Aug 9, 1988 - ... of mono- and dichlorinated aromatic com-. 342 pounds by aerobic bacteria is usually assumed to occur only after dearomatization (5, 14, 20).
Vol. 55, No. 2
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1989, p. 516-519
0099-2240/89/020516-04$02.00/0 Copyright C) 1989, American Society for Microbiology
Hydroxylation and Dechlorination of Tetrachlorohydroquinone by Rhodococcus sp. Strain CP-2 Cell Extracts MAX M. HAGGBLOM,t* DIETER JANKE,: AND MIRJA S. SALKINOJA-SALONEN
Department of General Microbiology, University of Helsinki, Mannerheimintie 172, SF-00300 Helsinki, Finland Received 9 August 1988/Accepted 14 November 1988
A cell extract of a polychlorophenol-degrading bacterium, Rhodococcus sp. strain CP-2, isolated from chlorophenol-contaminated soil, was shown to dechlorinate tetrachlorohydroquinone, the first intermediate in pentachlorophenol and 2,3,5,6-tetrachlorophenol degradation. Degradation of tetrachlorohydroquinone was catalyzed by a soluble enzyme(s). The reaction sequence for complete dechlorination involved hydroxylation and three reductive dechlorinations, producing 1,2,4-trihydroxybenzene. All chlorines were thus removed from the polychlorinated compound before ring cleavage.
Several aerobic bacteria that degrade polychlorinated phenols have been described (1, 7, 8, 13, 16, 27, 29, 34, 35, 40). The degradation of polychlorophenols has been shown to proceed via hydroxylation to chlorinated para-hydroquinones (3, 12, 13, 27, 33). Rhodococcus chlorophenolicus PCP-I further degrades tetrachlorohydroquinone (TeCH) through hydrolytic dechlorination and three reductive dechlorinations, producing 1,2,4-trihydroxybenzene (4), removing all chlorine atoms before ring cleavage. Bacterial degradation of most mono- and dichlorinated aromatic compounds, on the other hand, usually proceeds through chlorocatechols, with dechlorination occurring only after dearomatization (5, 14, 20). Dechlorination of monochlorinated aromatic compounds by replacement of the halogen with a hydroxyl group by several different bacteria has also been shown, however (15, 18, 19, 22, 28, 39). Rhodococcus sp. strain CP-2 (DSM 4598) was isolated from chlorophenol-contaminated soil after bioremediation (13, 37). It was shown to mineralize pentachlorophenol (PCP) and to degrade several other polychlorinated phenols, guaiacols, and syringols (13). The degradation of polychlorinated phenols was initiated by hydroxylation to chlorinated I para-hydroquinones. In this paper we describe the subsequent reactions leading to complete dechlorination of TeCH by cell extracts of Rhodococcus sp. strain CP-2. Rhodococcus sp. strain CP-2 was grown as described previously (13) in mineral salts medium, supplemented withI trace elements and vitamins, with glucose (1.0%, wt/vol) as the carbon source. For enzyme induction, PCP was addedl four times at 24-h intervals, with the concentration increasing from 10 to 40 ,uM. PCP-induced cells were collected by centrifugation and washed twice with S0rensen phosphate buffer, pH 7.0 (31). The same buffer was used throughout this work. Washed cells (4 g, wet weight) were suspended in 4 ml of buffer and broken by four passages through an X-press at -20°C. DNase type 1 (50 jig ml-'; Sigma Chemical Co., St. Louis, Mo.) was added to the melted extract and incubated for 1 h at room temperature. The crude extract was centrifuged at 100,000 x g for 30 min, and the superna'
Corresponding author. t Present address: Department of Microbiology, New York Uni-
*
versity Medical Center, 550 First Avenue, New York, NY 10016. t Present address: Academy of Sciences of the GDR, Central Institute of Microbiology and Experimental Therapy, Beutenbergstrasse 11, 6900 Jena, German Democratic Republic.
