| mapping of functions in the r-plasmid r388 by examination of deletion mutants generated in vitro. | mutant plasmids in which large segments of r388 dna are deleted were constructed in vitro from two r388::tna (tn801) plasmids, using the bamhi site of tna and the bamhi and bglii sites of r388. these deletion mutants permitted mapping of genetic functions into the restriction map of r388. | 1978 | 350713 |
| characterisation of tn1721, a new transposon containing tetracycline resistance genes capable of amplification. | r plasmid prsd1 contains tetracycline resistance (tet) genes in a 3.55 mdal-region capable of amplification by forming tandem repeats (mattes, burkardt and schmitt, molec. gen. genet., 1979). the repetitious tet element is itself part of a 7.2 mdal-transposon, named tn1721, as demonstrated by the following criteria; (i) tn1721 has been translocated to phage lambda. the resulting hybrid phage lambda tet contains the 7.2 mdal-insertion to the right of the attachment site, but not continguous with ... | 1979 | 377024 |
| characterization of sulfonamide resistance determinants and relatedness of bordetella avium r plasmids. | five plasmids, varying in size from 16 to 51.5 kb, were isolated from virulent strains of bordetella avium and compared by restriction endonuclease digestion and dna-dna hybridization. these plasmids confer resistance to streptomycin and sulfonamides, and three of the five also confer resistance to tetracycline, but they are not closely related. four of the plasmids, prl100, p4093, pcw, and pwam, carried determinants related to the heat-labile type i plasmid-mediated dihydropteroate synthase of ... | 1991 | 1661013 |
| structural and functional analysis of the origin of conjugal transfer of the broad-host-range incw plasmid r388 and comparison with the related incn plasmid r46. | we cloned and sequenced a 402 bp dna segment containing the origin of conjugal transfer (orit) of the incw plasmid r388. progressive deletions from each end of the sequence were assayed for orit activity. stepwise reductions in mobilization frequencies, representing the loss of functional elements, correlated with deletion of structural motifs in the sequence. a sequence of 330 bp of orit was sufficient for efficient mobilization. the first 86 bp of the sequence contains five tandemly repeated d ... | 1991 | 2038309 |
| general organization of the conjugal transfer genes of the incw plasmid r388 and interactions between r388 and incn and incp plasmids. | the complete conjugal transfer gene region of the incw plasmid r388 has been cloned in multicopy vector plasmids and mapped to a contiguous 14.9-kilobase segment by insertion mutagenesis. the fertility of the cloned region could still be inhibited by a coresident incp plasmid. the transfer region has been dissected into two regions, one involved in pilus synthesis and assembly (pilw), and the other involved in conjugal dna metabolism (mobw). they have been separately cloned. pilw also contains t ... | 1990 | 2170327 |
| the dhfri trimethoprim resistance gene of tn7 can be found at specific sites in other genetic surroundings. | the dhfri gene, mediating high-level trimethoprim resistance, was earlier found only on tn7. evidence is given here for an alternative location of this gene at a site identical to sites observed earlier for dhfrii on plasmid r388, dhfrv on plmo20, and aada on tn21. all these genes and dhfri are precisely inserted as discrete gtta-flanked elements at distinct loci in very conserved surrounding sequences. one of these dhfri insertions was observed to occur in association with a similarly inserted ... | 1990 | 2188588 |
| [new shuttle-type vectors for cloning in escherichia coli and agrobacterium tumefaciens]. | the vectors capable of replication in escherichia coli and agrobacterium tumefaciens have been constructed on the basis of the plasmid pub5502. the constructed vectors pva12, pva12-2, pva12-4 contain the mini-replicon and trimethoprim resistance gene (tp) of a broad host-range plasmid r388 (incw). the pva12 vector (8.8 kb) has been constructed by insertion of a kanamycin resistance gene (km) from the plasmid puc-4k into a psti site. it possesses 7 unique restriction sites for xhoi, smai, pvui, p ... | 1990 | 2233784 |
| transformation of penicillium chrysogenum to sulfonamide resistance. | penicillium chrysogenum has been transformed to sulfonamide resistance by vectors containing the dihydropteroate synthetase gene from plasmid r388 controlled by the promoter and terminator sequences of the p. chrysogenum trpc gene. transformation frequencies of four to ten transformants per microgram of vector dna were obtained. | 1989 | 2501160 |
| multiple high-frequency transpositions of tn5 in serratia marcescens 274 from a thermosensitive replication mutant of plasmid r388. | we have used the broad-host-range conjugative suicide plasmid vector described by sasakawa and yoshikawa [gene 56 (1987) 283-288] for transposon mutagenesis of serratia marcescens 274. we report multiple transposition events of tn5 from this vector. in addition, the unusual pattern of tn5 transposition might provide an insight into its regulation. | 1989 | 2553541 |
| location and characterization of two functions on rp1 that inhibit the fertility of the incw plasmid r388. | two fertility-inhibition functions which reduce r388 (incw) transfer were detected on rp1 (60 kb, incp). the respective genes, fiwa and fiwb, were mapped by transposon insertion mutagenesis to the regions between coordinates 32.8 to 31.7 kb (fiwa), and 59.8 to 0.8 kb (fiwb). the fiwa function occurs in a non-essential region of rp1 whereas fiwb is straddled by essential plasmid-maintenance and host-range determinants and apparently coincides (or overlaps) with the gene for tellurite-resistance. | 1989 | 2559940 |
| plasmid expression and maintenance during long-term starvation-survival of bacteria in well water. | strains of enteric bacteria and pseudomonads containing plasmid r388::tnl721 (tpr, tcr) or pro101 (hgr, tcr) were starved for over 250 days in sterile well water to evaluate effects of starvation-survival on plasmid expression and maintenance. viable populations dropped to between approximately 0.1 and 1% of the initial populations. escherichia coli(pro101) and pseudomonas cepacia(pro101) lost both viability and plasmid expression at a lower rate than strains containing r388::tnl721. three patte ... | 1989 | 2782868 |
