Peter Fortnagel

Biotransformation of 2,7-Dichloro- and 1,2,3,4-Tetrachlorodibenzo-p-Dioxin by Sphingomonas wittichii RW1

ABSTRACT Aerobic biotransformation of the diaryl ethers 2,7-dichlorodibenzo-p-dioxin and 1,2,3,4-tetrachlorodibenzo-p-dioxin by the dibenzo-p-dioxin-utilizing strain Sphingomonas wittichii RW1, producing corresponding metabolites, was demonstrated for the first time. Our strain transformed 2,7-dichlorodibenzo-p-dioxin, yielding 4-chlorocatechol, and 1,2,3,4-tetrachlorodibenzo-p-dioxin, producing 3,4,5,6-tetrachlorocatechol and 2-methoxy-3,4,5,6-tetrachlorophenol; all of these compounds were unequivocally identified by mass spectrometry both before and after N,O-bis(trimethylsilyl)-trifluoroacetamide derivatization by comparison with authentic standards. Additional experiments showed that strain RW1 formed a second metabolite, 2-methoxy-3,4,5,6-tetrachlorophenol, from the original degradation product, 3,4,5,6-tetrachlorocatechol, by methylation of one of the two hydroxy substituents.

Genetic and biochemical analysis of Shigella dysenteriae 1 O antigen polysaccharide biosynthesis in Escherichia coli K-12: structure and functions of the rfb gene cluster

The genetic organization and functions of the Shigella dysenteriae 1 rfb gene cluster, which specifies the somatic O antigen in this organism, have been studied in Escherichia coli K-12 by insertion and deletion mutagenesis of pSS9, a pBR322 hybrid containing the Shigella rfb genes. On the basis of the sensitivity/resistance to rough-specific bacteriophage T3 of E. coli K-12 derivatives containing mutant pSS9 plasmids, of the banding patterns and immunoreactivity of LPS isolated from such derivatives and electrophoresed on SDS-polyacrylamide gels, and of the sugar composition of the polysaccharide portion of the LPS determined by chemical analysis, six determinants for O antigen production were identified and localized. At least two determinants are involved in synthesis of TDP-rhamnose and the transfer of a rhamnose residue to the galactose-substituted core. One of these functions is probably TDP-rhamnose synthetase. A third function effects the transfer of a second rhamnose residue to the rha----gal-substituted core. A fourth function, for which evidence was obtained for two determinants (cistrons), is N-acetylglucosamine transferase, whereas a sixth determinant is necessary for extension of the first completed side chain repeat unit to the full O antigen polymer. These results confirmed the previously-determined chemical composition of the S. dysenteriae 1 O antigen and demonstrated that the order of the sugars is glcNAc----rha----rha----gal with gal as the first sugar linked to the core. Evidence was obtained for at least two transcriptional units in the rfb gene cluster and the approximate locations of two promoters are suggested. The detection of new electrophoretic species of LPS that may correspond to LPS biosynthetic intermediates, and the finding on the cell surfaces of structures corresponding to LPS core substituted with one or more O-specific sugars, appear to be novel findings.

Catabolism of 2,7-dichloro- and 2,4,8-trichlorodibenzofuran by Sphingomonas sp strain RW1

Due to their physicochemical and toxicological properties, polychlorinated dibenzofurans are regarded as a class of compounds providing reason for serious environmental concern. While the nonhalogenated basic structure dibenzofuran is effectively mineralized by appropriate bacterial strains, its polychlorinated derivatives are not. To elucidate the ability of the strain Sphingomonas sp RW1 to metabolize some of these chlorinated derivatives, we performed turnover experiments using 2,7-dichloro- and 2,4,8-trichlorodibenzofuran. As indicated by the oxygen-uptake rates determined for these two chlorinated dibenzofurans, Sphingomonassp RW1 can catabolize these chlorinated dibenzofurans yielding small quantities of oxidation products, which we isolated and subsequently characterized employing GC/MS and 1H- as well as 13C-NMR spectroscopy. In the case of 2,7-dichlorodibenzofuran, two metabolites accumulated, which we identified as 6-chloro- and 7-chloro-2-methyl-4H-chromen-4-one. The single metabolite isolated from the turnover experiments performed with 2,4,8-trichlorodibenzofuran was unequivocally identified as 6,8-dichloro-2-methyl-4H-chromen-4-one.

