Department of Biochemistry, Groningen
Biomolecular Sciences and Biotechnology Institute, University of
Groningen, 9747 AG Groningen, The Netherlands,1
and The Questor Centre, The Queen's University of Belfast,
Belfast BT9 5AG, United Kingdom2
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INTRODUCTION |
Synthetic haloalkanes form an
important class of environmental pollutants because of their widespread
use in industry and agriculture, persistence in the environment, and
potential carcinogenicity. The poor biodegradability of these chemicals
is mainly due to the inability of microorganisms to effectively
metabolize these unnatural compounds. Nevertheless, microbial
communities exposed to synthetic haloalkanes often respond by
expressing specific pathways that degrade these molecules in order to
exploit them as growth substrates. Since synthetic haloalkanes are
xenobiotic compounds of recent origin, the way in which genes have been
assembled to form functional catabolic pathways is an interesting
subject for studying microbial evolution and gene transfer.
Rhodococcus rhodochrous NCIMB13064, isolated in the United
Kingdom from a soil sample obtained from an industrial site which had
previously been exposed to chlorinated alkanes, is capable of utilizing
1-chlorobutane and several other haloalkanes as the sole carbon and
energy source (9). The cleavage of the carbon-halogen bond
in 1-chlorobutane, which is the key step in its catabolism, is
catalyzed by an inducible hydrolytic haloalkane dehalogenase (DhaA) and
results in the formation of n-butanol. This intermediate is
subsequently oxidized in two steps to n-butyric acid (Fig. 1), which can serve as a growth substrate
for many bacteria. The haloalkane dehalogenase gene (dhaA)
was shown to be located on the autotransmissible plasmid pRTL1
(22) and was cloned and sequenced (23).
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FIG. 1.
Catabolic pathways for haloaliphatics. (A)
1-Chlorobutane in R. rhodochrous NCIMB13064. (B)
1,3-Dichloropropene in P. pavonaceae 170. (C)
1,2-Dibromoethane in Mycobacterium sp. strain GP1.
Abbreviations: DhaA and DhaAf, haloalkane dehalogenases;
H-lyase, halohydrin halogen-halide lyase; CaaD, 3-chloroacrylic acid
dehalogenase; X, alcohol dehydrogenase cofactor; Y, aldehyde
dehydrogenase cofactor.
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The gram-negative 1,3-dichloropropene-utilizing bacterium
Pseudomonas pavonaceae 170, isolated in The Netherlands from
soil that was repeatedly treated with the nematocidic soil fumigant 1,3-dichloropropene, was shown by PCR amplification to possess a
haloalkane dehalogenase gene identical to the dhaA gene of
the gram-positive strain NCIMB13064 (35). In contrast to the
inducible production of DhaA in strain NCIMB13064, DhaA is
constitutively produced in strain 170 and catalyzes the first step in
the degradation of 1,3-dichloropropene (Fig. 1).
Recently, we demonstrated that the 1,2-dibromoethane-degrading organism
Mycobacterium sp. strain GP1, which was isolated by prolonged batch enrichment from a mixed bacterial culture, also contains a haloalkane dehalogenase gene (dhaAf)
that is very similar to the dhaA gene found in strain
NCIMB13064 (36). The haloalkane dehalogenase encoded by
dhaAf is identical to DhaA, except for three
amino acid substitutions and a 14-amino-acid extension at the C
terminus. Nucleotide sequence analysis indicated that the dhaAf gene was formed by a fusion of a
dhaA gene with the last 42 nucleotides of a hheB
gene, which encodes a haloalcohol dehalogenase (50). The
haloalkane dehalogenase (DhaAf) is constitutively produced
in strain GP1 and catalyzes the conversion of 1,2-dibromoethane to
2-bromoethanol, which is further metabolized via ethylene oxide (Fig.
1).
