Construction and characterization of derivatives carrying insertion mutations in F plasmid transfer region genes, trbA, artA, traQ, and trbB.
We devised a method for construction of insertion mutations in F plasmid tra region genes as a means of investigating the functions associated with previously uncharacterized loci. First, we constructed mutations in vitro, by insertion of a kanamycin resistance gene into a unique restriction site within a tra region fragment carried by a small, chimeric plasmid. Second, we crossed the insertion mutations, in vivo, onto a plasmid containing the complete F tra region sequence (either F lac, or pOX38, a Tra+ F plasmid derivative). Using this method, we obtained F lac mutant derivatives carrying KmR gene insertions in traQ, and a set of pOX38 mutant derivatives carrying a KmR gene insertion in trbA, artA, traQ, or trbB. Analysis of these derivatives showed that insertion of a kan gene at the NsiI site of traQ resulted in transfer deficiency, F-pilus-specific-phage resistance and an absence of detectable F-pilin subunit synthesis. Since the traQ mutants regained a wild-type phenotype when complemented with a traQ+ plasmid clone, we concluded that traQ expression is essential to transfer and F-pilus synthesis. However, pOX38 derivatives carrying kan gene inserts in genes trbA, artA, or trbB retained F-pilus-specific phage sensitivity and transferred at normal levels. Thus, these three gene products may not be essential for F-transfer from Escherichia coli K-12 under standard mating conditions.
Kathir
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P
Department of Medical Microbiology and Immunology, Texas A&M University, College Station 77843.
Ippen-Ihler
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K
D003001:Cloning, Molecular; D003227:Conjugation, Genetic; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics*; D005144:F Factor; D005798:Genes, Bacterial; D016254:Mutagenesis, Insertional; D015183:Restriction Mapping; D014169:Transformation, Bacterial
D004251:DNA Transposable Elements