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the mating pair formation system of plasmid rp4 defined by rsf1010 mobilization and donor-specific phage propagation.transfer functions of the conjugative plasmid rp4 (incp alpha) are distributed among distinct regions of the genome, designated tra1 and tra2. by deletion analyses, we determined the limits of the tra1 region, essential for intraspecific escherichia coli matings. the tra1 core region encompasses approximately 5.8 kb, including the genes traf, -g, -h, -i, -j, and -k as well as the origin of transfer. the tram gene product, however, is not absolutely required for conjugation but significantly incr ...19938407818
relaxase (trai) of incp alpha plasmid rp4 catalyzes a site-specific cleaving-joining reaction of single-stranded dna.conjugative dna transfer of the self-transmissible broad-host-range plasmid rp4 is initiated by strand- and site-specific cleavage at the nick site (nic) of the transfer origin (orit). cleavage results in covalent attachment of the plasmid-encoded relaxase (trai) to the 5'-terminal 2'-deoxycytidine residue at nic. we demonstrate that tyr22 is the center of the catalytic site of trai, mediating cleavage via formation of a phosphodiester between the dna 5' phosphoryl and the aromatic hydroxyl grou ...19938385350
stability of r-microbes: stabilization of plasmid vectors by the partitioning function of broad-host-range plasmid rp4.the genes for biosynthesis of the biodegradable polymer poly-beta-hydroxybutyric acid (phb) cloned from alcaligenes eutrophus h16 were used for synthesis of phb with recombinant escherichia coli strains. it was recognized that the phb-biosynthesis genes cause segregational instability to the plasmids used as vectors. recombinant phb-plasmids are rapidly lost from host cells and plasmid-free cells occur at high rates, even under conditions of selection for the plasmids. cloning the partitioning r ...19937763562
transfer of incp plasmids to extremely acidophilic thiobacillus thiooxidans.the broad-host-range incp plasmids rp4, r68.45, rp1::tn501, and and pub307 were transferred directly to extremely acidophilic thiobacillus thiooxidans from escherichia coli by conjugation at frequencies of 10 to 10 per recipient. the ability of t. thiooxidans to receive and express the antibiotic resistance markers was examined. the plasmid rp4 was transferred back to e. coli from t. thiooxidans at a frequency of 1.0 x 10 per recipient.199216348639
virulence plasmid pjm1 prevents the conjugal entry of plasmid dna into the marine fish pathogen vibrio anguillarum 775.studies involving the introduction of cloned homologous genes into vibrio anguillarum revealed that several plasmids could not be conjugally introduced into v. anguillarum 775(pjm1), but were transmissible to the pjm1-cured derivative h775-3. recombinant pbr322 plasmids containing v. anguillarum genomic dna inserts were mobilized from escherichia coli donors, using prk2013, into v. anguillarum h775-3 recipients at frequencies of 10(-6) to 10(-5) per recipient. when identical matings were perform ...19921336793
the dehalogenase gene dehi from pseudomonas putida pp3 is carried on an unusual mobile genetic element designated deh.as a result of the production of two dehalogenases (dehi and dehii), pseudomonas putida pp3 utilized halogenated alkanoic acids, such as 2-monochloropropionic acid (2mcpa), as sole sources of carbon and energy. the dehi gene (dehi) was carried on a mobile genetic element (deh) located on the chromosome of strain pp3. deh recombined with target plasmid dnas at high frequencies (e.g. 3.8 x 10(-4) per rp4.5 plasmid transferred). the regulated expression of dehi was detected in p. putida, pseudomona ...19921312533
a gene near the plasmid psa origin of replication encodes a nuclease.we have cloned and sequenced a gene (nuc) from the incw plasmid psa which shows amino acid sequence similarity to staphylococcal nuclease (ec 3.1.4.7) and to the parb locus of plasmid rp4. the 525 bp open reading frame encodes a 174-amino-acid potential polypeptide of 19.7 kda. expression of the gene was confirmed using an in vitro transcription-translation assay which produced a protein of identical size. nuclease activity was demonstrated using dna as the substrate in toluidine blue-dna agar p ...19921560781
korb protein of promiscuous plasmid rp4 recognizes inverted sequence repetitions in regions essential for conjugative plasmid transfer.we have constructed a rp4 korb overproducing strain and purified the protein to near homogeneity. korb is a dna binding protein recognizing defined palindromic 13-bp sequences (tttagcsgctaaa). inverted sequence repetitions of this type, designated ob, are present on rp4 12 times. ob-sequences are localized in replication and maintenance regions as well as in the regions tra1 and tra2 essential for conjugative transfer. all sites found in tra regions by computer search act as targets for specific ...19921579485
a directional, high-frequency chromosomal mobilization system for genetic mapping of rhizobium meliloti.a system for mapping of the rhizobium meliloti chromosome that utilizes transposon tn5-mob, which carries the mobilization site of incp plasmid rp4 (r. simon, mol. gen. genet. 196:413-420, 1984), was developed. insertions of tn5-mob that were located at particular sites on the r. meliloti chromosome were isolated and served as origins of high-frequency chromosomal transfer when incp tra functions were provided in trans. this approach is, in principle, applicable to any gram-negative bacterium in ...19921309521
sequence similarities between the rp4 tra2 and the ti virb region strongly support the conjugation model for t-dna transfer.transfer genes of the incp plasmid rp4 are grouped in two separate regions, designated tra1 and tra2. tra2 gene products are proposed to be mainly responsible for the formation of mating pairs in conjugating cells. to provide information relevant to understanding the function of tra2 gene products, the nucleotide sequence of the entire rp4 tra2 region is presented here. twelve open reading frames were identified in the tra2 core region, being essential for intraspecific escherichia coli matings. ...19921400366
sequence analysis and characterization of the mobilization region of a broad-host-range plasmid, ptf-fc2, isolated from thiobacillus ferrooxidans.the nucleotide sequence of a 5,317-bp fragment which includes the region required for mobilization of broad-host-range plasmid ptf-fc2 was determined. a region of approximately 3.5 kb was required for plasmid mobilization, and orit was localized on a 138-bp fragment. polypeptides which corresponded in size and location to several of the open reading frames were detected in an in vitro transcription-translation system. three open reading frames essential for plasmid mobilization and two which aff ...19921400173
[conjugative transfer of plasmid dna between bacteria in soil].conjugative transfer of plasmid rp4 between populations of azospirilla and between escherichia coli and azospirillum brasilense in nonsterile soil has been investigated. the process of genetic exchange was realized at the early stages of interpopulational interactions, further on the process intensity was obviously rather low. population dynamics of azospirilla transconjugates in soil depends on the presence or the absence of additional food substrate.19921474945
