Transposon mutagenesis of marine Vibrio spp.

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RESUMO

Coliphage P1 was used to transduce derivatives of transposons Tn5 and mini-Mu into marine Vibrio spp. Transposon Tn5 encoding tetracycline resistance (Tn5-132) was used to isolate mutants of Vibrio harveyi defective in genes for bioluminescence (lux). Insertion of transposon Tn5-132 into the lux gene region was demonstrated by intraspecific transduction with phage hv-1 and by Southern blot hybridization. Transposon mini-Mu, modified to specify tetracycline resistance, was employed to mutagenize genes for lateral flagella synthesis of Vibrio parahaemolyticus. Mini-Mu contains the lacZ structural gene, and transposition results in transcriptional fusion of Vibrio genes with the transposon lacZ gene. Thus, in these fusions, lacZ expression was proportional to the level of transcription of the target gene. Regulation of lateral flagella gene expression was studied in vivo by measuring beta-galactosidase activity, and conditions which activate transcription of these genes were identified. A method for gene cloning with transposon-induced mutations is discussed.

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