transcriptional level (Boehm et al, 2009). This observation was used as an entry point to address the molecular mechanism of c-di-GMP-regulated poly-GlcNAc biogenesis. To mimic the induced state of the Csr regulon, all assays were done in a partial loss-of-function csrA::Tn5 mutant strain background (Romeo et al, 1993), which will be referred to as control strain throughout this work. In order to monitor all Pga complex components individually, 3 × Flag-tagged versions of PgaA, PgaB, PgaC and PgaD were constructed. In the absence of the DGC YdeH, the protein levels of PgaD were reduced, while the levels of the other three Pga proteins remained constant, regardless of whether the pga operon was expressed from its native promoter with the 5′ UTR of pgaA or from the L-arabinose-dependent Para promoter with the 5′ UTR of araB (Figure 1B; Supplementary Figure 1A). Moreover, PgaD levels were strongly reduced in a ΔpgaC mutant, but were restored in a c-di-GMP-dependent manner when pgaC was expressed in trans and were further increased upon overexpression of pgaC (Figure 1C). PgaD levels were still c-di-GMP-dependent in cells expressing a pgaC active site mutant (D256N), arguing that PgaC protein but not PgaC GT activity is required to stabilize PgaD (Supplementary Figure 1B). Finally, expression of the heterologous DGC dgcA (Christen et al, 2006) strongly elevated PgaD levels in a ΔydeH mutant, but only when pgaC was present (Supplementary Figure 1C). The above data indicated that PgaC and c-di-GMP together control PgaD levels post-translationally. To substantiate this and to demonstrate that the effect is specific for PgaD, pgaD was replaced with yfiR, an unrelated gene from Pseudomonas aeruginosa. The observation that YfiR levels failed to fluctuate in response to c-di-GMP availability excludes the possibility that PgaD levels respond to a c-di-GMP-controlled promoter or to translation initiation control elements within pgaABC (Supplementary Figure 1D). Next, in vivo protein stability of PgaD–3 × Flag was determined under different c-di-GMP concentrations upon blocking de novo protein biosynthesis in exponentially growing cells. While PgaD remained stable over time in strains with normal or increased c-di-GMP levels (control strain and ΔydeH mutant expressing dgcA), the protein was rapidly degraded in strains with low cellular c-di-GMP concentrations (ΔydeH mutant and ΔydeH mutant expressing an active site mutant of dgcA) (Figure 1D; Supplementary Figure 1E). In summary, these data suggest that c-di-GMP positively modulates PgaD protein stability in a PgaC-dependent manner. C-di-GMP and PgaD together promote poly-GlcNAc-dependent biofilm formation The E. coli csrA::Tn5 mutant strain (control strain) forms biofilms under laboratory conditions that fully depend on the EPS adhesin poly-GlcNAc (Wang et al, 2004). To test if c-di-GMP is essential for poly-GlcNAc-dependent biofilm formation, multiple genes coding for potential DGCs (each containing a GGDEF domain) were successively deleted. Concomitant deletions of the two CsrA-controlled genes ydeH and ycdT (Jonas et al, 2008) resulted in a drastic reduction of biofilm formation, while a strain carrying a total of seven deletions (ΔydeH, ΔycdT, ΔyegE, ΔyfiN, ΔyhjK, ΔydaM, ΔyneF) completely lost the ability to form biofilms (Figure 1E). This strain showed a strongly reduced cellular c-di-GMP level in comparison to the control strain (Figure 1E) and will be referred to as Δ7 strain throughout this work. Importantly, both biofilm deficiency and c-di-GMP level could be complemented by reintroducing only ydeH into the bacterial genome (Figure 1E), supporting the idea that YdeH represents the major DGC responsible for poly-GlcNAc induction under these conditions (Boehm et al, 2009). In line with the data described above, PgaD protein was not detectable in the Δ7 mutant (Figure 1E). While c-di-GMP is required for normal PgaD levels under physiological conditions, overexpression of pgaD resulted in a biofilm induction both in the presence and in the absence of YdeH (Supplementary Figure 1F). However, the ΔydeH mutant never reached the same level of biofilm formation as the control strain, arguing that PgaD and c-di-GMP are synergistically needed for optimal biofilm formation. C-di-GMP enhances PgaC–PgaD interaction One scenario that could explain PgaC-dependent PgaD stability is a direct interaction of the two membrane proteins. Co-immunoprecipitation experiments using detergent-solubilized membranes revealed that PgaC and PgaD indeed form a stable complex that was resistant to high salt concentrations and up to 2 