The cloning and transposon Tn5 mutagenesis of the glnA region of Klebsiella pneumoniae: identification of glnR, a gene involved in the regulation of the nif and hut operons

Summary 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).