the inhibitory site of a diguanylate cyclase is a necessary element

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The Inhibitory Site of a Diguanylate Cyclase is a Necessary Element for Interaction and 1 Signaling with an Effector Protein 2

3 4 Kurt M. Dahlstrom1, Krista M. Giglio2, Holger Sondermann2, and George A. O’Toole1,* 5 6 1Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, 7 Hanover, New Hampshire 03755. 8 2Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, 9 New York 14853. 10 11 *To whom correspondence should be addressed 12 Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth 13 Rm 202 Remsen Building, Hanover, NH 03755 14 E-mail: [email protected] 15 Tel: (603) 650-1248 16 Fax: (603) 650-1728 17 18 Key words: DGC, GGDEF, inhibitory-site, c-di-GMP, signaling, biofilms, receptor, diguanylate 19 cyclase 20 21 22

JB Accepted Manuscript Posted Online 21 March 2016J. Bacteriol. doi:10.1128/JB.00090-16Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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Abstract 23 Many bacteria contain large cyclic diguanylate (c-di-GMP) signaling networks made of 24 diguanylate cyclases (DGCs) and phosphodiesterases that can direct cellular activities sensitive 25 to c-di-GMP levels. While DGCs synthesize c-di-GMP, many DGCs also contain an auto-26 inhibitory site (I-site) that binds c-di-GMP to halt excess production of this small molecule, thus 27 controlling the amount of c-di-GMP available to bind to target proteins in the cell. Many DGCs 28 studied to date have also been found to signal for a specific c-di-GMP related process, and 29 although recent studies have suggested that physical interaction between DGCs and target 30 proteins may provide this signaling fidelity, the importance of the I-site has not yet been 31 incorporated into this model. Our results from Pseudomonas fluorescens indicate that mutating 32 residues at the I-site of a DGC disrupts interaction with its target receptor. By creating varying 33 substitutions to a DGC’s I-site, we show that signaling between a DGC (GcbC) and its target 34 protein (LapD) is a combined function of I-site dependent protein-protein interaction and level of 35 c-di-GMP production. The dual role of the I-site in modulating DGC activity as well as 36 participating in protein-protein interactions suggests caution in interpreting the function of the I-37 site as only a means to negatively regulate a cyclase. These results implicate the I-site as an 38 important positive and negative regulatory element of DGCs that may contribute to signaling 39 specificity. 40 Importance. Some bacteria contain several dozen diguanylate cyclases (DGCs), nearly all of 41 which signal to specific receptors using the same small molecule, c-di-GMP. Signaling 42 specificity in these networks may be partially driven by physical interaction between DGCs and 43 their receptors, in addition to the auto-inhibitory site of DGCs preventing over-production of c-44 di-GMP. In this study, we show that disrupting the auto-inhibitory site of a DGC in 45


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Pseudomonas fluorescens can result in loss of interaction with its target receptor and reduced 46 biofilm formation, despite increased production of c-di-GMP. Our findings implicate the auto-47 inhibitory site as an important feature for signaling specificity both through regulation of c-di-48 GMP production, and as a necessary element for physical interaction between a diguanylate 49 cyclase and its receptor. 50


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Introduction 51 A major method of intracellular signaling in bacteria revolves around the second 52 messenger cyclic diguanylate (c-di-GMP). C-di-GMP is produced by diguanylate cyclases 53 (DGCs) and degraded by phosphodiesterases (PDEs) in networks that may range from one or a 54 few such proteins to several dozen (10). Effector proteins then sense c-di-GMP by binding it, and 55 promote some specific cellular action as a result. Importantly, many DGCs studied to date 56 promote only one or a small number of such processes (11, 14-16, 22), indicating that there must 57 be mechanisms in place that allow for specificity in signaling among the many members of a c-58 di-GMP network. 59

A key regulatory element in signaling specificity is the auto-inhibitory site (I-site) found 60 on many DGCs that locks the enzyme in an inactive state when bound to c-di-GMP. Many, but 61 not all, DGCs have a characteristic I-site that provides this kind of negative feedback regulation, 62 which has been described as characteristic of systems that require fast up-kinetics in their 63 enzymatic reactions (5). Studies focused on disrupting the I-site have previously found that 64 deregulation of a DGC leads to increased production of c-di-GMP and a concomitant increase in 65 c-di-GMP related phenotypes including increases in biofilm formation and decreases in motility 66 (5, 12). Additionally, effectors have been thought to provide one mechanism of signaling 67 specificity by differentially activating at varying levels of c-di-GMP as demonstrated in 68 Salmonella enterica Typhimurium (24), with a complementary study identifying varying 69 response curves for c-di-GMP dependent processes in Caulobacter crescentus (1). The apparent 70 function of the I-site is therefore twofold. First, the I-site allows for a rapid enzymatic response 71 to changing environmental factors in that the rate of c-di-GMP synthesis can be manipulated 72 without having to translate or degrade a given DGC. Second, the I-site can limit the total amount 73


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of c-di-GMP available to be read by effector proteins, providing one level of signaling 74 regulation. 75

Recently, a theme that has emerged in signaling specificity for large c-di-GMP networks 76 is physical interaction between DGCs, PDEs, and effectors (6, 15). By using physical proximity, 77 DGCs can successfully direct their signal to a specific effector target. It has remained an open 78 question to what extent, if any, this mode of c-di-GMP regulation is interwoven with the I-site-79 mediated control of DGC activity to govern signaling fidelity. 80

