Analysis of Membrane Protein UNIT 20.

12
Interactions with a Bacterial Adenylate
Cyclase–Based Two-Hybrid (BACTH)
Technique
Scot P. Ouellette,1,2 Gouzel Karimova,1 Marilyne Davi,1 and Daniel Ladant1
1
Unité de Biochimie des Interactions Macromoléculaires, Département de Biologie
Structurale et Chimie, Institut Pasteur, CNRS, UMR 3528, Paris, France
2
Division of Basic Biomedical Sciences, Sanford School of Medicine, University of South
Dakota, Vermillion, South Dakota

The bacterial two-hybrid (BACTH, for “Bacterial Adenylate Cyclase-based

Two-Hybrid”) technique is a simple and fast genetic approach to analyze
protein-protein interactions in vivo. In this system, the proteins of interest are
genetically fused to two complementary fragments from the catalytic domain
of Bordetella pertussis adenylate cyclase and co-expressed in strains of Es-
cherichia coli deficient in adenylate cyclase. Association of the hybrid proteins
restores synthesis of cyclic AMP (cAMP), which then triggers the expression of
catabolic operons such as the lactose operon or the maltose regulon. As BACTH
uses a cAMP second messenger, the association between the chimeric proteins
can take place at a distance from the transcription machinery. This technique is
therefore particularly appropriate for studying interactions involving integral-
membrane or membrane-associated proteins that may not be soluble in the
cytoplasm, and/or that may only associate in the plane of the membrane. This
unit describes the basic procedures to characterize protein-protein interactions
with the BACTH genetic system and to search for potential partners of known
proteins. 
C 2017 by John Wiley & Sons, Inc.

Keywords: protein interaction assay r two-hybrid technique r membrane pro-

tein r library screening r Escherichia coli r cAMP signaling

How to cite this article:

Ouellette, S. P., Karimova, G., Davi, M., & Ladant, D. (2017).
Analysis of membrane protein interactions with a bacterial
adenylate cyclase–based two-hybrid (BACTH) technique. Current
Protocols in Molecular Biology, 118,20.12.1–20.12.24.
doi: 10.1002/cpmb.36

INTRODUCTION
Protein-protein interactions are fundamental in all biological processes. Many genetic
techniques have been designed to characterize such interactions in living cells. All these
techniques (yeast two-hybrid, interaction trap, protein complementation, etc.) rely on
the co-expression of two (or more) hybrid proteins that can restore a phenotypic trait
upon association (see Chapter 20 in this manual; also see Fields & Song, 1989; Gyuris,
Golemis, Chertkov, & Brent, 1993; Stynen, Tournu, Tavernier, & Van Dijck, 2012).
We have devised a bacterial two-hybrid system, BACTH (Bacterial Adenylate Cyclase-
based Two-Hybrid), that works in Escherichia coli and involves a cAMP signaling
cascade (Karimova, Pidoux, Ullmann, & Ladant, 1998). In this system (Fig. 20.12.1),
Analysis of
Protein
Interactions

Current Protocols in Molecular Biology 20.12.1–20.12.24, April 2017 20.12.1

Published online April 2017 in Wiley Online Library (wileyonlinelibrary.com).
doi: 10.1002/cpmb.36
Copyright C 2017 John Wiley & Sons, Inc. Supplement 118
 Figure 20.12.1 Principle of BACTH two-hybrid technique. (A) The two fragments of B. pertussis
adenylate cyclase, T25 and T18, fused to protein X and Z which do not interact, are co-expressed
in E coli cya: they cannot assemble into a functional species and there is no cAMP synthesis.
(B) The T25 and T18 fragments are co-expressed as fusions with polypeptides X and Y that
can interact. Association of the hybrid proteins T25-X and T18-Y restores the adenylate cyclase
activity. (C) Cyclic AMP synthesized by the reconstituted enzyme binds to the catabolite activator
protein (CAP) and the cAMP/CAP complex can associate with specific promoter DNA and activates
transcription of catabolite operons (such as lac operon or mal regulon).

the proteins of interest are genetically fused to two fragments, T25 and T18, of the
catalytic domain of the adenylate cyclase (AC) toxin from Bordetella pertussis (Ladant
& Ullmann, 1999). These two fragments, when expressed as separate entities, are unable
to associate with each other and are inactive. However, when the T25 and T18 fragments
are fused to proteins that interact, heterodimerization of the chimeric polypeptides results
in enzymatic complementation and restores cAMP synthesis. In E. coli, cAMP is a
pleiotropic regulator of the expression of numerous catabolite genes, such as the lactose
operon or maltose regulon (Busby & Ebright, 1999). Hence, E. coli cya (i.e., deficient
in endogenous adenylate cyclase) strains are unable to ferment lactose or maltose. Co-
expression of interacting T25 and T18 hybrid proteins in these cya cells restores a Cya+
phenotype that is easily detectable on indicator or selective media (Karimova et al., 1998;
Karimova, Ullmann, & Ladant, 2000).

The BACTH system offers some interesting possibilities as compared to classical yeast
two-hybrid systems or other described E. coli hybrid systems. A main advantage of
the BACTH assay stems from the fact that it uses a signaling cascade involving a
diffusible messenger, cAMP. As a result, the association of the hybrid proteins can be
spatially separated from the transcriptional activation readout. Therefore, the BACTH
assay is capable of detecting interactions occurring in the cytosol, in the cytoplasmic
membrane, and even in the periplasm. We and others have used this system to characterize
a wide variety of interactions between proteins from various origins (e.g., bacterial,
viral, mammalian cells, plant cells) involved in many distinct biological functions (e.g.,
signaling, metabolism, cell division, secretion processes, pathogenesis). In particular, we
have used it to study the assembly of multi-molecular membrane-associated complexes
in bacteria, such as the bacterial cell division apparatus, secretion machineries, signaling
networks, etc. (Gauliard, Ouellette, Rueden, & Ladant, 2015; Georgiadou, Castagnini,
Karimova, Ladant, & Pelicic, 2012; Karimova, Dautin, & Ladant, 2005).
Membrane We describe here the main procedures and analytical methods to characterize protein-
Protein
Interaction protein interactions with the BACTH system. Basic Protocol 1 presents the standard
Analysis with methodology to characterize interactions between two proteins, while Alternate Proto-
BACTH
col 1 describes a variant of the BACTH system that is compatible with the Gateway
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cloning technology (Life Technologies, Thermo Fisher Scientific). Basic Protocol 2 il-
lustrates the application of the BACTH system in library screening in order to isolate
potential partners of a protein of interest, while Alternate Protocol 2 describes how to
use the BACTH Gateway system for such interaction traps.

DETECTION OF INTERACTION BETWEEN TWO DIFFERENT PROTEINS BASIC

WITH THE BACTH ASSAY PROTOCOL 1
The basic methodology to characterize interactions between two proteins with the
BACTH technique involves three main steps, as summarized in Figure 20.12.2.

Step 1: Construction of Recombinant BACTH Vectors

The genes encoding the two proteins of interest [e.g., X and Y in Fig. 20.12.2; usually
amplified by PCR using appropriate primers; UNIT 15.1 (Kramer and Coen, 2000)] are
sub-cloned into pKT25 (or pKNT25) and pUT18 (or pUT18C) vectors (described in
Table 20.12.1 and shown in Fig. 20.12.3) in frame with the T25 and T18 fragment
open reading frames using standard molecular biology techniques (see Chapter 3 in this
manual). Recombinant plasmids are purified with one of many commercial kits used
for mini-preparation of DNA, and verified by DNA sequencing (see Chapter 7 in this
manual).

Step 2: Phenotypic Assays of Interaction in Δcya E. coli Cells

Two different recombinant plasmids encoding the T25-X (or X-T25) and T18-Y (or Y-
T18) hybrid proteins are co-transformed into competent BACTH reporter cells (DHM1,
DHT1, or BTH101, see Table 20.12.2). Transformants are plated on indicator plates
(i.e., LB-X-gal agar or MacConkey/maltose agar) and incubated at 30°C (or 37°C) for

Analysis of
Figure 20.12.2 Detection of interaction between two proteins X and Y with the BACTH system. Protein
See text for detailed explanation. Interactions

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 Table 20.12.1 Standard BACTH Plasmid Vectors

Resistance and
Replication
Plasmids origin Main characteristics
pKT25 Kanamycin Low copy-number plasmid
p15A Expresses the T25 fragment under lac promoter control
MCSa at the 3 end of T25 for construction of in-frame fusions
to the C-terminus of T25
Karimova, Ullmann, & Ladant, 2001
pKNT25 Kanamycin Similar to pKT25 but with MCS located at the 5 end of T25
p15A coding region
For construction of fusions to the N-terminus of T25
Karimova, Dautin, & Ladant, 2005
pST25 Spectinomycin Similar to pKT25 but with a Spectinomycin resistant cassette
p15A instead of Kanamycin
For construction of fusions to the C-terminus of T25
Ouellette, Gauliard, et al., 2014
pSNT25 Spectinomycin Similar to pST25 but with MCS located at the 5 end of T25
p15A coding region
For construction of fusions to the N-terminus of T25
Ouellette, Gauliard, et al., 2014
pUT18 Ampicillin High copy-number plasmid
ColE1 expresses the T18 fragment under lac promoter control
MCS at the 5 end of T18 for construction of in-frame fusions
at the N-terminus of T18
Karimova, Ullmann, and Ladant, 2001
pUT18C Ampicillin Similar to pUT18 but with MCS at the 3 end of T18
ColE1 For construction of fusions to the C-terminus of T18
Karimova, Ullmann, & Ladant, 2001
pKT25-zip Kanamycin Positive control plasmid
P15A expresses T25 fused to the leucine zipper of GCN4
Karimova, Ullmann, & Ladant, 2001
pUT18C- Ampicillin Positive control plasmid
zip ColE1 expresses T18 fused to the leucine zipper of GCN4
Karimova, Ullmann, & Ladant, 2001
Gateway compatible BACTH vectors)b
pST25- Spectinomycin Similar to pST25 but with attR recombination sites at 3 end of
DEST Chloramphenicol T25 Orf
P15A For construction of fusions to the C-terminus of T25
Ouellette, Gauliard, et al., 2014
pSNT25- Spectinomycin Similar to pST25 but with attR recombination sites at 5 end of
DEST Chloramphenicol T25 Orf
P15A For construction of fusions to the N-terminus of T25
Ouellette, Gauliard, et al., 2014
pUT18C- Ampicillin Similar to pUT18C but with attR recombination sites at 3 end
DEST Chloramphenicol of T18 Orf
ColE1 For construction of fusions to the C-terminus of T18
Ouellette, Gauliard, et al., 2014
Membrane a MCS: multicloning site sequence.
Protein b The Gateway vectors must be propagated in E. coli strains that are resistant to the lethal effect of the CcdB toxin (i.e.,
Interaction harboring the CcdB resistant, gyrA462 gyrase mutation) such as the DB3.1 E. coli strain (see manufacturer’s guidelines
Analysis with at https://tools.thermofisher.com/content/sfs/manuals/gatewayman.pdf).
BACTH

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Figure 20.12.3 Schematic representation of BACTH plasmids. Light blue and green arrows
represent the open reading frames of T25 and T18 fragments, respectively, under the transcrip-
tional control of the lac promoter. Red and dark blue arrows correspond to the kanamycin- and
ampicillin-selectable markers, respectively. The yellow and brown boxes indicate the p15A and
ColE1 origins of replication, respectively. The hatched boxes schematized the multicloning se-
quences (MCS) used to insert foreign genes: some unique restriction sites are shown above the
nucleotide sequence and the encoded polypeptide sequences are shown below.

