Model systems: Studying molecular recognition using bacterial n-hybrid systems

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Model systems: Studying molecular recognition using bacterial n-hybrid systems

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James C. Hu Since the first description of the yeast two-hybrid system, related genetic assays for protein–protein interactions have become popular and powerful tools for structure–function analysis on the scale of individual proteins or whole proteomes. After a somewhat surprising lag, similar systems have recently been described for use in bacterial hosts. n-hybrid modifications of the original yeast system have been used to examine interactions with DNA, RNA and small molecules, and other modifications have improved throughput for genomic applications. Bacterial n-hybrid systems are being designed for a similar array of uses. Will the bacterial systems be as popular as the yeast n-hybrid systems? Only time will tell.

A large part of biochemistry and molecular biology is concerned with the binding properties of macromolecules, especially proteins (most of the rest involves what happens after a binding event has occurred). This can involve finding the substrates for an enzyme, the DNA or RNA sequences recognized by a regulatory factor, or which combinations of proteins comprise the subunits in a complex. Finding a protein that does not bind something else as part of its function would be considered remarkable, if not implausible. Thus, for most of us, the question of what our favorite proteins bind to is central to how we think about them. Now that we are well into the post-genomic era1, it is clear that questions based on the binding properties of proteins form the basis of much of functional genomics. Knowing where a protein is localized and when it is expressed is important, but to know what it does you have to find out what it binds. Using cells as test tubes

James C. Hu Dept of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843-2128, USA. e-mail: [email protected]

Hybrid systems for the study of protein interactions are binding assays based on reconstituting the activity of a protein from its parts. In the classical yeast n-hybrid systems2, a transcriptional activator is divided into DNA-binding and activation domains. In various one-hybrid forms, we can search for protein domains that can replace either the DNAbinding domain or the activation domain. In twohybrid and three-hybrid systems, we can test whether a pair of proteins or a ternary complex can replace the chain connectivity between the domains. Whether the hybrids reconstitute the tethering of the activation and DNA-binding domains, the subunits of a multimeric transcription factor3–5, or the activity of an enzyme from its subdomains6,7, all

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n-hybrid systems are basically binding assays that use cells as test tubes and cellular contents as complex reaction buffers. Given Arthur Kornberg’s famous advice to not ‘waste clean thoughts on dirty enzymes’, what is the basis for the popularity of these experimental systems, which contain thousands of proteins, other macromolecules and small molecules, in which the pH and ionic strength are not well defined, the usable temperature range is limited and the concentrations of the reactants are difficult to control, and the results of which are not quantitative? Although it is tempting to invoke a thinly disguised vitalism of studying interactions under physiological conditions, twohybrid studies do not recognize boundaries between species nor intracellular compartments. There is no single answer to the popularity of these systems, but contributing factors include: the power of cells not only to express proteins that are difficult to purify but also to keep them soluble; the universality of assays that do not depend on the natural activities of the proteins (beyond raw binding); the ability of phenotypes to reflect the activities of vanishingly small amounts of protein; and the power of genetics to sort quickly through large numbers of interactions. If you just want to know if the two soluble proteins you have already purified to homogeneity interact, using a two-hybrid system is a poor choice. Once we have made the leap to treating cells as test tubes, we still have many choices concerning the kind of cell to use. When the original description of the yeast two-hybrid system appeared2, it was clear that a similar kind of assay would have advantages in bacteria (Box 1). Shortly afterwards, Erin O’Shea, Peter Kim, Bob Sauer and I described a bacterial one-hybrid system to examine homotypic protein–protein interactions3, but general bacterial two-hybrid systems did not appear for several years. This gap is related to the nature of the Fields system and its relatives, which are based on the reconstitution of transcriptional activators from separate domains. Few of the well-studied bacterial activators had this kind of structure, and it was widely thought that activation could not be used to build a bacterial two-hybrid system, at least for σ70-dependent promoters. Two trends have led to a sudden proliferation of bacterial two-hybrids in

