Isolation and analysis of Escherichia coli mutants that allow increased replication of bacteriophage lambda.

Escherichia coli mutants were isolated that supported the growth of a lambda Ots and, in at least one case, a lambda Bts phage at the normally nonpermissive temperature of 39 degrees C. In one such strain, Ots and Bts suppression ability appeared to be a function of the guaB gene. Ots suppression by the mutant guaB strain was prevented if high levels of guanine or xanthine were present in the medium. No other base had any effect on Ots suppression in this strain. Other strains carrying spontaneous mutations resulting in guanine or xanthine auxotrophy (guaA or guaB lesions, respectively) all allowed lambda Ots replication at 39 degrees C; Ots suppression in these strains was also abolished by addition of guanine to the medium. Thus, reduced intracellular guanine levels resulting from guaA or guaB mutations appeared to suppress the inability of lambda Ots and, at least in some cases, Bts bacteriophage to form plaques at 39 degrees C. In burst size experiments, a guaB mutant produced a larger phage yield per infected cell of both lambda Ots and lambda O+ phage at 39 degrees C than did a similar guaB+ strain. It appeared that a lower-than-normal level of guanine (or a guanine derivative) in these cells may permit unusually efficient lambda replication. The fact that O+ and lambda Ots bursts in the guaB mutant were reduced significantly by addition of exogenous guanine to the medium is consistent with this suggestion. Another strain that suppresses the Ots allele has no known auxotrophic requirements, and suppression in this strain was unaffected by addition of guanine to the medium; however, addition of cytidine to the medium specifically eliminated Ots suppression in this strain. The mutation responsible for allowing Ots replication in this strain is unknown.     

Restriction of bacteriophage lambda by Escherichia coli K

Derivatives of phage lambda, for which the numbers and positions of the recognition sites for endonuclease R. Eco_k are known, were used as substrates for the Escherichia coli K restriction system in vivo and in vitro. A single unmodified recognition site was sufficient for a DNA molecule to be bound and broken by the K restriction enzyme. Although discrete fragments of DNA were not produced, the breaks were made preferentially in the proximity of the recognition site. Breakage of a DNA molecule with only one recognition site required a 10 to 40-fold higher concentration of restriction enzyme than breakage of a DNA molecule with two or more recognition sites, but these substrates were all equally effective in a binding assay for the enzyme.The polynucleotide kinase reaction provided no evidence for new 5′-terminal sequences generated by restriction in vitro; the 5′ termini were either refractory to the polynucleotide kinase reaction or had no sequence specificity.     

Requirement for maturation of Escherichia coli bacteriophage lambda

During infection a λ phage that is incapable of DNA replication requires recombination for maturation. If two prophages are situated in tandem, this requirement for DNA replication and recombination is bypassed. In physical experiments using the DNA cutting assay of Freifelder et al. (1973), the DNA of a sex factor containing one or two prophages defective in both excision and DNA replication is cut efficiently only when two prophages are in tandem. We interpret this to mean that λ can only be matured from a structure of greater than unit length, and hypothesize that the structure must contain two joined ends (AR-joints).     

Integration of bacteriophage lambda into the cryptic lambdoid prophages of Escherichia coli.

Bacteriophage lambda missing its chromosomal attachment site will integrate into recA+ Escherichia coli K-12 and C at the sites of cryptic prophages. The specific regions in which these recombination events occur were identified in both lambda and the bacterial chromosomes. A NotI restriction site on the prophage allowed its physical mapping. This allowed us to identify the locations of Rac, Qin, and Qsr' cryptic prophages on the NotI map of E. coli K-12 and, by analogy, to identify the cryptic prophage in E. coli C as Qin. No new cryptic prophages were detected in E. coli K-12.     

An Escherichia coli mutant unable to support site-specific recombination of bacteriophage lambda

We report the isolation of mutations in, and the characterization of, an Escherichia coli gene, hip, that is required for site-specific recombination of phage lambda. hip mutants are recessive and are located near minute 20 on the linkage map. The gene product is not vital to bacterial growth, since deletion mutants are viable. The absence of hip product reduces lambda integration to barely detectable levels and also reduces prophage excision, but less drastically. Certain mutations in the lambda int gene partially restore integration and excision in hip- hosts. Homologous recombination promoted by recA does not require hip function. In addition to their defect in site-specific recombination, hip mutants are unable to support lytic growth of phage Mu or of certain lambda mutants. Their pleiotropic phenotype closely resembles that of himA mutants, but complementation, mapping and DNA sequencing show that hip and himA are different genes.     