1 to 3 days. The cell phenotypes (i.e., Lac+ or Mal+ ) provide a direct, yet qualitative,
assessment of the interaction between protein X and Y.

Step 3: Quantification of Interactions by β-Galactosidase Assays

The efficiency of functional complementation between two hybrid proteins is quantified

by measuring the β-galactosidase activities in liquid cultures (either exponentially grow-
ing or overnight cultures) after cell permeabilization, using o-nitrophenol-β-galactoside
(ONPG) as a substrate.
Materials
BACTH plasmid vectors (see Table 20.12.1; store in TE buffer or distilled H2 O at
–20ºC)
Competent E. coli cells (e.g., XL1-Blue from Stratagene)
LB agar plates (see recipe)
Antibiotics (ampicillin, kanamycin, spectinomycin; see recipe for stock solutions)
Plasmid miniprep kit (e.g., Qiagen, Macherey-Nagel; also see UNIT 2.4, Wilson,
1997)
BACTH reporter strains (see Table 20.12.2; store in LB medium with 10% DMSO
at –80ºC); re-streak on LB-X-gal-IPTG plate (see recipe) before use and start
culture from an isolated white colony
LB-X-gal-IPTG agar plates (see recipe)
LB broth (see recipe)
Nalidixic acid (optional) Analysis of
Streptomycin (optional) Protein
Interactions
0.1 M CaCl2
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 Table 20.12.2 BACTH Reporter Strainsa

Strain Genotype Main characteristics

DHT1 F− , cya-854, ilv 691::Tn10, recA strain
recA1, endA1, gyrA96 (Nalr ), Displays a high BACTH complementation
thi1, hsdR17, spoT1, rfbD1, efficiency with fast growth
glnV44(AS) Requires addition of casamino acids for growth on
minimal medium (due to ilv phenotype)
Dautin et al., 2000
DHM1 F− , cya-854, recA1, endA1, recA strain
gyrA96 (Nalr ), thi1, hsdR17, An ilv+ DHT1 derivative
spoT1, rfbD1, glnV44(AS) Able to grow on minimal media plus sugars
Displays a lower complementation efficiency and
slower growth than the parental DHT1
Karimova, Dautin, and Ladant, 2005
BTH101 F− , cya-99, araD139, Rec+ strain
r
galE15, galK16, rpsL1 (Str ), Displays an intermediate BACTH
hsdR2, mcrA1, mcrB1 complementation efficiency with fast growth
Able to grow on minimal media plus sugars
Unpublished
a Thefrequencies of spontaneous reversion of these strains toward Lac+ or Mal+ phenotypes (due to cAMP/CAP
independent promoter mutations) are below 10−7 and 10−18 , respectively.

MacConkey/maltose agar plates (see recipe)

Isopropyl-β-D-thiogalactopyranoside (IPTG; see recipe for stock solution)
5× M63 minimal medium (see recipe)
0.05% (w/v) sodium dodecyl sulfate
Chloroform
PM2 medium (see recipe)
4 mg/ml o-nitrophenyl-β-D-galactopyranoside (ONPG), filter sterilized
1 M Na2 CO3
30° and 37ºC incubator for plates and shaking liquid cultures
Spectrophotometer for measuring OD600
42ºC water bath
96-well deep-well blocks (2.2- and 1.2-ml well volumes), sterile
Sterile toothpicks
AeraSeal sterile adhesive film (Dutscher, cat no. 760215) or Airpore Tape Strips
(Qiagen, cat. no. 19571)
Rotary shaker (for shaking deep-well 96-well blocks)
96-well flat-bottom microtiter plates, sterile
Microplate reader: Tecan or equivalent plate reader capable of kinetic
measurements at wavelengths 405 and 595 (or 600) nm
Spreadsheet software (e.g., Microsoft Excel)
Additional reagents and equipment for PCR (UNIT 15.1; Kramer & Coen, 2000),
plasmid miniprep (UNIT 2.4; Wilson, 1997), introduction of plasmid DNA into
cells (Sambrook, Russell, & Sambrook, 2006; Seidman, Struhl, Sheen, &
Jessen, 1997), restriction digestion (UNIT 3.1; Bloch & Grossmann, 1995), and
DNA sequencing (Chapter 7 in this manual)
Membrane Construction of recombinant BACTH vectors
Protein
Interaction Steps 1 to 3, below, describe how to clone the gene encoding protein X into a
Analysis with T25-encoding BACTH vector [i.e., pKT25, pKNT25, pST25, or pSNT25 (Karimova,
BACTH
Ullmann, & Ladant, 2001; Ouellette, Gauliard, Antosova, & Ladant, 2014)] and how
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to clone the gene encoding protein Y into a T18-encoding BACTH vector (pUT18C or
pUT18; Karimova et al., 2001).

1. Design appropriate primers to amplify genes X and Y (Kramer & Coen, 2000).
Include in these primers specific restriction sites (e.g., BamHI in 5 primer, KpnI
in 3 primer) to facilitate oriented cloning of the amplified DNA into the BACTH
vectors so that genes X and Y will be in frame with the T25 and T18 open reading
frames (ORF, see Fig. 20.12.3).
Other cloning strategies can be used, but require specific primer design for the application
(see e.g., Alternate Protocol 1).

2. PCR-amplify genes X and Y (from genomic DNA, cDNA, or other appropriate

sources) using a standard PCR protocol [UNIT 15.1 (Kramer and Coen, 2000); Sam-
brook et al., 2006)]. Purify the amplified DNA fragments with a standard PCR
purification kit. Digest the purified DNAs with the chosen restriction enzymes and
ligate them (with T4 DNA ligase) with the selected BACTH plasmids (e.g., pKT25
and pUT18C) similarly digested using the same enzymes.
3. Transform competent cells (e.g., XL1-Blue) with the constructs, and plate transfor-
mants on LB agar supplemented with appropriate antibiotics. Incubate plates at 30°C
for 24 to 36 hr. For each cloning experiment, purify plasmid DNAs from several
colonies by using a standard miniprep protocol (Wilson, 1997) or a commercial kit.
Check the recombinant plasmids by restriction analysis (UNIT 3.1; Bloch & Grossman,
1995) and/or DNA sequencing (Chapter 7 in this manual) to verify the correctness
of the constructions.
The BACTH vectors and recombinant plasmids are commonly propagated at 30°C in
standard E. coli K12 recA strains (such as XL1-Blue, Stratagene). As a host, we recom-
mend using an E. coli strain that overproduces the LacI repressor (i.e., lacIQ ) to prevent
expression of the hybrid proteins, and to grow the cells at 30°C (or below) to limit any
potential problems of instability of the recombinant plasmids.

Phenotypic assays of interaction in Δcya E. coli cells

Steps 4 to 6 describe how to co-transform the recombinant plasmids pKT25-X and
pUT18C-Y into competent Δcya E. coli cells (e.g., DHM1 or other BACTH reporter
strains; Table 20.12.2) and plate the transformed cells on indicator plates, i.e., LB-X-gal
agar or MacConkey/maltose agar. The plates are then incubated for 1 to 3 days and the
cell phenotype is recorded.

4. Prepare electro-competent or chemically competent E. coli Δcya cells (e.g., DHM1

strain) using a standard procedure (Miller, 1992; Sambrook et al., 2006).

a. To prepare competent cells with the CaCl2 technique (Sambrook et al., 2006),
first re-streak the cells (DHM1, DHT1, or BTH101) from a LB-DMSO stock
solution (kept at −80°C) on LB/X-gal/IPTG plates.
b. After overnight growth at 37°C, pick a white colony (i.e., cya) to inoculate 5 ml
of LB broth and grow overnight at 30°C.
If too many blue colonies (e.g., Lac+ revertants or other contaminants) are present,
re-streak the cell stock on LB/X-Gal/IPTG plates supplemented with 30 μg/ml nalidixic
acid (for DHT1 or DHM1) or 100 μg/ml streptomycin (for BTH101).
c. Using the overnight preculture (at 30°C), inoculate 0.4 liter LB broth and grow
with vigorous agitation at 37°C to reach an OD600 of 0.25 to 0.3. Cool on ice and
pellet the cells by centrifugation for 10 min at 3000 × g, 4°C.
It is critical to maintain cells, buffers, and vessels chilled at all stages of the process. Analysis of
Protein
Interactions

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 d. Wash the cells twice, each time in 50 ml ice-cold 0.1 M CaCl2 solution, re-
centrifuging each time, then resuspend them in 6 to 8 ml ice-cold 0.1 M CaCl2
and incubate overnight at 4°C.
The competency level (>106 cfu/μg) obtained with this procedure is sufficient for routine
transformations. See below for a protocol for preparing electrocompetent cells.

5. Co-transform the BACTH competent cells prepared in step 4 with the purified
pKT25-X and pUT18C-Y plasmid DNA. To do this:

a. Mix 50 μl of chemically competent DHM1 cells in a chilled microcentrifuge

tube with 5 to 10 ng of each plasmid, incubate about 30 min at 4°C, and then 2
min at 42°C. Add 1 ml of LB broth and further incubate the cell suspension 60
to 90 min at 30°C (for expression of antibiotic resistance).
b. In parallel, co-transform separate aliquots of cells with plasmids pKT25 and
pUT18C, to serve as a negative control (i.e., absence of interaction), and with
plasmids pKT25-zip and pUT18C-zip (that encode T25 and T18 fragments fused
to the GCN4 leucine-zipper dimerization motif, respectively, see Table 20.12.2)
for a positive control of cells expressing interacting hybrid proteins.
You can also co-transform other combinations of plasmids (e.g., pKT25-X and pUT18C,
and pKT25 and pUT18C-Y) to serve as additional negative controls.

