TN5-DIRECTED CLONING OF PQQ GENES FROM PSEUDOMONAS-FLUORESCENS CHA0 -MUTATIONAL INACTIVATION OF THE GENES RESULTS IN OVERPRODUCTION OF THEANTIBIOTIC PYOLUTEORIN

Pseudomonas fluorescens CHA0 produces several secondary metabolites, e .g., the antibiotics pyoluteorin (Pit) and 2,4-diacelylphloroglucinol (Phl), which are important for the suppression of root diseases caused by soil-borne fungal pathogens, A Tn5 insertion mutant of strain CHA0 , CHA625, does not produce Phl, shows enhanced pit production on malt agar, and has lost part of the ability to suppress black root rot in t obacco plants and take-all in wheat, We used a rapid, two-step cloning -out procedure for isolating the wild-type genes corresponding to thos e inactivated by the Tn5 insertion in strain CHA625. This cloning meth od should be widely applicable to bacterial genes tagged with Tn5, The region cloned from P. fluorescens contained three complete open readi ng frames, The deduced gene products, designated PqqFAB, showed extens ive similarities to proteins involved in the biosynthesis of pyrroloqu inoline quinone (PQQ) in Klebsiella pneumoniae, Acinetobacter calcoace ticus, and Methylobacterium extorquens. PQQ-negative mutants of strain CHA0 were constructed by gene replacement, They lacked glucose dehydr ogenase activity, could not utilize ethanol as a carbon source, and sh owed a strongly enhanced production of Plt on malt agar, These effects were all reversed by complementation with pqq(+) recombinant plasmids , The growth of a pqqF mutant on ethanol and normal Plt production wer e restored by the addition of 16 nM PQQ However, the Phl(-) phenotype of strain CHA625 was due not to the pqq defect but presumably to a sec ondary mutation, In conclusion, a lack of PQQ markedly stimulates the production of Pit in P. fluorescens.