(Received for publication, September 14, 1995; and in revised form, December 5, 1995)
From the
The chlorobenzene degradation pathway of Pseudomonas sp. strain P51 is an evolutionary novelty. The first enzymes of the pathway, the chlorobenzene dioxygenase and the cis-chlorobenzene dihydrodiol dehydrogenase, are encoded on a plasmid-located transposon Tn5280. Chlorobenzene dioxygenase is a four-protein complex, formed by the gene products of tcbAa for the large subunit of the terminal oxygenase, tcbAb for the small subunit, tcbAc for the ferredoxin, and tcbAd for the NADH reductase. Directly downstream of tcbAd is the gene for the cis-chlorobenzene dihydrodiol dehydrogenase, tcbB. Homology comparisons indicated that these genes and gene products are most closely related to those for toluene (todC1C2BAD) and benzene degradation (bedC1C2BA and bnzABCD) and distantly to those for biphenyl, naphthalene, and benzoate degradation. Similar to the tod-encoded enzymes, chlorobenzene dioxygenase and cis-chlorobenzene dihydrodiol dehydrogenase were capable of oxidizing 1,2-dichlorobenzene, toluene, naphthalene, and biphenyl, but not benzoate, to the corresponding dihydrodiol and dihydroxy intermediates. These data strongly suggest that the chlorobenzene dioxygenase and dehydrogenase originated from a toluene or benzene degradation pathway, probably by horizontal gene transfer. This evolutionary event left its traces as short gene fragments directly outside the tcbAB coding regions.
Bacteria that are able to use mono- or dicyclic aromatic compounds as their sole source of carbon and energy under aerobic growth conditions are present ubiquitously in the environment(1, 2) . Considering the potential use and importance of such bacteria to help to remove many man-made polluting compounds, it is necessary to study the genetic and biochemical variations found among these types of bacteria and to investigate their evolutionary development(3) . Only then can the limitations of existing metabolic pathways be understood and concepts be developed to select or engineer novel pathways(4, 5) . Of special interest will be to obtain degradation of highly recalcitrant chlorosubstituted aromatic compounds, such as chlorinated benzenes and biphenyls(6) .
A very important enzyme complex in the aerobic degradation of many aromatic compounds is the multicomponent aromatic ring dioxygenase(7, 8) . Aromatic ring dioxygenases, such as benzoate dioxygenase(9) , toluate dioxygenase(10) , naphthalene(11) , biphenyl, toluene(12) , or benzene dioxygenases(13) , are enzyme complexes with three- or four protein subunits. This complex catalyzes a redox reaction in which molecular oxygen is incorporated in the aromatic ring at the expense of the oxidation of NADH(7, 8, 12) . The resulting intermediate is a cis-dihydrodiol derivative of the aromatic ring structure. A dihydrodiol dehydrogenase then catalyzes the (formal) oxidation of the dihydrodiol to a dihydroxy derivative and regenerates the reduced NADH. The components of the aromatic ring dioxygenase consist of different electron transport proteins (a ferredoxin and a reductase, or a combined ferredoxin-NADH reductase) and the terminal oxygenase (also called hydroxylase component or iron sulfur protein), which is thought to determine the substrate specificity of the enzyme and to carry out the substrate activation(7, 8) . Despite their structural similarities, remarkable differences in substrate spectrum are found among the different aromatic ring dioxygenases(12, 14, 15) .
We have focused
on bacteria-degrading chlorinated benzenes, in particular Pseudomonas sp. strain P51. Upon growth on chlorobenzenes,
strain P51 induces enzyme activities which catalyze the conversion of
chlorobenzenes to chlorocatechols(16) . The genes for these
enzymes, the tcbAB genes, were cloned previously from a
catabolic plasmid present in strain P51 and were proposed to encode an
aromatic ring dioxygenase and a dihydrodiol dehydrogenase, similar to
the enzymes of other aromatic pathways(16) . Strain P51 also
contains the genes for a so-called chlorocatechol oxidative pathway,
which in strain P51 consists of an operon of four genes, tcbCDEF(17) , and a regulatory gene tcbR(18) . Interestingly, both gene clusters in strain
P51 are located on a transmissible plasmid pP51. Furthermore, the tcbAB genes itself are part of a transposable element,
Tn5280()(19) . We therefore strongly
believe that the chlorobenzene pathway is an evolutionary novelty in
bacteria, formed by a novel combination of two existing gene clusters,
perhaps through horizontal gene transfer.
