BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ARTICLE NO.
218, 908–915 (1996)
0161
Biodegradation of Polychlorinated Biphenyls by Rhizobia:
A Novel Finding
Mona Damaj and Darakhshan Ahmad1
Institut National de la Recherche Scientifique, INRS-Santé, Université du Québec,
Pointe-Claire, Québec, H9R 1G6, Canada
Received November 27, 1995
Metabolism of simple aromatic compounds in rhizobial strains has been a subject of study for a few decades,
due either to the significance of nutritional diversity in the inoculum survival during agricultural applications or
to the importance of plant phenolics in the microbe-plant cross-talk and signal-transduction. Here, we report the
capability of rhizobial strains to catabolize polychlorinated biphenyls (PCBs). In order to identify the genes in
these strains that mediate the catabolism of PCBs we used the bphABC genes from Comamonas testosteroni
strain B-356. Our results showed that genomic DNAs from all four rhizobial strains studied hybridized strongly
with the Comamonas-derived probe, indicating the presence of a similar genetic system. This is a novel and
interesting finding indicating for the first time, perhaps, of a role of rhizobia in recycling of aromatic compounds
in nature and, certainly, opening a new avenue to be explored in the field of bioremediation. © 1996 Academic Press,
Inc.
Among ubiquitous aromatic pollutants, polychlorinated biphenyls (PCBs) acquire one of the top
ranking positions. The extreme persistence of these xenobiotics in the environment led to the early
notion of the incapability of indegenous microorganisms to degrade and recycle these compounds
in nature. Later on, isolation and characterization of a variety of microorganisms from the environmental samples, capable of metabolising aerobically PCBs (1), has incited interest in using
biodegradation for the recycling of these xenobiotics. Although such attempts have proven useful
for the bioremediation of oil contaminated soil and water, the much more complex system of
biodegradation of PCBs still poses as a great challenge to the microbiologists and is an important
current research topic. Since, rhizobia, by definition and classification, are soil bacteria associated
with plant roots and rhizospere, they are naturally exposed to a range of aromatic exudates of roots
and thus may prove to possess interesting aromatic catabolic pathways and capabilities. However,
to our knowledge, they have not yet been considered and studied for use in bioremediation of
aromatic pollutants, perhaps because the focus always been oriented toward their N2-fixing feature,
and because the much advertized catabolic versatility of Pseudomonads since the work of deJong
on the growth of P. putida on 200 substrates, has taken much attention (2). Thus, as compared to
the extensively studied Pseudomonads, relatively few studies have investigated the aromatic catabolic functions in rhizobia, all oriented toward their ecological significance in agriculture, e.g., for
competition and survival of agricultural inoculum (3,4), for chemotaxis toward plants (5), N2fixation and nodulation (6), for catabolism of plant root products as agents or inducer molecules for
signal transduction between plant-microbe cross-talk (7), etc. The compounds studied include
simple aromatic compounds such as catechol, protocatechuate, benzoate, hydroxybenzoates, mandelate, gentisate (3–5, 8–11),different flavones (6), opines (12), octopine (7, 13). In the same
context, there are a few reports of aromatic ring-cleavage activities, one of the most crucial step in
aromatic metabolism, such as, catechol 1,2-dioxygenase and protocatechuate 3,4- or 4,5- dioxygenases (10,11,14,15) in free-living as well as bacteroid forms (9,16), a special c-ring fission
1
Corresponding author. Fax: (514)630-8850.
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activity (6). The present work shows, for the first time, the capability of degradation of biphenyl,
chlorobiphenyl and Aroclor 1242 by a rhizobial strain, Rhizobium meliloti Zb57.
MATERIALS AND METHODS
Bacterial strains, culture media and growth conditions. The bacterial strains used were Bradyrhizobium japonicum
USDA110, R. trifolii ANU543, Rhizobium meliloti Zb57, provided by D. P. S. Verma (Ohio State Biotechnology Center,
Ohio State University, Columbus, Ohio, USA), and R. leguminosarum RBL5560, a gift by C. Wijffelman (Institute of Plant
Molecular Science, Lieiden University, Holland). E. coli JM109 (17) and Comamonas testosteroni B-356 (18) were from
the culture collection of M. Sylvestre (INRS-Santé, Université du Québec, Canada).
