Isolation and characterization of lambda specialized transducing bacteriophages carrying Klebsiella pneumoniae nif genes

Seve lambda dnif specialized transducing bacteriophages were isolated from Escherichia coli strains containing plasmids carrying the his-nif region of Klebsiella pneumoniae. These phages collectively carry deoxyribonucleic acid for all of the genes in the nif regulon and adjacent deoxyribonucleic acid of K. pneumoniae. The phages were isolated by using Mu insertions in the nif region to direct the integration of lambda pMu phages in nif via formation of lambda pMu-Mu dilysogens which, upon induction, yielded lambda dnif phages. This procedure should be generally applicable for isolating lambda specialized transducing phages carrying genes from E. coli or other bacteria.     

Transducing lambda phages with Escherichia coli genes

The paper presents data on transducing lambdoid phages containing Escherichia coli genes. The major genetic techniques for isolating transducing phages (in vivo) are outlined. A combined table of best-studied transducing phages obtained by the methods of molecular genetics and genetic engineering lists phages genotype & basic literature references for the phages and their derivatives. The chromosome fragments of E. coli inserted in phage DNA are separately specified. Another table presents information about phages carrying E. coli fused operons and genes. The paper also provides detailed physical maps of three regions of the E. coli chromosome. The bibliography contains 300 items.     

Isolation of specialized transducing bacteriophages carrying deletions of the regulatory region of the Escherichia coli K-12 tryptophan operon.

A lysogen of a wild-type strain of Escherichia coli K-12 carrying a heat-inducible lambda-phi80 hybrid prophage was induced to yield transducing phages carrying all of the structural genes of the tryptophan operon. The presence or absence of elements of the trp regulatory region was determined by examining the effects of lambda genes N and cI on trp gene expression. The phages were further characterized by transduction studies and by examining anthranilate synthetase (EC 4.1.3.27) (TRYPE+D) synthesis in the presence of the lambda cI product. A number of phages deleted for the trp promoter were found. Recombination studies between trpOc bacteria and the transducing phages have yielded information that can be used to order the trp end points of some phages and to provide an estimate of the size of the trp promoter region.     

Isolation of specialized lambda transducing bacteriophages for flagellar genes (fla) of Escherichia coli K-12.

Specialized transducing lambda phages carrying the region III flagellar genes (fla) of Escherichia coli K-12 were isolated by a new method. A strain carrying both a cryptic lambda prophage near the his genes and a deletion of the attlambda gene was used as a starting strain. The lysogen of lambdacI857pga18-bio69 was isolated in which the prophage was integrated within the lambda cryptic genes by means of recombination with the residual lambda DNA. The strains with deletions starting within the prophage and ending in these fla genes were selected from among the heat-resistant survivors of the lysogen. They were then infected with heat-inducible and lysis-defective lambda phages and, thus, specialized transducing phage lines for hag and fla were obtained. High-frequency transfer lines of rare phages carrying the fla genes were isolated by inducing a strain carrying a heat-inducible lambda prophage near the his genes and selecting by transduction of a fla deletion strain. Preliminary characterization of these transducing phages is also reported.     

Organization of genes in the ftsA-envA region of the Escherichia coli genetic map and identification of a new fts locus (ftsZ).

Complementation tests have revealed that the mutation in the filamenting mutant PAT84 is distinct from ftsA and has been designated ftsZ. By isolating transducing phages carrying various amounts of the bacterial deoxyribonucleic acid in this region, it was possible to locate the ftsZ gene between ftsA and envA. It is concluded that these cell division genes are expressed independently of the neighboring murein genes.     

Isolation and characterization of nondefective transducing lambda bacteriophages carrying fla genes of Escherichia coli K-12.

