Mycobacterium tuberculosis Rv3586 (DacA) Is a
Diadenylate Cyclase That Converts ATP or ADP into c-di-
Yinlan Bai1, Jun Yang1, Xin Zhou2,3, Xinxin Ding2,3, Leslie E. Eisele2, Guangchun Bai1*
1Center for Immunology and Microbial Disease, Albany Medical College, Albany, New York, United States of America, 2Wadsworth Center, New York State Department of
Health, Albany, New York, United States of America, 3School of Public Health, State University of New York at Albany, Albany, New York, United States of America
Cyclic diguanosine monophosphate (c-di-GMP) and cyclic diadenosine monophosphate (c-di-AMP) are recently identified
signaling molecules. c-di-GMP has been shown to play important roles in bacterial pathogenesis, whereas information
about c-di-AMP remains very limited. Mycobacterium tuberculosis Rv3586 (DacA), which is an ortholog of Bacillus subtilis
DisA, is a putative diadenylate cyclase. In this study, we determined the enzymatic activity of DacA in vitro using high-
performance liquid chromatography (HPLC), mass spectrometry (MS) and thin layer chromatography (TLC). Our results
showed that DacA was mainly a diadenylate cyclase, which resembles DisA. In addition, DacA also exhibited residual ATPase
and ADPase in vitro. Among the potential substrates tested, DacA was able to utilize both ATP and ADP, but not AMP, pApA,
c-di-AMP or GTP. By using gel filtration and analytical ultracentrifugation, we further demonstrated that DacA existed as an
octamer, with the N-terminal domain contributing to tetramerization and the C-terminal domain providing additional
dimerization. Both the N-terminal and the C-terminal domains were essential for the DacA’s enzymatically active
conformation. The diadenylate cyclase activity of DacA was dependent on divalent metal ions such as Mg2+, Mn2+or Co2+.
DacA was more active at a basic pH rather than at an acidic pH. The conserved RHR motif in DacA was essential for
interacting with ATP, and mutation of this motif to AAA completely abolished DacA’s diadenylate cyclase activity. These
results provide the molecular basis for designating DacA as a diadenylate cyclase. Our future studies will explore the
biological function of this enzyme in M. tuberculosis.
Citation: Bai Y, Yang J, Zhou X, Ding X, Eisele LE, et al. (2012) Mycobacterium tuberculosis Rv3586 (DacA) Is a Diadenylate Cyclase That Converts ATP or ADP into c-
di-AMP. PLoS ONE 7(4): e35206. doi:10.1371/journal.pone.0035206
Editor: Tanya Parish, Queen Mary University of London, United Kingdom
Received February 6, 2012; Accepted March 10, 2012; Published April 17, 2012
Copyright: ? 2012 Bai et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by the Potts Memorial Foundation (to GB) and by grant CA092596 from the National Cancer Institute, National Institutes of
Health (to XD) (http://www.cancer.gov). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org.
Tuberculosis (TB) remains a global epidemic, with one-third of
the world’s population infected and approximately 9 million new
active cases annually . The TB epidemic is exacerbated by a
synergy with human immunodeficiency virus (HIV) and steadily
increasing rates of drug resistance [2,3]. The efficacy of the only
vaccine strain, Mycobacterium bovis BCG, varies from 0 to 80% in
preventing pulmonary TB . Therefore, new strategies for TB
therapy and novel vaccines for eradication of the infection are
urgently needed. A better understanding of the signaling
mechanism of Mycobacterium tuberculosis could facilitate these goals.
Several cyclic nucleotides have been shown to play important
roles in bacterial gene regulation and pathogenesis. These
nucleotides include cyclic adenosine monophosphate (cAMP),
cyclic guanosine monophosphate (cGMP), cyclic diguanosine
monophosphate (c-di-GMP) and cyclic diadenosine monophos-
phate (c-di-AMP) . cAMP has been well studied in a large
number of bacteria. This signaling molecule regulates gene
expression in response to diverse environmental conditions .
In M. tuberculosis, at least 15 adenylate cyclases have been identified
[7,8,9,10]. The M. tuberculosis complex bacteria are able to secrete
[11,12,13]; this event may play a role during infection . c-di-
GMP is another important second messenger that is widespread in
bacteria. It is synthesized from two GTP molecules by diguanylate
cyclase and can be converted into pGpG or GMP by various
phosphodiesterases [14,15,16]. c-di-GMP has been known to play
a role in the regulation of the biological cascades relevant to
bacterial pathogenesis [14,15,17,18,19,20].
c-di-AMP has recently been recognized as a signaling molecule.
