Strain BN6 Research Articles

Taxonomy and Antimicrobial Activity of an Actinomycete Strain Isolated from Swallow Bird Mud Nest

An actinobacterial strain designated as BN-6 was isolated from swallow bird mud nest by agar plate dilution method. It showed the morphological, physiological and biochemical characteristics of the genus Streptomyces. On the basis of 16S rRNA gene sequence analysis and physiological tests, this isolate was found to be most closely related with Streptomyces vinaceusdrappus NRRL2363. The strain BN-6 produced a broad spectrum active metabolite in Glucose Soybean meal (GS) medium and was extracted with HP Dianion resin. The resin extracted product showed activity against Gram-positive, Gram-negative bacteria, acid fast bacilli and fugal cultures, suggesting it broad spectrum characteristics. The bioautography on silica gel plates using Micrococcus luteus MTCC106 as test organism indicated the presence of a single metabolite having a Rf value of 3.2 and 9.1 in two different solvent systems. The characteristic peak obtained in UV–VIS spectrophotometer at 214 nm and chemical analysis indicates that it may be peptide class of metabolite.

Detection and characterization of conjugative degradative plasmids in xenobiotic-degrading Sphingomonas strains.

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.

Identification of quinoide redox mediators that are formed during the degradation of naphthalene-2-sulfonate by Sphingomonas xenophaga BN6.

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.

The function of cytoplasmic flavin reductases in the reduction of azo dyes by bacteria.

A flavin reductase, which is naturally part of the ribonucleotide reductase complex of Escherichia coli, acted in cell extracts of recombinant E. coli strains under aerobic and anaerobic conditions as an "azo reductase." The transfer of the recombinant plasmid, which resulted in the constitutive expression of high levels of activity of the flavin reductase, increased the reduction rate for different industrially relevant sulfonated azo dyes in vitro almost 100-fold. The flavin reductase gene (fre) was transferred to Sphingomonas sp. strain BN6, a bacterial strain able to degrade naphthalenesulfonates under aerobic conditions. The flavin reductase was also synthesized in significant amounts in the Sphingomonas strain. The reduction rates for the sulfonated azo compound amaranth were compared for whole cells and cell extracts from both recombinant strains, E. coli, and wild-type Sphingomonas sp. strain BN6. The whole cells showed less than 2% of the specific activities found with cell extracts. These results suggested that the cytoplasmic anaerobic "azo reductases," which have been described repeatedly in in vitro systems, are presumably flavin reductases and that in vivo they have insignificant importance in the reduction of sulfonated azo compounds.

Presence of two trans-o-hydroxybenzylidenepyruvate hydratase-aldolases in naphthalenesulfonate-assimilating Sphingomonas paucimobilis TA-2: comparison of some properties.

Two trans-o-hydroxybenzylidenepyruvate hydratase-aldolases named tHBP HA A and tHBP HA B were purified from a cell-free extract of naphthalenesulfonate-assimilating Sphingomonas paucimobilis (formerly Pseudomonas sp.) TA-2 to an electrophoretically homogeneous state by successive column chromatographies on DEAE-cellulose, DEAE-Toyopearl 650M, Sephacryl S-100, Hydroxyapatite, and Mono Q. These enzymes were similar to each other in molecular mass (ca. 37 kDa on SDS-PAGE, ca. 110 kDa on ultracentrifugation), thermal stability (<50 degrees C) and optimum pH (pH 9.0). However, they differed from each other in N-terminal amino acid sequences, pH stability, K(m) values for trans-o-hydroxybenzylidenepyruvate (tHBP), and inhibition by p-chloromercuribenzoic acid (PCMB). tHBP HA B had a homologous N-terminal amino acid sequence with tHBP HAs from Pseudomonas vesicularis DSM 6383 (strain BN6) and Sphingomonas aromaticivorans F119, and tHBP HA A had a homologous sequence with tHBP HAs of Pseudomonas putida strain OUS82, Pseudomonas sp. strain C18 and NAH7 plasmid. tHBP HA B was inhibited by PCMB, but tHBP HA A was not. Their K(m) values for tHBP were 9 and 3 M, respectively. tHBP HA B was stable in the range of pH 7.1 to pH 10.7, and tHBP HA A was stable in the range of pH 6.0 to 9.3.

