Phage-mediated Transduction Research Articles

The TGV trans genic vectors for single-copy gene expression from the Escherichia coli chromosome

Plasmid-based cloning and expression of genes in Escherichia coli can have several problems: plasmid destabilization; toxicity of gene products; inability to achieve complete repression of gene expression; non-physiological overexpression of the cloned gene; titration of regulatory proteins; and the requirement for antibiotic selection. We describe a simple system for cloning and expression of genes in single copy in the E. coli chromosome, using a non-antibiotic selection for transgene insertion. The transgene is inserted into a vector containing homology to the chromosomal region flanking the attachment site for phage lambda. This vector is then linearized and introduced into a recombination-proficient E. coli strain carrying a temperature-sensitive lambda prophage. Selection for replacement of the prophage with the transgene is performed at high temperature. Once in the chromosome, transgenes can be moved into other lysogenic E. coli strains using standard phage-mediated transduction techniques, selecting against a resident prophage. Additional vector constructs provide an arabinose-inducible promoter (P BAD ), P BAD plus a translation-initiation sequence, and optional chloramphenicol-, tetracycline-, or kanamycin-resistance cassettes. These Trans genic E. coli Vectors (TGV) allow drug-free, single-copy expression of genes from the E. coli chromosome, and are useful for genetic studies of gene function.

Facilitated diffusion of fructose via the phosphoenolpyruvate/glucose phosphotransferase system of Escherichia coli.

From mutants of Escherichia coli unable to utilize fructose via the phosphoenolpyruvate/glycose phosphotransferase system (PTS), further mutants were selected that grow on fructose as the sole carbon source, albeit with relatively low affinity for that hexose (K(m) for growth approximately 8 mM but with V(max) for generation time approximately 1 h 10 min); the fructose thus taken into the cells is phosphorylated to fructose 6-phosphate by ATP and a cytosolic fructo(manno)kinase (Mak). The gene effecting the translocation of fructose was identified by Hfr-mediated conjugations and by phage-mediated transduction as specifying an isoform of the membrane-spanning enzyme II(Glc) of the PTS, which we designate ptsG-F. Exconjugants that had acquired ptsG(+) from Hfr strains used for mapping (designated ptsG-I) grew very poorly on fructose (V(max) approximately 7 h 20 min), even though they were rich in Mak activity. A mutant of E. coli also rich in Mak but unable to grow on glucose by virtue of transposon-mediated inactivations both of ptsG and of the genes specifying enzyme II(Man) (manXYZ) was restored to growth on glucose by plasmids containing either ptsG-F or ptsG-I, but only the former restored growth on fructose. Sequence analysis showed that the difference between these two forms of ptsG, which was reflected also by differences in the rates at which they translocated mannose and glucose analogs such as methyl alpha-glucoside and 2-deoxyglucose, resided in a substitution of G in ptsG-I by T in ptsG-F in the first position of codon 12, with consequent replacement of valine by phenylalanine in the deduced amino acid sequence.

Gene transfer to mammalian cells using genetically targeted filamentous bacteriophage.

We have genetically modified filamentous bacteriophage to deliver genes to mammalian cells. In previous studies we showed that noncovalently attached fibroblast growth factor (FGF2) can target bacteriophage to COS-1 cells, resulting in receptor-mediated transduction with a reporter gene. Thus, bacteriophage, which normally lack tropism for mammalian cells, can be adapted for mammalian cell gene transfer. To determine the potential of using phage-mediated gene transfer as a novel display phage screening strategy, we transfected COS-1 cells with phage that were engineered to display FGF2 on their surface coat as a fusion to the minor coat protein, pIII. Immunoblot and ELISA analysis confirmed the presence of FGF2 on the phage coat. Significant transduction was obtained in COS-1 cells with the targeted FGF2-phage compared with the nontargeted parent phage. Specificity was demonstrated by successful inhibition of transduction in the presence of excess free FGF2. Having demonstrated mammalian cell transduction by phage displaying a known gene targeting ligand, it is now feasible to apply phage-mediated transduction as a screen for discovering novel ligands.

