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FEMS Microbiology Letters 204 (2001) 163^167 www.fems-microbiology.org Genetic data indicate that proteins containing the GGDEF domain possess diguanylate cyclase activity Nora Ausmees a; *, Raphael Mayer b , Haim Weinhouse c , Gail Volman c , Dorit Amikam d , Moshe Benziman c , Martin Lindberg a a c Department of Microbiology, Swedish University of Agricultural Sciences, SLU, Box 7025, S-75007 Uppsala, Sweden b InSight, Rehovot 76121, Israel Department of Biological Chemistry, Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 91904, Israel d Department of Biotechnology, Tel-Hai Academic College and Rambam Medical Center, Haifa 31096, Israel Received 21 June 2001 ; received in revised form 25 July 2001; accepted 13 August 2001 First published online 2 October 2001 Abstract A conserved domain, called GGDEF (referring to a conserved central sequence pattern), is detected in many procaryotic proteins, often in various combinations with putative sensory-regulatory components. Most sequenced bacterial genomes contain several different GGDEF proteins. The function of this domain has so far not been experimentally shown. Through genetic complementation using genes from three different bacteria encoding proteins with GGDEF domains as the only element in common, we present genetic data indicating (a) that the GGDEF domain is responsible for the diguanylate cyclase activity of these proteins, and (b) that the activity of cellulose synthase in Rhizobium leguminosarum bv. trifolii and Agrobacterium tumefaciens is regulated by cyclic di-GMP as in Acetobacter xylinum. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : GGDEF domain; Diguanylate cyclase; Cellulose synthesis; bis-(2P,5P)-Cyclic diguanylic acid 1. Introduction A novel regulatory mechanism involving bis-(2P,5P)-cyclic diguanylic acid (c-di-GMP) as an allosteric activator of a protein was ¢rst described in the cellulose synthesis context in Acetobacter xylinum [1], a bacterium in which the molecular mechanisms of biological cellulose synthesis were ¢rst revealed [2,3]. Genes encoding enzymes for the cellulose synthesis pathway have also been identi¢ed and characterized in Agrobacterium tumefaciens [4] and Rhizobium leguminosarum bv. trifolii [5]. Cellulose is the main cell wall polysaccharide in plants where it has a pronounced architectural role. Bacteria, however, seem to produce cellulose for various purposes and the biological advantages of this ability are not always obvious [6]. In A. xylinum the functions of cellulose involve gaining access to oxygen in liquid cultures and promoting colonization of solid substrates [7]. In Agrobacterium and Rhi- * Corresponding author. Tel. : +46 (18) 67 32 06; Fax: +46 (18) 67 33 92. E-mail address : [email protected] (N. Ausmees). zobium the cellulose ¢bres serve to aggregate and to anchor the bacteria on the plant surfaces, thus facilitating colonization and infection [8,9]. Regulatory elements involved in cellulose production have been characterized in Acetobacter and Rhizobium. In Acetobacter the cellulose synthesis is modulated by the opposing action of two enzymes, diguanylate cyclase (DGC) and c-di-GMP diesterase (PDEA), controlling the level of c-di-GMP in the cell [1,10]. DGC acts as a positive regulator by catalysing the formation of c-di-GMP, which speci¢cally activates the cellulose synthase enzyme. The phosphodiesterase protein PDEA1 cleaves c-di-GMP to the ine¤cient linear dinucleotide. Both proteins have similar domain architecture and consist of either a haembased oxygen or £avin-bound redox sensor domain in the N-terminus [11] followed by the so-called GGDEF and EAL domains of unknown function [10] (Fig. 1). In R. leguminosarum bv. trifolii cellulose synthesis is positively regulated by CelR2, a putative response regulator protein [5], which contains a GGDEF motif in its C-terminus (Fig. 1). No regulatory genes in the context of cellulose synthesis have been described in A. tumefaciens, but cellulose production in this bacterium responds 0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 3 9 4 - 9 FEMSLE 10142 17-10-01 Cyaan Magenta Geel Zwart 164 N. Ausmees et al. / FEMS Microbiology Letters 204 (2001) 163^167 Fig. 1. Domain architecture of the GGDEF proteins CelR2, PleD, DGC, PDEA and YhcK, discussed in this paper. RES, response regulator receiver domain; PAS, PAS