The use of λplac-Mu hybrid phages in Klebsiella pneumoniae and the isolation of stable Hfr strains

Abstract

Klebsiella pneumoniae 1033-5P14 and its P1-sensitive derivative KAY2026 were found to be resistant to lambda although they contained a LamB protein, active as a maltoporin. Sensitive derivatives could only be obtained after introduction of the pTROY9 plasmid which expresses lamB and the corresponding lambda receptor from Escherichia coli K12 at high levels. Lysogenic derivatives from such strains were shown to carry the phage at secondary att sites and to give high titer lysates when induced. The use of lambda plac-Mu hybrid phages allowed the isolation from several operons of lacZ fusions orientated in, or against, the direction of transcription. Such insertions could subsequently be used to isolate stable Hfr strains by allowing homologous recombination to take place between the lac genes in the inserted hybrid phages and those of plasmid F' ts114 lac+ zzf20::Tn10. The Hfr strains were able to transfer K. pneumoniae chromosomal genes and allowed the mapping of such genes. Characteristic differences between this conjugation system and that of Escherichia coli K12 are discussed. The insertions also allowed determination of the direction of transcription of the gut gene, the newly mapped scr gene and of the sor gene cluster encoding enzymes for the metabolism of D-glucitol, sucrose and L-sorbose.