Inhibition of Thymidine Transport in dnaA Mutants of Escherichia coli

DnaA protein is the initiator of chromosomal DNA replication in Escherichia coli. We report here our evidence that thymidine transport across cytoplasmic membranes in temperature-sensitive dnaA mutants is greatly decreased at a permissive temperature for growth of the mutants. Complementation analysis with a plasmid containing the wild type dnaA gene and P1 phage-mediated transduction confirmed that mutations in the dnaA gene were responsible for the phenotype. A low level of nucleoside transport in the dnaA mutant was specific for thymidine; transport activities for other nucleosides were much the same as those in wild type cells. Membrane vesicles prepared from the dnaA mutant showed much the same activity of thymidine transport as did those from the wild type cells. No significant difference in the activity of thymidine kinase, which is known to facilitate thymidine transport, was seen between the mutant and the wild type cells. An increase in the pool of dTTP, a negative regulator for thymidine kinase, was observed in the dnaA mutant. Based on these observations, we suggest that inhibition of thymidine transport in dnaA mutants is caused by increases in the dTTP pool.

Histone-like proteins are required for cell growth and constraint of supercoils in DNA

The gene hns of Escherichia coli K-12, which encodes the histone-like protein H-NS, was inactivated by insertion of a DNA (gene hph) encoding hygromycin phosphotransferase. The growth rates of two mutants lacking either one or the other of the histone-like proteins, HU and IHF, were not affected by introduction of the hns mutation. However, cells depleted of HU, IHF and H-NS simultaneously, could not be constructed by P1 phage-mediated transduction. These results, together with our previous finding that cells deficient in both HU and IHF are viable at 30–37°C [Kano and Imamoto, Gene 89 (1990) 133–137] showed that E. coli cells deficient in any two of these three histone-like proteins are viable at 30–37°C, and suggested that simultaneous deficiency of all three of the proteins is lethal. There were no detectable differences in the levels of superhelicity of the reporter plasmids isolated from cells deficient in either IHF or H-NS, or from wild-type cells, but about 15% decrease in negative superhelicity was detected for the reporter plasmid isolated from cells lacking HU and lacking both HU and H-NS. However, the cryptic bgl operon, whose expression was reported to be regulated through topological changes of cellular DNA, could not be activated in cells depleted of HU or IHF. The bgl operon was expressed in cells depleted of both HU and H-NS as well as in cells depleted of H-NS. These results indicate that expression of the bgl operon is not affected by a change in superhelical density of the cellular DNA and suggest that its expression depends on some physiological processes other than topological alteration of DNA.

Genetic mapping of the Escherichia coli gene for the stringent starvation protein and its dispensability for normal cell growth.

To determine its map position, the sSP gene was cloned into plasmid pBR322 and the recombinant plasmid was integrated into the chromosome of a polA mutant at the site of the sSP gene by homologous recombination. The chromosomal location of Ampr was then determined by P1 phage-mediated transduction. Thus, the sSP gene was mapped between gltB and glnF at min 69.5 on the Escherichia coli chromosome. Strains were constructed in which the sSP gene was brought under the control of the lac regulatory system. This indicated that the stringent starvation protein (SSP) is dispensable for growth, at least under normal culture conditions.

Interspecific transfer and expression of melanin gene(s) on cosmids in Streptomyces strains

Cosmid pR4C1 has been constructed from a multicopy plasmid pIJ365 and the cohesive ends site of an actinophage R4 (Morino et al. 1985). The cosmid can be transferred to various Streptomyces strains by R4 phage-mediated transduction. Melanin-synthesizing gene(s) originated from Streptomyces antibioticus was inserted into the cosmid and succesfully transferred by tranduction to 7 different strains out of 10 strains examined. The features of melanin-gene(s) expresion were examined in those cells. Tyrosinase activities from the melanin-gene(s) were detected in culture media and/or cell extracts although the extents of mel gene expression and tyrosinase excretion vary strain to strain. The cosmid transfer system described in this paper will be promising to survey suitable hosts for the maximum expression of cloned genes among Streptomyces strains.