Kinetics of bacteriophage lambda deoxyribonucleic acid infection of Escherichia coli

Barnhart, Benjamin J. (Los Alamos Scientific Laboratory, University of California, Los Alamos, N.M.). Kinetics of bacteriophage lambda deoxyribonucleic acid infection of Escherichia coli. J. Bacteriol. 90:1617-1623. 1965.-The kinetics of Escherichia coli K-12 infection by phage lambda deoxyribonucleic acid (DNA) were determined. An initial lag of 55 to 80 sec was found to be the time required for infecting DNA to become deoxyribonuclease-insensitive at 33 C. When cell-DNA interactions were stopped by washing away unbound DNA, the already bound DNA continued to infect the cell at rates described by linear kinetics with no apparent lag. Whereas the lag period was relatively insensitive to DNA and cell concentrations, both the lag and the subsequent linear portions of the rate curves were temperature-sensitive. Cell and DNA dose-response curves prescribed hyperbolic functions. Similarities between lambda DNA infection of E. coli and bacterial transformation systems are discussed.     

Recombination of bacteriophage lambda in recD mutants of Escherichia coli

RecBCD enzyme is centrally important in homologous recombination in Escherichia coli and is the source of ExoV activity. Null alleles of either the recB or the recC genes, which encode the B and C subunits, respectively, manifest no recombination and none of the nuclease functions characteristic of the holoenzyme. Loss of the D subunit, by a recD mutation, likewise results in loss of ExoV activity. However, mutants lacking the D subunit are competent for homologous recombination. We report that the distribution of exchanges along the chromosome of Red-Gam-phage lambda is strikingly altered by recD null mutations in the host. When lambda DNA replication is blocked, recombination in recD mutant strains is high near lambda's right end. In contrast, recombination in isogenic recD+ strains is approximately uniform along lambda unless the lambda chromosome contains a chi sequence. Recombination in recD mutant strains is focused toward the site of action of a type II restriction enzyme acting in vivo on lambda. The distribution of exchanges in isogenic recD+ strains is scarcely altered by the restriction enzyme (unless the phage contains an otherwise silent chi). The distribution of exchanges in recD mutants is strongly affected by lambda DNA replication. The distribution of exchanges on lambda growing in rec+ cells is not influenced by DNA replication. The exchange distribution along lambda in recD mutant cells is independent of chi in a variety of conditions. Recombination in rec+ cells is chi influenced. Recombination in recD mutants depends on recC function, occurs in strains deleted for rac prophage, and is independent of recJ, which is known to be required for lambda recombination via the RecF pathway. We entertain two models for recombination in recD mutants: (i) recombination in recD mutants may proceed via double-chain break--repair, as it does in lambda's Red pathway and E. coli's RecE pathway; (ii) the RecBC enzyme, missing its D subunit, is equivalent to the wild-type, RecBCD, enzyme after that enzyme has been activated by a chi sequence.     

Explanations accounting for transduction by bacteriophage lambda in maltose negative bacteriophage lambda resistant mutants of Escherichia coli K-12.

Summary Entry of DNA from phages particles into rMal^- mutants of Escherichia coli K-12 is shown to be due to two distinguishable processes. One, residual transduction, results from a low level expression of lamB. The other one, background transduction, is independent of gene lamB.Interpretations are presented for these results. It is proposed that residual transduction is due to a weak promoter pB3 located within or near the distal part of the gene preceeding lamB in the same operon. It is proposed that background transduction is due to a secondary receptor structure for phage . Finally a tentative hypothesis relating pB3 to insertion sequences is presented.     

Isolation of a lambda transducing bacteriophage carrying the relA gene of Escherichia coli.

In Escherichia coli the relA and pyrG loci are 99% cotransducible. On the basis of this knowledge, we have isolated lambdacI857S7dpyrG transducing bacteriophages carrying both the pyrG and relA genes. Single lysogens of this bacteriophage show basal levels of ppGpp that are 10-fold higher than normal. Stringent factor is present among the gene products synthesized by lambdadpyrG relA after infection of ultraviolet-killed cells, as analyzed by polyacrylamide gel electrophoresis. The intracellular content of stringent factor, as determined by enzymatic activity, rises 20-fold after induction of a single lysogen of lambdadpyrG relA. As measured by two-dimensional gel electrophoresis, the amount of stringent factor in an exponentially growing strain carrying a pyrG relA plasmid is at least 10-fold greater than in a normal strain. These data constitute strong evidence that stringent factor is the relA gene product.     

