Download Genetic data indicate that proteins containing the GGDEF domain

FEMS Microbiology Letters 204 (2001) 163^167
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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
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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
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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.
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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.
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