32] Phage-mediated transduction of cDNA expression libraries into mammalian cells

Publisher Summary This chapter describes methods for the transfer of cDNA expression libraries into cultured mammalian cells. The recent development of techniques for delivering DNA into cells and isolating appropriate mutant cells has made it possible to use the same approach in mammalian cells. In fact, several mammalian genes have already been cloned solely on the basis of genetic complementation of mutant cells by means of genomic DNA transfection. In mammalian cells, however, this approach has often been hampered by severe difficulties in recovering the transduced genes in Escherichia coli . In addition, mammalian genes are often large in size and they cannot be cloned intact in E. coli. As an alternative approach, the utilization of full length cDNAs for genetic complementation would avoid many of these problems. A system comprising techniques for efficient cloning of full length cDNAs and two vectors, a cDNA expression vector and a cDNA library transducing vector. In contrast to cellular genes, cloned cDNAs can be expressed in any desired host cells, bypassing the barrier of tissue and species-specificity, and in virtually any desired mode, including constitutive, inducible, reduced or elevated expression, by choosing an appropriate promoter. This unique property of eDNA expression permits additional cloning applications using normal cell hosts.

Isolation and genetic characterizations of Bacillus megaterium cobalamin biosynthesis-deficient mutants.

Ethanolamine is deaminated by the action of ethanolamine ammonia-lyase (EC 4.3.1.7), an adenosylcobalamin-dependent enzyme. Consequently, to grow on ethanolamine as a sole nitrogen source, Bacillus megaterium requires vitamin B12. Identification of B. megaterium mutants deficient for growth on ethanolamine as the sole nitrogen source yielded a total of 34 vitamin B12 auxotrophs. The vitamin B12 auxotrophs were divided into two major phenotypic groups: Cob mutants, which could use cobinamide or vitamin B12 to grow on ethanolamine, and Cbl mutants, which could be supplemented only by vitamin B12. The Cob mutants were resolved into six classes and the Cbl mutants were resolved into three, based on the spectrum of cobalt-labeled corrinoid compounds which they accumulated. Although some radiolabeled cobalamin was detected in the wild type, little or none was evident in the auxotrophs. The results indicate that Cob mutants contain lesions in biosynthetic steps before the synthesis of combinamide, while Cbl mutants are defective in the conversion of cobinamide to cobalamin. Analysis of phage-mediated transduction experiments revealed tight genetic linkage within the Cob class and within the Cbl class. Similar transduction analysis indicated the Cob and Cbl classes are weakly linked. In addition, cross-feeding experiments in which extracts prepared from mutants were examined for their effect on growth of various other mutants allowed a partial ordering of mutations within the cobalamin biosynthetic pathway.

The roles of HPr and FPr in the utilization of fructose by Escherichia coli

A mutant impaired in FPr activity was isolated. The altered gene ( fpr), which was located near min. 44 on the E. coli genome, was transferred by phage-mediated transduction to appropriate recipients that lack HPr ( ptsH), or Enzyme II man ( ptsM), or neither. The rates of growth on fructose of such transductants indicate that phosphate from PEP is transferred predominantly via FPr to fructose that enters the cells by Enzyme II fru, but that HPr can play a role in transferring phosphate to fructose taken up via Enzyme II man

Control of arg gene expression in Salmonella typhimurium by the arginine repressor from Escherichia coli K-12.

The regulation of synthesis of arg enzymes in Salmonella typhimurium by the arginine repressor of Escherichia coli K-12 has been reevaluated using a strain of S. typhimurium in which the argR gene was rendered nonfunctional by inserting the translocatable tetracycline-resistance element Tn10 into the argR gene. In contrast to previous studies, the introduction of the argR+ allelle of E. coli on an F-prime factor to the argR::Tn10 S. typhimurium strain reduced the synthesis of arg enzymes to essentially wild-type levels. The elevated levels of arg enzymes observed in other hybrid merodiploids may have been the consequence of the formation of hybrid repressor molecules. The readily scoreable phenotype of tetracycline resistance facilitated establishing linkage of cod and argR (0.6% cotransduction) by P22 phage-mediated transduction.

