Occurrence of the bacteriophage lambda receptor in some enterobacteriaceae.

In Escherichia coli K-12, the receptor for phage lambda is an outer membrane protein which inactivates the phage in vitro. Lambda receptor activity was found in extracts from all wild strains of E. coli tested, although most of them fail to support growth of the phage. In some cases this failure is due to a masking of the receptor in vivo, the bacteria being unable to adsorb the phage or to react with antireceptor antibodies. In other cases, adsorption does occur, and the nature of the block in phage growth was not investigated. Most Mal+ strains of Shigella have lambda receptor, whereas most Mal- strains do not have it. Synthesis of the lambda receptor in Shigella is thus presumably controlled by the positive regulator gene of the maltose regulon as is the case in E. coli K-12. Phage lambda adsorbs on many Mal+ strains of Shigella and even yields plaques on some of them, although at a low frequency. No lambda receptor activity could be found in extracts of several strains of Salmonella and Levinea.     

Genetic study of the effect of lambda phage on Mu phage production in Escherichia coli K-12 strains with Mu--lambda--Mu structures

The interaction of temperate bacteriophages Mu and lambda is studied during their simultaneous induction in specially constructed heterolysogenic strains of Escherichia coli bearing trimeric Mu--lambda--Mu structures. These strains were obtained by the MU-mediated integration of phage lambda circular genomes. Heterolysogenic strains of E. coli were used for studying phage lambda eliminating effect on Mu development with a simultaneous induction of prophages in the same cell. The results of the study allow the localization of the region of phage lambda genome incorporating gene (genes) lambda, which produces an eliminating effect on Mu development.     

Adsorptive capacity of bacteriophage Mu on Escherichia coli K-12 strains with deletions in the attlambda region]

Escherichia coli strains with deletions in att lambda region were obtained. The comparison of the extent of deletions with the sensitivity of the corresponding mutant clones to phage Mu showed that the gene controlling the sensitivity of E. coli K-12 to the phage Mu is located in nad A-gal region of the bacterial chromosome. It is shown that the resistance of E. coli strains which had lost the region of bacterial chromosome between nad A gene and genes of gal-operon have adsorption character. Deletion of the nad A-gal region does not affect the adsorption of other phages (lambda, P1 and T4). Thus, the gene, located in this region, is responsible for the specific adsorption of the phage Mu.     

Construction of lambda gt103, a derivative of phage lambda gt10 that has unique EcoRI, NotI, SacI and SpeI sites and retains positive selection for recombinants.

A phage vector, lambda gt103, that has unique EcoRI, NotI, SacI and SpeI sites within the imm434 cI repressor gene, was constructed by PCR-aided site-directed mutagenesis of lambda gt10 [Huynh et al., DNA Cloning Techniques: A Practical Approach, 1985, pp. 49-78]. This vector allows directional cloning and retains positive selection for recombinants on Escherichia coli C600hfl strains (since only phages with disrupted cI genes plate on this host). Libraries made with this phage vector can be efficiently screened for clones in which a part of the insert is homologous to probe DNAs derived from a plasmid-based library, without cross-hybridization.     

Evidence that the outer membrane protein gene nmpC of Escherichia coli K-12 lies within the defective qsr'' prophage.

Recombinants between phage lambda and the defective qsr' prophage of Escherichia coli K-12 were made in an nmpC (p+) mutant strain and in the nmpC+ parent. The outer membrane of strains lysogenic for recombinant qsr' phage derived from the nmpC (p+) strain contained a new protein identical in electrophoretic mobility to the NmpC porin and to the Lc porin encoded by phage PA-2. Lysogens of qsr' recombinants from the nmpC+ strain and lysogens of lambda p4, which carries the qsr' region, did not produce this protein. When observed by electron microscopy, the DNA acquired from the qsr' prophage showed homology with the region of the DNA molecule of phage PA-2 which contains the lc gene. Relative to that of the recombinant from the nmpC (p+) mutant, the DNA molecule of the recombinant from the nmpC+ parent contained an insertion near the lc gene. These results were supported by blot hybridization analysis of the E. coli chromosome with probes derived from the lc gene of phage PA-2. A sequence homologous to the lc gene was found at the nmpC locus, and the parental strains contained an insertion, tentatively identified as IS5B, located near the 3' end of the porin coding sequence. We conclude that the structural gene for the NmpC porin protein is located within the defective qsr' prophage at 12.5 min on the E. coli K-12 map and that this gene can be activated by loss of an insertion element.     

