| role of the origin of transfer in termination of strand transfer during bacterial conjugation. | conjugal transfer of the broad-host-range plasmid r1162 is initiated and terminated at the nic site within the 38-bp origin of transfer (orit). termination involves ligation of the transferred single strand by the plasmid-encoded moba protein. several different assays were used to identify the orit dna required for termination. for plasmids containing two orits, with transfer initiated at one and terminated at the other, the inverted repeat within orit is important for termination. deletion of t ... | 1992 | 1400216 |
| deletion of sites for initiation of dna synthesis in the origin of broad host-range plasmid r1162. | the origin of replication of the broad host-range plasmid r1162 contains two, oppositely facing initiation sites for dna synthesis. either of these sites can be deleted from an r1162 plasmid derivative. however, the resulting plasmids are unstable, maintained at a lower copy-number in the cell, and form dimers and other recombinants that are required for propagation of the plasmid. in vitro, a derivative lacking one initiation site is deficient in synthesis of the strand normally initiated from ... | 1990 | 2201775 |
| probing the activation of the replicative origin of broad host-range plasmid r1162 with tus, the e.coli anti-helicase protein. | the e.coli tus protein is an anti-helicase involved in the termination of chromosome replication. the binding site for this protein, ter, was cloned into derivatives of the broad host-range plasmid r1162. the ter site caused the orientation-specific termination of plasmid replication fork movement in cell extracts containing tus. plasmids were constructed so that two sites for initiation of r1162 replication flanked the iteron-containing domain of the origin. in these plasmids, the site next to ... | 1991 | 1923822 |
| an essential iteron-binding protein required for plasmid r1162 replication induces localized melting within the origin at a specific site in at-rich dna. | the r1162-encoded protein repib is essential for replication of the plasmid and binds specifically to iterons within the replicative origin. the protein causes the localized melting of dna (determined by sensitivity to p1 nuclease) at a site within the at-rich region of the origin, about 60 bp from the iteron binding sites and separated from them by a gc-rich tract. point mutations have been isolated in the at-rich dna. these mutations interfere with origin activity and also prevent the protein- ... | 1991 | 1885530 |
| a segment of a plasmid gene required for conjugal transfer encodes a site-specific, single-strand dna endonuclease and ligase. | the polypeptide encoded by a segment of a gene required for the conjugal mobilization of the broad host-range plasmid r1162 has been purified as a beta-galactosidase fusion protein. the hybrid protein binds specifically to a small, double-stranded dna fragment containing the origin of transfer (orit), and specifically cleaves orit single-stranded dna at the position cleaved during transfer. only one of the two dna strands is a substrate. a fraction of the digested dna is resistant to lambda exon ... | 1991 | 1850512 |
| site-specific recombination at orit of plasmid r1162 in the absence of conjugative transfer. | r1162 is efficiently comobilized during conjugative transfer of the self-transmissible plasmid r751. bacteriophage m13 derivatives that contain two directly repeated copies of orit, the site on r1162 dna required in cis for mobilization, were constructed. phage dna molecules underwent recombination during infection of escherichia coli, with the product retaining a single functional copy of orit. recombination was strand specific and depended on r1162 gene products involved in mobilization, but d ... | 1989 | 2644236 |
| copy-number of broad host-range plasmid r1162 is regulated by a small rna. | we have shown previously [kim, k. and meyer, r.j. (1985) j. mol. biol. 185,755-767] that copy-number of the broad host-range plasmid r1162 is controlled by the amounts of two proteins, encoded by cotranscribed genes comprising a region of the plasmid called repi. we have now demonstrated that expression of repi is negatively regulated by a 75 base rna that is complementary to a segment of the repi message. increased intracellular amounts of rna molecules that include this segment relieve the inh ... | 1986 | 2430262 |