Degradation of substituted naphthalenesulfonic acids by Sphingomonas xenophaga BN6.

Sphingomonas xenophaga BN6 was isolated from the river Elbe as a member of a multispecies bacterial culture which mineralized 6-aminonaphthalene-2-sulfonate. Pure cultures of strain BN6 converted a wide range of amino- and hydroxynaphthalene-2-sulfonates via a catabolic pathway similar to that described for the metabolism of naphthalene to salicylate by Pseudomonas putida NAH7 or Pseudomonas sp NCIB 9816. In contrast to the naphthalene-degrading pseudomonads, S. xenophaga BN6 only partially degraded the naphthalenesulfonates and excreted the resulting amino- and hydroxysalicylates in almost stoichiometric amounts. Enzymes that take part in the degradative pathway of the naphthalenesulfonates by strain BN6 were purified, characterized and compared with the isofunctional enzymes from the naphthalene-degrading pseudomonads. According to the enzyme structures and the catalytic constants, no fundamental differences were found between the 1,2-dihydroxynaphthalene dioxygenase or the 2'-hydroxybenzalpyruvate aldolase from strain BN6 and the isofunctional enzymes from the naphthalene-degrading pseudomonads. The limited available sequence information about the enzymes from strain BN6 suggests that they show about 40-60% sequence identity to the isofunctional enzymes from the pseudomonads. In addition to the gene for the 1,2-dihydroxynaphthalene dioxygenase, the genes for two other extradiol dioxygenases were cloned and sequenced from strain BN6 and the corresponding gene products were studied. S. xenophaga BN6 has also been used as a model organism to study the mechanism of the non-specific reduction of azo dyes under anaerobic conditions and to establish combined anaerobic/aerobic treatment systems for the degradation of sulfonated azo dyes. Furthermore, the degradation of substituted naphthalenesulfonates by mixed cultures containing strain BN6 was studied in continuous cultures and was described by mathematical models.

Distal cleavage of 3-chlorocatechol by an extradiol dioxygenase to 3-chloro-2-hydroxymuconic semialdehyde.

A 2,3-dihydroxybiphenyl 1,2-dioxygenase from the naphthalenesulfonate-degrading bacterium Sphingomonas sp. strain BN6 oxidized 3-chlorocatechol to a yellow product with a strongly pH-dependent absorption maximum at 378 nm. A titration curve suggested (de)protonation of an ionizable group with a pKa of 4.4. The product was isolated, purified, and converted, by treatment with diazomethane, to a dimethyl derivative and, by incubation with ammonium chloride, to a picolinic acid derivative. Mass spectra and 1H and 13C nuclear magnetic resonance (NMR) data for these two derivatives prove a 3-chloro-2-hydroxymuconic semialdehyde structure for the metabolite, resulting from distal (1,6) cleavage of 3-chlorocatechol. 3-Methylcatechol and 2,3-dihydroxybiphenyl are oxidized by this enzyme, in contrast, via proximal (2,3) cleavage.

Reduction of azo dyes by redox mediators originating in the naphthalenesulfonic acid degradation pathway of Sphingomonas sp. strain BN6

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 1,2-dihydroxynaphthalene, 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.

Localization of the Enzyme System Involved in Anaerobic Reduction of Azo Dyes by Sphingomonas sp. Strain BN6 and Effect of Artificial Redox Mediators on the Rate of Azo Dye Reduction

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.

Description of chlorophenol-degrading Pseudomonas sp. strains KF1T, KF3, and NKF1 as a new species of the genus Sphingomonas, Sphingomonas subarctica sp. nov.

