Characterization of dnaA gene carried by lambda transducing phage

Summary Specialized transducing phages dnaA were obtained by inducing lysogens in which tna was integrated at the tnaA region of the Escherichia coli chromosome; the tnA region is located in the vicinity of the dnaA gene. The dnaA ^- deletion derivatives of dnaA were isolated from the lysate of dnaA grown on bacteria carrying a transposon Tn3.The structures of various transducing phages thus obtained were determined by heteroduplex DNA mapping. From these results, the transducing fragment of 13.8-kb-long was divided into nine domains. Upon infection of UV-irradiated cells with the phage, production of polypeptides of 49 kD and 42 kD was specifically associated with infections by the dnaA and recF transducing phages. Polypeptides of 49 kD and 42 kD appeared to be coded for by dnaA and recF genes, respectively. The dnaA gene was assigned to the region of 2.8-kb-long which extends by 2.4 kb in the counterclockwise direction on the E. coli genetic map and 0.4 kb in the opposite direction, as measured from the nearest HindIII site close to the tnaA gene. The recF gene was also discovered to lie very close to dnaA in the order of tnaA-dnaA-recF.Merogenotes heterozygous for the dnaA gene were constructed by introducing F100-12 carrying dnaA into the recipients with different mutations at or near dnaA. For combinations, F(dnaA ⁺)/dnaA46 and F(dna ⁺)/dna-83, dnaA ⁺ was trans-dominant, whereas the dnaA ⁺ was recessive for F(dnaA ⁺)/dna-5. For F(dnaA ⁺)/dna-167, the result of the transdominance test was affected by the growth media employed; dnaA ⁺ was dominant on a -broth plate, and dna-167 was dominant on an M9-minimal plate. Thus, transdominance of dnaA ⁺ in heterozygotes is affected by difference in mutations and growth media.     

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.     

Endpoint distribution for deletions into imm lambda region forming p lambda CM replicons: phage lambda gene rex affects plasmid establishment

For the p lambda CM family of lambda-derived self-encapsidating plasmids, the rexB gene product facilitates plasmid establishment following injection into a new host cell. Temperature-stable chloramphenicol resistance (CmR at 40 degrees C) conferred by low-multiplicity infection with lambda::Tn9 cI857 lysates (Tn9 sites tested: 22.60 or 24.08 kb, in the b region, or 28.41 kb, in int) is usually due to a lambda::Tn9 plasmid (p lambda CM) formed by a deletion penetrating the lambda immunity region. These grow either as plasmids in the absence of, or lytic phages in the presence of N function supplied by a host such as lambda cI857 delta H1 lysogen MS1449. The 'groplaque' (plaque-shaped growth spot) assay, which selects for CmR growth in an MS1449 lawn at 32 degrees C after an initial plaquing period at 37 degrees C, reveals two distinguishable classes of p lambda CM isolates. All variants whose deletions extend into or beyond rexB give rise to visible CmR growth only after the temperature shift to 32 degrees C, and thus produce a hollow-centered 'donut' type of groplaque. In contrast, 16 out of 17 variants whose deletions fall short of rexB produce 'solid' groplaques which appear before the temperature shift. Tests of T4rII phage exclusion show the exceptional 17th variant to be Rex-, confirming the identification of rex as the lambda component whose loss results in the 'donut' groplaque morphology. More specific physiological tests showed that in the absence of Rex the establishment of a newly injected p lambda CM plasmid becomes temperature-sensitive (ts), while plasmid maintenance remains unaffected. This indicates that the role of Rex in plasmid survival is confined to the early stages of transduction, where it might either assist plasmid replication or retard host replication, to help the plasmid replicon achieve a copy number sufficient for stable transmission.     

