cloning and stable maintenance of 300-kilobase-pair fragments of human dna in escherichia coli using an f-factor-based vector.a bacterial cloning system for mapping and analysis of complex genomes has been developed. the bac system (for bacterial artificial chromosome) is based on escherichia coli and its single-copy plasmid f factor. it is capable of maintaining human genomic dna fragments of greater than 300 kilobase pairs. individual clones of human dna appear to be maintained with a high degree of structural stability in the host, even after 100 generations of serial growth. because of high cloning efficiency, easy ...19921528894
requirement of the escherichia coli dnaa gene product for plasmid f maintenance.there are dnaa protein-binding sites in at least one f origin of replication, and only potentially leaky dnaa(ts) mutations had ever been used in previous studies indicating that f replication was independent of the dnaa gene product. here we show that an escherichia coli dnaa::tn10 host which does not make a dnaa gene product cannot sustain autonomous or integrated f plasmid maintenance.19863020005
a plasmid rk2-based broad-host-range cloning vector useful for transfer of metagenomic libraries to a variety of bacterial species.the majority of microorganisms in natural environments are difficult to cultivate, but their genes can be studied via metagenome libraries. to enhance the chances that these genes become expressed we here report the construction of a broad-host-range plasmid vector (prs44) for fosmid and bacterial artificial chromosome (bac) cloning. prs44 can be efficiently transferred to numerous hosts by conjugation. it replicates in such hosts via the plasmid rk2 origin of replication, while in escherichia c ...200919459950
the incc korb region of rk2 repositions a mini-rk2 replicon in escherichia coli.analysis by fluorescence microscopy has established that plasmid rk2 in escherichia coli and other gram-negative bacteria is present as discrete clusters that are located inside the nucleoid at the mid- or quarter-cell positions. a mini-rk2 replicon containing an array of teto repeats was visualized in e. coli cells that express a tetr-eyfp fusion protein. unlike intact rk2, the rk2 mini-replicon (pcv1) was localized as a cluster at the cell poles outside of the nucleoid. insertion of the o(b1)i ...200717521722
energetics of the sequence-specific binding of single-stranded dna by the f factor relaxase domain.transfer of conjugative plasmids between bacteria requires the activity of relaxases or mobilization proteins. these proteins nick the plasmid in a site- and strand-specific manner prior to transfer of the cut strand from donor to recipient. trai36, the relaxase domain of trai from plasmid f factor, binds a single-stranded dna (ssdna) oligonucleotide containing an f factor sequence with high affinity and sequence specificity. to better understand the energetics of this interaction, we examined t ...200415123728
stability of a plasmid f trim in populations of a recombination-deficient strain of escherichia coli in continuous culture.populations of a reca derivative of escherichia coli ab1157 containing the plasmid f trim were grown in carbon-limited continuous culture at dilution rates of 0.1 h-1 to 0.4 h-1. the plasmid was lost after a lag, except in fermenter-experienced populations when it was retained. these results can be explained in terms of non-specific competition.19846380407
recombinant dna risk assessment studies in humans: efficacy of poorly mobilizable plasmids in biologic containment.recombinant dna risk assessment studies quantitated the mobilizability of "safe" plasmid pbr325, in comparison with readily mobilizable plasmid pjbk5 (chloramphenicol and tetracycline resistant). of 15 volunteers who became colonized after ingestion of 5 x 10(10) escherichia coli hs-4, a normal human flora strain containing pjbk5 and daily oral tetracycline, nine manifested transfer of pjbk5 to normal flora by means of triparental mating. in contrast, none of 12 other volunteers cocolonized with ...19836355313
location of an f-pilin pool in the inner membrane.polyacrylamide gel analysis of [35s]methionine-labeled membrane preparations from escherichia coli has revealed the presence of five polypeptides present only in the membranes of cells containing the conjugative plasmid f. in addition to the previously reported product of trat, polypeptides migrating with apparent molecular weights of 100,000, 23,500, 12,000, and 7,000 were resolved. membrane preparations from f traj mutants lacked these polypeptides, indicating that all of these proteins are tr ...19816111549
the ssb gene of plasmid colib-p9.the inci1 plasmid colib-p9 was found to carry a single-stranded dna-binding (ssb) protein gene (ssb) that maps about 11 kilobase pairs from the origin of transfer in the region transferred early during bacterial conjugation. the cloned gene was able to suppress the uv and temperature sensitivity of an ssb-1 strain of escherichia coli k-12. the nucleotide sequence of the colib ssb gene was determined, giving a predicted molecular weight of 19,110 for the ssb protein. sequence data show that colib ...19892651402
control of the ccd operon in plasmid f.the f sex factor plasmid of escherichia coli contains a pair of genes, ccda and ccdb, whose protein gene products are involved in an unusual feature of plasmid maintenance. the ccdb protein is a cytotoxin that becomes activated when the f plasmid is lost, thereby killing the f- segregant cells. in f+ cells, the ccda protein protects against the lethal effects of ccdb. in the present study we show that ccda and ccdb expressions are negatively autoregulated at the level of transcription. genetic s ...19892651399
characterization of the f-plasmid conjugative transfer gene trau.we characterized the trau gene of the escherichia coli k-12 conjugative plasmid f. plasmids carrying segments of the f transfer operon were tested for their capacity to complement f lac trau526. the protein products of trau+ clones were identified, and the nucleotide sequence of trau was determined. trau mapped between traw and trbc. it encodes a 330-amino-acid, mr36,786 polypeptide that is processed. ethanol caused accumulation of a precursor polypeptide; removal of ethanol permitted processing ...19902198250
structural and functional comparison between the stability systems pard of plasmid r1 and ccd of plasmid f.the stability determined by the systems pard of plasmid r1 and ccd of plasmid f is due to the concerted action of two proteins, a cytotoxin and an antagonist of this function. in this paper we report that ccda and kis proteins, the antagonists of the ccd and pard systems respectively, share significant sequence homologies at both ends. in kis, these regions seem to correspond to two different domains. despite the structural similarities, kis and ccda are not interchangeable. in addition we have ...19912017133
interaction of the expression of two membrane genes, acra and plsa, in escherichia coli k-12.the mutation acra1, leading to acriflavine sensitivity through disorganization of the plasma membrane, is located between proc and pure on the escherichia coli k-12 chromosome. gene plsa has been reported to determine biosynthesis of membrane phospholipid and to be located very near acra (1). genes acra and plsa fall into different cistrons and are arranged in the order proc-acra-plasa-pure. the genes were shown to interact with each other. introduction of acra mutation into a plsa temperature-s ...19751097404
the transfer operon of plasmid r1 extends beyond fino into the downstream replication genes.fino is the final gene in the 35.4 kb transfer operon of incfi plasmid f that is known to be involved in self-conjugative transfer. the genetic region distal to fino separates the conjugation and replication control modules of incfii plasmid r100 and carries uncharacterized genes not found in plasmid f. however, comparison of the r100 gene organization with database entries of f-like plasmids suggests its broad conservation. we determined the dna sequence of this region of incfii plasmid r1 and ...201021145348
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