| a series of tn5 variants with various drug-resistance markers and suicide vector for transposon mutagenesis. | a series of variants of transposon tn5 were constructed by replacement of the 2.7-kb central segment which encodes kanamycin resistance with various other resistance-coding genes: tetracycline, chloramphenicol, gentamicin, trimethoprim, streptomycin or ampicillin. a thermosensitive replication mutant of the broad-host-range transmissible plasmid r388 was also constructed for use as a suicide vector for the delivery of transposable elements. | 1987 | 2824292 |
| direct repeats flanking the bacteroides transposon tn4351 are insertion sequence elements. | the clindamycin-erythromycin resistance (ccr emr) region of the bacteroides transposon tn4351 is flanked by direct repeats. this study showed that the direct repeats are insertion sequence (is) elements. although both is elements can mediate transfer of the chloramphenicol (cmr) marker on pbr328 by cointegrate formation with the conjugal incw plasmid r388, is4351r-mediated transfer of cmr occurred at a consistently lower frequency than did the transfer mediated by is4351l. analysis of plasmids f ... | 1988 | 2826402 |
| transposon tn21 encodes a reca-independent site-specific integration system. | the incw plasmid r388 and the dna region of tn21 containing the smr and the sur genes are capable of reca-independent recombination. this recombination occurs at a relatively high frequency (up to 10(-4) recombinants per recipient molecule) and results in integration of the two plasmids. no detectable repeats are formed in the process. the crossover points have been confined to a 0.4-kb homologous segment in both plasmids which contains a 59-bp dna sequence presumably involved in the acquisition ... | 1988 | 2832705 |
| physical and genetic map of the incw plasmid r388. | | 1988 | 2853395 |
| [deletional and insertional analysis of the antitumorigenicity of the plasmid r388 in agrobacterium tumefaciens]. | the study of the plasmid r388 deletional derivatives has shown the antitumorigenicity determinant of the plasmid to be contained by the tra-operon. transposon tn5 has been used for selection of a number of insertional mutants having lost the antitumorigenic properties. the difference in the locations of tn5 inserts was found, all of the latter being localized within the tra-operon. the data obtained suggest the locus of the plasmid r388 responsible for its antitumorigenic properties to be locate ... | 1988 | 2855090 |
| suicide vector for transposon mutagenesis in pseudomonas solanacearum. | a suicide vector was constructed by cloning the transfer genes of the wide-host-range (incw group) plasmid r388 into the bamhi site of pbr325. this plasmid can deliver tn5 into pseudomonas solanacearum at frequencies ranging from 10(-6) to 10(-9) per recipient. | 1985 | 2993241 |
| site-specific recombination promotes linkage between trimethoprim- and sulfonamide resistance genes. sequence characterization of dhfrv and suli and a recombination active locus of tn21. | a new gene for trimethoprim resistance, dhfrv, found in several plasmid isolates with different characteristics, was sequenced and found to correspond to a peptide of 157 amino acids showing 75% similarity with the previously characterized, drug resistant dihydrofolate reductase of type i. the sequenced surroundings of dhfrv in plasmid plmo20, were found to be almost identical with genetic areas surrounding resistance genes in transposon tn21 and in r plasmid r388. the trimethoprim resistance ge ... | 1988 | 3054482 |
| predictive model of conjugative plasmid transfer in the rhizosphere and phyllosphere. | a computer simulation model was used to predict the dynamics of survival and conjugation of pseudomonas cepacia (carrying the transmissible recombinant plasmid r388:tn1721) with a nonrecombinant recipient strain in simple rhizosphere and phyllosphere microcosms. plasmid transfer rates were derived for a mass action model, and donor and recipient survival were modeled as exponential growth and decay processes or both. rate parameters were derived from laboratory studies in which donor and recipie ... | 1988 | 3355131 |
| characterization of a r plasmid-associated, trimethoprim-resistant dihydrofolate reductase and determination of the nucleotide sequence of the reductase gene. | the trimethoprim-resistant dihydrofolate reductase associated with the r plasmid r388 was isolated from strains that over-produce the enzyme. it was purified to apparent homogeneity by affinity chromatography and two consecutive gel filtration steps under native and denaturing conditions. the purified enzyme is composed of four identical subunits with molecular weights of 8300. a 1100 bp long dna segment which confers resistance to trimethoprim was sequenced. the structural gene was identified o ... | 1981 | 6261228 |
| tetracycline resistance transposon tn1721: reca-dependent gene amplification and expression of tetracycline resistance. | the 7.1-megadalton transposon tn1721 codes for inducible tetracycline resistance (tcr). the transposable element consists of a "minor transposon" (3.6 megadaltons) encoding functions required for transposition and a "tet region" (3.5 megadaltons) encoding resistance. multiple tandem repeats of the tet region can be generated by reca-dependent gene amplification. this feature of tn1721 has been used to analyze the relationship between gene dosage and tcr. derivatives of plasmid r388:tn1721 contai ... | 1981 | 6268611 |
| genetic and molecular characterization of tn21, a multiple resistance transposon from r100.1. | tge transposon tn21 has been transposed from r100.1 to plasmid pacyc184 and, from the resulting recombinants, to plasmid r388. the sites of insertion and the orientation of the element in several pacyc184::tn21 recombinants have been examined. restriction enzyme analysis of these recombinants has resulted in a detailed map of tn21; this is compared with the published maps of the relevant part of r100.1. heteroduplex analysis has shown short inverted repeat sequences at the ends of the element. w ... | 1982 | 6282806 |
| abnormal cointegrate structures mediated by gene b mutants of phage mu: their implications with regard to gene function. | a system where the transposition of mupapl (a derivative of phage mu carrying a determinant coding for ampicillin resistance) is followed from the small plasmid pml2 into the conjugative plasmid r388 has been used to investigate the influence on mu transposition of b, an early mu gene which is involved in normal phage dna synthesis. in the absence of active b protein a low level (about 1% of normal) of transposition was detected. roughly a third of these transpositional events was found to lead ... | 1982 | 6283319 |