This nucleotide is synthesized from ATP by diadenylate cyclase
and is linearized to pApA by c-di-AMP phosphodiesterase. The
diadenylate cyclase has been identified in Bacillus subtilis, Thermotoga
maritima [21,22], Listeria monocytogenes , Staphylococcus aureus 
and Streptococcus pyogenes . In B. subtilis, the cyclase is named as
DisA (or YacK) for DNA integrity scanning protein A, which is
involved in cell-cycle checkpoints. DisA forms a large octamer,
and each monomer consists of a nucleotide-binding domain and
two DNA binding domains . DisA converts ATP into c-di-
AMP, but does not utilize GTP as a substrate . In B. subtilis,
bacterial c-di-AMP levels are reduced in response to DNA
damage, which results in a delay of sporulation. This phenotype
can be corrected by supplementation of exogenous c-di-AMP .
PLoS ONE | www.plosone.org1April 2012 | Volume 7 | Issue 4 | e35206
A diadenylate cyclase and a c-di-AMP phosphodiesterase have
been characterized in S. aureus. Deletion of the phosphodiesterase
in this pathogen results in smaller bacterial size and alteration in
biofilm formation . B. subtilis YybT protein hydrolyzes c-di-
AMP and c-di-GMP into linear pApA and pGpG, respectively
. Bacterial c-di-AMP also modulates host immune responses.
It has been reported that c-di-AMP secreted by L. monocytogenes
represents a putative secondary signaling molecule that triggers a
cytosolic pathway of innate immunity . This response is likely
mediated by Sting, a host transmembrane protein [27,28]. In
addition, c-di-AMP has been recognized as an effective immuno-
adjuvant that promotes strong Th1/Th2/Th17 responses .
The M. tuberculosis Rv3586 protein is a putative DisA ortholog.
The two motifs (DGA and RHR) that are conserved in DisA
proteins of T. maritima and B. subtilis are also conserved in the
Rv3586 . The significance of c-di-AMP in other pathogens
implicates that characterization of the Rv3586 might provide new
insights into the biology of signal transduction in TB pathogenesis.
However, Rv3586 and c-di-AMP in mycobacteria have not been
experimentally explored. In this study, we show that the Rv3586
protein is functional as a diadenylate cyclase (Dac). Therefore, we
designated this protein DacA and its encoding gene dacA, based on
our results and the published records of other bacteria [23,24].
This is the first report describing the existence of a functional
diadenylate cyclase in M. tuberculosis.
Materials and Methods
This study was carried out in strict accordance with the
recommendations in the Guide for the Care and Use of
Laboratory Animals of the National Institutes of Health. The
protocol was approved by the Institutional Animal Care and Use
Committee of Albany Medical College (Permit Number: 11-
02005). All efforts were made to minimize suffering.
Bacterial strains and culture conditions
M. tuberculosis H37Rv was grown in Difco Middlebrook 7H9
medium (BD) supplemented with 0.5% glycerol, 10% oleic acid-
albumin-dextrose-catalase (OADC), 0.05% Tween-80, as previ-
ously described . Bacteria were grown to late log phase to
isolate genomic DNA. E. coli DH5a and BL21(DE3) were grown
in Luria-Bertani broth or on Luria-Bertani agar plates. Kanamy-
cin at 25 mg/ml was added for all recombinant strains. All cultures
were grown at 37uC.
Protein expression and purification
The M. tuberculosis dacA open reading frame (ORF) and the
truncated DNA fragments encoding the first 287 aa (DacA1–287)
and the last 219 aa (DacA140–358) of DacA were PCR amplified
using the primers listed in Table 1. The M. tuberculosis H37Rv
genomic DNA was used as a template. The PCR products for
DacA, DacA1–287and DacA140–358were cloned into pET28a(+)
vector (Novagen) between NcoI and HindIII sites to generate
pMBC1218, pGB067 and pGB068, respectively. These plasmids
were sequence verified and maintained in E. coli BL21(DE3).
Mutations of DGA (aa 72–74) and RHR (aa 105–107) motifs in
DacA were generated using SOEing PCR similarly as we reported
. Primers KM2948, JY199, JY200 and KM2949 (Table 1)
were used to replace DGA with AAL (DacADGA); primers
KM2948, JY178, JY179 and KM2949 (Table 1) were used to
replace DGA with AAA (DacADG); and primers KM2948, JY201,
JY202 and KM2949 (Table 1) were used to substitute RHR with
AAA (DacARHR). Since expression of both DacADGAand DacADG
was only detected in inclusion bodies, a point mutation
(DacAG73A) in DGA motif of DacA was generated using primers
KM2948, JY218, JY219 and KM2949 (Table 1). All the final PCR
products were digested with NcoI and HindIII, cloned into
pET28a(+) vector as described above, and verified by sequencing.
The plasmids for DacADGA, DacARHR, DacADGand DacAG73A
were designated as pGB125, pGB126, pGB137 and pGB141,
respectively. These plasmids were maintained in BL21(DE3) for
The expression of the proteins was induced with 0.05 mM
isopropyl b-D-1-thiogalactopyranoside (IPTG) for 3 h at room
temperature, except that DacARHRwas expressed at 16uC. The C-
His-tagged recombinant proteins were purified using a Ni-NTA
resin (Qiagen) with buffers as we previously reported [32,33].