or PAS and PAC domains together (conserved sequences involved in signal transduction sensing di¡erent stimuli, e.g. oxygen, redox or light); EAL, a conserved domain with unknown function, containing the conserved EAL motif. to environmental signals [8]. The GGDEF domain is detected in many procaryotic proteins and has got its name due to the conserved sequence pattern GG[DE][DE]F. According to the Pfam database the GGDEF domain is present in more than 80 di¡erent proteins, most of which also contain signalling or two-component regulatory domains [12]. The function of this domain has so far not been experimentally proven. By monitoring cellulose production in di¡erent wild-type and mutant strains of Rhizobium and Agrobacterium harbouring plasmids with cloned GGDEF genes from di¡erent bacteria, we have obtained data which strongly suggest that this domain is involved in the synthesis of c-di-GMP. 2. Materials and methods 2.1. Bacterial strains, plasmids and growth media R. leguminosarum bv. trifolii strain R200 (wild-type), strain R201 (a cellulose overproducing mutant of strain R200), strains R205 and R204 (cellulose-negative Tn5 mutants of R201 in celR2 and celA, respectively), are described in [5]. A. tumefaciens wild-type strain C58 was obtained from our strain collection at SLU. Cultivation of Rhizobium and Agrobacterium strains in TYC or YMBCongo red medium and matings were performed as in [5]. For motility analysis bacteria were stabbed into soft agar containing 1/10-strength TYC medium with 0.35% agar. Escherichia coli strain DH5K was used for cloning. Calco£uor staining of C58 derivatives was performed as in [8]. 2.2. Plasmids and DNA manipulations Broad host range plasmid pRK404A, a derivative of pRK404 [13] with a unique EcoRI site, was used to clone and introduce di¡erent GGDEF genes into Rhizobium and Agrobacterium strains. Ligations were performed using the Ready-to-go kit (Amersham Pharmacia Biotech). A derivative of cosmid 3F3 [10] and E. coli TG1 chromosomal FEMSLE 10142 17-10-01 DNA were used as templates to amplify the dgc1 and yhcK genes, respectively. Appropriate forward and reverse primers were purchased from Gibco BRL. The PCR product of dgc1 was puri¢ed, treated with T4 kinase, and ligated into PstI-cleaved and T4 polymerase-treated vector pRK404A. PCR with fragment- and vector-speci¢c primers was used to determine the orientation of the inserts. Plasmid pDgc1S contains the dgc1 gene downstream from the lac promoter and pDgc1AS contains the gene in the opposite orientation. The PCR product of yhcK was cleaved with BamHI and HindIII and ligated into pRK404A treated with the same enzymes resulting in the construct pYhcK, containing the yhcK gene and V200 bp upstream sequence downstream from the lac promoter. The clones were sequenced to detect PCR-introduced mutations using an ABI PRISM 337 DNA Sequencer (Perkin-Elmer). Sequences were analysed with the Vector NTI Suite software package (InforMax, Inc.). 3. Results and discussion The presence of GGDEF domains in all proteins known to be involved in the regulation of cellulose synthesis suggested to us that this domain plays a key role in this activity. Recently, in pro¢le-based extensive database searches sequence similarity was detected between the GGDEF domain and the eucaryotic adenylyl cyclase catalytic domain [14]. The authors deduced that the GGDEF domain is a regulatory enzyme involved in nucleotide cyclization, with the fold similar to that of the eucaryotic cyclase catalytic domain. To test the hypothesis that GGDEF domains in di¡erent proteins have a c-di-GMP synthase activity, genetic complementation experiments were performed using three di¡erent so-called GGDEF genes. These genes were celR2 (R. leguminosarum bv. trifolii), dgc1 (diguanylate cyclase gene from A. xylinum) and yhcK (E. coli), encoding proteins with a GGDEF domain as the only structural element in common. yhcK was arbitrarily chosen from the Cyaan Magenta Geel Zwart N. Ausmees et al. / FEMS Microbiology Letters 204 (2001) 163^167 database entries as a gene encoding a GGDEF protein so far not known to be related to cellulose synthesis and not possessing any other domains in common with DGC or CelR2. 