Chi activity during transduction-associated recombination.

Chi is a genetic element that stimulates phage lambda recombination by the Escherichia coli recBC pathway during lytic infection [Stahl, F. W. (1979) Annu. Rev. Genet. 13, 7--24]. Herein we show that chi in lambda prophage influences exchange distribution in P1 phage-mediated transduction and in conjugation. This demonstration encourages the view that chi may influence genetic exchange in E. coli in the total absence of lambda.

Transformation and transduction of Bacillus subtilis strains with the Bsu R restriction-modification system by means of modified and unmodified DNA of pUB110 plasmid

During transformation of B. subtilis cells with the Bsu R restriction-modification system by means of pUB110 plasmid restriction and modification of the plasmid DNA occurs. The effect of restriction on the transformation frequency is relatively weak, bringing about 20-fold decrease only. When using cells of a modifying recipient, the frequency of AR9 phage-mediated transduction of unmodified plasmid DNA is also relatively little decreased. The frequency of transduction by chromosomal markers, under the same conditions, falls much lower.

Isolation and characterization of multiflagellate mutants of Pseudomonas aeruginosa.

Multitrichously polar flagellated mutants were isolated from a monotrichously flagellated strain of Pseudomonas aeruginosa. The ability of the mutant cells to swarm in semisolid media at given gel strengths was increased by the multiflagellation. Observations of the mutant cells by electron microscopy revealed that the number of flagella produced per cell cycle was increased. F116 phage-mediated transduction showed that the multiflagellation occurred by a single mutation and that the mutation sites were linked to a fla cluster of this organism.

Definition of additional flagellar genes in Escherichia coli K12.

Twenty-nine flagellar genes in Escherichia coli K12 have previously been assigned to three regions of the genome. Flagellar region I is located between pyrC and ptsG, region II between aroD and uvrC, and region III between uvrC and his. In this study, flagellar mutants in Escherichia coli K12 were obtained by selection for resistance to the flagellotropic phage, chi. They were analyzed in complementation tests using P1 phage-mediated transduction. In addition to the fla genes already described, eight more flagellar genes were identified. This analysis defined six more fla genes in region I (flaU, etc.), one more in region II (flbB) and one more in region III (flbC). Region I was shown to include at least 12 fla cistrons. Complementation analysis with polar Mu phage-induced Fla- mutants and with lambda fla phage defined four transcriptional units in region I. These were: flaU, flbA-flaW-flaV-flaK-flaX-flaL-flaY-flaM, flaZ and flaS- flaT, with transcription proceeding from left to right. The flB gene was found to be part of an operon: flB-flaI in region II. In region III, a previously unidentified gene flbC was located between hag and flaN.

Lambda bacteriophage-mediated transduction of ColE1 deoxyribonucleic acid having a lambda bacteriophage-cohesive end site: selection of packageable-length deoxyribonucleic acid

An in vitro recombinant ColE1-cos lambda deoxyribonucleic acid (DNA) molecule, pKY96, has 70% of the length of lambda phage DNA. The process of lambda phage-mediated transduction of pKY96 generated a small amount of transducing phage particles containing ColE1-cos lambda DNA molecules of 80 or 101% of the length of lambda phage DNA, in addition to those containing original pKY96 DNA molecules. The newly isolated larger plasmid DNAs were transduced 100 times more efficiently than pKY96 DNA. Their structures were compared with that of a prototype pKY96 DNA, and the mechanism of the formation of these molecules is discussed.