The media used for rhizobial strains were TYc (19) and HM minimal medium containing Metals44 (20), biotin (0.5
mg/mL) and yeast extract (0.05%). Glucose (0.5%), biphenyl (BP) (0.05%), 4-chlorobiphenyl (4-CB) (0.05%), 2,29-, 3,39-,
4,49-dichlorobiphenyls (DCBs) (0.001%) and Aroclor 1242 (100 ppm, in hexane) were added according to the experimental
protocols. For strains USDA110 and ANU543 medium was supplemented with thiamine (1 mg/mL) and calcium pentothenate (2mg/mL), and for RBL5560 with kanamycine (25mg/mL). E. coli and C. testosteroni were grown in Luria-Bertani
(LB) broth (17). All incubations were done at 29°C, except for E. coli. Aroclor 1242 was gift to M. Sylvestre from Monsanto
Chemicals (St. Louis, MO, USA), and BP,4-CB and DCBs were all from Aldrich Chemical Company (Milwaki, WI, USA).
All other chemicals were of highest purity grade available.
Oxygen uptake measurements. Oxygen uptake measurements were done on washed resting-cell suspension at 29°C,
essentially using protocol described by Sondossi et al. (21), using YSI Model 5300 Biological Oxygen Monitor Electrode
(Yellow Springs Instrument Co. Inc., Ohio, USA). Log-phase cultures grown on minimal medium with YE and BP were
subcultured and grown overnight. Cells were, then, harvested, washed, resuspended in phosphate buffer (pH 7.1) to an OD
of 3 at 600 nm and kept aerated at room temperature by stirring for a few hours to reduce the endogenous oxygen uptake.
The viability of cells was verified at the beginning and at the end of the experiment, by comparing the uptake on succinate.
The reaction mixture contained 2.9 mL of phosphate buffer, 1.9 mL of cell-suspension and 0.1 mL of substrate (90mM) in
dimethyl sulfoxide.
Assays for biodegradation of PCBs, incubation, extraction and analyses. Biodegradation was assayed by measuring
substrate disappearance in resting-cell cultures. For experiments with 4-CB and DCBs, cells were grown on minimal
medium with BP, glucose and YE, harvested, washed, resuspended in the assay medium to an OD of 3 and incubated for
a period of 120 h. Aroclor degradation was assayed on cells grown on minimal medium with glucose and YE, washed,
resuspended into fresh assay medium (at a ratio of 1:10) and incubated for 11 days. Uninoculated and cultures with
UV-treated dead cells were used as control. Cultures were extracted with hexane and analysed on a Perkin-Elmer Sigma 3B
gas chromatograph with electron capture detector (22). A late eluting peak of Aroclor, congener no.110 (IUPC no.), resistant
to biodegradation, was used as internal standard and the assignment of congeners to peaks (Table 3) was based on previously
published data (22).
DNA manipulation techniques. The genomic DNAs were prepared essentially as described in the Current Protocols in
Molecular Biology (25) using proteinase K and CTAB. DNA dot blots were made on Nylon membranes using Hybri-Dot
Manifold (BRL). Hybridization was done using the digoxigenin DNA probe using Boehriger Mannheim DIG DNA
Labeling and Detection Kit.
RESULTS
Catabolism of Biphenyl and Chlorobiphenyls by Rhizobia
Several reports have mentioned catabolism of simple (monocyclic) aromatic compounds by
rhizobia. We attempted to find out whether their catabolic capability extends to BP as well. All four
rhizobial strains tested, B.japonicum USDA110, R.trifolii ANU543 R. leguminosarum RBL5560
and R.meliloti Zb57, did show, albeit with significant variability, the capability to grow on BP.
