Prophage lambda at unusual chromosomal locations. II. Mutations induced by bacteriophage lambda in Escherichia coli K12

An Escherichia coli strain deleted for the primary λ attachment site was lysogenized with λ at secondary sites. Some lysogens became mutants because of prophage insertion in the affected gene. Mutagenesis by phage λ is not random with respect to the gene affected: most mutants were pro, although certain other genes could be mutated at lower frequencies. In the case of several independent ilv and gal mutants, the sites of prophage insertion were in the same segment of the ilv region and galT gene respectively. The galT location may also be a preferred site for the insertion of DNAs other than prophage λ. Insertion of prophage λ within an operon can reduce the expression of operator-distal genes. A trpC λ insertion mutant expresses the operator-distal trpB function constitutively at a low level. This expression probably derives from a promoter located in the left arm of the prophage.     

Isolation of Plaque-Forming, Galactose-Transducing Strains of Phage Lambda

Plaque-forming, galactose-transducing lambda strains have been isolated from lysogens in which bacterial genes have been removed from between the galactose operon and the prophage by deletion mutation.—A second class has been isolated starting with a lysogenic strain which carries a deletion of the genes to the right of the galactose operon and part of the prophage. This strain was lysogenized with a second lambda phage to yield a lysogen from which galactose-transducing, plaque-forming phages were obtained. These plaque-forming phages were found to be genetically unstable, due to a duplication of part of the lambda chromosome. The genetic instability of these partial diploid strains is due to homologous genetic recombindation between the two identical copies of the phage DNA comprising the duplication. The galactose operon and the duplication of phage DNA carried by these strains is located between the phage lambda P and Q genes.     

Gross map location of Escherichia coli transfer RNA genes.

Chromosomal locations of Escherichia coli genes specifying more than 20 different transfer RNA species were determined by utilizing two different methods. One was based upon gene dosage effects caused by F′ factors. In 15 different F′ strains and their corresponding F^? strains, relative contents of individual tRNAs were measured after separating the tRNAs by two-dimensional polyacrylamide gel electrophoresis. Approximate doubling of the content of particular tRNA was found in individual F′ strains, as showing gross map location of the tRNA gene. The other method was based on the amplified synthesis of tRNAs occurring after prophage induction of λ lysogens. Synthesis of individual tRNAs was measured after the induction of λ phages integrated at five different bacterial sites. Characteristic overproduction of different tRNAs was observed in individual prophage strains. This finding also gave approximate map locations of tRNA genes close to the prophage sites. The mapping data obtained by the two methods were consistent with each other and also with the reported positions in the cases where previously mapped. On the basis of map location of the tRNA_f1^Met gene newly determined, the λ-transducing phage carrying the tRNA_f1^Met gene was found.     

Regulation of synthesis of ribosomal protein S20 in vitro

Summary In addition to the cryptic lambdoid prophage genes that are known to reside at the rac locus in Escherichia coli K12 strains, a second cryptic lambdoid prophage has been located near the gal operon. This prophage was shown to contain DNA that is homologous to the QSR genes of phage.     

Physical study of prophage excision and curing of lambda prophage from lysogenic Escherichia coli

A sex factor, F′450(λ), which can be isolated as a covalent circle of DNA, has been examined by alkaline sucrose gradient centrifugation of lysates of induced cells in order to study λ prophage excision. Thermal derepression of the prophage results in loss of F′450(λ) covalent circles, which is mediated by systems involved in excision and initiation of replication. When protocols known to result in prophage curing are used, the F′450(λ) is converted to an F′450 and a λ covalent circle; in normal excision leading to phage development, F′450 covalent circles are not found. We have shown that: (1) excision usually occurs later than initiation of DNA replication of the prophage so that the excised prophage is usually already replicated or in the act of replication; (2) the DNA growing points of the prophage leave the prophage and enter the bacterial DNA; (3) the int and xis genes are involved in the earliest detectable stage of the excision process, i.e. breakage of the DNA at the attachment region; (4) the xis gene product is involved in a weak non-specific nuclease activity in addition to its highly specific activity in excision; and (5) the excision system fails to attack a single attachment site.     

Genetic analysis of bacteriophage lambda strains which transduce the genes for leucine biosynthesis

Summary By means of two specific genetic tests as well as additional transduction studies, the genetic compositions of four different leucine-transducing lambda-phages and the two E. coli lysogens from which these phages originated were analyzed. Three of the phages, No. 267, 517 and 889, are of the bio-type, e.g. carry bacterial genes adjacent to prophage attachment element P. The former two contain a large portion of the E. coli leucine operon (genes leu B through leu D, see Fig. 2), the latter carries only gene leu B and part of leu C. Phage No. 518 is of the gal-type and carries at least part of the leu A gene. The two lysogens, No. 73 and 75, from which these phages arose, contain the prophage between two mutation sites in gene leu A and leu B, in an orientation that is opposite to the normal one for lambda.     

Characterization of the primary immunity region of the Escherichia coli linear plasmid prophage N15.

