APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1992, P. 1005-1010 Vol. 58, No.

3
0099-2240/92/031005-06$02.00/0

Metabolism of Dibenzo-p-Dioxin by Sphingomonas sp. Strain RW1

ROLF-MICHAEL WITTICH,' HEINZ WILKES,2 VOLKER SINNWELL,2
WITTKO FRANCKE,2 AND PETER FORTNAGELl*
Institut fiur Allgemeine Botanik, Abteilung fir Mikrobiologie, Ohnhorststrasse 18,
and Institut fiir Organische Chemie,2 Universitat Hamburg,
D-2000 Hamburg, Germany
Received 19 June 1991/Accepted 19 December 1991

In the course of our screening for dibenzo-p-dioxin-utilizing bacteria, a Sphingomonas sp. strain was isolated
from enrichment cultures inoculated with water samples from the river Elbe. The isolate grew with both the
biaryl ethers dibenzo-p-dioxin and dibenzofuran (DF) as the sole sources of carbon and energy, showing
doubling times of about 8 and 5 h, respectively. Biodegradation of the two aromatic compounds initially
proceeded after an oxygenolytic attack at the angular position adjacent to the ether bridge, producing
2,2',3-trihydroxydiphenyl ether or 2,2',3-trihydroxybiphenyl from the initially formed dihydrodiols, which
represent extremely unstable hemiacetals. Results obtained from determinations of enzyme activities and
oxygen consumption suggest meta cleavage of the trihydroxy compounds. During dibenzofuran degradation,
hydrolysis of 2-hydroxy-6-oxo-6-(2-hydroxyphenyl)-hexa-2,4-dienoate yielded salicylate, which was branched
into the catechol meta cleavage pathway and the gentisate pathway. Catechol obtained from the product of meta
ring fission of 2,2',3-trihydroxydiphenyl ether was both ortho and meta cleaved by Sphingomonas sp. strain
RW1 when this organism was grown with dibenzo-p-dioxin.

Polychlorinated dibenzo-p-dioxins (DDs) and dibenzo- 28°C on a rotary shaker at 150 rpm. Aliquots were trans-
furans (DFs) are unintentionally formed as contaminating ferred weekly from the culture to fresh medium. Subcultures
by-products during the manufacture of pesticides, incinera- were streaked onto nutrient medium, and after incubation at
tion of halogen-containing chemicals and of industrial and 28°C, single colonies were transferred onto mineral salts agar
domestic waste, and bleaching of paper pulp (3, 5, 31) and plates containing the above carbon source. Well-grown
thus have become widespread contaminants of the environ- colonies (plates without DD were used as controls) were
ment. retransferred into selective liquid medium, and after growth,
Dibenzo-p-dioxin, its monochloroderivatives, and diben- stock cultures were stored at -70°C until used for experi-
zofuran were shown to be cooxidized to ortho-dihydroxy- ments.
lated compounds by bacterial strains utilizing naphthalene or The identification of the strain was carried out by standard
biphenyl for growth (4, 18, 19). Cleavage of the aromatic laboratory procedures and those described in Bergey's Man-
nuclei of the target compounds, however, did not occur. ual (23); the guanine-plus-cytosine content of bacterial DNA
During our investigations of the microbial degradation of was determined as described previously by Frank-Ka-
diaryl ethers, a Pseudomonas sp., strain HH69, which menetskii (10). The isolate is deposited at the German
utilized DF as its sole source of carbon and energy and Collection of Microorganisms and Cell Cultures (no. 6014;
showed cometabolic activity toward DD and 3-chlorodiben- Deutsche Sammlung von Mikroorganismen und Zellkulturen
zofuran (8, 12, 13), was isolated. The degradation of DF was [DSM], Braunschweig, Germany).
initiated by an angular attack of a dioxygenase enzyme Growth experiments with and without (control) DD or DF
system followed by cleavage of the resulting unstable phe- were performed with 1-liter screw-cap, polytetrafluoroethyl-
nolic hemiacetal to yield 2,2',3-trihydroxybiphenyl, the lat- ene-sealed Erlenmeyer flasks containing the mineral salts
ter product being degraded in a manner similar to that of the medium (10% the nominal volume) and the appropriate
classical biphenyl pathway. Recently, cooxidation of some amount of substrate crystals as described above. At time
low-chlorinated DDs and DFs by a biphenyl-utilizing Alcali- points given in Fig. 1 and 2 (experimental section), flasks
genes sp. strain was reported (24, 25). However, complete were sacrificed from two parallel batches. For the estimation
biodegradation of DD has not yet been shown. Here, we of the cell number (CFU), 10-,u aliquots were plated on solid
describe for the first time the properties of a bacterium that acetate medium (10 mM) after appropriate dilution and
mineralizes DD. counted after about 1 week. The determinations of further
growth parameters such as turbidity and protein content of
MATERUILS AND METHODS the bacterial culture were performed as described previously
(8). For the estimation of the total amount of the carbon
Isolation, identification, and growth of bacteria. For enrich- source, including dissolved material, undissolved crystals,
ment and growth, a mineral salts medium which has been and the residue adsorbed on the cell surface and glass walls,
previously described was used (8). The enrichment cultures the entire contents of the Erlenmeyer flasks were extracted
were inoculated with water samples, supplemented with DD with an equal volume of chloroform. After drying with
(0.5 g/liter) as the substrate and actidione (0.1 g/liter) to anhydrous sodium sulfate, quantification was carried out by
suppress growth of protozoa, and incubated aerobically at high-performance liquid chromatography (HPLC). The re-
covery rate was always quantitative, as had been determined
in preceding experiments.
*
Corresponding author. Determination of oxygen uptake rates and enzyme activities.
1005
1006 WITTICH ET AL. APPL. ENVIRON. MICROBIOL.