A one-step miniprep for the isolation of plasmid DNA and lambda phage particles

Plasmid DNA minipreps are fundamental techniques in molecular biology. Current plasmid DNA minipreps use alkali and the anionic detergent SDS in a three-solution format. In addition, alkali minipreps usually require additional column-based purification steps and cannot isolate other extra-chromosomal elements, such as bacteriophages. Non-ionic detergents (NIDs) have been used occasionally as components of multiple-solution plasmid DNA minipreps, but a one-step approach has not been developed. Here, we have established a one-tube, one-solution NID plasmid DNA miniprep, and we show that this approach also isolates bacteriophage lambda particles. NID minipreps are more time-efficient than alkali minipreps, and NID plasmid DNA performs better than alkali DNA in many downstream applications. In fact, NID crude lysate DNA is sufficiently pure to be used in digestion and sequencing reactions. Microscopic analysis showed that the NID procedure fragments E. coli cells into small protoplast-like components, which may, at least in part, explain the effectiveness of this approach. This work demonstrates that one-step NID minipreps are a robust method to generate high quality plasmid DNA, and NID approaches can also isolate bacteriophage lambda particles, outperforming current standard alkali-based minipreps.     

Characterisation of a lambda transducing phage carrying the F conjugation gene traG

Summary A lambda transducing phage carrying the traGSTD genes of the E. coli K12 sex factor F was isolated by an in vivo technique, and characterized in ra complementation tests, by determining its restriction endonuclease fragment sizes, and by measuring heteroduplex molecules. The size and location on the F physical map of the tra transducing segment was thereby determined.Comparison of the proteins synthesized in UV-irradiated cells by this phage and by a derivative carrying the amber traG79 mutation, allowed the traG product to be identified as a protein of molecular weight 100,000. In the same experiments, the sizes of the traT and traD products made by the phage were also measured, being 25,000 and 85,000 daltons respectively.     

Localization of the metJBLF gene cluster of Escherichia coli in lambda met transducing phage

Summary The position of the metJBLF gene cluster in the transducing phage dmet102 was determined by ligation of its leftmost EcoRI fragment (102-1) to the BCDEF (nin5) EcoRI fragment of gtl (BC) and characterization of the resultant recombinant phage. The new transducing phage carries about 6kb of bacterial DNA which contains the entire met gene cluster including the promoter of its rightmost member metF. Reasonable estimates of the coding capacity required for the four genes indicate that most of the bacterial DNA of the recombinant phage is occupied by the met gene cluster.     

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.     

Gal transduction by phage lambda: on the origin and nature of LFT transducing genomes

Summary There are at least two classes of transducing particles made on the induction of normal lysogens: the first is capable of transducing by the insertion of the whole transducing genome into the host chromosome, so its genome must be capable of circularizing; the second transduces less well by insertion—perhaps not at all; if it does not transduce by insertion then its genome need not be linear.The formation of a transducing genome can be accomplished in three steps: (a) breaking the lysogenic bacterial chromosomes in two places, (b) joining the fragment ends together to form a circular structure, (c) opening the circle (by ter) to form a linear genome. If the resultant structure meets the requirements for packaging, it may be formed into a transducing phage, like a bougus .Any meaningful rearrangement of these steps in which step (b) is omitted or delayed leads to the formation of genomes, which are (1) unable to transduce by insertion (because both of its mature ends are unexposed) and (2) are on the average smaller than genomes which are capable of transducing by insertion (so the resultant transducing phage is less dense). Consequence (2) has been confirmed.We assume that the red function of catalyzes the joining of broken DNA molecules to each other. So red is responsible for rehealing the product of (a) back into a lysogenic chromosome and for catalyzing step (b), the healing of fragment ends into a circular structure. The much elevated level of stable transductants on induction of red ^– lysogens hereby is explained.Supported by grant E-2862 of the U.S.P.H.S. to Dr. Allan Campell.     

Chemotaxis in Escherichia coli: construction and properties of lambda tsr transducing phage.