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Box 1. General issues for the comparison of E. coli and S. cerevisiae n-hybrid systems Advantages of E. coli • Faster growth • Higher transformation efficiency • Nuclear localization not required • Domains with eukaryotic activation domains do not activate E. coli transcription • Fewer indirect interactions involving bridging by endogenous proteinsa Advantages of S. cerevisiae • Expression (sometimes) • Post-translational modification of eukaryotic proteins • Larger body of literature documenting problems and solutions • More indirect interactions involving bridging by endogenous proteinsa Problems for both • Fused proteins perturb structure and interactions of the bait and/or prey. aOf course, E. coli systems will report fewer indirect interactions than the yeast systems

when the source of the interacting proteins is eukaryotic; the opposite is likely to be seen with proteins from bacteria. However, as E. coli has fewer endogenous genes in the first place, on balance it will report fewer indirect interactions. Whether indirect interactions are advantages or disadvantages depends on what you are trying to determine; indeed, indirect interactions might allow faster characterization of functional networks than direct interactions alone.

recent years. First, two-hybrid systems built around other kinds of reconstitution reactions have appeared; some of which involve transcription factors4,5,8–11, whereas others involve soluble Table 1. The molecular basis for n-hybrid systems Hosta

Type of protein structure replaced by interacting proteins

Activity reconstituted

Refs

Polypeptide linker connecting domains

Transcriptional activation GAL4

Y

1

LexA–VP16

Y

16

LexA–B42

Y

16

cI–rpoA, cI–rpoZ

E

13,14

Zif–rpoA

E

18

Membrane localization of signaling proteins Normal protein–protein interaction

Promote reconstitution of protein fragments to form a folded protein

aAbbreviations:

Sos or Ras

Y

16

Transcriptional regulation λ repressor dimers

E

3

λ repressor cooperative interactions

E

10

LexA homodimers

E

5

LexA heterodimers

E

8

AraC activator

E

4

ToxR

E

11

Active enzyme or protein Ubiquitin

Y

16

Adenyl cyclase

E

7

Dihydrofolate reductase (DHFR)

E

6

E, Escherichia coli; Y, yeast.

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enzymes6,7,12. Second, Dove and Hochschild have shown that artificial activators can be built by connecting a DNA-binding protein to an RNA polymerase subunit13,14. Although this work was primarily motivated by a desire to resolve a basic question about the mechanisms of transcriptional activation, the resultant chimeric prokaryotic activators have the effective domain structure of the eukaryotic activators that inspired the yeast two-hybrid system. Table 1 outlines the different strategies used in some of the published yeast and bacterial hybrid systems. The features of each system have recently been compared12,15. In the meantime, the development of methods for use with the yeast system has forged ahead on several fronts. As in the development of the basic two-hybrid concept, it is likely that the future of the bacterial systems will both follow the lead of the yeast systems and branch out in new directions that exploit particular aspects of bacterial molecular genetics. Modifications of n-hybrid systems

The most dramatic modifications of the original twohybrid concept are the various n-hybrids that apply the ‘cell as a test tube’ concept to other kinds of interactions (Fig. 1)16. One-hybrid systems look for protein domains that replace, rather than connect, one of the pieces that is reconstituted by a two-hybrid system. For example, to identify proteins that recognize a specific DNA sequence, libraries of fusions to an activation domain are screened against a reporter containing the DNA sequence of interest upstream of an activatable promoter. The correct protein will recruit the fused activation domain to the promoter and give an increased transcription signal. The recruitment-based bacterial systems have already been adapted for this use17,18. The use of one-hybrids to identify activation domains in yeast actually preceded the two-hybrid system19. Because activation sequences are often found even in random DNA, this method is probably not very useful for screening libraries. However, it is commonly used to find the activation domains in a structure–function analysis of a specific gene of interest. The original λ repressor system and its relatives based on replacing a dimerization domain with interacting proteins are inherently one-hybrid systems. These are particularly well-suited for identifying homotypic protein–protein interactions from libraries. First, because the system scores selfinteraction, the number of clones needed to achieve saturating coverage scales with genome size rather than the square of genome size. Second, there is good evidence that two-hybrid systems under-represent homotypic interactions, at least among homodimers, owing to a self-squelching competition between baits fused to homodimeric DNA-binding domains and preys that have to compete from solution17,20. Finally, the repressor system can discriminate between homodimers and higher-order oligomers17.