516
tant was collected for further experiments. Enzyme assay was performed at a substrate concentration of 10 to 50 ,uM and 0.5 to 1.0 mg of cell protein, at a temperature of 28°C. Ascorbic acid (0.1%, wt/vol) was added to slow down abiotic oxidation of chlorohydroquinones (4). Protein concentration was assayed by the method of Bradford (6) with the Bio-Rad reagent kit (Bio-Rad Laboratories, Richmond, Calif.) and lysozyme (Sigma) for calibration. Substrates and reference compounds used in this study were as follows: TeCH and 2,5-dichlorohydroquinone (25DCH) were from Eastman Kodak Co., Rochester, N.Y. PCP, 1,2,3-trihydroxybenzene (123-THB), and 1,3,5-THB (135-THB) were from E. Merck AG, Darmstadt, Federal Republic of Germany. 1,2,4-THB (124-THB) was from Aldrich-Chemie (Steinheim, Federal Republic of Germany). Trichlorohydroquinone (TCH), 2,3-DCH (23-DCH), and 2,6DCH (26-DCH) were synthesized by J. Knuutinen, Univer-
sity of Jyvaskyla, Jyvaskyla, Finland, from chlorinated 4-hydroxybenzaldehydes (21) as described for chlorocatechols (J. Knuutinen, Ph.D. thesis, University of Jyvaskyla,
Jyvaskyla, Finland, 1984).
Turnover of chlorinated hydroquinones was determined by gas-liquid chromatography (GLC). For GLC analysis, the chlorinated hydroquinones were acetylated in buffer solution with 2,4,6-tribromophenol as an internal standard as described before (11) and extracted into heptane. Acetylated compounds were analyzed with a Carlo Erba Fractovap 2300 gas crhomatograph with a CP Sil 5 (Chrompack, Middelburg, The Netherlands) capillary column and a 63Ni electron capture detector. Metabolites from TeCH were identified as silylated derivatives by GLC-mass spectrometry (GLC-MS) as follows. Sample (100 RI) was evaporated to dryness in a vacuum evaporator, and 100 ,ul of bis(trimethylsilyl)trifluoroacetamide (E. Merck AG) was added to the dry sample. The mixture was incubated for 2 h at 70°C and then evaporated to dryness in an N2 stream at room temperature. The residue was redissolved in 30 pul of heptane and analyzed with a Hewlett-Packard HP 5880 gas chromatograph equipped with an HP 5970 A mass selective detector and an HP-1 (HewlettPackard, Co., Palo Alto, Calif.) capillary column. The production of metabolites was followed by GLC-MS with 135THB as an internal standard and selective ion monitoring of the following ions: TeCH, mle 390.0 and 392.0; dichlorotrihydroxybenzene, mle 410.1 and 412.1; monochlorotrihy-
VOL. 55, 1989
NOTES Metabolite Ml from TeCH
1001
IE
8e01 C
so
60j so 40
L
376
0
370
380
390
400
420
4 10
Metabolite M2 from TeCH 173
100 -
4 14
391
20
176
80-
vC60 41 s460 4
40
a
L
238
L20,
.,... 0-
253
11.
..,,...
240
260
280
288
36
305
1,.1,/
340 /
300
320
340
IL 360
1 380 rn/e
Metabolite M3 from TeCH
100;3
342
239
80o >
o
L
20'
14 7
11 9
L-100
179
-L
I
L.
211
-1-L
200
L
3 11 L
300
FIG. 1. Mass spectra of trimethylsilyl derivatives of m etabolites from TeCH.
droxybenzene, mle 376.1 and 378.1; and THB mle 2!39.1 and 342.1. We showed earlier that Rhodococcus sp. straiin CP-2 degraded polychlorinated phenols via hydroxylation to parahydroquinones, which were further degraded by ti te bacterium (13). As shown in this paper, TeCH was also degraded by cell extracts from PCP-induced cells of this balcterium. The initial rate of TeCH removal in the crude extrac t was 3.8 nkat/g of protein. The TeCH-degrading activity wa s located in the 100,000 x g supernatant of the crude cellI extract (specific rate of TeCH removal, 1.4 nkat/g of protein). Heating at 70°C for 30 min destroyed the activ,ity. The turnover of TeCH was thus mediated by a solub le, heatlabile enzyme(s). We next studied the substrate specificity of this cell extract. TeCH (10 ,uM) was completely consume(d by the 100,000 x g supernatant in 20 min. TCH was degra__ded at a substantially lower rate: 35% of a 10 puM initial conc(entration was consumed in 5 h. Less than 10% of the dichllorinated hydroquinones (23-DCH, 25-DCH, and 26-DCH) were removed in 5 h. In sterile medium, less than 109fo of the chlorohydroquinones was lost in 5 h under identic;al conditions. These results show that TeCH was readily coonsumed, TCH was consumed at a lower rate, and removal a f dichlorinated hydroquinones was insignificant. When cell extracts of PCP-induced Rhodoco(ccus sp. strain CP-2 degraded TeCH, there was a concomiitant formation of metabolites detectable by GLC-MS. The metabolites were identified as silylated derivatives by thleir mass