One example of signaling specificity achieved by physical interaction is the regulation of 81 biofilm formation by the inner membrane proteins GcbC and LapD in Pseudomonas fluorescens. 82 GcbC is a DGC that signals to the effector LapD. When bound to c-di-GMP, LapD changes 83 conformation to sequester a periplasmic protease called LapG, thus allowing the large adhesin 84 LapA to accumulate on the cell surface and thereby promoting biofilm formation (4, 20, 21). A 85 previous study from our lab used a mutagenic screen to identify a string of amino acids on the 86 surface of GcbC that appears to interact with a surface-exposed α-helix of LapD (6). While this 87 result paints a partial picture of how these two proteins interact, the residues identified to date are 88 not sufficient to account for the entirety of the interaction observed between these two proteins. 89

In this study, we demonstrate that mutations made to residues in the I-site of GcbC 90 disrupt interaction with LapD. Further, we find that I-site mutations that disrupt interaction 91 between GcbC and LapD fall into two groups. One group of mutations to the I-site causes large 92 spikes in c-di-GMP production resulting in the typical phenotype of an increase in biofilm 93 formation. The other group of mutations to the I-site results in reduced biofilm formation despite 94 similar, or modestly increased, c-di-GMP levels compared to the wild-type strain. These results 95


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suggest an important role for the I-site of GcbC in controlling c-di-GMP production, as well as 96 directly or indirectly mediating physical interaction with LapD. Collectively, this study 97 reinforces a model whereby the activation of some effector proteins is a combined function of c-98 di-GMP production and physical interaction, and adds to it evidence that the I-site is relevant to 99 mediating both of these processes. 100 Materials and Methods 101 Strains and Media: P. fluorescens Pfl01, P. aeruginosa PA14, and E. coli BTH101 were used 102 in this study (Table S1). E. coli S17-1 was used for cloning (26). P. aeruginosa and E. coli were 103 grown at 37o C except where indicated in the text. P. fluorescens was grown at 30 o C. All 104 overnight growth used lysogeny broth (LB), and all plates used 1.5% agar. The expression vector 105 pMQ72 was used for all biofilm, Congo red, and c-di-GMP quantification assays and was 106 induced by arabinose as described in the text. The expression vectors pUT18 and pKNT25 were 107 used for bacterial two-hybrid experiments and were induced with 0.5 mM isopropyl -D-1-108 thiogalactopyranoside (IPTG). Gentamycin (Gm) was used in conjunction with pMQ72 at 30 109 µg/ml for P. fluorescens on solid agar and 15 µg/ml in liquid, and 50 µg/ml for P. aeruginosa. 110 Kanamycin (Kn) and carbenicillin (Cb) were used at concentrations of 50 µg/ml in conjunction 111 with pKNT25 and pUT18, respectively. All sub-culturing was the result of picking a single P. 112 fluorescens colony and growing the culture overnight in 4 ml of LB with the appropriate 113 antibiotics before inoculating the sub-culture at a 1:75 ratio. Sub-cultures of 5 ml were grown on 114 a rotating wheel, while sub-cultures of 45 ml were grown in 250 ml flasks on a platform shaker 115 at 230 rpm. See Supplemental Tables 1 for a complete list of strains and plasmids, and 116 Supplemental Tables 2 for a list of primers used in this study. 117


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Bacterial Two-Hybrid Studies: BTH101 E. coli cells were co-electroporated with 50 ng of each 118 pUT18 and pKNT25 plasmid. Cells were recovered in LB at 37o C for 1 hr, and plated at a 1:4 119 dilution on LB plates amended with Kn, Cb, and IPTG. Plates were incubated at 30 o C for 15 120 hours and colonies were recovered from the plate by scraping. Recovered cells were diluted to an 121 optical density of approximately 0.5 at 600 nm, and a β-galactosidase assay was conducted as 122 described previously (6), on a published system (13). Briefly, cells were permeabilized using 123 chloroform and SDS followed by vortexing and a five minute incubation at 30o C. 800 µg of 124 ortho-Nitrophenyl-β-galactoside (ONPG) was added to each sample followed by another five 125 minute incubation at 30o C. The reaction was stopped using 500 µl of 1M Na2CO3, and the 126 optical density was read at 420 and 550 nm to calculate Miller Units. Each biological replicate 127 represents an independent co-transformation of BTH101 cells. 128 Congo Red Assay: Pseudomonas aeruginosa PA14 was transformed with various GcbC alleles 129 on the pMQ72 backbone. 1 ml of overnight culture was washed twice with 1 ml 20% sucrose, 130 before a final resuspension in 100 µl of 20% sucrose after which the desired plasmid was added 131 to 40 µl of the washed sample and electroporated, following a 1 hr recovery period in LB at 37o 132 C. The resulting transformants were picked from selective agar and grown in overnight LB 133 cultures supplemented with Gm, and 2.5 µl of culture was spotted onto 1.0% agar minimal 134 medium plates amended with Congo red dye. Plates were grown at 30o C for 36 hours. 135 Biofilm Assay: The biofilm assay was conducted as described previously (23). Briefly, strains of 136 P. fluorescens were grown overnight in 4 ml of liquid LB supplemented with Gm from LB agar 137 plates. Samples were normalized to cell optical density and 1.5 µl of normalized culture was 138 added to 100 µl of K10T-1 minimal medium in a 96 well U-bottom microtiter plate (Costar). 139 Plates were incubated inside a humidified chamber at 30o C for 6 hrs, after which supernatant 140