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Acknowledgements I thank Leonardo MariñoRamírez for invaluable help and Debby Siegele for critical comments on the manuscript; any omissions or errors are my own. Work in my lab related to this review was done with the support of the National Science Foundation (MCB9808474), the Robert A. Welch Foundation (A-1354) and the Advanced Research Program of the Texas Higher Education Coordinating Board (Award 999902-116).

TRENDS in Microbiology Vol.9 No.5 May 2001

Three-hybrid systems replace a protein–protein bridge with a ternary complex involving two proteins and a third molecule that may or may not be a protein. For example, an RNA bait can be constructed by combining a known RNA–protein interaction with a chimeric RNA that includes the target of the known RNA-binding protein with the target of a desired unknown21. This bait is used to screen or select clones from a library of prey hybrids. Other three-hybrids have been described using small molecules as the bridge. In most cases, this has been used to build molecular switches22,23, but it might be possible to use chimeric small molecules to identify the unknown targets of drugs. In cases where this would be possible, more direct biochemical approaches (e.g. affinity chromatography) are usually available. Although these three-hybrids have not yet been described in a bacterial system, in principle similar adaptations should work in bacterial backgrounds. The other class of advances in n-hybrid technology addresses technical issues for the identification of interacting proteins. To the outsider, these advances are less dramatic, but in the end might be more important for application of n-hybrids to genomics. For example, shortly after the description of the original Fields system, versions using different DNAbinding and activation domains appeared. Although these systems are superficially similar, each has its own characteristics, and combinations of hybrids with different DNA-binding domains (‘dual bait’) can be used to sort specific and non-specific interactions rapidly. Similarly, different DNA-binding domains have been used for bacterial one-hybrid and twohybrid systems4,5,8–11. Other advances improve the throughput for constructing and screening libraries for interactions. These include the use of homologous or site-specific recombination24,25 instead of restriction sites to build the libraries, and the use of interaction mating26 to perform combinatorial screens and selections. The site-specific systems are readily adaptable to the construction of libraries for bacterial systems, and recent advances in manipulating homologous recombination with linear DNA in Escherichia coli might also prove to be useful27. Although interaction mating might be possible with conjugative plasmids, transduction of phagemids is an easy alternative28. For any n-hybrid interaction that is identified, reverse approaches can provide useful information16. Mutational analysis can be used to localize and dissect the interaction surface. Here, I expect the higher transformation efficiencies of the bacterial systems will shine. Ligands that reverse n-hybrid interactions29,30

References 1 Eisenberg, D. et al. (2000) Protein function in the post-genomic era. Nature 405, 823–826 2 Fields, S. and Song, O. (1989) A novel genetic system to detect protein–protein interactions. Nature 340, 245–246 3 Hu, J.C. et al. (1990) Sequence requirements for coiled-coils: analysis with lambda http://tim.trends.com

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Bait Reconstitution of activity

Hybrid B RNA-binding domain

Hybrid A

RNA-binding domain Interaction

Chimeric RNA Hybrid B Reconstitution of activity Binding protein Interaction Hybrid A

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Fig. 1. Basis for three-hybrid systems using non-protein bridging interactions. (a) Scheme for three-hybrid systems to isolate proteins that bind a specific RNA target. A known RNA–protein interaction in the bait is used to tether a site for an unknown protein via a chimeric RNA. (b) Scheme for isolating proteins that bind a specific small-molecule ligand. Hybrid A and hybrid B could be from any two-hybrid system; however, most have been built around the yeast systems based on transcriptional activation.

can illuminate the biology of the system by identifying competing protein–protein interactions, or providing a means to identify lead compounds for drug development. Prokaryotic vs eukaryotic?