The tsr gene of Escherichia coli, located at approximately 99 min on the chromosomal map, encodes a methyl-accepting protein that serves as the chemoreceptor and signal transducer for chemotactic responses to serine and several repellents. To determine whether any other chemotaxis or motility genes were located in the tsr region, we constructed and characterized two lambda tsr transducing phages that each contain about 12 kilobases of chromosomal material adjacent to tsr. lambda tsr70 carries sequences from the promoter-proximal side of tsr; lambda tsr72 carries sequences from the promoter-distal side of tsr. Restriction maps of the bacterial inserts in these phages and Southern hybridization analyses of the bacterial chromosome indicated that the tsr gene is transcribed in the counterclockwise direction on the genetic map. Insert deletions were isolated in lambda tsr70 and transferred into the host chromosome to examine the null phenotype of tsr. All such strains exhibited wild-type swimming patterns and chemotactic responses to a variety of stimuli, but were specifically defective in serine taxis and other Tsr-mediated responses. In addition, UV programming experiments demonstrated that Tsr and several of its presumptive degradation products were the only bacterial proteins encoded by lambda tsr70 and lambda tsr72 that required host FlbB/FlaI function for expression. These findings indicate that there are probably no other chemotaxis-related genes in the tsr region. A series of tsr point mutations were isolated by propagating lambda tsr70 on a mutD host and used to construct a fine-structure map of the tsr locus. These mutations should prove valuable in exploring structure-function relationships in the Tsr transducer.     

The separation of phage promoter from bacterial lac promoter for beta-galactosidase expression in transducing phage lambda plac5.

The replication defective transducing phage λp$\text{lac}\text{5}\text{O}\text{29}\text{P}$3 carries a portion of the $\text{E.}\text{coli}\text{lac}$ operon in the $\text{b}$2 region of the lambda phage. This $\text{lac}$ operon segment contains the $\text{lac}$ promoter, the $\text{lac}$ operator, and the β-galactosidase $\text{z}$ gene, but does not contain the $\text{lac}$ repressor $\text{i}$ gene. The $\text{z}$ gene can be expressed from both the inserted $\text{lac}$ promoter and the phage promoter. When $\text{E.}\text{coli}$ strain 594 ($\text{z}$^?, $\text{i}$⁺) or JC6256 (Δ$\text{lac}$) is infected by λp$\text{lac}\text{5}\text{O}\text{29}\text{P}$3 in the absence of additional cyclic AMP, β-galactosidase synthesis is shown to be expressed from the phage promoter. When 594 (λ⁺) or JC6256 (λ⁺) is infected by λp$\text{lac}\text{5}\text{O}\text{29}\text{P}$3 in the presence of additional cyclic AMP and IPTG, β-galactosidase synthesis is shown to be expressed from the inserted $\text{lac}$ promoter.The ability to separate the phage promoter from the inserted $\text{lac}$ promoter for β-galactosidase expression will simplify the interpretation whenever λp$\text{lac}$5 is used.     

Selection of lambda Spi- transducing phages using the P2 old gene cloned onto a plasmid

The old gene product of the P2 prophage interferes with plaque formation by lambda wild type phage but allows lambda phages whose red and gam genes have been deleted to form small, visible plaques (the lambda Spi- phenotype). The old gene product also kills Escherichia coli recB or recC mutants. We have cloned the old gene into the high-copy-number plasmid pBR322, where it prevents plaque formation by both lambda Spi+ and lambda Spi- phages. We transferred a DNA fragment that carries the old gene to the low-copy-number plasmid pSC101 and found that lambda Spi- phages can be selected on strains that carry this plasmid. The plasmid-borne old gene kills E. coli recB mutants, providing a selection for old- mutants.     

A simple technique for the isolation of deletion mutants of phage lambda.