N15 is the only bacteriophage of Escherichia coli known to lysogenize as a linear plasmid. Clear-plaque mutations lie in at least two regions of the 46-kb genome. We have cloned, sequenced, and characterized the primary immunity region, immB. This region contains a gene, cB, whose product shows homology to lambdoid phage repressors. The cB3 mutation confers thermoinducibility on N15 lysogens, consistent with CB being the primary repressor of N15. Downstream of cB lies the locus of N15 plasmid replication. Upstream of cB lies an operon predicted to encode two products: one homologous to the late repressor of P22 (Cro), the other homologous to the late antiterminator of phi 82 (Q). The Q-like protein is essential for phage development. We show that CB protein regulates the expression of genes that flank the cB gene by binding to DNA at symmetric 16-bp sites. Three sites are clustered upstream of cB and overlap a predicted promoter of the cro and Q-like genes as well as two predicted promoters of cB itself. Two sites downstream of cB overlap a predicted promoter of a plasmid replication gene, repA, consistent with the higher copy number of the mutant, N15cB3. The leader region of repA contains terminators in both orientations and a putative promoter. The organization of these regulatory elements suggests that N15 plasmid replication is controlled not only by CB but also by an antisense RNA and by a balance between termination and antitermination.     

Xis and Fis proteins prevent site-specific DNA inversion in lysogens of phage HK022.

HK022, a temperate coliphage related to lambda, forms lysogens by inserting its DNA into the bacterial chromosome through site-specific recombination. The Escherichia coli Fis and phage Xis proteins promote excision of HK022 DNA from the bacterial chromosome. These two proteins also act during lysogenization to prevent a prophage rearrangement: lysogens formed in the absence of either Fis or Xis frequently carried a prophage that had suffered a site-specific internal DNA inversion. The inversion is a product of recombination between the phage attachment site and a secondary attachment site located within the HK022 left operon. In the absence of both Fis and Xis, the majority of lysogens carried a prophage with an inversion. Inversion occurs during lysogenization at about the same time as prophage insertion but is rare during lytic phage growth. Phages carrying the inverted segment are viable but have a defect in lysogenization, and we therefore suggest that prevention of this rearrangement is an important biological role of Xis and Fis for HK022. Although Fis and Xis are known to promote excision of lambda prophage, they had no detectable effect on lambda recombination at secondary attachment sites. HK022 cIts lysogens that were blocked in excisive recombination because of mutation in fis or xis typically produced high yields of phage after thermal induction, regardless of whether they carried an inverted prophage. The usual requirement for prophage excision was bypassed in these lysogens because they carried two or more prophages inserted in tandem at the bacterial attachment site; in such lysogens, viable phage particles can be formed by in situ packaging of unexcised chromosomes.     

Structural features of lambda site-specific recombination at a secondary att site in galT.

Integration of bacteriophage λ DNA into the chromosome of its E. coli host proceeds via a site-specific recombination between specific loci (att sites) on the phage and bacterial chromosomes. Infection of an E. coli host deleted for the primary bacterial att site results in λ integration with reduced efficiency at a number of different “secondary att sites” scattered around the E. coli chromosome. The first DNA sequence analysis of such a secondary att site, that occurring in the galT gene, is reported here, and several features pertinent to the mechanism of int-dependent site-specific recombination are discussed.Previous studies have shown that the crossover in int-dependent recombination must be somewhere within a 15 bp sequence (core region) common to the phage and primary bacterial att sites, as well as to the left and right prophage att sites which are at the junctures between prophage and host DNA. Comparison of the galT secondary prophage att sites with the primary prophage att sites allows determination of the analogous “core” region in the galT secondary att site. The 15 bp sequence thus identified shows an interrupted homology (8 out of 15) with the wild-type core. The extent and arrangement of nonhomologous bases allow precise placement of the crossover point for this recombination to the +4–+5 internucleotide bond of the core region.Sequences flanking the core region show no obvious homology with analogous sequences of the phage or primary bacterial att sites. Comparison of the galT left prophage att site with the analogous wild-type site is of particular interest and is discussed in relation to binding studies with purified int protein.     

A new gene of bacteriophage P22 which regulates synthesis of antirepressor

Two new mutants of bacteriophage P22 are described which define a new regulatory gene, arc (for antirepressor control). The properties of the arc mutants and of 31 phenotypic revertants indicate that the arc gene codes for a trans-acting protein whose primary role is to depress synthesis of P22 antirepressor protein during the lytic cycle of infection. Failure to regulate antirepressor production apparently leads secondarily to a lethal defect (i.e. failure to produce progeny phage).Although under certain conditions the arc function can be expressed by P22 prophages and can act as a weak barrier to superinfecting homologous phage, the arc product is neither necessary nor sufficient for maintenance of the prophage state or superinfection immunity in lysogens. Instead, as shown previously by others (Levine et al., 1975; Botstein et al., 1975), the prophage mnt gene product is responsible for repressing antirepressor synthesis, both by the prophage and by superinfecting phage.