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analysis of bacteriophage p1 immunity by using lambda-p1 recombinants constructed in vitro.we describe the dissection and reconstruction of a complex control circuit, the p1 immunity system, by a method that involves inserting ecori-generated fragments of p1 dna into lambda vectors that can then be sequentially inserted into a bacterial cell. using these techniques we have isolated lambda-p1 hybrid phages that express the products of p1 genes c1, c4, ant, and ban and, in appropriately constructed lysogens, confirmed the roles played by the first three of these products in phage immuni ...1978364485
suppression of a thermosensitive dnaa mutation of escherichia coli by bacteriophage p1 and p7. 1978372960
mapping of a new hem gene in escherichia coli k12.a new type of haem-deficient mutant was isolated in escherichia coli k12 by neomycin selection. the mutant, designated sasx38, accumulated uroporphyrin, coproporphyrin and protoporphyrin. since it possessed normal ferrochelatase activity, it was assumed to be deficient in protoporphyrinogen oxidase activity. the gene affected in the mutant was designated hemg. mapping of the hemg gene by phage p1-mediated transduction showed that it was located very close to the chlb gene (frequency of cotransdu ...1979390093
plasmid cloning vectors for the conjugal transfer of dna from escherichia coli to streptomyces spp.we have constructed cloning vectors for the conjugal transfer of dna from escherichia coli to streptomyces spp. all vectors contain the 760-bp orit fragment from the incp plasmid, rk2. transfer functions need to be supplied in trans by the e. coli donor strain. we have incorporated into these vectors selectable antibiotic-resistance markers (amr, thr, spr) that function in streptomyces spp. and other features that should allow for: (i) integration via homologous recombination between cloned dna ...19921628843
isolation and characterization of intermediates in site-specific recombination.cre, the site-specific recombinase from bacteriophage p1, catalyzes a recombination reaction between specific dna sequences designated as lox sites. the breakage and rejoining of partners during this recombination process must be highly concerted because it has not been possible to detect intermediates of the reaction with wild-type cre. several mutant cre proteins have been isolated that produce significant amounts of a possible intermediate product of the recombination reaction. the product ha ...19872821547
hyperproduction of the sigma subunit of rna polymerase in a mutant of escherichia coli.a mutant of escherichia coli k12 is described in which sigma and alpha subunits of the dna-dependent rna polymerase (ec 2.7.7.6) are produced at the rates much higher than in the normal strain. the rate of synthesis for sigma subunit was found to be at least 10-times higher, though the rapid degradation of sigma polypeptides accompanied with the accelerated synthesis precludes accurate estimation of the extent of hyperproduction. the alpha subunit synthesis was about 5-times higher in this mutan ...19751107814
localization of stx, a determinant essential for high-level production of shiga toxin by shigella dysenteriae serotype 1, near pyrf and generation of stx transposon mutants.hfr strains of shigella dysenteriae serotype 1 were constructed by transient integration of an rp4 plasmid derivative carrying transposon tn501 into the shigella chromosome through tn501-mediated cointegration. the hfr strains were mated with escherichia coli k-12 recipients carrying various auxotrophic markers, and e. coli recombinants which had received prototrophic shigella genes were selected. some of the e. coli transconjugants produced high levels of a cytotoxin which was neutralized by bo ...19873040592
integration of r plasmid rts1 to the gal region of the escherichia coli chromosome.an r plasmid rts1 was integrated into the gal region of the chromosome of escherichia coli xa-7012 (gale) strain by the directed transposition technique. the integration of the rts1 genome was confirmed mainly by conjugation studies and also by transduction experiments using phage p1. as a result, it was found that the integrated genome contained genes responsible for kanamycin resistance, conjugal transferability, and for autonomous replication. as reported previously, rts1 is temperature sensi ...19751090604
periplasmic protein related to the sn-glycerol-3-phosphate transport system of escherichia coli.two-dimensional gel electrophoresis of shock fluids of escherichia coli k-12 revealed the presence of a periplasmic protein related to sn-glycerol-3-phosphate transport (glpt) that is under the regulation of glpr, the regulatory gene of the glp regulon. mutants selected for their resistance to phosphonomycin and found to be defective in sn-glycerol-3-phosphate transport either did not produce glpt or produced it in reduced amounts. other mutations exhibited no apparent effect of glpt. transducti ...1976770459
chloramphenicol resistance mutation in escherichia coli which maps in the major ribosomal protein gene cluster.localized mutagenesis and selection for streptomycin resistance were utilized to isolate a chloramphenicol resistance mutation in escherichia coli k-12 linked to the stra (rpsl) locus. bacteriophage p1 transduction verified the map position of the new resistance mutation at 72 min, placing it within a dense cluster of ribosomal protein genes. the map position differs from that of known cmla and cmlb mutations, which map at 18 and 21 min, respectively. ribosomes prepared from chloramphenicol-resi ...1979374348
lysogenic conversion of pasteurella by escherichia coli bacteriophage p1 cm.bacteriophage p1 cm can convert pasteurella pestis or p. pseudotuberculosis to chloramphenicol resistance and phage restriction, but no viable phage was induced from converted pasteurella strains.19724553681