M urea (Figure 2A). When overexpressed, PgaC and PgaD could be co-purified even from membranes of a Δ7 strain (Figure 2B), arguing that under these conditions c-di-GMP is no longer required for PgaD stability. Together, this suggested that PgaC and PgaD form a stable complex in the cytoplasmic membrane, the formation of which is mediated by c-di-GMP under physiological conditions. Figure 2.C-di-GMP enhances PgaC–PgaD interaction. (A) PgaC–6 × His and PgaD–3 × Flag co-immunoprecipitate from detergent-solubilized membranes. Anti-Flag and protein A (mock) IPs were analysed by immunoblots using antibodies against the specific tags. The protein fraction that failed to bind to the beads is indicated (sn, supernatant). 2 M urea was present during the IP procedure as indicated. (B) Co-immunoprecipitation of PgaC and PgaD–3 × Flag from detergent-solubilized membranes of control strain and Δ7 mutant cells overexpressing pgaC and pgaD. IP samples were analysed by Coomassie staining. HC and LC mark heavy and light chains of IgG. (C) Bacterial two-hybrid (BacTH) analysis of PgaC–PgaD interaction. Presence of T18 and T25 fusions is indicated. Zip indicates the leucine zipper positive control. (D) BacTH analysis of c-di-GMP-stimulated PgaC–PgaD interaction. Left panel: Interaction in the presence of a plasmid-borne copy of dgcA or its active site mutant dgcAmut (D164N). Alleles were induced with 0.2% L-arabinose. Right panel: Interaction in strains lacking the DGC YdeH or multiple DGCs (Δ7). See Supplementary Figure 2 for the quantification of interaction strengths. (E) A PgaCD fusion protein is fully functional. Biofilm formation and protein levels of 3 × Flag-tagged PgaD or PgaCD fusion protein (PgaCDf) are indicated for the control strain (black bars) and a ΔydeH mutant (grey bars). Error bars are standard deviations of the mean of 3–6 replicas. Figure source data can be found in Supplementary data. Download figure Download PowerPoint To test if c-di-GMP is involved in PgaC–PgaD interaction, a bacterial two-hybrid (BacTH) assay was used that is based on the interaction-mediated reconstitution of the split cAMP signalling pathway in E. coli (Karimova et al, 1998). In this assay, full-length PgaC and PgaD showed a robust interaction (Figure 2C), while all truncated variants (e.g., predicted cytosolic parts) were negative (Supplementary Table 2). The interaction was stimulated by the ectopic expression of the heterologous DGC dgcA (Figure 2D; Supplementary Figure 2). Conversely, a step-wise reduction of the cellular c-di-GMP pool gradually lowered the interaction strength. PgaC–PgaD interaction was weakened upon deletion of ydeH and abolished in the Δ7 strain (Figure 2D; Supplementary Figure 2). These data further support the idea that c-di-GMP stimulates PgaC–PgaD interaction or complex stability. The above results can be interpreted in two different ways. C-di-GMP could regulate poly-GlcNAc production by determining PgaD stability and availability. Alternatively, c-di-GMP could promote PgaC–PgaD interaction with PgaD instability and degradation being a consequence of complex disintegration at low c-di-GMP concentrations. To be able to distinguish between these two possibilities, PgaD was 'stabilized' under low c-di-GMP conditions by directly fusing its N-terminus to the C-terminus of PgaC. Surprisingly, the resulting pgaCD fusion construct (pgaCDf) was fully functional and able to complement biofilm formation of a ΔpgaCD mutant in a c-di-GMP-dependent manner (Figure 2E). But in contrast to PgaD, the level of the PgaCD fusion protein (PgaCDf) was unaltered in a strain with lower c-di-GMP concentrations (Figure 2E). These findings reinforce the notion of a direct interplay between PgaC and PgaD and imply that PgaD instability at low c-di-GMP levels is not the cause for Pga control, but may simply result from weak protein interactions under these conditions. These data raise the question why the homologues of PgaC and PgaD exist as two separate proteins in all bacteria harbouring this EPS biogenesis system (see below). C-di-GMP acts as an allosteric activator of PgaCD GT activity In order to test whether c-di-GMP acts as an allosteric activator for the PgaCD GT complex, an in vitro activity assay was developed with membranes containing PgaCD. GT activity was determined indirectly using a modified enzyme-coupled spectrophotometric assay (Baykov et al, 1988) or directly by measuring UDP-GlcNAc consumption. In agreement with earlier data demonstrating that both PgaC and PgaD are needed for poly-GlcNAc synthesis in vivo (Wang et al, 2004; Itoh et al, 2008), UDP-GlcNAc