3.1. Three di¡erent GGDEF genes activate cellulose production in R. leguminosarum bv. trifolii R. leguminosarum bv. trifolii strain R200 grows as white colonies on YMB-C agar plates. Genes involved in cellulose production were therefore identi¢ed by subjecting a cellulose overproducing mutant strain R201 (bright red colonies on YMB-C plates, Fig. 2A) to Tn5 mutagenesis [5]. Tn5 insertion in the celR2 gene resulted in a cellulosenegative strain R205, which grows as white colonies on YMB-C plates (Fig. 2B). Plasmid pReg1, containing celR2, complements strain R205 (Fig. 2C) and causes overproduction of cellulose in strain R200 [5]. Plasmids pDgcS, pDgcAS and pYhcK, containing the dgc1 allele from A. xylinum in di¡erent orientations and the yhcK gene from E. coli, respectively, were introduced into strains R200 and R205. The YMB-C plate assay was used to screen the level of cellulose production of the transconjugants. All mentioned GGDEF plasmids complemented the function of the disrupted celR2 gene and restored a high cellulose production in R205 (Fig. 2). The GGDEF plasmids also induced cellulose overproduction in strain R200. However, transformants of strains R200 and R205 harbouring plasmid pDgcS, containing the dgc1 gene in sense orientation with respect to the lac promoter, were unstable and grew as red/white segmented colonies. The antisense dgc1 construction, pDgcAS, gave rise to uniformly red transconjugant colonies of strains R200 and R205. We have also previously observed weak transcription in the antisense direction relative to the lac promoter in pRK404A in Rhizobium [5]. The observation that the diguanylate cyclase gene dgc1 from A. xylinum induced cellulose overproduction in the wild-type R leguminosarum bv. trifolii strongly indicates that c-di-GMP acts as an activator of cellulose synthase also in this bacterium. Furthermore, the ability of the dgc1 gene to complement the disrupted celR2 gene suggests that these genes are likely to encode similar functions. The observation that the phenotypic e¡ect caused by the 165 yhcK gene was identical to that caused by the diguanylate cyclase gene brings to mind the possibility that also yhcK is involved in c-di-GMP production. Since the GGDEF domain is the only common element in these compatible proteins (CelR2, DGC and YhcK), this domain is the only candidate to possess the diguanylate cyclase activity. Alternatively, the similar complementation patterns of the three genes mentioned above could be explained by various secondary e¡ects, caused by overproduction of the respective proteins, which are not necessarily related to the c-di-GMP production. However, in this case one has to assume that these three proteins stimulate cellulose synthesis each in a di¡erent way via di¡erent domains, which makes this explanation rather constrained. Furthermore, at least in the case of CelR2 we have previously shown that the intact GGDEF domain is essential for the function of the protein. A derivative of celR2, truncated in the middle of the conserved GGDEF pattern, was not able to complement the celR2-defective strain R205 [5]. All DDGEF plasmids were also transferred to strain R204, in which the celA gene, encoding cellulose synthase protein, was disrupted by Tn5 [5]. As expected, these transconjugants remained white on YMB-C agar, con¢rming that the Congo red binding of the transconjugants harbouring a GGDEF plasmid re£ects cellulose production. 3.2. Cellulose synthesis in A. tumefaciens is also activated by GGDEF plasmids Amikam and Benziman have shown that c-di-GMP is produced naturally in A. tumefaciens where it stimulates cellulose synthase activity [15]. In contrast, other authors have reported that c-di-GMP does not a¡ect cellulose synthesis in A. tumefaciens [16]. We used the three GGDEF plasmids mentioned above to transform wild-type A. tumefaciens C58. Aggregation in liquid cultures and enhanced binding of the £uorescent dye Calco£uor have been shown to correlate with cellulose production in C58 [8]. Both characteristics were used to determine enhanced cellulose production of the transconjugants. Strain C58 grows in a homogeneous suspension during exponential growth (Fig. 3A) in TY medium, while transconjugants of strain C58 harbouring plasmids pReg1, pDgcAS or Fig. 2. The appearance of di¡erent R. leguminosarum bv. trifolii strains on YMB-Congo red plates. A: Cellulose overproducing strain R201; B: cellulose-negative mutant strain R205 (celR2 : :Tn5), harbouring the cloning vector pRK404A; C^E: transconjugants of strain R205 containing plasmids