the addition of lac+ chromosome fragments to the e. coli proa-prob-lac deletion 13 chromosome.escherichia coli with the proa-prob-lac deletion x111 (delta111) can be transduced with bacteriophage p1 propagated on a wild-type lac(+) donor. though the donor lac(+) genes cannot be integrated by replacement of the recipient delta111 marker, the transduction process has the characteristics generally associated with generalized transduction of bacterial genes. transduction does not require p1 helper infection, is stimulated by uv irradiation of transducing particles, and does require homology ...19724556177
a model for plasmid maintenance of bacteriophage p1.studies of the stability of p1 plasmid in a p1 cry escherichia coli lysogen have suggested a model for equipartition of plasmid copies. equipartition might be controlled by the detachment of p1 copies after replication, followed by their reattachment to membrane sites, in coordination with bacterial division.1978371479
chromosomal location of the mop (groe) gene necessary for bacteriophage morphogenesis in escherichia coli.the chromosomal location of a host gene, mop (groe), which is essential for the morphogenesis of several bacteriophages in escherichia coli, was determined by two- and three-factor transductional crosses using phage p1. cotransduction frequencies beteen mop and other markers were: aspa, 90%; ampa, 77%; frda, 73%; mel, 24%. the sequence of markers in the corresponding segment (mel to pura; 91.5 to 93.5 min) of the e. coli linkage map was shown to be mel--aspa--mop(groe)--ampa--frda--pur a.1978370345
partial reactivation of irradiated phage p1 by strain bs2 of escherichia coli. 19684880558
phage p1 modification of bacterial dna studied by generalized transduction. 19665331504
isolation and characterization of a glucosamine-requiring mutant of escherichia coli k-12 defective in glucosamine-6-phosphate synthetase.a mutant was isolated from escherichia coli k-12 which requires glucosamine or n-acetylglucosamine for growth. depriving the mutant of glucosamine resulted in a rapid loss of viability of the cells, followed by a decrease in the turbidity of the culture. when the mutant cells were resuspended in broth media containing 10% sucrose, the rod-shaped cells became spheroplasts. however, the presence of sucrose in the media did not prevent the cells from losing their viability. this mutant was shown to ...19715541523
[intergeneric conjugational hybridization of escherichia coli and salmonella typhimurium. 1. obtaining a salmonella hybrid possessing greater recipient activity in crosses with escherichia coli].intergeneric hybrids were selected from mating hfrh escherichia coli with f- salmonella typhimurium. the hybrid obtained from e. coli leu+ and pro+ genes possessed the increased recipient ability in the mating with e. coli hfrr1 (o--ilv--mete--ara). this hybrid lacked the ability to restrict the phage p1 dna propagated on e. coli k-12. the replacement of mutated uvra gene of salmonella for uvra+ gene of e. coli restore uvr+ phenotype of salmonella mutant.1977352802
in vitro packaging of a lambda dam vector containing ecori dna fragments of escherichia coli and phage p1.in this report we describe a coliphage lambda vector system for cloning endo r. ecori dna fragments. this system differs significantly from those previously described in two ways. first, restricted and ligated dna is encapsidated in vitro. second, with increasing lambda dna size in the range 78 to 100% that of wild-type, the efficiency of dna encapsidation into infectious phage particles markedly increases. for lambda wild-type dna the efficiency of in vitro packaging (10(6) to 10(7) plaques pro ...1977338419
the frequency of p1 transduction of the genes of escherichia coli as a function of chromosomal position: preferential transduction of the origin of replication.the frequencies with which the generalized transducing phage p1 transduced 26 selected markers on the e. coli. chromosome were measured. the frequencies were found to vary relative to argh+ = 1 from a maximum of 6.8 near the origin of replication to a minimum of 0.23 for a marker not far from the terminus. the low frequencies obtained for some markers were shown not to result from poor expression under the selective conditions employed. when plotted as a function of marker position on the chromo ...1977337128
naturally occurring plasmid carrying genes for enterotoxin production and drug resistance.escherichia coli strain 86, isolated from a piglet with diarrhea, carries plasmid-linked genes for resistance to tetracycline, streptomycin, and sulfonamides and for production of heat-labile and heat-stable enterotoxin. results of (i) genetic experiments involving conjugal transfer and phage p1-mediated transduction and (ii) physical experiments involving electron microscopic examination of plasmid dna and heteroduplex analysis show that a single conjugative plasmid carries the genes for drug r ...1977333581
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 ...197826660
expression and proteolytic processing of the dara antirestriction gene product of bacteriophage p1.the dara gene coding for one of the two bacteriophage p1 antirestriction functions is expressed late after infection or induction. the protein is made as a high-molecular-weight soluble precursor. this is proteolytically cleaved to the mature form, which is a structural component of the phage head. defective mutants of the phage have been found in which the synthesis of gpdara is normal but processing does not take place. these mutations all map to the same region of the p1 genome and we propose ...19873029955
bacteriophage p1-mediated generalized transduction in escherichia coli: structure of abortively transduced dna. 19806998107
evolution of l-1, 2-propanediol catabolism in escherichia coli by recruitment of enzymes for l-fucose and l-lactate metabolism.a mutant strain of escherichia coli capable of growth on l-1,2-propanediol was isolated previously. the mutant is characterized by constitutive production of a propanediol:nicotinamide adenenine dinucleotide (nad) oxidoreductase which is essential for the new growth property. in the present study, it is shown that phage p1 cotransduces the genetic locus conferring this property and the genes for the utilization of l-fucose. a further indication of a relationship between these two growth properti ...19744595205
