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. 1998 May 12;95(10):5752-6.
doi: 10.1073/pnas.95.10.5752.

A bacterial two-hybrid system based on a reconstituted signal transduction pathway

G Karimova  1 , J Pidoux, A Ullmann, D Ladant
Affiliations

A bacterial two-hybrid system based on a reconstituted signal transduction pathway

G Karimova et al. Proc Natl Acad Sci U S A. .

Abstract

We describe a bacterial two-hybrid system that allows an easy in vivo screening and selection of functional interactions between two proteins. This genetic test is based on the reconstitution, in an Escherichia coli cya strain, of a signal transduction pathway that takes advantage of the positive control exerted by cAMP. Two putative interacting proteins are genetically fused to two complementary fragments, T25 and T18, that constitute the catalytic domain of Bordetella pertussis adenylate cyclase. Association of the two-hybrid proteins results in functional complementation between T25 and T18 fragments and leads to cAMP synthesis. Cyclic AMP then triggers transcriptional activation of catabolic operons, such as lactose or maltose, that yield a characteristic phenotype. In this genetic test, the involvement of a signaling cascade offers the unique property that association between the hybrid proteins can be spatially separated from the transcriptional activation readout. This permits a versatile design of screening procedures either for ligands that bind to a given "bait," as in the classical yeast two-hybrid system, or for molecules or mutations that block a given interaction between two proteins of interest.

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Figures

Figure 1
Figure 1
Principle of an E. coli two-hybrid system based on functional complementation of CyaA fragments. (Upper) Schematic of the basic principle of in vivo complementation between the two fragments of the catalytic domain of B. pertussis adenylate cyclase. The two boxes represent the T25 and T18 fragments corresponding to amino acids 1–224 and 225–399 of the CyaA protein. In A, the full-length catalytic domain (residues 1–399), when expressed in E. coli, exhibits a basal calmodulin-independent activity that results in cAMP synthesis. In B, the two fragments, T25 and T18, when coexpressed as independent polypeptides, are unable to interact and no cAMP synthesis occurs. In C, the two fragments, fused to two interacting proteins, X and Y, are brought into close proximity, resulting in functional complementation followed by cAMP production. (Lower) Schematic of the readout of the complementation. cAMP, synthesized in an E. coli cya strain by the complementing T25 and T18 pairs, binds to the catabolite gene activator protein, CAP. The cAMP/CAP complex then can recognize specific promoters and switch on the transcription of the corresponding genes. These reporter genes can be either natural E. coli genes, such as lacZ or mal genes, or synthetic ones, such as antibiotic-resistance genes fused to a cAMP/CAP-dependent promoter.
Figure 2
Figure 2
Schematic representation of plasmids. The open boxes represent the ORFs of β-lactamase (bla) and chloramphenicol acetyl transferase (cat) genes. The solid boxes correspond to the ORF of cyaA′, with codon numbers indicated below. The hatched boxes correspond to the multicloning site sequences (MCS) that are fused at the indicated position of the cya ORF. The origin of replication of the plasmids is indicated by shaded boxes.
Figure 3
Figure 3
Screening of interacting proteins with the bacterial two-hybrid system. DHP1 cells were cotransformed with a mixture of plasmids pTI8, pT18-zip, and pT18-Tyr and either pT25 (A) or pT25-zip (B), plated on LB–X-gal agar plates containing 0.5 mM isopropyl-β-d-thiogalactopyranoside, ampicillin, and chloramphenicol, and incubated for 30 hr at 30°C. Note that the cya+ colonies are larger than the cya ones.
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