| recombination involving transposable elements: role of target molecule replication in tn1 delta ap-mediated replicon fusion. | donor dna molecules carrying tn1 or tn3 deletion mutants do not need to replicate in order to participate in replicon fusion recombination events during which the tn1/tn3 element is duplicated. we have assayed tn1 delta ap-mediated replicon fusion events involving plasmid r388 and the bacteriophage lambda-derived plasmid p lambda cm, and we find that the role of the recipient molecule is distinct. when p lambda cm carries tn1 delta ap, replicon fusion occurs in more than 1% of all cells assayed, ... | 1983 | 6300910 |
| plasmid r46 provides a function that promotes reca-independent deletion, fusion and resolution of replicon. | we report that plasmid r46 provides a function which promotes reca-independent deletion, replicon fusion, and resolution of the fusion. r46 belongs to the incompatibility group n and specifies resistance to ampicillin, tetracycline, streptomycin and sulfonamide. four kinds of deletion derivatives were observed by selection for susceptability to tetracycline from ampicillin-resistant clones. a common region, will be called alpha region thereafter, was postulated to be involved in these deletions. ... | 1984 | 6319964 |
| expression of plasmid r388-encoded type ii dihydrofolate reductase as a dominant selective marker in saccharomyces cerevisiae. | the r388 plasmid-encoded drug-resistant type ii dihydrofolate reductase gene (r . dhfr) was expressed in saccharomyces cerevisiae by fusing the r . dhfr coding sequence to the yeast trp5 promoter. yeast cells harboring these recombinant plasmids grew in media with 10 micrograms of methotrexate per ml and 5 mg of sulfanilamide per ml, a condition which inhibits the growth of wild-type cells. addition of a 390-base-pair fragment from the 3'-noncoding region of trp5 downstream from r . dhfr increas ... | 1984 | 6325876 |
| protein expression in escherichia coli minicells containing recombinant plasmids specifying trimethoprim-resistant dihydrofolate reductases. | deoxyribonucleic acid fragments containing the structural genes for several trimethoprim-resistant dihydrofolate reductases from naturally occurring plasmids were inserted into small cloning vehicles. the genetic expression of these hybrid plasmids was studied in purified escherichia coli minicells. the type i dihydrofolate reductase, encoded by plasmid r483 and residing within transposon 7 (tn7), had a subunit molecular weight of 18,000. the type ii dihydrofolate reductase, specified by plasmid ... | 1980 | 6988391 |
| nicking activity of trwc directed against the origin of transfer of the incw plasmid r388. | trwc is required for conjugal dna transfer of the broad host range plasmid r388. the purified protein shows in vitro dna helicase activity. here we report that it also has in vitro orit-endonuclease activity. trwc specifically nicks orit-containing supercoiled plasmid dna in the presence of mg2+, and the nicked dna can be visualized after treatment with sds. sequencing of the nicked dna showed a specific interruption of the lower dna strand on the r388 orit sequence. both the 5' and the 3' ends ... | 1995 | 7853404 |
| effects of pili rigidity and energy availability on conjugative plasmid transfer in aquatic environments. | conjugal transfer frequencies of nonconjugative plasmid pce325 associated with either the conjugative plasmid r388 (rigid pili) or r100-1 (flexible pili) were measured in waste water and seawater between two strains of escherichia coli k12. these strains were selected from three strains after estimating (i) their survival capacity in the two water types and (ii) the maintenance and expression of plasmid-located genes in the different strains. mobilization of plasmid pce325 was always below the d ... | 1993 | 7906598 |
| promiscuous dna transfer system of agrobacterium tumefaciens: role of the virb operon in sex pilus assembly and synthesis. | conjugative transfer of dna that occurs between bacteria also operates between bacteria and higher organisms. the transfer of dna between gram-negative bacteria requires initial contact by a sex pilus followed by dna traversing four membranes (donor plus recipient) using a transmembrane pore. accumulating evidence suggests that transfer of the t-dna from agrobacterium tumefaciens to plants may also occur via a conjugative mechanism. the virb operon of the ti plasmid exhibits close homologies to ... | 1994 | 7914664 |
| purification and biochemical characterization of trwc, the helicase involved in plasmid r388 conjugal dna transfer. | trwc is an essential protein in conjugative dna transfer of the broad-host-range plasmid r388. trwc was purified in two chromatographic steps from trwc-overproducing bacteria. the purification procedure resulted in > 90% pure trwc protein, which was free of contaminating nuclease activities. trwc behaved as a dimer in gel-filtration chromatography in the presence of 550 mm nacl, and had a pi of 10.1. the purified protein showed in-vitro ssdna-dependent nucleoside-5'-triphosphatase and dna helica ... | 1994 | 8001558 |
| requirements for mobilization of plasmids rsf1010 and cole1 by the incw plasmid r388: trwb and rp4 trag are interchangeable. | mobilization of plasmid rsf1010 by the incw plasmid r388 requires the genes involved in w pilus synthesis plus trwb. trag of the incp plasmid rp4 can substitute for trwb in rsf1010 mobilization by r388 but not in self-transfer of r388. this result suggests a dual specificity of trwb-like proteins in conjugation. the same genetic requirements were found for r388 to mobilize the unrelated plasmid cole1. | 1994 | 8021231 |
| diversity and relative strength of tandem promoters for the antibiotic-resistance genes of several integrons. | the integron is a new type of mobile element containing one or more antibiotic-resistance-encoding genes site-specifically integrated as cassettes. the integrated genes are expressed from a common promoter region located in an adjacent conserved segment. sequence analysis has revealed the existence of four versions of the integron promoters. in this study, we have determined the relative strength of the different integron promoters and compared their activity with that of the tac promoter. each ... | 1994 | 8181756 |