517
spectra and retention times in GLC. The mass spectra of the trimethylsilyl derivatives of the three metabolites detected are shown in Fig. 1, and their retention times in GLC are given in Table 1. The mass spectrum of metabolite M3 was identical to that of 124-THB, but differed from that of 123-THB and 135-THB (Fig. 2). Its retention time in GLC was also identical to that of 124-THB but not to those of 123-THB or 135-THB. Based on the differences in the fragmentation pattern of the different THB's (Fig. 2), the identity of metabolites Ml and M2 could be deduced, although no authentic reference compounds were available. The fragmentation and relative ion intensities of metabolites Ml and M2 were analogous to those of 124-THB. The molecular weight of M2 was 34 units higher than that of 124-THB, and its peak pattern corresponded to that of a compound containing one chlorine atom. The molecular weight of Ml was 34 units higher than that of M2, and its peak pattern corresponded to that of a compound containing two chlorine atoms. We therefore conclude that the metabolites produced from TeCH carry three hydroxyl groups in positions 1, 2, and 4. Metabolite Ml corresponded to a dichlorotrihydroxybenzene, metabolite M2 to a monochlorotrihydroxybenzene, and metabolite M3 to 124-THB. Figure 3 shows the turnover of 50 p.M TeCH and concomitant formation of metabolites by a cell extract from PCP-induced Rhodococcus sp. strain CP-2. The first metabolite, Ml (dichlorotrihydroxybenzene), produced by hydroxylation and dechlorination of TeCH, was found at a concentration of 35 nM. The two metabolites, M2 (monochlorotrihydroxybenzene) and M3 (124-THB), identified as dechlorination products of Ml, transiently accumulated to concentrations of 8 and 20 p.M, respectively. These results show that TeCH was degraded by hydroxylation to a chlorinated THB, followed by three reductive dechlorinations with formation of a nonchlorinated product, 124-THB (Fig. 4). From previous work we knew that PCP and 2,3,4,5-tetrachlorophenol were hydroxylated to TeCH by Rhodococcus sp. strain CP-2 (13). This pathway of chlorophenol degradation, involving para-hydroxylation followed by a second hydroxylation and three reductive dechlorinations, is identical to the one previously described for another polychlorophenol degrader, Rhodococcus chlorophenolicus PCP-I (3, 4). Rhodococcus sp. strain CP-2 was isolated from soil (13), whereas R. chlorophenolicus PCP-I was isolated from a lake sediment (1). These two strains have a different shade of yellow pigmentation and have different biochemical properties (1; M. Haggblom, L. Nohynek, K. Kronqvist, and M. Salkinoja-Salonen, manuscript in preparation). Both strains degrade a wide range of polychlorinated phenols, guaiacols, and syringols (2, 11-13). In TABLE 1. Mobilities of silylated reference compounds and metabolites from TeCH in GLC Retention time' (min)
Compound or metabolite
123-THB ........................................ 11.55 124-THB ........................................ 12.05 135-THB ............. .......................... 12.31 TeCH ........................................ 15.00 Ml ....................................... M2 .......................................
M3 ........................................ '
A capillary column (HP-1) and
increasing by 15°C min-'
a temperature program to 280°C, were used.
14.54 13.22
12.05 of 1 min at 50°C,
APPL. ENVIRON. MICROBIOL.
NOTES
518
1. 2. 3-Trihydroxybonzene
100
OH
OH
239
OH -
3
>1
C OHI t
,C I
I
12
OH
OH OH
JOH _
OH
OH
OH
OH
342
C
I
C1
OH
OH
U
69
c
FIG. 4. Suggested sequence for dechlorination of TeCH by Rhodococcus sp. strain CP-2.
c S
.Es ; 49
aL
20-
1 33 I I 9/
0
.\..,.I .L....
sp. (33). These authors suggested that reductively dechlorinated via TCH and 26-DCH. The reactions for complete dechlorination of the substrates were not elucidated. Dehalogenation of mono- and dichlorinated aromatic com-
Flavobacterium TeCH was
,/
100
390
290
'/Q 1. 2. 4-TrihydroxybenzQne
/
pounds by aerobic bacteria is usually assumed to
342
>1
239
499 L .199 20-
4
1
22, 23, 26, 36). I 19
147
\~~ L. L./
00
0-
179
100
/
1. 3. 5-Trihydroxybenzene
342
69
>
40
_
147
0
268
20
L
991
,. L,.
