Methods 58 (2012) 325–334

Contents lists available at SciVerse ScienceDirect

Methods
journal homepage: www.elsevier.com/locate/ymeth

The bacterial two-hybrid system based on adenylate cyclase reconstitution

in Escherichia coli
Aurélia Battesti a, Emmanuelle Bouveret b,⇑
a
NIH/NCI, 9000 Rockville Pike, Bethesda, MD 20892, USA
b
CNRS, Aix-Marseille University, LISM, 31 Chemin Joseph Aiguier, 13009 Marseille, France

a r t i c l e i n f o a b s t r a c t

Article history: The bacterial two-hybrid system based on the reconstitution of adenylate cyclase in Escherichia coli
Available online 24 July 2012 (BACTH) was described 14 years ago (Karimova, Pidoux, Ullmann, and Ladant, 1998, PNAS, 95:5752).
Communicated by Peter Uetz For microbiologists, it is a practical and powerful alternative to the use of the widely spread yeast
two-hybrid technology for testing protein–protein interactions. In this review, we aim at giving the
Keywords: reader clear and most importantly simple instructions that should break any reticence to try the tech-
Protein–protein interaction nique. Yet, we also add recommendations in the use of the system, related to its specificities. Finally,
Two-hybrid
we expose the advantages and disadvantages of the technique, and review its diverse applications in
Escherichia coli
Adenylate cyclase
the literature, which should help in deciding if it is the appropriate method to choose for the case at hand.
Calmodulin, b-galactosidase assay Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction 2. Principle and development of the bacterial adenylate cyclase

two hybrid system
As an alternative to the widely used yeast two-hybrid method,
several two-hybrid systems have been engineered in bacteria to The bacterial adenylate cyclase two hybrid (BACTH) system is
study protein–protein interactions. One of them, developed by based on the reconstitution of a regulatory cascade depending on
Karimova and collaborators in 1998, is based on the reconstitution cyclic adenosine 30 ,50 -monophosphate (cAMP). cAMP is synthe-
of adenylate cyclase activity in Escherichia coli [1]. This bacterial sized by a family of enzymes called adenylate cyclases. A peculiar
two-hybrid system takes advantage of the specific features of the member of this family is the adenylate cyclase of B. pertussis, the
adenylate cyclase toxin from Bordetella pertussis to detect interac- agent of the whooping cough. This enzyme is a toxin that becomes
tions between cytoplasmic proteins, as well as membrane proteins, fully active in the host after binding to calmodulin, a protein found
from prokaryotes but also from eukaryotes. Previous reviews only in eukaryotic organisms. Following its activation, the B. per-
describing extensively this technique have already been published tussis adenylate cyclase toxin alters the host’s functions by elevat-
[2–5]. However, recently, the BACTH system has been much more ing the level of cAMP in this organism [6]. The B. pertussis
frequently used in the molecular microbiology community and adenylate cyclase has been extensively studied (for review see
several improvements have been made on the technique. In this re- [7]). It is a large protein of 1706 amino acids with a catalytic activ-
view, we would like to give basic and easy protocols, that we use ity that resides in its 400 first amino acids [8] (Fig. 1). This catalytic
routinely in our laboratories, to allow everyone to apply rapidly domain itself can be divided in two sub-domains: a 25 kDa frag-
this technique to their research field. We will also describe the ment (residues 1–224) that contains the catalytic site and an
new tools available and summarize what was done in the last 18 kDa fragment (residues 225–399) that contains the main cal-
few years by using the BACTH system, discuss what is feasible, modulin binding site [9].
and underline the advantages and the successes of the technique Interestingly, when they are co-expressed in the presence of
to study protein–protein interactions. calmodulin, these two sub-domains interact and this interaction
restores the synthesis of cAMP [10]. Such observation highlights
the capacity of these two sub-domains to complement and recover
the adenylate cyclase activity when they are brought close from
Abbreviations: BACTH, bacterial adenylate cyclase two-hybrid; cAMP, cyclic
each other. The BACTH system is based on this complementation.
adenosine 30 ,50 -monophosphate.
⇑ Corresponding author. Fax: +33 04 91 71 21 24. Indeed, the proteins of interest are fused to the T25 or the T18
E-mail address: [email protected] (E. Bouveret). sub-units. If the two proteins of interest interact in an otherwise

1046-2023/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ymeth.2012.07.018
326 A. Battesti, E. Bouveret / Methods 58 (2012) 325–334

Fig. 1. Organization of B. pertussis adenylate cyclase and rational of the BACTH technique based on T18 and T25 domains reconstitution. The structure of the adenylate cyclase
domain is shown with colour corresponding to the T18 and T25 domain limits [59]. A: When the adenylate cyclase domain alone is expressed in E. coli cya cells, there is
residual cAMP synthesis. B: When T18 and T25 domains are produced separately, no cAMP is produced. C: T18 and T25 domains put close together by the interaction of
hybrid proteins X and Y restore adenylate cyclase activity. D: The technique functions for membrane proteins [17].

cya strain, they fulfill the role of calmodulin in bringing together 3.2.1. Vectors
T18 and T25 domains and thus the adenylate cyclase activity is re- Two different sets of compatible bacterial two-hybrid vectors
stored (Fig. 1). Newly synthesized cAMP interacts with the catabo- have initially been engineered to construct recombinant proteins
lite activator protein (CAP) and the cAMP/CAP complex binds to in frame with the T18 and T25 sub-units. The first set consisted
promoters and regulates transcription of several genes. Among in two vectors permitting to fuse the T18 domain at the C-terminal
these genes, the lactose and maltose catabolic operons are posi- and the T25 domain at the N-terminal end of the protein, with
tively regulated by cAMP/CAP, and their activation can be easily resistance to ampicillin and chloramphenicol respectively [1] (Ta-
detected by several assays in E. coli. ble 1). Both hybrids were under the control of the lacUV5 promoter.
The second set was complete, enabling the tagging with T18 and
3. Bacterial two-hybrid protocols T25 domains, either at the N-terminal or the C-terminal of the pro-
tein, with ampicillin and kanamycin resistances [13] (Table 1). In
3.1. Overview of the technique (Fig. 2) this second set of plasmids, the hybrid constructions are under
the control of the wild type lactose promoter. The choice of the
An E. coli strain deleted of the gene coding for the endogenous tag localization depends on the characteristics of each protein. Var-
adenylate cyclase (cya strain) is transformed by both plasmids ious tests can be performed to evaluate the incidence of the fusion
containing the T25 and T18 hybrids (Fig. 2A). Although B. pertussis on the protein functionality (see Section 3.6). The second set of
adenylate cyclase usually needs to bind calmodulin to be fully ac- vectors is available from Euromedex as a BACTH system kit (ref.
tive, it has been shown that in E. coli, a cryptic adenylate cyclase EUK001). Later, derived vectors containing a modified MCS or an
activity is detected even in absence of calmodulin [8]. There are additional flag tag have been designed (Table 1) [14,15]. In these
then several ways to detect a positive interaction within the plasmids, the choice of cloning sites is reduced, but the EcoRI
BACTH system. Indicator LB plates containing X-Gal, or MacConkey and XhoI sites are compatible with the widely used yeast two-hy-
plates containing lactose or maltose as the sole carbon source are brid vectors pEG202, pJG4-5, and pMW104 for example [16]. These
used for simple qualitative colorimetric assays (Fig. 2B). A positive plasmids are available through the Addgene plasmid repository
interaction is detected on LB/X-Gal plates by the appearance of a (http://www.addgene.org). For the constructions of the hybrids,
blue color, and on lactose/maltose MacConkey plates by the one has to make sure to add the stop codon in the cloned ORFs
appearance of a red color. For quantitative assays, direct cAMP when using N-terminal tags, and to be careful with the reading
measurement can be performed [11]. However, usually, an indirect frame for the C-terminal tags, because all the restriction sites are
b-galactosidase assay is used, following the classical Miller proto- not in the same reading frame. Strong positive controls are avail-
col [12]. b-galactosidase assays in 96-well plates have also been able, such as the pT18-zip and pKT25-zip plasmids containing a
developed to screen several conditions and interactions at the DNA sequence coding for a leucine zipper motif [1] (Table 1).
same time (see Section 3.4).
3.2.2. Strains
3.2. Materials Two E. coli strains are commonly used for BACTH experiments:
DHM1 [17] and BTH101. The genotype of BTH101 is F0 , cya-99,
We list below the material that will be needed to perform the araD139, galE15, galK16, rpsL1 (StrR), hsdR2, mcrA1, mcrB1, relA1
protocols that are described. (the relA1 mutations is not indicated in the Euromedex manual,
 A. Battesti, E. Bouveret / Methods 58 (2012) 325–334 327

