A RAPID METHOD FOR NAPHTHALENE DIOXYGENASE ASSAY IN WHOLE CELLS OF NAPHTHALENE CIS-DIHYDRODIOL DEHYDROGENASE BLOCKED PSEUDOMONAS-FLUORESCENS - SCREENING OF POTENTIAL INDUCERS OF DIOXYGENASE ACTIVITY

A rapid and sensitive photometric method was devised to assay naphthal ene dioxygenase in whole cells of Pseudomonas fluorescens NCIMB 40531, a strain derived from a naphthalene-metabolizing isolate by means of N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The naphthalene-assi milating pathway of NCIMB 40531 is functionally blocked at the level o f cis-1,2-dihydroxy-1,2-dihydronaphthalene dehydrogenase and therefore cis-1,2-dihydroxy-1,2-dihydronaphthalene (naphthalene dihydrodiol) is accumulated when cultures are supplied with naphthalene. This modifie d metabolism allowed dioxygenase to be assayed by monitoring product f ormation. Optimal conditions were selected to give linear optical dens ity vs time curves and reaction rates proportional to dry cell weight (DCW): specific activities of 0.125(+/-0.008) mu mol.min(-1).mgDCW(-1) were consistently obtained in cultures grown on succinate in the pres ence of naphthalene as inducer. By means of the developed assay, 62 co mpounds (mainly mono- and bicyclic aromatics) were screened as potenti al inducers of the dioxygenase activity, when added to the growth medi um at the concentration of 100 mg.l(-1): besides naphthalene, the high est activities were induced by 3-methylsalicylic acid (2-hydroxy-3-met hylbenzoic acid), O-acetylsalicylic acid and 5-chlorosalicylic acid wi th 0.198, 0.167 and 0.076 mu mol.min(-1.)mg DCW-1, respectively. Under the conditions used, no detectable dioxygenase activity was induced b y salicylic acid, which is recognized as the natural inducer of the en zyme in Pseudomonas.