The presence of a highly conserved dhaA gene in the
three phylogenetically different organisms R. rhodochrous NCIMB13064, P. pavonaceae 170, and
Mycobacterium sp. strain GP1 suggests that dhaA
has been distributed among these organisms by horizontal transfer. To
determine the size of the transferred DNA fragments and to identify the
mechanisms that were involved in the distribution process, we have
analyzed the DNA regions flanking the dhaA gene in these
three haloalkane-utilizing strains. The results suggest that the
haloalkane dehalogenase gene regions of strains 170 and GP1 originate
from a 1-chlorobutane catabolic gene cluster similar to the one that is
present on plasmid pRTL1 in strain NCIMB13064. Horizontal gene transfer
and integrase-dependent acquisition of existing DNA fragments harboring
the dhaA gene were probably the key steps during the
evolution of 1,3-dichloropropene- and
1,2-dibromoethane-degradative pathways. Furthermore, the
constitutive expression of dhaA in strains 170 and GP1, in
contrast to the inducible expression of dhaA in strain
NCIMB13064, is explained by the absence or inactivation of the
regulatory gene dhaR.
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MATERIALS AND METHODS |
Materials.
Restriction enzymes, Taq DNA
polymerase, T4 DNA ligase, the DNA-packaging kit, and materials used
for Southern blot hybridization were purchased from Boehringer Mannheim
(Mannheim, Germany). 1,2-Dibromoethane was supplied by Acros Organics
(Geel, Belgium). The oligonucleotides used as primers were supplied by
Eurosequence BV (Groningen, The Netherlands).
Bacterial strains, plasmids, and growth conditions.
The
characteristics of the 1,3-dichloropropene-degrading bacterium P. pavonaceae 170, formerly known as Pseudomonas cichorii 170 (44), and of the 1,2-dibromoethane-degrading organism
Mycobacterium sp. strain GP1 are given elsewhere (35,
36). R. rhodochrous NCIMB13064 contains the large
catabolic plasmid pRTL1 (22) and is able to use
1-chlorobutane as the sole carbon and energy source (9).
Escherichia coli strains HB101 (7) and JM101
(49) and plasmid pBluescript SK(
) (Stratagene, Leusden,
The Netherlands) were used for routine cloning experiments. E. coli HB101(pRK600) (13) was the helper strain used for
mobilizing pLAFR3/5- and pDSK519-derived plasmids in triparental
matings with the recipient Pseudomonas sp. strain GJ1
(17). Plasmid pDSK519 and cosmids pLAFR3 and pLAFR5 are
mobilizable broad-host-range vectors (19, 39). Recombinant
cosmid pLTL1k contains the 1-chlorobutane catabolic gene cluster of
strain NCIMB13064 (23) and was used as template for DNA
sequencing and as the source for some cloning and expression experiments described here. Recombinant cosmids pGP1-4B5, which contains the dhaAf gene region of strain GP1
(36), and pPC33, which contains the dhaA gene
region of strain 170 (this study), were used as templates for DNA sequencing.
R. rhodochrous NCIMB13064 and Mycobacterium sp.
strain GP1 were grown at 30°C on nutrient broth or on mineral medium
(17) supplemented with 5 mM n-propanol,
respectively. Pseudomonas and E. coli strains
were grown at 30°C on Luria-Bertani medium (38). When
required, Difco agar (15 g/liter) was added to the medium. LBZ medium,
used for qualitative dehalogenase activity determination, was solid
Luria-Bertani medium without NaCl. Antibiotics were added in the
following amounts: ampicillin, 100 µg/ml; kanamycin, 50 µg/ml;
chloramphenicol, 50 µg/ml; and tetracycline, 12.5 µg/ml. When
necessary, media were supplemented with
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside (X-Gal;
40 µg/ml) and isopropyl--D-thiogalactopyranoside
(IPTG; 0.4 mM).
DNA techniques.
General procedures for cloning,
transformation, and DNA manipulation were performed essentially as
described by Sambrook et al. (38). Triparental matings were
carried out as described by Janssen et al. (18). Isolation
of total genomic DNA from strains NCIMB13064, 170, and GP1 was
performed according to the phenol extraction procedure described
previously (35). The IS2112- and
IS1071-specific probes used in hybridization experiments
were obtained by PCR amplification by using primers and conditions as
previously described (21, 48). DNA fragments were purified by using the Qiaquick PCR purification kit (Qiagen). Southern blot
hybridization experiments were performed as described previously (21).