| recombination between directly repeated origins of conjugative transfer cloned in m13 bacteriophage dna models ligation of the transferred plasmid strand. | when two, directly-repeated copies of the origin of transfer (orit) of the conjugatively mobilizable, broad host-range plasmid r1162 are cloned into bacteriophage m13mp9 dna, they undergo recombination in the presence of one of the r1162-encoded proteins required for mobilization [meyer, r. (1989) j. bacteriol., 171, 799-806]. mutations in the outer arm of the inverted repeat within orit inhibit this recombination. these mutations also affect a late step in transfer. we propose that recombinatio ... | 1990 | 2362809 |
| two domains at the origin are required for replication and maintenance of broad-host-range plasmid r1162. | two domains at the replicative origin of broad-host-range plasmid r1162 are required in cis for plasmid maintenance in escherichia coli and for plasmid dna replication in cell extracts. increasing the distance between the domains reduces replication in vitro, without substantially changing plasmid dna content or stability in vivo. | 1987 | 2445734 |
| single-stranded circular dna generated from broad host range plasmid r1162 and its derivatives. | extraction of r1162 plasmid dna with the alkaline lysis method yields considerable amounts of single-stranded circular plasmid dna. destabilization of plasmid dna is stimulated by the r1162 mob region in cis. the formation of single-stranded circular dna is initiated at a specific site on the plasmid, presumably the origin of transfer (orit). | 1989 | 2675151 |
| unidirectional transfer of broad host-range plasmid r1162 during conjugative mobilization. evidence for genetically distinct events at orit. | a segment of r1162 dna containing genes for conjugative mobilization (mob) and the origin of transfer (orit) was integrated into the escherichia coli chromosome. bacterial genes were transferred in a polar fashion during conjugative mobilization of the integrated segment by a self-transmissible plasmid vector. the direction of transfer, together with the properties of mutated derivatives of orit, indicate that initial cleavage of orit, and subsequent religation after transfer, entail different m ... | 1989 | 2677391 |
| copy number of the broad host-range plasmid r1162 is determined by the amounts of essential plasmid-encoded proteins. | dna of the broad host-range plasmid r1162 contains a 1700 base-pair segment essential for plasmid maintenance. this region, repi, consists of two cotranscribed genes encoding polypeptides with molecular weights of 29,000 and 31,000. fusion of repi to the strong tac promoter results in greatly increased amounts of at least one of these polypeptides. in trans, this construction has two other properties: it can raise the copy number of r1162, and it can protect this plasmid from loss due to incompa ... | 1985 | 2932556 |
| the 20 bp, directly repeated dna sequence of broad host range plasmid r1162 exerts incompatibility in vivo and inhibits r1162 dna replication in vitro. | the broad host range plasmid r1162 contains a directly repeated, 20 bp dna sequence in the region of the plasmid required in cis for replication and maintenance. this sequence has been chemically synthesized and cloned, and shown to be sufficient for expression of plasmid incompatibility. the sequence also inhibits replication of r1162 dna in a cell-free system. the strengths of both these effects are determined by the number of direct repeats (drs) present, and are also affected to similar degr ... | 1987 | 2823058 |
| dna synthesis is initiated at two positions within the origin of replication of plasmid r1162. | dna synthesis of broad host-range plasmid r1162 is initiated from two positions, flanking a large (40 bp stem, 40 bp loop) inverted repeat. each start-point is located within a highly conserved, but oppositely oriented, 10 base-pair sequence. synthesis from the two positions converges within the intervening inverted repeat. an analysis of deletions suggests that both start positions must be present for synthesis. a model describing possible early events in replication of plasmid r1162 is present ... | 1987 | 3313280 |
| a 38 base-pair segment of dna is required in cis for conjugative mobilization of broad host-range plasmid r1162. | orit, the region required in cis for conjugative mobilization of broad host-range plasmid r1162, has been localized to a 38 base-pair segment of dna. the orit dna is also required for conjugation-dependent recombination. point mutations at the hinpi cleavage site within orit affect both mobilization and recombination, and the crossover location has been mapped to this site. an inverted repeat ten base-pairs from the recombination site is also involved in mobilization and recombination, and may b ... | 1987 | 3323533 |