| conjugation-independent, site-specific recombination at the orit of the incw plasmid r388 mediated by trwc. | plasmids containing a direct repeat of plasmid r388 orit are capable of site-specific recombination, which results in deletion of the intervening dna. this reaction occurs in the presence, but not in the absence, of the region of r388 implicated in dna processing during conjugation. this region contains three genes, trwa, trwb, and trwc. by using mutants of each of the three genes, it was demonstrated that only trwc is required for the orit-specific recombination. further analysis showed that th ... | 1994 | 8195075 |
| genetic organization of the conjugal dna processing region of the incw plasmid r388. | the region of the incw plasmid r388 involved in conjugal dna metabolism and mobilization (mobw) has been analyzed by tn5tac1 insertion mutagenesis, genetic complementation and dna sequencing. three genes (trwa, trwb and trwc) were mapped within mobw. they are transcribed from the same strand and away from orit. the predicted products of trwa, trwb and trwc are proteins of 121, 507 and 966 amino acids, respectively. the three proteins were visualized in a minicell expression system, showing appar ... | 1994 | 8289274 |
| a mutagenesis system utilizing a tn1722 derivative containing an escherichia coli-specific vector plasmid: application to pseudomonas species. | a novel transposon (tn) mutagenesis system for gram- non-enteric bacteria was developed which allowed rapid and one-step cloning of the mutated region in escherichia coli. the tn constructed was tn1722-299km, a tn1722 derivative containing a kmr gene and the entire sequence of an e. coli-specific plasmid, pacyc184. the hybrid plasmid consisting of tn1722-299km and the transfer genes of plasmid r388 was conjugally transferred from e. coli to pseudomonas putida or p. aeruginosa, and selection of t ... | 1993 | 8294012 |
| functional domains in protein trwc of plasmid r388: dissected dna strand transferase and dna helicase activities reconstitute protein function. | trwc is a bifunctional enzyme that displays two biochemical activities essential for plasmid r388 conjugation: orit-specific dna strand-transferase and dna helicase activities. we overproduced and purified different segments of the protein allowing us to map the relaxase and dna helicase activities to separate regions of the protein. a peptide comprising the n-terminal 275 amino acid residues of the protein was able to catalyze dna cleavage and strand-transfer reactions when using oligonucleotid ... | 1996 | 8950267 |
| orit-processing and regulatory roles of trwa protein in plasmid r388 conjugation. | trwa protein was purified from an overproducing escherichia coli strain and characterized as a 53 kda tetrameric dna-binding protein. gel shift assays showed that trwa bound specifically to the orit sequence of plasmid r388. dnase i footprinting analysis defined two dna regions within orit (sites a and b) that were protected by trwa. at low trwa concentrations only region a was protected (k(d) = 4 x 10(-8) m) while region b required higher trwa concentrations (k(d) = 4 x 10(-7) m). as a result o ... | 1997 | 9236121 |
| localization of the nic site of incn conjugative plasmid pcu1 through formation of a hybrid orit. | the n-type orit of plasmid pmur274 was cloned on a 474-bp rsai-sspi fragment, and the nucleotide sequence was determined. a comparison of the pmur274 orit sequence and the sequence of the orits of incn plasmid pcu1 and incw plasmid r388 demonstrated 57 and 28% identity, respectively. intramolecular, site-specific recombination between the pcu1 orit and the orit of pmur274 resulted in the formation of a hybrid orit containing one half of each parental sequence. the junction point of the hybrid oc ... | 1997 | 9294433 |
| trwd, a protein encoded by the incw plasmid r388, displays an atp hydrolase activity essential for bacterial conjugation. | a 1.7-kilobase pair segment from the conjugative transfer region of plasmid r388 dna was cloned and sequenced. it contained trwd, a gene essential for plasmid r388 conjugation, for expression of the conjugative w-pilus and for sensitivity to phage prd1. the deduced amino acid sequence of trwd showed homology to the pule/virb11 superfamily of potential atpases involved in various types of transport processes. a fusion of trwd with the glutathione s-transferase (gst) was constructed, and the resul ... | 1997 | 9325277 |
| a potential new gene (tcca) on incp plasmid rk2 and transposon tn1721: relationship of its product to the trwc relaxase/helicase of incw plasmid r388. | computational analysis of the fully sequenced 60-kb genome of broad-host-range incp alpha plasmid rk2 revealed a previously unreported potential protein-coding sequence, an 80-codon open reading frame (tcca), located in the region between the vegetative origin of replication (oriv) and the tetr gene of the tetracycline resistance determinant. the coding region is also present in the transposon tn1721 tet region, which is nearly identical to the tet region of rk2. remarkably, the predicted polype ... | 1997 | 9435024 |
| genetic analysis of the mobilization and leading regions of the incn plasmids pkm101 and pcu1. | the conjugative incn plasmids pkm101 and pcu1 have previously been shown to contain identical orit sequences as well as conserved restriction endonuclease cleavage patterns within their tra regions. complementation analysis and sequence data presented here indicate that these two plasmids encode essentially identical conjugal dna-processing proteins. this region contains three genes, trai, traj, and trak, transcribed in the same orientation from a promoter that probably lies within or near the c ... | 1999 | 10198024 |
| ihf protein inhibits cleavage but not assembly of plasmid r388 relaxosomes. | relaxosomes are specific nucleoprotein structures involved in dna-processing reactions during bacterial conjugation. in this work, we present evidence indicating that plasmid r388 relaxosomes are composed of origin of transfer (orit) dna plus three proteins trwc relaxase, trwa nic-cleavage accessory protein and integration host factor (ihf), which acts as a regulatory protein. protein ihf bound to two sites (ihfa and ihfb) in r388 orit, as shown by gel retardation and dnase i footprinting analys ... | 1999 | 10209739 |
| a small cryptic plasmid from ruminobacter amylophilus niah-3 possesses functional mobilization properties. | the complete nucleotide sequence of a small cryptic plasmid designated prao1, from the gram-negative ruminal bacterium ruminobacter amylophilus niah-3, was determined. the plasmid is a circular dna molecule, 2140 bp in size, with a gc content of 40%. computer-assisted analysis identified three open reading frames (orfs), one of which, orf3 (347 amino acids), displayed a high degree of amino acid identity with the mob proteins involved in conjugative mobilization and interplasmid recombination of ... | 1999 | 10564787 |
