Coliphage lambda gene expression is regulated temporally by systems of termination and antitermination of transcription. The lambda-encoded N protein (pN) acting with host factors (Nus) at sites (nut) located downstream from early promoters is the first of these systems to operate during phage development. We report observations on some of the components of this complex system that, in part, address the way in which these elements interact to render RNA polymerase termination-resistant. (1) The isolation of a conditionally lethal cold-sensitive nusA mutation demonstrates that NusA is essential for bacterial growth. (2) The effect on lambda growth in a host in which the Salmonella NusA protein is overproduced suggests that NusA is essential for N-mediated antitermination in phage lambda. (3) A truncated NusA product, representing only the amino two-thirds of the native protein, is active for both bacterial growth and pN action, indicating that the carboxy end of the molecule may not be a functionally important region. (4) lambda pN can function with the heterologous nut region from Salmonella typhimurium phage P22 when lambda pN is overproduced, demonstrating that lambda pN can function with the nut regions of other lambdoid phages. (5) A single base-pair change in the lambda nutR boxA sequence that was selected to permit a lambda derivative to utilize the Salmonella NusA protein restores lambda growth in the Escherichia coli nusA1 host.

We report the isolation of two mutations in the gyrB gene of Escherichia coli K12 obtained from an initial selection for resistance to coumermycin Al and a subsequent screening for bacteria that fail to support sitespecific recombination of phage A, i.e., Him-. These two mutations have a temperature-sensitive Himphenotype, supporting site-specific recombination efficiently at low temperature, but inefficiently at high temperatures. Like other Him mutants, the gyrB-him mutants fail to plate phage Mu; again this defect is observed only at high temperatures. Additional thermally sensitive characteristics have also been observed; growth of k as well as maintenance of the plasmids pBR322 and F' gal are reduced at high temperature. Restriction of foreign DNA imposed by a P1 prophage is also reduced in these mutants. The temperaturesensitive phenotypic characteristics imposed by both the gyrB-him-230(Ts) and gyrB-him-231(Ts) mutations correlate with in vitro studies that show decreased gyrase activity, especially at higher temperatures, and in vivo studies showing reduced supercoiling of A DNA in the mutants at high temperature. DNA gyrase is a type II topoisomerase that puts negative superhelical turns into DNA (reviewed in reference 13). The best-characterized gyrase, that of Escherichia coli, is composed of two subunits, A and B, encoded respectively by the gyrA (min 48) and gyrB (min 82) genes. Mutations that confer resistance to nalidixic acid and oxolinic acid map in the gyrA gene (15, 51), whereas mutations that confer resistance to

We have constructed Escherichia coli strains containing mutations at two different loci, both originally selected for failure to support X site-specific recombination: himA and gyrB-him(Ts). Although the gyrBhim(Ts) mutations by themselves reduce supercoiling at high temperature, the double mutants show a far greater effect on supercoiling.. Our studies show that growth of phage A is severely inhibited and that maintenance of plasmid pBR322 is extremely unstable in the double mutants. Physiological studies also reveal that the double mutants are isoleucine auxotrophs at 42°C. The fact that himA mutants are isoleucine auxotrophs at 42°C in the presence of leucine suggests that a significant component of the isoleucine auxotrophy of the double mutants is a result of the himA mutation. The himA gene encodes the ao subunit of a protein called the integration host factor. Since mutations in the hip or himD gene encoding ,B, the other subunit of the integration host factor, also result in isoleucine auxotrophy in the presence of leucine, we suggest that the integration host factor regulates the synthesis of at least one of the enzymes in the ilv pathway, acetohydroxyacid synthase I, which is encoded by the ilvB gene. Studies of the utilization of various sugars as the sole carbon source suggest that the integration host factor controls expression of some gene(s) involved in the utilization of xylose.An essential step in lysogeny by coliphage X is the integration of the phage genome into the bacterial chromosome. Integration occurs by a site-specific recombination reaction between a unique site on the phage genome (attP) and a unique site on the bacterial chromosome (attB) (reviewed in references 22 and 27a). This site-specific recombination reaction can proceed in the absence of both the phage Red and host Rec systems for generalized recombination.

Derivatives of phage lambda with the rightmost 3% of the genome (the QSR region) from the related phage phi 80 fail to grow at low temperatures (e.g., 32 degrees) in Escherichia coli hosts deficient in either protein component of IHF (integration host factor), the products of the himA and hip/himD genes. The abortive infection of lambda (QSR)80 in mutants defective for IHF was studied in detail. This infection is characterized by a lack of cell lysis and an inhibition of phage DNA replication after an initial period of normal synthesis. An inhibition of host DNA replication also occurs after a similar period of apparently normal synthesis, and the abortive lambda (QSR)80 infection is lethal to the host. An assay of beta-galactosidase activity in lambda (QSR)80-infected cells provided indirect evidence that RNA and protein synthesis continue late into the abortive infection. The defective growth is imposed by the product of the rha gene located in the (QSR)80 genetic material. Two-dimensional electrophoretic analysis of phage proteins produced in ultraviolet (uv)-irradiated phage-infected host cells has demonstrated the existence of a protein that is encoded or whose synthesis is regulated by the rha locus. Based on these findings, possible roles for a HimA-Hip/HimD-controlled rha product in a late stage of phi 80 development are discussed.