Fig. 2. BACTH protocol A: An E. coli cya strain is transformed by the two compatible plasmids carrying the hybrids with T25 and T18 domains. The interaction of proteins X
and Y brings back together domains T25 and T18 and restores cAMP synthesis. Diffusible cAMP then induces expression of lactose and maltose operons, and also increases by
a positive feedback the expression of the hybrid genes. B: The different steps of the protocol we routinely use in our laboratories are outlined. See text for the detail.

Table 1
Vectors for the BACTH. In the description column, the promoter (Plac or lacUV5), the MCS, and its position relative to the T18 and T25 domains are indicated. Full sequences of
most of these plasmids are included in the BACTH manual edited by Euromedex that can be freely downloaded on the Euromedex website. Sequences of pKT25linker,
pUT18Clinker, pKT25-Flag, and pUT18C-Flag can be found at the following url: http://lism.cnrs-mrs.fr/Bouveret/Pages/plasmids.html. MCS: multiple cloning site.

Name Tag Resistance Origin of Description References

localization replication
pT25 N-terminal Cam p15A Derived from pACYC184 vector. lacUV5–T25–MCS(PstI–BamHI–KpnI) [1]
pT18 C-terminal Amp ColE1 Derived from pBluescript IIKS. lacUV5–MCS(KpnI)–T18 [1]
pKT25 N-terminal Kan p15A Derived from pSU40. Plac–T25–MCS(PstI–XbaI–BamHI–SmaI–KpnI–EcoRI) [13]
pUT18C N-terminal Amp ColE1 Derived from pUC19. Plac–T18–MCS(PstI–SalI–XbaI–BamHI–SmaI–KpnI–EcoRI) [13]
pKNT25 C-terminal Kan p15A Derived from pSU40. Plac–MCS(HindIII–SphI–PstI–XbaI–BamHI–SmaI–KpnI–SacI– [17]
EcoRI)–T25
pUT18 C-terminal Amp ColE1 Derived from pUC19. Plac–MCS(HindIII–SphI–PstI–SalI–XbaI–BamHI–SmaI–KpnI– [13]
SacI–EcoRI)–T18
pT25-zip N-terminal Cam p15A Derived from pT25. Sequence coding for the leucine zipper region of the GCN4 [1]
yeast protein. Positive control
pT18-zip N-terminal Amp ColE1 Derived from pT18. Sequence coding for the leucine zipper region of the GCN4 [1]
yeast protein. Positive control
pKT25-zip N-terminal Kan p15A Derived from pKT25. Sequence coding for the leucine zipper region of the GCN4 [13]
yeast protein. Positive control
pUT18C-zip N-terminal Amp ColE1 Derived from pUT18C. Sequence coding for the leucine zipper region of the GCN4 [13]
yeast protein. Positive control
pKT25linker(pEB354) N-terminal Kan p15A Derived from pKT25. Plac–T25–MCS(PstI–XbaI–EcoRI–ClaI–XhoI) [15]
pUT18Clinker(pEB355) N-terminal Amp ColE1 Derived from pUT18C. Plac–T18–MCS(PstI–XbaI–EcoRI–EcoRV–XhoI) [15]
pKT25–Flag(pEB1029) N-terminal Kan p15A Derived from pKT25linker. Plac–T25–Flag–MCS(EcoRI–ClaI–XhoI) [14]
pUT18C– N-terminal Amp ColE1 Derived from pUT18Clinker. Plac–T18–Flag–MCS(EcoRI–EcoRV–XhoI) [14]
Flag(pEB1030)

but we have sequenced and verified it). The genotype of DHM1 is  IPTG (Isopropyl-b-D-thiogalactoside) stock at 0.1 M, filter steril-
F0 , cya-854, recA1, endA1, gyrA96 (NalR), thi1, hsdR17, spoT1, rfbD1, ized and kept at 20 °C.
glnV44(AS). Both strains are available in the BACTH system kit from  CaCl2 stock at 0.1 M kept at room temperature.
Euromedex (ref. EUK001). BTH101 grows faster and shows stron-  Glycerol stock at 80% kept at room temperature.
ger interaction signals than DHM1. However, some instability of  Antibiotic stock solutions: 25 mg/ml ampicillin and 10 mg/ml
plasmids can appear because it is Rec+, which is not the case for kanamycin kept at 4 °C.
DHM1. The authors have only experience in the use of BTH101,  Maltose 20%, sterilized by filtration, kept at room temperature
but other labs do use DHM1.  SDS (sodium dodecyl sulfate) 0.01%.
 Na2CO3 stock at 1 M.
3.2.3. Reagents and chemicals
Stock solutions: Z buffer: 8 g of Na2HPO412H2O, 3.125 g of NaH2PO4H2O,
0.375 g KCl, 0.123 g MgSO47H2O dissolved in 500 ml distilled
 X-Gal (5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside) water, adjusted to pH 7 if necessary. Immediately before use,
stock at 20 mg/ml in dimethyl formamide (DMF) kept at 20 °C. 1.35 ml b-mercaptoethanol is added. The buffer is kept at 4 °C.
328 A. Battesti, E. Bouveret / Methods 58 (2012) 325–334