Size-exclusion chromatography experiments were performed
with a Superdex 200 column (106300 mm) connected to a
Gradiphrac Automatic Sampler (Amersham Biosciences). The
column was equilibrated and eluted with the running buffer (10%
glycerol in PBS at pH 7.4) at a constant flow rate of 0.5 ml/min.
Molecular mass of the proteins was determined by using Gel
Filtration Standard (Bio-Rad) per the instruction in Gel Filtration
Principles and Methods (GE Healthcare). The protein concentra-
tions were then determined using BCA Protein Assay Kit (Thermo
Scientific). The purified proteins were stored in aliquots at 280uC.
Sedimentation velocity studies were performed using a Beck-
man Coulter XL-1 analytical ultracentrifuge and an An-60 Ti
rotor at 4uC as described earlier , except that the proteins were
analyzed in a buffer containing 10% glycerol in PBS. DacA and
DacA1–287were analyzed at 40 000 rpm, and DacA140–358was
analyzed at 50 000 rpm. The volume of protein samples was
400 ml, and the reference buffer volume was 420 ml. The viscosity
and the density of the buffer and the partial specific volume of the
proteins were determined using the SEDNTERP software. The
data were analyzed by the c(s) and the c(M) methods found in
SEDFIT, a program developed by Schuck . Because the
molecular distribution consisted of a single major peak, the c(M)
method was used to estimate the molecular mass of the main
High-performance liquid chromatography (HPLC)
Determination of DacA’s enzymatic activities using HPLC was
performed as reported [22,37] with minor modification. Briefly,
reaction mixtures (10 ml) contained 40 mM Tris-HCl (pH 7.5),
10 mM MgCl2, 100 mM NaCl and nucleotide as specified. The
reaction was initiated by adding 2.5 mM protein and was
incubated at 37uC for 1 h. The reaction was then terminated by
adding 1 ml of 0.5 M EDTA, followed by a 1:5 dilution with water.
Finally, 20 ml of each sample was injected and separated by
reverse-phase HPLC with a C18 column (25064.6 mm, Vydac)
using a Waters 625 LC system equipped with a 996 Photodiode
Array Detector and a 717 Autosampler (Waters). Samples were
eluted using the same buffers and program as reported .
Nucleotides were monitored at 254 nm. c-di-AMP and pApA
standards were purchased from BioLog. ATP, GTP, ADP and
AMP were purchased from Sigma.
Mass spectrometry (MS)
The reaction mixture (10 ml) as described for HPLC analysis
was diluted 50 times, and 10 ml was analyzed using an LC/UV/
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MS system consisting of an Agilent 1200 separation module, an
Agilent 1260 photodiode array detector (Agilent) and an ABI
4000 Q-Trap mass spectrometer (Applied Biosystems). The
chromatographic separation of products was achieved on a 5-
mm Gemini C18 (15062.0 mm) column (Phenomenex). The
mobile phase consisted of solvent A (10 mM ammonium acetate
in water) and solvent B (100% acetonitrile). The samples were
eluted, at a flow rate of 0.2 ml/min, with 100% A for 5 min,
followed by linear increases from 0% B to 100% B between 5 and
10 min, and then 100% B for a further 2.5 min. The enhanced
full mass scan (EMS) was conducted at a mass range of 100 to
1000 amu with a scan rate of 1000 Da/s, and the mass
spectrometer was operated in a negative ion mode with an
electrospray ionization source. The parameters for the chamber
were as follows: curtain gas, 50 psi; heated nebulizer temperature
400uC; ion spray voltage, 24500 V; gas 1, 50 psi; gas 2, 50 psi,
declustering potential, 250 V; collision energy 210 eV; CAD
Hydrolysis of nucleotides and thin layer chromatography
For TLC samples, reaction mixture (10 ml) contained 40 mM
Tris-HCl (pH 7.5), 10 mM MgCl2, 100 mM NaCl, 100 mM
unlabeled ATP and 0.01 mCi/ml of [a-33P]ATP (MP Biomedicals).
The reaction was initiated by adding proteins as specified and
incubated at 37uC for various time periods as indicated in Results.
An aliquot of 2.5 ml was removed at each time point and
immediately mixed with an equal volume of 0.5 M EDTA. One
microliter of this mixture was finally spotted onto a pre-coated
polyethyleneimine-cellulose plate (Sigma) and was separated with
a solvent containing 1:1.5 (v/v) saturated (NH4)2SO4and 1.5 M
KH2PO4(pH 3.6) for 1 h. The dried plate was exposed on a
phosphor screen, scanned with a Storm 860 PhosphorImager
(Molecular Dynamics), and analyzed using ImageQuant software
(Molecular Dynamics). For nucleotide standards, one microliter
from a 5-mM stock of each unlabeled nucleotide was spotted onto
a TLC plate and separated using the same solvent. Image was
taken under 254 nm UV light.