ultraviolet sensitivity gene of escherichia coli b.the ultraviolet sensitivity gene of escherichia coli b was introduced into a k-12 recipient by transduction with phage p1. the uvs gene of e. coli b is cotransducible with the proc locus of k-12, is closely linked to tsx, is not linked to lacz, and only rarely to pure. the transductants are mucoid, filamentous on irradiation, and show plating-medium response. the order of markers is lacz proc tsx uvs pure.19684870274
transposition of tn4551 in bacteroides fragilis: identification and properties of a new transposon from bacteroides spp.tn4551, a clindamycin resistance (ccr) transposon from the r plasmid pbi136, was cloned onto an escherichia coli-bacteroides shuttle vector which could replicate normally in e. coli but was maintained unstably in bacteroides fragilis. to aid in cloning and to ensure maintenance of tn4551 in e. coli, a kanamycin resistance determinant (kmr) was inserted in the transposon. the transposon-bearing shuttle vector pfd197 was transformed into b. fragilis 638, and putative insertions of tn4551::kmr were ...19873038840
naturally occurring r.colbm plasmids belonging to the incfiii incompatibility group.two escherichia coli strains isolated from urinary tract infections were resistant to streptomycin, kanamycin, neomycin, tetracycline and sulphonamides. the strains also produced colicins b and m. the resistance to streptomycin, kanamycin and neomycin and the ability to produce colicins b and m could be transferred to an e. coli k12 recipient. resistance and colicinogeny markers were transferred together by conjugation, and did not segregate even after interrupted mating or phage p1-mediated tra ...19807014768
the c1 repressor of bacteriophage p1. i. isolation of the c1 protein and determination of the p1 dna region to which it binds. 19807001033
cloning and complementation analysis of the "frizzy" genes of myxococcus xanthus.fruiting-body formation in myxococcus xanthus involves the aggregation of cells into raised mounds, where they sporulate. "frizzy" mutants fail to aggregate into mounds, but rather aggregate into "frizzy" filaments (d.r. zusman 1982). the frizzy mutations (frz) were found to be genetically linked. the region of dna carrying the frz genes was cloned in escherichia coli by selecting for the kanamycin resistance element present on a transposon tn5 insertion linked to the frz genes. phage p1 mediate ...19852984519
requirement of the escherichia coli dnaa gene function for integrative suppression of dnaa mutations by plasmid r 100-1.the phenotype of escherichia coli dnaa missense and nonsense mutations was integratively suppressed by plasmid r100-1. the suppressed strains, however, could not survive when the dnaa function was totally inactivated. this was demonstrated by the inability of replacing the dnaa allele in the suppressed strain by a dnaa::tn10 insertion using phage p1-mediated transduction. when the intact dnaa+ allele was additionally supplied by a specialized transducing phage, lambda imm21 dnaa+, which integrat ...19882851703
the c1 repressor of bacteriophage p1 operator-repressor interaction of wild-type and mutant repressor proteins.the c1 repressor gene of bacteriophage p1 and the temperature-sensitive mutants p1c1.100 and p1c1.162 was cloned into an expression vector and the repressor proteins were overproduced. a rapid purification procedure was required for the isolation of the thermolabile repressor proteins. identification of the highly purified protein of an apparent molecular weight of 33,000 as the product of the c1 gene was verified by (i) the coincidence of partial amino acid sequences determined experimentally t ...19892678004
genetic analysis of the lytic replicon of bacteriophage p1. ii. organization of replicon elements.the region of bacteriophage p1 dna containing a lytic (vegetative) replicon has been identified by cloning p1 fragments into a phage lambda vector. we present the sequence of that replicon. using a novel fusion vector containing two p1 loxp recombination sites, we have developed a transformation assay for replicon function and have used that assay to identify some of the components of the p1 lytic replicon. among those components is a transcription promoter, p53, whose activity is essential for ...19892661830
isolation of a formamidopyrimidine-dna glycosylase (fpg) mutant of escherichia coli k12.the fpg+ gene of escherichia coli coding for formamidopyrimidine-dna glycosylase was previously cloned on a multicopy plasmid. the plasmid copy of the fpg+ gene was inactivated by cloning a kanamycin resistance gene into the open reading frame, yielding the fpg-1::knr mutation. this mutation was transferred to the chromosome in the following steps: (i) linearization of the plasmid bearing the fpg-1::knr mutation and transformation of competent bacteria (recb recc sbcb); (ii) selection for chromo ...19892651883
the antirepressor of phage p1. isolation and interaction with the c1 repressor of p1 and p7.two antirepressor proteins, ant1 and ant2, of molecular weight 42 and 32 kda, respectively, are encoded by p1 as a single open reading frame, with the smaller protein initiating at an in-frame start codon. another open reading frame, icd, 5' upstream of and overlapping ant1 is required for ant1 expression. using appropriate ant gene-carrying plasmids we have overproduced and purified ant1/2 in the form of a protein complex and ant2 as a single protein. sequence analysis confirmed the n-terminal ...19938224242
three additional operators, op21, op68, and op88, of bacteriophage p1. evidence for control of the p1 dam methylase by op68.the repressor of bacteriophage p1, encoded by the c1 gene, is responsible for maintaining the p1 prophage in the lysogenic state. previously, 11 c1 repressor binding sites or operators scattered over the whole genome of p1 have been found. from sequence analysis an asymmetric, 17-base pair consensus sequence, attgctctaataaattt, was derived. using a synthetic 15-base-long oligodeoxyribonucleotide as operator probe, we have identified three additional operators. we have mapped the operators at the ...19892536753