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was decanted from the wells and stained for 20 minutes with 125 µl of 0.1% crystal violet, 141 followed by three washes in distilled water and drying overnight. Wells were then photographed 142 and dissolved for quantification using 150 µl of a de-stain solution (45:45:10 143 water:methanol:glacial acetic acid). 125 µl was transferred to a flat-bottomed 96 well plate to 144 read at OD 550 nm. 145 Quantification of c-di-GMP: Strains of P. fluorescens were struck on LB plates supplemented 146 with Gm, and colonies were picked for overnight growth in liquid LB supplemented with Gm. At 147 16 hours, 200 µl of the overnight culture was added to 5 ml of K10 minimal medium 148 supplemented with 0.2% arabinose. The sub-cultures were aerated and grown for 6 hrs at 30o C. 149 Samples were then pelleted and nucleotides were extracted using 0.5 ml of an extraction buffer 150 (40:40:20 methanol:acetonitrile:ddH2O + 0.1 N formic acid) at -20o C for 1 hr as described 151 previously (18). Samples were then centrifuged, and 8 µl of NH4HCO3 was added to the 152 supernatant, followed by drying under a vacuum to dryness. Samples were resuspended in 200 µl 153 of HPLC-grade water. Samples were analyzed by LC-MS via the Michigan State University 154 Mass Spectrometry Facility, compared to standard curve of known c-di-GMP concentration, and 155 results were normalized to the dry weight of the bacterial pellets that generated the extractions. 156 Each biological replicate represents a separate colony picked from the struck out plate. 157 Western Blot Analysis: Overnight cultures of P. fluorescens were sub-cultured 1:75 in 45 ml of 158 K10 minimal medium supplemented with 0.1% arabinose. Sub-cultures were shaken for 6 hrs 159 and pelleted by centrifugation followed by resuspension in lysis buffer (20 mM Tris pH 8.0 and 160 25 mM MgCl2) and sonication. Samples were normalized to protein concentration via BCA 161 assay, and 6 µg of protein sample was loaded onto a 10% polyacrylamide gel. Blotting was 162 conducted with an anti-HA antibody (Covance) at 4o C overnight in 3% BSA. 163


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Statistics: Error bars throughout the study represent standard deviation, and p values were 164 calculated using a two-tailed t-test. 165


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Results 166 The I-site of GcbC is necessary for interaction with LapD. A previous study from our 167 lab reported the structure for the GGDEF domain of the P. fluorescens diguanylate cyclase GcbC 168 (6). Using this crystal structure, we are able to verify that GcbC likely has a primary inhibitory 169 site (Fig. 1A-C) with the previously characterized RXXD motif (3, 5). The RXXD motif is found 170 at residues R410 to D413 of GcbC. Notably, R409 also makes contact with a c-di-GMP 171 molecule; the residue at position 409 is not highly conserved among DGCs, and it is somewhat 172 unusual for it to participate in c-di-GMP binding. In addition to the primary I-site, GcbC also 173 contains a secondary I-site at position R366 that completes GcbC’s regulatory c-di-GMP binding 174 site (Fig. 1A-C). In our crystal structure of GcbC bound to c-di-GMP, two molecules of c-di-175 GMP were found to be bound between the primary and secondary I-sites of this inactive dimer 176 (6), following a convention described for other DGCs known to contain both a primary and 177 secondary I-site (3, 7-9, 25). Finally, three more residues were found to coordinate the formation 178 of the I-site pocket where R363 of each monomer of GcbC forms a polar contact with residues 179 E360 and E429 of the other GcbC monomer within the dimer complex of this protein (Fig. 1A-180 C). 181

This crystal structure was previously used to understand how the diguanylate cyclase 182 GcbC physically interacts with the effector protein LapD in order to promote biofilm formation. 183 Specifically, a mutagenic bacterial two-hybrid (B2H) screen was conducted to identify alleles of 184 GcbC that failed to interact with LapD (6). In this assay, wild-type versions of GcbC and LapD 185 interact as judged by the blue color formation on medium supplemented with X-gal (see 186 Materials and Methods for details of the B2H assay). E. coli containing the wild-type allele of 187 the lapD gene and mutagenized library of gcbC alleles were screened on plates supplemented 188


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with X-gal, and colonies that failed to turn blue were identified. The gcbC-containing plasmids 189 from these colonies were isolated and sequenced, yielding 15 isolates carrying mutations in the 190 gcbC gene that failed to interact normally with LapD (6). While some of these isolates provided 191 information that led to the identification of a string of amino acids on a surface-exposed α-helix 192 of GcbC that contacts a similarly surface-exposed -helix of LapD, not all mutations mapped to 193 these helical regions. Furthermore, the previously identified residues on these surface-exposed α-194 helices of GcbC and LapD were not sufficient to fully account for binding between these two 195 proteins (6). 196

Suspecting that interaction between GcbC and LapD may depend upon additional 197 structural elements, we reanalyzed the mutagenic dataset using the crystal structure of GcbC. A 198 surprising finding was the number of isolates that implicated the GcbC I-site as a necessary 199 element of interaction. Of the original 15 mutants isolated in our screen, 7 had mutations that 200 map near the I-site of GcbC (Table 1). Two isolates with mutations near the primary I-site were 201 found, R402W and I404T, and five isolates were identified with mutations in the vicinity 202 containing the secondary I-site residue and the residues that interact in the inhibited GcbC 203 GGDEF domain dimer, including Q355R, S356P, and W361R (Fig. 1B-C). The Q355R and 204 W361R mutations were found twice each, suggesting that these specific mutations are 205 responsible for the observed phenotype among the multiple mutations found in the isolates. 206

