Mutagenesis and chromosome mobilization in Hyphomicrobium facilis B-522.
Spontaneously derived antibiotic-resistant mutants of Hyphomicrobium facilis B-522, a restricted facultative methylotroph, occurred at a high frequency on agar plates with low antibiotic concentrations. Mutants specifically defective in methanol oxidation have been obtained using an allyl alcohol direct selection technique. By chemical mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine in the presence of chloramphenicol several stable auxotrophic mutants could be isolated: three leucine auxotrophs, two threonine auxotrophs, and two leucine-methionine double auxotrophic mutants. Optimal conditions for transposon mutagenesis have been developed by comparing several transposon delivery vectors. With the suicide plasmid pRK2013 as a vector, the tetracycline resistance conferring transposon Tn5-132 was introduced into the genome of H. facilis B-522. The following insertion mutants have been obtained: leu-3::Tn5-132, ilv-1::Tn5-132, and pur-1::Tn5-132. Broad host range IncP-1 plasmids could be successfully transferred by interspecific matings. Chromosome mobilization was demonstrated with the conjugative IncP-1 plasmids RP1, R68.45, pMO60, and H. facilis 2189 (leu-2, met-1, mox-1, nfs-1, str-12) as recipient strain. Transconjugants occurred at frequencies ranging from 10(-6) to 10(-8) for each marker.
Gliesche
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CG
Institut für Allgemeine Mikrobiologie, Universität Kiel, Germany.
Hirsch
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P
D001419:Bacteria / Q000235:genetics*; D002876:Chromosomes, Bacterial; D003227:Conjugation, Genetic; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D016254:Mutagenesis, Insertional; D010957:Plasmids; D011550:Pseudomonas aeruginosa / Q000235:genetics
D004251:DNA Transposable Elements