Oxygen uptake rates of resting cell suspensions with DD,

structurally related compounds and their (potential) metab-
olites, and the activities of enzymes present in cell-free
preparations were determined as previously described (8).
The meta-cleaving activities of crude cell extracts for the
oxidation of 2,3-dihydroxybiphenyl and 2,2',3-trihydroxybi-
phenyl and the hydrolysis of their reaction products were
estimated by the procedures reported by Furukawa et al.
(11), Ishigooka et al. (15), and Omori et al. (22). For
calculation of the formation of the assumed metabolite
2 - hydroxy - 6 - oxo - 6 - (2 - hydroxyphenyl) - hexa - 2,4 - dienoate
from 2,2',3-trihydroxybiphenyl (8), the molar absorption
coefficient (11.100 cm2 mol-1 at 432 nm) of the meta cleav-
age product of 2,3-dihydroxybiphenyl (15) had to be used
because no reference sample was available. The conversion
of 2,2',3-trihydroxydiphenyl ether by cell extracts was mon-
itored by following substrate consumption with HPLC anal-
ysis of the assay medium described above for the biphenyl
2,3-dioxygenase activity, since the formation of a yellow
product suitable for optical enzyme assays was not detect- 0 20 60 100
able. Estimations of salicylate hydroxylase, catechol oxy- Time [h]
genases, and gentisate dioxygenase were performed as pre- FIG. 1. Growth of Sphingomonas sp. strain RW1 with DD. A
viously described (8). Assays were performed with an mineral salts medium was supplemented with DD crystals as the
Uvikon 930 spectrophotometer (Kontron Instruments, Ech- sole source of carbon and energy at a concentration of 5 mM. Two
ing, Germany) at 25°C. parallel sets of Erlenmeyer flasks were inoculated with an exponen-
Isolation of metabolites. Metabolites which were excreted tially growing preculture at late log phase which was freed from
substrate crystals by filtration. After an appropriate time, parallel
into the medium by growing or cometabolizing cultures were batches were worked up for determination of substrate consumption
extracted and purified as previously described (8). For the and formation of biomass, as described in Materials and Methods.
preparation of 2,2',3-trihydroxydiphenyl ether or the respec-
tive trihydroxybiphenyl, DF-grown resting cells were incu-
bated in the presence of a 0.1 mM concentration of the
potent meta cleavage inhibitor 3-chlorocatechol (2) and a short rods (about 1 by 3 ,um) were immotile, as judged by
large excess (about 5 g/liter) of mortar-ground, crystalline microscopic investigations; however, less than 1% of cells
DD or DF to ensure the sufficient dissolution kinetics needed were found to be motile when the organism was grown in
for good bioavailability of the substrates. Product formation nutrient broth. Further investigations (which were per-
was followed by HPLC. formed by DSM) allowed assignment to the Pseudomonas
Analytical methods. Monitoring of culture supernatants by paucimobilis group upon determination of the ubiquinone
HPLC, gas chromatographic analyses of extracted metabo- and fatty acid patterns. Recently, this group was transferred
lites, and identification of purified compounds by mass to the new genus Sphingomonas by Yabuuchi et al. (35).
spectrometry and 'H and '3C nuclear magnetic resonance Further work by DSM on definite assignment to a more
(NMR) spectroscopy were carried out as previously de- distinct species is in progress.
scribed (8). Growth of bacteria with aromatic compounds. The follow-
Chemicals. 2,3-Dihydroxybiphenyl was from Wako Chem- ing compounds were utilized by Sphingomonas sp. RW1
icals, Neuss, Germany. DD, 2-hydroxydibenzo-p-dioxin, 2- for growth: DD, DF, 2-hydroxydibenzofuran, 2-acetoxy-
acetoxydibenzo-p-dioxin, 2-acetoxydibenzofuran, 3-chloro- dibenzofuran, 2-hydroxybiphenyl, 2,3-dihydroxybiphenyl,
catechol, 4-chlorocatechol, 2,2',3-trihydroxybiphenyl, and 2,2'-dihydroxybiphenyl, 2,2' ,3-trihydroxybiphenyl, 2,2',3-
2-(2-hydroxyphenoxy)-muconate were prepared as de- trihydroxydiphenyl ether, benzoate, salicylate, gentisate, 5-
scribed previously (8, 13, 27). All other chemicals were of methylsalicylate, phenylacetate, phenylmalonate, DL-man-
the highest purity commercially available. delate, phenylglyoxylate, 2-hydroxyphenylglyoxylate, and
catechol. Benzene, naphthalene, biphenyl, 4-hydroxybi-
RESULTS phenyl, diphenyl ether, fluorene, fluoren-9-one, xanthene,
xanthen-9-one, isomeric hydroxy- and methylbenzoates or
Enrichment, isolation, and characterization of the DD- methylsalicylates other than the above, 2-hydroxyphenylac-
utilizing organism. With water samples from the Elbe River etate, phenol, the isomeric methylphenols, phenoxyacetate,
collected downstream from the city of Hamburg as the and 2-(2-hydroxyphenoxy)-muconate, the ortho cleavage
inoculum, enrichment cultures were started by mixing 100 product of 2,2',3-trihydroxydiphenyl ether (13), were not
ml of the samples with 300 ml of a mineral salts medium used for growth. However, spontaneous mutants able to
supplemented with 0.1 g of DD crystals as the substrate. utilize biphenyl were isolated after 4 weeks of incubation in
After about 5 weeks, utilization of the substrate became the presence of this compound at a rate of about 10'.
visible by the increasing turbidity of the enrichment culture. Generation times during growth with DD (Fig. 1) were 8 h
Strain RW1 was isolated from subcultures and initially at 28°C during the early exponential phase, and those with
identified as a Pseudomonas species on the basis of the DF were 5 h when the substrate was at a concentration of 5
classification schemes of Bergey's Manual. The strain was mM. Significantly shorter generation times were obtained for
gram negative and catalase and oxidase positive and grew both substrates when these carbon sources were offered in
under strictly aerobic conditions. The guanine-plus-cytosine excess.
content of bacterial DNA was estimated to be 67 mol%. The Figure 2 shows the simultaneous degradation of DD and
VOL. 58, 1992 METABOLISM OF DIBENZO-p-DIOXIN 1007