three escherichia coli heat shock proteins are required for p1 plasmid dna replication: formation of an active complex between e. coli dnaj protein and the p1 initiator protein.dna containing the plasmid origin of bacteriophage p1 is replicated in vitro by a protein fraction prepared from uninfected escherichia coli supplemented with purified p1 repa protein. it has previously been shown that the reaction required the e. coli dnaa initiator protein, the dnab helicase, dnac protein, rna polymerase, and dna gyrase. i show here that three e. coli heat shock proteins, dnaj, dnak, and grpe, are directly involved in p1 plasmid replication. purified dnaj, dnak, and grpe prote ...19902181445
generation of a 50,000-member human dna library with an average dna insert size of 75-100 kbp in a bacteriophage p1 cloning vector.a bacteriophage p1 cloning system that permits the isolation and amplification of cloned dna fragments as large as 100 kbp was described previously. we have now utilized a similar system to generate a 50,000-member human dna library with dna inserts ranging in size from 75 to 100 kbp. two major obstacles were overcome in constructing the library. the first concerned the mcrab restriction system of escherichia coli, which degrades dna containing mec and interferes with the recovery of cloned huma ...19901964591
a tn3 derivative that can be used to make short in-frame insertions within genes.a tn3 derivative was constructed to make small in-frame insertions within genes. the transposon contains the ura3 gene, the teta gene, a truncated lacz, and phage p1 loxp recombination sites at either end. insertions that have fused lacz to an open reading frame are lac+ because they express the truncated lacz. in the presence of the phage p1 cyclization recombinase cre, the transposon can delete the ura3, teta, and lacz genes between the two loxp sites. the remaining short imperfect palindrome ...19911647034
d-arabinose metabolism in escherichia coli b: induction and cotransductional mapping of the l-fucose-d-arabinose pathway enzymes.d-arabinose is degraded by escherichia coli b via some of the l-fucose pathway enzymes and a d-ribulokinase which is distinct from the l-fuculokinase of the l-fucose pathway. we found that l-fucose and d-arabinose acted as the apparent inducers of the enzymes needed for their degradation. these enzymes, including d-ribulokinase, appeared to be coordinately regulated, and mutants which constitutively synthesized the l-fucose enzymes also constitutively synthesized d-ribulokinase. in contrast to d ...19883056899
chromosomal location of mutations affecting the regualtion of biotin synthesis in escherichia coli.the chromosomal locations of biotin regulatory mutations, bira, bior, and dhbb, of escherichia coli are determined by transduction using phage p1. all mutant genes are mapped between bfe and supm.19751097077
dual regulatory control of a particle maturation function of bacteriophage p1.a unique arrangement of promoter elements was found upstream of the bacteriophage p1 particle maturation gene (mat). a p1-specific late-promoter sequence with conserved elements located at positions -22 and -10 was expected from the function of the gene in phage morphogenesis. in addition to a late-promoter sequence, a -35 element and an operator sequence for the major repressor protein, c1, were found. the -35 and -10 elements constituted an active escherichia coli sigma(70) consensus promoter, ...200111418548
transductional analysis of chromosome replication time.following transduction of exponentially growing cultures of escherichia coli with phage p1, cells with recombinant phenotype begin to increase in number after an initial lag of about one generation time. we show that transductants for markers located at different positions on the chromosome begin to increase at different times, in reverse order to that in which they are replicated. the period over which this happens is equal in duration to the time taken to replicate the chromosome and we have u ...19873325777
genetic mapping of xtha, the structural gene for exonuclease iii in escherichia coli k-12.the genes xtha, pnca, and pabb were ordered relative to others by two- and three-factor transductional crosses with bacteriophage p1. the genes studied span 2 min (2%) of the genetic map of escherichia coli k-12 in the clockwise sequence phes-pfkb-xtha-pnca-gap-pabb-fadd. eleven independently derived xth mutations were examined; all were known to affect exonuclease iii and its associated endonuclease ii activity, and all were mapped in the xtha region. pnca mutations were found to confer resista ...1976780339
productive interaction between the chromosome partitioning proteins, para and parb, is required for the progression of the cell cycle in caulobacter crescentus.in caulobacter crescentus the partitioning proteins para and parb operate a molecular switch that couples chromosome partitioning to cytokinesis. homologues of these proteins have been shown to be important for the stable inheritance of f-plasmids and the prophage form of bacteriophage p1. in c. crescentus, parb binds to sequences adjacent to the origin of replication and is required for the initiation of cell division. additionally, parb influences the nucleotide-bound state of para by acting a ...200312603730
genetic location of genes encoding enterobacterial common antigen.a new rff mutation (rff-726) of escherichia coli is described which affects the biosynthesis of the enterobacterial common antigen. this mutation was detected in an rfe-defective strain. a tn10 insertion near the rfe locus was isolated to facilitate further mapping. both mutations rfe and rff were mapped by transduction with bacteriophage p1, giving the gene order ilv rfe rff uvrd mete. the f' factor f14 was able to complement both mutations rfe and rff, whereas the f' factor f16 could complemen ...19853894334
transduction of linked genetic characters of the host by bacteriophage p1. 195513267987
bacteriophage p1 carries two related sets of genes determining its host range in the invertible c segment of its genome.the bacteriophage p1 genome carries an invertible c segment consisting of 3-kb unique sequences flanked by 0.6-kb inverted repeats. host range mutations of p1 have been mapped in the c segment region. p1 derivatives carrying insertions and deletions in the left half of the c segment in one of two orientations termed c(+) do not affect the plaque-forming ability on escherichia coli k12 and e coli c, whereas those having insertions in the right half of the c segment fail to form plaques on these h ...19846100576