Gram-negative polychlorophenol-degrading bacterial strains KF1T (T = type strain), KF3, and NKF1, which were described previously as Pseudomonas saccharophila strains, were studied by chemotaxonomic, genetic, and physiological methods and by electron microscopy and compared with selected xenobiotic compound-degrading bacteria. These strains contained sphingolipids with d-18:0, d-20:1, and d-21:1 as the main dihydrosphingosines, ubiquinone 10 as the main respiratory quinone, and spermidine as the major polyamine, and the DNA G + C content was 66 mol%. The cellular fatty acids included about 60% octadecenoic acid, 9% 2-hydroxymyristic acid, 14% cis-9-hexadecenoic acid, and 10% hexadecanoic acid. These strains exhibited less than 97% 16S ribosomal DNA sequence similarity to all of the other taxa studied. In the DNA-DNA reassociation studies the highest levels of reassociation between these strains and previously described species were less than 40%. Thin sections of cells of strains KF1T, KF3, and NKF1 were examined by electron microscopy, and the results showed that the cells had peculiar concentrically arranged layered membranous blebs that extruded from the outer membrane, especially at the cell division points. On the basis of the results of this study, polychlorophenol-degrading strains KF1T, KF3, and NKF1 are considered members of a new species of the genus Sphingomonas, Sphingomonas subarctica. The polycyclic aromatic hydrocarbon-degrading organism Sphingomonas paucimobilis EPA 505 was closely related to Sphingomonas chlorophenolica as determined by chemotaxonomic, phylogenetic, and physiological criteria. The xenobiotic compound degraders Alcaligenes sp. strain A175 and Pseudomonas sp. strain BN6 were identified as members of species of the genus Sphingomonas.

Conversion of substituted naphthalenesulfonates by Pseudomonas sp. BN6

The range of substituted naphthalenesulfonates which are metabolized by Pseudomonas sp. BN6 were investigated. Resting cells from strain BN6 oxidized 1- and 2-naphthalenesulfonate, 1-hydroxynaphthalene-2-sulfonate, 2,6-naphthalenedisulfonate and all monosulfonated naphthalene-2-sulfonates which carry one or two substitutents in the positions 4-, 5-, 6-, 7- or 8- of the naphthalene ring-system. With the exception of (substituted) 4- or 5-amino- and 4-hydroxynaphthalene-2-sulfonates these compounds were converted to the corresponding salicylates. Strain BN6 did not oxidize substituted naphthalene-1-sulfonates, 3-substituted naphthalenesulfonates and substituted naphthalenedisulfonates. Turnover of 4-amino- or 4-hydroxynaphthalene-2-sulfonates resulted in the accumulation of the corresponding naphthoquinones in the culture medium. Thus, degradation of 4-amino- and 4-hydroxynaphthalenesulfonates was restricted by the rapid autoxidation of the substituted 1,2-dihydroxynaphthalenes formed as metabolites. Catabolic activities of strain BN6 for naphthalenesulfonates were induced by salicylate, 3- or 6-hydroxysalicylate, and 3-, 4- or 5-aminosalicylate but not by 4- and 5-hydroxysalicylate. All naphthalenesulfonates that were not converted into the corresponding salicylates, were found to be inefficient as effectors. It was therefore concluded that (substituted) salicylates are the inducers of the relevant enzymes. The degradation of 2-naphthalene-sulfonate by a pure culture of strain BN6 was prevented by the toxicity of the dead-end product salicylate. Substituted salicylates were less toxic and allowed growth of strain BN6 in axenic culture with various substituted naphthalenesulfonates.

5-Hydroxyquinoline-2-Carboxylic Acid, a Dead-End Metabolite from the Bacterial Oxidation of 5-Aminonaphthalene-2-Sulfonic Acid

5-Aminonaphthalene-2-sulfonate (5A2NS) is converted by strain BN6 into 5-hydroxyquinoline-2-carboxylate (5H2QC). The authenticity of this new compound is confirmed by nuclear magnetic resonance and mass spectrometry. Its formation is explained by a spontaneous cyclization of the hypothetical metabolite 6'-amino-2'-hydroxybenzalpyruvate. The formation of 5H2QC as a dead-end product of 5A2NS prevents NADH regeneration so that 5A2NS oxidation is limited by the internal NADH pool.