6. Plate different amounts of the transformation mixtures (to have about 100 to 300
colonies/plate) on either MacConkey-maltose-IPTG or LB-X-gal-IPTG agar plates
(plus appropriate antibiotics). Incubate the plates for 24 to 48 hr at 30°C.
On MacConkey/maltose/IPTG, the cells expressing interacting hybrid proteins form red
colonies: the Cya+ bacteria are able to ferment maltose—this leads to an acidification of
the medium and triggers a color change of the phenol red dye present in the MacConkey
medium (Fig. 20.12.2). The cells expressing non-interacting proteins (i.e., Cya- ), unable
to ferment maltose, form white or pale pink colonies.
On LB-X-Gal-IPTG, the cells expressing interacting hybrid proteins will form blue
colonies (Fig. 20.12.2), as the expression of the lacZ gene (encoding β-galactosidase)
is positively controlled by cAMP/CAP. In contrast, cells expressing non-interacting pro-
teins will remain white (or pale blue). IPTG (0.5 mM) is added to the medium to increase
β-galactosidase expression (Miller, 1992).
These two types of indicator media are equally effective in monitoring protein-protein
interactions. Discrimination between Cya+ and Cya− cells may be slightly easier on
MacConkey/maltose medium as compared to LB-X-gal/IPTG. The latter is simpler to
prepare, is readily available in any molecular biology laboratory, and is appropriate for
most studies.
Note that after a prolonged incubation (4 to 5 days) at 30°C, even negative colonies
(i.e., Cya− ) may exhibit a weak red (on MacConkey-maltose) or light blue (on LB-X-
gal) phenotype, especially in the center of the colonies (the periphery usually remains
colorless). In addition, do not streak too many colonies on the indicator plates (maximum
300 to 500 per plate); otherwise, it may be difficult to detect the positive clones.
BACTH complementation may also be tested at 37°C, although, in many cases, the
complementation is less efficient at this temperature than at 30°C.
This analysis of the cell phenotypes (i.e., Lac+ or Mal+ ) already provides a direct,
yet qualitative (i.e., + or −), result on the tested interactions. A comparative assay of
the interactions among a variety of different proteins (for example test of mutagenic
variants of a given protein) can thus be performed by direct scoring of the phenotypes
Membrane of the different transformants spotted or replicated on the same indicator plate, together
Protein with positive (pKT25-zip/pUT18C-zip) and negative (pKT25/pUT18C) controls. This
Interaction qualitative assay of interactions has frequently been used in many published studies.
Analysis with
BACTH However, a more quantitative evaluation of the efficiency of functional complementation

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 between two hybrid proteins is obtained by measuring the β-galactosidase activities in
permeabilized cells (see below).

Quantification of functional complementation between hybrid proteins by

β-galactosidase assays
In E. coli, the expression of the lacZ gene (encoding β-galactosidase) is positively
controlled by cAMP/CAP, and therefore the measurement of β-galactosidase activities
in liquid cultures of bacterial cells offers a convenient means to quantify the interaction
between hybrid proteins. Many methods for β-galactosidase assays have been described
(Miller, 1992; Sambrook et al., 2006). These β-galactosidase activity assays can be
conveniently carried out in a 96-well microtiter plate, as follows.

7. In a 96-well microtiter plate (2.2-ml 96-well sterile storage plate or deep-well block),
distribute 300 to 400 μl/well of sterile LB broth supplemented with 0.5 mM IPTG
and appropriate antibiotics.
8. With a sterile toothpick, inoculate single colonies into individual wells.
Usually, eight independent colonies from each set of transformations (i.e., expressing a
given pair of T25 and T18 hybrid proteins) are tested in parallel. Be sure to include
at least four wells without inoculation to serve as background measurements for the
microplate reader.

9. Seal the plate with a microporous tape sheet (e.g., AirPore from Qiagen) to allow
gas exchange, and incubate overnight at 30°C on a rotary shaker.
Depending upon the type of shaker used, it may be necessary to tilt the microtiter plate
to increase culture agitation in the wells.

10. After a minimum of 16 hr of growth, dilute the cultures 5-fold by adding an appro-
priate volume of M63 medium to the deep-well microplate.
11. Transfer 175 μl of the diluted cultures into a flat-bottom 96-well microtiter plate.
Record the absorbance at 595 nm (A595 ) with a microplate reader and export data to
a spreadsheet program.
12. Transfer 200 μl of the diluted cultures into a new microtiter plate (1.2-ml polypropy-
lene 96-well storage block). Add 7 μl of 0.05% SDS (sodium dodecyl sulfate) and
10 μl of chloroform to permeabilize the cells. Mix vigorously by pipetting up and
down several times, then leave the plate under a fume hood at room temperature for
30 to 40 min to allow chloroform evaporation.
13. In a new microtiter plate, distribute 105 μl/well of PM2 buffer containing 100 mM
2-mercaptoethanol (added fresh just before use) and 0.1% (w/v) o-nitrophenol-β-
galactoside (ONPG). Start the enzymatic reactions by adding 20-μl aliquots of the
permeabilized cell suspensions (see step 10). Incubate the plate at room temperature
for 20 to 30 min or until sufficient yellow color has developed (note the time). In
parallel, perform control assays with 20 μl of M63 medium instead of permeabilized
cells.
14. Stop the enzymatic reaction by adding 50 μl of 1 M Na2 CO3 . Record the absorbance
at 405 nm (A405 ) with a microplate reader and export data to a spreadsheet program
15. Analyze data with appropriate software (e.g., Microsoft Excel or other spreadsheet
program). For each well, calculate the enzymatic activity, A (in relative units),
according to:

A = 1000 × (A405 in sample wells − A405 in control wells) / (A595 in sample wells Analysis of
Protein
Interactions
−A595 in control wells) /time (min) of incubation
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 Figure 20.12.4 BACTH analysis of interactions between a subset of E. coli Fts proteins. The E.
coli cya DHM1 cells were transformed with BACTH plasmids expressing the indicated fusions (‘-‘
designates the unfused T25 or T18 fragments, and Zip designates the GCN4 leucine zipper moiety
used as positive control). The β-galactosidase activities (expressed in relative units) were mea-
sured on several (4 to 8) independent colonies for each transformants (with standard deviations
generally within 20% of the mean), as described in the text. Note that in most cases, comple-
mentation between the T25 and the T18 hybrids is detected in both configurations, i.e., when the
given Fts proteins are fused to either T25 or T18, although the β-galactosidase measurements
may vary significantly between the two configurations. An extreme case is FtsW, for which only
the T25-FtsW hybrid appears to be functional. We hypothesize that T18-FtsW, which is expressed
from the high-copy vector pUT18C, may be overexpressed under the assay conditions, and this
might prevent its proper insertion in the inner membrane (FtsW is multipass membrane protein
with 10 membrane-spanning segments).

With this calculation, the results are expressed in relative units (RU) of β-galactosidase
activity (this corresponds to roughly 1/6 of the classical Miller units). It is therefore
essential to include, in the series of assays, negative and positive controls, i.e., bacteria
that express non-interacting (e.g., T25 and T18 only or fused to proteins that do not or
should not interact with the protein of interest) and interacting (e.g., T25-zip and T18-zip,
or other positive controls) hybrid proteins, respectively. Under routine conditions, the
β-galactosidase activity measured with the positive controls (T25-zip/T18-zip) is defined
as 100% activity, while the β-galactosidase activities of the negative controls (T25/T18)
should be below 2% to 3% of positive control activity (Gauliard et al., 2015; Karimova,
Davi, & Ladant, 2012; Karimova, Robichon, & Ladant, 2009; Ouellette, Gauliard, et al.,
2014). The β-galactosidase activities in cells expressing the hybrid proteins of interest
should be at least 4 to 5 times higher than the background level to be considered an
indication of a positive interaction in BACTH assays (Gauliard et al., 2015; Karimova
et al., 2005, 2012).
Figure 20.12.4 shows the results of a BACTH analysis of interactions among a subset of
the E. coli proteins involved in cell division (so-called Fts proteins). These proteins are
assembled at the future cell division site into a multimolecular complex called a divisome
that catalyzes the cell septation. Many of these proteins are integral membrane proteins,
and their interaction properties have been difficult to assess. Our BACTH study (Karimova
et al., 2005) confirmed previously known interactions and provided experimental support
Membrane for several suspected interactions (e.g., associations of FtsN with FtsA, FtsI and FtsQ)
Protein that were postulated from prior genetic and/or physiological evidence. Moreover, these
Interaction data suggest that most Fts proteins are able to associate with multiple partners. These
Analysis with interactions appeared to be specific, as no complementation was detected between any
BACTH
of the Fts hybrids and other proteins tested as controls, including the native T25 or T18
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Supplement 118 Current Protocols in Molecular Biology
 fragments or T25 or T18 fusions to the leucine zipper or to unrelated membrane proteins
(data not shown).
The BACTH assay was further applied to delineate, through a deletion mapping analysis,
the regions responsible for the association of specific cell-division proteins with their
partners (Karimova et al., 2005). Finally, we also showed that the interactions between
two Fts hybrid proteins could be modulated (strengthened or diminished) by co-expression
of a third Fts partner (Karimova et al., 2005).
The measurement of the β-galactosidase activities in liquid cultures of cells expressing
hybrid proteins, described above, offers a rapid and easy way to quantify the efficiency
of functional complementation between two different hybrid proteins. Note that it could
also be possible to measure the production of cAMP, a direct readout from the adenylate
cyclase activity resulting from the association of the two complementing T25 and T18
hybrids (Karimova et al., 1998, 2000), using commercial kits for cAMP determination
(ELISA or radioimmunoassays). However, these kits are rather expensive, and the cAMP
assay is much more tedious and time consuming than the standard β-galactosidase assay,
so these cAMP measurements are rarely performed.