Mutant of Escherichia coli that instantaneously loses the ability to adsorb lambda bacteriophage upon exposure to high temperature.

lad (lambda adsorption), an Escherichia coli mutant that loses the ability to adsorb lambda phage immediately after a shift to high temperature (e.g., 42 C), was isolated. This property for phage adsorption is irreversible and has been observed with phage lambda and 21 but not with phages 434, phi 170, and phi 80. A crude receptor preparation, extracted from lad cells will cholate-ethylenediaminetetraacetic acid by the procedure of Randall-Hazelbauer and Schwartz (1973), inactivated the phage lambda only at low temperature.     

Regulation of glutamine synthetase formation in Escherichia coli: characterization of mutants lacking the uridylyltransferase.

A lambda phage (lambdaNK55) carrying the translocatable element Tn10, conferring tetracycline resistance (Tetr), has been utilized to isolate glutamine auxotrophs of Escherichia coli K-12. Such strains lack uridylyltransferase as a result of an insertion of the TN10 element in the glnD gene. The glnD::Tn10 insertion has been mapped at min 4 on the E. coli chromosome and 98% contransducible by phage P1 with dapD. A lambda transducing phage carrying the glnD gene has been identified. A glnD::Tn10 strain synthesizes highly adenylylated glutamine synthetase under all conditions of growth and fails to accumulate high levels of glutamine synthetase in response to nitrogen limitation. However, this strain, under nitrogen-limiting conditions, allows synthesis of 10 to 20 milliunits of biosynthetically active glutamine synthetase per mg of protein, which is sufficient to allow slow growth in the absence of glutamine. The GlnD phenotype in E. coli can be suppressed by the presence of mutations which increase the quantity of biosynthetically active glutamine synthetase.     

Plasmid-Controlled Variation in the Content of Methylated Bases in Bacteriophage Lambda Deoxyribonucleic Acid

The N(6)-methyladenine (MeAde) and 5-methylcytosine (MeC) contents in deoxyribonucleic acid (DNA) of bacteriophage lambda has been analyzed as a function of host specificity. The following facts have emerged: (i) lambda grown on strains harboring the P1 prophage contain ca. 70 more MeAde residues/DNA molecule than lambda grown either in the P1-sensitive parent, or in a P1 immune-defective lysogen which does not confer P1 modification; (ii) lambda grown on strains harboring the N-3 drug-resistance factor contain ca. 60 more MeC residues/DNA molecule than lambda grown on the parental strain lacking the factor; (iii) lambda grown in Escherichia coli B strains is devoid of MeC, whereas lambda grown in a B (N-3) host contains a high level of MeC; (iv) the MeAde content in lambda DNA is not affected by the N-3 factor. These results suggest that P1 controls an adenine-specific DNA methylase, and that the N-3 plasmid controls a cytosine-specific DNA methylase. The N-3 factor has been observed previously to direct cytosine-specific methylation of phage P22 DNA and E. coli B DNA in vivo; in vitro studies presented here demonstrate this activity.     

Negative dominance in gene lamB: random assembly of secreted subunits issued from different polysomes

lamB is the structural gene for the lambda receptor, an oligomeric outer membrane protein from Escherichia coli K12 involved in phage lambda adsorption. We show that, under certain conditions, in a strain diploid for gene lamB, all the missense lamB mutations conferring lambda resistance that we have tested are dominant with respect to wild-type. We propose a model which allows a quantitative interpretation of the data. It is based on negative complementation at the level of oligomerisation. Wild-type and mutant subunits would assemble at random forming homo- and hetero-oligomers. Only wild-type homo-oligomers would be efficient for phage inactivation. For some classes of missense mutations the hetero-oligomers would have the capacity to bind, but not to inactivate the phage. The model confirms that active lambda receptor is a trimer and implies that for this secreted protein there is no preferential assembly of subunits originating from the same polysome.     