was only turned over to poly-GlcNAc and UDP by membranes of cells expressing pgaC and pgaD (Figure 3A). Following incubation of active membranes with substrate for several hours, a slimy and viscous reaction product was visualized by light microscopy (Figure 3B). Immunoblot analysis with an anti-poly-GlcNAc antibody confirmed the identity of the reaction product (Supplementary Figure 3A). Experiments to determine the substrate affinity of the PgaCD GT complex revealed a Km for UDP-GlcNAc of 270.5±37.2 μM (Figure 3C). To test if PgaCD GT activity is stimulated by c-di-GMP, initial reaction velocities were measured at varying c-di-GMP concentrations in the presence of a constant UDP-GlcNAc concentration of 50 μM. Under these conditions, c-di-GMP stimulated GT activity >20-fold and curve fitting indicated a c-di-GMP concentration for half-maximal initial velocity (Kact) of 62.2±7.2 nM (Figure 3D). This induction was highly specific as the addition of GTP failed to activate the enzyme and furthermore, the c-di-GMP-mediated activity was fully dependent on the PgaCD machinery (Supplementary Figure 3B). The basal enzymatic GT activity in the absence of exogenously added c-di-GMP correlated with the cellular c-di-GMP concentration of the strain used for pgaCD overexpression and membrane preparation. Almost no basal activity was detected for membranes originating from the Δ7 mutant (Supplementary Figure 3B). A Lineweaver-Burk plot analysis integrating initial reaction velocity data at different UDP-GlcNAc concentrations in the presence of a non-saturating and a saturating c-di-GMP concentration resulted in fitted lines converging close to the x axis, indicating that c-di-GMP affects the Vmax rather than the Km of the enzyme complex (Figure 3E). Figure 3.C-di-GMP allosterically stimulates PgaCD glycosyltransferase activity in vitro. (A) GT activity depends on an intact PgaCD complex. Enzyme activities were determined using control strain membranes containing PgaC, PgaD or both proteins in the presence (2 mM) or absence of the substrate UDP-GlcNAc. A representative data set of two independent experiments is shown. (B) Microscopic analysis of the viscous poly-GlcNAc reaction product. Membranes were incubated with 30 mM UDP-GlcNAc for 5 h at 30°C. Scale bars are indicated: 15 μm. (C) Determination of the PgaCD Km for UDP-GlcNAc. Membranes of a Δ7 mutant containing PgaC and PgaD were incubated with increasing concentrations of UDP-GlcNAc in the presence of 1 μM c-di-GMP. Data represent an average of two independent experiments with standard deviations. (D) Stimulatory effect of c-di-GMP on PgaCD GT activity (Kact). Membranes of a Δ7 mutant containing PgaC and PgaD were incubated with increasing concentrations of c-di-GMP in the presence of 50 μM UDP-GlcNAc. A representative data set of two independent experiments is shown. (E) Lineweaver-Burk plot analysis of PgaCD GT activity. Membranes of a Δ7 mutant containing PgaC and PgaD were incubated with increasing concentrations of UDP-GlcNAc in the presence of a non-saturating (0.03 μM) and a saturating (1 μM) c-di-GMP concentration. Negative reciprocal Km is indicated. A representative data set of two independent experiments is shown. GraphPad Prism was used for curve fitting and linear regression. a.u., arbitrary unit. Download figure Download PowerPoint In summary, these data strongly suggest that c-di-GMP acts as a direct allosteric activator of the PgaCD GT complex. Concomitant binding of c-di-GMP to both PgaC and PgaD The above in vitro assays argued for a direct role of c-di-GMP as an allosteric activator of PgaCD GT activity. To corroborate these findings, c-di-GMP binding to the PgaCD complex was tested by using a c-di-GMP capture compound (cdG-CC). This molecule consists of a c-di-GMP moiety that is asymmetrically modified at the 2′ hydroxyl of one ribose with a linker connecting to a photo-reactive and a biotin sorting group (Nesper et al, 2012). The PgaCD complex was specifically and competitively captured by the cdG-CC from membrane preparations (Figure 4A). An excess of c-di-GMP, but not GTP, gradually competed with cdG-CC binding. While the PgaCD complex and the PgaCD fusion protein were specifically pulled down, no specific binding was observed when membranes were used that only contained PgaC or PgaD (Figure 4B). Although some residual binding to the cdG-CC was observed under these conditions, the addition of an excess of c-di-GMP failed to compete with this interaction (Figure 4B). When membranes were used that contained 3 × Flag-tagged variants of both PgaC and PgaD, both proteins showed specific