Challenge phage provide a powerful method for genetically dissecting DNA-protein interactions by using the phage P22 ant gene as the reporter gene. Under appropriate conditions, expression of the ant gene determines the lysis-lysogeny decision of P22. This provides a positive selection for and against DNA-binding: repression of ant can be selected by requiring growth of lysogens, and mutants that cannot repress ant can be selected by requiring lytic growth of the phage. Thus, placing ant gene expression under the control of a specific DNA-protein interaction provides very strong genetic selections for regulatory mutations in the DNA-binding protein and DNA-binding site that either increase or decrease the apparent strength of a DNA-protein interaction in vivo. The challenge phage selection provides a general method for identifying critical residues involved in DNA-protein interactions. Challenge phage selections have been used to genetically dissect many different prokaryotic and eukaryotic DNA-binding interactions.
P22 regulation. P22 has two regulatory regions that control the decision between lysis and lysogeny. The immC region encodes the c2 repressor which regulates transcription from PL and PR. The regulation of P22 by the c2 repressor is virtually identical to the regulation of phage lambda by cI repressor (see Ptashne, M. 1992. A Genetic Switch. Cell Press). However, P22 has a second regulatory region, immI, which is not present on phage lambda. The immI region indirectly regulates the lysis/lysogeny decision by modulating the activity of the c2 repressor. The immI region contains the genes ant and mnt. The ant gene encodes antirepressor, a protein that binds noncovalently to the c2 repressor and inactivates it, thus expression of ant prevents lysogeny. Expression of the ant gene is negatively regulated by the Mnt protein (maintenance of lysogeny). ant is expressed from the Pant promoter.
How is this complex regulatory network coordinated? Early after infection, expression from Pant results in a burst of Ant synthesis. If Mnt protein is made, it binds to an operator site which overlaps Pant, and turns off ant expression. If the phage infects a lysogen that is producing the Mnt repressor, Mnt represses ant expression on the incoming phage, allowing the superinfecting phage to lysogenize the cell. Thus, the binding of Mnt repressor to Omnt regulates ant expression and thereby controls the decision between lysis and lysogeny.
In addition to Ant, other physiological factors can also affect the lysis/lysogeny decision mediated by c2 repressor. One important variable is the multiplicity of infection (MOI). A high multiplicity of infection (MOI) favors the lysogenic pathway: when the MOI is greater than 10, over 95% of the infecting phage form lysogens. However, if the MOI is too high, the incoming phage may titrate repressor binding to Omnt, resulting in lytic growth of the phage.
Challenge phage. Challenge phage are derivatives of P22 with a mnt::Kan mutation and a substitution of any desired DNA-binding site for Omnt. Thus, binding of a protein to the substituted Omnt site controls the decision between lysis and lysogeny for the challenge phage: if no protein is bound to the site ant will be expressed and the host cells will lyse, but if a protein binds to the site ant expression will be repressed yielding Kmr lysogens. This provides a very tight, direct selection for studying specific DNA-protein interactions. The efficiency of lysogeny is typically between 10-1 to 10-2 if the host produces a protein that can bind to the substituted Omnt site, but the efficiency of lysogeny is less than 10-7 if the protein cannot bind to the substituted Omnt site.
Expression of DNA binding proteins. Most specific DNA binding proteins are expressed at low levels in vivo. To express sufficient levels of a DNA binding protein for use with challenge phage, it is often necessary to express the DNA binding protein under control of a regulated promoter. A useful approach is to clone the structural gene for the DNA binding protein downstream of the tac promoter/operator (Ptac). Ptac is a strong hybrid promoter composed of the -35 region of the trp promoter and the -10 region of the lacUV5 promoter/operator (Amman et al. 1983). Expression of Ptac is repressed by the LacI protein. The lacIq allele is a promoter mutation that expresses the LacI repressor at high levels, resulting in strong repression of Ptac unless the inducer IPTG is added. IPTG inactivates the LacI repressor. Thus, the amount of expression from Ptac is proportional to the concentration of IPTG added: low concentrations of IPTG result in relatively low expression from Ptac and high concentrations of IPTG result in high expression from Ptac. By varying the IPTG concentration the amount of a DNA binding protein expressed from Ptac can be varied over several orders of magnitude.
In vivo DNA-binding assays. When a DNA-binding protein is expressed from Ptac, the relative affinity of a DNA-binding protein for the binding site on the challenge phage in vivo can be quantitated by measuring the frequency of lysogeny in media with different concentrations of IPTG (and hence different concentrations of the DNA-binding protein).
Selection for DNA-binding site mutations. Challenge phage form lysogens on a host due to repression of ant by a DNA-binding protein which prevents RNA polymerase from binding to Pant. It is possible to select for operator constitutive mutations in the DNA-binding site by isolating challenge phage mutants that are not repressed by the DNA-binding protein (and thus form plaques on a lawn of cells that express the DNA-binding protein). This provides a very strong selection for mutations that affect the DNA-protein interaction in vivo
Although mutations in a DNA-binding site are usually rare, the resulting rare plaques can be easily selected from a population of over 108 infecting phage. The frequency of point mutations may be enhanced by mutagenizing the phage. It is useful to use several mutagens with diffent specificities to obtain a wide spectrum of mutations. Using this approach, it is possible to quickly isolate a large number of operator constitutive mutations in the substituted DNA-binding site. The resulting mutations define nucleotides in the DNA-binding site that are necessary for recognition by the DNA-binding protein.
Selection for DNA-binding protein mutants. Mutations that specifically affect the amino acids of DNA-binding protein that are involved in recognition of a DNA-binding site can be obtained in two ways. (1) By selecting for Kan resistant lysogens at a suboptimal concentration of IPTG, it is possible to select for mutants of DNA-binding proteins with increased affinity for DNA. (2) By selecting for Kmr lysogens from operator constitutive challenge phage mutants, it is possible to isolate second site suppressor mutant DNA-binding proteins that recognize the mutant site. Such second site suppressor mutations may either increase the affinity of the DNA-binding protein for each of the DNA-binding sites (extended specificity mutants) or alter the site specificity of the DNA-binding protein (altered specificity mutants).