Publications

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footprinting studies of specific complexes formed by repa, a replication initiator of plasmid pcu1, and its binding site.the basic replicon of plasmid pcu1 contains three different replication origins. replication initiated from the orib origin requires pcu1-encoded protein repa. previously, information analysis of 19 natural repa binding sequences predicted a 20-bp sequence as a repa binding site. guanines contacting repa in the major groove of dna have also been determined. in this study, we used the missing-nucleoside method to determine all of the bases relevant to repa binding. the importance of some thymine ...200010986243
physical and genetic organization of the incn-group plasmid pcu1.a restriction endonuclease-cleavage map of the incn group plasmid pcu1 was constructed. deletion mutants of the plasmid were obtained by in vivo or in vitro methods. comparison of the restriction maps of these mutants to that of pcu1 enables one to assign the known functions of the plasmid to particular regions on the plasmid dna. for different enzymes, the number and distribution of restriction sites on pcu1 is compared to that of other incn and related plasmids.19816269961
determination of the binding sites of repa, a replication initiator protein of the basic replicon of the incn group plasmid pcu1.a 2kb dna region of the broad-host-range plasmid pcu1 carries all of the information essential for the stable maintenance of the plasmid and to express the same host-range specificity. it was predicted that the protein required to initiate replication from at least one of the three origins of the plasmid is encoded by the longest open-reading frame (orf239) of the three overlapping in-frame orfs located within the 2 kb region. the product of orf239 has been named repa. the initiator protein was ...19957877179
tyrosine partners coordinate dna nicking by the salmonella typhimurium plasmid pcu1 relaxase enzyme.conjugative plasmid transfer results in the spread of antibiotic resistance genes and virulence factors between bacterial cells. plasmid transfer is dependent upon the dna nicking activity of a plasmid-encoded relaxase enzyme. tyrosine residues within the relaxase cleave the dna plasmid nic site in a highly sequence-specific manner. the conjugative resistance plasmid pcu1 encodes a relaxase with four tyrosine residues surrounding its active site (y18,19,26,27). we use activity assays to demonstr ...201121439279
structure and mode of action of kika, a genetic region lethal to klebsiella oxytoca and associated with conjugative antibiotic-resistance plasmids of the incn group.transmission of conjugative plasmids of the incn group into klebsiella oxytoca, but not into escherichia coli, results in the marked reduction of viability of the recipients. in the plasmid pcu1 a 500-bp locus called kika has the major role in determining this phenotype. expression of two open reading frames (orf104 and orf70) is required for the kik+ phenotype and they can function in cis or in trans. orf104 encodes a 8.9-kda soluble protein which is translocated into the periplasm. orf70 encod ...19968812785
isolation and characterization of kika, a region on incn group plasmids that determines killing of klebsiella oxytoca.transfer of the incn group plasmid pcu1 from escherichia coli to klebsiella oxytoca by conjugation kills a large proportion (90 to 95%) of the recipients of plasmid dna, whereas transfer to e. coli or even to the closely related enterobacter aerogenes does not. two regions, kika and kikb, have been identified on pcu1 that contribute to the kik (killing in klebsiellas) phenotype. we have localized the kika region to 500 bp by deletion analysis and show by dna-dna hybridization that kika is highly ...19921569033
lethality and survival of klebsiella oxytoca evoked by conjugative incn group plasmids.the transmission of plasmid pcu1 (or other incn group plasmid) into a population of klebsiella oxytoca cells reduces the viability of the population. a 2,400-bp region adjacent to traa is responsible for this phenotype and includes two regions, called kika and kikc. klebsiella cells which received this region and survived were found to acquire a chromosomal mutation which renders them immune to killing even after the plasmid is cured from the cells. to obtain insight into the mode of this appare ...19957592409
three clustered origins of replication in a promiscuous-plasmid replicon and their differential use in a pola+ strain and a delta pola strain of escherichia coli k-12.a 1,197-bp region of the broad-host-range plasmid pcu1 is adequate for its replication. analysis of replicating molecules containing this region reveals three clustered origins of vegetative replication and replication proceeds bidirectionally from each in a theta mode. in an escherichia coli polymerase i deletion mutant, utilization of one of these three origins was not detected. the potentiality for origin utilization may therefore be a determinant of replicon host range.19921459961
host ranges of the incn group plasmid pcu1 and its minireplicon in gram-negative purple bacteria.the bacterial host ranges of the conjugatively self-transmissible incn group plasmid pcu1 and its mobilizable miniderivative, pcu785, were examined. species of the gram-negative purple bacteria were chosen for this study. conjugative mobilization of pcu785 into a wide variety of bacteria was facilitated by the presence of orit of the broad-host-range plasmid rk2 in pcu785. although the host range of the incn tra system of pcu1 is broad, the host range of its replicon is limited. however, the pcu ...198816347740
use of a dna polymerase iii bypass mutant of escherichia coli, pcba1, to isolate potentially useful mutations of a complex plasmid replicon.the essential replicon region of plasmid pcu1 has, within 1.2 kb, two origins of replication that can function in the absence of escherichia coli dna polymerase i and one that requires this polymerase. to isolate mutants in the replicon pathway that uses the poli-dependent origin in the presence of the two other origins, we examined the feasibility of exploiting e. coli strains carrying a polymerase c bypass mutation (pcba) and which can survive and form colonies with the polymerase activity of ...19968693027