Lambda phage shuttle vectors for analysis of mutations in mammalian cells in culture and in transgenic mice

Foreign DNA sequences contained in lambda bacteriophage genomes integrated in mammalian DNA can be efficiently rescued into infectious phage particles by treatment of the mammalian DNA with lambda-packaging extracts prepared in E. coli. This system provides for rapid, non-selective recovery of stably integrated, chromosomal sequences into lambda phage for subsequent analysis in bacterial systems. Since rescue is prior to selection, mutations can be recovered from intact animals made transgenic for the phage-target gene sequences. Such approaches allow study of physiologically relevant aspects of mammalian mutagenesis at the molecular level.     

Host/vector interactions which affect the viability of recombinant phage lambda clones

A class of recombinant phage lambda clones are recovered from human genomic libraries on Escherichia coli recB21 recC22 sbcB15 cells, which fail to form plaques on wild-type cells. We report experiments which address the mechanism of this inhibition. The introduction of the recombination-stimulating sequence chi into one such clone allows growth of this phage on Rec+ cells. In addition, the insertion of lambda gam+ gene into a rec+-inhibited clone results in the ability of the phage to form plaques on wild-type cells. Since lambda Gam protein is an inhibitor of host RecBC enzyme, we tested a collection of such phage for growth on a variety of hosts altered in RecBC function. Host permissiveness correlated with the inactivation of the RecBC nucleolytic activities and not with the recombinational activities. These observations suggest that the inserted DNA sequences of these phage limit the production of packageable chromosomes. This conclusion is easily reconciled with our current knowledge of the interaction of the host recombination systems with lambda replication and encapsidation. Based on these experiments we have constructed strains, both recombination-proficient and recombination-deficient, which serve as improved hosts for the recovery of genomic sequences which are otherwise inhibitory to the growth of phage lambda.     

Development of a short-term, in vivo mutagenesis assay: the effects of methylation on the recovery of a lambda phage shuttle vector from transgenic mice.

Transgenic mice suitable for the in vivo assay of suspected mutagens at the chromosome level have been constructed by stable integration of a lambda phage shuttle vector. The shuttle vector, which contains a beta-galactosidase (beta-gal) target gene, can be rescued from genomic DNA with in vitro packaging extracts. Mutations in the target gene are detected by a change in lambda phage plaque color on indicator agar plates. Initial rescue efficiencies of less than 1 plaque forming unit (pfu)/100 micrograms of genomic DNA were too low for mutation analysis. We determined the cause of the low rescue efficiencies by examining primary fibroblast cultures prepared from fetuses of lambda transgenic animals. The rescue efficiency of 5-azacytidine-treated cells increased 50-fold over non-treated controls indicating that methylation was inhibiting rescue. The inhibitory role of methylation was supported by the observation that mcr deficient E. coli plating strains and mcr deficient lambda packaging extracts further improved lambda rescue efficiency. Present rescue efficiencies of greater than 2000 pfu/copy/micrograms of genomic DNA represent a 100,000-fold improvement over initial rescue efficiencies, permitting quantitative mutational analysis. The background mutagenesis rate was estimated at 1 x 10(-5) in two separate lineages. Following treatment with the mutagen N-ethyl-N-nitrosourea (EtNU), a dose dependent increase in the mutation rate was observed in DNA isolated from mouse spleen, with significant induction also observed in mouse testes DNA.     

Genetic and physiological studies of an Escherichia coli locus that restricts polynucleotide kinase- and RNA ligase-deficient mutants of bacteriophage T4.

The RNA ligase and polynucleotide kinase of bacteriophage T4 are nonessential enzymes in most laboratory Escherichia coli strains. However, T4 mutants which do not induce the enzymes are severely restricted in E. coli CTr5X, a strain derived from a clinical E. coli isolate. We have mapped the restricting locus in E. coli CTr5X and have transduced it into other E. coli strains. The restrictive locus seems to be a gene, or genes, unique to CTr5X or to be an altered form of a nonessential gene, since deleting the locus seems to cause loss of the phenotypes. In addition to restricting RNA ligase- and polynucleotide kinase-deficient T4, the locus also restricts bacteriophages lambda and T4 with cytosine DNA. When lambda or T4 with cytosine DNA infect strains with the prr locus, the phage DNA is injected, but phage genes are not expressed and the host cells survive. These phenotypes are unlike anything yet described for a phage-host interaction.     

