Showing papers by "Stephen C. Winans published in 1985"
TL;DR: A mutation of a cloned gene that has been made by introducing a transposon or some other selectable genetic determinant can be crossed into the gene's original replicon by linearizing the cloned DNA and transforming a recB recC sbcB mutant.
Abstract: A mutation of a cloned gene that has been made by introducing a transposon or some other selectable genetic determinant can be crossed into the gene's original replicon by linearizing the cloned DNA and transforming a recB recC sbcB mutant. A number of applications of this method are described with genes of either chromosomal or plasmid origin.
326 citations
TL;DR: The conjugal transfer system of the broad-host range IncN plasmid pKM101 was analyzed genetically and 11 complementation groups required for transfer were identified, including traC, which is of special interest in that mutations at this locus can be partially suppressed if, prior to mating, cells carrying a traC mutant plasmide are incubated with cells which elaborate sex pili but are unable to transfer their plasmids.
Abstract: The conjugal transfer system of the broad-host range IncN plasmid pKM101 was analyzed genetically Its organization differed significantly from that of the F plasmid The tra genes are located in three regions, each between 3 and 4 kilobases in length All of the genes in the first two regions are required for sensitivity to "donor-specific" phage which bind to the plasmid-mediated sex pilus, and these genes therefore are involved in the synthesis, and possibly retraction, of the sex pilus The plasmid9s origin of transfer was localized to a 12-kilobase region at an extreme end of the transfer region Using two different methods, we have identified 11 complementation groups required for transfer One of these, traC, is of special interest in that mutations at this locus can be partially suppressed if, prior to mating, cells carrying a traC mutant plasmid are incubated with cells which elaborate sex pili but are unable to transfer their plasmids One possible explanation for this is that pilus-elaborating cells can donate traC gene product to a traC mutant in a form that can be reused
128 citations
TL;DR: Two pKM101-encoded loci have been identified which elaborate products that are potentially lethal to the bacterial cell, and both korA and korB are required to control the lethality of either kil gene.
Abstract: Two pKM101-encoded loci (designated kilA and kilB) have been identified which elaborate products that are potentially lethal to the bacterial cell. The lethal effects of each of these products is inhibited by two other plasmid-encoded loci, designated korA and korB (for kil override). Both korA and korB are required to control the lethality of either kil gene. In the presence of korA and korB both kil genes have other phenotypes: kilB is necessary for conjugal transfer, whereas kilA is responsible for the small-colony morphology on defined media that is characteristic of pKM101-containing strains (the Slo phenotype).
37 citations
TL;DR: The fip-deficient Tn5 insertion derivatives of pKM101 were isolated and did not inhibit either the synthesis of the IncP plasmid's sex pilus or its ability to mediate entry exclusion against other IncP Plasmids.
Abstract: IncN plasmids, including pKM101, strongly inhibit the conjugal transfer of cohabiting IncP plasmids. We localized the pKM101 DNA sufficient for this phenomenon to a 1.1-kilobase region (denoted fip). Two fip-deficient Tn5 insertion derivatives of pKM101 were isolated; neither affected other pKM101-mediated functions. fip did not inhibit either the synthesis of the IncP plasmid's sex pilus or its ability to mediate entry exclusion against other IncP plasmids.
28 citations
TL;DR: PKM101 renders its host a poor recipient in conjugal matings with genetically distinguishable derivatives of itself and a gene linked to, or coincident with, the region needed for vegetative plasmid replication also inhibited establishment of related plasmids under certain conditions.
Abstract: pKM101 renders its host a poor recipient in conjugal matings with genetically distinguishable derivatives of itself. The gene(s) primarily responsible for this, denoted eex, is located in between genes required for both conjugal transfer and sensitivity to donor-specific bacteriophage, although it itself is not necessary for transfer. A gene linked to, or coincident with, the region needed for vegetative plasmid replication also inhibited establishment of related plasmids under certain conditions. Construction of an operon fusion between eex and the Escherichia coli lac promoter has shown that this gene is transcribed in a clockwise fashion on the circular map of pKM101. To date, we have not been able to visualize a protein product(s) of the eex gene(s).
27 citations
01 Jan 1985
TL;DR: A number of plasmic loci have been found tocontain genes whose products arepotentially lethal to thebacterial host, and thesegeneshave beenimplicated inenhancing plasmid stability bycoupling hostcell division toplasmid replication.
Abstract: TwopKM101-encoded loci (designated kilA andkiLB) havebeenidentified whichelaborate products that are potentially lethal tothebacterial cell. Thelethal effects ofeachofthese geneproducts isinhibited bytwoother plasmid-encoded loci, designated korAandkorB(for kiloverride). BothkorAandkorBarerequired tocontrol thelethality ofeither kit gene.InthepresenceofkorAandkorBbothkil geneshaveother phenotypes: kilBis necessaryforconijugal transfer, whereas kiL4 isresponsible forthesmall-colony morphology on defined media that ischaracteristic ofpKM101-containing strains (the Slophenotype). A numberofplasmic& havebeenfound tocontain genes whoseproducts arepotentially lethal tothebacterial host. TheIncPplasmid RK2 hasthree suchgenes, designated kilA, kilB, andkilC(11). Thelethality ofthese genesis normally inhibited bythree other plasmid-encoded genes, designated korA,korB, andkorC(for kiloverride). korA alone issufficient tocontrol thelethality ofkilA(30). In contrast, korAandkorBtogether arerequired tocontrol the lethality ofkilB (D.H.Bechhofer andD.H.Figurski, manuscript inpreparation), andsimilarly, korAandkorCmust acttogether toprevent thelethal phenotype ofkilC (30). In astrain containing arhomutation, korCalone issufficient to control kilC. Ithasbeenspeculated thatkorApositively effects transcription ofkorC, possibly byantitermination. TheDNA sequence ofthekorAgenehasbeendetermined (1).'- TheF episome hasalsobeenreported tocontain two genes, onehaving thephenotype ofakil geneandtheother having thephenotype ofakorgene(20). Thesegeneshave beenimplicated inenhancing plasmid stability bycoupling hostcell division toplasmid replication. pKM101isa35.4-kilobase (kb)self-transmissible IncN
2 citations