In the final analysis, will the bacterial systems be better than the yeast systems? This question is probably meaningless. Each pairwise comparison of n-hybrid systems will have strengths and weaknesses that are based not only on the host used (Table 1), but also on the general approach and the detailed quirks of the particular systems involved. From the yeast twohybrid literature, it is already apparent that different results can be obtained using different combinations of DNA-binding domains and activation domains. Different results are often seen just by reversing the identities of bait and prey in the same system. Thus, different systems are not better or worse, they are just different, and will generate complementary information. As it is impractical to use all systems for all problems, the complementarity can probably be maximized by comparing results from one eukaryotic and one prokaryotic n-hybrid system.

repressor–GCN4 leucine zipper fusions. Science 250, 1400–1403 4 Bustos, S. and Schleif, R. (1993) Functional domains of the AraC protein. Proc. Natl. Acad. Sci. U. S. A. 90, 5638–5642 5 Schmidt-Dörr, T. et al. (1991) Construction, purification, and characterization of a hybrid protein comprising the DNA binding domain of

the LexA repressor and the Jun leucine zipper: a circular dichroism and mutagenesis study. Biochemistry 30, 9657–9664 6 Pelletier, J. et al. (1998) Oligomerization domain-directed reassembly of active dihydrofolate reductase from rationally designed fragments. Proc. Natl. Acad. Sci. U. S. A. 95, 12141–12146

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7 Karimova, G. et al. (1998) A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proc. Natl. Acad. Sci. U. S. A. 95, 5752–5756 8 Dimitrova, M. et al. (1998) A new LexA-based genetic system for monitoring and analyzing protein heterodimerization in Escherichia coli. Mol. Gen. Genet. 257, 205–212 9 Kornacker, M.G. et al. (1998) Gene activation by the AraC protein can be inhibited by DNA looping between AraC and a LexA repressor that interacts with AraC: possible applications as a two-hybrid system. Mol. Microbiol. 30, 615–624 10 Hays, L.B. et al. (2000) Two-hybrid system for characterization of protein–protein interactions in E. coli. Biotechniques 29, 288–296 11 Kolmar, H. et al. (1995) Membrane insertion of the bacterial signal transduction protein ToxR and requirements of transcription activation studied by modular replacement of different protein substructures. EMBO J. 14, 3895–3904 12 Ladant, D. and Karimova, G. (2000) Genetic systems for analyzing protein–protein interactions in bacteria. Res. Microbiol. 151, 711–720 13 Dove, S.L. et al. (1997) Activation of prokaryotic transcription through arbitrary protein–protein contacts. Nature 386, 627–630 14 Dove, S.L. and Hochschild, A. (1998) Conversion of the omega subunit of Escherichia coli RNA

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polymerase into a transcriptional activator or an activation target. Genes Dev. 12, 745–754 Legrain, P. and Selig, L. (2000) Genome-wide protein interaction maps using two-hybrid systems. FEBS Lett. 480, 32–36 Vidal, M. and Legrain, P. (1999) Yeast forward and reverse ‘n’-hybrid systems. Nucleic Acids Res. 27, 919–929 Hu, J.C. et al. (2000) Escherichia coli one- and two-hybrid systems for the analysis and identification of protein–protein interactions. Methods 20, 80–94 Joung, J.K. et al. (2000) A bacterial two-hybrid selection system for studying protein–DNA and protein–protein interactions. Proc. Natl. Acad. Sci. U. S. A. 97, 7382–7387 Ruden, D.M. et al. (1991) Generating yeast transcriptional activators containing no yeast protein sequences. Nature 350, 250–252 Newman, J.R. et al. (2000) A computationally directed screen identifying interacting coiled coils from Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U. S. A. 97, 13203–13208 SenGupta, D.J. et al. (1996) A three-hybrid system to detect RNA–protein interactions in vivo. Proc. Natl. Acad. Sci. U. S. A. 93, 8496–8501 Licitra, E. and Liu, J. (1996) A three-hybrid system for detecting small ligand–protein receptor interactions. Proc. Natl. Acad. Sci. U. S. A. 93, 12817–12821

