A systematic survey for the presence of plasmids in 17 different xenobiotic-degrading Sphingomonas strains was performed. In almost all analyzed strains, two to five plasmids with sizes of about 50 to 500 kb were detected by using pulsed-field gel electrophoresis. A comparison of plasmid preparations untreated or treated with S1 nuclease suggested that, in general, Sphingomonas plasmids are circular. Hybridization experiments with labeled gene probes suggested that large plasmids are involved in the degradation of dibenzo-p-dioxin, dibenzofuran, and naphthalenesulfonates in S. wittichii RW1, Sphingomonas sp. HH69, and S. xenophaga BN6, respectively. The plasmids which are responsible for the degradation of naphthalene, biphenyl, and toluene by S. aromaticivorans F199 (pNL1) and of naphthalenesulfonates by S. xenophaga BN6 (pBN6) were site-specifically labeled with a kanamycin resistance cassette. The conjugative transfer of these labeled plasmids was attempted with various bacterial strains as putative recipient strains. Thus, a conjugative transfer of plasmid pBN6 from S. xenophaga BN6 to a cured mutant of strain BN6 and to Sphingomonas sp. SS3 was observed. The conjugation experiments with plasmid pNL1 suggested a broader host range of this plasmid, because it was transferred without any obvious structural changes to S. yanoikuyae B1, Sphingomonas sp. SS3, and S. herbicidovorans. In contrast, major plasmid rearrangements were observed in the transconjugants after the transfer of plasmid pNL1 to Sphingomonas sp. HH69 and of pBN6 to Sphingomonas sp. SS3. No indications for the transfer of a Sphingomonas plasmid to bacteria outside of the Sphingomonadaceae were obtained.

The effect of different artificial redox mediators on the anaerobic reduction of azo dyes by Sphingomonas sp. strain BN6 or activated sludge was investigated. Reduction rates were greatly enhanced in the presence of sulfonated anthraquinones. For strain BN6, the presence of both cytoplasmic and membrane-bound azo reductase activities was shown. Various bacterial strains reduce azo dyes under anaerobic conditions. The most generally accepted hypothesis for this phenomenon is that many bacterial cells possess a rather unspecific cytoplasmic azo reductase which transfers electrons under anaerobic conditions via (soluble) flavins to the azo dyes (7, 12, 30, 32). Recently, it was shown that the naphthalenesulfonate-degrading Sphingomonas sp. strain BN6 converted 2-naphthalenesulfonate to some kind of redox mediator (19). It was suggested that these redox mediators enabled the strain under anaerobic conditions to transfer redox equivalents to the azo dyes. In the present study, the location of the enzyme system which is responsible for the reduction of azo dyes by whole cells was determined. The effect of artificial redox mediators on the anaerobic reduction of azo dyes by whole cells from different bacterial strains was also studied. Bacterial strains and media. The isolation and characterization of Sphingomonas sp. strain BN6 (DSM 6383) and the composition of the minimal media have been described before (15, 27). Reduction of amaranth by resting cells. Strain BN6 was grown in a mineral medium with glucose (10 mM). Cells were harvested by centrifugation and resuspended in 50 mM Na-Kphosphate (pH 7.4) buffer to an optical density at 546 nm of 11. This corresponded to a protein concentration of approximately 1.1 g/liter. The reaction mixture contained, in a final volume of 7.2 ml, 80 mol of glucose, 400 mol of Na-K-phosphate buffer (pH 7.4), and different concentrations of the redox mediators. This cell suspension was transferred to a rubber-stoppered serum bottle (30 ml). Oxygen was removed from the medium by at least 15 2-min cycles of evacuation and flushing with nitrogen gas. The reaction was started by the injection of 0.8 ml from an anaerobic stock solution of amaranth (5 or 10 mM). The reduction of the azo dye was determined spectrophotometrically at a of 520 nm. Experiments with activated sludge. The activated sludge was obtained from the aerobic part of the sewage treatment plant of the University of Stuttgart (Büsnau). Particulate material was collected by centrifugation (8,500 ϫ g, 30 min), resuspended in Na-K-phosphate buffer (50 mM; pH 7.4), and passed through a sieve with a mesh size of 1.0 mm to eliminate

The anaerobic reduction of azo dyes by Sphingomonas sp. strain BN6 was analyzed. Aerobic conversion of 2-naphthalenesulfonate (2NS) by cells of strain BN6 stimulated the subsequent anaerobic reduction of the sulfonated azo dye amaranth at least 10-fold. In contrast, in crude extracts, the azo reductase activity was not stimulated. A mutant of strain BN6 which was not able to metabolize 2NS showed increased amaranth reduction rates only when the cells were resuspended in the culture supernatant of 2NS-grown BN6 wild-type cells. The same increase could be observed with different bacterial strains. This suggested the presence of an extracellular factor which was formed during the degradation of 2NS by strain BN6. The addition of 1,2dihydroxynaphthalene, the first intermediate of the degradation pathway of 2NS, or its decomposition products to cell suspensions of the mutant of strain BN6 (2NS ؊) increased the activity of amaranth reduction. The presence of bacterial cells was needed to maintain the reduction process. Thus, the decomposition products of 1,2-dihydroxynaphthalene are suggested to act as redox mediators which are able to anaerobically shuttle reduction equivalents from the cells to the extracellular azo dye.