Metal ion and pH dependence
The assay conditions used for metal ion screening were: 40 mM
Tris-HCl (pH 7.5), 100 mM NaCl, 2 mM [metal2+], 2.5 mM
DacA and 2 mM ATP or 0.5 mM ADP. For pH analysis,
reactions consisted of 100 mM NaCl, 10 mM MgCl2, 2.5 mM
DacA, 2 mM ATP or 0.5 mM ADP, and 40 mM Tris-HCl at
pH 6.0, 6.5, 7.0, 7.5 and 8.0, respectively. Reactions were
incubated for 1 h at 37uC, terminated by adding 1 ml of 0.5 M
EDTA and analyzed by HPLC. The peak areas of c-di-AMP were
compared and presented as arbitrary units.
Preparation of polyclonal antibody against DacA
Five female BALB/c mice (Taconic) were immunized subcuta-
neously with 50 mg of purified DacA emulsified 1:1 with Alum
(Thermo Scientific) in 100 ml and boosted twice biweekly with the
same amount of the protein and the adjuvant. The specificity of
serum was analyzed by Western blot with the purified DacA
protein. The protein was blotted onto polyvinylidene fluoride
(PVDF) membranes and sequentially probed with the anti-DacA
antibody we generated and with a peroxidase-conjugated goat
anti-mouse IgG secondary antibody (Thermo Scientific). Peroxi-
dase detection was carried out with the ECL Western blotting
detection reagents and analysis system (Thermo Scientific).
Cross-linking of protein
Cross-linking of purified DacA, DacA1–287and DacA140–358was
performed as described earlier [32,39] with slight modification.
Each protein was diluted in cross-linking buffer (50 mM sodium
phosphate, pH 7.4, 20% glycerol, 5 mM MgCl2) to 1.5 mM, and
was then incubated with glutaraldehyde at a final concentration of
35 mM for 1 h at room temperature. The reaction was quenched
by the addition of SDS-PAGE sample buffer, and a portion of
each protein sample was separated on a 10% SDS-PAGE gel. The
protein was transferred onto a PVDF membrane and visualized
after Western blot with the anti-DacA antibody.
ATP binding assay
The ATP binding with DacA, DacA1–287, DacA140–358,
DacAG73Aand DacARHRwas analyzed using a gel mobility shift
assay. Briefly, the reaction mixture (10 ml) contains 40 mM Tris-
Table 1. Primers used for protein expression in this studya.
ProteinPrimer Oligo sequence (59 to 39)
DacA KM2948 GCGCCATGGAGCACGCTGTGACTCGTCCGACC
a, Primer JY078 was used in combination with KM2948, and primer JY077 was used in combination with KM2949. The mutations for the respective amino acids in
primers JY178 to JY219 are indicated in bold.
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HCl (pH 7.5), 100 mM NaCl and 2 mg of protein either in the
presence or in the absence of 10 mM ATP. The reaction mixtures
were prepared and incubated on ice for 10 min. Samples were
then loaded onto a 6% polyacrylamide native gel and separated
with 0.56TBE at 4uC. Proteins were visualized by staining with
0.25% Coomassie Brilliant Blue G250.
Bioinformatics and structural modeling
The amino acid sequence of M. tuberculosis DacA was aligned
with that of T. maritima DisA using the Clustal W method of
MegAlign software (DNAStar). For structural modeling, the crystal
structure of T. maritima DisA was obtained from the entry 3C23 of
Protein Data Bank (PDB)  using the Cn3D program of the
National Center for Biotechnology Information (NCBI, version
Oligomerization of DacA
We expressed and purified the C-terminal His-tagged DacA in
E. coli to high homogeneity. The apparent molecular weight of this
protein was about 43 kDa on a SDS-PAGE gel (Fig. 1A), which is
consistent with the calculated molecular weight (41.4 kDa). Gel
filtration analysis with the purified protein revealed that DacA
formed a highly stable octamer with an estimated molecular mass
of 330 kDa (Fig. 1B), which is equivalent to the calculated
molecular weight of octamerized DacA (331.2 kDa). In addition,
sedimentation velocity study of DacA demonstrated a molecular
mass of ,307 kDa (Fig. 1C), supporting the notion that DacA
exists as an octamer.
DacA is an ortholog of DisA as a diadenylate cyclase
Amino acid sequence alignment revealed that M. tuberculosis
DacA shares 42% identity with B. subtilis DisA. The two putative
motifs of DisA, DGA and RHR, are conserved in DacA. These
analyses suggest that DacA is a putative diadenylate cyclase. We
first performed an enzymatic analysis of DacA with ATP by using
HPLC. The results showed that DacA converted ATP into a
major product with the same retention time as that of the product
of DisA, which is c-di-AMP (Fig. 2A). This result was also
supported by HPLC of the c-di-AMP standard (Fig. 2A).