Integration of specialized transducing bacteriophage lambda cI857 St68 h80 dgnd his by an unusual pathway promotes formation of deletions and generates a new translocatable element.

Molecular and genetic studies have revealed that several illegitimate recombinational events are associated with integration of the specialized transducing bacteriophage lambda cI57 St68 h80 dgnd his into either the Escherichia coli chromosome or into a plasmid. Most Gnd+ His+ transductants did not carry the prophage at att phi-80, and 10% were not immune to lambda, i.e., "nonlysogenic." Integration of the phage was independent of the phage Int and Red gene products and of the host's general recombination (Rec) system. In further studies, bacterial strains were selected which carried the phage integrated into an R-factor, pSC50. Restriction endonuclease analysis of plasmid deoxyribonucleic acid (DNA) purified from these strains showed that formation of the hybrid plasmids resulted from recombination between a single region of pSC50 and one of several sites within the lambda-phi 80 portion of the phage. Furthermore the his-gnd region of the phage, present in the chromosome of one nonlysogenic transductant, was shown to be able to translocate to pSC50. Concomitant deletion of phage DNA sequences or pSC50 DNA was frequently observed in conjunction with these integration or translocation events. In supplemental studies, a 22- to 24-megadalton segment of the his-gnd region of the chromosome of a prototrophic recA E. coli strain was shown to translocate to pSC50. One terminus of this translocatable segment was near gnd and was the same as a terminus of the his-gnd segment of the phage which translocated from the chromosome of the nonlysogenic transductant. These data suggest that integration of lambda cI857 St 68 h80 dgnd his may be directed by a recombinationally active sequence on another replicon and that the resulting cointegrate structure is subject to the formation of deletions which extend from the recombinationally active sequence. Translocation of the his-gnd portion of the phage probably requires prior replicon fusion, whereas the his-gnd region of the normal E. coli chromosome may comprise a discrete, transposable element.     

Construction from Mu d1 (lac Apr) lysogens of lambda bacteriophage bearing promoter-lac fusions: isolation of lambda ppheA-lac.

Bacteriophage Mu d1 (lac Aprr) was used to obtain strains of Escherichia coli K-12 in which the lac genes are expressed from the promoter of pheA, the structural gene for the enzyme chorismate mutase P-prephenate-dehydratase. A derivative of bacteriophage lambda which carries the pheA-lac fusion was prepared; the method used is generally applicable for the construction, from Mu dl lysogens, of specialized transducing lambda phage carrying the promoter-lac fusions. A restriction enzyme cleavage map of lambda ppheA-lac for the enzymes HindIII and PstI is presented.     

Luria effect in bacteriophages and the role of the products of recA+ and lexA+ genes in its induction

An analysis of UV-damages accumulation in the phages as revealed by delay of intracellular growth is represented using temperate lambda phage. The maximum of growth delay of phage lambda at given UV-dose was found with lambda red+, infecting Escherichia coli AB1886 uvrA strain. The growth delay was absent, when a strain RH-1 uvrA-recA- was infected with UV-irradiated phage lambda red3. A moderate growth delay was obtained with the phages lambda red+, infecting E. coli RH-1 uvrA-recA- or phage lambda red3, infecting E. coli AB1886 uvrA-. THe growth delay was also absent when wild type, recA- and uvrA mutants of E. coli were infected with phage lambda after 8-metnoxypsoralen + light (lambda > 310 nm) treatment. It is known that the crosslinks appear to be the DNA defects which give rise to the observed biological inactivation following psoralen + light treatment. However, a considerable growth delay of phage lambda, treated by 8-metnoxypsoralen + light, was only found under condition of crosslinks repair (W-reactivation and prophage-reactivation). The results obtained are best explained by the assumption that the growth delay reflects the time required for the postreplication repair (RecA, LexA, Red) of any lethal UV-lesion.     