electron microscopy study of early lytic replication forms of bacteriophage p1 dna.p1 replication intermediates were isolated from the intracellular dna of lytically infected cells and analyzed by electron microscopy. at early times in infection replication intermediates were mainly of two types, circular theta- and sigma-shaped molecules plus a small proportion of linear bubble-shaped molecules. at later times in infection sigma molecules were the predominant replicating form. in contrast, sigma molecules were rarely found in recombination deficient, reca, infected cells. the ...19836359666
generalized transduction between salmonella typhi and salmonella typhimurium by phage j2 and characterization of the j2 plasmid in escherichia coli.phage j2, a p1-like phage in salmonella typhi, was heteroimmune to phage p1 and existed in the lysogenic state as a plasmid of molecular size 58.6 mdal. the phage j2 plasmid was incompatible with the p1 plasmid (incy group). a j2-sensitive mutant of salmonella typhimurium lt2 was isolated by transduction of j2ap phage into lt2 followed by curing of the prophage. the mutant was used to demonstrate transduction between s. typhi and s. typhimurium by phage j2.19836363617
a new family of mobilizable suicide plasmids based on broad host range r388 plasmid (incw) and rp4 plasmid (incpalpha) conjugative machineries and their cognate escherichia coli host strains.we describe the construction of the psw family of conditionally replicating plasmids which are based on the incx oriv origin (oriv(r6kgamma)) of replication that is dependent on the pir-encoded protein. we constructed several escherichia coli derivatives expressing pir from different chromosomal loci, and the pir gene could be transduced by phage p1 to any e. coli strain. these chromosomal constructions generate dapa and thya knockouts, which lead to diaminopimelate or thymidine auxotrophies, re ...200515748991
a dnab analog function specified by bacteriophage p7 and its comparison to the similar function specified by bacteriophage p1.evidence is presented that bacteriophage p7 specifies an analog of the e. coli dna replication protein, dnab. as in the related bacteriophage p1 (d'ari et al., 1975; ogawa, 1975), in lysogens of p7, the production of the analog protein is repressed and constitutive mutants could be isolated. such constitutive mutants could suppress efficiently the thermosensitivity of several dnab(ts) mutations and also rescue a strain carrying a dnab amber mutation. while neither p7 nor the mutant p1bacban (def ...19806993853
high-affinity arabinose transport mutants of escherichia coli: isolation and gene location.the gene araf, the product of which is the l-arabinose-binding protein--a component of the high-affinity l-arabinose transport system, was located on the escherichia coli linkage map at 45 min. we established this location using bacteriophage p2 eductates and bacteriophage p1 cotransduction frequencies with the adjacent genetic loci, his (histidine biosynthesis) and mgl (methylgalactoside transport). in addition, we isolated a number of mutants that phenotypically exhibited altered high-affinity ...19817024251
structural and functional analysis of tn4430: identification of an integrase-like protein involved in the co-integrate-resolution process.the 4149-bp transposon tn4430 from bacillus thuringiensis is delineated by 38-bp inverted repeats and codes for a 113-kd protein that shares homology with the transposases (tnpa) of tn3, tn21 and tn501. through transpositional recombination, this protein generates the formation of co-integrates between both donor and target replicons, with duplication of tn4430 molecules. these features are characteristic of transposons of the tn3 family (class ii elements). the second step of the transposition ...19882842151
the assembly of large bacs by in vivo recombination.we have developed a method for recombining bacterial artificial chromosomes (bacs) and p1 artificial chromosomes (pacs) containing large genomic dna fragments into a single vector using the cre-lox recombination system from bacteriophage p1 in vivo. this overcomes the limitations of in vitro methods for generating large constructs based on restriction digestion, ligation, and transformation of dna into escherichia coli cells. we used the method to construct a human artificial chromosome vector o ...200011112344
genetic studies of h group plasmids by bacteriophage p1 transduction.bacteriophage p1 transduction was used to study the incompatibility group h1 plasmid prg1251, molecular weight 120 x 10(6), and the incompatibility group h2 plasmid psd114, molecular weight 166 x 10(6). the order of resistance (r) determinants on psd114 was deduced from transduction and segregation experiments to be chloramphenicol-tetracycline-kanamycin-streptomycin. resistance to tellurium and to coliphages, which are properties also encoded by many h2 plasmids, were not transduced with the ot ...19817011519
[lethal and mutagenic action of uv light on salmonellae carrying wild or mutant alleles of the escherichia coli lexa-gene].to elucidate the reasons for the absence of uv-mutability in salmonella typhimurium, the lexa gene of escherichia coli has been transduced by phage p1 into s. typhimurium. the functioning of lexa+ allele of e. coli in the chromosome of salmonella failed to cause uv-mutability of the hybrid. the transfer of pkm101 plasmid into the lexa+ hybrid mediates the expressed uv mutability and uv-protective plasmid effect. this plasmid harboured by the lexa hybrid fails to increase uv-resistance and mutabi ...19817014362
a site-specific, conservative recombination system carried by bacteriophage p1. mapping the recombinase gene cin and the cross-over sites cix for the inversion of the c segment.the bacteriophage p1 genome carries an invertible c segment consisting of 3-kb unique sequences flanked by 0.6-kb inverted repeats. with insertion and deletion mutants of p1 derivatives the site-specific recombinase gene cin for c inversion) has been mapped adjacent to the c segment and the cix sites (for c inversion cross-over) have been located at the outside ends of the inverted repeats. inversion of the c segment functions as a biological switch and controls expression of the gene(s) respons ...19826327269