Interestingly, we noticed that three minor peaks were also present
in the reaction of DacA with ATP (Fig. 2A), but not in a control
reaction that was carried out in the absence of ATP (not shown),
suggesting that DacA may have activities other than that of a
diadenylate cyclase. The identities of the DacA-catalyzed products
were detected using LC/UV/MS. The total ion chromatogram
(TIC) of EMS analysis displayed four products derived from ATP
(Fig. 2B). Extracted ion chromatogram (EIC) processing of the
EMS dataset for m/z 346, 426, 675 and 657 revealed the identities
of the products, which are consistent with the molecular ion of
AMP, ADP, pApA and c-di-AMP, respectively. The identities of
the products were confirmed based on coelution with authentic
standards under the same LC conditions (Fig. 2C) or by
comparing the retention times with those of purified standards
We further analyzed [a-33P]-labeled nucleotides in the catalytic
reaction by using TLC (Fig. 3A), in which the order of migration
away from the origin (Rf) was c-di-AMP, AMP, ADP and ATP, as
determined through comparisons with unlabeled standards
(Fig. 3B). By mixing DacA with [a-33P]ATP, c-di-AMP was
formed in a time-dependent manner (Fig. 3A). The reaction rate of
DacA was slower than DisA (Fig. 3A,C). At ,50 min, the
production of c-di-AMP catalyzed by DacA was equivalent to that
by DisA, which was saturated at ,6 min (Fig. 3A, C). This result
indicates that DacA is ,5 to 10-fold less active in synthesis of c-di-
AMP than that of the DisA control.
ADP and AMP were also detected using TLC from the reaction
catalyzed by DacA, while AMP was detected in the reaction with
DisA (Fig. 3A), similar to the HPLC data (Fig. 2A). These
nucleotides are likely secondary products produced by DacA or
DisA. Noticeably, the ADP in DacA reactions and the AMP in
DisA reactions were more abundant at 10 to 40 min than those at
50 to 60 min (Fig. 3A), indicating that these nucleotides may also
be used as substrates by the respective diadenylate cyclase. In the
presence of increasing amounts of DacA in the reaction, the c-di-
AMP production, but not the yield of the secondary products, was
clearly dependent on the enzyme concentration (Fig. 3D, E).
In addition to ATP, we also determined the enzymatic activity
of DacA with GTP as a potential substrate using HPLC. We did
Figure 1. Purification and oligomerization of DacA. (A) SDS-PAGE of purified DacA. Lane M, EZ-Run Pre-stained Rec Protein Ladder (Fischer
Scientific); lane 1, purified DacA. (B) Gel filtration chromatograph of DacA. The molecular weights (in kDa) and the retention volumes of the standards
are indicated on the top. (C) Analytical ultracentrifugation of DacA. Molecular mass of DacA was estimated using the c(M) method.
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not find any peak other than GTP, indicating that the activity of
DacA is specific to ATP, but not GTP (data not shown).
Taken together, these results indicate that the major activity of
DacA is as a diadenylate cyclase, while the minor activities may
include those of ATPase and ADPase.
DacA utilizes both ATP and ADP as substrates
It has been well known that c-di-GMP can be hydrolyzed into
pGpG and GMP by various phosphodiesterases. Many diguanylate
cyclases also have a c-di-GMP phosphodiesterase domain. In this
study, we noticed that in the reaction with ATP and DacA, AMP,
ADP, pApA and c-di-AMP were all present as products (Fig. 2).
These products might be converted directly from either ATP or c-
di-AMP. Therefore, we determined the enzymatic activity of DacA
with ADP, AMP, c-di-AMP and pApA, respectively. Interestingly,
DacA converted ADP into AMP, pApA and c-di-AMP, although
the yield of c-di-AMP from ADP was much lower compared with
that from ATP (Fig. 4A). In contrast, no additional product was
detected in reactions of DacA with AMP, c-di-AMP or pApA
(Fig. 4A). These results suggest that DacA does not have c-di-AMP
phosphodiesterase activity. Based on our results, we have proposed
a model of DacA’s activities (Fig. 4B), which shows that DacA
catalyzes the conversion of both ATP and ADP into c-di-AMP.
Meanwhile, DacA also produces ADP, AMP and pApA.
Effect of metal ion and pH on DacA’s activity
The effect of metal ion on the diadenylate cyclase activity of
DacA was analyzed using six divalent ions: Mg2+, Mn2+, Co2+,
Ni2+, Ca2+and Fe2+. When ATP was provided, the production of
c-di-AMP was detected in the reactions with Mg2+, Mn2+or Co2+,
but not with the other ions. Under our testing conditions, DacA
preferred Mn2+.Mg2+.Co2+as co-factors (Fig. 5A). When ADP
was utilized, the diadenylate cyclase activity was also detected in
the presence of Mg2+, Mn2+or Co2+, but not with the other ions
(Fig. 5B). More c-di-AMP was converted from ADP in the
presence of Mg2+than in the presence of Mn2+or Co2+(Fig. 5B).
As controls, DacA did not show any enzymatic activity in the
absence of any divalent metal ion when either ATP or ADP was
provided (Fig. 5A, B).