TABLE 1. Specific rates of oxygen uptake with aromatic

compounds by resting cells of Sphingomonas sp. strain RW1°
Specific oxygen uptake rate
(nmoles of 02/min/mg of
Compound protein) after growth on:
Acetate DD DF
04 DD 87 273 312
2-Hydroxydibenzo-p-dioxin 29 90 118
cm
2-Acetoxydibenzo-p-dioxin 83 213 230
C]
r-
DF 99 302 338
2-Hydroxydibenzofuran 106 119 120
2-Acetoxydibenzofuran 113 217 206
2-Methoxydibenzofuran 83 100 89
Biphenyl 43 14 49
2,3-Dihydroxybiphenyl 840 920 1,128
2,2',3-Trihydroxybiphenyl 359 478 553
Time[h] 2,2',3-Trihydroxydiphenyl ether 122 198 212
FIG. 2. Simultaneous degradation of DD and DF by Sphingomo- Diphenyl ether 56 37 49
nas sp. strain RW1. Equal amounts of DD and DF, each at a
Naphthalene 31 18 22
concentration of 10 mM, were added to a mineral salts medium. Anthraquinone 5 <2 4
Inoculation of the medium with an acclimated, late-exponential- Fluoren-9-one 46 32 51
phase culture pregrown with this substrate mixture and work-up of Dibenzothiophene 45 22 41
the parallel batches were carried out as described in the legend to Xanthene 41 22 48
Fig. 1. Xanthen-9-one 48 49 55
Benzoate 7 <2 <2
Catechol 438 451 621
Salicylate 152 174 188
DF by an acclimated culture of Sphingomonas sp. RW1 and Gentisate 25 29 28
shows that DF seemed to be the preferred substrate. This a Cells were grown in the presence of the above carbon sources, harvested
observation will depend on the degree of the bioavailability by centrifugation, washed with 55 mM phosphate buffer (pH 7.2), and
of the substrates; the concentrations of dissolved DD and resuspended in this buffer (E578 = 1). Stock solutions of water-insoluble
DF were about 0.4 and 5 mg/liter of the mineral salts medium compounds were made up with dimethyl sulfoxide. The final concentration in
at 28°C, as was determined by HPLC. the assay was always 1 mM; the concentrations of hydroxy derivatives of DD,
DF, biphenyl, and diphenyl ether were 0.1 mM (each) to prevent toxic effects
Oxygen uptake rates by resting cells. Washed cell suspen- to cells. Rates represent the means of at least two independently performed
sions of strain RW1 grown on acetate, DD, and DF were experiments and are corrected for endogenous respiration.
used for oxygen uptake rate studies. Growth-supporting
compounds and their substituted derivatives, some potential
metabolites, and structurally related compounds were
tested. Results are shown in Table 1. DD- and DF-grown 2,3-dihydroxybiphenyl 1,2-dioxygenase, (ii) that responsible
cells showed relatively high activities during the oxidation of for hydrolysis of its reaction product [2-hydroxy-6-oxo-6-(2-
their aromatic substrates; of their respective acetoxy deriv- hydroxyphenyl)-hexa-2,4-dienoate hydrolase (HOPDA hy-
atives; and to a lesser extent, of the corresponding hydroxy drolase)], (iii) catechol 2,3-dioxygenase, and (iv) catechol
derivatives. Very high activities were found during the 1,2-dioxygenase were determined with cell extracts of DD-,
oxidation of 2,3-dihydroxybiphenyl, 2,2',3-trihydroxybiphe- DF-, and acetate-grown cells. Activities of salicylate hy-
nyl, and even 2,2',3-trihydroxypdiphenyl ether. The intense droxylases (forming catechol and gentisate) were not detect-
yellow color which developed during oxidation of the di- and able in crude extracts.
trihydroxylated biphenyls and which was stable for more Results shown in Table 2 indicate that catechol 1,2-
than 15 min, however, was not observed during oxidation of dioxygenase was induced solely during growth with DD as
the above ether. the substrate. This enzyme also showed minute activity
Acetate-grown cells, used as the control, exhibited rela- during the cleavage of both isomeric methylcatechols. Ad-
tively low but significant levels during the oxidation of the ditionally, significant levels of catechol 2,3-dioxygenase
aromatic heterocycles DD, DF, and most of their above were present, showing high rates during the oxidation of
derivatives. Activities during the oxidation of 2,3-dihydrox- catechol and 3-methylcatechol; 4-methylcatechol was
ybiphenyl, 2,2',3-trihydroxybiphenyl, catechol, salicylate, cleaved at a low rate.
and some of their derivatives were as high as those found for Activities of catechol 2,3-dioxygenase found in DF-grown
cells grown on selective aromatics. These results gave cells were only slightly higher than those found in DD-grown
evidence of inducible initial enzymes present at significant cells. The activity of this enzyme was also present in
constitutive levels, whereas the sequence of the consecutive acetate-grown cells. Very high activities during the meta
enzymes seemed to be constitutive. Benzoate-grown resting cleavage of 2,3-dihydroxybiphenyl and 2,2',3-trihydroxybi-
cells exhibited high rates for these substrates and for cate- phenyl and during hydrolysis of the respective reaction
chol but not during the oxidation of DD, DF, 2,3-dihydrox- products by HOPDA hydrolase were found. No activity,
ybiphenyl, 2,2',3-trihydroxybiphenyl, and the respective tri- however, and no development of a yellow color caused by
hydroxydiphenyl ether, providing evidence of repression or the accumulation of meta cleavage products were detectable
elimination of the degradative pathway for both diaryl ether when 2,2',3-trihydroxydiphenyl ether was used as the sub-
compounds (data not shown). strate in this spectrophotometric assay. The activity for this
Determination of enzyme activities in crude extracts. The substrate was therefore determined by HPLC, following the
enzyme activities of (i) the catabolic meta-cleaving enzyme decline of the substrate concentration. The estimation of
1008 WITTICH ET AL. APPL. ENVIRON. MICROBIOL.