pReg1, pDgcAS and pYhcK, respectively. Plates were photographed with a digital camera Olympus C-820L and images were processed with Adobe Photoshop v5.0.2 software. FEMSLE 10142 17-10-01 Cyaan Magenta Geel Zwart 166 N. Ausmees et al. / FEMS Microbiology Letters 204 (2001) 163^167 Fig. 3. Exponential phase TY cultures of A. tumefaciens C58 harbouring di¡erent GGDEF plasmids. A: Control, containing the vector pRK404A without insert; B: pDgcAS or pReg1; C: pYhcK. Cultures were poured into wells of a tissue culture plate and photographed as in Fig. 2. pYhcK £occulated heavily (Fig. 3B,C). Strong £uorescence was observed in and around these aggregates in the microscope after staining with Calco£uor (data not shown). Plasmid pYhcK caused the strongest aggregation in the liquid culture (Fig. 3C). These results support the data presented by Amikam and Benziman that c-di-GMP acts as an allosteric activator in cellulose synthesis also in A. tumefaciens. Interestingly, it was recently reported that E. coli and Salmonella typhimurium produce cellulose as one component of their extracellular matrix, which contributes to the multicellular behaviour of these bacteria [17]. It is remarkable that a GGDEF protein, called AdrA, is involved in the activation of cellulose synthesis also in these bacteria [17,18]. The authors speculate that the AdrA protein activates cellulose production by direct interaction with one or several proteins of the cellulose biosynthetic pathway. In the light of data presented in this paper, it seems likely that this activation is accomplished by c-di-GMP acting as an allosteric activator of cellulose synthase also in these bacteria. 3.3. CelR2 and PleD One of the few GGDEF proteins with characterized function is the PleD protein from Caulobacter crescentus, sharing the same domain architecture and 65% sequence similarity with CelR2 [5]. The divK-pleD operon is a key regulatory element in synchronizing changes in the cell morphology with cell division in Caulobacter, where switches between £agellated swarmer cells and adhesive stalked cells take place in every cell cycle. DivK is an essential cell division regulator [19] with dynamic localization pattern during the cell cycle [20]. It is intriguing that Rhizobium also contains the same genetic structure: the celR2 gene is preceded by celR1, encoding a response regulator protein with more than 80% sequence similarity to DivK [5]. The PleD protein has several regulatory functions in the polar morphogenesis of Caulobacter, and mutations in pleD cause pleiotropic e¡ects [21]. Recently it was shown that PleD is required for e¤cient removal of a £agellar anchor protein FliF during the swarmer-tostalked cell transition [22]. Considering the striking simi- FEMSLE 10142 17-10-01 larity between CelR2 and PleD we asked the question whether disruption of celR2, besides a¡ecting the levels of cellulose production, also a¡ects motility in Rhizobium. The parent strain R201 and the CelR2-defective mutant strain R205 were compared considering their ability to spread in soft agar. Also wild-type strain R200 and another Tn5 mutant strain R204 (celA : :Tn5) were included in the test. No signi¢cant di¡erence could be detected in the size of the swarms (data not shown). Obviously, despite the striking similarity in structure, CelR2 and PleD ful¢l di¡erent functions in Rhizobium and Caulobacter, respectively. In accordance with the hypothesis presented in this paper, PleD should, like CelR2, be a regulatory enzyme activated by phosphorylation to produce c-di-GMP. The activation of di¡erent target proteins by c-di-GMP would explain both the reported multiple functions of PleD in Caulobacter and the di¡erent phenotypic e¡ects of PleD and CelR2 in Caulobacter and Rhizobium, respectively. Acknowledgements This work was supported by grants from the Swedish Council for Forestry and Agriculture Research (201.0652/ 00) and Carl Trygger's Foundation for Scienti¢c Research to M.L. and a Faculty scholarship from Behm's Fund to N.A. References [1] Ross, P., Weinhouse, H., Aloni, Y., Michaeli, D., Weinberger-Ohana, P., Mayer, R., Braun, S., de Vroom, E., van der Marel, G.A., van Boom, J.H. and Benziman, M. (1987) Regulation of cellulose synthesis in Acetobacter xylinum by cyclic diguanylic acid. Nature 325, 279^281. [2] Wong, H.C., Fear, A.L., Calhoon, R.D., Eichinger, G.H., Mayer, R., Amikam, D., Benziman, M., Gelfand, D.H., Meade, J.H. and Emerick, A.W. (1990) Genetic organization of the cellulose synthase operon in Acetobacter xylinum. Proc. Natl. Acad. Sci. USA 87, 8130^ 8134. [3] Saxena, I.M., Lin, F.C. and Brown Jr., R.M. (1990) Cloning and sequencing of the cellulose synthase catalytic subunit gene of Acetobacter xylinum. Plant Mol. Biol. 15, 673^683. [4] Matthysse, A.G., White, S. and Lightfoot, R. (1995) Genes required for cellulose synthesis in Agrobacterium tumefaciens. J. Bacteriol. 