Glucose dehydrogenase from Bacillus subtilis expressed in Escherichia coli I: purification, characterization and comparison with glucose dehydrogenase from Bacillus megaterium

Escherichia coli containing the Bacillus subtilis glucose dehydrogenase gene on a plasmid (prL7) was used to produce the enzyme in high quantities. Gluc-DH-S was purified from the cell extract by (NH4)2SO4-precipitation, ion-exchange chromatography and Triazine-dye chromatography to a specific activity of 375 U/mg. The enzyme was apparently homogenous on SDS-PAGE with a subunit molecular mass of 31.5 kDa. Investigation of Gluc-DH-S was performed for comparison with the corresponding properties of Gluc-DH-M. The limiting Michaelis constant at pH 8.0 for NAD+ is Ka = 0.11 mM and for D-glucose Kb = 8.7 mM. The dissociation constant for NAD+ is Kia = 17.1 mM. Similar to Gluc-DH-M, Gluc-DH-S is inactivated by dissociation under weak alkaline conditions at pH 9.0. Complete reactivation is attained by readjustment to pH 6.5. Ultraviolet absorption, fluorescence and CD-spectra of native Gluc-DH-S, as well as fluorescence- and CD-backbone-spectra of the dissociated enzyme were nearly identical to the corresponding spectra of Gluc-DH-M. The aromatic CD-spectrum of dissociated Gluc-DH-S was different, representing a residual ellipticity of tryptophyl moieties in the 290-310 nm region. Density gradient centrifugation proved that this behaviour is due to the formation of inactive dimers in equilibrium with monomers after dissociation. In comparison to Gluc-DH-M, the kinetics of inactivation as well as the time-dependent change of fluorescence intensity at pH 9.0 of Gluc-DH-S showed a higher velocity and a changed course of the dissociation process.

New bacterial degradation of the biaryl ether structure

We report here the oxygenolytic cleavage of the ether bond occurring as the initial enzymatic step of high regioselectivity in the course of the bacterial attack on such aromatic compounds. Their complete catabolism is achieved by subsequent cleavage of the aromatic rings and further known reactions

16S ribosomal RNA gene sequence characterization and phylogenetic analysis of a dibenzo‐p‐dioxin‐degrading isolate within the new genus Sphingomonas

Partial 16S rRNA gene sequence comparisons have been used to determine the phylogenetic placement of the Elbe River isolate RW1, the first described bacterium capable of complete mineralization of dibenzo‐p‐dioxin. Sequence similarities, cluster analysis and signature positions demonstrate that RW1 groups with other species of Sphingomonas as a distinct, new species of this genus.

Biotransformation of diphenyl ether by the yeastTrichosporon beigelii SBUG 752

Trichosporon beigelii SBUG 752 was able to transform diphenyl ether. By TLC, HPLC, GC, GC-MS, NMR- and UV-spectroscopy, several oxidation products were identified. The primary attack was initiated by a monooxygenation step, resulting in the formation of 4-hydroxydiphenyl ether, 2-hydroxydiphenyl ether and 3-hydroxydiphenyl ether (48:47:5). Further oxidation led to 3,4-dihydroxydiphenyl ether. As a characteristic product resulting from the cleavage of an aromatic ring, the lactone of 2-hydroxy-4-phenoxymuconic acid was identified. The possible mechanism of ring cleavage to yield this metabolite is discussed.

Immunoblotting procedure for the analysis of electrophoretically-fractionated bacterial lipopolysaccharide

A procedure is described for the efficient transfer of fractionated bacterial lipopolysaccharide (LPS) from SDS-polyacrylamide gels to nitrocellulose filters, and its subsequent display by a peroxidase-linked antibody. The method is sensitive, and reveals and resolves high molecular weight LPS molecules having side chain lengths of up to and greater than 30 repeat units. It is also useful for the rapid analysis of LPS in bacterial outer membrane preparations.

Genetic and biochemical analysis of Shigella dysenteriae 1 O antigen polysaccharide biosynthesis in Escherichia coli K-12: 9 kb plasmid of S. dysenteriae 1 determines addition of a galactose residue to the lipopolysaccharide core

Production of the somatic antigen, O-specific polysaccharide of Shigella dysenteriae 1 is determined by the chromosomal rfb gene cluster and the rfp gene located on the 9 kb plasmid pHW400 carried by this organism. When transferred to Escherichia coli K-12, which produces lipopolysaccharide consisting only of core oligosaccharide linked to lipid A, rfp gene-containing plasmids caused modification of the core oligosaccharide leading to the appearance of core molecules with new electrophoretic mobilities. Chemical analysis of the modified core has shown that it is substituted with a galactose residue which is the first sugar of the O-polysaccharide repeat unit.