The mutations identified could feasibly disrupt the I-site through both structural changes 207 or via the result of substitutions that directly interfere with a nearby residue’s ability to bind c-di-208 GMP, which led us to hypothesize that the I-site of GcbC is necessary for interaction with the 209 effector LapD. However, because most isolates from our screen contained multiple mutations, 210 and because the exact residues mutated could not be directly shown to impact the GcbC I-site, 211


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we made targeted, non-conservative substitutions to several canonical I-site residues to verify by 212 bacterial two-hybrid that disruption of the I-site residues results in a decrease in interaction 213 between GcbC and LapD. 214

The use of the B2H assay has been previously validated for the GcbC-LapD interaction 215 through a coprecipitation analysis (6), thus we used this method to assess the impact of the 216 directed I-site mutations on interaction of these two proteins. Three I-site substitutions were 217 initially identified in GcbC that resulted in reduced interaction with LapD. GcbC-E429R 218 demonstrated a modest, non-significant reduction in interaction with LapD, while GcbC proteins 219 with mutations in E360R and D413K showed almost complete loss of interaction with LapD (Fig 220 2A). Residues E360 and E429 are engaged in a protein-protein interface between two monomers 221 of GcbC, which is a prerequisite to form the inactive, c-di-GMP-bound dimer, while D413 is part 222 of the primary I-site and binds c-di-GMP directly. The decrease in interaction between LapD and 223 GcbC associated with these changes to I-site residues in GcbC suggest that disruption either of 224 GcbC’s ability to form the I-site or the I-site’s ability to bind c-di-GMP leads to loss of 225 interaction with LapD. Importantly, a previous study demonstrated that neither GcbC’s ability to 226 produce c-di-GMP nor LapD’s ability to bind c-di-GMP were required for protein-protein 227 interaction, eliminating the catalytic state of GcbC as a confounding factor to interaction between 228 these two proteins (6). 229 I-site mutations in GcbC result in decreased biofilm formation. While disruption to 230 the I-site of GcbC lead to an apparent loss of interaction with LapD, we wanted to directly test 231 what impact these mutations would have on the functional relationship between these two 232 proteins in terms of their ability to govern biofilm formation. We therefore set out to test biofilm 233 formation when GcbC has a mutated I-site using the mutated alleles described in the previous 234


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section. To this end, we built HA-tagged versions of wild-type and the mutant versions of GcbC 235 on a plasmid and expressed these constructs in a background of P. fluorescens lacking its native 236 DGC sub-network that normally governs biofilm formation, referred to as the ∆DGC strain. In 237 this way, the wild-type or mutant version of GcbC would be the sole DGC responsible for 238 promoting biofilm formation in our assay conditions (22). 239

We found that in each case examined, variants of GcbC mutated in the I-site resulted in a 240 significant decrease in biofilm formation compared to the wild-type GcbC (Fig. 2B), suggesting 241 that the loss of physical interaction between GcbC and LapD as a result of the disrupted I-site is 242 physiologically important to in vivo functionality of these two proteins. To confirm that that the 243 mutant versions of GcbC are stably expressed, whole-cell lysates normalized by protein 244 concentration were examined by Western blot. In each case, the wild-type and mutant variants of 245 GcbC appeared to be stably expressed at a level comparable to wild-type GcbC (Fig. 2C), 246 indicating that protein instability is not responsible for the loss of biofilm formation observed. 247

Previous studies have suggested that disruption to a DGC’s I-site leads to an inability to 248 form the inactive dimer conformation (9), and that a DGC will still readily adopt its active dimer 249 conformation, leading to c-di-GMP over-production (12). However, we could not immediately 250 rule out the possibility that the mutant variants of GcbC experienced a loss of ability to produce 251 c-di-GMP, and thus their catalytic ability is assessed below. 252 Disruption of the I-site of GcbC leads to deregulation of c-di-GMP production. Our 253 initial results that mutating the I-site of GcbC reduced biofilm formation was somewhat 254 surprising, given that other studies have found I-site disruption leads to over-production of c-di-255 GMP and enhanced biofilm formation relative to proteins with functional I-sites (5, 12). To 256


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analyse the impact of these I-site mutations on c-di-GMP levels, we employed two different 257 assays. First, we over-expressed each allele of GcbC heterologously in Pseudomonas aeruginosa 258 PA14 and grew the resulting transformants on a minimal medium supplemented with Congo red. 259 This assay has been shown to detect c-di-GMP production via c-di-GMP dependent Pel 260 polysaccharide production; this polysaccharide binds to the Congo red dye resulting in a red 261 colony. P. aeruginosa was used for this experiment due to its ability to produce Pel in response 262 to c-di-GMP, which is a process P. fluorescens lacks. Expression of GcbC produces a very light 263 red color compared to wild-type P. aeruginosa (Fig. 3A). The D413K substitution in the I-site 264 produced a somewhat darker red tint compared to wild-type GcbC. Furthermore, the E360R and 265 E429R substitutions produced very pronounced red colors, consistent with elevated levels of c-266 di-GMP production (Fig. 3A). 267 We next wanted to test c-di-GMP production in vivo to determine relative amounts of c-268 di-GMP produced in P. fluorescens carrying wild-type GcbC or the I-site mutant variants. We 269 again grew each strain in a minimal medium and overexpressed each allele of GcbC, performed a 270 nucleotide extraction on the resulting cells, and analyzed the resulting samples using mass 271 spectrometry for c-di-GMP levels normalized to total cellular dry weight. We saw similar levels 272 of c-di-GMP produced by the wild-type strain and the ∆DGC strain expressing GcbC from a 273 plasmid (Fig. 3B). We further saw a significant reduction in c-di-GMP levels for the ∆DGC 274 strain lacking its native DGC sub-network compared to the ∆DGC strain expressing GcbC from 275 a plasmid. Conversely, the strain carrying the pGcbC-GGAAF plasmid, wherein the catalytic 276 residues are mutated to prevent c-di-GMP production, as reported previously (22), showed no 277 increase in c-di-GMP production compared to the ∆DGC strain (Fig. 3B). The D413K mutant 278 produced a similar level of c-di-GMP as wild-type GcbC (Fig. 3B). The E429R mutant showed a 279