TABLE 2. Specific activities of catabolic enzymes in cell extracts H +93 93.125 109,109 M+
of Sphingomonas sp. strain RW1° 100- HO '. J OH 1
to04 OH
Sp act (pmol/min/mg of HO I H + 109
Enzyme and substrate protein) after growth on:
Acetate DD DF
OH 1
Catechol 1,2-dioxygenase OH
Catechol <0.002 0.143 <0.002 50-
3-Methylcatechol <0.002 0.048 <0.002
4-Methylcatechol <0.002 0.041 <0.002
M+
Catechol 2,3-dioxygenase -co M+
Catechol 0.065 0.127 0.215 -H20
-H20
3-Methylcatechol 0.087 0.194 0.428 -H -H
4-Methylcatechol 0.006 0.014 0.018
l
11. hl l
LL..1 -I.- ---i - -., L
2,3-Dihydroxybiphenyl 1,2- 50 70 90 110 130 150 170 190 210 m/z
dioxygenase
2,3-Dihydroxybiphenyl 1.292 1.981 2.238 FIG. 3. 70 eV mass spectrum of 2,2',3-trihydroxydiphenyl ether.
2,2',3-Trihydroxybiphenyl 0.417 0.639 0.873 They axis represents the relative percent.
2,2',3-Trihydroxydiphenyl ether" ND 0.018 0.020
HOPDA hydrolase The pure product was subsequently analyzed by mass and
2-Hydroxy-6-oxo-6-phenyl-hexa- 0.256 0.729 0.672 NMR spectroscopy. In the mass spectrum of 2,2',3-trihy-
2,4-dienoate droxydiphenyl ether (Fig. 3), the molecular ion at mlz = 218
2-Hydroxy-6-oxo-6-(2-hydroxy- 0.189 0.431 1.184
phenyl)-hexa-2,4-dienoate (base peak) is accompanied by two major fragment ions at
mlz = 94 and mlz = 110, formed upon stabilization of the
Gentisate dioxygenase ND 0.279 0.432 respective a-cleavage products by hydrogen transfer. Two
a Enzyme activities of crude cell extracts were recorded by spectrophoto-
minor fragment ions at m/z = 199 and m/z = 171 are formed
metrical standard methods. Data represent means of at least 2 experiments. upon successive loss of 18 (H20), 1 (H), and 28 (CO) amu.
ND, not determined. 'H NMR data (400.13 MHz, CD3OD/C6D6 = 100:15; tetram-
'
Conversion of the trihydroxydiphenyl ether by crude extracts was deter- ethylsilane as internal standard) (Fig. 4) were as follows: 8 =
mined by HPLC. 6.36 (H-4, J4,5 = 8.0 Hz, J4,6 =1.6 Hz), 6-60 (H-5, 5,6= 8.0
Hz), 6.66 (H-6), 6.73 (H-5',J6' = 8.0 Hz), 6.83 (H-6'), 6.93
(H-4',I J4,5, = 7.1Hz, J4',6' = 1.5 Hz), and 6.98 (H-3', J3, 4'
oxygen uptake rates of cell extracts with 2,2',3-trihydroxy- = 8.0 Hz, J3,5 = 1.8 Hz) ppm. The following 13C NMR data
biphenyl and the respective ether as the enzyme substrates, (100.62 MHz, solvent and internal standard as above) were
however, yielded specific rates of 460 versus 122 nmol/ obtained: 8 = 111.1 (C-4), 111.9 (C-6), 117.7 (C-3'), 119.9