| characterization of atp and dna binding activities of trwb, the coupling protein essential in plasmid r388 conjugation. | trwb is the conjugative coupling protein of plasmid r388. trwbdeltan70 contains the soluble domain of trwb. it was constructed by deletion of trwb sequences containing trwb n-proximal transmembrane segments. purified trwbdeltan70 protein bound tightly the fluorescent atp analogue tnp-atp (k(s) = 8.7 microm) but did not show measurable atpase or gtpase activity. a single atp binding site was found per trwb monomer. an intact atp-binding site was essential for r388 conjugation, since a trwb mutant ... | 1999 | 10593894 |
| two active-site tyrosyl residues of protein trwc act sequentially at the origin of transfer during plasmid r388 conjugation. | protein trwc is the relaxase-helicase responsible for the initiation and termination reactions of dna processing during plasmid r388 conjugation. site-directed mutagenesis was used to change to phenylalanine each of a set of four conserved tyrosyl residues in the sequence of the n-terminal relaxation domain of the protein. simultaneous mutation of both y18 and y26 was required to abolish in vitro cleavage and strand-transfer reactions catalyzed by protein trwc on oligonucleotides containing the ... | 2000 | 10653694 |
| effects of yoghurt intake on plasmid transfer and colonisation with transconjugants in the digestive tract of mice associated with human faecal flora. | this study deals with the effects of yoghurt intake on wild-type and recombinant plasmid transfer from an exogenous escherichia coli k12-derivative donor strain to an endogenous recipient strain in the digestive tract of mice associated with human faecal flora. we showed that the self-transmissible plasmid r388 was efficiently transferred to recipient strain pg1 in mice associated with human faecal flora (hff-pg1) and that the resulting transconjugants (pg1-r388) became established at a high and ... | 2000 | 10719205 |
| two atypical mobilization proteins are involved in plasmid clodf13 relaxation. | the mobilization region of plasmid clodf13 was localized to a 3.6 kb dna segment that was analysed by transposon mutagenesis and dna sequencing. analysis of the dna sequence allowed us to identify two mobilization genes and the clodf13 origin of conjugative transfer (orit), which was localized to a 661 bp segment at one end of the mobilization (mob) region. thus, the overall organization was orit-mobb-mobc. plasmid clodf13 dna was isolated mainly as a relaxed form that contained a unique strand ... | 2001 | 11251827 |
| solving a 300 kda multimeric protein by low-resolution mad phasing and averaging/phase extension. | the structure of the conjugative coupling protein trwbdeltan70 from escherichia coli plasmid r388 was solved using two crystal forms. this large multimeric membrane protein of 437 residues per monomer is involved in cell-to-cell single-strand dna transfer. diffraction data to 2.4 a were available from trigonal crystals obtained from ammonium sulfate and to 2.5 a from monoclinic crystals grown from tartrate. a single tantalum bromide (ta(6)br(12)(2+)) derivative of the trigonal form, which presen ... | 2001 | 11375499 |
| conjugative plasmid protein trwb, an integral membrane type iv secretion system coupling protein. detailed structural features and mapping of the active site cleft. | bacterial conjugation is an example of macromolecular trafficking between cells and responsible for the spreading of antibiotic resistance among bacteria. it involves translocation of single-stranded dna across membranes through a type iv secretion system. a coupling protein links the dna-processing nucleoprotein complex, the relaxosome, with the transport apparatus during cell mating. in escherichia coli plasmid r388 such a protein is trwb, a basic integral inner-membrane nucleoside-triphosphat ... | 2002 | 11748238 |
| trwd, the hexameric traffic atpase encoded by plasmid r388, induces membrane destabilization and hemifusion of lipid vesicles. | trwd, a hexameric atp hydrolase encoded by plasmid r388, is a member of the pule/virb11 protein superfamily of traffic atpases. it is essential for plasmid conjugation, particularly for expression of the conjugative w pilus. in the present study, we analyzed the effects that trwd produced on unilamellar vesicles consisting of cardiolipin and phosphatidylcholine in equimolar amounts. trwd induced dose-dependent vesicle aggregation and intervesicular mixing of the lipids located in the outer monol ... | 2002 | 11872717 |
| bacterial persistence within erythrocytes: a unique pathogenic strategy of bartonella spp. | the genus bartonella comprises human-specific and zoonotic pathogens responsible for a wide range of clinical manifestations, including carrion's disease, trench fever, cat scratch disease, bacillary angiomatosis and peliosis, endocarditis and bacteremia. these arthropod-borne pathogens typically parasitise erythrocytes in their mammalian reservoir host(s), resulting in a long-lasting haemotropic infection. we have studied the process of bartonella erythrocyte parasitism by tracking green fluore ... | 2002 | 11890558 |
| characterization and movement of the class 1 integron known as tn2521 and tn1405. | two putative transposons, tn2521 and tn1405, carrying determinants for the pse-4 beta-lactamase and for resistance to streptomycin, spectinomycin, and sulfonamides were previously isolated from the chromosome of pseudomonas aeruginosa dalgleish. detailed mapping and determination of the complete sequence of tn2521 revealed that it is a class 1 integron, here renamed in33, with a backbone structure identical to that of in4 from tn1696. in33 contains two gene cassettes, blap1 and aada1, replacing ... | 2002 | 11959558 |
| structure and role of coupling proteins in conjugal dna transfer. | type iv secretory systems are transmembrane bacterial multiprotein complexes. they are pivotal for conjugation, bacterial-induced plant tumour formation, toxin secretion and mammalian pathogen intracellular activity. these systems are involved in the spread of antibiotic resistance genes among bacteria by enabling conjugative dna transfer. when such translocons transport dna, they require the assistance of multimeric integral inner membrane proteins, the type iv coupling proteins. its structural ... | 2002 | 12066890 |