To test this idea further, we wanted to characterize the genes for the chlorobenzene dioxygenase and for the cis-chlorobenzene dihydrodiol dehydrogenase of Pseudomonas sp. strain P51 in detail. We wanted to analyze further the capability of the enzymes to convert several different aromatic compounds. This would make it possible to compare the enzymes both genetically and biochemically with related enzymes from other aromatic degradation pathways. The data in the paper indicate clearly that the chlorobenzene dioxygenase and the cis-chlorobenzene dihydrodiol dehydrogenase resemble most the toluene/benzene-type enzymes. Furthermore, evidence is presented to show the relics of the horizontal gene transfer events which may have lead to the imprecise excision of a genetic element containing the genes for an aromatic ring dioxygenase and dihydrodiol dehydrogenase from a tod-like operon.
Figure 1: Physical map of the region of the tcbAB genes and of the different cloned DNA fragments described in this study. The different hatchings in the physical map indicate the size and location of the ORFs derived from the DNA sequence analysis. The black ends on both sides of the physical map indicate the start of the insertion elements, IS1066 (left) and IS1067 (right). The arrows below show the size and location of the DNA sequences which were determined from the M13mp18-derived subclones. Relevant restriction sites are indicated on the DNA, but not all vector-located restriction sites are shown. Restriction sites within brackets point to the NcoI sites which were introduced by PCR to facilitate cloning. The open arrows in front of the cloned DNA fragments symbolize the different promoters of the used vector, i.e. T7, T7 promoter of pET8c; lac, lac promoter of pUC18.
The pET8c-derived plasmids did not, unfortunately, express active Tcb enzymes in E. coli (see below). Therefore, we constructed a clone containing the complete tcbAaAbAcAd gene sequence in pUC19. Plasmid pTCB115 was hereto cut with MluI and BamHI, and a 4.7-kb fragment was isolated and ligated with a 3.0-kb MluI-BamHI fragment of pTCB60. After transformation, this resulted in plasmid pTCB130. We then removed the complete 4.2-kb XbaI-BamHI insert of plasmid pTCB130 and ligated this fragment into pUC19, cut with XbaI and BamHI. This resulted in plasmid pTCB144. Colonies of E. coli containing this plasmid turned blue green when growing on LB plates at 30 °C, which indicates synthesis of active dioxygenase in the cells(11) .
cis-Chlorobenzene dihydrodiol dehydrogenase activity was
tested in E. coli DH5 (pTCB149). Cultures were grown on
50 ml of LB medium at 37 °C to an A
of 1.0,
after which the cells were harvested and a cell extract was prepared as
described previously(16) . The reaction mixture for dihydrodiol
dehydrogenase activity contained 0.65 ml of 20 mM sodium
phosphate buffer, pH 7.5, 25 µl of 20 mM NAD
solution, 50 µl of the cell extract, and 50 µl of
dihydrodiol substrate. As substrates we used the supernatants of the
whole cell incubations after 2 h (see above). The assay mixture was
incubated at 37 °C, and the change in A
was
measured on a spectrophotometer. When no more changes in A
were observed, the assay mixture was analyzed
on HPLC to check for the disappearance of the dihydrodiol and the
presence of the dihydroxy compound.
Dihydrodiol intermediates were extracted from supernatants of the whole cell incubations after 2 h with an equal volume of ethyl acetate and dried with sodium sulfate. Samples were derivatized with BSTFA and subjected to GC-MS analysis as described elsewhere(22) .
Figure 2: DNA sequence of the coding strand of the 5,451-bp SphI-MluI fragment containing the tcbAB genes and the predicted amino acid translation of the ORFs encoding the individual protein subunits. Relevant restriction sites are indicated, as well as DNA sequences which could function as ribosome binding sites (in bold). The putative start codon of the ORF with homology to the sequence of todE is shown downstream of tcbB. The sequence in italics, starting at position 5,338, indicates the border repeat sequence of IS1067. The stop codon, which is introduced by the insertion of IS1067 in this ORF, is shown underlined at position 5,353.