It is well known that environmental pH may affect the structures
and activities of enzymes. In this study, we determined the
diadenylate cyclase activity of DacA at pH 6.0, 6.5, 7.0, 7.5 and
8.0, respectively, using either ATP or ADP as a substrate. In the
presence of ATP, DacA produced more c-di-AMP at a basic pH
than at an acidic pH; the activity was increased by almost two fold
at pH 8.0, compared to that at pH 6.0 (Fig. 5C). However, when
ADP was provided, no difference was detected by changing pH
from 6.0 to 8.0 (Fig. 5D).
Figure 2. Determination of DacA’s activities using HPLC and LC-MS. (A) Analysis of the products from reaction of ATP with DacA using HPLC.
Reaction of ATP with DisA was included as a positive control. The reactions were carried out as described in the Methods. ATP, c-di-AMP, ADP, AMP
and pApA standards were also analyzed under the same conditions. (B and C) LC/UV/MS profiles of the products formed by DacA with ATP. The
products were detected by monitoring EMS at mass range from 100 to 1000 amu (B) or monitored by UV absorption at 254 nm (C).
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Both the N-terminal and the C-terminal domains are
required for DacA’s activity
Based on the sequence analysis using the Web CD Search
cgi) and the sequence alignment with DisA , DacA possesses
three domains (Fig. 6A). To determine which domain is important
for DacA’s activity, we generated two truncated forms of DacA,
DacA1–287and DacA140–358, which lacks the C-terminal domain
and the N-terminal domain, respectively. We then determined the
oligomerization of these proteins and their enzymatic activity as a
diadenylate cyclase. The apparent molecular weights of DacA1–287
and DacA140–358were about 37 kDa and 27 kDa, respectively, on
a SDS-PAGE gel (Fig. 6B), which are consistent with their
calculated molecular weights (33.8 kDa and 26.3 kDa, respective-
ly). Ultracentrifugation analysis revealed that DacA1–287corre-
sponded to a stable tetramer with an estimated molecular mass of
150 kDa (Fig. 6C). DacA140–358was detected with an estimated
molecular mass of 42 kDa (Fig. 6C), which is between a monomer
and a dimer. Similar results, for either DacA1–287or DacA140–358,
were obtained using gel filtration (data not shown). To further
determine the oligomerization of DacA140–358, we treated
DacA140–358with glutaraldehyde and then analyzed by Western
blot with the anti-DacA antibody. A dominant dimer-sized band
was detected in the glutaraldehyde-treated sample, whereas the
untreated sample was detected exclusively as a monomer (Fig. 6D).
Therefore, DacA140–358is stable as a dimer at the native condition.
According to the structural study of DisA and the sequence
similarity between DacA and DisA , ATP might interact with
DacA at the N-terminal domain. In this study, we analyzed the
ATP binding by DacA using a gel mobility shift assay. In this
assay, no divalent ion was provided, thus no c-di-AMP could be
formed from ATP. In the absence of ATP, both DacA and DacA1–
287showed multiple bands in a native gel. However, with the
presence of ATP in the protein samples, all the proteins migrated
Figure 3. Determination of DacA’s activities using TLC. (A)
Separation of nucleotides generated from [a-33P]ATP by DacA and DisA.
The positions of ATP, ADP, AMP and c-di-AMP are indicated based on
the Rfof each standard analyzed in panel B under the same conditions.
(B) Separation of nucleotide standards using TLC. Spots 1–5 are ATP,
ADP, AMP, c-di-AMP and pApA, respectively. (C) Quantitation of c-di-
AMP production. The relative intensity of c-di-AMP generated by DacA
or DisA at various time points as in panel A was analyzed using the
ImageQuant software. Data shown are representative of two repeat
experiments. (D) Production of c-di-AMP with various concentrations of
DacA at 30 min of incubation. Reactions contain 2-fold serial diluted
DacA protein as indicated on the top of the TLC graph (in log2mg). ‘‘N’’
indicates a control with no protein, and ‘‘Ctl’’ contains 1 mg DisA as a
positive control. ‘‘0’’ equals 1 mg of protein. (E) Quantitation of ATP
depletion and c-di-AMP production by DacA from panel D. Data shown
are representative of two repeat experiments.
Figure 4. Catalytic activities of DacA with different nucleotides.
(A) Reaction of DacA with ADP, AMP, c-di-AMP or pApA. Samples were
separated by HPLC. The peaks in ‘‘DacA+ADP’’ are labeled according to
the retention time of each standard as shown in Fig. 2A. (B) Reactions
catalyzed by DacA using ATP as a substrate, based on the results shown
in Fig. 2 and Fig. 4A. ‘‘A’’ stands for adenosine, and ‘‘P’’ stands for
phosphate. The thickness of arrows denotes priority of reaction, and the
thickest arrow shows the major catalytic reaction.
M. tuberculosis Rv3586 Is a Diadenylate Cyclase
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as a single band. Additionally, DacA migrated faster in the
presence of ATP than in the absence of ATP (Fig. 6E), suggesting
that ATP interacts with DacA and likely alters DacA’s conforma-
tion. A similar result was observed with DacA1–287, but not
DacA140–358(Fig. 6E), indicating that the N-terminal domain of
DacA is responsible for ATP binding.