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small but non-significant increase in c-di-GMP levels compared to the strain expressing the wild-280 type GcbC, while the E360R mutant showed a significant increase in c-di-GMP production 281 compared to the wild-type GcbC, mirroring the results found in the Congo red assay (Fig. 3A & 282 3B). Taken together, these results suggest that these mutations made to the I-site of GcbC do not 283 result in lower levels of biofilm formation due to lower c-di-GMP production, and that the 284 individual mutations result in levels of c-di-GMP production ranging from 1 to 1.6 times that of 285 the wild-type GcbC. 286 Targeted I-site mutations result in elevated levels of biofilm formation. For two of 287 the I-site mutants we constructed (E429R and E360R), there appears to be reduced biofilm 288 formation despite elevated levels of c-di-GMP production. Interestingly, previous studies have 289 found disruptions to DGC I-sites to result in an increase in both c-di-GMP and a concomitant 290 increase in c-di-GMP regulated processes. Given that our results show a DGC I-site mutant that 291 produces more c-di-GMP is less able to stimulate biofilm formation, we wanted to better 292 understand the relationship between the necessity of physical interaction for productive signaling 293 and the amount of c-di-GMP produced. 294

The mutations to the I-site we have tested up to this point created relatively modest (<2-295 fold) increases in total cellular c-di-GMP. Inspection of the crystal structure of GcbC indicated 296 that residue D413 coordinates the base of only one of the c-di-GMP molecules of the intercalated 297 c-di-GMP dimer bound at the I-site. In contrast, close-by residues R409 and R410 coordinate the 298 same base as D413 but these arginine residues are also involved in π-stacking interactions 299 involving the entire c-di-GMP dimer (Fig. 1B). Hence, one would expect that mutating residue 300 D413 may have a more moderate effect than mutations at positions R409 or R410, or that these 301 residues are in part functionally redundant and thus only yielding partial effects. Residues E429 302


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and E360 each contribute to protein-protein interactions between GcbC protomors in the dimer, 303 again having an overlapping function with each other, possibly reducing the impact of either 304 mutation alone. We hypothesized that if we made more severe disruptions to the I-site of GcbC, 305 we would create a DGC that over-produced c-di-GMP to a greater degree. Previous studies 306 established that mutations at the secondary I-site also yielded a more active DGC (8). We 307 therefore selected residues R366 and R363 for mutagenesis studies. R366 is the sole residue in 308 the secondary I-site of GcbC contacting c-di-GMP, and this residue engages in interactions with 309 both c-di-GMP molecules, suggesting that its disruption would have a profound effect on c-di-310 GMP binding and DGC regulation (Fig. 1B). R363 was chosen as it is intimately involved in 311 GbcC protein-protein interactions, similar to residues like E360 and E429 (Fig. 1B). In fact, 312 R363 interacts directly with both E360 and E429, rendering this position ideal for a targeted 313 mutation predicted to severely interfere with the formation of an inactive, c-di-GMP-binding-314 competent conformation. We therefore tested R366E and R363E mutant variants of GcbC for 315 their impact on LapD-GcbC protein-protein interaction and biofilm disruption. 316 Since our previous results suggest that the ability to form the I-site is necessary for GcbC 317 to interact with LapD, we hypothesized that these mutations would result in minimal interaction 318 with LapD. When tested by bacterial two-hybrid, we saw significant reduction in interaction 319 between these mutant versions of GcbC and the wild-type LapD proteins (Fig. 4A). We next 320 tested these GcbC mutants for their ability to promote biofilm formation in vivo from the same 321 expression construct used previously. Both R366E and R363E resulted in a significant increase 322 in biofilm formation, suggesting that more severe disruptions to the I-site of GcbC result in the 323 expected finding of increased c-di-GMP resulting in increased biofilm formation (Fig. 4B). 324


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Finally, we again tested these mutant proteins for expression stability by Western blot; each 325 mutant appeared to be as stably expressed as wild-type GcbC (Fig. 4C). 326 Mutations R366E and R363E at the I-site of GcbC result in significant increase in c-327 di-GMP production. We next wanted to verify that the reason the R366E and R363E mutations 328 behave more like other reported I-site mutations was in fact due to an increase in c-di-GMP 329 production. To this end, we again used the Congo red assay by heterologously expressing each 330 mutant construct in P. aeruginosa PA14. We found that the colony biofilms produced by the 331 mutant variants of GcbC were considerably more red and wrinkly than the wild-type GcbC-332 expressing colonies, indicating much higher levels of c-di-GMP production (Fig. 5A). To 333 confirm c-di-GMP over-expression in vivo in P. fluorescens, we subcultured strains expressing 334 each construct, and again performed a nucleotide extraction that was analyzed by mass 335 spectrometry. We found that strains carrying either the R366E or R363E mutations produced 336 levels of c-di-GMP that were approximately 16 and 20 times higher, respectively, than for those 337 strains expressing the wild-type GcbC (Fig. 5B). These results suggest that more severe 338 mutations at the I-site of GcbC results in greater levels of c-di-GMP production than when 339 mutating I-site residues with potentially more subtle and perhaps redundant functions, a finding 340 consistent with the biofilm results discussed above.341


