Degradation of aromatic hydrocarbons by Sphingomonas paucimobilis strain EPA505.

To determine the substrate range capability of Sphingomonas paucimobilis strain EPA505, a number of aromatic compounds were tested as potential growth substrates. Strain EPA505 grew on phenanthrene, naphthalene, fluoranthene, toluene, benzoic acid, 2,3- and 3,4-dihydroxybenzoic acids, 1-chloro-2,4-dinitrobenzene, anthracene, 2-hydroxy-3-naphthoic acid and 1-hydroxy- 2-naphthoic acid, salicylic acid, and catechol. Strain EPA505 was unable to grow on coumarine 3-carboxylic acid, naphthalene dicarboxylic acid, acenaphthene, chrysene, pyrene, benzo[b]fluoranthene, and fluorene. Catabolic products were not detected or identified when the bacterium was incubated with coumarine 3-carboxylic acid, naphthalene dicarboxylic acid, acenaphthene, chrysene, or benzo[b]fluoranthene. Dihydroxypyrene, the ortho ring fission product of pyrene, and 10-hydroxy-1- phenanthroic acid were detected when the bacterium was incubated with pyrene. The open rings of benzo[b]fluoranthene, hydroxyacephenanthroic acid, hydroxyacephenanthrene, and phenanthrene anhydride, catabolites of benzo[b]fluoranthene degradation, were detected with Tn5 mutants of EPA505. With strain EPA505, both 9-fluorenone and an open ring fission product accumulated during incubation with fluorene. Other catabolites beyond the open ring of fluorene were detected, specifically dihydroxyfluorene, hydroxy-9-fluorenone, dihydroxy-9-fluorenone, hydroxyindane, and a putative glutathione-conjugated benzylanhydride. Benzylanhydride appeared to be a final end product of fluorene degradation by strain EPA505.

Convergent and divergent points in catabolic pathways involved in utilization of fluoranthene, naphthalene, anthracene, and phenanthrene by Sphingomonas paucimobilis var. EPA505.

Catabolic pathways for utilization of naphthalene (NAP), anthracene (ANT), phenanthrene (PHE), and fluoranthene (FLA) by Sphingomonas paucimobilis EPA505 were identified. Accumulation of catabolic intermediates was investigated with three classes of Tn5 mutants with the following polycyclic aromatic hydrocarbon (PAH)-negative phenotypes; (class I NAP(-) PHE(-) FLA(-), class II NAP(-) PHE(-), and class III FLA(-)). Class I mutant 200pbhA had a Tn5 insertion within a meta ring fission dioxygenase (pbhA), and a ferredoxin subunit gene (pbhB) resided directly downstream. Mutant 200pbhA and other class I mutants lost the ability to catalyze the initial dihydroxylation step and did not transform NAP, ANT, PHE, or FLA. Class I mutant 401 accumulated salicylic acid, 2-hydroxy-3-naphthoic acid, 1-hydroxy-2-naphthoic acid, and hydroxyacenaphthoic acid during incubation with NAP, ANT, PHE, or FLA, respectively. Class II mutant 132pbhC contained the Tn5 insertion in an aldolase hydratase (pbhC) and accumulated what appeared to be meta ring fission products: trans-o-hydroxybenzylidene pyruvate, trans-o-hydroxynaphylidene pyruvate, and trans-o-hydroxynaphthyl-oxobutenoic acid when incubated with NAP, ANT, and PHE, respectively. When mutant 132pbhC was incubated with 1-hydroxy-2-naphthoic acid, it accumulated trans-o-hydroxybenzylidene pyruvate. Class III mutant 104ppdk had a Tn5 insertion in a pyruvate phosphate dikinase gene that affected expression of a FLA-specific gene and accumulated a proposed meta ring fission product; trans-o-hydroxyacenaphyl-oxobutenoic acid during incubation with FLA. Trans-o-hydroxyacenaphyl-oxobutenoic acid was degraded to acenaphthenone that accumulated with class III mutant 611. Acenaphthenone was oxidized via incorporation of one molecule of dioxygen by another oxygenase. 2,3-Dihydroxybenzoic acid was the final FLA-derived catabolic intermediate detected. Analysis of PAH utilization mutants revealed that there are convergent and divergent points involved in NAP, ANT, PHE, and FLA utilization by S. paucimobilis EPA505.

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

Sphingomonas paucimobilis var. EPA505 utilizes fluoranthene (FLA), naphthalene (NAP), and phenanthrene (PHE) as sole carbon sources for energy and growth. A genetic library of EPA505 was constructed using mini-Tn5 promoter reporter genes encoding for tetracycline resistance (tc(p-)) or luminescence (luxAB(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 classIII (fla(-)). Four of five mutants in classI did not express dioxygenase function, whereas one classI mutant and all classII and classIII mutants retained dioxygenase activity. In Tn5 tc(p-) classI mutants 200 and 394 (dioxygenase negative) and classII mutant 132 (dioxygenase positive), promoter reporter was expressed when induced with FLA, NAP, PHE, other polycyclic aromatic hydrocarbons (PAHs), and several proposed PAH-derived catabolites. The Tn5 tc(p-) derived classIII mutant 104 was induced only with PAHs and not with PAH-derived catabolites. DNA sequence analysis of cloned regions of classI mutant 200 revealed that Tn5 inserted into a gene that shared (96%) DNA sequence homology with 2,3-dihydroxybiphenyl 1,2-dioxygenase that is designated pbhA. Nucleotide sequences downstream of pbhA shared (84%) homology to a Rieske-type ferredoxin subunit gene of a multicomponent dioxygenase designated pbhB. The Tn5 tc(p-) in classII mutant 132 occurred within sequences that shared (74%) homology with a trans-o-hydroxybenzylidene-pyruvate hydratase-aldolase gene (pbhC). Sequence analysis of the region proximal to this gene revealed a putative promoter that contained a binding site for a LysR transcriptional activator. In classIII mutant 104, the Tn5 tc(p-) resided within a region that shared 94% nucleotide homology to that of a pyruvate phosphate dikinase gene known to be involved in cellular uptake of glucose. The FLA-specific catabolic gene disrupted in mutant 104 was designated phbD. Functional and sequence analyses of promoter probe mutants allowed identification of four genes necessary for the utilization of PAHs that are controlled by at least two promoters that are affected by a wide range of aromatic compounds.