M63 medium: 13.6 g KH2PO4, 2 g (NH4)2SO4, 500 lg FeSO4 7H2O overnight at 30 °C with shaking. The next day, 2 ll of each culture
and 245 mg MgSO47H2O in 1 liter of distilled water, adjusted to are dropped on LB-X-Gal plates or on MacConkey/maltose or lac-
pH 7 with KOH. tose plates (see Section 3.2.3). The plates are then incubated at
LB (Luria–Bertani) broth: 10 g NaCl, 10 g tryptone, 5 g yeast ex- 30 °C. MacConkey/Maltose or lactose plates can be incubated sev-
tract in 1 liter of distilled water. Adjust pH to 7 with NaOH, and eral days until a red coloration appears. Alternatively to the color-
autoclave. Plates are prepared by adding 1.5% agar. imetric assay on reporter plates, b-galactosidase assay is
M63/Maltose plates: M63 medium supplemented with 0.2% performed on the same overnight liquid cultures, following the
maltose, kanamycin (30 lg/ml), ampicillin (50 lg/ml), Vitamin B1 classical Miller protocol [12] that we will not describe here. It
(0.5 lg/ml), IPTG (0.5 mM) and X-Gal (40 lg/ml) and 1.5% agar. has to be noted that the signal obtained by following this protocol
MacConkey plates: For the MacConkey/maltose plates, suspend with liquid precultures and drops on plates (Fig. 2B), gives higher
Difco™ MacConkey agar base powder (ref: 281810) in distilled signals than when the bacteria are directly plated on the reporter
water (following the indications on the bottle), and dissolve by plates after transformation, or when the bacteria are reisolated
boiling with frequent agitation. Autoclave at 121 °C for 15 min. on the reporter plates from the transformation plates.
The medium can also be used directly after 5 min boiling, without
autoclaving. Add ampicillin (100 lg/ml), kanamycin (50 lg/ml), 3.4. b-Galactosidase assay in 96-well arrays (Fig. 3)
IPTG (0.5 mM), and 1% maltose. For the MacConkey/lactose plates,
use the standard Difco™ MacConkey agar (ref: 212123) that al- When using the BACTH technique, the number of assays to be
ready contains lactose, and omit IPTG. performed grows exponentially even when not so many clones
LB–X-Gal plates: LB agar supplemented with ampicillin (100 lg/ are tested. Indeed, for a given pair of proteins, one wants to test
ml), kanamycin (50 lg/ml), X-Gal (40 lg/ml), and IPTG (0.5 mM). all the combinations possible between ORF fused to T18 or T25 se-
ONPG (O-nitrophenyl-b-D-galactoside): 4 mg/ml of ONPG diluted quences, and at the N-terminus or the C-terminus (this results in
in Z buffer, prepared just before use. already eight assays just for two partners). Furthermore, due to
some variability in the detection of interactions, it is necessary to
3.2.4. Specific material for the 96-well b-galactosidase assay perform replicates. Here, we describe the standard b-galactosidase
assay in 96-well plates that we use routinely, doing 3, 4, or 6 rep-
 2.2 ml 96-well polypropylene block, licates for each pair to be tested. A microplate spectrophotometer
 standard 96-well microplates, is required for this protocol. We use a TECAN machine, which per-
 multi-channel pipette, mits the direct injection of the ONPG in the plate, and the measure-
 a spectrophotometer for 96-well microplates. ment at 420 nm at different time points (see Fig. 3).
This b-galactosidase assay was adapted to 96-well array by
3.3. Basic protocol to test the interaction between a given pair of modification of the Griffith and Wolf’s method [18]. The bacteria
proteins are grown in a 2.2 ml 96-well polypropylene block, each well con-
taining 0.6 ml of LB medium supplemented with 50 lg/ml kana-
Here is the basic protocol that we use routinely in our laborato- mycin, 100 lg/ml ampicillin, and 0.5 mM IPTG. The block is
ries (Fig. 2B). In addition to testing the pair of proteins of interest, positioned in an incubator with an inclination of about 45° in order
positive controls are performed with T25-zip and T18-zip plas- to enhance cell agitation. After overnight growth at 30 °C, 50 ll of
mids. Negative controls are performed with any pair of proteins culture from each well are transferred with a multi-channel pip-
that do not interact, for example the two empty T18 and T25 plas- ette into a flat-bottom microtiter plate already filled with 150 ll
mids, or better, each protein of interest against the empty T18 or LB, in order to measure the OD600nm in a microplate reader. In par-
T25 plasmids. allel, 200 ll of each culture are transferred into a glass tube con-
Transformation. BTH101 (or DHM1) competent cells are pre- taining 800 ll of Z buffer (see Section 3.2.3). One drop of SDS
pared by treatment with CaCl2. Cells are grown in LB at 37 °C until 0.01% and two drops of chloroform are added, and the glass tubes
OD600nm is comprised between 0.3 and 0.5. Cells are collected by are mixed thoroughly during 10 s to permeabilize the cells. After
centrifugation 10 min at 4000 rpm and the supernatant is dis- letting chloroform settle down at the bottom of the tube, aliquots
carded. The cell pellet is suspended in ½ volume of ice-cold CaCl2 of 50 ll are transferred into a 96-well flat-bottom microplate con-
50 mM and incubated 20 min on ice. Cells are again collected by taining 150 ll of Z buffer and pre-equilibrated at 28 °C in the
centrifugation, and the supernatant is discarded. The cell pellet is microplate reader. Then, 40 ll of ONPG 0.4 % are dispensed and
suspended in 1/10 volume of ice–cold CaCl2 50 mM, 15% glycerol the enzymatic reaction is carried out at 28 °C for 15–20 min with
and incubated 20 min on ice. Aliquots of 500 ll can be stored at measurement of OD420nm every 2 min in the microplate reader.
80 °C or used immediately. For each assay, 0.5 ll (prepared with The relative b-galactosidase activity in each of the 96 samples is
a standard miniprep kit) of the two plasmids carrying the T25 and then calculated by simple Excel file manipulation. It corresponds
T18 fusions are added to 100 ll of competent cells in an ice cold to ((OD420nm at time t2–OD420nm at time t1)/t2–t1 (min))/OD600nm.
1.5 ml tube and incubated on ice during 20 min. The cells are then The t2 and t1 time points are chosen to be located in the linear part
heat shocked at 42 °C during 1.5 min followed immediately by of the kinetic.
1 min on ice. Then, 1 ml LB is added and cells are placed at 37 °C One drawback of this approach is that the intensity of colora-
for recovery for 1 h. Cells are collected by centrifugation at tion of the ONP formed by the reaction is lower at the pH of the
8000 rpm for 3 min, and 1 ml of supernatant is discarded. The pel- Z buffer as compared to that after classical addition of Na2CO3
let is suspended in the remaining 100 ll of LB and cells are spread [12]. This might lead to missing some weak interactions. Yet, the
on LB plates containing 100 lg/ml ampicillin and 50 lg/ml kana- advantage is that the b-galactosidase activities can be accurately
mycin. Plates are incubated at 30 °C for 48 h. determined in parallel on multiple samples presenting a wide var-
Hybrid expression and interaction assay. After incubating the iation in enzymatic activities (e.g. negative and positive controls):
plates for 2 days at 30 °C, 3 ml of LB containing 100 lg/ml ampicil- the activities of samples expressing a high level of the b-galactosi-
lin, 50 lg/ml kanamycin and 0.5 mM IPTG are inoculated with sev- dase can be determined from the early time points of the kinetics,
eral clones from the transformation plate (several clones are whereas those expressing a low level can be accurately determined
picked in order to reduce heterogeneity, see Section 3.6). Tripli- after a prolonged incubation (as the variation in OD420nm is larger
cates are performed for each pair of plasmids. Cultures are grown at these times). Furthermore, there is no need to perform a blank
 A. Battesti, E. Bouveret / Methods 58 (2012) 325–334 329

