Abstract

Pseudomonas sp. strain PY002, isolated from soil, was mutagenized with a transposon Tn5(21). To screening of siderophore biosunthesis defective mutant, 138 kanamycin resistant mutants were tested of growth on MKB medium supplemented with iron chelator(bipydidyl and EDDHA) and in vitro antibiosis. Among 138 mutants, 32 mutants do not excreted a siderophore and lose their antibiotic activity. So, these mutants were designated Flu^-Sid^-. A gene bank of DNA from Pseudomonas sp. strain PY002 was constructed using the broad-host range conjugative cosmid pLAFR3. The recombinant cosmids contained insert DNA averaging 21 kb in length and the frequence of transduction into E. coli HB101 per 1㎍ of insert DNA was 9 × 10³. Nonfluorescent mutants were complemented by mating the gene bank en masse and identifying the 108 fluorescent transconjugants. Restriction enzyme analysis of these complemented transconjugants revealed three different types and they were named pCOM61, pCOM91 and pCOM97. Sizes of their insert DNA were 30kb, 26kb and 28kb, respectively.

• Keywords: Pseudomonas sp. strain PY002m siderophore biosynthesis defective mutantm complementation; cloning