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Discussion 342 Physical interaction is an emerging theme in DGC signalling specificity. In E. coli, the 343

DGC YdaM has been shown to physically interact with and stimulate the Mer-Like transcription 344 factor MlrA (15). Additionally, the DGC HmsT and PDE HmsP co-localize to impact biofilm 345 development in Yersinia pestis (2). In this study we demonstrated that GcbC must be able to 346 form an intact auto-inhibitory site in order to properly interact with and functionally signal to the 347 c-di-GMP receptor LapD. We determined that nearly any kind of disruption to GcbC’s I-site – be 348 it to the primary I-site, secondary I-site, or residues supporting dimerization of the inactive, c-di-349 GMP bound GcbC – results in a decrease in interaction with LapD. We further demonstrated that 350 while mutations in the I-site of GcbC predicted to result in loss of binding of this dinucleotide 351 based on previous studies (3, 5) resulted in a canonical increase in c-di-GMP levels and biofilm 352 formation, mutating I-site residues with predicted less severe c-di-GMP-binding defects resulted 353 in reduced biofilm formation despite production of equal or greater amounts of c-di-GMP 354 compared with the wild-type DGC. We also note that interaction between GcbC and LapD 355 appears to be very sensitive to any change made to the GcbC I-site, likely causing loss of a 356 specific signaling between this DGC and its effector. As we make mutations with greater impact 357 on I-site function, more c-di-GMP is produced, which correlates with higher promotion of 358 biofilm formation. This increase in biofilm formation may occur by activating other targets 359 including motility regulators, or may directly compensate for loss of GcbC-LapD interaction 360 through production of very high levels of c-di-GMP. We further note that there does not appear 361 to be any correlation between c-di-GMP produced, and the ability of GcbC to interact with 362 LapD. Our data suggest a model whereby, in addition to its known role in regulating c-di-GMP 363


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output, the I-site also regulates signaling specificity in that it is required for GcbC to functionally 364 interact with its receptor. 365

An important question remaining is how the I-site of GcbC physically contributes to the 366 protein-protein interaction of GcbC and LapD. While our results support a model that makes the 367 formation of a functional I-site necessary for GcbC-LapD interaction, it is not immediately 368 apparent how these residues are contributing to the interaction. One possibility is that GcbC must 369 enter its auto-inhibited conformation in order to appropriately interact with LapD. If this model 370 were correct, it would strongly tie regulation of the cyclase activity by c-di-GMP to interaction 371 with its effector. Perhaps linking such control of DGC activity to receptor interaction allows the 372 tuning of enzymatic activity or product availability to the level necessary to signal specifically to 373 LapD without signaling to other potential targets, thus providing one possible explanation for 374 how dozens of cyclases may signal simultaneously in the same cell without significant cross-talk. 375 Alternatively, mutating I-site residues may simply disrupt larger structural elements, resulting in 376 more gross structural changes that would impact GcbC-LapD interaction; while possible, we find 377 such a scenario unlikely. Every independent, I-site related mutation we tested appeared necessary 378 for full interaction between GcbC and LapD. Further, the mutant proteins are expressed at 379 similar levels as the wild-type form, lacking any sign of global protein instability. Additionally, 380 the active dimer of GcbC appears to be able to form successfully in each I-site mutant we have 381 created, as evidenced by normal or elevated levels of c-di-GMP production. Thus, it appears that 382 the inability to adopt the auto-inhibited state, rather than a general structural defect, is likely 383 responsible for the observed loss of interaction between GcbC I-site mutants with LapD. We also 384 cannot rule out the possibility that c-di-GMP at the I-site is itself part of the interaction interface. 385


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Further, we note the I-site is on the opposite face of GcbC as is the α5GGDEF helix, 386 previously shown to bind LapD’s EAL domain (6). If these two elements work synergistically, it 387 is possible that the I-site also targets the EAL domain of LapD, which may indicate that only a 388 catalytically quieted version of GcbC is able to bind LapD under some conditions. Alternatively, 389 disruption of the I-site may abolish GcbC-LapD interaction by causing other interacting residues, 390 such as those in the α5GGDEF helix, to come out of register with their interface partners in LapD, 391 though we note that no previous mutations or combination thereof have had as profound an 392 impact on GcbC-LapD interaction as some of the I-site mutants. Intriguingly, the GcbC-LapD 393 interaction has been shown to endure regardless of the activation state of LapD (6), allowing for 394 the possibility that different surfaces of GcbC are in play and operate independently of one 395 another depending on the differential structural conformation taken by an active versus inactive 396 LapD (19). A final possibility is that some of the I-site residues or their neighbors are physically 397 contacting a surface on LapD, which may itself resemble the points of contact these residues 398 make with c-di-GMP. Crystallographic studies that focus on a GcbC-LapD co-crystal structure 399 will be necessary to sort among these and other possibilities. 400

Our study calls for renewed attention to the I-site as an important regulatory element for 401 DGCs. In the case of GcbC, the I-site appears to function both as a regulatory mechanism to 402 control c-di-GMP output and as a structural element which allows interaction between GcbC and 403 LapD. Our results also highlight that physical interaction between a DGC and an effector protein 404 can be an important part of proper signaling, and that the ability to produce, or mildly over-405 produce, c-di-GMP cannot always make up for loss of physical interaction. In an effort to 406 replicate the concept that a disrupted inhibitory-site will lead to an increase in the c-di-GMP 407 dependent phenotype being studied, we constructed the R363E and R366E mutants which 408