Crude extracts and dehalogenase assays.
Pseudomonas
and E. coli cells were harvested in the late-exponential
growth phase by centrifugation (10 min at 10,000 × g), were washed with 1 volume of 50 mM Tris-sulfate buffer (pH 8.2), and
were disrupted at 4°C in an appropriate amount of this buffer by
sonication (10 s per ml of suspension at a 70 W output in a Vibra cell
sonicator). A crude extract was obtained by centrifugation (45 min at
16,000 × g).
Haloalkane dehalogenase activities were measured by incubating an
appropriate amount of cell extract with 3 ml of 5 mM 1,2-dibromoethane in 50 mM Tris-sulfate buffer (pH 8.2) at 30°C. Halide liberation was
monitored colorimetrically as described previously (20). All
dehalogenase activities are expressed as units per miligram; 1 U was
defined as the amount of enzyme that catalyzes the production of 1 µmol of halide per min. Protein concentrations were estimated with
Coomassie brillant blue by using bovine serum albumin as the standard.
Enzyme assays were carried out twice, and the differences in specific
activities were less than 10%.
Cloning of the dhaA gene region from P. pavonaceae 170.
A genomic library of P. pavonaceae 170 was constructed by using cosmid vector pLAFR3
according to a previously described procedure (39).
Individual cosmid clones were screened for dehalogenase activity by
monitoring halide production upon incubation with 1,2-dibromoethane.
Out of 5,000 E. coli HB101 clones tested, two dehalogenase-positive clones were found. Recombinant cosmids pPC8 and
pPC33 encoding haloalkane dehalogenase were isolated from these two
HB101 clones and were digested with BamHI. Both cosmids had
a 3-kb BamHI fragment in common. The 3-kb BamHI
fragment of pPC33 was ligated into the BamHI site of
pBluescript SK(). The ligation mixture was used to transform cells of
E. coli JM101, and transformants were plated on LBZ plates
containing ampicillin, X-Gal, and IPTG. Ampicillin-resistant white
colonies displaying haloalkane dehalogenase activity with
1,2-dibromoethane were selected. Plasmid DNA (pSK45) of one of these
colonies was isolated and used as template for DNA sequencing. The
nucleotide sequences of DNA regions flanking the 3-kb BamHI
fragment were determined by using cosmid pPC33 directly as a template
for DNA sequencing.
Nucleotide sequencing and analysis.
DNA sequencing was
performed as previously described (21, 36). The nucleotide
sequence data were analyzed by using the programs supplied in the
DNASTAR software package (DNASTAR Inc., Madison, Wis.) or those
supplied in the PC/GENE software package (Genofit, Geneva,
Switzerland). Searches for nucleotide and amino acid sequence
similarities were carried out by using the BLAST program (3)
and the DDBJ, EMBL, and GenBank databases. Protein sequences were
aligned by using CLUSTAL W (41), and alignments of
nucleotide sequences were made by using LALIGN (Institut de Génétique Humaine, Montpellier, France).
Nucleotide sequence accession numbers.
The nucleotide
sequence data of the haloalkane dehalogenase gene regions from R. rhodochrous NCIMB13064, P. pavonaceae 170, and
Mycobacterium sp. strain GP1 have been submitted to the
DDBJ, EMBL, and GenBank databases under accession no. L49435, AJ250371, and AJ250372, respectively.
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RESULTS AND DISCUSSION |
Sequence analysis of the dhaA gene region from R. rhodochrous NCIMB13064.
The dhaA gene of the
1-chlorobutane-degrading bacterium R. rhodochrous NCIMB13064
is located on the 100-kb plasmid pRTL1 (23). A 13.1-kb DNA
fragment of pRTL1, including a 8.2-kb region upstream and a 4-kb region
downstream of dhaA, was sequenced (Fig.