cdG-CC binding. A fraction of the PgaC–PgaD heterodimers withstood boiling in SDS sample buffer and appeared as a distinct band on the immunoblot, emphasizing the remarkable stability of these complexes (Figure 4B). Probing cdG-CC samples with an antibody against the biotin moiety of the capture compound revealed that the cdG-CC was covalently crosslinked to both PgaC and PgaD in a competitive way, suggesting that c-di-GMP is able to directly interact with both components of the complex (Supplementary Figure 4A and B). Figure 4.Specific binding of c-di-GMP requires PgaC and PgaD. (A) Immunoblot of PgaD captured from membranes containing PgaC and PgaD–3 × Flag. Presence of cdG-CC and competing nucleotides is indicated. (B) Immunoblots of PgaC, PgaD and PgaCD fusion protein (PgaCDf) captured from membranes containing PgaC and PgaD–3 × Flag (first panel), PgaCDf–3 × Flag (second panel), PgaC–3 × Flag (third panel), PgaD–3 × Flag (fourth panel) or PgaC–3 × Flag and PgaD–3 × Flag (fifth panel). Presence of cdG-CC and competing nucleotides is indicated. SDS-resistant heterodimeric PgaCD complexes are indicated (PgaCD). (C) Specific labelling of PgaC and PgaD with [33P]c-di-GMP. Membranes containing PgaC–3 × Flag and PgaD–3 × Flag were UV crosslinked in the presence of [33P]c-di-GMP and competing nucleotides as indicated. Coomassie staining (left panel) and autoradiography (right panel) are shown. HC and LC mark heavy and light chains of IgG. SDS-resistant heterodimeric PgaCD complexes are indicated (PgaCD). (D) Absence of PgaD abolishes c-di-GMP binding. Membranes containing PgaC–3 × Flag and PgaD–3 × Flag (left panels) or PgaC–3 × Flag (right panel) were UV crosslinked in the presence of [32P]c-di-GMP and competing nucleotides as indicated. Only autoradiographies are shown. Figure source data can be found in Supplementary data. Download figure Download PowerPoint To corroborate these findings, UV light-induced crosslinking experiments with radiolabelled c-di-GMP were performed (Christen et al, 2006). In good agreement with the data obtained with the capture compound, PgaC and PgaD were specifically and competitively labelled with [33P]c-di-GMP when both proteins were present in the membrane fraction (Figure 4C). An excess of c-di-GMP, but not GTP, efficiently outcompeted the [33P]c-di-GMP crosslink to both proteins. It is interesting to note that PgaC labelling was generally much stronger than PgaD labelling. Again, specific c-di-GMP binding and radiolabelling was only observed in membranes containing both proteins, but was lost for PgaC when PgaD was not present (Figure 4D). Interestingly, the presence of the substrate UDP-GlcNAc increased the specific binding of c-di-GMP, indicating some form of communication between the GT active site and the allosteric c-di-GMP binding pocket within the PgaCD complex (Supplementary Figure 4C and D). Altogether, these data suggest that the PgaCD GT complex represents a novel type c-di-GMP receptor, where ligand binding to two individual proteins promotes their stable interaction and subsequent activation. Constitutive mutations in pgaD uncouple PgaCD activity from c-di-GMP To more closely define the c-di-GMP binding site in PgaD, variants with C-terminal truncations were analysed for their ability to stimulate biofilm formation. Although biofilm formation gradually decreased with deletions extending towards the second transmembrane helix, c-di-GMP stimulation was sustained in truncations extending to amino acid R78 (Figure 5A and B). This argued that c-di-GMP binds to a region within the first 78 amino acids of PgaD consisting of only two transmembrane helices with short flanking regions in the cytoplasm, thus suggesting that c-di-GMP modulates the interaction of PgaC and PgaD in the vicinity of the cytoplasmic membrane. To test this hypothesis, we set up a genetic screen to isolate mutations in pgaC and pgaD that facilitate biofilm formation in the absence of c-di-GMP. Error-prone PCR mutagenesis and screening for biofilm-forming colonies in the Δ7 strain using Congo Red agar plates led to the isolation of several constitutive mutants (Supplementary Table 3). With one exception, all mutations in PgaD clustered within a short conserved region between the second transmembrane helix and residue R78 (Figure 5A). Two of the activating pgaD alleles (N75D,K76E and L73Q,K76E,R78C) firmly locked biofilm formation at an intermediate level independently of the availability of c-di-GMP (Figure 5C). In both cases, this constitutive phenotype required the presence of multiple
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