identification of the repressor and repressor bypass (antirepressor) polypeptides of bacteriophage p1 synthesized in infected minicells.p1 infected minicells synthesize approximately 50 phage-encoded polypeptides. phage expression is temporally controlled, demonstrating phage polypeptides synthesized both early and late after infection. the p1 repressor, gpcl1 (mr = 33,000), repressor bypass polypeptide, gpreb a (mr = 27,500) and cistron 10 product, (gp10) (mr = 64,000), have been identified by infection of minicells with p1 amber mutants. the beta-lactamase gene product (gpbla) carried by the closely related p7 and the chloramp ...19806991877
decrease in the linking number of plasmid dna in dnaa mutants of escherichia coli.we made use of agarose gel electrophoresis in the presence of chloroquine to examine the linking number of plasmids in temperature-sensitive dnaa mutants, including dnaa5 and dnaa46 mutants. the linking number of dna prepared from dnaa mutants growing at 37 degrees c was lower than that from wild type cells yet there was no significant difference when cells were grown at 28 degrees c. complementation analysis with a plasmid containing the wild type dnaa gene and phage p1-mediated transduction co ...19968573119
doc of prophage p1 is inhibited by its antitoxin partner phd through fold complementation.prokaryotic toxin-antitoxin modules are involved in major physiological events set in motion under stress conditions. the toxin doc (death on curing) from the phd/doc module on phage p1 hosts the c-terminal domain of its antitoxin partner phd (prevents host death) through fold complementation. this phd domain is intrinsically disordered in solution and folds into an alpha-helix upon binding to doc. the details of the interactions reveal the molecular basis for the inhibitory action of the antito ...200818757857
glutathione s-transferase-sspa fusion binds to e. coli rna polymerase and complements delta sspa mutation allowing phage p1 replication.bacteriophage p1 is unable to form plaques on e. coli hosts lacking a functional sspa gene. however, sspa mutants can be infected by p1, resulting in the synthesis of p1 early gene products and accumulation of p1 dna, but without p1 late gene product formation or host lysis. overexpression of the stringent starvation protein (sspa) as a glutathione-s-transferase fusion results in complementation of the sspa mutation and production of viable viral particles as in sspa+ strains. this suggests that ...19948198564
mutation of the htrb gene in a virulent salmonella typhimurium strain by intergeneric transduction: strain construction and phenotypic characterization.the htrb gene product of haemophilus influenzae contributes to the toxicity of the lipooligosaccharide. the htrb gene encodes a 2-keto-3-deoxyoctulosonic acid-dependent acyltransferase which is responsible for myristic acid substitutions at the hydroxy moiety of lipid a beta-hydroxymyristic acid. mass spectroscopic analysis has demonstrated that lipid a from an h. influenzae htrb mutant is predominantly tetraacyl and similar in structure to lipid iv(a), which has been shown to be nontoxic in ani ...19979287009
interaction of the dnak and dnaj chaperone system with a native substrate, p1 repa.dnak, the hsp70 chaperone of escherichia coli interacts with protein substrates in an atp-dependent manner, in conjunction with dnaj and grpe co-chaperones, to carry out protein folding, protein remodeling, and assembly and disassembly of multisubunit protein complexes. to understand how dnaj targets specific proteins for recognition by the dnak chaperone system, we investigated the interaction of dnaj and dnak with a known natural substrate, bacteriophage p1 repa protein. by characterizing repa ...200212237299
functional mapping of cre recombinase by pentapeptide insertional mutagenesis.cre is a site-specific recombinase from bacteriophage p1. it is a member of the tyrosine integrase family and catalyzes reciprocal recombination between specific 34-bp sites called loxp. to analyze the structure-function relationships of this enzyme, we performed large scale pentapeptide insertional mutagenesis to generate insertions of five amino acids at random positions in the protein. the high density of insertion mutations into cre allowed us to identify an unexpected degree of functional t ...200415218019
the anti-immunity system of phage-plasmid n15: identification of the antirepressor gene and its control by a small processed rna.n15 is a temperate virus of escherichia coli related to lambdoid phages. however, unlike all other known phages, the n15 prophage is maintained as a low copy number linear dna molecule with covalently closed ends. the primary immunity system at the immb locus is structurally and functionally comparable to that of lambdoid phages, and encodes the immunity repressor cb. we have characterized a second locus, imma, in which clear plaque mutations were mapped, and found that it encodes an anti-immuni ...199910594823
[expression of genes of prophage p1 escherichia coli in cells of phytopathogenic erwinia].it is shown that the temperate coliphage p1cmcts100 can transduce resistance to chloramphenicol in the cells of phytopathogenic bacteria erwinia horticola and e. carotovora subsp. atroseptica. in the latter case the extrachromosomal dna is inherited by recipient cells as the authentic prophage p1. the prophage p1 expresses rectification-modification ecop1, as well as the genes of lysogenic conversion in phytopathogenic erwinia. here the character of superinfection of transductants of e. atrosept ...200616786627
suppression of the lexc (ssba) mutation of escherichia coli by a mutant of bacteriophage p1.a new mutant of bacteriophage p1 designated lxc that suppresses the phenotype of lexc and ssba mutants of escherichia coli was isolated and characterized. the properties of lexc mutants suppressed by the lxc mutation include temperature sensitive growth at 42 degrees c, sensitivity to ultraviolet light and alkylating agents, and a nonmutagenic response following exposure to ultraviolet irradiation. a bac mutant of bacteriophage p1 that suppresses the temperature sensitivity of dnab mutants does ...19827050621
