The cloning and transposon Tn5 mutagenesis of the glnA region of Klebsiella pneumoniae: Identification of glnR, a gene involved in ... View Full Text


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Article Info

DATE

1981-10

AUTHORS

F. J. de Bruijn, F. M. Ausubel

ABSTRACT

A 15 kilobase HindIII fragment of Klebsiella pneumoniae DNA containing the glnA gene was cloned into the plasmid vector pACYC184. The resulting plasmid, pFB51, complements glnA- mutations in Escherichia coli and K. pneumoniae. pFB51 also complements the GlnR phenotype of a Klebsiella pneumoniae gln regulatory mutant (KP5060) defined by the restoration of Hut+ and Nif+ (histidine utilization and nitrogen fixation) phenotypes to this strain. Three recombinant plasmids containing subsegments of the 15 kb HindIII fragment were derived from pFB51. Plasmid pFB514 which contains a spontaneous 4 kb delection of K. pneumoniae DNA from pFB51 is more stable than pFB51 and is still able to complement glnA- mutations and the GlnR- phenotype of KP5060. Plasmids pFB53 and pFB54, which contain a 6.5 kb SalI DNA fragment from pFB51 recloned into pACYC184 in opposite orientations, complement glnA- mutations but not the GlnR- phenotype of KP5060. Plasmids pFB514 and pFB53 were mutagenized by transposon Tn5 resulting in a total of 92 Tn5 insertions in the cloned fragments. Utilizing these insertion mutations, a correlated physical and genetic map was constructed by determining the physical location of each Tn5 insertion and by analyzing the ability of each Tn5 mutated plasmid to complement a glnA- strain of E. coli and a glnA- GlnR- strain of K. pneumoniae. Two classes of Tn5 insertions with an altered Gln phenotpye were obtained. One cluster of insertions spanning a 1.3 kb region abolished complementation of the glnA- mutations. A second 2 kb cluster of Tn5 insertions, immediately adjacent to the first cluster, abolished the ability of pFB514 plasmid to complement the GlnR- phenotype, while glnA- complementation was unaffected. We propose that the second cluster of Tn5 insertions define a DNA region coding for a positive regulatory factor for nitrogen fixation (nif) and histidine utilization (hut) genes (glnR). More... »

PAGES

289-297

Identifiers

URI

http://scigraph.springernature.com/pub.10.1007/bf00270631

DOI

http://dx.doi.org/10.1007/bf00270631

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1045836499

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/6120440


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