In Escherichia coli K-12, 11 fla genes and a hag gene are located between his and uvrC, making two clusters at map positions 42.5 and 43.0 min. Nondefective transducing lambda phages for these genes were isolated. Low-frequency-transducing donors were constructed starting from lysogens of lambda cI857 in which the prophage is integrated at a secondary attachment site at 44 min on the E. coli map. Two strategies were used to delete the region between the prophage and the fla genes. Deletion mutants of the supD locus between fla and the prophage were isolated by selecting for loss of Su1+, an allele of supD. A strain with a deletion starting within the prophage and ending at a position close to the fla genes was isolated from heat-resistant derivatives of the lysogen. A lysogen of lambda b2 was then constructed in which the prophage had integrated at the site of the defective prophage by means of recombination with residual lambda deoxyribonucleic acid. From low-frequency-transducing lysate of the donor strains thus constructed, either directly or in combination with a procedure that extends the loci transduced, various lambda pfla's were isolated. lambda pflaL1 carries all nine fla genes at 43 min, and lambda pflaH14 carries hag and two fla genes at 42.5 min.     

Genetic and physical studies of lambda transducing bacteriophage carrying the beta subunit gene of the Escherichia coli ribonucleic acid polymerase.

The prophage lambdac1857 was inserted into the bfe gene located near rif (the structural gene for the beta subunit of deoxyribonucleic acid [DNA]-dependent ribonucleic acid polymerase) on the Escherichia coli chromosome. Induced lysates (low-frequency transducing lysates) of such a lysogen contained defective lambda phage particles (lambdadrif+) that can specifically transduce the wild-type rif+ gene. Upon transduction into a recipient strain carrying recA, heterogenotes harboring both the wild-type and the mutant rif genes were isolated. Rec+ derivatives of these heterogenotes produce high-frequency transducing lysates that contain lambdadrif+ and normal active phages at a ratio of 1 to 2. The results of marker rescue experiments and of density determination with several transducing phages indicate that most of the late genes are deleted and replaced by a segment of the chromosomal DNA carrying the bfe-rif region. The length of the chromosomal segment seems to vary between approximately 0.5 and 0.6% of the total bacterial DNA among the three independently isolated lambdadrif+ phages. Electron microscopy of heteroduplex DNA consisting of one strand from lambdadrif+-6 and the other from lambdaimm-21 phages directly confirmed that most of the phage DNA of the "left arm" was replaced by the bacterial DNA. The heteroduplex study also demonstrated that the integration of prophage lambda into the bfe region occurred at the normal cross-over point within the phage attachment site.     

Molecular cloning of four tricarboxylic acid cyclic genes of Escherichia coli.

A fragment of DNA (3.1 kilobases [kb]) from a ColE1 Escherichia coli DNA hybrid plasmid containing the bacterial citrate synthase gene (gltA) was subcloned in both orientations into phage lambda vectors by in vitro recombination. The resulting phages were able to transduce gltA and, as prophages, complemented the lesion of a gltA mutant, showing that a functional gltA gene is contained in the 3.1-kb fragment. The segment of E. coli DNA cloned in these lambda gltA phages was extended in vivo by prophage integration and aberrant excision in the gltA region. Plaque-forming derivatives, carrying up to three additional tricarboxylic acid cycle genes, succinate dehydrogenase (sdh), 2-oxoglutarate dehydrogenase (sucA), and dihydrolipoamide succinyltransferase (sucB), were isolated and characterized by their transducing and complementing activities with corresponding mutants, and the order of the genes was confirmed as gltA-sdh-sucA-sucB. Physical maps of a variety of the transducing phages showed that the four tricarboxylic acid cycle genes are contained in a 12.8-kb segment of bacterial DNA. The four gene products, plus a possible succinate dehydrogenase small subunit, were identified in postinfection labeling studies, and the polarities of gene expression were defined as counterclockwise for gltA and clockwise for sdh, sucA, and sucB, relative to the E. coli linkage map.     

Isolation and characterization of lambda transducing bacteriophages for the su1+ (supD minus) amber suppressor of Escherichia coli.