We describe a simple technique for isolating deletion mutants of phage lambda and use it to dissect a cloned fragment of foreign DNA. The technique is based on our previous finding that the normally essential product of lambda head gene D is dispensible for phage growth if the DNA content of the phage is less than 82% that of lambda wild-type (Sternberg and Weisberg, 1977). A significant fraction of the few phage that form plaques when a D amber mutant is plated on a nonsuppressing host contains deletions that reduce the phage chromosome size to less than 82% that of wild-type. It is possible to isolate deletions ranging in size from less than 1.5 kb to 14 kb (3 to 27% of wild-type lambda), and the size range can be restricted by an appropriate choice of the DNA content of the starting phage. This method, unlike the older EDTA or heat resistance methods, permits the scoring of deletions because of the absence of phenotypic variants. We investigated the effect of several host and phage mutations on deletion frequency and type and have determined that a host polA mutation increases the frequency of deletions about 30-50-fold without changing the type of deletions. A host mutD mutation or thymine deprivation increases deletion frequency about 10-fold. In contrast, a host ligts mutation has no effect on the frequency of deletions. We have also determined that the size of the smallest lambda chromosome packageable in a plaque-forming phage particle is 72-73% that of lambda wild-type.     

Evidence for positive regulation of plasmid prophage P1 replication: integrative suppression by copy mutants.

Like low-copy-number plasmids including P1 wild type, multicopy P1 mutants (P1 cop, maintained at five to eight copies per chromosome) can suppress the thermosensitive phenotype of an Escherichia coli dnaA host by forming a cointegrate. At 40 degrees C in a dnaA host suppressed by P1 cop, the only copy of P1 is the one in the host chromosome. Trivial explanations of the lack of extrachromosomal copies of P1 cop have been eliminated: (i) during integrative suppression, the P1 cop plasmid does not revert to cop+; (ii) the dnaA+ function of the host is not required to maintain P1 cop at a high copy number; and (iii) integrative recombination does not occur within the region of the plasmid involved in regulation of copy number. Since there are no more copies of the chromosomal origin (now located within the integrated P1 plasmid) than in a P1 cop+-suppressed strain, the extra initiation potential of the P1 cop is not used to provide multiple initiations of the chromosome. When a P1 cop-suppressed dnaA strain was grown at 30 degrees C so that replication could initiate from the chromosomal origin as well as from the P1 origin, multicopy supercoiled P1 DNA was found in the cells. This plasmid DNA was lost again when the temperature was shifted back to 40 degrees C.     

Construction of recombinant plasmid carrying the lambda DNA fragment responsible for prophage integration.

The recombinant DNA molecules were constructed from plasmid RSF2124 and the EcoRI fragment of lambda DNA containing the genes responsible for prophage integration. The presence of these genes in recombinant plasmids was detected genetically. lambda int-gene was shown to be expressed in either orientation of insertion in the plasmid. We found that recombinant plasmid was able to integrate into chromosome of lambda lysogens. The integration of plasmid into host chromosome was demonstrated by contransduction of chromosome and plasmid markers using generalized transducer P1 and by specialized transduction with lambda phages.     

A specialized transducing phage, lambda psrlA, for the sorbitol phosphotransferase of Escherichia coli K12

A specialized transducing phage for the srlA gene, specifying the sorbitol-specific Enzyme II of the phosphoenolpyruvate:sugar phosphotransferase system, was constructed and its DNA was analysed by restriction endonuclease digestion. Phage construction involved four steps: (1) integration of lambda into the srlA gene; (2) selection of phage carrying (a) the left and (b) the right end of the srlA gene by means independent of the function of the new DNA acquired; (3) reconstitution of the srlA gene in a dilysogen of these two phage; and (4) the excision, using the heteroimmune lambdoid phage 21, of a plaque-forming srlA+ phage from the dilysogenic chromosome. Comparison of the DNA restriction digests of the transducing phage with those of its parents and of wild-type lambda revealed fragments consisting partly of lambda and partly of Escherichia coli DNA. The junction points in the intermediate phage define a site that must lie within the reconstituted gene of the final phage. This technique should be of general application in relating genes, cloned by our method, to DNA sequences.