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.
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.
The regulation of the transport of leucine, isoleucine, and valine in Escherichia coli B/r was studied in a mutant with a complete deletion of the leucine biosynthetic operon and a temperature-sensitive leucyl-tRNA synthetase [L1leucine:tRNAI-u ligase (AMP-forming), EC 6.1.1.4].Under conditions of excess leucine and a functional leucyltRNA synthetase transport activity was repressed. Shifting the culture to a temperature at which the activation of leucine to an appropriate tRNA species became growth-rate-limiting led to a large increase in the high-affinity transport of leucine, isoleucine, and valine (system LIV-I) while the uptake of histidine and proline was unchanged. A similar increase was observed for branched-chain amino-acid binding protein activity. (1) showed that the level of leucine transport could be lowered by growth of Escherichia coil with exogenous leucine. This study has been confirmed in a number of laboratories since that time (2-4). During the same period of time extensive studies (5, 6) indicated that some aminoacyl-tRNA synthetases are important both as components in protein synthesis and as a part of the regulatory system by which certain biosynthetic operons are repressed by their cognate amino acids. The independence of the regulatory systems for transport and biosynthesis of the branched-chain amino acids has been established (7), although the regulatory systems for these two cellular processes appear to share some common component(s). The availability of a temperature-sensitive mutant for the leucyltRNA synthetase [L-leucine:tRNAI-eu ligase (AMP-forming), EC 6.1.1.4] provided an opportunity to determine if this enzyme was involved in the regulation of both transport and biosynthesis. The results of the present communication indicate that leucine must be activated to its respective leucyltRNAs to repress branched-chain amino-acid transport. MATERIALS AND METHODSBacteria. Two strains of E. coil B/r were used: strain EB143 (ara-leuAllOl, leuSl) and its isogenic parental strain, EB144 (ara-leuAllOl). Strain EB144 was derived by selection for growth on leucine-containing media at 440 following a Pjbt transduction of strain EB143. The lysate was obtained from a wild-type B/r strain. The previously characterized ara-leuAllOl deletion (8) growing logarithmically for at least three generations before assays were performed. The culture density at harvesting was 0.12-0.15 mg/ml (dry weight). For small scale experiments, temperature changes were made by the addition of small volumes of cells to prewarmed media at the desired temperature. For the isolation of shock fluid (10), 1 liter cultures were grown at 360 to a density of 0.2 mg/ml (dry weight), and then transferred to a shaking water bath at 41°. The temperature of the culture was equilibrated in 10-15 min.Biochemical Assays. Cell extracts were prepared for enzyme assays as previously described (7). Enzyme assays were performed within 1-2 hr after harvest. Whole cell transport was assayed as described (11) RESULTSBranched-chain a...
The ability of indole derivatives to facilitate RNA polymerase transcription of the L-arabinose operon in Escherichia coli was shown to require the catabolite activator protein (CAP) as well as the araC gene product. Adenosine 3',5'-monophosphate (cAMP) was not obligatory for araBAD transcription when the cells were grown in the presence of 1 mM indole-3-acetic acid or in the presence of indole-3-acetamide, indole-3-propionic acid, indole-3-butyric acid, or 5-hydroxyindole-3-acetic acid. However, these indole derivatives were unable to circumvent the cAMP requirement for the induction of the lactose and the maltose operons. Catabolite repression occurred when glucose was added to cells grown in the presence of L-arabinose and 1 mM indoleacetic acid or 1 mM cAMP. This effect was reversed at higher concentrations of indoleacetic acid or cAMP. The induction and the catabolite repression phenomena were quantitated by measuring the differential rate of synthesis of L-arabinose isomerase (the araA gene product). These results indicated that indole metabolites from various living systems may regulate gene expression and may be involved in "metabolite gene regulation."The induction of the L-arabinose regulon-araBAD, araE, and araF-in Escherichia coli requires the protein product (P2) of the araC gene, the catabolite activator protein (CAP), and adenosine 3',5'-monophosphate (cAMP) (1-8). Hence, mutants with an inactive product for the crp gene (the gene coding for CAP), the araC gene, or the cya gene (the gene coding for adenylate cyclase) are unable to utilize L-arabinose as a carbon source.The existence of araCl mutants that can circumvent the requirement for cAMP in the induction of the L-arabinose operon (2) and the complexity of various regulatory interlocks (9-11) led to the idea that low molecular weight metabolites other than the ubiquitous cAMP and guanosine 3',5'-monophosphate might function at the genetic level in cell regulation. Recently, we demonstrated with cya deletion strains of E. coli (12) that specific concentrations of imidazoleacetic acid, a compound structurally related to the amino acid histidine, could circumvent the necessity for cAMP in the induction of the araBAD structural genes. In addition, preliminary evidence indicated that a metabolite of tryptophan, indole-3-acetic acid (IAA), could function in the "metabolite gene regulation" of eukaryotic cells and various genera of bacteria. *
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