Fig. 3. b-galactosidase assay in 96-well plates. The different steps of the protocol we routinely use in our laboratories are outlined. See text for the detail.

reaction, neither to correct with the OD550nm, because these terms 3.5.2. Protocol
disappear in the subtraction between the two time points. In the following, we will consider that the bait protein is fused
To conclude, any type of settings that can be implemented in with the T18 domain and, therefore, that the screen is performed
the lab for high throughput b-galactosidase assay should be used. against a genomic library prepared in a pT25 vector.
For example, a similar protocol for b-galactosidase assay in 96-well Transformation and selection. BTH101 strain is transformed
plates applied to BACTH has been published recently [19]. Finally, using the CaCl2/heat shock method (see Section 3.3) by the plasmid
in parallel, 2 ll of the overnight cultures from the 96-well polypro- expressing the T18-bait hybrid protein. Then, cells are prepared for
pylene block may also be directly replicated on indicator plates DNA transformation by electroporation. Briefly, a 100 ml culture is
using a multi-channel pipette. Indeed, the indicator plates often grown until an OD600nm around one. Then, cells are centrifuged
display higher sensitivity than the b-galactosidase assay. 10 min at 4000 rpm at 4 °C. The resulting pellet is suspended in
50 ml of cold water and cells are centrifuged again. This step is re-
peated 2 times. Finally cells are suspended in 0.3 ml of cold
3.5. Library screening water + 10% glycerol and incubated on ice for 20 min. Cells can
be stored at 80 °C or be used immediately. Sixty microliters of
3.5.1. Principle cells are electroporated with 50–100 ng of the T25 library, 1 ml
The BACTH system is a simple and rapid tool to look for new of LB is added and cells are incubated 90 min at 30 °C with shaking
partners of a protein of interest. For this purpose, T18 and T25 for recovery. After incubation, cells are centrifuged 3 min at
two-hybrid libraries have already been constructed with genomic 8000 rpm and the pellet is washed with 1 ml M63 medium (see
DNA from E. coli [20,21] but also from Mycobacterium tuberculosis Section 3.2.3). This step is repeated 4 times. After the last wash,
[22]. The screen is based on the ability of the cells to grow on min- OD600nm of the sample is measured and about 106 cells are spread
imal medium containing 0.2% lactose or maltose as the sole carbon on selection plates and incubated at 30 °C. The selective plates con-
source. Indeed, a cell devoid of adenylate cyclase cannot activate tain M63 medium, 0.2% maltose, kanamycin (30 lg/ml), ampicillin
the catabolic lactose and maltose operons and by consequence is (50 lg/ml), IPTG (0.5 mM) and X-Gal (40 lg/ml). Thus, a positive
not able to use them as carbon source. On the contrary, bacteria interaction not only allows the growth of the colony but also the
containing a positive interaction are able to grow on this medium. appearance of a blue color. It can take 5–10 days for colonies to ap-
This gives a positive selection procedure for the isolation of clones pear on the plates (Fig. 4A).
containing interacting proteins. In the section below, we will call Analysis of the selected clones. Colonies growing on the selection
‘‘bait’’ the protein of interest and ‘‘preys’’ the candidate proteins se- plates are re-streaked on the same medium or on MacConkey/
lected by screening the BACTH library. Maltose or lactose plates (Fig. 4B). If the signal is still positive,
330 A. Battesti, E. Bouveret / Methods 58 (2012) 325–334

Fig. 4. Genomic library screening by BACTH. Each step of the procedure is illustrated. A: After 5 to 10 days at 30 °C, blue positive clones appear on the selective medium. B:
Bacteria are re-isolated on selective medium. C: A classical interaction test is performed with the mix of plasmids obtained from bacteria at step B. D: After purification of the
prey plasmids, interaction is tested against a control vector or against the bait. E: Positive clones obtained at step D are sequenced and the prey is identified. F: The full-length
prey sequence is cloned in pT18 and pT25 and the interaction with the bait is again tested.

the DNA mix of the two T18 and T25 plasmids expressing the bait the BACTH vectors and check the interaction with the bait protein
and the prey proteins is extracted, and the interaction is tested in the various available combinations of plasmids (Fig. 4F). If a po-
again in new BTH101 cells, following the standard protocol (see sitive signal is still detected, the interaction can then be demon-
Section 3.3) (Fig. 4C). Then, to recover only the T25 plasmid coding strated by another method as for example co-purification
for the selected prey protein, E. coli cells are transformed by the experiments [20,22]. For this purpose, we have designed a set of
mix of two plasmids, and cells are incubated on plates containing vectors for BACTH (Table 1) and for purification with 6His, CBP,
only kanamycin. The T25-prey plasmid is then purified. To validate or TAP N-terminal tags, which all have in frame restriction sites
the prey, the T18-bait and the T25-prey plasmids are again used to [14]. It is then easy to transfer the insert from the two hybrid vec-
transform BTH101 and tested for interaction (see Section 3.3) tors to the affinity purification vectors, in order to confirm the
(Fig. 4D). BTH101 is also transformed by the T25-prey and the con- interaction by purification or pull-down experiments. The cloning
trol empty T18 plasmid, to ensure that the prey is not a ‘‘sticky’’ or sites of these modified BACTH vectors are also compatible with
‘‘autoactivating’’ protein by itself. If the interaction is positive and yeast two-hybrid vectors (see Section 3.2.1). Yeast two hybrid
specific of the presence of the bait, the prey insert is sequenced and can also be a second technique to validate an interaction found
identified by BLAST search (Fig. 4E). The interaction must then be with BACTH. The advantage to test an interaction in a heterologous
confirmed. organism is to determine if the interaction is direct between the
two partners or involves other proteins. Detection of a positive
3.5.3. Good practice to validate an interaction interaction, in this case in yeast, would imply that the interaction
The identification of a partner by screening a library with between the two partners is direct.
BACTH, or by testing interactions between proteins of interest, is Functional link. The establishment of a functional link is also
only the beginning of a series of experiments that need to be per- necessary to prove the physiological significance of an interaction.
formed in order to validate the interaction and then establish its If a homologous protein involved in another physiological path-
physiological relevance. In this section, we wish to describe the way exists, it is a good control to test the specificity of the inter-
good practice approach that has been already extensively de- action [24]. Mutants affecting the interaction can also be studied:
scribed in the case of the yeast two-hybrid technique, for which it could be mutants from both partners, known to be defective for
much more experience has been gathered [23]. The doctrine con- their functions and tested for their ability to interact. The reverse
sists in confirming the interaction by another technique, accumu- is also true: mutants impaired in their interaction with the pro-
lating evidence that the interaction is specific (no interaction tein partner can be tested to check if they are affected in their
with homologs for example), isolating mutations breaking and functions.
then restoring the interaction, and any other data showing its In the same order of idea, mapping the protein domain involved
physiological relevance. in the interaction may also give useful information to understand
Confirmation of the interaction. Frequently, the plasmid isolated the link between the two proteins. In this case, interactions are
during the screening of the BACTH library does not contain the full tested between one protein and several truncated forms of the pro-
coding sequence of the protein but just a fragment. The first step to tein partner [24].
validate the interaction is to clone the full-length prey protein in
 A. Battesti, E. Bouveret / Methods 58 (2012) 325–334 331