Figure 3:
PILEUP clustering of an amino acid
sequence alignment predicted from the gene sequence of a number of
aromatic ring dioxygenases for the terminal oxygenase large subunits
(at a gap creation penalty of 3.0 and a gap extension cost of
0.1)(24) . The right part of the figure shows the
genetic organization of the aromatic ring dioxygenases and surrounding
genes. Horizontal bars indicate the size and location of the
ORFs on the DNA of these organisms. Similar hatchings and shadings represent homologous genes and derived gene products.
Symbols: checkered box, large subunit of the terminal
oxygenase (ISP); waffled box, small subunit of
the terminal oxygenase (ISP
); dotted box,
ferredoxin; white on black lined box, reductase; diagonally lined box, dihydrodiol dehydrogenase; shaded
box, meta-cleavage enzyme. The figure does not show the
location of all genes within a particular gene cluster. ben genes, Acinetobacter calcoaceticus(9) ; xyl, P. putida mt2(3, 10) ; dox, (29) ; ndo, P. putida NCIB9816(26, 27) ; nah, P. putida G7(26) ; pah,(28) ; bph, P.
pseudoalcaligenes KF707(30, 48) ; PcBph, bph genes of Pseudomonas sp. strain
LB400(32, 49) ; Kks102Bph, bph genes
of P. paucimobilis strain KKS102(31) ; bed, P. putida ML2(33) ; tod, P. putida F1(23, 46) ; tcb, Pseudomonas sp. strain P51; bnz, P. putida 136-R3(13) ; RgBph, bph genes of Rhodococcus globerulus P6(35) ; RsBph, bph genes of Rhodococcus sp.
RHA1(34) .
It is interesting that to a large extent the gene organization within the clusters has been conserved as well, but clearly differs between them. For example, the benzene/toluene family has the gene order: large subunit of the terminal oxygenase, small subunit, ferredoxin, reductase, and, in three cases, dehydrogenase. The group of the Gram-positive biphenyl dioxygenases, which appeared to be the cluster closest to that of the benzene/toluene dioxygenases, has an identical organization of the genes encoding the core dioxygenase, but differs in genes located downstream. The family of the Gram-negative biphenyl dioxygenases has a genetic organization comparable to that of the benzene/toluene family, but in two cases contain an extra ORF between the genes for the terminal oxygenase and that for the ferredoxin. Also in these cases, the upstream regions lack homology between each other or with the corresponding regions of the benzene/toluene family. The biphenyl dioxygenase of strain KKS102 lacks the extra ORF, but also lacks the gene for the reductase at this position (Fig. 3). A stronger difference is found with the family of the naphthalene dioxygenases. Here the gene order is reductase, ferredoxin, large subunit of the terminal oxygenase, small subunit, dehydrogenase. In the dox-encoded system, no (clustered) gene for a reductase was described(29) , but this may similarly be located directly upstream. The naphthalene reductases (i.e. nahAa and pahAa) are also substantially shorter than those of the others (329 amino acids versus approximately 410) and biochemically different (7) . In the case of the benzoate and toluate dioxygenase, the largest difference in gene order with the others is found in the presence of a gene for the combined ferredoxin and reductase function (i.e. benC and xylZ)(8, 9) .
Figure 4:
SDS-PAGE of the cell extracts from E.
coli BL21(DE3) strains containing the different plasmids with tcbAB genes. Lanes: 1, pET8c; 2, pTCB147 (tcbAb, tcbAc, and deletion of tcbAa); 3, pTCB120 (tcbAc, tcbAd, and tcbB); 4, pTCB117 (tcbAc and a deletion of tcbAd); 5, pTCB116 (tcbAc, tcbAd,
and a deletion of tcbB); 6, pTCB115 (tcbAa, tcbAb, tcbAc, and small region of tcbAd); 7, pTCB114 (tcbAa and tcbAb with a
frameshift mutation); 8, pTCB113 (tcbAa). Symbols: Aa, gene product of tcbAa; Aa, product of the interrupted tcbAa gene; Ab, product of tcbAb; Ac, product of tcbAc; Ad, product of tcbAd; Ad
, product of the interrupted tcbAd; B, product of tcbB; B
, product of the
interrupted tcbB gene. Migration of the molecular mass
standards is indicated in kilodaltons on the right
side.