dissection of incp conjugative plasmid transfer: definition of the transfer region tra2 by mobilization of the tra1 region in trans.we constructed a transfer system consisting of two compatible multicopy plasmids carrying the transfer regions tra1 and tra2 of the broad-host-range incp plasmid rp4. in this system, the plasmid containing the tra1 region with the origin of transfer (orit) was transferred, whereas additional functions essential for the conjugative process were provided from the tra2 plasmid in trans. the tra2 region, as determined for matings between escherichia coli cells, maps between coordinates 18.03 and 29. ...19921556069
acquisition of a sucrose utilization system in escherichia coli k-12 derivatives and its application to industry.an escherichia coli strain, b-62, that was isolated from a clinical source and was epidemiologically unrelated to e. coli k-12 was the source of chromosomal dna for a sucrose utilization system (scr+) in the construction of a plasmid, pst621. the cloned insert of a gene encoding scr+ in pst621 conferred a sucrose-positive phenotype onto transformed cells of e. coli k-12 derivatives. sucrase activity of the transformants was as high as that which would correspond to a "gene dosage effect" of a ve ...19921622287
trak protein of conjugative plasmid rp4 forms a specialized nucleoprotein complex with the transfer origin.conjugative transfer of the self-transmissible incp plasmid rp4 requires the product of the rp4 trak gene. by using the phage t7 expression system, the trak gene product was efficiently overproduced and purified to near homogeneity. trak encodes a basic protein (pi = 10.7) of 14.6 kda that, as shown by dna fragment retention assay, interacts exclusively with its cognate transfer origin. the apparent equilibrium constant k(app) for the complex of trak and orit-dna was estimated to be 4 nm. footpr ...19921324929
[mu-induced deletions and mutations of erwinia carotovora chromosomes, including resident prophages e105 and 59].the plasmid rp4::mu cts62 in stably inherited by erwinia carotovora 268 strain. under the conditions of thermoinduction bacteriophage mu is segregated and completely eliminated more intensively than in escherichia coli cells. at thermoinduction the transposition of bacteriophage mu cts62 into different chromosomal sites takes place, causing the induction of chlorate resistant and auxotrophic mutants with the frequency of 10(-4). two clones deficient in production of 2 of the 4 resident prophages ...19921406759
a natural mutant of plasmid rp4 that confers phage resistance and reduced conjugative transfer.a natural isolate of rp4 (prc#116) acquired from the stanford university plasmid reference center differed from the wild-type incompatibility group p plasmid in several respects. cells of escherichia coli harboring prc#116 were resistant to the incp pili-specific bacteriophage prd1 and gu5, and transferred this plasmid at a lower efficiency than the wild-type rp4. phage sensitivity was restored, and transfer considerably improved in prc#116+ bacteria transformed with plasmid constructs containin ...19921587464
the divergent promoters mediating transcription of the par locus of plasmid rp4 are subject to autoregulation.the partitioning region of broad-host-range plasmid rp4 contains four genes (para, parb, parc, and pard) that encode products essential for partition activity. two divergently arranged promoters located in the intercistronic region between parc and pard mediate transcription of these genes. the transcriptional initiation sites for both promoters were determined by primer extension. transcriptional fusions were used to show that para, parb, and parc are combined in an operon, while pard constitut ...19921508044
nucleotide sequence and organization of genes flanking the transfer origin of promiscuous plasmid rp4.the nucleotide sequence of the relaxase operon and the leader operon which are part of the tra1 region of the promiscuous plasmid rp4 was determined. these two polycistronic operons are transcribed divergently from an intergenic region of about 360 bp containing the transfer origin and six close-packed genes. a seventh gene completely overlaps another one in a different reading frame. conjugative dna transfer proceeds unidirectionally from orit with the leader operon heading the dna to be transf ...19911665997
a thiostrepton-inducible expression vector for use in streptomyces spp.a shuttle expression vector containing the thiostrepton-inducible streptomyces lividans promoter, ptipa, and the origin of transfer from plasmid rp4 was constructed. cassettes containing a promoterless xyle gene upstream from a hyg gene were used to demonstrate thiostrepton-inducible expression from ptipa in both s. lividans and streptomyces ambofaciens, ptipa was estimated to be induced 60-fold or more in streptomyces ambofaciens.19911879704
construction of a mobilizable cloning vector for site-directed mutagenesis of gram-negative bacteria: application to rhizobium leguminosarum.a mobilizable cloning vector was constructed from defined fragments to serve as a suicide plasmid for site-directed mutagenesis. the new vector, pkok4, closely resembles plasmid pbr325. however, the inverted duplication existing in the latter was not introduced. the useful cloning sites of pbr325 (ecori, hindiii, ecorv, bamhi, sali, psti and pvui) were retained and are located in one of the three resistance markers, apr, cmr or tcr, respectively. also, in pkok4 the cmr gene retains its own promo ...19912013412
characterization and classification of actinobacillus (haemophilus) pleuropneumoniae plasmids.actinobacillus (haemophilus) pleuropneumoniae plasmids were characterized and classified. they were isolated from a pleuropneumoniae strains different in serotype, year isolated, or location from which isolated. six of 8 plasmids encoded streptomycin (sm) and sulfonamide (su) resistance (smsu). one of the other plasmids, pvm105, encoded ampicillin (ap) resistance and another, phm0, encoded no drug resistance. all smsu plasmids were transferred to escherichia coli strains by transformation. among ...19911785724
electroporation and conjugal plasmid transfer to members of the genus aquaspirillum.electroporation methods and conjugal matings were used to transfer several plasmid vectors to aquaspirillum dispar and aquaspirillum itersonii. the incompatibility p class plasmid rp4 was conjugally transferred from escherichia coli hb101 to these spirilla, and the transconjugants subsequently donated the molecule to plasmid-free e. coli and a. dispar strains via conjugal matings. high-voltage electrotransformation was used to transfer plasmids pucd2, psa151 and rp4 to a. dispar and a. itersonii ...19911907445
competition between strains of escherichia coli with and without plasmid rp4 during chemostat growth.escherichia coli strains j53(nal) and j53(rp4) were grown together in glucose-limited continuous cultures. based on the measured growth kinetic constants of the two strains, take-over of the cultures by j53(rp4) was predicted. however, in practice, an initial period of predominance by j53(rp4) was always followed by a prolonged period in which relative numerical proportions of the two strains oscillated widely. this period of oscillation was removed or greatly reduced when the difference between ...19911913355