Finally, a functional link can also be established by overproduc- tion of the hybrids can also be tested by fractionation experiments,
ing or deleting one of the protein and see if it has any effect on the especially for membrane proteins.
function of the protein partner. Tricks to improve the detection of an interaction. One critical step
to detect an interaction can be the incubation time of the transfor-
mation plates. Although the required incubation time is certainly
3.6. Technical recommendations dependent of the strength of the interaction, we have noticed that
incubation shorter than 48 h might not be sufficient to accumulate
In this part of the review, we will go through the different steps enough cAMP and detect a signal. Sometimes, cAMP accumulation
of the BACTH technique to give some indications and tricks to im- in bacteria containing interacting hybrids can be directly inferred
prove the different assays. from the aspect of the colonies on the LB agar plates. Indeed, after
Construction of the fusion plasmids. The two plasmids of the prolonged growth, the colonies have irregular outlines and a
BACTH system express the fusion proteins from wild type lactose rougher aspect than the parental cya strain (A. Battesti, personal
promoter. However, the LacI repressor is not encoded by the plas- communication). Moreover, overnight cultures corresponding to
mids, which might lead to the accumulation of mutations during a positive interaction usually reach a higher OD600nm than the neg-
the cloning process if the fusions provoke growth disadvantage. ative ones.
Therefore, during the construction of the plasmids, it is better to The different indicator plates used to perform qualitative assay
use E. coli strains that have a high level expression of LacI (lacIq), have different sensitivities. We observed that the MacConkey/lac-
such as XL1-Blue. Glucose may also be added to the growth med- tose plates give slightly higher signals than the MacConkey/malt-
ium to further repress expression. This might be very important ose plates, and both MacConkey plates are much more sensitive
when constructing genomic libraries. than the LB-XGal plates because they can be incubated longer at
Hybrid production. As for every technique based on the produc- 30 °C with the negative control remaining white. Besides, the Mac-
tion of recombinant proteins, it is necessary to prove that the re- Conkey reporter plates will often allow the detection of a weak
combinant hybrid protein is correctly produced. Expression of interaction that cannot be detected by b-galactosidase assay. Fur-
the hybrid DNA is under the control of the lac promoter. During thermore, even if we recommend our standard conditions (LB,
BACTH experiment, it is induced by IPTG addition. However, lac IPTG, Ampi/Kana) for b-galactosidase assay, one has to keep in
expression also requires the presence of the cAMP/CAP complex mind that an interaction can be detected or not depending on
[25]. This implies that the cAMP produced when there is an inter- the medium used. In this regard, some authors reported the use
action contributes to the promoter induction. On the contrary, of richer medium [29], and some have evidenced a clear effect of
when only one plasmid is present in the cya strain, or when there the medium on their specific interaction [30].
is no interaction between the two proteins tested, the level of Due probably in part to the expression regulation by cAMP but
induction of the two-hybrid proteins is low because of the lack also to the difference in copy number between the two plasmids,
of cAMP. Thus, correct production of the hybrid protein should the BACTH system presents certain heterogeneity. Indeed, if cells
not be tested in the BTH101 or DHM1 strains but rather in a cya+ are directly plated on indicator plates after recovery, a mix be-
strain. On the contrary, two interacting proteins can contribute tween white and red colonies can be observed. We have found that
to signal amplification through cAMP synthesis, and also to each starting cultures with several colonies help to cope with this issue.
other stabilization and, by consequence, to a better detection. Finally, the detection of an interaction can sometimes be im-
In most two-hybrid systems, recombinant protein production proved by the deletion of already known interactants (A. Battesti,
and stability are verified by Western blot using antibodies against personal communication). This can be tested for example if an
the fused domains. However, such antibodies directed against T18 interaction initially detected via a BACTH library screen cannot
and T25 domains of adenylate cyclase were not clearly proposed in be reproduced. Indeed, as the medium used to do the screen is a
the first description of BACTH. To fill this gap, we first exploited the harsh condition for the cell, some secondary mutations can appear
property of the T18 sub-unit of the B. pertussis adenylate cyclase to in the genome allowing the interaction between two proteins. In
bind calmodulin [10]. The T18-fused recombinant proteins can some circumstances the expression of a third protein can inversely
therefore be detected by Western blot using biotinylated calmod- have a positive effect if this protein is needed to form a more stable
ulin, for example by using the CBP detection kit from Stratagene complex or stabilize an interaction [17,31] (see Section 5.2).
[14]. Additionally, calmodulin binding property allows the direct
purification of T18-tagged proteins on calmodulin coated beads
4. Advantages/disadvantages
[26]. We have also developed two new vectors with a Flag epitope
inserted at the N-terminus, between the T18 and T25 domains and
When considering the potential drawbacks of a technique, it of-
the ORF of interest (Table 1). The addition of a Flag tag in the two-
ten happens that its disadvantages might turn out to be advantages
hybrid constructions does not affect the interaction between the
and vice versa. This is especially true for the BACTH, as shown by
two partner proteins [14]. Now, several antibodies have been
the following examples.
developed against the adenylate cyclase of B. pertussis, and some
of them can be used to detect hybrid proteins from the BACTH sys-
tem. For example, the monoclonal 3D1 antibody from Santa Cruz 4.1. Expression system
recognizes the T18 sub-unit [19,26]. The use of b300 polyclonal
antibody from Santa Cruz to detect the T25 domain has also been In order to correctly analyze the results of a BACTH experiment,
reported, however we do not have experience with it [27]. one has to take into account the principle guiding recombinant
Hybrid functionality. Together with verifying correct production protein expression. The two hybrid proteins are expressed from
of the hybrid protein, proving that this hybrid protein is still func- two compatible plasmids that have distinct replication origins:
tional can be a plus. However, the possibility to do so is specific of the pT25 plasmids have a p15A origin (low copy number), while
each research project. When mutants are available and a pheno- the pT18 plasmids have a ColE1 origin (high copy number). How-
type can be detected, one can test complementation by the hybrid ever, the same promoter in the two plasmids, i.e. the wild type pro-
vectors. For example T18- or T25-SpoT recombinant proteins were moter of the lactose operon, drives the expression of the hybrids.
functional in spoT mutant strains [24] and T18-ACP was functional This results in different relative amounts of the two hybrid pro-
in an acpP thermosensitive mutant strain [28]. The correct localiza- teins that are produced. Because the stoichiometry between two
332 A. Battesti, E. Bouveret / Methods 58 (2012) 325–334