Specialized lambda transducing phages for the sul+ (supD-) amber suppressor in Escherichia coli K-12 have been isolated, using a secondary site lambda-cI857 lysogen in which we have shown the prophage to be closely linked to sul+.sul+ transducing particles were detected frequently, at 10-5 per plaque-forming unit, in lysates prepared from the secondary-site lysogens. High-frequency transducing lysates were obtained from several independently isolated sul+ transductants and were analyzed by CsCl equilibrium density gradient centrifugation. The transducing phages are defective; marker rescue analysis indicates that the lambda-N gene is not present. In lambda-cI857DELTANdSul+, a bio-type transducing phage, the genes specifying recombination and excision functions have been replaced by bacterial deoxyribonucleic acid.     

Molecular characterization of ilvC specialized transducing phages of Escherichia coli K-12

A series of lambda defective ilvC specialized transducing phage has been isolated which carry regions of isoleucine and valine structural and regulatory genes derived from the ilv cluster at minute 83 on the linkage map of the chromosome of Escherichia coli K-12. The ilv genes carried by these phages and their order have been determined by transduction of auxotrophs. The ilvC+ lysogen of an ilvC- strain gave rise, after heat induction of the lysogen, to transducing particles which carried the wild-type allele of the cya-marker. Further experiments have shown that the lambda defective ilvC phages were able to cotransduce a rho-15ts mutation as well as a rep-5 mutation. Hence, the order of the clockwise excision of the ilv cluster was found to be ilvC-rho-rep-cya. Enzyme levels in strains carrying the lambda defective ilvC phages indicated the the ilvC gene was not altered by the insertion of lambda into the ilv cluster. The isolation and digestion of lambda defective ilvC DNA by EcoRI and HindIII restriction endonucleases demonstrated that the specialized transducing phages carried part of the genome from the E. coli K-12 chromosome.     

Use of lambda pMu bacteriophages to isolate lambda specialized transducing bacteriophages carrying genes for bacterial chemotaxis.

A general method for constructing lambda specialized transducing phages is described. The method, which is potentially applicable to any gene of Escherichia coli, is based on using Mu DNA homology to direct the integration of a lambda pMu phage near the genes whose transduction is desired. With this method we isolated a lambda transducing phage carrying all 10 genes in the che gene cluster (map location, 41.5 to 42.5 min). The products of the cheA and tar genes were identified by using transducing phages with amber mutations in these genes. It was established that tar codes for methyl-accepting chemotaxis protein II (molecular weight, 62,000) and that cheA codes for two polypeptides (molecular weights, 76,000 and 66,000). Possible origins of the two cheA polypeptides are discussed.     

Deletion mapping and heterogenote analysis of a mutation responsible for osmosis-sensitive growth, spectinomycin resistance, and alteration of cytoplasmic membrane in Escherichia coli

Lambda transducing phages carrying Escherichia coli deoxyribonucleic acid of various lengths from the aroE-rpsL region were lysogenized into the F'3 plasmid and were used for heterogenote analysis of YM101, a sucrose-dependent, spectinomycin-resistant mutant of E. coli. Three characteristics of the mutant strain, resistance to spectinomycin, sucrose dependence of growth, and lack of I-19 protein in the cytoplasmic membrane, were shown to be the result of a mutation in a region designated delta 53-spcl. This region extends over 3.6-kilobase pairs and is located within a cluster of ribosomal genes. The mutation is recessive to the wild-type allele.     

Location of the Escherichia coli K-12 ruv gene affecting septum formation after inhibition of deoxyribonucleic acid synthesis.

Escherichia coli ruv gene was located at 36.1 min on the chromosome by P1 transduction experiments and the gene order his - supD - uvrC, dar4 - ruv - eda - fadD - pps was proposed. Complementation analysis by an F' factor carrying genes in the his region indicated that ultraviolet light sensitivity genes, ruv and uvrC, consist of different cistrons and wild-type alleles of these genes are dominant over the mutant alleles.     