detection of plasmid transfer from pseudomonas fluorescens to indigenous bacteria in soil by using bacteriophage phir2f for donor counterselection.the transfer of a genetically marked derivative of plasmid rp4, rp4p, from pseudomonas fluorescens to members of the indigenous microflora of the wheat rhizosphere was studied by using a bacteriophage that specifically lyses the donor strain and a specific eukaryotic marker on the plasmid. transfer of rp4p to the wheat rhizosphere microflora was observed, and the number of transconjugants detected was approximately 10 transconjugants per g of soil when 10 donor cells per g of soil were added; tr ...199116348599
conjugal transfer of megaplasmid 2 between rhizobium meliloti strains in alfalfa nodules.a dna fragment containing the rp4 mob function, as well as the gentamicin and spectinomycin resistance genes, was inserted by gene replacement onto the megaplasmid 2 (pm2) of rhizobium meliloti 0540 (inf eps), resulting in pg101 (inf eps). the self-transfer of pm2 and the mobilization of pm2 by plasmid rp4-4 were investigated during conjugation between pg101 and r. meliloti 2526 (nod). in filter conjugations, pm2 was readily mobilized by rp4-4. in addition to this, the self-transfer of one megap ...199016348248
influence of glycine betaine on the transfer of plasmid rp4 between escherichia coli strains in marine sediments.the conjugative transfer of plasmid rp4 between two strains of escherichia coli in a sterile marine sediment was enhanced by the presence of glycine betaine (frequency increased 20 to 40 times). the conjugation was also facilitated by the osmoprotection of donor cells. glycine betaine is a universal osmolyte and has been found in marine sediments at high concentrations. so this phenomenon could have epidemiological and sanitary importance by increasing the possibility of dissemination of some pl ...19901366575
[the genetic transfer, heredity and phenotypic expression of plasmid rp4::mu cts genes in bacillus cereus].the transcipients were obtained in intrageneric matings of e.coli donor harbouring the plasmid pr4::mu cts 62 with bac. cereus gp7 recipient cells with the frequency 10(-9). the transcipient clone bac. cereus 682 was selected having stably inherited and expressed the hybrid plasmid pr4::mu cts 62 genes for antibiotic resistance and temperature sensitivity. production of the bacteriophage mu cts 62 particles was not registered in the bacillary transcipient cells. the plasmid rp4::mu cts 62 genes ...19902114185
mechanism of integration of the broad-host-range plasmid rp4 into the chromosome of myxococcus xanthus.the site-specific recombination mechanism through which the plasmid rp4 has been previously shown to integrate into the chromosome of myxococcus xanthus has been investigated further. once integrated in one of the numerous chromosomal sites from two different strains, through a precise site on the plasmid, the latter can be excised either precisely or after a definite 14.5-kb deletion. in some cases, the integration is followed by different dna rearrangements that yield a higher rate of excision ...19902120716
plasmid rp4 host-lethal function kila and its repressor kora sequences are part of the cryptic tellurite resistance transposon tn521.the normally silent 4.5 kb tellurite resistance transposon tn521 of rp4 has been shown to carry sequences from both the flanking kila and kora loci of this broad host range plasmid. the major portion of both of these sequences were used as probes to examine dna homology in southern transfer hybridization experiments with plasmid recipients of tn521 chosen from varying incompatibility groups. in the case of every recipients molecule analyzed using either probe, dna homology was observed.19902165965
mobilization of escherichia coli r1 silver-resistance plasmid pjt1 by tn5-mob into escherichia coli c600.escherichia coli r1 is an ag(+)-resistant strain that, as we have shown recently, harbours at least two large plasmids, pjt1 (83 kb) and pjt2 (77 kb). tn5-mob was introduced into the e. coli r1 host replicon via conjugation on membrane filters. the transfer functions of plasmid rp4-4 were provided in this process and tn5-mob clones mated with e. coli c600 yielded ag(+)-resistant transconjugants. this mobilization procedure allowed transfer and expression of pjt1 ag+ resistance in e. coli c600. p ...19902169281
in vitro assembly of relaxosomes at the transfer origin of plasmid rp4.during initiation of conjugative transfer of dna containing the transfer origin (orit) of the promiscuous plasmid rp4, the proteins trai, traj, and trah interact and assemble a specialized nucleoprotein complex (the relaxosome) at orit. the structure can be visualized on electron micrographs. site- and strand-specific nicking at the transfer origin in vitro is dependent on the proteins trai and traj and on mg2+ ions. substrate specificity is directed exclusively towards the cognate transfer orig ...19902168553
partitioning of broad-host-range plasmid rp4 is a complex system involving site-specific recombination.the broad-host-range plasmid rp4 encodes a highly efficient partitioning system (par) that was previously mapped within the 6.2-kb psti c fragment. the essential functions were assigned to a region of 2.2 kb between fiwa and is21 (is8). on the basis of the nucleotide sequence data of the entire par locus and of in vitro and in vivo expression studies, three distinct loci encoding polypeptides of 9, 18, and 24 kda were identified. evidence for the expression of another polypeptide was found. a pu ...19902172207
[erwinia carotovora as a recipient system for the natural hybrid plasmid rp4::mu cts62].the plasmid rp4::mu cts62 is transferred from escherichia coli cells into a recipient strain erwinia carotovora 268 by conjugation with the frequency 1.5-5 x 10(-7) per donor cell. the maximal frequencies of transfer are obtained by cultivation of donor and recipient cells for 3-5 h on the filters. structural and functional validity of the plasmid in transconjugants is expressed in preservation of all antibiotic-resistant markers of rp4, thermosensitivity to growth at 42 degrees c as well as spo ...19902175013
cloning and analysis of rfb gene synthesizing the mannan 0 side chain of escherichia coli 09 lipopolysaccharide.the rfb gene encoding the proteins responsible for the synthesis of the repeating units (o side-chain) of escherichia coli 09 lipopolysaccharide was cloned into a conjugative plasmid rp4::minimu and was expressed in e. coli k-12.19902183549
covalent association of the trai gene product of plasmid rp4 with the 5'-terminal nucleotide at the relaxation nick site.formation of relaxosomes is the first step in the initiation of transfer dna replication during bacterial conjugation. this nucleoprotein complex contains all components capable of introducing a site- and strand-specific nick at a cognate transfer origin (orit) on supercoiled plasmid dna, thus providing the substrate for generation of the strand to be transferred. characterization of the terminal nucleotides at the orit nick site revealed that relaxation occurs by hydrolysis of a single phosphod ...19902191955
silver accumulation and resistance in escherichia coli r1.e. coli r1 contains at least 2 large plasmids (83 and 77 kb) while e. coli s1 was previously cured of the 83 kb plasmid. transformation using artificial competence, high-voltage electroporation, and plasmid mobilization experiments with the mobilizing plasmid rp4, failed to ascertain the 83 kb plasmid was responsible for ag(+)-resistance. silver accumulation by an ag(+)-sensitive e. coli s1 strain was 5-fold higher than an ag(+)-resistant e. coli r1 strain. the ag(+)-resistant e. coli r1 strain ...19902202786