| integrative suppression of dnaa46 by broad host-range plasmid r1162. | replication of plasmid r1162 dna does not require the product of the dnaa gene. an integrated copy of the plasmid can suppress the temperature-sensitive dnaa46 allele when (1) additional plasmid copies are present in the cytoplasm and (2) an inactive replication origin, generated by deletion, is also present in the chromosome. we propose that the inactive origin sets the rate of initiation of chromosome replication at a level compatible with cell viability, possibly by providing additional bindi ... | 1988 | 2853828 |
| genetic organization of plasmid r1162 dna involved in conjugative mobilization. | dna involved in the mobilization of broad-host-range plasmid r1162 was localized to a region of 2.7 kilobases within coordinates 3.4 to 6.1 kilobases on the r1162 map. by examining the transfer properties of plasmids containing cloned fragments of dna from within this region, we showed that at least four trans-active products and a cis-active site (orit) were involved in mobilization. a cloned dna fragment of 155 base pairs was capable of providing full orit activity. this fragment was located w ... | 1986 | 3525520 |
| identification and sequence of the basic replication region of a broad-host-range plasmid isolated from thiobacillus ferrooxidans. | the minimum region required for replication of the broad-host-range thiobacillus ferrooxidans plasmid ptf-fc2 in escherichia coli was shown to be contained on a 2.05-kilobase fragment of dna. a 184-base-pair fragment that was required in cis for plasmid replication was identified. this region was also involved in plasmid incompatibility. nucleotide sequencing of this region revealed three perfectly conserved 22-base-pair tandemly repeated sequences. a comparison of this region with the equivalen ... | 1989 | 2708316 |
| broad host-range plasmid r1162: replication, incompatibility, and copy-number control. | | 1985 | 2990408 |
| directly repeated, 20-bp sequence of plasmid r1162 dna is required for replication, expression of incompatibility, and copy-number control. | dna required in cis for the replication of the broad-host-range plasmid r1162 is located on two contiguous hpaii fragments of 210 and 370 bp. the latter of these contains three and one-half, perfectly conserved, 20-bp directly repeated sequences. the significance of these for plasmid replication, incompatibility, and copy-number control was examined by generating deletions into these repeats and testing the properties of the remaining dna. we conclude from the results that the direct repeats are ... | 1986 | 3513218 |
| broad-host-range incp-4 plasmid r1162: effects of deletions and insertions on plasmid maintenance and host range. | r1162 is an 8.7-kilobase (kb) broad-host-range replicon encoding resistance to streptomycin and sulfa drugs. in vitro deletion of 1.8-kb dna between coordinates 3.0 and 5.3 kb did not affect plasmid maintenance, but a tn1 insertion at coordinate 6.3 kb led to a recessive defect in plasmid maintenance. the only cis-acting region necessary for plasmid replication appears to lie between the tn1 insertion at coordinate 6.3 kb and a second tn1 insertion at coordinate 6.5 kb. all r1162 sequences betwe ... | 1982 | 6288654 |
| nucleotide sequence and functional properties of dna encoding incompatibility in the broad host-range plasmid r1162. | a 370 base pair (bp) fragment of r1162 dna encoding the incompatibility determinant has been cloned and sequenced. the dna is located between 6.1 and 6.5 on the r1162 map, near the origin of replication. the sequence contains three perfectly conserved 20 bp direct repeats, with 11 bp of this sequence repeated a fourth time. the direct repeat unit shows some homology with that of another, unrelated broad host-range plasmid, rk2. the cloned dna has two other properties: it lowers the copy number o ... | 1984 | 6377013 |
| the use of plasmid r1162 and derivatives for gene cloning in the methanol-utilizing pseudomonas am1. | a physical map for plasmid r1162 (sm, su, incp4) was constructed. neither ecori, psti nor ecai cut within a region essential for replication, molbilization or streptomycin resistence. plasmid r1162 can replicate in e. coli as well as in pseudomonas species and shows a strong dependence for dna polymerase i in e. coli. by rp4 induced mobilization, r1162 can be transferred from e. coli to pseudomonas am1. a hybrid plasmid pfg7 (mw=8.4 x 10(6), sm, su, ap, tc) was constructed between pbr322 and r11 ... | 1980 | 6248728 |
