A. The integrase proteins of phages lambda and HK022 are site specific recombinases that are closely related in structure and function, but recognize different nucleotide sequences in the core regions of their substrates, the attachment sites of the two phages. In order to locate the parts of the proteins that discriminate between the substrates, we selected mutations that allowed lambda Int to recombine HK022 attachment sites. Most of the altered proteins had relaxed specificity; i.e., they retained the ability to recombine the original attachment sites. Several such mutations change amino acids in or near the active site of the recombinase, and several more are identical to or are located near a previously characterized mutation that is known to decrease recombinase specificity and also its requirement for the accessory factors Xis and IHF. We suggest that members of first class increase catalytic activity and members of the second increase the stability of an unstable recombinational intermediate. One mutation altered specificity; i.e., it no longer recombined the original sites. We found that the mutation changes Asn/99 of lambda Int to Asp, the corresponding HK022 residue. We have also constructed and analyzed numerous recombinant proteins that contain sequences from both recombinases. The analysis shows that several different regions of the proteins contain determinants of the HK022/lambda specificity difference. B. E. coli Integration Host Factor (IHM is a site-specific DNA binding and bending protein that is a member of a widespread and highly conserved prokaryotic family, the histone-like proteins. It is a heterodimer of two nonidentical but related subunits. Both subunits are required for IHF activity, and mutations that inactivate either one have numerous phenotypes including defects in phage and bacterial site specific recombination, gene expression, and DNA packaging, and plasmid maintenance. The pleiotropic action of IHF has suggested that it regulates the processes it is involved in, and we have therefore looked for conditions that change its intracellular levels. We have found, contrary to previous indications, that IHF is abundant in exponentially growing cells: the concentration of both subunits is comparable to that of ribosomal proteins (c20,000 copies/cell), and these levels are similar in minimal and broth grown cells. Further analysis shows that there is a 3-5 fold increase in the level of both IHF subunits as the cells enter stationary phase and a corresponding decrease when they are diluted into fresh medium and begin to grow. This change in the amount of the IHF subunits appears not to be due to transcriptional regulation because it is seen even when the IHF genes are transcribed from an exogenous promoter. We therefore suggest that IHF levels fluctuate as a result of changes in translational yield or protein stability. C. Lambdoid phage HK022, like its relatives, contains transciption terminators in its early operons and expresses genes located downstream of these terminators by antiterminating transcription. However, we have been unable to locate any HK022-encoded antitermination function analogous to the N proteins of other lambdoid phages. To see which host proteins, if any, are required for antitermination, we isolated E. coli mutants that are resistant to infection with HK022 and its derivatives, but sensitive to infection with all other phages tested. All of the mutations are located in the rpoC gene, which encodes the Beta subunit of RNA polymerase core enzyme, and one of them was extensively characterized. This mutation strongly reduces the expression of an HK022 early promoter (Pr), and also prevents antitermination of the residual transcript. It has no detectable effect on the analogous promoter from phage lambda nor on any E. coli promoter tested. These results suggest that the core of RNA polymerase plays a role in recognizing the HK022 early promoters, and also has a role in HK022 antitermination. Promoter activation may involve direct interaction of the Beta subunit with the DNA or, alternatively, interaction of the Beta subunit with an unidentified DNA binding protein that itself recognizes the promoters. It is tempting to speculate that the inability of the mutant RNA polymerase to antiterminate is related to its defect in promoter recognition.