[isolation of transmissible cointegrates of yersinia pestis plasmids pyv and pyt with the plasmid rp4::mu cts 62, incp1].the transmissible cointegrates of the yersinia pestis plasmids pyv and pyt with the broad host range plasmid rp4::mu cts62 of the incompatibility group incp have been constructed by the in vivo recombination. the cointegrative plasmid pkr14 (pyv76 omega rp4::mu cts62) conferred on the transconjugants the properties of ca2(+)-dependence at 37 degrees c, v-antigen synthesis, rp4 plasmid markers (apr, kmr, tcr), immunity to the lysis by the bacteriophage mu cts62 and incompatibility with the homolo ...19902233780
[the multiple effect of plasmid rp4::mu cts 62 in transcipient bacilli].transfer of conjugative hybrid plasmid rp4::mu cts 62 from escherichia coli into bac. cereus, bac. thuringiensis, bac. mesentericus and bac. polymyxa cells led to the multiple effects on the structure and physiology of bacillus cells. it has resulted in a decrease of the bacillus vitality, in the accelerated autolytic decay of cells, in the delay of cell growth and reproduction rate in liquid and solid media, in the disruption of ultrastructure of the cell membrane and its surface layer.19902273994
[the infection of bacilli by mu cts phage integrated into the plasmid].the infection of bacillus thuringiensis, b. cereus, b. mesentericus and b. polymyxa strains with temperate e. coli bacteriophage mu cts62 integrated into plasmid rp4 under conditions of conjugative transfer is shown possible. the investigated strains of bacilli are not able to produce intact phage particles but they acquire the thermosensitive property determined by the phage genome. gel electrophoresis and blot hybridization of dna have confirmed the transfer of mu cts62 genome or a part of it ...19902145498
two mechanisms necessary for the stable inheritance of plasmid rp4.plasmid rp4 is stably maintained in strains of escherichia coli and other gram-negative bacteria. inactivation of the plasmid primase gene (pri) or removal of the pstic fragment gave rp4 derivatives that are slightly unstably maintained in e. coli. removal of the tn 1 multimer resolution system (res and tnpr) did not lead to any detectable plasmid loss. removal of all three of these regions, however, gave rise to pnj5000 which is lost at high frequency. we have dissected the mechanisms causing t ...19892699039
direct selection for curing and deletion of rhizobium plasmids using transposons carrying the bacillus subtilis sacb gene.we have constructed derivatives of the transposon tn5 carrying the mob site (orit) of plasmid rp4, and an npti-sacb-sacr cassette [ried and collmer, gene 57 (1987) 239-246]. the mob site, in conjunction with the antibiotic-resistance markers carried on the transposons, allows identification of transposon inserts in cryptic plasmids by mobilisation to other strains. the sacb-sacr genes allow direct selection for the loss or curing of plasmids, because only strains which no longer contain an activ ...19892548927
traj protein of plasmid rp4 binds to a 19-base pair invert sequence repetition within the transfer origin.transfer of plasmid rp4 during bacterial conjugation requires the plasmid-encoded traj protein, which binds to the transfer origin (fürste, j. p., pansegrau, w., ziegelin, g., kröger, m., and lanka, e. (1989) proc. natl. acad. sci. u.s.a. 86, 1771-1775). as indicated by traj mutants, the traj protein is a constituent of the relaxosome, the initiation complex of transfer dna replication. the traj gene maps adjacent to the transfer origin (orit). the structural gene consists of a 372-base pair seq ...19892663846
anaerobic growth of escherichia coli on glycerol by importing genes of the dha regulon from klebsiella pneumoniae.the dha regulon of klebsiella pneumoniae specifying fermentative dissimilation of glycerol was mobilized by the broad-host-range plasmid rp4:mini mu and introduced conjugatively into escherichia coli. the recipient e. coli was enabled to grow anaerobically on glycerol without added hydrogen acceptors, although its cell yield was less than that of k. pneumoniae. the reduced cell yield was probably due to the lack of the coenzyme-b12-dependent glycerol dehydratase of the dha system. this enzyme in ...19892559947
lack of expression of rp4-specified beta-lactamase in azospirillum brasilense.plasmid rp4, which normally confers resistance to ampicillin (apr), tetracycline (tcr), and kanamycin (kmr) to its hosts, failed to express enhanced apr when transferred from escherichia coli to azospirillum brasilense which has its own intrinsic beta-lactamase. even in a beta-lactamase-deficient mutant, a. brasilense rg-d16, no increase in beta-lactamase or significant apr appeared following transfer of rp4. however, a. brasilense rg (rp4) and a. brasilense rg-d16 (rp4) did exhibit tcr kmr. whe ...19892699042
survival of and plasmid stability in pseudomonas and klebsiella spp. introduced into agricultural drainage water.cell survival and plasmid stability in pseudomonas fluorescens r2f and pseudomonas putida cym 318 containing respectively, plasmid rp4 and prk2501, and klebsiella aerogenes nctc 418 harboring plasmid pbr322 were studied in sterile and nonsterile agricultural drainage water under both aerobic and anaerobic conditions and in the absence and presence of added nutrients. both pseudomonas strains survived well in sterile drainage water incubated aerobically, with or without added nutrients. however, ...19892590305
[change in the ultrastructure of the cell wall of bacillus cereus, determined by rp4 mucts62 plasmid].the influence of plasmid rp4 mucts62, heterologous for b. cereus, on the growth rate of b. cereus strains ga 682 and 319 obtained in our earlier experiments and on changes in the ultrastructure of their cell walls in comparison with b. cereus initial strains gp 7 and dsm 318 has been studied. plasmid rp4 mucts62 with a wide spectrum of action has been found not only to determine the functional signs of resistance to antibiotics and thermal sensitivity in the heterologous host, but also to take p ...19892499144
conjugative transfer of promiscuous incp plasmids: interaction of plasmid-encoded products with the transfer origin.to characterize protein-dna interactions involved in the initiation of conjugative transfer replication, we isolated and sequenced the transfer origins (orit) of the promiscuous incp plasmids rp4 and r751. the central initiating event at the transfer origin of a conjugative plasmid is the cleavage at a unique site (nic) of the strand to be transferred to a recipient cell. this process can be triggered after the assembly of "relaxosomes" (plasmid dna-protein relaxation complexes), requiring plasm ...19892538813
establishment of gene transfer systems for and construction of the genetic map of a marine vibrio strain.two gene transfer systems were established for a marine bacterium, vibrio sp. strain 60. one was generalized transduction with a newly isolated bacteriophage, as3, and the other was conjugal gene transfer by the use of newly constructed transposon-facilitated recombination (tfr) donors. as3 transduced various chromosomal markers at frequencies of 10(-4) to 10(-6). tfr donors, which were constructed by introducing transposon tn10 into both plasmid rp4 and the chromosome, mediated the polarized tr ...19892539353