Whole cells of E. coli (pTCB144) were incubated with different aromatic substrates in
minimal medium in order to produce the cis-dihydrodiols. E. coli (pTCB144) cells rapidly produced one single metabolite
as detected by HPLC, when incubated with 1,2-dichlorobenzene, toluene,
biphenyl, or naphthalene (Fig. 5). No conversion of benzoate was
detected with these cells. The UV spectra of the intermediates of
toluene, naphthalene, and biphenyl incubation on HPLC were in agreement
with the values published previously for the
corresponding dihydrodiols(36, 37, 38) . For
the product of 1,2-dichlorobenzene incubation, we observed a UV
spectrum similar to that of toluene, although with a
at 272 nm. Further information on the identity of the four
intermediates was obtained by GC-MS analysis of the BSTFA-derivatized
form (Fig. 6). All four mass spectra gave a similar
fragmentation pattern with the molecular ions showing at m/z 324, 270, 332, and 306 for the products of dichlorobenzene (Fig. 6A), toluene (Fig. 6B), biphenyl (Fig. 6C), and naphthalene (Fig. 6D),
respectively. The usually dominant (M - 15)
ion
(loss of one of the methyl groups of the trimethylsilyl moiety) is
absent from all the mass spectra, but loss of OSi(CH
)
(molecular mass of 89) is apparent in all of them. The ions at m/z 191
([(CH
)SiOCHOSi(CH
)
]
),
147
([(CH
)
SiOSi(CH
)
]
),
and 73 ([(CH
)
Si]
)
dominate the four spectra. In the case of the product of
dichlorobenzene, the ion at m/z 289 is formed by the loss of
chlorine (mass of 35) from the molecular ion. Following the line of
evidence for the formation of cis-dihydrodiols from aromatic
compounds by toluene and naphthalene
dioxygenase(36, 37, 38) , for example, all
our results are in agreement with the proposed cis-dihydrodiol
structures which would be formed during conversion of toluene,
1,2-dichlorobenzene, biphenyl, and naphthalene by the chlorobenzene
dioxygenase.
Figure 5: Formation of dihydrodiol intermediates by washed whole cells of E. coli (pTCB146). Shown are mean values of peak areas of the intermediates as measured on HPLC from two independent incubations. Formation of the dihydrodiols excreted in the supernatant was measured as an increase in absorbance at the wavelength of the respective absorption maximum, i.e. 262 nm for naphthalene dihydrodiol, 265 nm for toluene dihydrodiol, 272 nm for 1,2-dichlorobenzene dihydrodiol, and 303 nm for biphenyl dihydrodiol.
Figure 6: Electron ionization mass spectra of the trimethylsilyl derivatives of the products of whole cell incubations with E. coli (pTCB144) and the following substrates. A, 1,2-dichlorobenzene; B, toluene; C, biphenyl; D, naphthalene. The structural formula of the proposed product has been drawn above the respective mass spectrum. The fragmentation pattern is discussed in the text.
The dihydrodiols were then incubated with cell extracts of E. coli (pTCB149), which expresses the dihydrodiol dehydrogenase, and analyzed by HPLC. In the case of 1,2-dichlorobenzene, we found that the dihydrodiol was converted to one single product. This product cochromatographed with authentic 3,4-dichlorocatechol, and the UV spectra of the two compounds were identical. Biphenyl dihydrodiol was enzymatically converted to a compound with an identical retention time and UV spectrum as 2,3-dihydroxybiphenyl and, similarly, toluene dihydrodiol to a compound with identity to 3-methylcatechol. In the case of naphthalene 1,2-dihydrodiol, the presumed product 1,2-dihydroxynaphthalene could not be detected, as it became autooxidized quickly as described earlier(39, 40) .