partner proteins can be critical for their interaction, the interaction 4.4. Reporter detection
has to be tested in the two possible combinations of T18 and T25
fusions (it is not rare to detect an interaction in only one case). The direct consequence of an interaction in the BACTH is recon-
In order to resolve this problem, authors have designed new plas- stitution of adenylate cyclase activity, and ideally that is this activ-
mids with the same copy number [32]. The issue may also be re- ity that should be measured to quantify the level of interaction.
solved by putting the two hybrid genes on the same plasmid. Even if cAMP levels can be measured [11], usually the output of
The use of the wild type Plac promoter itself may come as a sur- the BACTH assay is the level of expression of the lactose and malt-
prise. Because of the positive feedback loop driven by the cAMP ose operons. Therefore, colorimetric assay on reporter plates or
(see Section 3.6), it is only if an interaction occurs that the cAMP quantification of b-galactosidase activity is only an indirect mea-
level will rise and allow full expression of the hybrid proteins. This sure of the interaction, involving a complex signaling cascade. In-
results in a threshold effect, which may be the strength of the tech- deed, lactose and maltose operons are subjected to complex
nique, by keeping the rate of false interactions due to sticky pro- regulation and are affected by a variety of signals in addition to
teins or misfolding very low. Indeed, in order to avoid this auto- cAMP [25]. Because the assay depends on wild type promoters
activation, BACTH plasmids containing a mutant Plac promoter and reporter genes, there is the possibility of indirect effects on
not inducible by cAMP (lacUV5) have been constructed, but it in- the expression level caused by the recombinant proteins. For
creased the rate of detection of false positive interactions (D. La- example, we have been blocked in our studies on enzymes in-
dant, personal communication). On the other hand, this auto- volved in stringent response, because (p)ppGpp affects lactose
amplification might be the reason for the heterogeneity in interac- and maltose operon transcription (Battesti and Bouveret,
tion that is observed for some interactions in the b-galactosidase unpublished).
assays (see Section 3.5). This is therefore a feature of the technique One advantage of the use of wild type lactose and maltose oper-
that is both advantageous and disadvantageous. on expression as the output for BACTH is the possibility to perform
positive selection of interacting proteins on minimal media con-
taining only lactose or maltose. This is especially powerful for li-
4.2. Recombinant protein production and false positive brary screening (see Section 3.5). Inversely, this setup
theoretically permits a reverse system, in which it is this time
As it is the case for any two-hybrid technique, independently of the absence of interaction that is positively selected, by incubating
the principle of interaction detection, production of recombinant cells in presence of lambda phage. Indeed, the lambda phage recep-
proteins may cause problems. The fusion to T18 or T25 domains tor LamB is encoded within the maltose operon, and its absence
may cause the protein to be badly folded, unstable, or simply pre- would render the cells resistant to lambda phage. This may prove
vent the interaction with its partners. Furthermore, proteins may to be useful to identify residues involved in an interaction, how-
have intrinsic tendency to interact with any protein, what is often ever we are not aware of any studies that would have used this
called ‘‘sticky proteins’’. system.
Currently, no cases of auto-activators in the BACTH, meaning
proteins that alone are able to bind to T18 or T25 domains have 4.5. The host is E. coli
been reported in the literature. In our experience, we did not find
such auto-activators either, but they might exist, as calmodulin- The BACTH relies on the expression of recombinant proteins in
like proteins have been described in bacteria [33]. It would be E. coli cells. Therefore, for use with eukaryotic proteins, this might
interesting to list all the sticky proteins and also the potential cause a problem if proteins have to be post-translationally modi-
auto-activators that will certainly appear from library screening fied. Yet, it can be viewed as an advantage to get rid of indirect
studies. However, there have not been so many screens published interactions [40] and it might be the only solution to study mem-
till now, and unfortunately authors usually do not list the false brane protein interactions. On the contrary, for studies of bacterial
positives. proteins, it is not possible to exclude that the detected interaction
is indirect, especially for bacteria closely related to E. coli. Yet, for
researchers working on E. coli, this technique is very powerful. In-
4.3. BACTH can be used with membrane proteins deed, it permits the dissection of complexes, by playing with the
genetic background of the strain used for the BACTH [41], or by
One clear advantage of the BACTH technique is the possibility to modifying the growth conditions [30].
study membrane protein interactions. Indeed, because the output
of the interaction is a diffusible cAMP signaling molecule, the inter-
action can take place anywhere in the cell, hence at the membrane. 5. What can be done with BACTH?
Providing that the hybrid proteins are correctly inserted in the
membrane, with the T18 and T25 domains facing the cytoplasm, In order for the reader to know if the technique will be helpful
the interaction can be detected (Fig. 1D). This has been first de- for her or his research, we will try here to summarize the types of
scribed for multiple interactions between Fts proteins of E. coli proteins and interactions that have been studied successfully so far
[17], and then for several other membrane proteins [21,34–36]. by BACTH. We will also give examples of the questions that can be
The interaction can be reduced down to interaction between trans- asked by playing with the method.
membrane segments alone fused to T18 or T25 domains. Conse-
quently, BACTH has been the technique of choice to study small 5.1. Subjects covered (types of organisms and proteins)
transmembrane peptides involved in the regulation of membrane
proteins [37–39]. However, we observed that for membrane pro- Quite expectedly, most publications referring to BACTH deal
teins, unspecific weak signal of interaction is more often observed with bacterial protein interactions. Indeed, studies have been per-
than for soluble proteins, maybe due to overcrowding of the mem- formed not only in E. coli, but also in any bacterial phylum. How-
brane. In this case, it is therefore very important to perform spec- ever, as demonstrated in the founder paper [1], this technique
ificity controls with unrelated proteins. For example, MalF and can also be successfully applied to eukaryote protein interactions.
MalG hybrids that interact strongly with each other can be good For example, interactions between Laminin proteins of mouse [42]
controls [17]. or between proteins of the yeast Set1 complex have been evi-
 A. Battesti, E. Bouveret / Methods 58 (2012) 325–334 333