However, they all seem to required some supplement of YE (0.02–0.05%) for growth. Also, as
compared to C. testosteroni (a well studied BP/PCB-metabolising strain (18) the cell yields were
significantly low (data not presented). GCMS analysis of extract of resting culture, yellowishpinkish-brown in colour, did not elucidate the identity of any metabolic intermediates or end
products, except 4-hydroxy biphenyl. However, the presence of yellowish brown colour in the
organic extract of culture does indicate the presence of phenolic transformation products or their
polymerized derivatives.
In order to find out if the BP-degradation system of rhizobial strains studied, along with the
capability to grow on BP, also possesses the capability to oxidize chlorinated biphenyls, we
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TABLE 1
Oxygen Uptake Rates by R. meliloti Zb57
Cells Grown with Biphenyl
Substrate used for
oxygen uptake
Oxygen
uptake ratea
Succinate
Biphenyl
2-Chlorobiphenyl
3-Chlorobiphenyl
4-Chlorobiphenyl
Benzoate
Protocatechuate
Catechol
34.5
(27.6)b
(41.4)b
(41.4)b
(41.4)b
(34.5)b
0
(34.5)b
13.3
16.1
20.7
16.6
18.9
18.4
a
Values expressed as nmol of O2 consumed/
min/ml.
b
Produced S-shaped curves. The initial O2
uptake rates are given in parentheses.
measured the oxygen uptake on monochlorobiphenyls in strain Zb57, one of the best BP-utilizing
strain among the rhizobial strains tested. Results presented in Table 1 show that initial oxygen
uptake rates in cells grown with BP were even higher on monochlorinated BPs than on BP only.
Since complete degradation of BP/PCB implies the catabolism of benzoic acid/chlorobenzoic acid
(BA/CBA) and catechol and protocatechuate degradation pathways are also intricately related to
BP/PCB pathway, we also measured oxygen uptake rates on these substrates. There was oxygen
consumption in these cells with BA and catachol but not with protocatechuate (Table 1).
Since we were not able to detect any transformation products or metabolic intermediates from
BP or 4-CB resting-cell cultures, we attempted to measure the consumption of substrate by
measuring its disappearence. With 4-CB as substrate a 15% decrease was observed as compared to
the dead-cell control, while in cultures with a mixture of DBCs, containing 2,29- 3,39- and 4,49DCB, the decrease observed, respectively for the three DBCs, were 8%, 9% and 8%, and 3%, 8%
and 10% when supplemented with glucose (Table 2). These results indicate the presence of an
aerobic BP/PCB degradation system in Rhizobium meliloti strain Zb57.
Homology of Genomic DNA of Rhizobial Strains with bph Gene Cluster of C. testosteroni
B-356 and with xylE of pTOL of P. putida
In an attempt to determine whether the BP/PCB degradation system in these rhizobial strains
shares genetic homology with the known systems, we probed the total cellular DNA dot-blots with
TABLE 2
Disappearance of Substrate in Resting-Cell
Culture of R. meliloti Zb57
Incubation medium
% decrease
of substrate
HM
only
HM with
glucose
4-CBa
2,2-CBb
3,39-CBb
4,49-CBb
0
8
9
10
15
3
8
8
a
Culture supplemented with 4-CB.
Culture supplemented with 2,29-, 3,39- and
4,49-DCBs.
b
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FIG. 1. Chemiluminescent dot blots showing hybridization of genomic DNA with the B-356 bphABC gene probe (4.2
kb Bg/II fragment). Lanes: 1, P.putida KT2440; 2, C. testosteroni B-356; 3, pDA1 (#); 4, R. japonicum USDA110; 5, R.
trifolii ANU543; 6, R. leguminosarum RBL5560; 7, R. meliloti Zb57 and 8, E. coli JM109. Amounts of DNA blotted were
1.5, 1.0 and 0.5 mg for rows A, B and C, respectively.
a 4.2 kb BglII fragment from C. testosteroni B-356 bearing a substantial part of pbh gene cluster,
bphABC (18). As shown in Figure 1, all four strains hybridized strongly with C. testosteroniderived probe, indicating a high degree of homology.
We also probed these genomic DNA dot-blots for homology with gene coding catechol 2,3
dioxygenase, the ring-cleavage enzyme, of the well known toluene degradation system of plasmid
pTOL, using a 2 kb xhoI fragment bearing the xylE. All four strains showed strong genetic
homology to the xylE gene as well (data not shown).