| purification and properties of trwb, a hexameric, atp-binding integral membrane protein essential for r388 plasmid conjugation. | trwb is an integral membrane protein linking the relaxosome to the dna transport apparatus in plasmid r388 conjugation. native trwb has been purified in monomeric and hexameric forms, in the presence of dodecylmaltoside from overexpressing bacterial cells. a truncated protein (trwbdeltan70) that lacked the transmembrane domain could be purified only in the monomeric form. electron microscopy images revealed the hexameric structure and were in fact superimposable to the previously published atomi ... | 2002 | 12244053 |
| non-permanent proteins in membranes: when proteins come as visitors (review). | the present review introduces the concept of 'non-permanent membrane proteins', to encompass the wide variety of proteins that are not found in a stable membrane-bound form under physiological conditions, yet they interact with the membrane at some stage of their specific course of action. non-permanent membrane proteins can be codified by the cell's own genome, or else they may arise from a foreign genome. non-permanent membrane proteins can be classified, according to the reversibility of the ... | 2002 | 12512770 |
| a bacterial trwc relaxase domain contains a thermally stable alpha-helical core. | the trwc protein is the relaxase-helicase responsible for the initiation and termination reactions of dna processing during plasmid r388 conjugation. the trwc-n275 fragment comprises the 275-amino-acid n-terminal domain of the protein that contains the dna cleavage and strand transfer activities (the relaxase domain). it can be easily purified by keeping a cell lysate at 90 degrees c for 10 min. infrared spectroscopy shows that this domain has a predominantly alpha/beta structure with some amoun ... | 2003 | 12837798 |
| conjugative coupling proteins interact with cognate and heterologous virb10-like proteins while exhibiting specificity for cognate relaxosomes. | conjugative coupling proteins (cps) are proposed to play a role in connecting the relaxosome to a type iv secretion system (t4ss) during bacterial conjugation. here we present biochemical and genetic evidence indicating that the prototype cp, trwb, interacts with both relaxosome and type iv secretion components of plasmid r388. the cytoplasmic domain of trwb immobilized in an affinity resin retained trwc and trwa proteins, the components of r388 relaxosome. by using the bacterial two-hybrid syst ... | 2003 | 12925737 |
| a bacterial conjugation machinery recruited for pathogenesis. | type iv secretion systems (t4ss) are multicomponent transporters of gram-negative bacteria adapted to functions as diverse as dna transfer in bacterial conjugation or the delivery of effector proteins into eukaryotic target cells in pathogenesis. the generally modest sequence conservation between t4ss may reflect their evolutionary distance and/or functional divergence. here, we show that the establishment of intraerythrocytic parasitism by bartonella tribocorum requires a putative t4ss, which s ... | 2003 | 12940985 |
| role of the transmembrane domain in the stability of trwb, an integral protein involved in bacterial conjugation. | trwb is an integral membrane protein encoded by the conjugative plasmid r388. trwb binds atp and is essential for r388-directed bacterial conjugation. the protein consists of a cytosolic domain, which contains an atp-binding site, and a transmembrane domain. the complete protein has been purified in the presence of detergents, and in addition, the cytosolic domain has also been isolated in the form of a soluble truncated protein, trwbdeltan70. the availability of intact and truncated forms of th ... | 2004 | 14699106 |
| coupling factors in macromolecular type-iv secretion machineries. | type iv secretion systems (t4sss) are bacterial multiprotein organelles specialised in the transfer of (nucleo)protein complexes across cell membranes. they are essential for conjugation, bacterial-induced tumour formation in plant cells, as observed in agrobacterium, toxin secretion, like in bordetella and helicobacter, cell-to-cell translocation of virulence factors, and intracellular activity of mammalian pathogens like legionella. by enabling conjugative dna delivery, these systems contribut ... | 2004 | 15134575 |
| dna binding properties of protein trwa, a possible structural variant of the arc repressor superfamily. | conjugative dna processing of plasmid r388 requires the concerted action of two proteins, the relaxase-helicase trwc and the relaxase enhancer trwa. trwa can be aligned with dna binding proteins belonging to the ribbon-helix-helix (rhh) protein family. to further analyse trwa function, the structural domains of the protein have been identified and dissected by limited proteolysis. two stable domains were found that resulted to be, according to dna binding experiments and oligomerization analysis ... | 2004 | 15450172 |
| predicted hexameric structure of the agrobacterium virb4 c terminus suggests virb4 acts as a docking site during type iv secretion. | the agrobacterium t-dna transporter belongs to a growing class of evolutionarily conserved transporters, called type iv secretion systems (t4sss). virb4, 789 aa, is the largest t4ss component, providing a rich source of possible structural domains. here, we use a variety of bioinformatics methods to predict that the c-terminal domain of virb4 (including the walker a and b nucleotide-binding motifs) is related by divergent evolution to the cytoplasmic domain of trwb, the coupling protein required ... | 2005 | 15668378 |
| trwb, the coupling protein involved in dna transport during bacterial conjugation, is a dna-dependent atpase. | bacterial conjugation is an example of macromolecular trafficking between cells, based on the translocation of single-stranded dna across membranes through a type iv secretion system. trwbdeltan70 is the soluble domain of trwb, an essential integral membrane protein that couples the relaxosome (a nucleoprotein complex) to the dna transport apparatus in plasmid r388 conjugation. trwbdeltan70 crystallographic structure revealed a hexamer with six equivalent subunits and a central channel. in this ... | 2005 | 15919815 |
| site-specific recombinase and integrase activities of a conjugative relaxase in recipient cells. | conjugative relaxases are the proteins that initiate bacterial conjugation by a site-specific cleavage of the transferred dna strand. in vitro, they show strand-transferase activity on single-stranded dna, which suggests they may also be responsible for recircularization of the transferred dna. in this work, we show that trwc, the relaxase of plasmid r388, is fully functional in the recipient cell, as shown by complementation of an r388 trwc mutant in the recipient. trwc transport to the recipie ... | 2005 | 16260740 |