min/mg of protein. Experiments performed with whole cells (C-6'), 120.1 (C-5), 120.9 (C-5'), 125.0 (C-4'), 137.5 (C-2),
and extracts of strain RW1 with 2-(2-hydroxyphenoxy)- 146.1 (C-i'), 146.7 (C-1 or C-3), 147.9 (C-1 or C-3), and 149.1
muconate, the ortho cleavage product of 2,2',3-trihydroxy- (C-2') ppm. The assignment of protons and carbons is based
diphenyl ether, showed no activity. on increment calculations as well as on H,H-COSY (1) (Fig.
3-Chlorocatechol was an efficient competitive inhibitor of 5), H,C-COSY (33), and H,C-COLOC (17) correlations. The
the catechol 2,3-dioxygenase of Sphingomonas sp. strain coupling pattern of the protons is confirmed by the H,H-
RW1 (Km = 0.83 mM for catechol); the inhibitor constant (Ki) COSY correlation showing two independent spin systems
with crude extracts of DD- and DF-grown cells for determi- consisting of three and four protons, respectively. The
nations of enzyme activity was always 30 FM. 4-Chlorocate-
chol exhibited a less inhibitory character (Ki = 74 p,M).
Isolation and identification of metabolites from DD. Trihy- 5
OH OH
_I
droxydiphenyl ether and catechol were identified as metab- 3'
olites in ethyl acetate extracts of the spent supernatant of a _
DD-grown culture by gas chromatography-mass spectrome- 6' 3' ~
4~
try analysis. For large-scale production of the trihydroxy- E-l
6 4'
S3 ~6'
rO
6- 4

diphenyl ether, washed cells of DD- or DF-grown cultures m

were incubated in the presence of excess DD (5 g/liter) to
ensure sufficient bioavailability of the dissolved substrate 4'
5.
and 3-chlorocatechol (0.1 mM) to achieve inhibition of the 5'
4l
r 4 l >
4I
2,3-dihydroxybiphenyl 1,2-dioxygenase (3-phenylcatechol
2,3-dioxygenase). The formation of polar products, one of
which was easily identified as catechol, and the maintenance
of 3-chlorocatechol concentration were monitored by
HPLC. When the rate of product formation decreased, DD
crystals were removed by filtration, and after further incu-
bation for 1 h, cells were pelleted by centrifugation. The
supernatant was extracted with ethyl acetate, dried over 7.0 6.9 6.8 6.7 6.6 6.5 6.4 6.3 ppm
Na2SO4, and concentrated to dryness. The residue was
dissolved in methanol and purified by preparative HPLC. FIG. 4. 'H NMR spectrum of 2,2',3-trihydroxydiphenyl ether.
VOL. 58, 1992 METABOLISM OF DIBENZO-p-DIOXIN 1009

ck,sz)
X11 d k J
AAih
(ppm)