transfer and properties of some natural and suicide replicons in pasteurella multocida.we tested the transfer of several plasmids and transposons from escherichia coli to pasteurella multocida by filter mating. two plasmids, prktv5 (prk2013::tn7) and puw964 (prktv5::tn5), were derived from prk2013--a narrow-host-range plasmid with the broad-host-range incp conjugation genes. most p. multocida transconjugants obtained with prktv5 had tn7 insertions in the chromosome but some had insertions of the whole plasmid. by contrast, all the transconjugants obtained with puw964 had insertion ...19892561489
isr1, a transposable dna sequence resident in rhizobium class iv strains, shows structural characteristics of classical insertion elements.isr1 is a small transposable element, identified in rhizobium class iv strains by its high frequent mutagenic insertion into plasmid rp4. hybridization studies showed that isr1 is present in, multiple copies in rhizobium class iv strains. nucleotide sequence analysis revealed that isr1 has a length of 1260 bp and is characterized by perfect inverted repeats of 13 nucleotides followed by a stretch of 28/29 nucleotides with imperfect homology. the insertion under study generated a target site dupl ...19892544911
transfer of incp plasmids into stigmatella aurantiaca leading to insertional mutants affected in spore development.derivatives of the broad-host-range plasmid rp4, containing the wild-type or modified transposon tn5 were transferred by conjugation to various stigmatella aurantiaca isolates. the transposons and in some cases fragments of the plasmid as well were integrated into the chromosome. thus, insertional mutants have been obtained affected in spore formation in liquid culture.19882853291
[compatibility of transposable phages of escherichia coli and pseudomonas aeruginosa. i. co-development of phages mu and d3112 and integration of phage d3112 into rp4::mu plasmid in pseudomonas aeruginosa cells].the possibility of using a model system (which included rp4::mu plasmid and d3112 phage in pseudomonas aeruginosa cells) for analysis of compatibility of transposable escherichia coli phage mu and p. aeruginosa phage d3112, as phages and transposons, was studied. no interaction was observed during the vegetative growth of phages. the majority of the hybrid rp4::mu plasmids lost the mu dna after insertion of d3112 into rp4::mu. the phenomenon was not a result of transposition immunity. we conside ...19882840341
[sos-induction of the rp4 plasmid tet-determinant].induction of the tetracycline-resistance genes function by the inducer of the dna-repair and mutability sos-system, uv-light, has been tested. activity of the tet-genes residing on the plasmid rp4 in escherichia coli cells has been shown to be inducible by the low doses of tetracycline as well as by the mutagenic doses of uv-light. the induction was quantitatively registered by measuring the activity of beta-galactosidase of bacteriophage mud1 (ap, lac) inserted into the tet-genes of the plasmid ...19882848194
construction of transposons carrying the transfer functions of rp4.the transfer genes and origin of transfer of the wide host range plasmid rp4 have been cloned into the transposons tn1 and tn5. the newly constructed transposons can be used to mutagenize bacterial plasmids or the chromosome in species such as escherichia coli or rhizobium. it is then possible to mobilize the plasmid or chromosome using the transfer functions provided in cis by the transposon. these constructs may aid chromosome mapping in many gram-negative species by allowing the wider use of ...19882854282
mini-mulac transposons with broad-host-range origins of conjugal transfer and replication designed for gene regulation studies in rhizobiaceae.novel mini-mu derivatives were constructed, carrying a truncated laczya operon fused to the terminal 117 bp of the mu s-end, for the isolation of translational lac fusions by mini-mu-mediated insertion mutagenesis. different selectable markers (chloramphenicol resistance; gentamycin resistance) were introduced to allow selection for mini-mu insertions in different replicons and bacterial strains. a mini-mulac derivative carrying the site for conjugal transfer of plasmid rp4 (orit) and the origin ...19882838383
transfer of the ti plasmid from agrobacterium tumefaciens into escherichia coli cells.we have screened strains of agrobacterium tumefaciens for spontaneous mutants showing constitutive transfer of the nopaline ti plasmid ptic58 during conjugation. the ti plasmid derivatives obtained could be transferred not only to a. tumefaciens but also to e. coli cells. the ti plasmid cannot survive as a freely replicating plasmid in e. coli, but it can occasionally integrate into the e. coli chromosome. however, insertion in tandem of plasmids carrying fd replication origins (pfd plasmids) in ...19883049936
effects of lincomycin on synthesis of tem beta-lactamase by escherichia coli.sub-inhibitory concentrations of lincomycin slightly inhibit growth of escherichia coli carrying plasmid rp4 and cause a 2-fold increase in tem-2 beta-lactamase. to analyze this effect, cultures were pulse-labeled with [3h]leucine, chased with non-radioactive leucine and immunoprecipitated with anti-beta-lactamase antiserum. the synthesis rate of beta-lactamase was two times higher in inhibited cultures than in control cultures. no significant decrease of labeled enzyme occurred during the 30 mi ...19883290174
physical and functional mapping of two cointegrate plasmids derived from rp4 and tol plasmid pdk1.cointegrate plasmids were formed in vivo between the broad-host-range r-plasmid rp4 and two catabolic plasmids derived from pseudomonas putida hs1. one of these was the wild-type plasmid pdk1 encoding the complete inducible toluene/xylene (tol) catabolic pathway and one was pdkt1, a deletion derivative of pdk1 selected after growth of hs1 on benzoate and supporting growth on only toluene. the two plasmids formed, pdk2 and pdkt2 respectively, each consisted of a complete rp4 replicon in which was ...19883076182
expression of degradative genes of pseudomonas putida in caulobacter crescentus.the recombinant plasmid rp4-tol was transferred into caulobacter crescentus at a high frequency, and the plasmid was maintained for at least 50 generations. c. crescentus cells which contained rp4-tol grew on all the aromatic compounds that the plasmid normally allowed pseudomonas putida to grow on. reciprocal transfers from c. crescentus donor to p. putida or escherichia coli recipients were less efficient and occurred at frequencies of approximately 10(-3). some representative tol-specified en ...19873597317
[genetic determination of the vibriocinogenicity trait in vibrio cholerae of the el tor biotype].in two different strains of cholera vibrios two reca-dependent plasmids, pvib i (1.9-2.2 md) and pvib ii (5.2-5.8 md), have been detected. these plasmids determine the synthesis of vibriocin, coagulase and fibrinolysin, which has been established by the cotransformation of the dna of plasmids pvib i and pbr322 and by the transfer mobilization with the use of plasmid rp4.19873303760
[localization of structural genes of vibrio cholerae cholerae toxin using the recombinant plasmid rp4omega elt].the recombinant plasmid rp4 omega elt carrying escherichia coli heat-labile enterotoxin elt genes with 70-80% homology with genes vct of vibrio cholerae has been constructed. we used this plasmid to determine localization of the cholerae toxin genes vct on the map of vibrio cholerae cholerae. two types of the donors were revealed in matings of 10 strains of v. cholerae cholerae 569b/rp4 omega elt with the polyauxotrophic recipients rv31 and rv175: some strains had enhanced frequency of mobilizat ...19873319775