atp hydrolysis is required for dna cleavage by ecopi restriction enzyme.the type iii restriction endonuclease ecopi, coded by bacteriophage p1, cleaves unmodified dna in the presence of atp and magnesium ions. we show that purified ecopi restriction enzyme fails to cleave dna in the presence of non-hydrolyzable atp analogs. more importantly, this study demonstrates that ecopi restriction enzyme has an inherent atpase activity, and atp hydrolysis is necessary for dna cleavage. furthermore, we show that the progress curve of the reaction with ecopi restriction enzyme ...19957723013
mutator and antimutator effects of the bacteriophage p1 hot gene product.the hot (homolog of theta) protein of bacteriophage p1 can substitute for the escherichia coli dna polymerase iii theta subunit, as evidenced by its stabilizing effect on certain dnaq mutants that carry an unstable polymerase iii epsilon proofreading subunit (antimutator effect). here, we show that hot can also cause an increase in the mutability of various e. coli strains (mutator effect). the hot mutator effect differs from the one caused by the lack of theta. experiments using chimeric theta/ ...200616885451
the rpoe gene encoding the sigma e (sigma 24) heat shock sigma factor of escherichia coli.previous work has established that the transcription factor sigma e (sigma 24) is necessary for maintaining the induction of the heat shock response of escherichia coli at high temperatures. we have identified the gene encoding sigma e using a genetic screen designed to isolate trans-acting mutations that abolish expression from either htra or rpohp3, two promoters recognized uniquely by sigma e-containing rna polymerase. such a screen was achieved by transducing strains carrying a single copy o ...19957889935
assembling new escherichia coli strains by transduction using phage p1.a protocol is described that allows the transfer of genetic material from one escherichia coli strain to another using bacteriophage p1. p1 transduction can be used to construct new bacterial strains containing multiple alleles, to restore a locus to wild type, to move specific genetic markers from one strain to another, to relocate different mutant genes to a common genetic background, and to evaluate second-site suppression of a mutant allele. because of these abilities, p1 transduction remain ...201121815092
plasmid addiction genes of bacteriophage p1: doc, which causes cell death on curing of prophage, and phd, which prevents host death when prophage is retained.p1 lysogens of escherichia coli carry the prophage as a stable low copy number plasmid. the frequency with which viable cells cured of prophage are produced is about 10(-5) per cell per generation. here we show that a significant part of this remarkable stability can be attributed to a plasmid-encoded mechanism that causes death of cells that have lost p1. in other words, the lysogenic cells appear to be addicted to the presence of the prophage. the plasmid withdrawal response depends on a gene ...19938411153
phenotypes of dnaa mutants of escherichia coli sensitive to phenothiazine derivatives.the activation of dnaa protein by cardiolipin is inhibited by fluphenazine in vitro. we therefore examined the sensitivity of temperature-sensitive dnaa mutants of escherichia coli to fluphenazine and other phenothiazine derivatives. among the eight dnaa mutants tested, dnaa5, dnaa46 dnaa602, and dnaa604, mutants with mutations in the putative atp binding site of dnaa protein, showed higher sensitivities to phenothiazine derivatives than did the wild-type strain. the dnaa508 and dnaa167 mutants, ...19968804395
visualization of bacteriophage p1 infection by cryo-electron tomography of tiny escherichia coli.bacteriophage p1 has a contractile tail that targets the conserved lipopolysaccharide on the outer membrane surface of the host for initial adsorption. the mechanism by which p1 dna enters the host cell is not well understood, mainly because the transient molecular interactions between bacteriophage and bacteria have been difficult to study by conventional approaches. here, we engineered tiny e. coli host cells so that the initial stages of p1-host interactions could be captured in unprecedented ...201121745674
structure of the escherichia coli dna polymerase iii epsilon-hot proofreading complex.the epsilon subunit of escherichia coli dna polymerase iii possesses 3'-exonucleolytic proofreading activity. within the pol iii core, epsilon is tightly bound between the alpha subunit (dna polymerase) and subunit. here, we present the crystal structure of epsilon in complex with hot, the bacteriophage p1-encoded homolog of , at 2.1 a resolution. the epsilon-hot interface is defined by two areas of contact: an interaction of the previously unstructured n terminus of hot with an edge of the epsi ...200616973612
altered directionality in the cre-loxp site-specific recombination pathway.the site-specific recombinase cre must employ control mechanisms to impose directionality on recombination. when two recombination sites (locus of crossing over in phage p1, loxp) are placed as direct repeats on the same dna molecule, collision between loxp-bound cre dimers leads to excision of intervening dna. if two sites are placed as inverted repeats, the intervening segment is flipped around. cre catalyzes these reactions in the absence of protein co-factors. current models suggest that dir ...200111492999
escherichia coli sspa is a transcription activator for bacteriophage p1 late genes.the stringent starvation protein a (sspa), an escherichia coli rna polymerase (rnap)-associated protein, has been reported to be essential for lytic growth of bacteriophage p1. unlike p1 early promoters, p1 late promoters are not recognized by rnap alone. a phage-encoded early protein, lpa (late promoter activator protein, formerly called gp10), has been shown to be required for p1 late transcription in vivo. here, we demonstrate that sspa is a transcription activator for p1 late genes. our resu ...200312791143
