Identification of four structural genes and two putative promoters necessary for utilization of naphthalene, phenanthrene and fluoranthene by Sphingomonas paucimobilis var. EPA505.

Sphingomonas paucimobilis var. EPA505 utilizes fluoranthene (FLA), naphthal ene (NAP), and phenanthrene (PHE) as sole carbon sources for energy and gro wth. A genetic library of EPA505 was constructed using mini-Tn5 promoter re porter genes encoding for tetracycline resistance (tc(p-)) or luminescence (lux-AB(p-)). Out of 2250 Tn5 mutants, ten were deficient in utilization of FLA, NAP, and/or PHE as sole carbon sources. Three classes of Tn5 mutants were defined: classI (nap(-)phe(-)fla(-)), classII (nap(-)phe(-)), and clas sIII (fla(-)). Four of five mutants in classI did not express dioxygenase f unction, whereas one classI mutant and all classII and classIII mutants ret ained dioxygenase activity. In Tn5 tc(p-) classI mutants 200 and 394 (dioxy genase negative) and classII mutant 132 (dioxygenase positive), promoter re porter was expressed when induced with FLA, NAP, PHE, other polycyclic arom atic hydrocarbons (PAHs), and several proposed PAM-derived catabolites. The Tn5 tc(p-) derived classIII mutant 104 was induced only with PAHs and not with PAM-derived catabolites. DNA sequence analysis of cloned regions of cl assI mutant 200 revealed that Tn5 inserted into a gene that shared (96%) DN A sequence homology with 2,3-dihydroxybiphenyl 1,2-dioxygenase that is desi gnated pbhA. Nucleotide sequences downstream of pbhA shared (84%) homology to a Rieske-type ferredoxin subunit gene of a multicomponent dioxygenase de signated pbhB. The Tn5 tc(p-) in classII mutant 132 occurred within sequenc es that shared (74%) homology with a trans-o-hydroxybenzylidene-pyruvate hy dratase-aldolase gene (pbhC). Sequence analysis of the region proximal to t his gene revealed a putative promoter that contained a binding site for a L ysR transcriptional activator. In classIII mutant 104, the Tn5 tc(p-) resid ed within a region that shared 94% nucleotide homology to that of a pyruvat e phosphate dikinase gene known to be involved in cellular uptake of glucos e. The FLA-specific catabolic gene disrupted in mutant 104 was designated p hbD. Functional and sequence analyses of promoter probe mutants allowed ide ntification of four genes necessary for the utilization of PAHs that are co ntrolled by at least two promoters that are affected by a wide range of aro matic compounds. (C) 2000 Elsevier Science B.V, All rights reserved.