| properties of r1162, a broad-host-range, high-copy-number plasmid. | regions of plasmid dna encoding characteristic properties of the incq (p-4) group plasmid r1162 were identified by mutagenesis and in vitro cloning. coding sequences sufficient for expression of incompatibility and efficient conjugal mobilization by plasmid r751 were found to be linked to the origin of dna replication. in contrast, there was a region remote from the origin, and active in trans, that was required for plasmid maintenance. a derivative that was temperature sensitive for stability w ... | 1982 | 6279561 |
| conjugal mobilization of plasmid dna: termination frequency at the origin of transfer of plasmid r1162. | conjugal transfer of plasmid r1162 is initiated and terminated at a 38-bp origin of transfer (orit). plasmids containing two directly repeated copies of orit were used to determine the actual frequency of termination at this site. this frequency was calculated both for oritnic, a mutated origin that cannot act as the initiation site of transfer, and for an unmutated orit where transfer had been initiated. in both cases, the termination frequency decreased as the distance between the initiation a ... | 1994 | 7928956 |
| specific binding of moba, a plasmid-encoded protein involved in the initiation and termination of conjugal dna transfer, to single-stranded orit dna. | moba protein, encoded by the broad host-range plasmid r1162, is required for conjugal mobilization of this plasmid. the protein is an essential part of the relaxosome, and is also necessary for the termination of strand transfer. in vitro, moba is a nuclease specific for one of the two dna strands of the origin of transfer (orit). the protein can cleave this strand at the same site that is nicked in the relaxosome, and can also ligate the dna. we show here that purified moba protein forms a comp ... | 1993 | 8233790 |
| the origin of greater-than-unit-length plasmids generated during bacterial conjugation. | in gram-negative bacteria, the general mechanism of conjugal plasmid transfer, which is probably similar for many different groups of plasmids, involves the transfer of a single plasmid dna strand with 5' to 3' polarity. transfer is initiated by nicking of the duplex dna at a particular site, i.e. the origin of transfer (orit). we constructed plasmids containing two directly repeated copies of orit, derived from the broad-host-range plasmid r1162 and flanking the lac operator. the number of laco ... | 1993 | 8446031 |
| acquisition of resistance genes by the incq plasmid r1162 is limited by its high copy number and lack of a partitioning mechanism. | r1162 is a representative member of the broad-host-range incq group of multicopy plasmids. lower-copy-number derivatives of r1162 were constructed in vitro and shown to be unstable, indicating that partitioning of plasmid copies at cell division is due to random distribution and not to an active partitioning mechanism. however, the normal copy number of r1162 reduces cell fitness during growth in broth and favors the emergence of unstable, lower-copy-number variants. as a result, plasmid-borne a ... | 1997 | 9294457 |
| the relaxosome protein mobc promotes conjugal plasmid mobilization by extending dna strand separation to the nick site at the origin of transfer. | the frequency of conjugal mobilization of plasmid r1162 is decreased approximately 50-fold if donor cells lack mobc, one of the plasmid-encoded proteins making up the relaxosome at the origin of transfer (orit). the absence of mobc has several different effects on orit dna. site- and strand-specific nicking by moba protein is severely reduced, accounting for the lower frequency of mobilization. the localized dna strand separation required for this nicking is less affected, but becomes more sensi ... | 1997 | 9302013 |
| stabilization of the relaxosome and stimulation of conjugal transfer are genetically distinct functions of the r1162 protein mobb. | mobb is a small protein encoded by the broad-host-range plasmid r1162 and required for efficient mobilization of its dna during conjugation. the protein was shown previously to stabilize the relaxosome, the complex of plasmid dna and mobilization proteins at the origin of transfer (orit). we have generated in-frame mobb deletions that specifically inactivate the stabilizing effect of mobb while still allowing a high rate of transfer. thus, mobb has two genetically distinct functions in transfer. ... | 1999 | 10094690 |