Purification and properties of 2'-hydroxybenzalpyruvate aldolase from a bacterium that degrades naphthalenesulfonates

2'-Hydroxybenzalpyruvate aldolase catalyzes the cleavage of 2'-hydroxybenzalpyruvate to salicylaldehyde and pyruvate. This reaction is part of the degradative pathways for naphthalene and naphthalenesulfonates by bacteria. 2'-Hydroxybenzalpyruvate aldolase has been purified to homogeneity from a bacterium that degrades naphthalenesulfonates (strain BN6). The enzyme has a molecular weight of about 120,000 and is composed of identical subunits with a molecular weight of about 38,500. Thus the enzyme appears to exist as a trimeric oligomer. The NH2-terminal amino acid sequence did not show significant homology to other published amino acid sequences. Extensive loss of enzyme activity occurred when the enzyme was incubated with 2'-hydroxybenzalpyruvate in the presence of sodium borhydride. This suggested the intermediate formation of a stable Schiff base between enzyme and substrate. 2'-Hydroxybenzalpyruvate aldolase was inhibited by p-chloromercuribenzoate and by the reaction product salicylaldehyde. The enzyme converted 2'-hydroxybenzalpyruvate, 2',4'- and 2',6'-dihydroxybenzalpyruvate.

Mineralization of the sulfonated azo dye Mordant Yellow 3 by a 6-aminonaphthalene-2-sulfonate-degrading bacterial consortium

Under anaerobic conditions the sulfonated azo dye Mordant Yellow 3 was reduced by the biomass of a bacterial consortium grown aerobically with 6-aminonaphthalene-2-sulfonic acid. Stoichiometric amounts of the aromatic amines 6-aminonaphthalene-2-sulfonate and 5-aminosalicylate were generated and excreted into the medium. After re-aeration of the culture, these amines were mineralized by different members of the bacterial culture. Thus, total degradation of a sulfonated azo dye was achieved by using an alternating anaerobic-aerobic treatment. The ability of the mixed bacterial culture to reduce the azo dye was correlated with the presence of strain BN6, which possessed the ability to oxidize various naphthalenesulfonic acids. It is suggested that strain BN6 has a transport system for naphthalenesulfonic acids which also catalyzes uptake of sulfonated azo dyes. These dyes are then gratuitously reduced in the cytoplasm by unspecific reductases.

Purification and characterization of a 1,2-dihydroxynaphthalene dioxygenase from a bacterium that degrades naphthalenesulfonic acids

1,2-Dihydroxynaphthalene dioxygenase was purified to homogeneity from a bacterium that degrades naphthalenesulfonic acids (strain BN6). The enzyme requires Fe2+ for maximal activity and consists of eight identical subunits with a molecular weight of about 33,000. Analysis of the NH2-terminal amino acid sequence revealed a high degree of homology (22 of 29 amino acids) with the NH2-terminal amino acid sequence of 2,3-dihydroxybiphenyl dioxygenase from strain Pseudomonas paucimobilis Q1. 1,2-Dihydroxynaphthalene dioxygenase from strain BN6 shows a wide substrate specificity and also cleaves 5-, 6-, and 7-hydroxy-1,2-dihydroxynaphthalene, 2,3- and 3,4-dihydroxybiphenyl, catechol, and 3-methyl- and 4-methylcatechol. Similar activities against the hydroxy-1,2-dihydroxynaphthalenes were also found in cell extracts from naphthalene-degrading bacteria.

Bacterial communities degrading amino- and hydroxynaphthalene-2-sulfonates.

A 6-aminonaphthalene-2-sulfonic acid (6A2NS)-degrading mixed bacterial community was isolated from a sample of river Elbe water. The complete degradation of this xenobiotic compound may be described by a mutualistic interaction of two Pseudomonas strains isolated from this culture. One strain, BN6, could also grow on 6A2NS in monoculture, however, with accumulation of black polymers. This organism effected the initial conversion of 6A2NS into 5-aminosalicylate (5AS) through regioselective attack of the naphthalene skeleton in the 1,2-position. 5AS was totally degraded by another member of the community, strain BN9. After prolonged adaptation of strain BN6 to growth on 6A2NS, this organism readily converted all naphthalene-2-sulfonates with OH- or NH2-substituents in the 5-, 6-, 7-, or 8-position. The corresponding hydroxy- or aminosalicylates were excreted in stoichiometric amounts, with the exception that the metabolite from 5A2NS oxidation was not identical with 6AS.