At6.4

6.5

!"Iffis 6.6

OH
Li 6.7

6.8 WHO@]
"IR GI OH
-6.9

7.0

(ppm) 7.0 6.9 6.8 6.7 6.6 6.5 6.4

FIG. 5. H,H-COSY contour plot of 2,2',3-trihydroxydiphenyl

ether.
OH
OH

determined coupling constants indicate the presence of a

1,2-di- and a 1,2,3-trisubstituted aromatic nucleus. The
higher extent of high field shift observed for all protons of the L OH J
trisubstituted nucleus corresponds with the expected shield- FIG. 6. Proposed pathway for the degradation of DD by Sphin-
ing effect of oxygen substituents. With respect to the 13C gomonas sp. strain RW1. Catechol is ortho as well as meta cleaved.
NMR experiment, data for C-1 and C-3 are interchangeable
because of the symmetry of the 1,2,3-trisubstituted aromatic
nucleus. cleavage of the substrate and suggests meta cleavage of the
Experiments performed with the structurally related com- isolated 2,2',3-trihydroxydiphenyl ether. Our suggestion is
pound fluoren-9-one yielded the known angular dihydrodiol based on the fact that 2-(2-hydroxyphenoxy)-muconate, the
(6, 9) irrespective of whether cells were grown with DF or product of ortho ring fission, was neither utilized for growth
DD. nor oxidized at all. A further indication of meta cleavage was
the inhibitory action of 3-chlorocatechol. This inhibitor was
DISCUSSION also utilized for the preparation of 2,2',3-trihydroxybiphenyl
by Strubel et al. (29). meta cleavage of analog 2,3-dihydrox-
Bacterial strains of the P. paucimobilis group now trans- ydiphenyl ether has been shown by Pfeifer et al. (26),
ferred to the new genus Sphingomonas (35) are potent proceeding by hydrolysis and intramolecular transesterifica-
microorganisms degrading aromatic compounds of environ- tion, forming phenol and the lactone of 2-hydroxymuconate.
mental concern (12, 16, 21). Our DF-utilizing Pseudomonas meta cleavage of 2,2',3-trihydroxydiphenyl ether may occur
sp. strain HH69 (8) was also assigned to this new genus after at either side of the catechol structure between C-1 and C-2,
recent investigations of its patterns of fatty acids and ubi- yielding the 6-(2-hydroxyphenyl) ester of 2-hydroxymuconic
quinone (performed by DSM). Studies of RNA of the latter acid, or between C-3 and C-4, producing 2-hydroxy-3-(2-
organism, however, should provide evidence of its relation- hydroxyphenoxy)muconic acid semialdehyde. Neither com-
ship to the gram-positive Brevibactenum family (14). Bacte- pound has yet been described; their chemical structures
ria of both systematic groups have been shown to degrade indicate considerable thermodynamic instability. The ester
DF by an oxygenolytic attack at the angular position (6-9, may be easily saponified and does not necessarily cause any
13, 30, 34) and diphenyl ether by a similar reaction (28). yellow color, which is generally regarded as an indication of
Biodegradation of diphenyl ether by a Pseudomonas cepacia meta cleavage and which could not be observed during our
strain, however, was shown to be initiated by 2,3-dioxygen- experiments. Since the aldehyde should show a yellow
ation, following the classical biphenyl pathway (26). Further- color, we, like Pfeifer et al. (26), consider the ester to be a
more, angular dioxygenation for effective dibenzothiophene good candidate for the initial product of the degradation of
degradation was demonstrated for a Brevibactenum sp. (32). 2,2',3-trihydroxydiphenyl ether. Details of initial reactions
Metabolism or cooxidation of the heterocycles diben- of the degradation of (halogenated) diaryl ethers will be
zothiophene (20), DF (4), DD (18, 19), and low-chlorinated published separately.
congeners (19, 24, 25) followed classical schemes and never
led to complete degradation or mineralization. The pathway ACKNOWLEDGMENTS
postulated for DD degradation by our newly isolated Sphin- The technical assistance of Karen Gerstandt and Angela Jordan is
gomonas sp. strain, shown in Fig. 6, demonstrates ring gratefully acknowledged. We thank Gaby Stehr and Stefan Schmidt
1010 WITTICH ET AL. APPL. ENVIRON. MICROBIOL.