| functional interactions between type iv secretion systems involved in dna transfer and virulence. | this paper reports an analysis of the functional interactions between type iv secretion systems (t4ss) that are part of the conjugative machinery for horizontal dna transfer (ct4ss), and t4ss involved in bacterial pathogenicity (pt4ss). the authors' previous work showed that a conjugative coupling protein (t4cp) interacts with the virb10-type component of the t4ss in order to recruit the protein-dna complex to the transporter for conjugative dna transfer. this study now shows by two-hybrid analy ... | 2005 | 16272374 |
| unsaturated fatty acids are inhibitors of bacterial conjugation. | this report describes a high-throughput assay to identify substances that reduce the frequency of conjugation in gram-negative bacteria. bacterial conjugation is largely responsible for the spread of multiple antibiotic resistances in human pathogens. conjugation inhibitors may provide a means to control the spread of antibiotic resistance. an automated conjugation assay was developed that used plasmid r388 and a laboratory strain of escherichia coli as a model system, and bioluminescence as a r ... | 2005 | 16272375 |
| transconjugation between bacteria in the digestive tract of the cutworm peridroma saucia. | transconjugants arising from transfer of plasmid r388::tn1721 between donor and recipient strains of enterobacter cloacae were detected in samples from the digestive tracts and fecal pellets of variegated cutworms (peridroma saucia). | 1990 | 16348197 |
| effect of parental growth on dynamics of conjugative plasmid transfer in the pea spermosphere. | plasmid transfer rates for the conjugative plasmid r388::tn1721 from pseudomonas cepacia (donor) to pseudomonas fluorescens (recipient) on agar media, in broth, and in microcosms containing sterile or nonsterile soil, in the presence or absence of germinating pea seeds, were determined. donors, recipients, and transconjugants were enumerated on selective media after 1 day on agar or in broth culture and over a 7-day period in soil or pea spermosphere microcosms. donor and recipient growth rates ... | 1995 | 16535110 |
| conjugative transfer can be inhibited by blocking relaxase activity within recipient cells with intrabodies. | horizontal transfer of antibiotic resistance genes carried by conjugative plasmids poses a serious health problem. as conjugative relaxases are transported to recipient cells during bacterial conjugation, we investigated whether blocking relaxase activity in the recipient cell might inhibit conjugation. for that purpose, we used an intrabody approach generating a single-chain fv antibody library against the relaxase trwc of conjugative plasmid r388. recombinant single-chain fv antibodies were en ... | 2007 | 17163977 |
| the atpase activity of the dna transporter trwb is modulated by protein trwa: implications for a common assembly mechanism of dna translocating motors. | conjugative systems contain an essential integral membrane protein involved in dna transport called the type iv coupling protein (t4cp). the t4cp of conjugative plasmid r388 is trwb, a dna-dependent atpase. biochemical and structural data suggest that trwb uses energy released from atp hydrolysis to pump dna through its central channel by a mechanism similar to that used by f1-atpase or ring helicases. for dna transport, trwb couples the relaxosome (a dna-protein complex) to the secretion channe ... | 2007 | 17599913 |
| analysis of dna processing reactions in bacterial conjugation by using suicide oligonucleotides. | protein trwc is the conjugative relaxase responsible for dna processing in plasmid r388 bacterial conjugation. trwc has two catalytic tyrosines, y18 and y26, both able to carry out cleavage reactions using unmodified oligonucleotide substrates. suicide substrates containing a 3'-s-phosphorothiolate linkage at the cleavage site displaced trwc reaction towards covalent adducts and thereby enabled intermediate steps in relaxase reactions to be investigated. two distinct covalent trwc-oligonucleotid ... | 2007 | 17660746 |
| ppsy: a vector for the stable cloning and expression of streptomycete single gene phenotypes in escherichia coli. | ppsy is a 12kb cloning vector derived from the incw plasmid r388, which provides a rapid and easy way to stably clone phenotypes encoded in dna segments <10kb. in the present study three different genes were amplified by pcr, cloned into pgem-t easy and sub-cloned into the ecori site of ppsy. the first gene, vioa, is a fad-dependent l-tryptophan amino acid oxygenase from the high g+c gram-negative bacterium chromobacterium violaceum. vioa is involved in the synthesis of the indolocarbazole antit ... | 2008 | 18406459 |
| atpase activity and oligomeric state of trwk, the virb4 homologue of the plasmid r388 type iv secretion system. | type iv secretion systems (t4ss) mediate the transfer of dna and protein substrates to target cells. trwk, encoded by the conjugative plasmid r388, is a member of the virb4 family, comprising the largest and most conserved proteins of t4ss. virb4 was suggested to be an atpase involved in energizing pilus assembly and substrate transport. however, conflicting experimental evidence concerning virb4 atp hydrolase activity was reported. here, we demonstrate that trwk is able to hydrolyze atp in vitr ... | 2008 | 18539740 |
| changing the recognition site of a conjugative relaxase by rational design. | trwc is a relaxase protein, which starts and finishes dna processing during bacterial conjugation in plasmid r388. trwc recognizes a specific sequence of dna (25 nucleotides) in the donor cell: the nic-site. as a model example, a single transversion c24g in nic avoids dna processing by trwc. using this simple model, our objective was to obtain a proof of principle that trwc specificity can be changed. several structures of dna-trwc complexes were used as reference to design a focused saturation ... | 2009 | 19039782 |
| escherichia coli genes affecting recipient ability in plasmid conjugation: are there any? | how does the recipient cell contribute to bacterial conjugation? to answer this question we systematically analyzed the individual contribution of each escherichia coli gene in matings using plasmid r388 as a conjugative plasmid. we used an automated conjugation assay and two sets of e. coli mutant collections: the keio collection (3,908 e. coli single-gene deletion mutants) and a collection of 20,000 random mini-tn10::km insertion mutants in e. coli strain dh5alpha. the combined use of both col ... | 2009 | 19203375 |