Our studies with the Tcb dioxygenase showed that it is not specific for catalyzing the conversion of 1,2-dichlorobenzene only, but capable of converting toluene, naphthalene, and biphenyl. Benzoate was not converted by the Tcb dioxygenase, which is like other characterized three-component aromatic ring dioxygenases(8, 12) . The outcome of the whole cell incubations suggests that naphthalene and biphenyl are converted even faster than 1,2-dichlorobenzene and toluene. However, we do not know in what way solubility of these compounds, different uptake, and excretion rates influence this outcome. We cannot exclude that other products, such as other stereoisomers or monohydroxylated rings, were formed in the incubations, since no attempts were made to determine a mass balance in the whole cell incubations. Furthermore, we did not determine the absolute stereoconfiguration of the products. HPLC analysis, however, suggested the formation of single intermediates. UV and mass spectrum of these compounds were in agreement with the structures of cis-dihydrodiols, as published previously(36, 37, 38) . In the subsequent enzyme incubations, we obtained good evidence that the TcbB dihydrodiol dehydrogenase converts all of these cis-dihydrodiols to dihydroxy intermediates (i.e. 3,4-dichlorocatechol, 3-methylcatechol, 2,3-dihydroxybiphenyl, and 1,2-dihydroxynaphthalene). These results strongly suggest that the formation of dihydrodiols and dihydroxy compounds by the Tcb dioxygenase and TcbB dihydrodiol dehydrogenase proceed as expected from the general line for dioxygenation(7, 8, 12) .
It is not so clear which subunit of the aromatic ring dioxygenases determines the substrate specificity of the enzyme and why the toluene/benzene subclass enzymes have such a wide substrate spectrum. The remarkable potential of the Tod enzyme to catalyze incorporation of oxygen into a wide range of aromatic substrates has been well studied and explored(12, 41) . For instance, the Tod dioxygenase oxidizes biphenyl, the main substrate of the group of bph-encoded enzymes. On the other hand, biphenyl dioxygenase from P. pseudoalcaligenes KF707 does not oxidize toluene(14, 42) . This limitation supposedly arose in the small subunit of Bph dioxygenase, because when hybrid enzymes between Tod and Bph were constructed, some were found (e.g. BphA1TodC2AB) that gained both the Bph and Tod substrate range. The Bph-dioxygenases were mostly studied for their capability to convert (poly-) chlorinated biphenyls. For example, differences in polychlorinated biphenyls-congener specificity were found between the Bph dioxygenase from Pseudomonas sp. strain LB400 and P. pseudoalcaligenes KF707, which in this case were attributed to changes in the large subunit of the terminal oxygenase(15) . It will be interesting to study in more detail whether the Tcb dioxygenase has acquired any new substrate specificities which enable it to convert higher chlorinated aromatic compounds more efficiently.
A major gene rearrangement has probably taken place in Pseudomonas sp. strain P51. In this microorganism the genes for the aromatic ring dioxygenase and the dihydrodiol dehydrogenase were most likely transposed from their original position by the activity of two insertion elements(19) . The relics of this process can still be seen on the DNA (Fig. 7). Upstream of the gene tcbAa there are parts of a gene similar to todF(46) , and downstream of tcbB there is a large part of a gene for a meta-cleavage enzyme similar to todE(23) . It may have been a necessity for the organism to inactivate such a meta-cleavage enzyme since these are known in some microorganisms to interfere with the metabolically productive ortho-cleavage of chlorinated catechols, which arise as intermediates in chlorobenzene degradation(47) . We believe that the strong genetic and biochemical similarity between the tcbAB and the tod system is good evidence to assume that the tcbAB genes originated in a microorganism which could use toluene or benzene as the sole carbon and energy source.
Figure 7: Comparison of the tcbAB genes and part of the tod gene clusters. Shown is a physical map of the most important features in this comparison. Blocks indicate size and location of ORFs on the DNA encoding the different proteins. Similar shadings or hatchings mean homologous regions in both gene clusters. Directly upstream of tcbAa there are 35 bp (indicated in black) with homology to the upstream region of todC1. Some 30 bp further upstream is a region of 201 bp with homology to todF, although from the start of this gene. Percentages of identity in nucleotide sequence between the different gene regions are given in the figure. Downstream of tcbB is the part of the gene with homology to todE. The sizes and locations of the ORFs for tcbAb, tcbAc, and tcbAd, respectively, todC2, todB, and todA are not shown in detail here.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U15298[GenBank].