inorganic polyphosphate essential for lytic growth of phages p1 and fd.transduction frequency with phage p1 had been observed to be very low in escherichia coli k-12 mutants lacking the operon (ppk1-ppx) responsible for the synthesis of inorganic polyphosphate (poly p). we now find that these mutants, for lack of poly p, are lysogenic for p1 and when infected with phage p1 produce only approximately 1% the number of infective centers compared with the wt host. both phage adsorption and release were unaffected. the host-encoded p1 late-gene transcriptional activator ...200717261797
the bacteriophage p1 hot gene, encoding a homolog of the e. coli dna polymerase iii theta subunit, is expressed during both lysogenic and lytic growth stages.the bacteriophage p1 hot gene product is a homolog of the theta subunit of e. coli dna polymerase iii. previous studies with hot cloned on a plasmid have shown that hot protein can substitute for theta, as evidenced by its stabilizing effect on certain dnaq mutator mutants carrying an unstable pol iii proofreading subunit (epsilon subunit). these results are consistent with hot, like theta, being a replication protein involved in stabilizing the intrinsically unstable epsilon proofreading functi ...200717482649
genome of bacteriophage p1.p1 is a bacteriophage of escherichia coli and other enteric bacteria. it lysogenizes its hosts as a circular, low-copy-number plasmid. we have determined the complete nucleotide sequences of two strains of a p1 thermoinducible mutant, p1 c1-100. the p1 genome (93,601 bp) contains at least 117 genes, of which almost two-thirds had not been sequenced previously and 49 have no homologs in other organisms. protein-coding genes occupy 92% of the genome and are organized in 45 operons, of which four a ...200415489417
pepa and argr do not regulate cre recombination at the bacteriophage p1 loxp site.in the lysogenic state, bacteriophage p1 is maintained as a low copy-number circular plasmid. site-specific recombination at loxp by the phage-encoded cre protein keeps p1 monomeric, thus helping to ensure stable plasmid inheritance. two escherichia coli dna-binding proteins, pepa and argr, were recently reported to be necessary for maintenance or establishment of p1 lysogeny. pepa and argr bind to regulatory dna sequences upstream of the cole1 cer recombination site to regulate site-specific re ...200818226834
phage like it hot: solution structure of the bacteriophage p1-encoded hot protein, a homolog of the theta subunit of e. coli dna polymerase iii.dna polymerase iii, the main replicative polymerase of e. coli, contains a small subunit, theta, that binds to the epsilon proofreading subunit and appears to enhance the enzyme's proofreading function--especially under extreme conditions. it was recently discovered that e. coli bacteriophage p1 encodes a theta homolog, named hot. the (1)h-(15)n hsqc spectrum of hot exhibits more uniform intensities and less evidence of conformational exchange than that of theta; this uniformity facilitates a de ...200415576035
effect of deletion mutation on the recombination activity of cre recombinase.cre recombinase from bacteriophage p1 is widely used in both in vitro and in vivo dna manipulations. based on a structural and functional analysis, three deleted cre mutants were constructed and expressed in escherichia coli. mutated recombinases were purified and their recombination activities were determined in vitro. our results revealed that the mutant with amino-terminal deletion retains the recombination activity as high as wild type cre; however, the carboxy-terminal deletion and the midd ...200515912212
escherichia coli transcription factor yncc (mcbr) regulates colanic acid and biofilm formation by repressing expression of periplasmic protein ybim (mcba).quorum-sensing signal autoinducer 2 (ai-2) stimulates escherichia coli biofilm formation through the motility regulator mqsr that induces expression of the putative transcription factor encoded by yncc. here, we show that yncc increases biofilm formation by repressing overproduction of the exopolysaccharide identified as colanic acid (corroborated by decreasing mucoidy and increased sensitivity to bacteriophage p1 infection). differential gene expression and gel shift assays demonstrated that yn ...200818309357
a novel role for site-specific recombination in maintenance of bacterial replicons.if daughter copies of unit-copy replicons recombine with each other, a replicon dimer results that cannot be partitioned equally to daughter cells at cell division. we present evidence that dimer formation interferes with plasmid equipartition in the case of a miniplasmid derived from the unit-copy plasmid prophage of bacteriophage p1. asymmetric partition occurs, leading to a relatively high rate of loss of the plasmid from the growing population. in contrast, the wild-type p1 plasmid is mainta ...19817026049
crystallization of doc and the phd-doc toxin-antitoxin complex.the phd/doc addiction system is responsible for the stable inheritance of lysogenic bacteriophage p1 in its plasmidic form in escherichia coli and is the archetype of a family of bacterial toxin-antitoxin modules. the his66tyr mutant of doc (doc(h66y)) was crystallized in space group p2(1), with unit-cell parameters a = 53.1, b = 198.0, c = 54.1 a, beta = 93.0 degrees . these crystals diffracted to 2.5 a resolution and probably contained four dimers of doc in the asymmetric unit. doc(h66y) in co ...200818997335
structure of the theta subunit of escherichia coli dna polymerase iii in complex with the epsilon subunit.the catalytic core of escherichia coli dna polymerase iii contains three tightly associated subunits, the alpha, epsilon, and theta subunits. the theta subunit is the smallest and least understood subunit. the three-dimensional structure of theta in a complex with the unlabeled n-terminal domain of the epsilon subunit, epsilon186, was determined by multidimensional nuclear magnetic resonance spectroscopy. the structure was refined using pseudocontact shifts that resulted from inserting a lanthan ...200616740953
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