[isolation of the r'his plasmids of vibrio cholerae].v. cholerae strain vt5104 capable of donor activity in conjugation has been constructed by the genetic technique based on plasmid rp4::mucts62 integration into v. cholerae chromosome due to plasmid homology with mucts62 inserted into the chromosome. the gene for histidine synthesis has been mobilized and transferred into the recipient cells from vt5104 donor. the conjugants obtained are able to efficiently transfer his+ gene included into the plasmid structure in conjugation with eltor recipient ...19873474038
cloning of the cyo locus encoding the cytochrome o terminal oxidase complex of escherichia coli.the structural genes encoding the cytochrome o terminal oxidase complex (cyo) of escherichia coli have been subcloned into the multicopy plasmid pbr322 after the mu-mediated transposition of the gene locus from the bacterial chromosome onto the conjugative r plasmid rp4. introduction of cyo plasmids into strains (cyo cyd) lacking both terminal oxidases restored the ability of the strains to grow aerobically on nonfermentable substrates. strains carrying the cyo plasmids produced 5 to 10 times mo ...19873036778
mini-mu transposition of bacterial genes on the transmissible plasmid.using the prm30 plasmid, an aps deletion derivative of broad host range plasmid rp4 with integrated new minimu 5 (11 kb), we followed the transfer of escherichia coli chromosomal genes to the recipient strain. the minimu 5-mediated transposition of chromosomal genes occurs onto the plasmid with integrated minimu 5 rather than onto the "recipient" plasmid pnh602. the plasmid dna in recipient cells was detected by electrophoresis. one of the acquired hybrid plasmids ptb2 was analyzed genetically a ...19872826318
mode of insertion of the broad-host-range plasmid rp4 and its derivatives into the chromosome of myxococcus xanthus.the mode of insertion of the broad-host-range plasmid rp4 into the chromosome of myxococcus xanthus strain dz1 has been analyzed. the plasmid integrated in numerous sites of the chromosome and generated insertional mutations. there is a hot spot of integration located between 31.5 and 34.5 kb clockwise from the ecori site of the plasmid. in the absence of this segment the insertion can, however, take place, but much less efficiently. the presence of transposable elements on the plasmid decreases ...19872829249
nucleotide sequence of the kanamycin resistance determinant of plasmid rp4: homology to other aminoglycoside 3'-phosphotransferases.the kanamycin resistance determinant of the broad-host-range plasmid rp4 encodes an aminoglycoside 3'-phosphotransferase of type i. the nucleotide sequence of the kanamycin resistance gene (kmr) and the right end of the insertion element is8 of plasmid rp4 has been determined. the gene (816 bp) is located between is8 and the region (tra 1) encoding plasmid factors mediating bacterial conjugation. kmr and tra 1 are transcribed toward each other. the nucleotide sequence has been compared to five r ...19872832861
the association behaviour of beta-lactamases. sedimentation equilibrium studies in ammonium sulphate solutions.the beta-lactamases (ec 3.5.2.6) from tem plasmid rp4, bacillus licheniformis 749/c and enterobacter cloacae p99 were studied in solution over a wide concentration range by equilibrium sedimentation. though crystal symmetries indicate that all three enzymes are potentially dimeric in their crystal forms, in 50 mm-sodium cacodylate at ph 6.5 the enzymes show only a small tendency to associate, indicated by a weight-average mr (mw) at 3% (w/v) concentration about 9% greater than that of the monome ...19863492196
[characteristics of derivatives of the plasmid rp4 in a broad range of hosts with altered properties of maintenance and inheritance].hydroxylamine-induced mutants of the plasmid ppd6 (8.4 kb) were isolated which are resistant to high doses of tetracycline. one of the plasmids studied--ppd21 is a multicopy mutant, another one, ppd12 is a dimeric form of the ppd6 plasmid. the ppd12 plasmid is very unstable, its derivative, ppd13 spontaneous mutant acquiring stability but not the ability to resolve dna multimeric forms into monomeric forms. multicopy bireplicon ppd619 plasmid was constructed by joining in vitro ppd6 and puc19 pl ...19863026896
[structural-functional organization of r-plasmid pbs222 with a broad range of bacterial hosts].the broad host-range plasmid pbs222 is compatible with broad host-range plasmids of all known incompatibility groups and codes for tetracycline resistance. pbs222 is efficiently mobilized by inc p-1 plasmid rp4 and is also capable of conjugal transfer with low efficiency to different gramnegative microorganisms. the size of the plasmid (17.2 kb) has been determined and its physical map has been constructed. the plasmid harbours the unique sites for restriction endonucleases bglii, hindiii, hpai, ...19863027554
[molecular cloning and functional analysis of dna regions of plasmid rp4 determining the incompatibility properties].sau3a-generated dna fragments determining incompatibility functions of the plasmid rp4 were cloned on the vectors ptk16 and pbr322. inc+ recombinant plasmids were divided into two types: 1) expressing incompatibility only towards the homologous rp4 replicon, 2) expressing incompatibility - both towards the homologous rp4 replicon and towards the heterologous replicons of plasmids r906 and r751. for one member of the first type plasmids it was shown that the cloned inc+-specific insertion derived ...19863021575
[effective, plasmid rp4-dependent replication-transposition of dna of the transposable phage d3112 of pseudomonas aeruginosa in a heterologous escherichia coli system].the processes of replication and transposition of pseudomonas aeruginosa transposable phage d3112 in cells of escherichia coli (d3112) and e. coli (rp4::d3112) were studied. d3112 genome is a "silent cassette" ("conex-phage"--conditionally expressible) in e. coli cells incubated at 42 degrees c. two compulsory conditions for d3112 genome expression are incubation at 30 degrees c and the presence in cells of rp4 plasmid. processes of replication and transposition in e. coli are coupled. rp4 plasm ...19863021578
role and specificity of plasmid rp4-encoded dna primase in bacterial conjugation.the role of the dna primase of incp plasmids was examined with a derivative of rp4 containing tn7 in the primase gene (pri). the mutant was defective in mediating bacterial conjugation, with the deficiency varying according to the bacterial strains used as donors and recipients. complementation tests involving recombinant plasmids carrying cloned fragments of rp4 indicated that the primase acts to promote some event in the recipient cell after dna transfer and that this requirement can be satisf ...19863522540
lincomycin stimulates synthesis of tem-2 beta-lactamase by escherichia coli.lincomycin increased the tem-2 beta-lactamase activity of escherichia coli k-12 cells carrying plasmid rp4 at a concentration which slightly inhibited cell growth. in a control culture beta-lactamase activity reached its maximal level in late log phase, whereas when lincomycin was present beta-lactamase activity continued to increase into the stationary phase. lincomycin (100 micrograms/ml) inhibited both cell growth and protein synthesis by about 35% but stimulated beta-lactamase activity 2.5-f ...19863530127