23 Belshaw, P.J. et al. (1996) Controlling protein association and subcellular localization with a synthetic ligand that induces heterodimerization of proteins. Proc. Natl. Acad. Sci. U. S. A. 93, 4604–4607 24 Liu, Q. et al. (1998) The univector plasmid-fusion system, a method for rapid construction of recombinant DNA without restriction enzymes. Curr. Biol. 8, 1300–1309 25 Walhout, A.J. et al. (2000) Protein interaction mapping in C. elegans using proteins involved in vulval development. Science 287, 116–122 26 Finley, R.L., Jr and Brent, R. (1994) Interaction mating reveals binary and ternary connections between Drosophila cell cycle regulators. Proc. Natl. Acad. Sci. U. S. A. 91, 12980–12984 27 Murphy, K.C. (1998) Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli. J. Bacteriol. 180, 2063–2071 28 Vershon, A.K. et al. (1986) Isolation and analysis of Arc repressor mutants: evidence for an unusual mechanism of DNA binding. Proteins: Struct. Funct. Genet. 1, 302–311 29 Colas, P. et al. (1996) Genetic selection of peptide aptamers that recognize and inhibit cyclindependent kinase 2. Nature 380, 548–550 30 Park, S.H. and Raines, R.T. (2000) Genetic selection for dissociative inhibitors of designated protein–protein interactions. Nat. Biotechnol. 18, 847–851

The biofilm matrix – an immobilized but dynamic microbial environment Ian W. Sutherland The biofilm matrix is a dynamic environment in which the component microbial cells appear to reach homeostasis and are optimally organized to make use of all available nutrients. The major matrix components are microbial cells, polysaccharides and water, together with excreted cellular products. The matrix therefore shows great microheterogeneity, within which numerous microenvironments can exist. Although exopolysaccharides provide the matrix framework, a wide range of enzyme activities can be found within the biofilm, some of which will greatly affect structural integrity and stability.

Ian W. Sutherland Institute of Cell & Molecular Biology, Edinburgh University, Rutherford Building, Mayfield Road, Edinburgh, UK EH9 3JH. e-mail: [email protected]

With the realization that many, perhaps even the majority of microorganisms exist naturally as biofilms, interest in these phenomena has increased considerably. Not only are biofilms found in a very wide range of natural and artificial environments, they also provide their component microbial cells with an almost infinite range of constantly changing microenvironments. The matrix can almost be considered as an immobilized enzyme system in which the milieu and the enzyme activities are constantly changing and evolving to an approximately steady state. This steady state can then be radically altered by applying physical forces such as high shear, or via

external or internal reactions that cause the detachment and loss of regions of the biofilm. It must also be remembered that, because of the wide range of environments in which biofilms are found, it is extremely difficult to generalize about their structure and physiological activities1. The nature of the matrix, as exemplified by Wimpenny2, is thus dependent on both intrinsic and extrinsic factors. Intrinsic factors arise in accordance with the genetic profile of the component microbial cells; extrinsic factors include the physico-chemical environment in which the biofilm and its matrix are located, which, inevitably, is constantly influenced by solute transport and solute diffusion gradients. Sufficient information on biofilms and their structure is now available to permit the construction of realistic models3. Three variants have been suggested – heterogeneous mosaic, dense confluent and penetrated water channel – and all could be correct given the wide variety of biofilms that have been studied. Biofilms are found in the majority of environments, natural or artificial, where a surface is

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