| relaxase dna binding and cleavage are two distinguishable steps in conjugative dna processing that involve different sequence elements of the nic site. | trwc, the relaxase of plasmid r388, catalyzes a series of concerted dna cleavage and strand transfer reactions on a specific site (nic) of its origin of transfer (orit). nic contains the cleavage site and an adjacent inverted repeat (ir(2)). mutation analysis in the nic region indicated that recognition of the ir(2) proximal arm and the nucleotides located between ir(2) and the cleavage site were essential for supercoiled dna processing, as judged either by in vitro nic cleavage or by mobilizati ... | 2010 | 20061574 |
| functional dissection of the conjugative coupling protein trwb. | the conjugative coupling protein trwb is responsible for connecting the relaxosome to the type iv secretion system during conjugative dna transfer of plasmid r388. it is directly involved in transport of the relaxase trwc, and it displays an atpase activity probably involved in dna pumping. we designed a conjugation assay in which the frequency of dna transfer is directly proportional to the amount of trwb. a collection of point mutants was constructed in the trwb cytoplasmic domain on the basis ... | 2010 | 20363945 |
| the mechanism and control of dna transfer by the conjugative relaxase of resistance plasmid pcu1. | bacteria expand their genetic diversity, spread antibiotic resistance genes, and obtain virulence factors through the highly coordinated process of conjugative plasmid transfer (cpt). a plasmid-encoded relaxase enzyme initiates and terminates cpt by nicking and religating the transferred plasmid in a sequence-specific manner. we solved the 2.3 a crystal structure of the relaxase responsible for the spread of the resistance plasmid pcu1 and determined its dna binding and nicking capabilities. the ... | 2010 | 20448025 |
| reconstitution in liposome bilayers enhances nucleotide binding affinity and atp-specificity of trwb conjugative coupling protein. | bacterial conjugative systems code for an essential membrane protein that couples the relaxosome to the dna transport apparatus, called type iv coupling protein (t4cp). trwb is the t4cp of the conjugative plasmid r388. in earlier work we found that this protein, purified in the presence of detergents, binds preferentially purine nucleotides trisphosphate. in contrast a soluble truncated mutant trwbδn70 binds uniformly all nucleotides tested. in this work, trwb has been successfully reconstituted ... | 2010 | 20647001 |
| nuclear targeting of a bacterial integrase that mediates site-specific recombination between bacterial and human target sequences. | trwc is a bacterial protein involved in conjugative transfer of plasmid r388. it is transferred together with the dna strand into the recipient bacterial cell, where it can integrate the conjugatively transferred dna strand into its target sequence present in the recipient cell. considering that bacterial conjugation can occur between bacteria and eukaryotic cells, this protein has great biotechnological potential as a site-specific integrase. we have searched for possible trwc target sequences ... | 2010 | 21037296 |
| membrane insertion stabilizes the structure of trwb, the r388 conjugative plasmid coupling protein. | trwb is an integral membrane protein that plays a crucial role in the conjugative process of plasmid r388. we have recently shown [vecino et al., biochim. biophys. acta 1798(11), 2160-2169 (2010)] that trwb can be reconstituted into liposomes, and that bilayer incorporation increases its affinity for nucleotides and its specificity for atp. in the present contribution we examine the structural effects of membrane insertion on trwb, by comparing the protein in reconstituted form and in the form o ... | 2011 | 21211515 |
| autoinhibitory regulation of trwk, an essential virb4 atpase in type iv secretion systems. | type iv secretion systems (t4ss) mediate the transfer of dna and protein substrates to target cells. trwk, encoded by the conjugative plasmid r388, is a member of the virb4 family, comprising the largest and most conserved proteins of t4ss. in a previous work we demonstrated that trwk is able to hydrolyze atp. here, based on the structural homology of virb4 proteins with the dna-pumping atpase trwb coupling protein, we generated a series of variants of trwk where fragments of the c-terminal doma ... | 2011 | 21454654 |
| the stb operon balances the requirements for vegetative stability and conjugative transfer of plasmid r388. | the conjugative plasmid r388 and a number of other plasmids carry an operon, stbabc, adjacent to the origin of conjugative transfer. we investigated the role of the stba, stbb, and stbc genes. deletion of stba affected both conjugation and stability. it led to a 50-fold increase in r388 transfer frequency, as well as to high plasmid loss. in contrast, deletion of stbb abolished conjugation but provoked no change in plasmid stability. deletion of stbc showed no effect, neither in conjugation nor ... | 2011 | 21625564 |
| Transfer of R388 derivatives by a pathogenesis-associated type IV secretion system into both bacteria and human cells. | Bacterial type IV secretion systems (T4SSs) are involved in processes such as bacterial conjugation and protein translocation to animal cells. In this work, we have switched the substrates of T4SSs involved in pathogenicity for DNA transfer. Plasmids containing part of the conjugative machinery of plasmid R388 were transferred by the T4SS of human facultative intracellular pathogen Bartonella henselae to both recipient bacteria and human vascular endothelial cells. About 2% of the human cells ex ... | 2011 | 21908662 |
| plasmid segregation without partition. | low-copy number plasmids need a segregation mechanism to assort one half of the plasmid copies to each daughter cell during cell division. this can be achieved directly by partitioning plasmid copies through a mechanism reminiscent of eukaryotic mitosis. briefly, plasmid copies are paired around a centromere-like site, and then separated toward the daughter cells at cell division. partition mechanisms are used by a majority of well-studied plasmids. they involve two proteins, a dna-binding prote ... | 2011 | 22312593 |