molecular cloning of the plasmid rp4 primase region in a multi-host-range tacp expression vector.plasmid rp4 primase was overproduced by utilizing autoregulated high-level expression vector systems in escherichia coli and in four other gram-negative bacterial species. analysis of the products in e. coli revealed that in addition to the two primase polypeptides of 118 and 80 kda the pri region of rp4 encodes two smaller proteins of 16.5 and 8.6 kda. the transcript for the four rp4-specified products is polycistronic. the vector system used in e. coli is based on the plasmid pkk223-3 (brosius ...19863549457
mobilization of thiobacillus ferrooxidans plasmids among escherichia coli strains.nonconjugative thiobacillus ferrooxidans plasmids were mobilized at high frequencies among escherichia coli strains by the incp plasmid rp4 and at low frequencies by the incn plasmid r46, but not by the incw plasmid psa. the mobilization region of a nonconjugative t. ferrooxidans plasmid was located on a 5.3-kilobase t. ferrooxidans dna fragment.19853890747
[stability and dynamics of r-plasmids in escherichia coli populations in continuous cultivation].the stability of the conjugative plasmid rp4 and the nonconjugative plasmid pbs94 in escherichia coli c600 cells containing both plasmids was studied in continuous cultivation under chemostat and ph-stat conditions. the plasmids remained stable in the cells of the bacterial population for 100 generations, and no cells were found without the plasmids. the competition between strains with and without the plasmids in a mixed culture resulted in the removal of the plasmid-free strain from the popula ...19853892245
trimethoprim resistance plasmids in escherichia coli isolated from cases of diarrhoea in cattle, pigs and sheep.a total of 1572 isolates of escherichia coli obtained from the faeces of young farm animals with diarrhoea over the period 1980-1983 were screened for resistance to trimethoprim (tp). resistance to tp was detected in 263/954 (28%) of bovine isolates, 59/441 (13%) of porcine isolates and 15/177 (9%) of ovine isolates. seventy-five resistant isolates from separate outbreaks of infection on farms within a 25 mile radius of nottingham were examined in detail. sixty-eight (91%) of the 75 isolates wer ...19853897163
[oxidation of n-alkanes by pseudomonas aeruginosa strain carrying the plasmid pbs251].pseudomonas aeruginosa pao8 cannot use n-alkanes or their respective alcohols as a sole carbon source. however, it can grow on n-alkanes when plasmid pbs251 is transferred into its cells. the hybrid plasmid pbs251 is a plasmid rp4 containing genes which control the capability to grow on n-alkanes of the c6-c12 series. studies of n-alkane oxidation by p. aeruginosa pao8 carrying pbs251 have shown that this plasmid controls the inducible alkane and alcohol oxidizing activities; the subsequent step ...19853937960
[characteristics of reca-independent recombination of plasmids in e. coli cells producing restriction endonuclease ecori].the restriction endonuclease ecori dependent recombination of compatible plasmids has been studied in reca cells of escherichia coli. plasmid rp4 and the isogenic cole1 type plasmids psa14 or psa25, differing in restriction-modification rm ecori genes, have been used to study this type of recombination. ecori dependent recombination of plasmids is demonstrated in reca cells and, thus, is independent of general system of homologous recombination. the classes of recombinant plasmids isolated from ...19853025706
inhibition of conjugal transfer of r plasmids by nitrofurans.nifurzide is a nitrofuran with antibacterial activity. as nitrofurans have been reported to interact with dna, we tested the ability of nifurzide to inhibit plasmid transfer. inhibition of plasmid transfer between escherichia coli strains was obtained for ten plasmids belonging to nine incompatibility groups. the same effect was observed when plasmid rp4 was harboured in six different members of the enterobacteriaceae. inhibition depended on the reduction of the -no2 group of nifurzide and was o ...19853906037
transfer of plasmid rp4 to myxococcus xanthus and evidence for its integration into the chromosome.the broad-host-range plasmid rp4 and its derivative r68.45 were transferred to myxococcus xanthus dk101 and dz1; rp4 was maintained integrated in the chromosome. loss of plasmid markers occurred during the growth of the transconjugants, which could be prevented by selective pressure with oxytetracycline. the integrated plasmid was transferred back to escherichia coli often as rp4-prime plasmids carrying various segments of the m. xanthus chromosome. it also mediated chromosomal transfer between ...19853918015
[transfer of prophage mu into methylotrophic bacteria in the plasmid rp4].bacteriophage mu genome has been transferred into the cells of pseudomonas methanolica and methylobacterium sp. skf240, that are naturally resistant to the bacteriophage, as a fragment of a hybrid plasmid rp4::mu cts62. temperature induction of the bacteriophage results in host cell lysis. plasmid rp::mu cis62 is maintained in methylotrophic cells presenting a cointegrative structure. the genetic and electrophoretic, analyses of the dna isolated from transconjugant cells have confirmed the concl ...19852948119
[interaction of yersinia pestis bacteria with bacteriophage mu].yersinia pestis cells are shown to be sensitive to bacteriophage mu cts62 infection. lysis of bacteria has been shown to be more efficient on solid nutrient medium than in lb broth. 10(-5) pfu per ml is the maximal concentration of bacteriophage particles yielded from the broth cultures of bacteria. moi 0.1 has been used to obtain such yields of bacteriophage. lysogenization of yersinia pestis cells has not been achieved when the standard methods of bacteriophage infection have been used. it was ...19852948125
genetic analysis of tn7 transposition.the purpose of this work was to localize the dna regions necessary for the transposition of tn7. several deletions of tn7 were constructed by the excision of dna fragments between restriction sites. the ability of these deleted tn7s to transpose onto the recipient plasmid rp4 was examined. all the deleted tn7s isolated in this work had lost their transposing capability. the possibility of complementing them was studied using plasmids containing all or part of tn7. two deleted tn7s could not be c ...19852984518
f factor inhibition of conjugal transfer of broad-host-range plasmid rp4: requirement for the protein product of pif operon regulatory gene pifc.by the use of deletions, point mutations, and gene fusions, we show that the protein product of the f factor pifc gene is responsible for f factor inhibition of plasmid rp4 conjugal transfer. deletion analysis of pif sequences carried by psc101-f chimeric plasmids demonstrated that removal of all or part of the pifc coding sequence greatly decreased or abolished the ability of these plasmids to inhibit rp4 transfer. amber mutations in the pifc gene eliminated inhibition in an su- host strain but ...19852993231
[effective method of transduction with virulent phage pf16 using specific mutants of pseudomonas putida ppg1].a procedure of simple selection of pseudomonas putida ppg1 mutants is described. the mutants can be used for transduction with virulent pf16 phage, giving reliable results. the frequency of transduction of chromosomal markers ilv and trp was 10(-6). also, transduction of plasmid rp4 with phage pf16 was shown with the frequency of 10(-7).19853926609
