Transposon mutagenesis in Acinetobacter calcoaceticus RAG-1.
Molecular genetic studies of Acinetobacter spp. have been greatly limited by the lack of a method for transposon mutagenesis. In this study, a genetically engineered derivative of Tn10, mini-Tn10PttKm, was conjugally transferred in plate matings from Escherichia coli SM10[(lambda pir)(pLOFPttKm)] to Acinetobacter calcoaceticus RAG-1. Transfer frequencies were dependent on mating ratios and varied from 7.9 x 10(-8) to 3.4 x 10(-7) per recipient cell. The 27 lipase-deficient transconjugants which were isolated exhibited several different phenotypes, including gelatinase mutants, esterase mutants, and putative auxotrophs. Southern blot analysis confirmed the insertion of the mini-Tn10PttKm transposon in single, unique sites in five transconjugants. Four of five lipase mutants contained single insertions of mini-Tn10PttKm in the same chromosomal restriction fragments. To our knowledge, this is the first report of the use of a transposon for direct, generalized mutagenesis in Acinetobacter spp.
Leahy
J G
JG
Department of Microbiology, University of Maryland, College Park 20742.
Jones-Meehan
J M
JM
Pullias
E L
EL
Colwell
R R
RR
D016954:Acinetobacter calcoaceticus / Q000235:genetics*; D015139:Blotting, Southern; D003227:Conjugation, Genetic; D003470:Culture Media; D004251:DNA Transposable Elements; D016254:Mutagenesis, Insertional
D003470:Culture Media; D004251:DNA Transposable Elements