GATEWAY CLONING OF GENES ENCODING PROTEINS OF INTEREST ALTERNATE

INTO BACTHGW VECTORS PROTOCOL 1
We recently implemented the BACTH system to make it compatible with the Gate-
way cloning technology (Thermo Fisher Scientific). This technique is convenient for
transferring genes of interest, initially cloned in a so-called “entry clone,” into a vari-
ety of “destination vectors,” by using a site-specific recombination reaction (Hartley,
Temple, & Brasch, 2000; Sands & Brent, 2016). We have designed BACTH-Gateway
destination vectors (BACTHGW ), pST25-DEST, pSNT25-DEST, and pUT18C-DEST
(Ouellette, Gauliard, et al., 2014), which can be used to easily subclone genes from
any Gateway entry clone (Table 20.12.1 and Fig. 20.12.5). A precise description
of the Gateway cloning techniques can be found on the manufacturer’s Web site
(https://tools.thermofisher.com/content/sfs/manuals/gatewayman.pdf). Here, we briefly
describe the procedure: (i) to generate an entry clone with a gene of interest; and then
(ii) to transfer the gene of interest into the BACTHGW vectors.

Figure 20.12.5 Schematic representation of BACTHGW plasmids. Light blue and green arrows
represent the open reading frames of T25 and T18 fragments, respectively, under the transcrip-
tional control of the lac promoter. Orange and dark blue arrows correspond to the spectinomycin-
and ampicillin-selectable markers, respectively. The yellow and brown boxes indicate the p15A
and ColE1 origins of replication, respectively. The hatched boxes indicate the position of insertion
of the Gateway recombination cassette that contains a chloramphenicol resistance marker (CmR ), Analysis of
the CcdB toxin, and the two attR recombination sites. Note that these vectors must be propagated Protein
in E. coli strains resistant to CcdB toxin (e.g., DB3.1 E. coli strain). Interactions

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Current Protocols in Molecular Biology Supplement 118
 Additional Materials (also see Basic Protocol 1)
pDONR221 plasmid (Thermo Fisher Scientific)
BP Clonase II enzyme (Thermo Fisher Scientific)
Proteinase K
BACTHGW destination vectors:
pST25-DEST
pSNT25-DEST
pUT18C-DEST
LR Clonase enzyme mix (Thermo Fisher Scientific)
Additional reagents and equipment for agarose gel electrophoresis (UNIT 2.5A;
Voytas, 2000)
Clone genes of interest into a Gateway “entry” plasmid
Also see https://tools.thermofisher.com/content/sfs/manuals/gatewayman.pdf.

1. Design oligonucleotide primers to amplify the genes of interest. To do this, append

to the gene-specific sequences (usually corresponding to the 5 and 3 extremities
of the open reading frame, indicated below by XXX . . . ) the “Gateway specific”
sequences (lowercases, underlined, corresponding to the specific attB sites necessary
for recombination reaction), as illustrated below:

5 primer (the bold ATG corresponds to the initiation codon of the open reading
frame): 5 -GCCGCacaagtttgtacaaaaaagcaggctTTATGXXXXXXXX
3 primer (the stop codon can be removed if desired):
5 -GCGGAccactttgtacaagaaagctgggtTXXXXXXXX
Refer to the manufacturer’s guidelines for further explanation on the design of
primers for Gateway cloning.
2. Use PCR (Kramer & Coen, 2000) to amplify the genes of interest (from genomic
DNA, cDNA, or other appropriate sources) using the designed Gateway primers, and
purify the PCR products with a standard PCR purification kit (see Basic Protocol 1).
Gel purify using agarose gel electrophoresis (UNIT 2.5A; Voytas, 2000), if necessary.
3. Mix the purified PCR products (150 ng) with pDONR221 plasmid (150 ng), add
BP Clonase II enzyme according to the manufacturer’s instructions, and incubate 2
hr at room temperature to allow the BP recombination reaction. Longer incubation
times can be used for larger gene inserts. Add 2 μg of proteinase K to terminate the
recombination reaction and transform the mixture into E. coli XL1 competent cells.
Select the transformants on LB plates supplemented with 50 μg/ml of kanamycin
(Ouellette, Gauliard, et al., 2014).
4. Purify plasmid DNA from three or four independent clones from each cloning as
described in Basic Protocol 1 and check the recombinant plasmids by restriction
analysis and DNA sequencing.
In the resulting plasmids (pDONR-gene X), the genes of interest are flanked by attL
recombination sites, and can be easily transferred into Gateway destination vectors, by
an “LR” reaction (Ouellette, Gauliard, et al., 2014; Sands & Brent, 2016)

Gene transfer from the Gateway “Entry” plasmids into the BACTHGW destination
vectors
Membrane The genes cloned into the Gateway “entry” plasmids (pDONR-gene X) can be easily
Protein transferred into the BACTHGW destination vectors using an “LR” reaction as follows.
Interaction
Analysis with
BACTH

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Supplement 118 Current Protocols in Molecular Biology
 5. Mix the entry pDONR-gene X plasmid (150 ng) with the appropriate BACTHGW
destination vectors (150 ng), pST25-DEST, pSNT25-DEST, or pUT18C-DEST. Add
the LR Clonase enzyme mix (see manufacturer’s guidelines), and incubate 1 to 2 hr
at room temperature.
6. Add proteinase K (see step 3) to terminate the reaction and transform the mixture in
E. coli XL1 competent cells. Select the transformants on LB plates supplemented
with the appropriate antibiotic (spectinomycin or ampicillin).
7. Purify plasmid DNA from two to three independent clones from each cloning, and
check the recombinant plasmids by restriction analysis and/or DNA sequencing.
The resulting plasmids encode the T25 or T18 fragments fused in frame to the gene
of interest (gene X), via short additional peptidic linkers that are encoded by the attB
recombination sequences. We verified that these additional small linkers do not affect
BACTH complementation efficacy (Ouellette, Gauliard, et al., 2014).

APPLICATION OF BACTH FOR SCREENING FOR INTERACTING BASIC

PARTNERS OF A TARGET PROTEIN PROTOCOL 2
The BACTH system can be used to screen libraries in order to isolate potential partners
of a protein of interest (e.g., protein X, classically designated as “bait”; Zervos, Gyuris, &
Brent, 1993). Selection of bacteria expressing interacting proteins can easily be performed
by plating transformants on a selective medium that consists of a synthetic minimal
medium supplemented with maltose as a unique carbon source: as mal regulon (involved
in maltose catabolism) expression is strongly dependent upon cAMP/CAP activation,
only Cya+ bacteria can utilize maltose as a carbon source (Karimova et al., 1998, 2005,
2012). Hence, only the cells that express interacting hybrid proteins are able to grow on
this minimal medium (Fig. 20.12.2). X-gal and IPTG are usually added to the selective
medium to facilitate the early visualization of the Cya+ colonies, as these cells are also
Lac+ and turn blue in the presence of X-gal.

The first step consists of the construction of a library of genomic DNA (or cDNA)
fragments inserted in one of the BACTH vectors (pKT25 or pUT18C). Numerous ap-
proaches can be used for this critical step (Miller, 1992; Sambrook et al., 2006). Im-
portantly, the quality—i.e., the complexity or number of distinct clones present in the
library—determines the success in isolating relevant clones. Then, the pool of DNA
(e.g., constructed in pKT25) is used to transform BACTH host cells harboring the
complementary BACTH vector expressing the bait hybrid protein (e.g., pUT18C-X).
The co-transformants are plated on M63 minimal medium plus maltose and antibiotics
(+ X-Gal and IPTG). After a few days of growth at 30°C, cells expressing a potential
partner of X should form blue colonies (i.e., Cya+ ).
Materials
BACTH plasmid vectors (see Table 20.12.1)
Restriction endonuclease SmaI (also see UNIT 3.1; Bloch & Grossmann, 1995)
Shrimp alkaline phosphatase (Thermo Fisher Scientific, cat. no. 783901000UN)
ElectroMAX DH10B cells (Thermo Fisher Scientific; or other highly competent
E. coli recipient cells)
LB agar plates (see recipe)
Antibiotics (ampicillin, kanamycin, spectinomycin; see recipe for stock solutions)
LB broth (see recipe)
Plasmid miniprep kit (e.g., Qiagen, Macherey-Nagel; also see UNIT 2.4, Wilson,
1997) Analysis of
10% (v/v) glycerol Protein
5× M63 minimal medium (see recipe) Interactions

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Current Protocols in Molecular Biology Supplement 118
 M63/maltose plates (see recipe) supplemented with kanamycin (25 μg/ml),
ampicillin (50 μg/ml), IPTG (0.5 mM), and X-Gal (40 μg/ml)
Bath sonicator
30° and 37ºC incubator for plates and shaking liquid cultures
Spectrophotometer for determining OD600
Refrigerated centrifuge
Electroporator (e.g., BioRad) and electroporation cuvettes (1-mm)
30ºC water bath
Additional reagents and equipment for use of mung bean nuclease (UNIT 3.12;
Nichols, 2011) and DNA polymerases (UNIT 3.5; Kucera & Nichols, 2008),
restriction digestion (UNIT 3.1; Bloch & Grossmann, 1995), cloning into BACTH
vectors (Basic Protocol 1), and DNA sequencing (see Chapter 7 in this manual)
Construction of a DNA library in BACTH vector pKT25
A simple procedure used in the laboratory to construct a library of genomic E. coli
chromosomal DNA fragments in the pKT25 vector is provided below (other experimental
details can be found in Karimova et al., 2012; see other units in this manual for basic
molecular biology techniques).

1. Prepare genomic DNA (or cDNA) from the organism(s) of interest.

Note that when we prepared libraries from E. coli, we used a Δcya derivative of the
reference strain MG1655 in order to avoid cloning the wild-type cya gene, which will
restore a Cya+ phenotype.

2. Fragment the genomic DNA (50 μg) by sonication using a bath sonicator to
obtain mean size range of 500 to 1,500 bp (check by agarose gel analysis; UNIT 2.5A;
Voytas, 2000).
Sonication time and power settings should be determined empirically.