177, 1069^1075. [5] Ausmees, N., Jonsson, H., Hoglund, S., Ljunggren, H. and Lindberg, M. (1999) Structural and putative regulatory genes involved in cellulose synthesis in Rhizobium leguminosarum bv. trifolii. Microbiology 145, 1253^1262. [6] Ross, P., Mayer, R. and Benziman, M. (1991) Cellulose biosynthesis and function in bacteria. Microbiol. Rev. 55, 35^58. [7] Williams, W.S. and Cannon, R.E. (1989) Alternative environmentalroles for cellulose produced by Acetobacter xylinum. Appl. Environ. Microbiol. 55, 2448^2452. [8] Matthysse, A.G., Holmes, K.V. and Gurlitz, R.H. (1981) Elaboration of cellulose ¢brils by Agrobacterium tumefaciens during attachment to carrot cells. J. Bacteriol. 145, 583^595. [9] Smit, G., Kijne, J.W. and Lugtenberg, B.J. (1987) Involvement of both cellulose ¢brils and a Ca2 -dependent adhesin in the attachment Cyaan Magenta Geel Zwart N. Ausmees et al. / FEMS Microbiology Letters 204 (2001) 163^167 [10] [11] [12] [13] [14] [15] of Rhizobium leguminosarum to pea root hair tips. J. Bacteriol. 169, 4294^4301. Tal, R., Wong, H.C., Calhoon, R., Gelfand, D., Fear, A.L., Volman, G., Mayer, R., Ross, P., Amikam, D., Weinhouse, H., Cohen, A., Sapir, S., Ohana, P. and Benziman, M. (1998) Three cdg operons control cellular turnover of cyclic di-GMP in Acetobacter xylinum: genetic organization and occurrence of conserved domains in isoenzymes. J. Bacteriol. 180, 4416^4425. Chang, A.L., Tuckerman, J.R., Gonzalez, G., Mayer, R., Weinhouse, H., Volman, G., Amikam, D., Benziman, M. and Gilles-Gonzalez, M.A. (2001) Phosphodiesterase A1, a regulator of cellulose synthesis in Acetobacter xylinum, is a heme-based sensor. Biochemistry 40, 3420^3426. Bateman, A., Birney, E., Durbin, R., Eddy, S.R., Finn, R.D. and Sonnhammer, E.L. (1999) Pfam 3.1: 1313 multiple alignments and pro¢le HMMs match the majority of proteins. Nucleic Acids Res. 27, 260^262. Ditta, G., Schmidhauser, T., Yakobson, E., Lu, P., Liang, X.W., Finlay, D.R., Guiney, D. and Helinski, D.R. (1985) Plasmids related to the broad host range vector, pRK290, useful for gene cloning and for monitoring gene expression. Plasmid 13, 149^153. Pei, J. and Grishin, N.V. (2001) GGDEF domain is homologous to adenylyl cyclase. Proteins 42, 210^216. Amikam, D. and Benziman, M. (1989) Cyclic diguanylic acid and cellulose synthesis in Agrobacterium tumefaciens. J. Bacteriol. 171, 6649^6655. FEMSLE 10142 17-10-01 167 [16] Matthysse, A.G., Thomas, D.L. and White, A.R. (1995) Mechanism of cellulose synthesis in Agrobacterium tumefaciens. J. Bacteriol. 177, 1076^1081. [17] Zogaj, X., Nimtz, M., Rohde, M., Bokranz, W. and Romling, U. (2001) The multicellular morphotypes of Salmonella typhimurium and Escherichia coli produce cellulose as the second component of the extracellular matrix. Mol. Microbiol. 39, 1452^1463. [18] Romling, U., Rohde, M., Olsen, A., Normark, S. and Reinkoster, J. (2000) AgfD, the checkpoint of multicellular and aggregative behaviour in Salmonella typhimurium regulates at least two independent pathways. Mol. Microbiol. 36, 10^23. [19] Hecht, G.B., Lane, T., Ohta, N., Sommer, J.M. and Newton, A. (1995) An essential single domain response regulator required for normal cell division and di¡erentiation in Caulobacter crescentus. EMBO J. 14, 3915^3924. [20] Jacobs, C., Hung, D. and Shapiro, L. (2001) Dynamic localization of a cytoplasmic signal transduction response regulator controls morphogenesis during the Caulobacter cell cycle. Proc. Natl. Acad. Sci. USA 98, 4095^4100. [21] Sommer, J.M. and Newton, A. (1989) Turning o¡ £agellum rotation requires the pleiotropic gene pleD : pleA, pleC, and pleD de¢ne two morphogenic pathways in Caulobacter crescentus. J. Bacteriol. 171, 392^401. [22] Aldridge, P. and Jenal, U. (1999) Cell cycle-dependent degradation of a £agellar motor component requires a novel-type response regulator. Mol. Microbiol. 32, 379^391. Cyaan Magenta Geel Zwart