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.     

Structural studies of lambda transducing bacteriophage carrying bacterial deoxyribonucleic acid from the metBJLF region of the Escherichia coli chromosome.

The structures of several lambda dmet and related lambda darg transducing phage were studied by restriction fragment mapping and electron microscopic measurements of homoduplexes and heteroduplexes. A new transducing phage (lambda dmet141), in which metF is the only functional gene of the cluster, was isolated. In contrast, lambda dmet117, which expresses the entire metBJLF cluster, has only 3 kilobases more bacterial deoxyribonucleic acid (DNA) than lambda dmet141. An EcoRI restriction fragment of lambda dmet117, which carries the leftmost 6 kilobases of the bacterial DNA insert, was isolated and shown to contain a functional copy of metB. Small structural differences at the attachment sites of some of the phage were shown to result from different sites of lambda integration in the two parent insertion lysogens.     

Recognition properties of the beta subunit of Escherichia coli ribonucleic acid polymerase.

Changes in the phage protein patterns obtained by gel electrophoresis of extracts from phage S13 and phiX174 infection of rifampin-resistant hosts suggest that the beta subunit of ribonucleic acid polymerase of Escherichia coli has a function in the recognition of promoter or terminator sites or both. The altered protein patterns also provide information on the location of some ribonucleic acid polymerase recognition signals in S13 deoxyribonucleic acid. There is a promoter site before gene A, which lies either in gene H or between H and A. There is evidence for a promotor between genes C and D or in gene C. There is either a terminator or a promoter somewhere between the end of gene D and the beginning of gene F.     

Isolation and characterization of a lambda transducing bacteriophage carrying the cysE gene of Escherichia coli K-12

A specialized lambda transducing phage carrying the cysE and gpsA genes of E. coli K-12 has been isolated. The transducing phage has been separated from the helper phage on equilibrium gradients and has been shown to be defective. Evidence is presented that the phage kil gene is not expressed.     

Specialized transducing bacteriophage lambda carrying the structural gene for a major outer membrane matrix protein of Escherichia coli K-12.

A specialized transducing phage lambda carrying the structural gene for the OmpF protein, an outer membrane matrix protein, was isolated. The phage carries the 20.5--21-min region of the Escherichia coli K-12 chromosome and carries asnS, ompF, and aspC genes.     

Specialized lambda transducing bacteriophage which carries hisS, the structural gene for histidyl-transfer ribonucleic acid synthetase.

A number of specialized lambda transducing bacteriophages which carry the Escherichia coli gene guaB were isolated from E. coli. One of these bacteriophages, lambda cI857 Sam7 d guaB-2, also carries hisS, the structural gene for histidyl-transfer ribonucleic acid synthetase (EC 6.1.1.21). Histidyl-transfer ribonucleic acid synthetase activities in induced and uninduced lysogens carrying lambda d guaB-2 indicate that the phage carries the entire structural gene and that the gene is under the control of an E. coli promoter. These conclusions were confirmed by the in vivo production of a protein encoded by the phage which comigrates with authentic histidyl-transfer ribonucleic acid synthetase on two-dimensional polyacrylamide gels.     

Isolation of specialized transducing bacteriophage lambda carrying genes of the L-arabinose operon of Escherichia coli B/r.

A heat-inducible lysis-defective phage lambda (lambdacI857S7) has been integrated at multiple sites within the L-arabinose region (araCOIBAD) of a strain of Escherichia coli K-12 deleted for the normal lambda attachment site (lambdaattdelta). The lambda phage has become integrated with opposite orientations at two different loci within the aratb gene and with the "normal" orientation (clockwise N-RA-J) at a single site in the araC gene. The burst size, spontaneous-curing frequencies, and number of prophage harbored by each of the ara secondary-site lysogens have been determined. From these secondary-site lysogens it has been possible to generate plaque-forming ara-transducing phage (lambdapara) and defective ara-transducing phage (lambdadara), as well as defective leucine-transducing particles (lambdadleu). The construction and characterization of these lambdaara-transducing phage and their derivatives which carry genetically defined portions of the L-arabinose region are presented.     

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.     