3. Repair the fragment ends by treatment with the mung bean nuclease (UNIT 3.12;
Nichols, 2011) together with a mixture of T4 DNA polymerase and Klenow fragment
(plus dNTP; UNIT 3.5; Kucera & Nichols, 2008) to fill in the extremities and create
blunt ends.
4. In parallel, digest the pKT25 vector (10 μg) with SmaI (UNIT 3.1; Bloch & Gross-
mann, 1995), dephosphorylate it with shrimp alkaline phosphatase according to the
manufacturer’s instructions, and purify the linearized vector by agarose gel elec-
trophoresis (UNIT 2.5A; Voytas, 2000).
5. Ligate the blunt-ended DNA fragments with the SmaI-digested pKT25 vector.
6. Transform the mixture into electrocompetent ElectroMAX DH10B cells and
plate aliquots on several LB agar/kanamycin plates (no more than 5–10 × 104
clones/plate). Also, plate diluted aliquots (e.g., at 102 , 104 or 106 dilution in LB
broth) on separate plates in order to estimate the efficacy of transformation and the
overall number of clones.
For example, in a prior study (Karimova et al., 2012), about 5 × 105 independent clones
were obtained. Note that among these clones, only 1/6 of the clones potentially harbor
DNA fragments inserted in pKT25 in the correct orientation and in frame with the T25
fragment.

Membrane 7. After growth to confluence, pool all the colonies and purify the plasmid DNA to be
Protein used as a stock for the BACTH DNA library. Note that the DNA should be eluted
Interaction
Analysis with from the purification column in water if you plan to use electrocompetent cells for the
BACTH library screen (see below). To verify the quality of insert cloning, prepare miniprep
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Supplement 118 Current Protocols in Molecular Biology
 DNA (use kit or see Wilson, 1997) from about 20 to 30 isolated colonies and analyze
the DNA either by performing restriction analysis, DNA sequence analysis, or PCR
analysis (using primers flanking the cloning sites).
This approach will provide both an estimate of the frequency of DNA insertion (higher
is better; at least more than 70% of plasmids should have an insert) as well as the
distribution of sizes of the DNA inserts.

Clone the gene encoding the “bait” protein X into pUT18C and transform the
resulting pUT18C-X plasmid into BACTH host cells
8. Clone the gene encoding protein X into one of the BACTH vectors (e.g., pUT18C,
pUT18, or pUT18C-DEST) to generate “bait” plasmid pUT18C-X that codes for the
T18-X hybrid protein (see Basic Protocol 1).
9. Transform the resulting pUT18C-X into a BACTH strain (e.g., DHM1; see Basic
Protocol 1).
10. Prepare electrocompetent cells from the resulting transformants DHM1/pUT18C-X
as follows:

a. Inoculate freshly re-isolated cells in 1 liter of LB containing 100 μg/ml

ampicillin and grow them with vigorous shaking at 37°C until the OD600
reaches 0.5 to 0.7.
b. Chill the cells on ice and pellet them by centrifugation for 15 min at 4,000 × g,
4°C.
c. Wash the cells at least three times with ice-cold water using the centrifugation
conditions above and resuspend them in 10 ml of 10% (v/v) glycerol in
double-distilled water (all operations should be performed under sterile
conditions).
Transform DHM1/pUT18C-X competent cells with the DNA library
11. Transform electro-competent DHM1/pUT18C-X cells with the BACTH DNA li-
brary. To do this:

a. Transfer 50 μl of electrocompetent DHM1/pUT18C-X cells into an electropora-

tion cuvette (1 mm wide) previously equilibrated on ice.
b. Add 50 to 100 ng of DNA from the BACTH library. Mix gently and incubate 4
to 5 min at 4°C.
c. Place the cuvette in electroporator set at 2.5 KV, 100  capacitance, and trigger
the electroporation.
d. Immediately add 1 ml of LB broth (at room temperature) to the cuvette, and
further incubate the cells at 30°C for 1 to 1.5 hr.
12. Collect the cells by centrifugation for 5 min at 3000 to 4000 × g, 20º to 25ºC, and
wash them several times with M63 medium using the same centrifugation conditions
to remove all nutrients from the rich LB medium.
13. Plate the electroporated cells (approximately 1 × 106 transformants/plate) on M63
minimal medium agar supplemented with maltose (0.2%), kanamycin (25 μg/ml),
ampicillin (50 μg/ml), IPTG (0.5 mM), and X-gal (40 µg/ml, to facilitate the detec-
tion of Cya+ clones that are Mal+ and also Lac+ ).
Note that when using DHT1 as a reporter strain, casamino acids should be added to the
M63/maltose/antibiotics plates at a concentration of 50 μg/ml to allow growth, as this
strain is ilv− (i.e., unable to synthesize isoleucine and valine).
14. Incubate plates at 30°C for 4 to 8 days until appearance of blue, Cya+ colonies. Re-
Analysis of
isolate these colonies on fresh plates of M63 agar plus maltose, antibiotics, IPTG, Protein
and X-Gal (as above). Interactions

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Current Protocols in Molecular Biology Supplement 118
 15. Purify the pKT25 plasmids from the Cya+ colonies and further characterize the
DNA inserts by sequencing (see Chapter 7 in this manual).
To discard potential false positive clones that could arise as a result of spontaneous rever-
sion of the host strain toward a Mal+ phenotype (see Troubleshooting), it is recommended
to re-transform the selected pKT25 plasmids into the electrocompetent DHM1/pUT18C-
X cells and repeat the screening procedure (i.e., steps 11 to 14) to verify that the hybrid
proteins they encode confer an authentic Cya+ phenotype.
In principle, the gene encoding a potential partner of X, or a fragment of its coding
sequence, should be inserted in the correct orientation and in frame with the T25 coding
region. In some cases, however, genes cloned out of frame may still be expressed in
part as an authentic fusion with the T25 due to transcription/translation slippage. Such
out-of-frame clones might be found when the encoded gene has a tendency to be toxic
for the host cells (either intrinsically or when expressed as a fusion protein—here to the
T25 fragment).
We have applied this screening procedure to isolate novel components of the E. coli cell
division machinery (Karimova et al., 2005, 2009, 2012). Other laboratories have also
applied the BACTH system for screening partners and constructed libraries using their
own protocols; the reader may refer to these articles for further inspiration (Chen et al.,
2010; Houot, Fanni, de Bentzmann, & Bordi, 2012; Kleinschnitz et al., 2011; Pfeiffer &
Jendrossek, 2011).

ALTERNATE APPLICATION OF BACTHGW IN LIBRARY SCREENING

PROTOCOL 2
Many libraries of full-length open reading frames (ORFs) or cDNA from various organ-
isms or tissues cloned into the Gateway entry vectors are now available commercially or
through academic consortia. These collections of genes can be easily transferred into the
BACTHGW destination vectors using the standard Gateway recombineering technique
(see above). The overall process is efficient enough to envisage the overall transfer of a
complete library of ORFs from an organism (e.g., E. coli) into the BACTHGW destination
vectors. This BACTH ORF library can then be used for searching potential partners of a
bait of interest as described above.

These tools may also be applied in systematic exploration of the protein interaction
networks in a given organism using an array approach such as described in the clas-
sical Finley and Brent study (Finley & Brent, 1994). For this, each individual ORF is
transferred into both pST25-DEST (or pSNT25-DEST) and pUT18C-DEST (see above),
to create two distinct collections of BACTHGW plasmids. Then, these plasmids are
co-transformed in a pairwise manner to systematically probe all possible interactions
between the complete set of ORFs or cDNA of an organism. Such approaches have been
widely applied to many different eukaryotes using the yeast-two hybrid technology. It
should now be possible to similarly analyze protein-interaction networks of bacteria with
the BACTHGW assay. Indeed, in our laboratory we have exploited this approach to iden-
tify novel interactions between proteins from the pathogenic bacterium Chlamydia tra-
chomatis (Gauliard et al., 2015; Ouellette, Karimova, Subtil, & Ladant, 2012; Ouellette,
Rueden, et al., 2014).

REAGENTS AND SOLUTIONS

Use deionized, distilled water in all recipes and protocol steps. For common stock
solutions, see APPENDIX 2.
Antibiotic stock solutions
Membrane
Protein Prepare stock solutions in distilled H2 O of:
Interaction
Analysis with ampicillin (100 g/liter)
BACTH kanamycin (50 g/liter)
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Supplement 118 Current Protocols in Molecular Biology
 spectinomycin (50 g/liter)
Filter-sterilize through a 0.22-μm syringe filter and store at −20°C up to 2 years
Prepare stock solution of chloramphenicol (30 g/l) in ethanol
All are used at 1/1000 dilution in broth or agar medium (e.g., ampicillin at
100 µg/ml, kanamycin at 50 µg/ml, etc.) unless otherwise indicated.
5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal)
Prepare a stock solution of 20 mg/ml in 100% dimethylformamide (DMF). Store up
to 2 years at −20°C in the dark.
Isopropyl-β-D-thiogalactopyranoside (IPTG) stock solution
Prepare a 50 mM stock solution of IPTG in water. Filter-sterilize and store up to 2
years at −20°C.
LB broth
10 g of NaCl
10 g tryptone
10 g yeast
Dissolve in 1 liter deionized H2 O
Adjust pH to 7.0 with NaOH
Autoclave
Store up to 3 months at 4°C
LB agar plates and LB-X-gal-IPTG agar plates
Add 15 g of agar per liter of LB broth (see recipe) and autoclave. When the medium
has cooled down below 45°C, add appropriate antibiotics (see recipe for stock solu-
tions), and, if needed, 40 µg/ml of X-gal (from 20 mg/ml stock; see recipe), and 0.5
mM IPTG (from 50 mM stock; see recipe). Pour into 9- to 10-cm petri plates.
M63 minimal medium, 5×
10 g (NH4 )2 SO4
68 g KH2 PO4
2.5 mg FeSO4 .7H2 O
Deionized H2 O to 1 liter
Adjust pH to 7.0
Autoclave
Store up to 6 months at 4°C
M63/maltose plates
Add 15 g of agar to 800 ml H2 O and autoclave. When the medium has cooled down
below 45°C, add 200 ml sterile 5× M63 medium, 0.2% to 0.4% (w/v) of glucose-free
maltose (Sigma-Aldrich; diluted from a 20% stock solution in water sterilized by
filtration), 40 µg/ml X-gal (from 20 mg/ml stock; see recipe), 0.5 mM IPTG (from
50 mM stock; see recipe), the appropriate antibiotics at half the usual concentrations
(i.e., 50 µg/ml of ampicillin, 25 µg/ml of kanamycin; see recipe for antibiotic stock
solutions), and pour into 9- to 10-cm petri plates. When needed (i.e., with DHT1),
add casamino acids at a final concentration 50 µg/ml. Store up to 1 month at 4ºC.
MacConkey/maltose agar plates
Add 40 g of MacConkey agar (note that MacConkey agar base medium is not of
equal quality; we recommend MacConkey agar from Difco, cat. no. 216830) in 1
liter of deionized H2 O and autoclave. When the medium has cooled down below
45°C, add appropriate antibiotics (see recipe for stock solutions), 1% of glucose-free Analysis of
Protein
maltose (Sigma-Aldrich; diluted from a 20% stock solution in water sterilized by Interactions