0
d,
.. LA-,
100
19 1
\
,1
\
22 1
I,
.1.
,.,
I I*, 360
290
FIG. 2. Mass spectra of trimethylsilyl derivatives of
L . z
io/r
50 M
conditions and required
an exog-
Apajalahti, M. Laureaus, and M.
Salkinoja-Salonen, Abstr. Annu. Meet. Am. Soc. Microbiol. 1988, K101, p. 223). The reaction
sequence
of PCP and 2,3,5,6-tetrachloro-
phenol degradation in Rhodococcus sp. strain CP-2 and Rhodococcus chlorophenolicus PCP-I thus involves parahydroxylation into TeCH (3, 13), followed by a second hydroxylation and three reductive dechlorinations producing nonchlorinated product, 124-THB (Fig. 4) (4). All chlorine substitutents were thus removed before ring cleavage. A similar pathway has also been observed for 2,4,5-trichlorophenoxyacetic acid degradation by Pseudomonas cepacia AC1100. 2,4,5-Trichlorophenol, the initial intermediate in a
2,4,5-trichlorophenoxyacetic acid degradation by P. cepacia
snols.
\
C
*lj30
a)
\
0
.0
E
0=~~
PM o LiE
o:N 4-E0 0° Production of metabolites from TeCH
Rhodococcus sp. strain CP-2.
by cell
We thank Juha Knuutinen and co-workers for synthesis of chlorinated hydroquinones not commercially available and Riitta Boeck for her help in the laboratory. This study was supported by the Technology Development Centre (TEKES) and by the Academy of Finland (M. Salkinoja-Salonen and D. Janke). M. Haggblom is a holder of a young scientist's fellowship from the University of Helsinki.
0,1 o 0
LITERATURE CITED 1. Apajalahti, J. H. A., P. Karpanoja, and M. S. Salkinoja-Salonen. 1986. Rhodococcus chlorophenolicus sp. nov., a chlorophenol-
time (h) FIG. 3.
bacterium has also
AC1100 (16), was hydroxylated first into 25-DCH and then into 5-chloro-1,2,4-THB (U. Sangodkar, P. Chapman, and A. M. Chakrabarty, personal communication). This pathway through para-hydroquinones and 124-THBs may thus be common for bacterial degradation of polychlorinated phe-
10.
C
was favored by anaerobic enous reductant (4; J.
isomeric
both strains the enzyme(s) for chlorohydroquinone dechlorination is specific for a polychlorinated substrate. T was only slowly and DCHs were insignificantly trans oCH formphed which shows that these are not intermediates in chi [orophenol metabolism. A different pathway of TeCH degradation has bceen proposed for a PCP-degrading bacterium, KC-3 (27)1, and a
mpn
of 2,4-dichloroben-
nolicuis PCP-I, the reductive dechlorination system of TeCH
l
281
THBs.
0
dehalogenation by an aerobic
Anaerobic consortia from sludge have been shown to reductively dechlorinate both mono- and polychlorinated aromatic compounds (24, 25, 30). Reductive dechlorination of 3-chlorobenzoate by an obligate anaerobic bacterium, DCB-1, was also described (9, 30). The enzyme(s) for reductive dechlorination has so far been obtained as a cell extract from aerobic Rhodococcus strains only, Rhodococcus sp. strain CP-2 and R. chlorophenolicus PCP-I (4). These were not inactivated by molecular oxygen. In R. chlorophe-
300
3
C
only
been described (38).
200
rn/e
S
Reductive
zoate to 4-chlorobenzoate
311
211
/
occur
after dearomatization (5, 14, 20). However, hydrolytic dechlorination of monochlorinated benzoates has been observed for both gram-negative and gram-positive strains (15, 18, 19, 22, 28, 39) and demonstrated in cell extracts (17, 19,
extracts of
mineralizing actinomycete. Int. J. Syst. Bacteriol. 36:246-251. 2. Apajalahti, J. H. A., and M. S. Salkinoja-Salonen. 1986. Degradation of polychlorinated phenols by Rhodococcus chlorophenolicus Appl. Microbiol. Biotechnol. 25:62-67.
NOTES
VOL. 55, 1989
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23.
24. 25.
26.
27.
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519
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