Plaque assay for lambda transducing phage carrying the E. coli metB gene

A halo plaque assay has been developed for the detection of nondefective lambda transducing phage carrying functional alleles of the metB gene of Escherichia coli K12. The assay is based upon the production of phage plaques on lawns of metB- bacterial cells which are supplemented with limiting amounts of methionine and upon the subsequent transduction of methionine-starved cells in the lawn surrounding the plaques. The resulting prototrophic transductants give rise to a halo of bacterial growth surrounding the plaque. A precise genotype can be ascribed to the characteristic morphologies of selected haloes. This technique has general application for all biosynthetic markers.     

A specialized transducing lambda phage carrying the Escherichia coli genes for phenylalanyl-tRNA synthetase.

A lambda phage has been isolated which specifically transduces the Escherichia coli pheS and pheT genes coding for the alpha and beta subunits of the phenylalanyl-tRNA synthetase (PRS). This phage transduces with high frequency (i) several temperature-sensitive PRS mutants to thermoresistance and (ii) a p-fluorophenylalanine resistant PRS mutant to sensitivity against this amino-acid analog. The in vitro PRS activities of such lysogens suggest that the alpha and beta subunits coded by the transducing phage complement the mutant host PRS-subunits in vivo by means of formation of hybrid enzymes.The transducing lambda phages were also used to infect UV light irradiated cells. The SDS-gel electrophoretic analysis of the proteins synthesized in such cells revealed that the phage codes at least for four different E. coli proteins. Two proteins with molecular weights of 94,000 and 38,000 daltons cross-reacted with an anti PRS serum and were thus identified as the beta and alpha subunits of PRS, respectively. A third protein with a molecular weight of 22,000 daltons is identical with the ribosomal initiation factor IF3 (Springer et al., 1977b). The other protein (Mr 78,000) is still unidentified.     

Defective and plaque-forming lambda transducing bacteriophage carrying penicillin-binding protein-cell shape genes: genetic and physical mapping and identification of gene products from the lip-dacA-rodA-pbpA-leuS region of the Escherichia coli chromosome

A series of defective lambda transducing phage carrying genes from the lip-leuS region of the Escherichia coli chromosome (min 14 on the current linkage map) has been isolated. The phage defined the gene order as lac---lip-dacA-rodA-pbpA-leuS---gal. These included the structural genes for penicillin-binding protein 2 (pbpA) and penicillin-binding protein 5 (dacA) as well as a previously unidentified cell shape gene that we have called rodA. rodA mutants were spherical and very similar to pbpA mutants but were distinguishable from them in that they had no defects in the activity of penicillin-binding protein 2. The separation into two groups of spherical mutants with mutations that mapped close to lip was confirmed by complementation analysis. The genes dacA, rodA, and pbpA lie within a 12-kilobase region, and represent a cluster of genes involved in cell shape determination and peptidoglycan synthesis. A restriction map of the lip-leuS region was established, and restriction fragments were cloned from defective transducing phage into appropriate lambda vectors to generate plaque-forming phage that carried genes from this region. Analysis of the proteins synthesized from lambda transducing phage in ultraviolet light-irradiated cells of E. coli resulted in the identification of the leuS, pbpA, dacA, and lip gene products, but the product of the rodA gene was not identified. The nine proteins that were synthesized from the lip-leuS region accounted for 57% of its coding capacity. Phage derivatives were constructed that allowed about 50-fold amplification of the levels of penicillin-binding proteins 2 and 5 in the cytoplasmic membrane.     

Genetic studies on the beta subunit of Escherichia coli RNA polymerase. II. Evidence that large N-terminal amber fragments of the beta subunit interfere with RNA polymerase function

Summary A collection of 95 independent, spontaneously-occurring mutants carrying amber lesions that affect expression of the gene, rpoB, has been isolated (see accompanying paper (Nene and Glass 1982)). Certain rpoB amber mutations act in trans, preventing a functional allele present on an F plasmid from acting at high temperature. Two such temperature-sensitive rpoB(Am) strains are shown to produce large, N-terminal amber fragments. The possibility that these truncated polypeptides are the cause of this trans-dominant conditional-lethal phenotype is supported by analysis of fragment levels in thermoresistant survivors: the nonsense fragments are degraded at a significantly faster rate (half-lives 1.4- to 2.6-fold reduced) in Ts⁺ derivatives likely to carry second-site mutations within rpoB. We suggest that the fragments interfere with RNA polymerase function by interacting with one or more of the polymerase subunits.