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Current Protocols in Molecular Biology Supplement 118
 filtration), 0.5 mM IPTG (from 50 mM stock; see recipe), and pour into 9- to 10-cm
petri plates. Store up to 1 month at 4ºC.
PM2 medium
70 mM Na2 HPO4
30 mM NaH2 PO4
1 mM MgSO4
0.2 mM MnSO4
pH should be 7.0
Store with the above components up to 2 years at room temperature
Add 100 mM of 2-mercaptoethanol just before use
COMMENTARY
Background Information
The BACTH system is an easy way to char-
acterize interactions between two proteins in
bacteria. This system works in an Escherichia
coli adenylate cyclase-deficient strain (cya)
and is based on the reconstitution of a signal
transduction pathway coupled to the produc-
tion of a regulatory molecule, cAMP, that trig-
gers the transcriptional activation of catabolic
operons (or specific reporter genes), giving rise
to a selectable phenotype. Even though many
other two-hybrid systems and/or complemen-
tation assays have been developed in various
hosts (Stynen et al., 2012), the BACTH as-
say has become a popular technique to charac-
terize protein interactions, particularly in the
microbial field. The reasons for its wide use
include the following:
1. This genetic test is carried out in E. coli,
and makes use of simple and common tech-
niques in bacteriology and molecular biology.
Thus, this assay can be readily carried out by
most researchers, with no need for any specific
equipment or training courses. The BACTH
assay is therefore a method of choice to answer
the simple question: do the two proteins X and
Y interact in vivo? For this, one just needs to
clone the coding genes into BACTH vectors
and then transform the recombinant plasmids
into cya cells, and a result is obtained in few
days (see below).
2. The use of E. coli as a host greatly fa-
cilitates the screening as well as the charac-
terization of the interacting proteins. The high
efficiency of transformation achieved in E. coli
enables the analysis of libraries of high com-
plexity, and the hybrid proteins can then be
easily expressed to confirm potential interac-
tion by in vitro binding assays.
BACTH can also be applied to carry out
Membrane
Protein simple structure-to-function analysis in or-
Interaction der to delineate key regions and/or amino
Analysis with acid residues involved in a given interaction
BACTH
20.12.18
Supplement 118
(Falord, Karimova, Hiron, & Msadek, 2012;
Georgiadou et al., 2012; Karimova et al., 2001,
2005; Mitchell et al., 2007; Ouellette, Rueden,
et al., 2014; Robichon, Karimova, Beckwith,
& Ladant, 2011). Specific mutations [deletion,
point mutations, random mutations (e.g., gen-
erated by error-prone PCR or other mutagenic
procedures)] can be introduced into one hy-
brid and BACTH assays can then be carried
out to detect/select variants unable to interact
with its partner(s). Identification of such muta-
tions is interesting for defining the molecular
determinants of a given interaction. It is also
important to establish the relevance of the in-
teraction at both functional (e.g., does it affect
protein activity or stability) and physiological
levels (e.g., does the interaction affect organ-
ism survival or for example the pathogenicity
of the bacterium).
Furthermore, in the case of prokaryotic pro-
teins, it is also possible in some instances to
assay the biological function of the hybrid pro-
teins, i.e., to monitor the activity of the X moi-
ety in a T25-X chimeric protein. In that case,
one can directly explore the correlation be-
tween the activity (or activities) of a protein
with its interacting capacities.
As stated above, one advantage of the
BACTH system, as it involves a cAMP signal-
ing cascade, is that the interaction between two
hybrid proteins can occur at a distance from
the transcriptional activation readout (due to
the rapid diffusion of cAMP). Hence, BACTH
assays can detect interactions that occur in a
variety of subcellular locations in the bacterial
cell, as illustrated in Figure 20.12.6.
The BACTH system has been extensively
used to analyze molecular interactions be-
tween membrane proteins, which have been
traditionally difficult to study with the standard
yeast two-hybrid system. It has been instru-
mental in characterizing numerous membrane-
associated multi-molecular structures such as
Current Protocols in Molecular Biology
Figure 20.12.6 Different types of interaction monitored by BACTH assay. Schematic organization
of some hybrid complexes successfully detected with BACTH assay and localized either in the
cytosol or at the inner membrane (im). FtsA, FtsQ, FtsW, FtsX, FtsZ, represent E. coli cell division
proteins; MalE, MalF, two subunits of the E. coli maltose transport system; Zip, the GCN4 leucine
zipper; Rsd, the E. coli regulator of sigma D and σ70, the principal σ factor of E. coli.