During aerobic degradation of naphthalene-2-sulfonate (2NS), Sphingomonas xenophaga strain BN6 produces redox mediators which significantly increase the ability of the strain to reduce azo dyes under anaerobic conditions. It was previously suggested that 1,2-dihydroxynaphthalene (1,2-DHN), which is an intermediate in the degradative pathway of 2NS, is the precursor of these redox mediators. In order to analyze the importance of the formation of 1,2-DHN, the dihydroxynaphthalene dioxygenase gene (nsaC) was disrupted by gene replacement. The resulting strain, strain AKE1, did not degrade 2NS to salicylate. After aerobic preincubation with 2NS, strain AKE1 exhibited much higher reduction capacities for azo dyes under anaerobic conditions than the wild-type strain exhibited. Several compounds were present in the culture supernatants which enhanced the ability of S. xenophaga BN6 to reduce azo dyes under anaerobic conditions. Two major redox mediators were purified from the culture supernatants, and they were identified by high-performance liquid chromatography-mass spectrometry and comparison with chemically synthesized standards as 4-amino-1,2-naphthoquinone and 4-ethanolamino-1,2-naphthoquinone.Wastewaters from textile industries are often highly colored due to residual dyestuff from the dyeing processes. It has been estimated that up to 60% of the total dyestuff used in the dyeing processes may be found in the wastewaters (3). Only small amounts of these dyes are removed by conventional aerobic biological wastewater treatment systems (37, 46). On the other hand, it is well known that azo dyes are reduced anaerobically by different microorganisms, which usually results in the generation of colorless aromatic amines (2,5,7,12,14,51,56). These amines are in most cases recalcitrant under anaerobic conditions (6). Therefore, it has been repeatedly suggested that two-stage anaerobic-aerobic treatment systems should be used to reduce the azo dyes anaerobically and to mineralize the amines formed in a subsequent aerobic stage (1,36,38,45,51,54). The first successful example of mineralization of a sulfonated azo dye by an anaerobic-aerobic treatment process involved a 6-aminonaphthalene-2-sulfonate-degrading mixed bacterial culture. This culture consisted of the naphthalenesulfonate-degrading strain Sphingomonas xenophaga BN6 in a mutualistic coculture with other bacterial strains (21,34,50). Recently, it was found that strain BN6 uses a new mechanism for conversion of azo dyes. During aerobic degradation of naphthalene-2-sulfonate (2NS), redox mediators are released, which mediate the reduction of azo dyes under anaerobic conditions (27). It was proposed that these redox mediators shuttle electrons from the cells to the azo dyes, which results in purely chemical, extremely nonspecific, extracellular reductive cleavage of the azo bond. Aerobic preincubation of strain BN6 with 2NS increased subsequent anaerobic reduction of the azo dyes almost 20-fold. It was suggested that 1,2-dihydroxynaphthalene (1,2-DHN), which is the ...

Sphingomonas xenophaga BN6 degrades various (substituted) naphthalenesulfonates to the corresponding (substituted) salicylates. A gene cluster was identified on the plasmid pBN6 which coded for several enzymes participating in the degradative pathway for naphthalenesulfonates. A DNA fragment of 16 915 bp was sequenced which contained 17 ORFs. The genes encoding the 1,2-dihydroxynaphthalene dioxygenase, 2-hydroxychromene-2-carboxylate isomerase, and 29-hydroxybenzalpyruvate aldolase of the naphthalenesulfonate pathway were identified on the DNA fragment and the encoded proteins heterologously expressed in Escherichia coli. Also, the genes encoding the ferredoxin and ferredoxin reductase of a multi-component, ring-hydroxylating naphthalenesulfonate dioxygenase were identified by insertional inactivation. The identified genes generally demonstrated the highest degree of homology to enzymes encoded by the phenanthrene-degrading organism Sphingomonas sp. P2, or the megaplasmid pNL1 of the naphthalene-and biphenyl-degrading strain Sphingomonas aromaticivorans F199. The genes of S. xenophaga BN6 participating in the degradation of naphthalenesulfonates also shared the same organization in three different transcriptional units as the genes involved in the degradation of naphthalene, biphenyl, and phenanthrene previously found in Sphingomonas sp. P2 and S. aromaticivorans F199. The genes were flanked in S. xenophaga BN6 by ORFs which specify proteins that show the highest homologies to proteins of mobile genetic elements.