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resulted in >15-fold increase in c-di-GMP pools, and demonstrated this large increase in 409 production of the cyclic dinucleotide is sufficient to overcome loss of interaction. Intriguingly, 410 despite an apparent increase in c-di-GMP production of over an order of magnitude compared to 411 the wild-type for the R363E and R366E mutations (Fig. 5), we observed only an ~40% increase 412 in biofilm formation compared to the wild-type strain. This observation underscores the 413 importance of physical interaction to the potency of signaling between this DGC-effector pair 414 while raising questions about how PDEs may be involved to keep distal DGCs from impacting 415 effectors. Together the R363E and R366E substitutions demonstrate that choosing mutations to 416 make to a DGC’s inhibitory site should be undertaken with care, as very severe disruptions may 417 result in exaggerated phenotypes that lack biological meaning. On the other hand, we do note 418 that dramatically increased global levels of c-di-GMP overcome loss of specific DGC-effector 419 interaction to promote biofilm formation. While the impact of high c-di-GMP levels may not 420 have physiological relevance in the case of GcbC, it leaves open the possibility for global levels 421 of c-di-GMP overriding local DGC signaling events, potentially providing some of the other 422 DGCs or PDEs with a means to regulate biofilm formation in P. fluorescens that does not require 423 interaction with LapD. For example, expression of the RapA phosphodiesterase reduced LapD-424 dependent biofilm formation, but RapA does not appear to interact with the receptor based on 425 B2H assays (17, Collins and O’Toole, unpublished data). 426

Finally, the differing sets of results we produced with the targeted mutations to the I-site 427 of GcbC underscores the need to interpret observations regarding perturbing the DGC’s I-site 428 with care. Our results show some mutations to the I-site of a DGC result in exaggerated 429 phenotypes as a consequence of large (order-of-magnitude) increases in c-di-GMP. This finding 430 suggests the role of an I-site is to reduce the signal a DGC is broadcasting to its receptor, or other 431


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non-cognate receptors. However, our results also suggest that the I-site may have an additional 432 function to affirmatively associate a DGC’s signal to its target, and thus provides true 433 modulation to the DGC-effector signaling event. Future studies will be necessary to determine if 434 the I-sites of other DGCs provide similar functionality with effector proteins, or if there may be 435 some interplay between DGCs and PDEs utilizing I-sites. 436

Additionally, many DGCs lack a traditional I-site altogether, and it is largely unknown 437 how these enzymes regulate themselves or specify an effector, although one recent study has 438 suggested an alternative inhibitory state such DGCs may enter (27). While it is possible that 439 proteins with primary inhibitory sites using the RXXD motif favor specificity through 440 interaction, there is at least one example of a DGC lacking an I-site interacting with LapD in P. 441 fluorescens (Collins and O’Toole, unpublished data). This result could suggest that some DGCs 442 contribute to the global pool of c-di-GMP and are subject to other modes of regulation, while 443 other DGCs, such as GcbC, engage in more localized signaling. 444

Our results on the role of the I-site for the ability of GcbC to effectively interact with and 445 signal to LapD add to work that demonstrates physical interaction as a mechanism of signaling 446 specificity between DGCs and their effectors. Collectively, it provides a model that suggests 447 protein complexes can exist in large c-di-GMP networks, and that the regulatory state of a given 448 DGC may be integrated with the proximity of its effector to modulate its signal. The finding that 449 a canonical regulatory site of a DGC is involved in effector signaling reinforces the notion that 450 these systems are highly organized, and provides a focal point for future studies aimed at 451 signaling specificity in c-di-GMP networks. 452


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Acknowledgements. This work was supported by grants R01-AI097307 (HS/GAO), Fleming 453 Fellowship (KMG), and T32-GM08704 (KMD), and by the NSF grant MCB-9984521 (GAO). 454 Part of this work is based upon research conducted at the Cornell High Energy Synchrotron 455 Source (CHESS), which is supported by the National Science Foundation and the National 456 Institutes of Health/National Institute of General Medical Sciences under NSF award DMR-457 1332208, using the Macromolecular Diffraction at CHESS (MacCHESS) facility, which is 458 supported by award GM-103485 from the National Institute of General Medical Sciences, 459 National Institutes of Health. 460


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545 546


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Figure Legends 547 Figure 1. Architecture of GcbC’s I-site. A) Model for the negative-feedback inhibition of 548 GcbC by c-di-GMP. The yellow circles and asterisks denote the active and inhibitory sites, 549 respectively, on the GGDEF domain. B) The crystal structure of GcbC shows a dimer of the 550 GGDEF domains binding two molecules of c-di-GMP at the canonical I-site located at the GcbC 551 dimer interface. GcbC contains a primary I-site with the canonical R410xxD413 motif, which in 552 GcbC is extended by another arginine residues at position 409, and a secondary I-site that uses a 553 single arginine residue (R366). Primary and secondary I-sites are located on adjacent dimers, 554 with a c-di-GMP dimer bridging the interface. Additionally, the GcbC dimer uses protein-protein 555 interface made up of a series of glutamic acid and arginine residues. The catalytic residues are 556 highlighted in yellow. The GcbC crystal structure was originally published by Dahlstrom, et al. 557 (6). C) Mutations predicted to disrupt the I-site or GcbC domain dimers listed in Table 1 were 558 mapped onto the crystal structure of GcbC as cyan spheres. Most substitutions identified in this 559 screen appear to disrupt the ability of GcbC to form an inactive dimer by preventing the I-site 560 from being occupied with c-di-GMP. 561 562 Figure 2. Disruption of GcbC’s I-site leads to loss of interaction with LapD and loss of 563 biofilm formation. A) A bacterial-two hybrid experiment measuring interactions between LapD 564 and several variants of GcbC. E. coli BTH101 cells were grown on selective media at 30o C for 565 15 hours, scraped from the plate, and analyzed for β-galactosidase production. Error bars 566 represent standard deviation of three biological replicates made up of three technical replicates 567 per biological replicate. *** p < 0.001 compared to wild-type GcbC. B) The same GcbC 568