Towards single-copy gene expression systems making gene cloning physiologically relevant: lambda InCh, a simple Escherichia coli plasmid-chromosome shuttle system

We describe a simple system for reversible, stable integration of plasmid-borne genes into the Escherichia coli chromosome. Most ordinary E. coli strains and a variety of pBR322-derived ampicillin-resistant plasmids can be used. A single genetic element, a lambda phage, is the only specialized vector required. The resultant strains have a single copy of the plasmid fragment inserted stably at the lambda attachment site on the chromosome, with nearly the entire lambda genome deleted.     

Lambda phagemids and their transducing properties

Two recombinant lambda DNAs, lambda gt::pMB9 and lambda NM::pBR322, containing, respectively, the pMB9 and pBR322 replicon were constructed and characterized. Both constructs (phagemid DNAs) transfect Escherichia coli cells, producing mature infectious phage progenies. Alternatively, drug-resistant colonies of transductants can be selected upon infection with these phages (phagemid particles) that maintain phagemid DNA in the cell in the form of covalently closed circular plasmids. The efficiency of transduction for nonlysogenic E. coli strains with lambda gt::pMB9 phage producing lambda repressor cIts ranges from 10(-7) to 10(-2) transductant colonies per input phage, depending on the temperature and strain used, while lambda NM::pBR322 phage carrying imm21 transduces with a frequency of up to 1. This means that each lambda NM::pBR322 phagemid particle is capable of establishing itself in the cell as a nonlethal plasmid, permitting formation of a resistant bacterial colony. The maximal level of transduction with lambda gt::pMB9 was obtained when E. coli cells lysogenic for lambda were used. Thus, we believe that the efficiency of transduction is determined by the turn-on of the phage repressor in the transductant. In addition, we have found that all lambda gt::pMB9-containing transductants under certain conditions harbor precisely excised pMB9; excision of pBR322 from lambda NM::pBR322 has not been observed.     

Temperature Sensitivity of Maltose Utilization and Lambda Resistance in Escherichia coli B

Escherichia coli B strains that have acquired the malB region from E. coli K-12 are able to utilize maltose and to adsorb phage lambda when grown at 30 C, but when grown at 40 C they do not absorb phage lambda and are devoid of amylomaltase activity. These Mal(ts) Lam(ts) cells can be mutated or transduced to become able to grow on maltose at 40 C, but they still have no detectable amylomaltase activity nor functional lambda receptors at that temperature. This Mal(40) phenotype is governed by a gene located near or at malA. It is suggested that the temperature sensitivity of both characters results from a defect in malT. However, transduction of malA from E. coli B to E. coli K-12 results in a wild-type phenotype, whereas E. coli B cells that have acquired malA from E. coli K-12 donors are still temperature sensitive for both amylomaltase and lambda-receptor production.     

Isolation and characterization of specialized lambda transducing bacteriophage carrying the metBJF methionine gene cluster.

Secondary attachment site lysogens of Deltaatt(lambda)Deltappc-argECBH strains of Escherichia coli with lambdacI857 integrated into the bfe gene (88 min) were isolated. Of 20 such lysogens examined, 2 produce lysates with transducing phage containing the metBJF gene cluster (87 min). Reintroduction of the ppc-argECBH chromosome segment (which lies between the bfe and met genes) into these strains virtually abolishes the production of met transducing phage. All of the phage examined have lost essential genes from the left arm of the lambda chromosome. Approximately 85% of the phage appear to have the same genetic composition, containing the metBJF gene cluster, but not the closely linked gene cytR, and having lost phage genes G and J. Analytical CsCl density gradient centrifugation of five representatives of this major class of phage shows four of them to have identical densities (lighter than lambda), while the fifth cannot be resolved from lambda. The four apparently identical phage were isolated from three separate lysates, which suggests the existence of preferred sites for illegitimate recombination on the bacterial and phage chromosomes. Three specialized transducing phage that carry cytR in addition to metB, metJ, and metF have also been studied. Each of these viruses has a different amount of phage deoxyribonucleic acid. Two of them have less deoxyribonucleic acid than lambda, whereas the third has about the same amount. The metB, metF, and cytR genes of the transducing phage have been shown to function in vivo. The phage-borne metB and metF genes are subject to metJ-mediated repression.