Degradation of Aroclor 1242 by R. meliloti Zb57
In order to test the efficiency of BP/PCB degrading system of R. meliloti Zb57 on commercial
preparation of PCBs, we assayed the degradation of Aroclor 1242 (composition given in Table 3,
TABLE 3
Assignment of Congeners to Peaks of Aroclor 1242 Analyzed on GC-ECD
Peak
No.a
Congener
No.b
No. of
chlorines
Peak
No.a
Congener
No.b
No. of
chlorines
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
4, 10
7, 9
6
5, 8
19
18
15, 17
24, 27
16, 32
34
29
26
25
28, 31
20, 33, 53
22, 51
45
46
52
47, 49
2
2
2
2
3
3
2,3
3
3
3
3
3
3
3
3,4
3,4
4
4
4
4
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
3,4
4
4
4
4
4
4
4, 5
5
4, 5
5
5
5
5
5
5
5
5
5, 6
5, 6
21
22
48
44
4
4
43
44
37, 42
41, 64, 71
40
67
58, 63
74
70
66, 95
91
56, 60, 89
90, 101
99
112
83
97
87
85
110
82, 151
106, 123,
149
105
138, 160,
163, 164
5
6
a
Aroclor 1242 peak numbers and corresponding congeners that were identified in our chromatographic system following Barriault and Sylvestre (22).
b
Numbers refer to IUPAC enumeration. Congeners in bold characters are major components of the
peak.
911
FIG. 2. Biodegradation of Aroclor 1242 by resting-cell cultures of R.meliloti ZB57. ECD chromatograms of A, BP + Aroclor (unextracted control); B, BP + Aroclor (after extraction
control); C, BP + Aroclor + UV-treated dead cells (control used for calculation of biodegradation); D, E and F are cultures with Aroclor, Aroclor + BP and Aroclor + 4-CB, respectively.
Arrowheads indicate peaks of internal standard. (For details see footnote of Table 4).
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ref. 22), in glucose-grown cells in media supplemented either with glucose only, or glucose and BP
or glucose and 4-CB. Comparison of GC-ECD chromatograms (a representative set of spectra is
presented in Figure 2), showed degradation of PCB congeners containing up to five chlorine atoms
(Table 4) under all three experimental conditions. No significant difference in total degradation was
found between cultures incubated with and without BP, total degradation being 38% and 40%,
respectively. However, in cultures incubated with 4-CB the degradative performance was only
26%.
DISCUSSION
Two microbial systems for removal and recycling of PCBs are known to operate in nature, the
well studied aerobic biodegradation system and the relatively much less studied and understood,
anaerobic dechlorination system (1, 24). The aerobic system has been identified in several gram
positive and gram negative bacteria, but rhizobia. The main objective of this work was to study
ability of rhizobial strains to degrade aromatic hydrocarbons, more specifically PCBs. The results
strongly illustrated that all four rhizobial strains studied are naturally bestowed with a PCB
biodegradation system genetically similar to the BP/PCB dioxygenase system found in Pseudomonads. However, the degradative performance of R. meliloti Zb57, the most efficient degrader
among the four strains studied, was much less than known for pseudomonal strains (22) under the
experimental set up used. Moreover, the Aroclor degradation was not significantly affected by the
presence of BP, a known inducer of BP/PCB system in Pseudomonads and the presence of 4-CB
TABLE 4
Degradation of Aroclor 1242 by R. meliloti Zb57 in Glucose Minimal Medium
Percent degradation with
Peak
No.a
4
6,7
9
14
15
16
19
20
22, 23
24
28
29
30
32
Congeners
2,3 & 2,49
4,49 & 2,29, 4 &
2,29,5
2,29,3 & 2,49,6
2,4,49 & 2,49,5
2,3,39 & 29,3,4 &
2,29,5,69
2,3,49 & 2,29,4,69
2,29,5,59
2,29,4,49 & 2,29,4,59
3,4,49 & 2,29,3,49 &
2,29,3,59
2,29,3,4 & 2,3,49,6
& 2,39,49,6
2,4,49,5
2,39,49,5
2,39,4,49 &
2,29,3,59,6
2,29,3,4,69 &
2,3,4,49 & 2,3,3949
Total degradationb
IUPAC
No.