2). The dhaA gene sequence and
the analysis of insertion element IS2112, which is located
approximately 6 kb upstream of dhaA (Fig. 2), were described
previously (21, 23).
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FIG. 2.
Organization of the haloalkane dehalogenase gene regions
of R. rhodochrous NCIMB13064, P. pavonaceae 170, and Mycobacterium sp. strain GP1. Genes are shown as hatched
boxes, and arrows indicate the direction of transcription. Identical
hatching indicates identical genes. Incomplete ORFs are indicated by a
cross through the arrow. The borders of the conserved DNA segments in
strains 170 and GP1, which are highly similar to segments of the
1-chlorobutane-catabolic gene cluster of strain NCIMB13064, are
indicated by vertical arrows. The two deletions within the conserved
region of strain 170 are indicated by vertical dotted lines. The
42-nucleotide extension of the dhaA ORF in strain GP1, which
is the result of a fusion between the dhaA segment and
haloalcohol dehalogenase HheB encoding DNA (36), is
indicated by a shaded box. EcoRI restriction sites (E) are
shown.
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Two complete open reading frames (ORFs), designated invA and
dhaR, were found upstream of dhaA (Fig. 2 and
3). InvA shares extensive similarity with
proteins belonging to the invertase family of site-specific
recombinases. The inversion reaction is a site-specific recombination
between inverted repeat sequences which flank the invertable DNA
fragment and is carried out by invertases. Several examples of
invertable DNA that can serve as a genetic switch between the
expression of alternative sets of genes have been described
(14). InvA is most similar to the invertases Pin of E. coli (34) and Hin of Salmonella enterica serovar Typhimurium (51) (Table
1). The high amino acid sequence identity
with these DNA invertases implies a common phylogenetic origin,
although invertase action has yet to be demonstrated for InvA.
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FIG. 3.
Partial nucleotide sequence of the
1-chlorobutane-degradative gene cluster found in pRTL1 of R. rhodochrous NCIMB13064. The borders of the conserved DNA segment
in Mycobacterium sp. strain GP1 are indicated by vertical
arrows. DNA stretches identical to the nucleotide sequences flanking
the dhaA gene in P. pavonaceae 170 are
underlined. Inverted and directed repeats are indicated by horizontal
arrows above the sequence. Palindromic sequences are shown in
boldface.
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Database searches with the deduced amino acid sequence of the
dhaR gene revealed no proteins with significant similarity
to the entire sequence of the dhaR product. However, DhaR
contains a region near the N terminus which resembles the
helix-turn-helix (HTH) DNA binding motifs of a number of
transcriptional regulators (10, 30). Considerable similarity
was found with the HTH motifs of the Streptomyces
autoregulator receptors ArpA, BarA, and FarA (Fig.
4), which were proposed to act as
repressor-type regulators for secondary metabolism and morphogenesis
(28, 29, 45). The overall sequence similarity to these three
Streptomyces regulators is low (Table 1).
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FIG. 4.
Alignment of the N-terminal amino acid sequences of
FarA, BarA, ArpA, and DhaR. Amino acids conserved in all four proteins
are indicated by asterisks, whereas amino acids conserved in three out
of four proteins are indicated by dots. The sequences corresponding to
helix 2 and helix 3 in the HTH DNA binding motif are boxed.
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Cells of strain NCIMB13064 grown in n-butanol do not possess
DhaA activity, but growth on 1-chlorobutane induces the expression of
the dehalogenase DhaA (9). The presence of a region
resembling the HTH DNA binding motifs in DhaR and the localization of
its encoding ORF directly upstream of dhaA suggest that DhaR
modulates transcription of dhaA by binding directly to its
promoter region in response to 1-chlorobutane. Sequence analysis of the
dhaR-dhaA intergenic region revealed the presence of two
identical directly repeated sequences of 13 bp (Fig. 3). The repeated
sequences contain a 10-bp palindromic sequence (TGACCGGTCA)
and are both part of a larger (imperfect) palindrome. The same
13-bp repeated sequence was also found upstream of dhaR
(Fig. 3). The presence of these putative binding sites for DhaR
upstream of both the dhaR and dhaA genes suggests
that this protein regulates its own expression and that of DhaA.