| the moba-linked primase is the only replication protein of r1162 required for conjugal mobilization. | cells newly transformed with plasmid r1162 dna were used as donors in conjugal matings to determine if the plasmid replication genes are necessary for transfer. an intact system for vegetative replication is not required for transfer at normal frequency, but the plasmid primase, in the form linked to the nickase, must be present in donor cells. | 1999 | 10217797 |
| replication of a plasmid lacking the normal site for initiation of one strand. | the origin of replication of the plasmid r1162 contains an initiation site for the synthesis of each dna strand. when one of these sites (oril) is deleted, synthesis on the corresponding strand is no longer initiated efficiently in vitro by the r1162-encoded replication proteins, and the plasmid is no longer stably maintained in the cell. however, in vivo the two strands of the plasmid duplex molecule are active at a similar level as templates for dna synthesis, and newly synthesized copies of e ... | 1996 | 8759850 |
| localized denaturation of orit dna within relaxosomes of the broad-host-range plasmid r1162. | the broad-host-range, multicopy plasmid r1162 is efficiently mobilized during conjugation by the self-transmissible plasmid r751. the relaxosome, a complex of plasmid dna and r1162-encoded proteins, forms at the origin of transfer (orit) and is required for mobilization. transfer is initiated by strand- and site-specific nicking of the dna within this structure. we show by probing with potassium permanganate that orit dna is locally melted within the relaxosome, in the region from the inverted r ... | 1995 | 8801426 |
| the primase of broad-host-range plasmid r1162 is active in conjugal transfer. | the broad-host-range plasmid r1162 is conjugally mobilized at high frequency by the incp-1 plasmid r751 but is poorly mobilized by pox38, a derivative of the f factor. in both cases, the origin of transfer (orit) and the mob proteins of r1162 are required, indicating that these plasmids are mobilized by similar mechanisms. r1162 encodes a primase, essential for vegetative replication of the plasmid, that is made both as a separate protein and as the carboxy-terminal domain of moba, one of the r1 ... | 1996 | 8955311 |
| recognition of orit for dna processing at termination of a round of conjugal transfer. | conjugal transfer of plasmid dna is terminated when the transferred strand, linearized at the 38 base-pair origin of transfer (orit), is recircularized. for the plasmid r1162, it is the protein moba, covalently linked to the linear strand, that rejoins the ends by a reversible transesterification reaction. we have identified from those oligonucleotides with a partially degenerate orit base sequence, subpopulations bound by moba that undergo transesterification, and support efficient termination ... | 2000 | 10903855 |
| elements in the co-evolution of relaxases and their origins of transfer. | the central elements in the conjugative mobilization of most plasmids are the relaxase and its cognate origin of transfer (orit). the relaxase of the plasmid r1162, together with its orit, belong to a large and widely distributed family of related relaxase/orit pairs. several of the properties of these elements are considered for r1162 and for other members of this family with a view to understanding how systems for mobilization might have evolved. | 2005 | 15737398 |
| mechanisms of strand replacement synthesis for plasmid dna transferred by conjugation. | a single strand of plasmid dna is transferred during conjugation. we examined the mechanism of complementary strand synthesis in recipient cells following conjugative mobilization of derivatives of the incq plasmid r1162. a system for electroporation of donor cells, followed by immediate mating, was used to eliminate plasmid-specific replicative functions. under these conditions, escherichia coli recipients provided a robust mechanism for initiation of complementary strand synthesis on transferr ... | 2005 | 15866925 |
| relaxed specificity of the r1162 nickase: a model for evolution of a system for conjugative mobilization of plasmids. | the primary dna processing protein for conjugative mobilization of the plasmid r1162 is the transesterase moba, which acts at a unique site on the plasmid, the origin of transfer (orit). both moba and orit are members of a large family of related elements that are widely distributed among bacteria. each orit consists of a highly conserved core and an adjacent region that is required for binding by its cognate moba. the sequence of the adjacent region is important in determining the specificity o ... | 2003 | 12775691 |