To test whether the truncated proteins still have diadenylate
cyclase activity, these proteins were incubated with ATP followed
by HPLC analysis. When 2.5 mM protein was used, no enzymatic
activity was detected with either protein (data not shown). At
10 mM protein, DacA1–287showed weaker activities than that of
DacA, while DacA140–358did not show any enzymatic activity
(Fig. 6F). These data suggest that both the N-terminal and the C-
terminal domains of DacA are required for DacA’s activities.
However, the catalytic domain is located at the N-terminus of the
RHR motif is essential for ATP binding and the
diadenylate cyclase activity
It has been shown structurally that the functional motifs (DGA
and RHR) of DisA interact with c-di-AMP . These motifs are
also conserved in DacA (Fig. 7A). According to structural
modeling using the DisA protein of T. maritima, these motifs may
be in contact with ATP (Fig. 7B). In this study, we substituted the
residuals within DGA and RHR motifs of DacA to determine the
function of these motifs in the ATP binding and the diadenylate
cyclase activity. Mutation of DGA to AAL (DacADGA) or AAA
(DacADG) resulted in expression within inclusion bodies. Structural
studies with DisA showed that the glycine in the DGA motif that
interacts with c-di-AMP . Therefore, we mutated this glycine
to alanine (DacAG73A) and RHR to AAA (DacARHR). We purified
DacAG73A and DacARHR to high homogeneity; the protein
showed the same apparent molecular weight as the native DacA
(Fig. 7C). The interaction of ATP with these two proteins was
analyzed using gel mobility shift assay. The result showed that the
mobility of DacAG73Awas identical to the native DacA either in
the presence or absence of ATP. In contrast, the mobility of
DacARHR was not shifted in the presence of ATP (Fig. 7D),
indicating that the RHR motif is important in the interaction with
ATP. The diadenylate cyclase activity of DacAG73Aand DacARHR
was also determined using HPLC. The result showed that
mutation of RHR in DacA completely abolished the production
of c-di-AMP (Fig. 7E), whereas DacAG73Aretained the diadenylate
cyclase activity, suggesting that RHR, but not the glycine in DGA,
is essential for DacA’s diadenylate cyclase activity.
In this study, we identified M. tuberculosis DacA as a diadenylate
cyclase similar to the B. subtilis DisA protein, as predicted .
DacA also exhibited residual ATPase and ADPase activities, which
have not been described with the DisA proteins of B. subtilis and T.
maritima . There may be several possible explanations for the
ATPase and the ADPase activities of DacA. When one molecule of
c-di-AMP is synthesized from two ATP molecules, b- and c-
phosphates are eventually removed, and only a-phosphate remains
in c-di-AMP (Fig. 4B). Hydrolysis of b- and c-phosphate by DacA
could be catalyzed either by the specific structure of ATPase and
ADPase, or more likely as a part of the diadenylate cyclase activity.
Thus, ADP might be an intermediate product, which can be
utilized by DacA to produce c-di-AMP. Furthermore, the ATPase
Figure 5. Effect of divalent metal ions and pH on DacA’s activities. (A and B) Effect of metal ions on c-di-AMP production catalyzed by DacA
in the presence of 2 mM ATP (A) or 0.5 mM ADP (B). (C and D) Effect of pH on c-di-AMP production catalyzed by DacA in the presence of 2 mM ATP
(C) or 0.5 mM ADP (D). Note that less ADP was used in the reactions compared with ATP, and thus the arbitrary units between reactions with ATP and
ADP are not directly comparable.
M. tuberculosis Rv3586 Is a Diadenylate Cyclase
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and the ADPase activities are unlikely due to contaminations in
the purified DacA. Our data clearly showed that, with higher
concentration of DacA, the yields of ADP and AMP became lower
(Fig. 3D), rather than proportionally higher, as would be expected
if the activity was due to contaminations in the DacA preparation.
Additionally, GTPase activity has been previously observed in a
GGDEF domain protein , and an atypical GGDEF domain of
YybT exhibited unexpected ATPase activity as well .
Many diguanylate cyclases also have a phosphodiesterase
domain, which converts c-di-GMP to pGpG. However, a
phosphodiesterase domain was not predicted from the DisA
structural study, whereas B. subtilis YybT protein functions partly
as c-di-AMP phosphodiesterase . In our reactions with DisA
and DacA, we detected several nucleotides in addition to c-di-
AMP that were generated from ATP. In particular, the presence of
AMP and trace amount of pApA led us to determine whether
DacA also has a phosphodiesterase activity. By providing c-di-
AMP in the reaction, we demonstrated that DacA was unable to
hydrolyze c-di-AMP. Therefore, DacA does not have c-di-AMP
phosphodiesterase activity. The AMP and pApA products that we
detected in the reactions might be intermediates formed during
synthesis of c-di-AMP. In addition, M. tuberculosis Rv0805 has been
reported as a phosphodiesterase of certain cyclic nucleotides
[40,41]. In this study, we also incubated Rv0805 protein with c-di-
AMP and analyzed the products using TLC. Our preliminary
study showed that this protein could not hydrolyze c-di-AMP
either (data not shown). Therefore, the phosphodiesterase of c-di-
AMP in M. tuberculosis remains unknown.