denced by BACTH [40]. Interaction between viral proteins has also interaction in the case of ternary complexes. Examples of the dif-
been reported [43], and this suggests that the technique may be ferent possibilities are nicely shown in the case of Fts division pro-
used for studying interactions between virus and host proteins. teins in E. coli [17].
Even if the examples are scarce (yet it is not certain that we are
aware of all of them), they indicate that the technique can be used  Characterization of an interaction
for eukaryote and viral protein studies.
In the last ten years, about hundred papers have been published Because of the simplicity of the method and different possibili-
making use of the BACTH. Interestingly, there are clearly recurrent ties of screening for gain or loss of interaction, the method can be
processes that are studied in bacteria using this technique. It con- used for dissecting a given interaction that has been detected.
cerns proteins involved in cell division in E. coli, Synechocystis, Moreover, the characterization of the residues or domain involved
Bacillus subtilis or other bacteria [17,21,44,45]. Two-component can be part of validating the interaction (see Section 3.5.3). Follow-
system proteins have also been repeatedly studied by this tech- ing studies are examples of such careful dissection experiments.
nique [46,47]. It has also been the technique of choice to study The domains and residues involved in the interaction between
newly identified small proteins that form transmembrane segment Crl and SigmaS of E. coli were identified in both proteins by trunca-
[37–39]. Two explanations can be given for these preferred sub- tion and mutagenesis experiments [48,49]. The same was done for
jects: first, the BACTH appears as the perfect approach for rapid characterizing the interaction between SpoT and Acyl Carrier Pro-
screening of membrane protein interactions (see Section 4.3). Sec- tein [24,26]. The dimerization of CbpA protein was dissected by
ond, when protein interactions have been reported once, they are truncation and alanine scanning mutagenesis, permitting the iden-
often studied by the same approach by the other scientists in the tification of a hydrophobic surface involved in the dimerization
same field of research. Because there are still not so many interac- [30]. A screen for a gain of interaction between membrane proteins
tion studies with BACTH that are published, both explanations cer- TrwE and TrwB have permitted the isolation of point mutations
tainly apply here. indicating an interaction between the transmembrane regions of
BACTH has been used successfully for screening genomic li- these proteins [50]. Finally, the association of cell division proteins
braries of E. coli and M. tuberculosis [20–22]. Other studies should FtsL and FtsB through leucine zipper motifs has been dissected
be published soon of screens performed in E. coli or in P. aeruginosa [19].
(A. Battesti, unpublished; L. Houot, 2012, in press). Some studies
also systematically checked the protein–protein interactions be-  Other applications based on T18/T25 reconstitution
tween an important set of proteins of interest in a systematic
way [17,36,44]. In this kind of approach, b-galactosidase assay in The ability to detect the reconstitution of adenylate cyclase
96-well plate might prove to be very useful. from T18 and T25 domains association is amenable to other appli-
cations than protein–protein interaction studies. This system is in-
5.2. Technical developments and applications deed well adapted to screen for the synthesis of a full-length
polypeptide consisting of the fused T18 and T25 domains. For
When browsing the literature, several original use of the example, by inserting linkers between the T18 and T25 sequences,
BACTH, besides simply testing a pair of proteins or screening a li- it is possible to screen genetically for events such as premature co-
brary, have been reported. These approaches are a promise for dons [51] or presence of specific protease cleavage sites [52,53].
powerful developments and applications.
6. Other bacterial two-hybrid systems
 Chemical screening to identify inhibitors
This review is dedicated to the BACTH technique. However, it is
Recently, Paschos et al. screened a compound collection in order
important to mention that other bacterial two-hybrid systems are
to find molecules that inhibit the dimerization of VirB8, assayed by
available. The major alternatives to the BACTH in E. coli are tech-
BACTH [29]. About 30,000 molecules were screened, demonstrat-
niques that are based on the activation of transcription, similar
ing here again the suitability of the BACTH for high throughput
to the classical yeast two-hybrid [54,55]. One complete system
studies.
called BacterioMatchÒII is distributed by Stratagene. In this system,
one of the proteins is fused to a component of the RNAP (either a or
 Influence of the genetic background
x subunit), while the other is fused to specific DNA binding do-
main (Zif or kcI). In this case, engineered test reporters (genes cod-
For researchers working in E. coli, it is possible to test the effect
ing for resistance to antibiotics or for b-galactosidase) contain the
of gene deletions in the BTH101 strain on the interactions that are
Zif or kcI binding box in their promoter sequence. These techniques
studied. It is for example possible to abolish transient interaction
conserve most of the advantages of working in E. coli, yet they do
by removing an intermediate involved in the interaction. Indeed,
not allow studying membrane protein interactions. However, they
interactions between some enzymes of molybdenum cofactor syn-
are powerful to study interactions involved in gene regulation [56].
thesis were abolished when the first step of synthesis was mutated
Furthermore, we want to point out the existence of dedicated plas-
[41]. Similarly, the interactions between nitrate reductase A and
mids for Gateway cloning [57], which should unable high through-
the enzymes involved in molybdenum cofactor synthesis strictly
put screening of interactions, in order to describe interactomes
require the presence of mature Moco (abolished in moa or mob mu-
such as what is done with yeast two-hybrid. Even without using
tants) [31]. Because the technique is amenable to high throughput
these Gateway plasmids, a high throughput study of Mycobacte-
studies, screening mutant library obtained by transposon muta-
rium tuberculosis interactome has been performed using this tech-
genesis or on the reverse, screening an expression library, should
nique [58].
be possible. Both positive and negative effects could then be
screened. For example, the production of the two proteins NarH
and NarJ restores the interaction between NarG and the Mo pro- 7. Concluding remarks
teins [31]. This is a typical case of three-hybrid: on a single plas-
mid, a hybrid protein is expressed together with a non hybrid. We have described the current applications of the BACTH sys-
This could lead either to competition or to enhancement of the tem. Its use has increased in the recent years in the community
334 A. Battesti, E. Bouveret / Methods 58 (2012) 325–334