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mutations from A were tested for their ability to promote biofilm formation by P. fluorescens. 569 Strains were cultured overnight and inoculated into a 96 well plate of minimal medium K10 and 570 grown for 6 hrs at 30o C, followed by staining with 0.1% crystal violet. Error bars represent 571 standard deviation of four biological replicates made up of eight technical replicates per 572 biological replicate. **p<0.01,*** p < 0.001 compared to wild-type GcbC. C) The strains used in 573 panel B were sub-cultured in a shaking flask for 6 hrs in 45 ml of minimal medium K10, and 574 cells were lysed by sonication. Whole cell lysates were normalized to total protein 575 concentrations, and 6 µg of protein was analyzed by Western blot to evaluate protein stability of 576 wild-type GcbC and its mutant variants. An anti-HA antibody was used to detect GcbC. 577 578 Figure 3. Mutations to the I-site of GcbC disrupt c-di-GMP regulation. A) Wild-type GcbC 579 and its mutant variants were heterologously expressed in P. aeruginosa PA14. Strains were 580 grown overnight at 37o C, and 2.5 µl was spotted onto a minimal medium plate containing 581 Congo Red dye. Expression was induced with 0.1% arabinose. Colony biofilms were grown at 582 30o C for 36 hrs and photographed. Stronger red coloration indicates more c-di-GMP production. 583 Note the redder tint of the strain expressing GcbC (D413K), and the darker red coloration of 584 strains expressing GcbC(E360R) and GcbC(E429R) compared to wild-type GcbC. B) The same 585 P. fluorescens strains used for the biofilm assay in Fig. 2B were sub-cultured in rotating tubes for 586 6 hours and pelleted. A nucleotide extraction was performed on the pellets, and the resulting 587 organic phase was analyzed by mass spectroscopy (see Materials and Methods). Expression was 588 induced with 0.2% arabinose. Error bars represent standard deviation of three biological 589 replicates. * p < 0.05 compared to wild-type GcbC. 590


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591 Figure 4. Mutation of non-redundant residues in the GcbC I-site promotes biofilm 592 formation despite loss of interaction with LapD. A) Residues with apparent non-redundant 593 function based on the GcbC crystal structure were mutated in a non-conservative manner and the 594 resulting GcbC proteins were tested for their ability to interact with LapD. BTH101 cells were 595 scraped from selective plates containing 0.5 mM IPTG at 15 hours of growth at 30o C. A β-596 galactosidase assay was conducted on the recovered cells, and the results are expressed in Miller 597 units. Error bars represent standard deviation of three biological replicates made up of three 598 technical replicates per biological replicate. *** p <0.001 compared to wild-type GcbC. The 599 negative and wild type control results are the same as shown in Figure 2A to aid in comparison. 600 B) The same mutations as in panel A were tested for their ability to promote biofilm formation 601 by P. fluorescens. Cells were grown overnight and inoculated into a 96 well plate containing the 602 minimal medium K10 and grown for 6 hours, and were stained with 0.1% crystal violet. Error 603 bars represent standard deviation of four biological replicates made up of eight technical 604 replicates per biological replicate. *** p <0.001 compared to wild-type GcbC. C) In order to 605 ensure that levels of biofilm formation were not low due to altered protein level, 6 µg of protein-606 normalized lysate generated from the strains used in B were analyzed by Western blot for GcbC 607 stability. The membrane was blotted with an anti-HA antibody. 608 609 Figure 5. Mutations made to non-redundant residues in the I-site of GcbC correspond with 610 greatly increased levels of c-di-GMP production. A) To determine if the increases in biofilm 611 observed in Figure 4B is the result of increased c-di-GMP levels, Congo red assays were used as 612


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an indication of c-di-GMP production. Mutant and wild-type variants of GcbC were 613 heterologously expressed in P. aeruginosa PA14 and grown on Congo red plates supplemented 614 with 0.1% arabinose. Colonies were grown for 36 hrs at 30o C. Note both the redness and 615 wrinkling features of the R366E and R363E mutations indicating high levels of c-di-GMP 616 production. B) The strains used for the biofilm assay in Figure 4B were sub-cultured and 617 subjected to a nucleotide extraction followed by quantification by mass spectroscopy. Expression 618 was induced with 0.2% arabinose. Error bars represent standard deviation of three biological 619 replicates. *** p <0.005 compared to wild-type GcbC. Note: the wild type, ∆DGC, pGcbC, and 620 pGcbC GGAAF strain results are the same as those shown in Figure 3C and were also included 621 here for ease of comparison. 622


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Table 1. GcbC mutants that disrupt interaction between GcbC and LapD in a bacterial 623 two-hybrid screen. 624

Isolate Number GcbC Mutants

1 I179T N244Y Q355Ra

2

M153V S212G H266N I404T

3 L88Q

W361R A482T

4 E290G S356P H390Q

5

S24G I123T L310P

R402W

6 W361R

7 Q355R

a The mutation in each sequenced allele of GcbC predicted to disrupt the I-site is highlighted in 625 bold. 626


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the inhibitory site of a diguanylate cyclase is a necessary element

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