DisA forms a large octamer, and each monomer contains three
domains including an N-terminal catalytic domain and a C-
terminal HhH domain . The structural study of DisA has
shown that two tetramers interact via domain 1. The main
interaction for a four-fold symmetry is mediated by domain 2, but
also includes domains 1 and 3 . In the present study, DacA
was purified as an octamer, which is comparable to the
oligomerization of DisA. Deletion of the C-terminal domain of
DacA revealed a tetramer, while deletion of the N-terminal
domain of DacA exhibited a dimer. These data suggest that in the
oligomerization of DacA, the N-terminal domain contributes to
tetramerization, and the C-terminal HhH domain is responsible
for additional dimerization. This model deviates from the DisA
model that has been reported , possibly because DacA differs
According to the sequence alignment with DisA and our
mutagenesis results of DacA, the catalytic moiety of DacA is
located at the N-terminal domain. Furthermore, the RHR motif is
essential for DacA’s ATP-binding and the enzymatic activity.
Deletion of the N-terminal catalytic domain of DacA completely
abolishes DacA’s ATP binding and the enzymatic activity.
Surprisingly, the activities of DacA are dramatically reduced by
Figure 6. Function of the N-terminal and the C-terminal domains of DacA in oligomerization and enzymatic activity. (A) Schematic
representation of the primary structures of DacA, DacA1–287and DacA140–358, as indicated with black lines. DacA1–287lacks the C-terminal HhH
domain, while DacA140–358lacks the N-terminal Dac domain. (B) SDS-PAGE of purified DacA1–287and DacA140–358. Lane M, MW marker; lanes 1 and 2
are purified DacA1–287and DacA140–358, respectively. (C) Analytical ultracentrifugation of DacA1–287and DacA140–358. (D) Cross-linking of DacA140–358
with glutaraldehyde. Lane M, MW marker; lane 1, untreated DacA140–358; and lane 2, glutaraldehyde-treated DacA140–358. Lanes 1 and 2 were analyzed
using Western blot with the anti-DacA antibody. (E) ATP binding by DacA, DacA1–287and DacA140–358. Proteins, either in the presence (+) or absence
(2) of ATP, were separated by electrophoresis with a native gel and stained with Coomassie Brilliant Blue. (F) Enzymatic activity of 10 mM DacA1–287
and DacA140–358analyzed using HPLC.
M. tuberculosis Rv3586 Is a Diadenylate Cyclase
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deletion of the HhH domain, while the tetrameric catalytic
domains and the ATP binding ability are still retained. This
suggests that the enzymatically active conformation is required,
and that the HhH domain plays a role in stabilizing the active
conformation. Therefore, we have proposed a structural model of
DacA based on our results (Fig. 7F). In this model, we hypothesize
that the removal of the C-terminal domain results in a
conformational alteration, which significantly reduces the diade-
nylate cyclase activity, but retains the capability to bind ATP. On
the other hand, deletion of the N-terminal domain or mutation of
the RHR motif abolishes the ATP-binding and the diadenylate
The activities of DacA are strictly dependent on divalent metal
ions. Generally, the catalytic activities are exhibited in the
presence of Mg2+, Mn2+or Co2+. These divalent ions are co-
factors for enzymes, such as adenylate cyclases and phosphodies-
terases [16,42]. In M. tuberculosis, Mg2+or Mn2+is needed for the
activity of Cya (Rv1625c), an adenylate cyclase . We have also
detected that Rv0805 has a preference for Mn2+or Co2+(Bai and
McDonough, manuscript submitted). These results suggest that
such cations play important roles in bacterial signaling. Further-
more, similar to several enzymes, such as YybT and Rv0805 ,
the diadenylate cyclase activity of DacA is more active at a basic
pH, rather than at an acidic or a neutral condition. The biological
equivalence of the pH effect warrants further investigation.
The roles of c-di-GMP in bacterial pathogenesis have been well
established. However, little is known about the function of c-di-
AMP. Our future studies will explore the role of c-di-AMP in the
biology and pathogenesis of M. tuberculosis.
We thank Dr. Samir El Qaidi for helpful discussions and Dr. Gregor Witte
for generously providing the DisA expression plasmid. We acknowledge the
technical assistance from the laboratories of Drs. Kathleen McDonough
and Hongmin Li at the Wadsworth Center, New York State Department
of Health. We are grateful to the Biochemistry Core of the Wadsworth
Center for analytical ultracentrifugation and HPLC analyses.
Conceived and designed the experiments: GB YB. Performed the
experiments: YB JY XZ LEE GB. Analyzed the data: YB GB XZ XD.
Contributed reagents/materials/analysis tools: GB XD LEE. Wrote the
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