Bakterielle Mineralisierung von Dibenzofuran, Dibenzo-p-dioxin und 1,2,4,5-Tetrachlorbenzol in Böden

ZusammenfassungDer Abbau von Dibenzofuran (DBF), Dibenzo-p-dioxin (DBD) und 1,2,4,5-Tetrachlorbenzol (TeCB) durch die Bakterienstämme Sphingomonas sp. HH 69, Sphingomonas sp. RW 1 und Pseudomonas sp. PS 14 wurde mittels der Radio-Tracer-Methode in Flüssigkulturen, besonders aber in Mustern unterschiedlicher Böden, quantitativ untersucht. Die Versuchsergebnisse zeigen, daß die Reinkulturen die Substanzen DBF, DBD und TeCB sowohl in belüfteten Flüssigkulturen als aber auch, bei günstigen pH-Werten, in heterogenen, schadstoffarmen Böden innerhalb weniger Tage bis zu 80% in CO2 überführten. Geschwindigkeit und Grad der Mineralisation von DBF, DBD und TeCB waren bei niedriger und hoher Kontamination der Böden gleich gut, d.h. über weite Bereiche unabhängig von der im Boden vorhandenen Substanzkonzentration. Jedoch bestand zwischen der Startkonzentration an aktiven Zellen (Startkeimzahl) und der Geschwindigkeit des Abbaus der Substanzen im Boden verständlicherweise ein enger Zusammenhang; so wurde durch die Erhöhung der Startkeimzaahl im Boden der Abbau von DBF, DBD bzw. TeCB deutlich beschleunigt.Dagegen fand unter sonst gleichen Voraussetzungen in stärker sauren Böden (pH-Werte < 4) kein signifikanter Abbau von DBF, DBD bzw. TeCB zu CO2 statt. Diese Schwierigkeit konnte erwartungsgemäß durch das Einmischen von CaCO3 in die sauren Böden (Kalken) beseitigt werden.Leicht verwertbare Stoffe wie Pepton, Triolein und Glukose, die den schadstoffarmen Versuchsböden als besondere Kohlenstoff- und Energiequelle zugesetzt wurden, verzögerten die Mineralisation der Substanzen DBF, DBD und TeCB—wenn überhaupt—nur wenig. Jedoch konnten die Bakterienstämme ihre speziellen Fähigkeiten, die Substanzen DBF, DBD und TeCB zu mineralisieren, in bereits mit Schadstoffgemischen belasteten Böden nur begrenzt entfalten und wiesen je nach Art der Grundbelastung schr unterschiedliche Abbauleistungen auf. Das heißt, die Wirksamkeit der eingesetzten Abbauspezialisten in Böden hing u.a. von Art und Konzentration der dort insgesamt vorhandenen Kontaminanten ab, also von dem jeweils dort herrschenden chemischen Milieu.AbstractQuantitative degradation of dibenzofuran (DBF), dibenzo-p-dioxin (DBD), and 1,2,4,5-tetrachlorbenzene (TeCB) by the bacterial strains sphingomonas sp. HH 69, sphingomonas sp. RW1 and pseudomonas sp. PS 14 was investigated by radio-tracer techniques in liquid cultures and especially in samples of different soils. Pure strains converted DBF, DBD and TeCB up to 80% to CO2 within few days. This degradation occurred not only in aerated liquid cultures but also in heterogeneous soils, with low levels of other pollutatns at favourable pH-values. Rate and degree of the mineralization of DBF, DBD and TeCB were independent of the DBF-, DBD-, or TeCB-concentration in the soils within a broad range. There was obviously a close correlation between the starting concentration of active cells (starting bacterial count) and the rate of degradation of the test substances in soil; by raising the starting bacterial count in the soil samples, mineralization of DBF, DBD, and TeCB, respectively, was clearly accelerated.However, under nearly the same coniditions in more acid soils (pH-values < 4) no significant degradation of DBF, DBD, and TeCB to CO2 took place. As expected, this difficulty can be overcome by mixing CaCO2 into the acid soils (liming). Easily utilizable substances like peptone, triolein, and glucose added as special carbon and energy sources to low contaminated soils, had only a small—if any—effect on the mineralization of DBF, DBD, and TeCB.In soils contaminated by a mixture of pollutants, the bacterial strains could develop their degradation capacity only to a limited extent and showed different degradation effects depending on the basic type of contamination. The efficiency of the degradation specialists was thus dependent on the “Chemical Environment” (type and concentration of contaminants present).