No.
of Cl
No
supplement
BP
4-CB
5, 8
15, 17, 18
2
2, 3
57 ± 10
55 ± 3
68 ± 5
60 ± 3
36 ± 20
46 ± 23
16, 32
28, 31
20, 33, 53
3
3
3, 4
53 ± 0
60 ± 3
50 ± 3
56 ± 4
59 ± 5
48 ± 6
26 ± 12
46 ± 3
39 ± 6
22, 51
52
47, 49
37, 42, 44
3, 4
4
4
3, 4
55 ± 0
46 ± 0
47 ± 0
42 ± 5
55 ± 0
46 ± 0
47 ± 0
42 ± 5
32 ± 4
28 ± 0
30 ± 0
32 ± 10
41, 64, 71
4
41 ± 3
39 ± 6
24 ± 3
74
70
66, 95
4
4
4, 5
34 ± 6
30 ± 8
44 ± 6
34 ± 6
27 ± 4
40 ± 6
22 ± 0
22 ± 4
38 ± 3
56, 60, 89
4, 5
30 ± 8
26 ± 3
20 ± 0
38%
40%
26%
Percent degradation was calculated relative to UV-treated dead-cell control. Values are given as the means of 3 replicates
± SD.
a
Aroclor 1242 peak numbers on the chromatogram (see Table 3). Peaks not degraded are not listed.
b
Total degradation was calculated considering the molar ratio of each congener in Aroclor 1242 and presented as percent
total degradation.
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was inhibitory. The reasons could be varied. Such as, inefficient induction of genes (several distinct
modules of genes in rhizobia are induced by plant phenolics, O2-tension, cell density signals, and
other environmental signals bringing out total personality changes) (7, 25, 26, 27), genetic background of rhizobia in terms of different biochemical modules present and their interdependent
regulation (for example, the degradative systems for chlorobenzoates are well known to interfere
positively or negatively with PCB degradation system in a complex and yet not well understood
mechanism) (28), flux of metabolic intermediates, end products and their non-specific transformation products (29), etc. Although we have shown the presence of BP/PCB degradation genes in
rhizobia, we have not been yet able to postulate the biochemical pathway effective during the
observed degradation in the absence identification of metabolic products. And therefore, it is
certainly possible that the degradation observed is a cumulative result of more than one system
present (several cleavage enzymes or isoenzyme), or yet of a completely different system (such as,
a monooxygenase or a dehalogenase). The flexible and versatile life-style of rhizobia, the freeliving aerobic, microaerobic and fermentative, the anaerobic free-living-denitrifying and the symbiotic forms, their ability to survive under stressed environmental conditions, such as draught,
flood, metal contamination, etc., make this finding all more interesting and pertinent. Recently
several studies have pointed out the significance of phytoremediation (30), importance of rhizosphere in decontamination and recycling of pollutants (31, 32). Studies showing the horizontal
transfer of plasmid pJP4, bearing genes for murcury resistance and 2,4-D degradation into rhizobia
in nonsterile soil (33), mobilization into R. trifolii and subsequent cometabolism of herbicide 2,4-D
in soil (34), and the enhancement of microbial PCB degradation in soil in presence of a variety of
individual chemicals that are plant compounds (35), may indicate toward an existing role of
rhizobia in environmental maintenance or of its possibility in future.
ACKNOWLEDGMENTS
This work was partly supported by NSERC research grant to D.A. and an INRS post-doctoral fellowship to M.D. We are
grateful to D. P. S. Verma and C. Wijffelman for providing us the bacterial strains, M. Sylvestre, D. Barriault, Y. Hurtubise,
C. Pelltier, I. Guillematte, F. Shareck and M. Sondossi for their expert help and advices and D. Lacoste and H. Faucher for
drawings and secreterial assistance.
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