Specialized transduction with lambda plac5: dependence on recA and on configuration of lac and att lambda.

The construction of lambda plac5 transducing phages carrying various lacZ alleles is described. Genetically disabled (N- N- P-) lambda plac transducing the phages were used to study the dependence of specialized transduction on host RecA function and on the location of the lacZ gene in the recipient strain. In the absence of site-specific recombination at att lambda, transduction was completely dependent on host RecA function. Regardless of the configuration of att lambda, lambda plac transducing phages recombined at a 20- to 50-fold higher frequency with F42 lac than with a lac gene located in the cellular chromosome. Deletion mutants of lacZ in the recipient strain were used to show that the probability of lac recombination resulting from lambda plac infection is apparently proportional to the amount of homology between the parental lacZ genes.     

A specialised transducing phage of P22 for which the ability to form plagues is associated with transduction of the proAB region

Summary The multisite mutantproAB47 has been used to isolate specialised transducing phages of P22 for theproA andproB genes. Several of the preparations contain phages that form small plaques, and many of the properties of these suggest that the small plaques are formed by the transducing phages. Preliminary studies on the transduction of theproAB region by one such phage suggest that it forms fairly stable heterogenotes, both from apro point mutant and fromproAB47. The fact that the latter mutant is missing the phage attachment site results in a delay in integration of the specialised transducing phage.     

Relation between Px phages and generalised transducing particles of phage P22

Summary P22 mutants with an altered ability to form generalised transducing particles were tested for their ability to form the hybrid Px phages during growth on Py-lysogenic strains of Salmonella typhimurium. The mutant which produces more (less) transducing particles produces also more (less) Px phages. It is suggested that Px particles might be regarded as self-replicating generalised transducing particles.     

Formation of lambda transducing phage in vitro: involvement of DNA gyrase

We found that transducing phages carrying the gal or bio regions of the Escherichia coli genome were formed during in vitro packaging of endogenous lambda DNA. Structural analysis of the transducing phage genomes indicated that they were formed by abnormal excision of lambda prophage. Formation of transducing phages was stimulated by oxolinic acid, an inhibitor of DNA gyrase, implying that DNA gyrase participates in the abnormal excision of lambda prophage. When pBR322 DNA was added to the reaction mixture, transducing phages into which pBR322 had been inserted were produced at a high frequency. This reaction was also stimulated by oxolinic acid. Sequence analyses revealed that pBR322 is inserted into the sites of abnormal excision of the prophage. These results show that transducing phages can be formed by DNA gyrase-dependent illegitimate recombination in an in vitro system and that secondary recombination takes place frequently at the site where the first recombination occurs.     

New transducing phage with RNA polymerase beta- and beta'-subunit genes derived from a hybrid phage lambda att80: isolation, genetic analysis and physical mapping]

A hybrid lambda att 80 phage with the genetic structure lambda (A-J) phi 80 (att-int-xis) imm lambda..cI857s7 is shown to be a convenient vector for creating transducing phages. On the one hand, the restriction analysis indicates that it has 3 restriction sites for EcoRI in comparison with 5 and 9 sites for parental phages lambda and phi 80 respectively. On the other hand, its buoyant density is less than that of phage lambda and under centrifugation it is easier separated from the phage transducing particles. When lambda att 80 prophage was excluded from the bfe locus of Escherichia coli, transducing phages with genes of two RNA polymerase beta-subunits (rpoB and rpoC) were isolated. To identify the latter, a convenient genetic test was worked out. A physical map of lambda att 80 drifd 35 transducing phage, carrying rpoB and rpoC genes has been constructed using endonucleases EcoRI and HindIII. A comparison of this map and the corresponding maps of transducing phages lambda drifd 18 and lambda drifd 47, studied earlier, led to the discovery of two integration sites of phage lambda within the locus bfe spaced apart by about 1800 nucleotide pairs. At all the sites both phages (lambda and lambda att 80) have integrated in the locus bfe in the counter clockwise order.