of microbiologists, and we hope this review will be helpful for [25] W.S. Reznikoff, Mol. Microbiol. 6 (1992) 2419–2422.
[26] S. Angelini, L. My, E. Bouveret, PLoS ONE 7 (2012) e36111.
newcomers to the technique. It is rapid, easy, and very reliable,
[27] M.L. Workentine, L. Chang, H. Ceri, R.J. Turner, FEMS Microbiol. Lett. 292
with few reported case of false positives or false negatives. How- (2009) 50–56.
ever, due to some drawbacks that have been exposed here, there [28] A. Battesti, E. Bouveret, J. Bacteriol. 191 (2009) 616–624.
is still space for improvement, such as designing other reporters [29] A. Paschos, A. den Hartigh, M.A. Smith, V.L. Atluri, D. Sivanesan, R.M. Tsolis, C.
Baron, Infect. Immun. 79 (2011) 1033–1043.
than b-galactosidase, like GFP or antibiotic resistance, and the con- [30] S. Cosgriff, K. Chintakayala, Y.T. Chim, X. Chen, S. Allen, A.L. Lovering, D.C.
struction of other plasmids for Gateway cloning for example. Grainger, Mol. Microbiol. 77 (2010) 1289–1300.
[31] A. Vergnes, K. Gouffi-Belhabich, F. Blasco, G. Giordano, A. Magalon, J. Biol.
Chem. 279 (2004) 41398–41403.
Acknowledgments [32] A.J. Jervis, J. Green, J. Bacteriol. 189 (2007) 2930–2932.
[33] J. Michiels, C. Xi, J. Verhaert, J. Vanderleyden, Trends Microbiol. 10 (2002) 87–
First, we are deeply grateful to Daniel Ladant and Gouzel Karim- 93.
[34] M.E. Maxson, A.J. Darwin, Mol. Microbiol. 59 (2006) 1610–1623.
ova who invented and developed the BACTH system. We thank [35] Y. Hara, M. Seki, S. Matsuoka, H. Hara, A. Yamashita, K. Matsumoto, Genes
Laetitia My, Julie Viala, Rim Maouche, all past members of the Genet Syst. 83 (2008) 433–442.
Bouveret team, and Susan Gottesman and Olivier Genest for sup- [36] M. Georgiadou, M. Castagnini, G. Karimova, D. Ladant, V. Pelicic, Mol.
Microbiol. (2012).
port and discussion. We would like to thank all the BACTH users [37] Y. Eguchi, J. Itou, M. Yamane, R. Demizu, F. Yamato, A. Okada, H. Mori, A. Kato,
in our laboratories, from whom we got back experience and discus- R. Utsumi, Proc. Natl. Acad. Sci. USA 104 (2007) 18712–18717.
sion. Research in EB laboratory is funded by CNRS and ANR. AB is [38] E. Alix, A.B. Blanc-Potard, EMBO J. 27 (2008) 546–557.
[39] A.M. Lippa, M. Goulian, PLoS Genet. 5 (2009) e1000788.
funded by the NIH.
[40] P.M. Dehe, B. Dichtl, D. Schaft, A. Roguev, M. Pamblanco, R. Lebrun, A.
Rodriguez-Gil, M. Mkandawire, K. Landsberg, A. Shevchenko, A. Shevchenko,
References L.E. Rosaleny, V. Tordera, S. Chavez, A.F. Stewart, V. Geli, J. Biol. Chem. 281
(2006) 35404–35412.
[1] G. Karimova, J. Pidoux, A. Ullmann, D. Ladant, Proc. Natl. Acad. Sci. USA 95 [41] A. Magalon, C. Frixon, J. Pommier, G. Giordano, F. Blasco, J. Biol. Chem. 277
(1998) 5752–5756. (2002) 48199–48204.
[2] G. Karimova, A. Ullmann, D. Ladant, Methods Enzymol. 328 (2000) 59–73. [42] M.M. Edwards, E. Mammadova-Bach, F. Alpy, A. Klein, W.L. Hicks, M. Roux, P.
[3] G. Karimova, A. Ullmann, D. Ladant, Int. J. Med. Microbiol. 290 (2000) 441–445. Simon-Assmann, R.S. Smith, G. Orend, J. Wu, N.S. Peachey, J.K. Naggert, O.
[4] G. Karimova, D. Ladant, A. Ullmann, Int. J. Med. Microbiol. 292 (2002) 17–25. Lefebvre, P.M. Nishina, J. Biol. Chem. 285 (2010) 7697–7711.
[5] G. Karimova, D. Ladant 2005. A bacterial two-hybrid system based on a cAMP [43] N. Dautin, G. Karimova, D. Ladant, J. Virol. 77 (2003) 8216–8226.
signaling cascade, in: E. Golemis, P.D. Adams, (Eds.), Protein–Protein [44] M. Marbouty, C. Saguez, C. Cassier-Chauvat, F. Chauvat, Mol. Microbiol. (2009).
interactions, a molecular cloning manual, CSHL Press, (2005) 499–515. [45] R.A. Daniel, M.F. Noirot-Gros, P. Noirot, J. Errington, J. Bacteriol. 188 (2006)
[6] D.L. Confer, J.W. Eaton, Science 217 (1982) 948–950. 7396–7404.
[7] D. Ladant, A. Ullmann, Trends Microbiol. 7 (1999) 172–176. [46] A.L. Goodman, M. Merighi, M. Hyodo, I. Ventre, A. Filloux, S. Lory, Genes Dev.
[8] P. Glaser, D. Ladant, O. Sezer, F. Pichot, A. Ullmann, A. Danchin, Mol. Microbiol. 23 (2009) 249–259.
2 (1988) 19–30. [47] M. Sivaneson, H. Mikkelsen, I. Ventre, C. Bordi, A. Filloux, Mol. Microbiol. 79
[9] D. Ladant, J. Biol. Chem. 263 (1988) 2612–2618. (2011) 1353–1366.
[10] D. Ladant, S. Michelson, R. Sarfati, A.M. Gilles, R. Predeleanu, O. Barzu, J. Biol. [48] V. Monteil, A. Kolb, J. D’Alayer, P. Beguin, F. Norel, J. Bacteriol. 192 (2010)
Chem. 264 (1989) 4015–4020. 1075–1087.
[11] G. Karimova, D. Ladant, cAMP assays, CSH Protoc. 2007 (2007) pdb.prot4739. [49] V. Monteil, A. Kolb, C. Mayer, S. Hoos, P. England, F. Norel, J. Bacteriol. 192
[12] J.H. Miller, A short course in bacterial genetics: a laboratory manual and (2010) 6401–6410.
handbook for Escherichia coli and related bacteria, Cold Spring Harbor [50] H.D. de Paz, D. Larrea, S. Zunzunegui, C. Dehio, F. de la Cruz, M. Llosa, J.
Laboratory Press, Plainview, N.Y., 1992. Bacteriol. 192 (2010) 2655–2669.
[13] G. Karimova, A. Ullmann, D. Ladant, J. Mol. Microbiol. Biotechnol. 3 (2001) 73– [51] S.M. Real, D.M. Marzese, L.C. Gomez, L.S. Mayorga, M. Roque, BMC Biotechnol.
82. 6 (2006) 38.
[14] A. Battesti, E. Bouveret, Proteomics 8 (2008) 4768–4771. [52] N. Dautin, G. Karimova, A. Ullmann, D. Ladant, J. Bacteriol. 182 (2000) 7060–
[15] D. Gully, E. Bouveret, Proteomics 6 (2006) 282–293. 7066.
[16] J. Gyuris, E. Golemis, H. Chertkov, R. Brent, Cell 75 (1993) 791–803. [53] M. Obrist, F. Narberhaus, J. Bacteriol. 187 (2005) 3807–3813.
[17] G. Karimova, N. Dautin, D. Ladant, J. Bacteriol. 187 (2005) 2233–2243. [54] S.L. Dove, A. Hochschild, Genes Dev. 12 (1998) 745–754.
[18] K.L. Griffith, R.E.J. Wolf, Biochem. Biophys. Res. Commun. 290 (2002) 397–402. [55] S.L. Dove, J.K. Joung, A. Hochschild, Nature 386 (1997) 627–630.
[19] C. Robichon, G. Karimova, J. Beckwith, D. Ladant, J. Bacteriol. 193 (2011) 4988– [56] B.E. Nickels, Methods 47 (2009) 53–62.
4992. [57] S.L. Karna, X. Zogaj, J.R. Barker, J. Seshu, S.L. Dove, K.E. Klose, Biotechniques 49
[20] F. Domain, X.R. Bina, S.B. Levy, Mol. Microbiol. 66 (2007) 383–394. (2010) 831–833.
[21] G. Karimova, C. Robichon, D. Ladant, J. Bacteriol. (2008) 333–346. [58] Y. Wang, T. Cui, C. Zhang, M. Yang, Y. Huang, W. Li, L. Zhang, C. Gao, Y. He, Y. Li,
[22] L.I. Klepp, M. Soria, F.C. Blanco, M.V. Bianco, M.P. Santangelo, A.A. Cataldi, F. F. Huang, J. Zeng, C. Huang, Q. Yang, Y. Tian, C. Zhao, H. Chen, H. Zhang, Z.G. He,
Bigi, BMC Mol. Biol. 10 (2009) 3. J. Proteome Res. 9 (2010) 6665–6677.
[23] R. Brent, R.L.J. Finley, Annu. Rev. Genet. 31 (1997) 663–704. [59] Q. Guo, Y. Shen, Y.S. Lee, C.S. Gibbs, M. Mrksich, W.J. Tang, EMBO J. 24 (2005)
[24] A. Battesti, E. Bouveret, Mol. Microbiol. 62 (2006) 1048–1063. 3190–3201.