the incn plasmid replicon: two pathways of dna polymerase i-independent replication.the 2,053-bp broad-host-range incompatibility group n replicon of plasmid pcu1 has two components: a region of 1,200 bp that is sufficient for its replication in escherichia coli pola+ and pola- hosts and a regulatory region called the group i iteron region that contains 13 39-bp iterons. within the 1,200-bp region, there are three replication origins, two of which, called orib and oris, function in pola+ and pola- hosts and a third, called oriv, which functions only in pola+ hosts. the region a ...19948002601
the orit region of the conjugative transfer system of plasmid pcu1 and specificity between it and the mob region of other n tra plasmids.the orit region of the conjugative incn plasmid pcu1 has been localized to a 669-bp sequence extending from pcu1 coordinates 8.48 to 9.15 kb. the nucleotide sequence of this region was determined. the region is at-rich (69% at residues), with one 19-bp and one 81-bp sequence containing 79% or more at residues. prominent sequence features include one set of thirteen 11-bp direct repeats, a second set of two 14-bp direct repeats, six different inverted repeat sequences ranging from 6 to 10 bp in s ...19921309528
inverted repeats in the dna of plasmid pcu1.renaturable regions in the dna strands of the n group plasmid pcu1 have been visualized as stem-loop structures by electron microscopy. four such distinct structures are described, the smallest of which is within the loop of a larger one. the region of pcu1 in which these structures occur has several restriction sites. this and the availability of plasmid deletions and recombinants has permitted the mapping of these structures relative to one another and to the restriction and functional map of ...19836338000
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 ...201020448025
development and application of molecular tools in the study of incn-related plasmids from lakewater sediments.homology to incn, p, q and w inc regions was investigated amongst 114 hg(2+)-resistant or antibiotic-resistant bacteria isolated from lakewater sediments. no hybridisation signals were found with inc p, q and w probes, and only one plasmid, plv1402, hybridised to the incn probe. pcr primers designed to conserved regions in the replicon of the incn plasmid pcu1 and the related beta replicon from pgsh500 were used to amplify a 978-bp fragment from plv1402, with sequence analysis showing a close re ...200010802172
n conjugative transfer system of plasmid pcu1.the mating and plasmid dna transfer functions in escherichia coli k-12 strains that are determined by the incn group plasmid pcu1 are specified by a single 19.4-kilobase (kb) segment of the 39-kb plasmid. analysis of this tra region by transposon tn5 mutagenesis and by complementation tests indicated that a subset of tra comprising six complementation groups (presumably transcription units) determines sensitivity to the n pilus-specific bacteriophages ike and prd1. deletion derivatives and molec ...19852863255
regions on plasmid pcu1 required for the killing of klebsiella pneumoniae.plasmid pcu1 was kik+ (promotes killing of klebsiella pneumoniae). all tn5 insertions within the tra region of pcu1 were kik-. two other regions, kika and kikb, were needed. they may be separated on different plasmids, but both must be mobilized into klebsiella pneumoniae. establishment of one kik region in k. pneumoniae followed by receipt of the second did not lead to killing. kik was therefore intracellular and required concerted and transient action of both regions.19852993244
analysis of pcu1 replication origins: dependence of oris on the plasmid-encoded replication initiation protein repa.the broad-host-range replicon of the plasmid pcu1 has three origins of vegetative replication called orib, oris, and oriv. in the multi-origin replicon, individual origins can distinguish among replication factors provided by the host. it has been found that during replication in escherichia coli pola(-) host, oris was the only active origin of a mutant pcu1 derivative bearing a mutation in the gene encoding replication initiation protein repa. to further investigate the capacity of oris to func ...200312726768
isolation and structure of the replicon of the promiscuous plasmid pcu1.evidence is presented to indicate that a pvuii fragment of approx. 2 kb isolated from the 39-kb incn-group plasmid pcu-1 contains all plasmid-borne determinants for stable maintenance as an extrachromosomal element in escherichia coli k-12. the fragment was sequenced. the features of this sequence include a group of 13 direct tandem repeats of 37 bp and a second group of two other direct repeats of 30 bp flanking a third partial member of this group. in addition, for a 19-bp sequence that overla ...19872828186
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 ...19902170327
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 ...19979294433
conditionally lethal genes in the n pilus region of plasmid pcu1.plasmid genes or regions that are conditionally lethal to escherichia coli have been called kil and those lethal to klebsiella but not to e. coli have been called kik. both classes of genes are found in or close to the n pilus region of the plasmid pcu1 and the closely related plasmid pkm101. here we describe two new and overlapping lethal genes that are located between kika and traa of the plasmid pcu1 and display host specificity. kilc is lethal in e. coli and klebsiella while kikc is lethal o ...199910413666
mutations within the replicon of the incn plasmid pcu1 that affect its escherichia coli pola-independence but not its autonomous replication ability.the minimal replicon of the incompatibility n group plasmid pcu1 is contained within a 2-kb dna region of the plasmid. the ability of this region and of the deletion derivatives thereof, that are capable of autonomous maintenance, to direct polypeptide synthesis was examined. two proteins of 27 and 5.5 kda are encoded by the minimal replicon. polypeptide chain-terminating mutations within the predicted open reading frame for the 27-kda polypeptide abolished the synthesis of this polypeptide and ...19902205534
cloning of a plasmid region specifying the n transfer system of bacterial conjugation in escherichia coli.hindiii restriction sites were created artificially by the insertion of the transposon tn5 into the incn plasmid pcu1 near a presumptive end of its conjugal transfer region (tra). this allowed cloning of an entire and continuous 19.4-kb region of this plasmid that specifies the n transfer system. the cloning vector was the nonconjugative plasmid pacyc184. the recombinant plasmid was as efficient in transfer as the parental n plasmid. other clones and deletions extending into the tra region allow ...19836303904
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