for help in the determination of the G+C content of DNA and peptides by heteronuclear shift correlation via small coupling
Christa Adami for preparing the photoprints. constants with broad decoupling in t1(COLOC). J. Magn. Res.
This research was supported by grant no. 0318896B from the 57:331-336.
Bundesminister fur Forschung und Technologie. 18. Klecka, G. M., and D. T. Gibson. 1979. Metabolism of dibenzo-
p-dioxin by a Pseudomonas species. Biochem. J. 180:639-645.
REFERENCES 19. Klecka, G. M., and D. T. Gibson. 1980. Metabolism of dibenzo-
1. Aue, W. P., E. Bartholdi, and R. R. Ernst. 1975. Two-dimen- p-dioxin and chlorinated dibenzo-p-dioxins by a Beijerinckia
sional spectroscopy. Application to nuclear magnetic reso- species. Appl. Environ. Microbiol. 39:288-296.
nance. J. Chem. Phys. 64:2229-2246. 20. Laborde, A. L., and D. T. Gibson. 1977. Metabolism of diben-
2. Bartels, I., H.-J. Knackmuss, and W. Reineke. 1984. Suicide zothiophene by a Beijerinckia species. Appl. Environ. Micro-
inactivation of catechol 2,3-dioxygenase from Pseudomonas biol. 34:783-790.
putida mt-2 by 3-halocatechols. Appl. Environ. Microbiol. 21. Mueller, J. G., P. J. Chapman, B. 0. Blattmann, and P. H.
47:500-505. Pritchard. 1990. Isolation and characterization of a fluoran-
3. Buser, H.-R. 1986. Polybrominated dibenzofurans and dibenzo- thene-utilizing strain of Pseudomonas paucimobilis. Appl. En-
p-dioxins: thermal reaction products of polybrominated diphe- viron. Microbiol. 56:1079-1086.
nyl ether flame retardants. Environ. Sci. Technol. 20:404-408. 22. Omori, T., K. Sugimura, H. Ishigooka, and Y. Minoda. 1986.
4. Cerniglia, C. E., J. C. Morgan, and D. T. Gibson. 1979. Purification and some properties of a 2-hydroxy-6-oxo-6-phenyl-
Bacterial and fungal oxidation of dibenzofuran. Biochem. J. hexa-2,4-dienoic acid hydrolyzing enzyme from Pseudomonas
180:175-185. cruciviae S93 Bi involved in the degradation of biphenyl. Agric.
5. Czuczwa, J. M., and R. A. Hites. 1984. Environmental fate of Biol. Chem. 50:931-937.
combustion-generated polychlorinated dioxins and furans. En- 23. Palleroni, N. J. 1984. Pseudomonas, p. 141-198. In N. R. Krieg
viron. Sci. Technol. 18:444-450. and J. G. Holt (ed.), Bergey's manual of systematic bacteriol-
6. Engesser, K. H., V. Strubel, K. Christoglou, P. Fischer, and ogy, 9th ed. The Williams and Wilkins Co., Baltimore.
H. G. Rast. 1989. Dioxygenolytic cleavage of aryl ether bonds: 24. Parsons, J. R., C. Ratsak, and C. Siekerman. 1990. Biodegrada-
1,10-dihydro-1,10-dihydroxyfluoren-9-one, a novel arene dihy- tion of chlorinated dibenzofurans by an Alcaligenes strain, p.
drodiol as evidence for angular dioxygenation of dibenzofuran. 377-380. In 0. Hutzinger and H. Fiedler (ed.), Dioxin '90,
FEMS Microbiol. Lett. 65:205-210. Organohalogen Compounds 1, EPRI-Seminar, Bayreuth, Ger-
7. Fortnagel, P., H. Harms, R.-M. Wittich, S. Krohn, H. Meyer, many.
and W. Francke. 1989. Cleavage of dibenzofuran and dibenzo- 25. Parsons, J. R., and M. C. M. Storms. 1989. Biodegradation of
p-dioxin ring systems by a Pseudomonas bacterium. Naturwis- chlorinated dibenzo-p-dioxins in batch and continuous cultures
senschaften 76:222-223. of strain JB1. Chemosphere 19:1297-1308.
8. Fortnagel, P., H. Harms, R.-M. Wittich, S. Krohn, H. Meyer, V. 26. Pfeifer, F., S. Schacht, J. Klein, and H. G. Trfiper. 1989.
Sinnwell, H. Wilkes, and W. Francke. 1990. Metabolism of Degradation of diphenylether by Pseudomonas cepacia. Arch.