the bacterial cell division apparatus, a vari-
ety of secretion machineries (type II, type III,
Type IV, Type VI, Tat), membrane-associated
biosynthetic complexes, or diverse signaling
systems (Fransen et al., 2002; Cisneros, Bond,
Pugsley, Campos, & Francetic, 2012; Falord
et al., 2012; Gauliard et al., 2015; Georgiadou
et al., 2012; Jack et al., 2004; Karimova et al.,
1998, 2001, 2005; Ouellette et al., 2012; Zoued
et al., 2016).
The BACTH system has been shown to
detect interaction between a wide diver-
sity of integral membrane proteins having a
variable number of transmembrane segments
and/or topology, as well as with peripheral or
membrane-associated polypeptides. The main
constraint, however, is that the T25 and T18
fragments have to be localized in the cytosol
to have access to ATP. Therefore, when testing
integral membrane proteins, the knowledge (or
prediction) of their topology should guide the
design of hybrid proteins: there is no reason
to construct fusions where the T25 or T18
moieties would be appended to a polypeptide
extremity localized in the periplasm (although
results with such aberrant design have been
published in the literature). If there is no prior
knowledge of the topology of the membrane
proteins, then the different types of fusions (fu-
sions of the cyclase fragments to either N or C
extremities of the proteins) should be tested.
We recently implemented the BACTH sys-
tem to extend its application to the study of
interactions occurring within the periplasm or
at the extra-cytoplasmic side of the inner mem-
brane. For this, we designed a series of vec-
Current Protocols in Molecular Biology
tors in which a DNA sequence encoding a sin-
gle transmembrane segment (derived from the
E. coli OppB protein) was grafted between the
T25 or T18 moieties and the MCS (Ouellette,
Gauliard, et al., 2014; Ouellette, Rueden, et al.
2014). Thus, the T25/T18 fragments can be
localized in the cytosol while the fused pro-
teins are targeted to the periplasm (see Fig.
20.12.6). These tools can be used to monitor
interactions occurring in the periplasmic space
or to probe interactions with integral mem-
brane proteins that have their extremities lo-
calized in the periplasm. Interestingly, we also
showed that this approach could serve to verify
experimentally whether an in silico–predicted
transmembrane domain is indeed functional
for insertion into the membrane (Ouellette,
Gauliard, et al., 2014).
Another interesting application of the
BACTH two-hybrid system is in drug
discovery, where the technique can be easily
adapted for high-throughput screening assays
in order to identify small molecules that
either abolish or reinforce a given interaction
(Paschos et al., 2011). One obvious advantage
of using BACTH (or other bacterial) assays
for drug screening is that the effects of the
small molecules are monitored in conditions
that resemble the physiological situations. In
particular, for anti-bacterial compounds, one
of the main constraints limiting the efficacy of
many drugs is the impermeability of the bacte-
rial envelopes. The BACTH assay should thus
be particularly appropriate for searching for Analysis of
novel classes of antibiotics targeting protein- Protein
Interactions
protein interactions involved in a variety of
20.12.19
Supplement 118
 essential and prokaryotic-specific processes
(cell division, secretory machineries, toxin
production, biosynthetic pathways, etc.). As
antimicrobial resistance (AMR) is becoming
a very serious global public health threat
(https://www.cdc.gov/drugresistance/index
.html), novel approaches to elaborate effective
antibiotics become a most urgent need.
Critical Parameters and
Troubleshooting
False negative results
There can be notable differences between
the efficacies of complementation observed
with different combinations of hybrid proteins
made from the same interacting polypeptides
but fused to either end of the T25 or T18 frag-
ments (e.g., T25-X/T18-Y may be strongly
positive while X-T25/T18-Y or T25-Y/T18-X
are not). This variation may be due to the
differential stability of the various hybrid pro-
teins or, more likely, to structural constraints
within the heterodimeric complexes that
keep T18 and T25 from interacting correctly
(Fransen et al., 2002; Karimova et al., 2001,
2005). Because functional complementation
depends on the spatial proximity between the
adenylate cyclase (AC) fragments, as can be
seen in 3-D structure of AC (Guo et al., 2005),
the steric constraints imposed by the structural
arrangement of the hybrid protein complexes
may affect, and eventually prevent, the AC
enzymatic activity. This can be the case if the
hybrid proteins interact in such a way that the
fused T25 and T18 fragments are held too far
apart to properly associate in an active com-
plex, as illustrated for example in a study of
the interaction between different subdomains
of the Bacillus stearothermophilus tyrosyl-
tRNA synthetase (the 3-D structure of which
is known; Karimova et al., 2001). Hence, the
absence of a functional complementation be-
tween two hybrid proteins does not necessarily
mean that the two proteins do not associate.
This potential drawback, which is true for
all two-hybrid or protein complementation
approaches (leading to so-called false negative
results), should always be kept in mind.
Therefore, unless there is a specific consid-
eration (e.g., the topology of an integral mem-
brane protein; see above), it is wise to explore
all possible combinations of hybrid proteins
to increase the chance of successful detection
Membrane
Protein of an interaction. For this, the genes encod-
Interaction ing proteins X and Y should be cloned into
Analysis with all four vectors, pKT25, pKNT25, pUT18C,
BACTH
and pUT18, in order to generate all possible
20.12.20
Supplement 118
fusions, e.g., X and Y fused to either the N or
C-terminus of either T25 or T18.
Another source of failure to detect in-
teractions in the BACTH assay is the lack
of specific components and/or modifications
of the hybrid proteins in the E. coli envi-
ronment. This is the case for interactions
between eukaryotic proteins, which can be
dependent upon phosphorylation or other se-
lective post-translational modifications (e.g.,
acylation or glycosylation) that are performed
by eukaryotic-specific kinases or other ded-
icated enzymes (Ouellette, Gauliard, et al.,
2014). Other interactions may also require spe-
cific lipids, metabolites, or macromolecules
that might not be present in E. coli (Gauliard
et al., 2015). Although this limitation is shared
by all bacterial complementation or two-
hybrid assays, in certain cases, these systems
could nevertheless be exploited to screen for
the specific components (modifying enzymes,
protein partners, etc.) needed for a given inter-
action, as illustrated in Erce, Abeygunawar-
dena, Low, Hart-Smith, & Wilkins (2013).
False positive results
False positive results are another main
drawback of all two-hybrid or protein com-
plementation techniques: they correspond to
cells that exhibit a complementation-positive
phenotype (i.e., Lac+ , Mal+ ), although they
co-express hybrid proteins that do not inter-
act.
With the BACTH system, this can arise
from two main sources:
1. The host cells acquire a Lac+ or Mal+
phenotype independently of the expressed hy-
brid proteins. This could be due to: (i) spon-
taneous mutations of the CAP gene toward
a cAMP-independent phenotype (so-called
CRP*; may occur at 10−8 /10−9 frequency);
(ii) spontaneous cAMP-independent lac pro-
moter mutations (occur at frequencies around
10−7 /10−8 ). These false positive clones can be
easily identified upon retransformation of their
plasmids into new competent cya cells.
Note that the BACTH host strains carry
deletions of (or within) the cya gene and,
therefore, cannot revert to a cya+ phenotype
while the frequency of spontaneous cAMP-
independent Mal+ mutations (<10−18 ) is be-
low any detectable threshold.
2. Cloning in one of the BACTH vectors of
a polypeptide fragment that exhibits adenylate
cyclase enzymatic activity in E. coli. This in-
deed happened in our initial screens, using a
library prepared from a wild-type E. coli strain
(e.g., cya+). This can be easily remedied (the
Current Protocols in Molecular Biology
plasmid alone should confer a cya+ pheno-
type) and, if the library is made from bacterial
DNA, can be circumvented by using a cya
strain.
Another kind of false positive result may
arise with certain polypeptides that exhibit
“sticky” or aggregating properties; that is, they
will associate (or aggregate) with many other
hybrid proteins in a relatively nonspecific man-
ner. As such, these are not truly “false posi-
tive,” as there is an authentic association be-
tween the two hybrid proteins, even though it
is likely irrelevant from a physiological point
of view. We observed such behavior, in partic-
ular, with certain integral membrane proteins
that have a tendency to generate positive com-
plementation signal with other membrane pro-
teins. Under these conditions, it is beneficial to
test a protein of interest for interactions against
unrelated proteins that should not display an
interaction in vivo.
One possible approach to probe the speci-
ficity or selectivity of the interaction is to per-
form competition experiments. For this, one of
the protein partners (e.g., protein X) is over-
expressed in trans in addition to the two hy-
brid proteins T25-X and T18-Y. If the inter-
action between X and Y is specific, then the
overexpressed X component should compete
for binding T18-Y and displace the forma-
tion of the active complex T25-X/T18-Y (Ka-
rimova et al., 2005). This is illustrated in Fig-
ure 20.12.7, where we compare the efficien-
cies of complementation between T18-FtsI
and T25 fused to various Fts proteins. Strong
β-galactosidase activities (red bars) were mea-
sured in cells co-expressing T18-FtsI and T25-
FtsL, T25-FtsQ, or T25-FtsW, in agreement
with prior measurements (Fig. 20.12.4). How-
ever, when the FtsL protein was also simul-
taneously overexpressed in the same cells in
addition to the T18-FtsI and the various T25
fusions (blue bars), the interaction between
T18-FtsI and T25-FtsL was completely abol-
ished as expected (free FtsL competes with
T25-FtsL for binding to T18-FtsI), while the
others were not affected (Karimova et al.,
2005).
Anticipated Results
The BACTH system has been used in many
laboratories worldwide and has proven to be a
simple, reliable, and efficient two-hybrid tech-
nology to study protein-protein interactions in
vivo and to characterize proteins from a wide
variety of organisms. This technique is partic-
ularly attractive for the study of interactions
involving membrane proteins. In the bacterial
Current Protocols in Molecular Biology
world, the BACTH system has been instru-
mental in characterizing the molecular inter-
actions between components of membrane-
associated multi-molecular machineries such
as the cell-division apparatus, many different
signaling complexes, a variety of secretion
systems, and other biological processes in-
volved in bacterial pathogenesis. The BACTH
system has also been exploited in library
screening as well as in drug screening to iden-
tify small molecules able to blunt a specific
interaction. The recent implementations of the
BACTH system to make it compatible with the
Gateway ‘recombineering’ cloning technique
(BACTH-Gateway system or BACTHGW ), as
well as the modification of the BACTH plas-
mids to enable the characterization of interac-
tions between extracytoplasmic proteins, will
further expand the applicability and versatility
of the BACTH system for the study of protein-
protein interactions.
Time Considerations
The time needed will largely depend on the
objective of the research, that is, whether one
wishes to characterize interactions between
a given set of proteins (Basic Protocol 1 or
Alternate Protocol 1) or to perform a library
screen (Basic Protocol 2 or Alternate Protocol
2) to identify potential partners of a protein of
interest.
Characterization of protein-protein interac-
tions with the BACTH system (i.e., using Ba-
sic Protocol 1 or Alternate Protocol 1 with
the BACTH Gateway system) can be easily
carried out within 2 weeks. Once the experi-
ments have been planned, the design and syn-
thesis of the oligonucleotide primers for PCR
will take 1 to 3 days (depending on the com-
pany). During this time, the BACTH vectors
can be purified (standard minipreps are usually
enough) and prepared for cloning. The com-
petent BACTH host cells can also be prepared
at that time. Once the oligonucleotide primers
are available, the genes of interest can be am-
plified by PCR, purified, and cloned into the
BACTH vectors within the next 3 to 4 days.
After verification of sequence fidelity of the re-
combinant plasmids, they can be transformed
into competent BACTH cells. Results will be
apparent in 2 to 3 days. The efficiency of com-
plementation between the hybrid proteins can
then be quantified by β-galactosidase assays
(1 to 2 days).
Identification of potential partners of a pro-
tein of interest through a BACTH library Analysis of
screening experiment (i.e., Basic Protocol 2 or Protein
Interactions
Alternate Protocol 2) will naturally take much
20.12.21
Supplement 118
 Figure 20.12.7 Competition experiments. Red bars: the E. coli cya DHM1 cells were transformed
with BACTH plasmids expressing T18-FtsI and the indicated T25-fusions (‘no’ designates the
unfused T25). Blue bars: the cells were transformed with BACTH plasmids expressing the same
indicated T25-fusions and with a pUT18C derivative that co-expresses the T18-FtsI fusion and,
independently, the FtsL polypeptide (see Karimova et al. (2005) for experimental details). The
β-galactosidase activities (expressed in relative units) were measured as described above.