Secondary attachment site for bacteriophage lambda in the guaB gene of Escherichia coli.

lambda gua transducing bacteriophages were used to identify and sequence the secondary attachment site for lambda in the guaB gene of Escherichia coli. The sequence matched the primary core sequence at nine positions, and a putative integrase binding-site overlapped the left core-arm junction. Recombinational crossover occurred between nucleotides -3 and +2 of the core region.     

Effects of high incubation temperature upon the cell wall of Escherichia coli

Hoffman, Heiner (New York University, New York, N.Y.), Jon Valdina, and Michael E. Frank. Effects of high incubation temperature upon the cell wall of Escherichia coli. J. Bacteriol. 91:1635-1637. 1966.-Escherichia coli cells grown at 45 C were found to swell when suspended in distilled water, and were broken down when subjected to egg white lysozyme. Cells grown at 37 C were unaffected by these reagents. It appears, therefore, that cultivation at the upper limits of the temperature growth range leads to cell wall damage. It is suggested that this reaction may account for the favorable therapeutic effects of artificially elevated body temperature in certain infectious diseases.     

Oligomerization of the bacteriophage lambda S protein in the inner membrane of Escherichia coli.

Western blot (immunoblot) analysis of cell extracts from induced bacteriophage lambda lysogens probed with S-protein-specific antibody (raised against an S--beta-galactosidase fusion protein) demonstrated that the bacteriophage lambda S protein begins to appear 10 min after phage induction and is localized to the inner membrane at all times during the lytic cycle. Between 100 and 1,000 molecules of S protein per cell were present at the time of phage-induced lysis. Western blots of chemically cross-linked membranes from induced lysogens showed a ladder of bands at 18, 24, 32, and 42 kilodaltons (the S-protein monomer ran at 8 kilodaltons) that reacted with anti-S-protein antibody. Thus, the S protein appears to reside in the inner membrane as a multimer, and the molecular weights of the cross-linked species are consistent with those of S-protein homopolymers. Sodium dodecyl sulfate-resistant dimers were also detected when S protein was purified by immunoprecipitation.     

Kinetics of induction of error-prone repair of bacteriophage lambda by temperature shift in an Escherichia coli dnaB mutant.

Summary Preincubation at 42^o, before infection at permissive temperature by phage , of an Escherichia coli dnaB mutant, provokes a significant increase in survival and mutagenesis of ultraviolet irradiated phage as well as mutagenesis of untreated phage. Similarly to UV irradiation and many chemical mutagens, the inhibition of DNA synthesis by temperature shift of this dnaB mutant induces SOS repair. This work shows that replication blockage in bacterial DNA is not only mutagenic for bacterial DNA itself (Witkin, 1975) but also for normally replicating DNA, probably due to induction of diffusible products.     

Stimulation of groE synthesis in Escherichia coli by bacteriophage lambda infection

We found that infection of Escherichia cell by lambda results in at least a twofold stimulation in the rate of synthesis of one of the products of groE. To determine what lambda-coded factors were responsible for this stimulation, numerous phage lambda mutants carrying bio substitutions were analyzed for their ability to stimulate groE synthesis. Our results revealed that the main factor(s) which is responsible for stimulating groE synthesis is located between the endpoints of the lambda bio69 and lambda bio252 substitutions, a region of DNA coding for bet, gam, kil, and cIII.     

Maltose transport in Escherichia coli K-12: involvement of the bacteriophage lambda receptor.

Mutants affected in lamB, the structural gene for phage lambda receptor, are unable to utilize maltose when it is present at low concentrations (less than or equal 10 muM). During growth in a chemostat at limiting maltose concentrations, the lamB mutants tested were selected against in the presence of the wild-type strain. Transport studies demonstrate that most lamB mutants have deficient maltose transport capacities at low maltose concentrations. When antibodies against purified phage lambda receptor are added to a wild-type strain, transport of maltose at low concentrations is significantly reduced. These results strongly suggest that the phage lambda receptor molecule is involved in maltose transport.