| molecular handcuffing of the relaxosome at the origin of conjugative transfer of the plasmid r1162. | the assembly of plasmid-encoded proteins at a unique site (orit) on the plasmid r1162, to form a complex called the relaxosome, is required for conjugative transfer of the plasmid and for negative regulation of neighboring promoters. two-dimensional chloroquine gel electrophoresis was used to show that orits are physically coupled at the relaxosome. this interaction requires all the relaxosome proteins, which are assembled into a structure resulting in a decrease in the average linking number of ... | 2003 | 12907717 |
| moba, the dna strand transferase of plasmid r1162: the minimal domain required for dna processing at the origin of transfer. | moba is a dna strand transferase encoded by the plasmid r1162 and required for plasmid dna processing during conjugal transfer. the smallest active fragment was identified using phage display and partial enzymatic digestion of the purified protein. this fragment, consisting of approximately the first 184 amino acids, is able to bind and cleave its normal dna substrate, the origin of transfer (orit). smaller fragments having one of these activities were not obtained. an active intermediate consis ... | 2002 | 11839744 |
| the structure of the minimal relaxase domain of moba at 2.1 a resolution. | the plasmid r1162 encodes proteins that enable its conjugative mobilization between bacterial cells. it can transfer between many different species and is one of the most promiscuous of the mobilizable plasmids. the plasmid-encoded protein moba, which has both nicking and priming activities on single-stranded dna, is essential for mobilization. the nicking, or relaxase, activity has been localized to the 186 residue n-terminal domain, called minmoba. we present here the 2.1 a x-ray structure of ... | 2007 | 17157875 |
| the r1162 relaxase/primase contains two, type iv transport signals that require the small plasmid protein mobb. | the relaxase of the plasmid r1162 is a large protein essential for conjugative transfer and containing two different and physically separate catalytic activities. the n-terminal half cleaves one of the dna strands at the origin of transfer (orit) and becomes covalently linked to the 5' terminal phosphate; the c-terminal half is a primase essential for initiation of plasmid vegetative replication. we show here that the two parts of the protein are independently transported by the type iv pathway. ... | 2007 | 17880426 |
| the r1162 mob proteins can promote conjugative transfer from cryptic origins in the bacterial chromosome. | the mobilization proteins of the broad-host-range plasmid r1162 can initiate conjugative transfer of a plasmid from a 19-bp locus that is partially degenerate in sequence. such loci are likely to appear by chance in the bacterial chromosome and could act as cryptic sites for transfer of chromosomal dna when r1162 is present. the r1162-dependent transfer of chromosomal dna, initiated from one such potential site in pectobacterium atrosepticum, is shown here. a second active site was identified in ... | 2009 | 19074386 |
| effect of divalent ions on the minimal relaxase domain of moba. | the moba protein encoded by plasmid r1162 plays an important role in conjugative mobilization between bacterial cells. it has two functional domains, the n-terminal relaxase domain and c-terminal primase domain. the n-terminal 186 residues (minmoba) is the minimal domain required for relaxase activity. we investigated the effects of different divalent metallic cations on minmoba activity measuring dna binding, dna nicking, and protein denaturation experiments. the results show that divalent cati ... | 2009 | 19527679 |
| functional organization of mobb, a small protein required for efficient conjugal transfer of plasmid r1162. | in most bacteria, transfer of plasmid dna by conjugation is essentially a specialized form of type iv secretion (12). the relaxase, a plasmid-encoded protein, cleaves one of the plasmid dna strands at a unique locus called the origin of transfer (orit) (6). cleavage takes place by a transesterification, with the protein becoming covalently linked to the dna by a tyrosyl 5'-phosphodiester bond. the relaxase, with or without its attached dna, is targeted to the type iv secretion machinery and secr ... | 2011 | 21622757 |