more time, the main component being by far
the construction of a library of DNA fragments
in one of the BACTH vectors. This step could
take at least 2 to 4 weeks depending on exper-
tise and the specificity of the project, while the
library screening per se may take about 1 week.
This does not include, of course, the down-
stream experiments needed to further validate
the isolated candidates. This can be done, first,
by re-cloning the prey genes into BACTH
vectors for a confirmatory assay using Basic
Protocol 1, and then through a variety of com-
plementary experimental approaches includ-
ing pull-down, cross-linking, co-localization,
biochemical assays, and genetic assays.
Acknowledgements
This work was supported by Institut Pasteur
and the Centre National de la Recherche Sci-
entifique (CNRS UMR 3528, Biologie Struc-
turale et Agents Infectieux).
Conflicts of Interest
D.L. is owner of a patent covering
the BACTH two-hybrid technology (Bac-
terial multi-hybrid system and applications
thereof—ref US 6333154 B1).
Literature Cited
Bloch, K. D., & Grossmann, B. (1995). Digestion
of DNA with restriction endonucleases. Cur-
rent Protocols in Molecular Biology, 31, 3.1.1–
Membrane 3.1.21.
Protein Busby, S., & Ebright, R. H. (1999). Transcription
Interaction activation by catabolite activator protein (CAP).
Analysis with
BACTH Journal of Molecular Biology, 293, 199–213.
doi: 10.1006/jmbi.1999.3161
20.12.22
Supplement 118
Chen, C., Banga, S., Mertens, K., Weber, M. M.,
Gorbaslieva, I., Tan, Y., . . . Samuel, J. E.
(2010). Large-scale identification and transloca-
tion of type IV secretion substrates by Coxiella
burnetii. Proceedings of the National Academy
of Sciences of the United States of America, 107,
21755–21760. doi: 10.1073/pnas.1010485107
Cisneros, D. A., Bond, P. J., Pugsley, A. P., Cam-
pos, M., & Francetic, O. (2012). Minor pseu-
dopilin self-assembly primes type II secretion
pseudopilus elongation. The EMBO Journal, 31,
1041–1053. doi: 10.1038/emboj.2011.454
Dautin, N., Karimova, G., Ullmann, A., & Ladant,
D. (2000). Sensitive genetic screen for pro-
tease activity based on a cyclic AMP sig-
naling cascade in Escherichia coli. Jour-
nal of Bacteriology, 182, 7060–7066. doi:
10.1128/JB.182.24.7060-7066.2000
Erce, M. A., Abeygunawardena, D., Low, J. K. K.,
Hart-Smith, G., & Wilkins, M. R. (2013). In-
teractions affected by arginine methylation in
the yeast protein-protein interaction network.
Molecular & Cellular Proteomics, 12, 3184–
3198. doi: 10.1074/mcp.M113.031500
Falord, M., Karimova, G., Hiron, A., & Msadek,
T. (2012). GraXSR proteins interact with
the VraFG ABC transporter to form a
five-component system required for cationic an-
timicrobial peptide sensing and resistance in
Staphylococcus aureus. Antimicrobial Agents
and Chemotherapy, 56, 1047–1058. doi:
10.1128/AAC.05054-11
Fields, S., & Song, O. (1989). A novel genetic sys-
tem to detect protein-protein interactions. Na-
ture, 340, 245–246. doi: 10.1038/340245a0
Finley, R. L. J., & Brent, R. (1994). Interaction
mating reveals binary and ternary connections
between Drosophila cell cycle regulators. Pro-
ceedings of the National Academy of Sciences of
the United States of America, 91, 12980–12984.
doi: 10.1073/pnas.91.26.12980
Current Protocols in Molecular Biology
Fransen, M., Brees, C., Ghys, K., Amery, L.,
Mannaerts, G. P., Ladant, D., & Van Veld-
hoven, P. P. (2002). Analysis of mammalian
peroxin interactions using a non-transcription-
based bacterial two-hybrid assay. Molecu-
lar & Cellular Proteomics, 1, 243–252. doi:
10.1074/mcp.M100025-MCP200
Gauliard, E., Ouellette, S. P., Rueden, K. J., &
Ladant, D. (2015). Characterization of inter-
actions between inclusion membrane proteins
from Chlamydia trachomatis. Frontiers in Cel-
lular and Infection Microbiology, 5, 13. doi:
10.3389/fcimb.2015.00013
Georgiadou, M., Castagnini, M., Karimova, G.,
Ladant, D., & Pelicic, V. (2012). Large-
scale study of the interactions between pro-
teins involved in type IV pilus biology
in Neisseria meningitidis: Characterization of
a subcomplex involved in pilus assembly.
Molecular Microbiology, 84, 857–873. doi:
10.1111/j.1365-2958.2012.08062.x
Guo, Q., Shen, Y., Lee, Y.-S., Gibbs, C. S.,
Mrksich, M., & Tang, W.-J. (2005). Struc-
tural basis for the interaction of Bordetella
pertussis adenylyl cyclase toxin with calmod-
ulin. The EMBO Journal, 24, 3190–3201. doi:
10.1038/sj.emboj.7600800
Gyuris, J., Golemis, E., Chertkov, H., & Brent, R.
(1993). Cdi1, a human G1 and S phase protein
phosphatase that associates with Cdk2. Cell, 75,
791–803. doi: 10.1016/0092-8674(93)90498-F
Hartley, J. L., Temple, G. F., & Brasch, M. A.
(2000). DNA cloning using in vitro site-specific
recombination. Genome Research, 10, 1788–
1795. doi: 10.1101/gr.143000
Houot, L., Fanni, A., de Bentzmann, S., &
Bordi, C. (2012). A bacterial two-hybrid
genome fragment library for deciphering
regulatory networks of the opportunistic
pathogen Pseudomonas aeruginosa. Microbiol-
ogy (Reading, England), 158, 1964–1971. doi:
10.1099/mic.0.057059-0
Jack, R. L., Buchanan, G., Dubini, A., Hatzixanthis,
K., Palmer, T., & Sargent, F. (2004). Coordinat-
ing assembly and export of complex bacterial
proteins. The EMBO Journal, 23, 3962–3972.
doi: 10.1038/sj.emboj.7600409
Karimova, G., Dautin, N., & Ladant, D. (2005).
Interaction network among Escherichia coli
membrane proteins involved in cell division
as revealed by bacterial two-hybrid analysis.
Journal of Bacteriology, 187, 2233–2243. doi:
10.1128/JB.187.7.2233-2243.2005
Karimova, G., Davi, M., & Ladant, D. (2012).
The beta-lactam resistance protein Blr, a small
membrane polypeptide, is a component of
the Escherichia coli cell division machinery.
Journal of Bacteriology, 194, 5576–5588. doi:
10.1128/JB.00774-12
Karimova, G., Pidoux, J., Ullmann, A., & Ladant,
D. (1998). A bacterial two-hybrid system based
on a reconstituted signal transduction pathway.
Proceedings of the National Academy of Sci-
ences of the United States of America, 95, 5752–
5756. doi: 10.1073/pnas.95.10.5752
Current Protocols in Molecular Biology
Karimova, G., Robichon, C., & Ladant, D. (2009).
Characterization of YmgF, a 72-residue in-
ner membrane protein that associates with
the Escherichia coli cell division machinery.
Journal of Bacteriology, 191, 333–346. doi:
10.1128/JB.00331-08
Karimova, G., Ullmann, A., & Ladant, D. (2000).
A bacterial two-hybrid system that exploits a
cAMP signaling cascade in Escherichia coli.
Methods in Enzymology, 328, 59–73. doi:
10.1016/S0076-6879(00)28390-0
Karimova, G., Ullmann, A., & Ladant, D. (2001).
Protein-protein interaction between Bacillus
stearothermophilus tyrosyl-tRNA synthetase
subdomains revealed by a bacterial two-hybrid
system. Journal of Molecular Microbiology and
Biotechnology, 3, 73–82.
Kleinschnitz, E.-M., Heichlinger, A., Schirner,
K., Winkler, J., Latus, A., Maldener, I.,
. . . Muth, G. (2011). Proteins encoded by
the mre gene cluster in Streptomyces coeli-
color A3(2) cooperate in spore wall synthesis.
Molecular Microbiology, 79, 1367–1379. doi:
10.1111/j.1365-2958.2010.07529.x
Kramer, M. F., & Coen, D. M. (2000). Enzymatic
amplification of DNA by PCR: Standard pro-
cedures and optimization. Current Protocols in
Molecular Biology, 56, 15.1.1–15.1.14.
Kucera, R. B., & Nichols, N. M. (2008). DNA-
dependent DNA polymerases. Current Proto-
cols in Molecular Biology, 84, 3.5.1–3.5.19.
Ladant, D., & Ullmann, A. (1999). Bordetella per-
tussis adenylate cyclase: A toxin with multiple
talents. Trends in Microbiology, 7, 172–176. doi:
10.1016/S0966-842X(99)01468-7
Miller, J. H. (1992). A short course in bacterial
genetics: A laboratory manual and handbook
for Escherichia coli and related bacteria. Cold
Spring Harbor, NY: Cold Spring Harbor Labo-
ratory Press.
Mitchell, J. E., Oshima, T., Piper, S. E., Webster, C.
L., Westblade, L. F., Karimova, G., . . . Lee, D. J.
(2007). The Escherichia coli regulator of sigma
70 protein, Rsd, can up-regulate some stress-
dependent promoters by sequestering sigma 70.
Journal of Bacteriology, 189, 3489–3495. doi:
10.1128/JB.00019-07
Nichols, N. M. (2011). Endonucleases. Current
Protocols in Molecular Biology, 93, 3.12.1–
3.12.7.
Ouellette, S. P., Gauliard, E., Antosova, Z., &
Ladant, D. (2014). A Gateway-compatible bac-
terial adenylate cyclase-based two-hybrid sys-
tem. Environmental Microbiology Reports, 6,
259–267. doi: 10.1111/1758-2229.12123
Ouellette, S. P., Karimova, G., Subtil, A., & Ladant,
D. (2012). Chlamydia co-opts the rod shape-
determining proteins MreB and Pbp2 for cell
division. Molecular Microbiology, 85, 164–178.
doi: 10.1111/j.1365-2958.2012.08100.x
Ouellette, S. P., Rueden, K. J., Gauliard, E., Persons,
L., de Boer, P. A., & Ladant, D. (2014). Analy- Analysis of
sis of MreB interactors in Chlamydia reveals a Protein
RodZ homolog but fails to detect an interaction Interactions
20.12.23
Supplement 118
 with MraY. Frontiers in Microbiology, 5, 279.
doi: 10.3389/fmicb.2014.00279
Paschos, A., den Hartigh, A., Smith, M. A.,
Atluri, V. L., Sivanesan, D., Tsolis, R. M., &
Baron, C. (2011). An in vivo high-throughput
screening approach targeting the type IV se-
cretion system component VirB8 identified in-
hibitors of Brucella abortus 2308 proliferation.
Infection and Immunity, 79, 1033–1043. doi:
10.1128/IAI.00993-10
Pfeiffer, D., & Jendrossek, D. (2011). Interac-
tion between poly(3-hydroxybutyrate) granule-
associated proteins as revealed by two-hybrid
analysis and identification of a new phasin
in Ralstonia eutropha H16. Microbiology
(Reading, England), 157, 2795–2807. doi:
10.1099/mic.0.051508-0
Robichon, C., Karimova, G., Beckwith, J., &
Ladant, D. (2011). Role of leucine zipper
motifs in association of the Escherichia coli
cell division proteins FtsL and FtsB. Jour-
nal of Bacteriology, 193, 4988–4992. doi:
10.1128/JB.00324-11
Sambrook, J., Russell, D. W., & Sambrook,
J. (2006). The condensed protocols from
molecular cloning: A laboratory manual. Cold
Spring Harbor, NY: Cold Spring Harbor Labo-
ratory Press.
Sands, B., & Brent, R. (2016). Overview
of post Cohen-Boyer methods for sin-
gle segment cloning and for multiseg-
ment DNA assembly. Current Protocols in
Molecular Biology, 113, 3.26.1–3.26.20. doi:
10.1002/0471142727.mb0326s113
Membrane
Protein
Interaction
Analysis with
BACTH
20.12.24
Supplement 118
Seidman, C. E., Struhl, K., Sheen, J., & Jessen, T.
1997. Introduction of plasmid DNA into cells.
Current Protocols in Molecular Biology, 37,
1.8.1–1.8.10.
Stynen, B., Tournu, H., Tavernier, J., & Van Di-
jck, P. (2012). Diversity in genetic in vivo
methods for protein-protein interaction stud-
ies: From the yeast two-hybrid system to the
mammalian split-luciferase system. Microbiol-
ogy and Molecular Biology Reviews, 76, 331–
382. doi: 10.1128/MMBR.05021-11
Voytas, D. (2000). Agarose gel electrophoresis.
Current Protocols in Molecular Biology, 51,
2.5A.1–2.5A.9.
Wilson, K. 1997. Preparation of genomic DNA from
bacteria. Current Protocols in Molecular Biol-
ogy, 00, 2.4.1–2.4.5.
Zervos, A. S., Gyuris, J., & Brent, R. (1993). Mxi1,
a protein that specifically interacts with Max to
bind Myc-Max recognition sites. Cell, 72, 223–
232. doi: 10.1016/0092-8674(93)90662-A
Zoued, A., Durand, E., Brunet, Y. R., Spinelli, S.,
Douzi, B., Guzzo, M., . . . Cascales, E. (2016).
Priming and polymerization of a bacterial con-
tractile tail structure. Nature, 531, 59–63. doi:
10.1038/nature17182
Internet Resources
http://www.euromedex.com
Euromedex: Commercial supplier of the BACTH
system.
https://www.thermofisher.com.
Supplier for Gateway ‘recombineering’ cloning
technique.
Current Protocols in Molecular Biology