transfer of transposable drug-resistance elements tn5, tn7, and tn76 to azotobacter beijerinckii: use of plasmid rp4::tn76 as a suicide vector.transposable elements tn5, tn7, and tn76 were transferred to azotobacter beijerinckii. evidence was obtained for the transposition of tn5 but cells of the majority of presumptive transposition isolates had abnormal morphologies and rapidly lost viability when subcultured. data are presented that indicate that plasmid rp4::tn76 behaves as a suicide vector upon transfer to this host, allowing the isolation of a. beijerinckii::tn76 isolates at a high frequency. nitrogen-fixing mutants and leucine a ...19852999852
[gene mutation and transfer caused by plasmid rp4::mu in vibrio cholerae]. 19852943082
[hybrid plasmid pbs251 containing genes for n-alkane degradation].the strain of pseudomonas aeruginosa bs316 utilizing h-alkanes of the c6-c12 series (alk+) harbours the 96 md plasmid pbs250. the use of plasmid rp4 to mobilize alk+ markers in conjugal transfer to pseudomonas aeruginosa and pseudomonas putida has resulted in isolation of transconjugants resistant to antibiotics (due to genes coded by plasmid rp4) and capable of growth on h-alkanes. a transconjugant from this series harbours plasmid pbs251, a derivative of plasmid rp4 containing the genes for oc ...19853025683
[plasmid recombination stimulated by restriction endonuclease ecori in vivo: formation of recombinant plasmids in reca+-cells of e. coli].the possible participation of restriction endonuclease ecori in recombination of compatible nonhomologous plasmids in e. coli cells has been studied. to study the process, plasmids rp4 and r245 have been transferred by conjugation into the recipient cells of e. coli harbouring one of isogenic plasmids, psa14 and psa25, different for the genes coding restriction endonuclease ecori. the genetic analysis of transconjugant phenotypes, coded by the plasmids, has permitted to register the recombinant ...19853025697
[expression of pseudomonas aeruginosa phage transposons in pseudomonas putida ppg1 cells. ii. zygotic induction--a necessary condition for the formation of defective lysogens].the transfer of hybrid plasmid rp4::pt (where pt is the genome of a transposable phage specific for pseudomonas aeruginosa) into recipient cells of p. putida strain ppg1 occurs with the same frequency as into p. aeruginosa, the homologous host for pt. approximately 1/3 of all ppg1 exconjugants carrying rp4 markers lost the capability to produce viable pt phage. in contrast, in a cross with homologous recipient p. aeruginosa all exconjugant clones contained nondefective prophages in the hybrid pl ...19853002910
genetic rearrangement of plasmids: in vivo recombination between a dehalogenation plasmid and multiple-resistance plasmid rp4 in pseudomonas sp.when moraxella plasmid puo1 encoding haloacetate dehalogenase and mercury resistance coexisted with incp-1 plasmid rp4 in pseudomonas sp., genetic exchange between the plasmids often occurred, probably by site-specific recombination. the recombinant plasmids obtained were classified into four groups on the basis of phenotype. representative plasmids for each group were analyzed for dna composition and function, and the mechanism for the formation of these plasmids was sought. they were inherited ...198516346824
[introduction of the hybrid plasmid rp4::d3112 into pseudomonas putida cells requires the presence of specific mutation in the phage genome].the wild type of d3112, a transposable phage of pseudomonas aeruginosa can not be introduced as a portion of the hybrid plasmid rp4::d3112 into pseudomonas putida cells. it is only possible when phage d3112 carries mutations designated lpc (lethal for p. putida and escherichia coli). analysis of heteroduplex molecules between dnas of phages d3112w+ and d3112lpc demonstrated the absence of nonhomology regions, which suggests that lpc is a point mutation. the lpc2 mutation was located within the i ...19846086454
[properties of potential vectors--derivatives of the broad-host--range plasmid rp4].nonconjugative deletion and recombinant derivatives of the rp4 plasmid are constructed. the plasmids can be used as vectors because they have relatively small molecular weights, unique cleavage sites for enzymes ecori, xhoi, bamhi, psti, kpni, bglii, salgi and hindiii (the plasmids prp401 and prp417 having six of these sites), and easily tested phenotypes (tcr, apr and gal+). in addition, all of them retain the broad host range property. also, the plasmid prp420 is a multicopy derivative capable ...19846094306
high frequency mobilization of gram-negative bacterial replicons by the in vitro constructed tn5-mob transposon.a dna fragment of the broad host range plasmid rp4 carrying the cis-acting dna recognition site for conjugative dna transfer between bacterial cells (mobsite) was cloned into the kanamycin-neomycin resistance transposon tn5. using conventional transposon mutagenesis techniques the new transposon, called tn5-mob, can easily be inserted into the host dna of gram-negative bacteria. a host replicon carrying tn5-mob is then mobilizable into any other gram-negative species if the transfer functions of ...19846094969
involvement of kil and kor genes in the phenotype of a host-range mutant of rp4.plasmid prp761 is a derivative of the promiscuous plasmid rp4, which has a tn76 insert 1.8 kb from its ecori site within the trfb region (barth 1979). this mutation was pleiotropic, having three effects: the plasmid is unstably maintained in e. coli, it reduces the growth rate of its host and it has suffered a reduction in host-range. we show that prp761 has reduced expression from both its kora and korb genes and that tn76 has inserted between them. fragment exchange experiments showed that thi ...19846097793
replication defective rp4 plasmids recovered after chromosomal integration.phh6000 is a composite replicon made by the in vitro ligation of the incp plasmid rp4 to a fragment of bacteriophage lambda capable of autonomous replication. derivatives were selected in which it had integrated into the escherichia coli chromosome by homologous recombination with the resident lambda prophage, and plasmids were subsequently regenerated from the integrated molecules. although of the same molecular size as phh6000, all had altered properties: those recovered from the chromosome of ...19846231650
[selection, analysis and mapping of mutations in the gene for resistance to kanamycin in plasmid rp4].we have tested possibilities of escherichia coli strains dependent on drugs streptomycin and paromomycin for selection of spontaneous mutations in the rp4 kan gene specifying resistance to aminoglycosids--kanamycin, neomycin and paromycin. a set of kan gene mutations were obtained, classified ad mapped.19846389260
genetic manipulation of the restricted facultative methylotroph hyphomicrobium x by the r-plasmid-mediated introduction of the escherichia coli pdh genes.the inability of hyphomicrobium x to grow on compounds such as pyruvate and succinate is most likely due to the absence of a functional pyruvate dehydrogenase (pdh) complex. further support for this was sought by studying the effect of the introduction of the escherichia coli pdh genes in hyphomicrobium x on the pattern of substrate utilization by the latter organism. these genes were cloned by in vivo techniques using the broad-host range conjugative plasmid rp4::mucts. plasmid rp4 derivatives ...19846393893
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