The bacterial two-hybrid system based on adenylate cyclase reconstitution in Escherichia coli
Introduction
As an alternative to the widely used yeast two-hybrid method, several two-hybrid systems have been engineered in bacteria to study protein–protein interactions. One of them, developed by Karimova and collaborators in 1998, is based on the reconstitution of adenylate cyclase activity in Escherichia coli [1]. This bacterial two-hybrid system takes advantage of the specific features of the adenylate cyclase toxin from Bordetella pertussis to detect interactions between cytoplasmic proteins, as well as membrane proteins, from prokaryotes but also from eukaryotes. Previous reviews describing extensively this technique have already been published [2], [3], [4], [5]. However, recently, the BACTH system has been much more frequently used in the molecular microbiology community and several improvements have been made on the technique. In this review, we would like to give basic and easy protocols, that we use routinely in our laboratories, to allow everyone to apply rapidly this technique to their research field. We will also describe the new tools available and summarize what was done in the last few years by using the BACTH system, discuss what is feasible, and underline the advantages and the successes of the technique to study protein–protein interactions.
Section snippets
Principle and development of the bacterial adenylate cyclase two hybrid system
The bacterial adenylate cyclase two hybrid (BACTH) system is based on the reconstitution of a regulatory cascade depending on cyclic adenosine 3′,5′-monophosphate (cAMP). cAMP is synthesized by a family of enzymes called adenylate cyclases. A peculiar member of this family is the adenylate cyclase of B. pertussis, the agent of the whooping cough. This enzyme is a toxin that becomes fully active in the host after binding to calmodulin, a protein found only in eukaryotic organisms. Following its
Overview of the technique (Fig. 2)
An E. coli strain deleted of the gene coding for the endogenous adenylate cyclase (cya− strain) is transformed by both plasmids containing the T25 and T18 hybrids (Fig. 2A). Although B. pertussis adenylate cyclase usually needs to bind calmodulin to be fully active, it has been shown that in E. coli, a cryptic adenylate cyclase activity is detected even in absence of calmodulin [8]. There are then several ways to detect a positive interaction within the BACTH system. Indicator LB plates
Advantages/disadvantages
When considering the potential drawbacks of a technique, it often happens that its disadvantages might turn out to be advantages and vice versa. This is especially true for the BACTH, as shown by the following examples.
What can be done with BACTH?
In order for the reader to know if the technique will be helpful for her or his research, we will try here to summarize the types of proteins and interactions that have been studied successfully so far by BACTH. We will also give examples of the questions that can be asked by playing with the method.
Other bacterial two-hybrid systems
This review is dedicated to the BACTH technique. However, it is important to mention that other bacterial two-hybrid systems are available. The major alternatives to the BACTH in E. coli are techniques that are based on the activation of transcription, similar to the classical yeast two-hybrid [54], [55]. One complete system called BacterioMatch®II is distributed by Stratagene. In this system, one of the proteins is fused to a component of the RNAP (either α or ω subunit), while the other is
Concluding remarks
We have described the current applications of the BACTH system. Its use has increased in the recent years in the community of microbiologists, and we hope this review will be helpful for newcomers to the technique. It is rapid, easy, and very reliable, with few reported case of false positives or false negatives. However, due to some drawbacks that have been exposed here, there is still space for improvement, such as designing other reporters than β-galactosidase, like GFP or antibiotic
Acknowledgments
First, we are deeply grateful to Daniel Ladant and Gouzel Karimova who invented and developed the BACTH system. We thank Laetitia My, Julie Viala, Rim Maouche, all past members of the Bouveret team, and Susan Gottesman and Olivier Genest for support and discussion. We would like to thank all the BACTH users in our laboratories, from whom we got back experience and discussion. Research in EB laboratory is funded by CNRS and ANR. AB is funded by the NIH.
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