Repair of ultraviolet-light damaged ColE1 factor carrying Escherichia coli genes for guanine synthesis

Hybrid ColE1 plasmids called ColE1-coslambda-qua A or ColE1-coslambda-gal can be efficiently tranduced into various E. coli K-12 cells through packaging into lambda phage particles. Using these plasmids, repair of ultraviolet-light (UV) damaged ColE1 DNAs was studied in various UV sensitive E. coli K-12 mutants. (1) The host mutations uvrA and uvrB markedly reduced host-cell reactivation of UV-irradiated ColE1-coslambda-guaA. (2) Pre-existing hybrid ColE1 plasmids had no effect on the frequency of lambda phage-mediated transduction of another differentially marked hybrid ColE1 DNAs. (3) ColE1-coslambda-guaA and ColE1-coslambda-gal DNAs could temporarily but not stably co-exist in E. coli K-12 recA cells. (4) The presence of ColE1-coslambda-gal in uvrB cells promoted the repair of super-infected UV-irradiated ColE1-coslambda-guaA about 7-fold. (5) The same ColE1-coslambda-gal plasmid in a uvrB recA double mutant did not have this promoting effect. These results indicate that the effect of resident hybrid ColE1 plasmids is manifested by the host recA+ gene function(s) and suggest that ColE1 plasmid itself provides norecA+-like functions.

Packaging of ColE1 DNA having a lambda phage cohesive end site

The mechanism of lambda phage-mediated transduction of hybrid colicin E1 DNAs of various lengths was studied, and factors influencing the formation of these transducing particles were investigated. The results were as follows: 1. The presence of a cohesive end site of lambda phage (coslambda) on colicin E1 DNA was essential for packaging of the DNA. 2. Packaging of colicin E1 DNAs, which carry coslambda with molecular sizes corresponding to 68% of that of lambda phage DNA, was observed in the absence of all known recombination functions of E. coli K-12 and of lambda phage. 3. Hybrid colicin E1 DNAs having coslambda with molecular sizes corresponding to 28% of that of lambda phage DNA were packaged within lambda phage particles as trimers; hybrid DNAs with coslambda of 40 and 47% of the length of lambda phage DNA were packaged as dimers; and those with molecular sizes of 68% of that of lambda phage DNA were packaged mostly as monomers. These results demonstrated that two factors are essential for the packaging of DNAs within lambda phage particles; the presence of coslambda on the DNA molecule and an appropriate size of DNA.

The genetics of mycobacteria and mycobacteriophages — A review

The genus Mycobacterium, despite its medical importance, has so far received relatively little attention from bacterial geneticists. Nevertheless examples of the transfer of genes from one strain to another by means of transformation, phage-mediated transduction and direct cellular contact have been reported. The modification of strains by experimental or naturally occurring lysogeny has been studied in some detail and there is evidence that phage may contribute significantly to variation within species. Mycobacteriophages have been the subject of genetic analyses and have proved of value in the study of certain aspects of host-induced phage modification. In addition phages are being used to develop typing systems for use in epidemiological studies. It is evident that much of interest and value awaits discovery by means of genetic analysis of the mycobacteria and their phages and hopefully the next decade or so will bring great advances in this subject.

Transduction in Klebsiella.

PHAGE-MEDIATED transduction has been reported in strains of four genera of Enterobacteriaceae: Salmonella, Escherichia, Shigella, and Proteus1. Clarke2 described possible transduction of a single Klebsiella pneumoniae auxotroph to prototrophy, but this has not been confirmed. A search of the literature failed to reveal any other report of transduction involving a member of the genus Klebsiella. Indeed, there does not seem to be any previous report of a system permitting transfer of chromosomal genetic determinants between strains of Klebsiella. This is rather surprising, for genetic studies might be expected to extend greatly the considerable amount of information accumulated as a result of biochemical studies with organisms of the Klebsiella group (often still referred to as Aerobacter aerogenes despite the taxonomic confusion which this name perpetuates3). Examples of studies which might well be enhanced by the availability of a system of genetic analysis are those on exopolysaccharide biosynthesis by Klebsiella strains (unpublished work of I. W. S. and J. F. W.); nitrate assimilation4; polyphosphate accumulation and degradation5; and many others. This communication describes the isolation of a phage capable of effecting transduction in certain strains of Klebsiella aerogenes.