dibenzofuran by Pseudomonas sp. strain HH69 and the mixed Microbiol. 152:512-519.
culture HH27. Appl. Environ. Microbiol. 56:1148-1156. 27. Sander, P., R.-M. Wittich, P. Fortnagel, H. Wilkes, and W.
9. Fortnagel, P., R.-M. Wittich, H. Harms, S. Schmidt, S. Franke, Francke. 1991. Degradation of 1,2,4-trichloro- and 1,2,4,5-tetra-
V. Sinnwell, H. Wilkes, and W. Francke. 1989. New bacterial chlorobenzene by Pseudomonas strains. Appl. Environ. Micro-
degradation of the biaryl ether structure. Naturwissenschaften biol. 57:1430-1440.
76:523-524. 28. Schmidt, S. (Universitit Hamburg). Personal communication.
10. Frank-Kamenetskii, M. D. 1971. Simplification of the empirical 29. Strubel, V., K.-H. Engesser, P. Fischer, and H.-J. Knackmuss.
relationship between melting temperature of DNA, its GC 1991. 3-(2-Hydroxyphenyl)catechol as substrate for proximal
content, and concentration of sodium ions in solution. Biopoly- meta ring cleavage in dibenzofuran degradation by Brevibacte-
mers 10:2623-2624. rium sp. strain DPO 1361. J. Bacteriol. 173:1932-1937.
11. Furukawa, K., J. R. Simon, and A. M. Chakrabarty. 1983. 30. Strubel, V., H. G. Rast, W. Fietz, H.-J. Knackmuss, and K. H.
Common induction and regulation of biphenyl, xylene/toluene, Engesser. 1989. Enrichment of dibenzofuran utilizing bacteria
and salicylate catabolism in Pseudomonas paucimobilis. J. with high cometabolic potential towards dibenzodioxin and
Bacteriol. 154:1356-1362. other anellated aromatics. FEMS Microbiol. Lett. 58:233-238.
12. Harms, H., H. Wilkes, V. Sinnwell, R.-M. Wittich, K. Figge, W. 31. Swanson, S. E., C. Rappe, J. Malmstrom, and K. P. Kringstad.
Francke, and P. Fortnagel. 1991. Transformation of 3-chloro- 1988. Emissions of PCDDs and PCDFs from the pulp industry.
dibenzofuran by Pseudomonas sp. HH69. FEMS Microbiol. Chemosphere 17:681-691.
Lett. 81:25-30. 32. van Afferden, M., S. Schacht, J. Klein, and H. G. Truper. 1990.
13. Harms, H., R.-M. Wittich, V. Sinnwell, H. Meyer, P. Fortnagel, Degradation of dibenzothiophene by Brevibacterium sp. DO.
and W. Francke. 1990. Transformation of dibenzo-p-dioxin by Arch. Microbiol. 153:324-328.
Pseudomonas sp. strain HH69. Appl. Environ. Microbiol. 56: 33. Wilde, J. A., and P. H. Bolton. 1984. Suppression of homonu-
1157-1159. clear couplings in heteronuclear two-dimensional spectroscopy.
14. Hofle, M. (Gesellschaft fir Biotechnologische Forschung, Braun- J. Magn. Res. 59:343-346.
schweig). Personal communication. 34. Wittich, R.-M., S. Schmidt, and P. Fortnagel. 1990. Bacterial
15. Ishigooka, H., Y. Yoshida, T. Omori, and Y. Minoda. 1986. degradation of 3- and 4-carboxybiphenyl ether by Pseudomonas
Enzymatic dioxygenation of biphenyl-2,3-diol and 3-isopropyl- sp. NSS2. FEMS Microbiol. Lett. 67:157-160.
catechol. Agric. Biol. Chem. 50:1045-1046. 35. Yabuuchi, E., I. Yano, H. Oyaizu, Y. Hashimoto, T. Ezaki, and
16. Katayama, Y., S. Nishikawa, A. Murayama, M. Yamasaki, N. H. Yamamoto. 1990. Proposals of Sphingomonas paucimobilis
Morohoshi, and T. Haraguchi. 1988. The metabolism of biphenyl gen. nov. and comb. nov., Sphingomonasparapaucimobilis sp.
structures in lignin by the soil bacterium (Pseudomonas pauci- nov., Sphingomonas yanoikuyae sp. nov., Sphingomonas ad-
mobilis SYK-6). FEBS Lett. 233:129-133. haesiva sp. nov., Sphingomonas capsulata comb. nov., and two
17. Kessler, H., C. Griesinger, J. Zarbock, and H. R. Loosli. 1984. genospecies of the genus Sphingomonas. Microbiol. Immunol.
Assignment of carbonyl carbons and sequence analysis in 34:99-119.