However, promoter sequences for Rhodococcus genes are not
well characterized (24), and the promoter responsible for
dhaA expression has not been identified.
Two ORFs, designated adhA and aldA, were found
downstream of dhaA (Fig. 2 and 3). AdhA shares considerable
similarity with several proteins that belong to the alcohol
dehydrogenase subgroup which contains the NAD(P)- and zinc-dependent
long chain alcohol dehydrogenases (Table 1). The highest similarity was
found with putative alcohol dehydrogenases from Mycobacterium
tuberculosis H37Rv (Table 1). Structural and catalytic residues
that are conserved among almost all microbial alcohol dehydrogenases
(37) are also present in AdhA. AldA shares considerable
similarity with putative aldehyde dehydrogenases from M. tuberculosis H37Rv and with several known NAD(P)-dependent
aldehyde dehydrogenases (Table 1). The cysteine residue (C302, family
position) that is strictly conserved among all available sequences of
the aldehyde dehydrogenase superfamily, and is proposed to be the
active site nucleophile (6, 12), is also conserved in AldA.
Curragh and coworkers (9) showed that 1-chlorobutane was
metabolized by strain NCIMB13064 via n-butanol and
n-butyric acid (Fig. 1). 1-Chlorobutane is converted to
n-butanol by DhaA (23). It therefore appears very
likely that adhA and aldA, which are located
downstream of dhaA (Fig. 2), encode the alcohol and aldehyde dehydrogenases that are involved in the oxidative conversion of n-butanol to n-butyric acid. The genes for the
initial steps in the degradation of 1-chlorobutane thus appear to be
located in a cluster on plasmid pRTL1.
The extent of the conserved dhaA gene fragments in
P. pavonaceae 170 and Mycobacterium sp. strain
GP1.
When the haloalkane dehalogenase gene regions of P. pavonaceae 170 and R. rhodochrous NCIMB13064 are
compared, the conserved DNA fragment in strain 170 can be seen to
include a region of about 1.3 kb that harbors little more than the
coding region of the dhaA gene (Fig. 2). The dhaA
genes of strains 170 and NCIMB13064 are completely identical. The first
37 nucleotides upstream of the start codon of dhaA are also
identical in both strains, after which there is a deletion of 98 nucleotides in the Pseudomonas sequence when compared to the
Rhodococcus sequence (Fig. 2 and 3). Upstream of this
deletion, the sequences continue to be identical for 268 nucleotides
and then abruptly become completely unrelated. The 98-nucleotide
deletion in the Pseudomonas sequence includes exactly the
DNA sequence between the 13-bp directed repeat as well as one of the
repeated sequences itself (Fig. 3). The formation of this deletion may
be explained by DNA strand slippage, which allows one repeated sequence
to mispair with the complement of the other (2).
The two sequences are identical for only 77 nucleotides downstream of
the dhaA gene. The following 60 nucleotides in the
Pseudomonas sequence (nucleotides 3600 to 3659) are
identical to a fragment downstream of aldA (nucleotides
12313 to 12372) in the Rhodococcus sequence (Fig. 3). This
suggests that a large deletion of about 3.2 kb, including the alcohol
and aldehyde dehydrogenase genes, has occured in the
Pseudomonas sequence when compared to the
Rhodococcus sequence (Fig. 2). No similarity between the two
sequences was found further downstream.
When the haloalkane dehalogenase gene regions of
Mycobacterium sp. strain GP1 and R. rhodochrous
NCIMB13064 are compared, the conserved DNA fragment in strain GP1
appears to include a region of about 2.7 kb which harbors
dhaA, dhaR, and part of invA (Fig. 2).
The similarity of the dehalogenase gene region of strain GP1 to that of
strain NCIMB13064 starts at nucleotide 17 of the invA ORF
and ends exactly before the stop codon of the dhaA ORF (Fig.
3). This DNA segment in strain GP1 is identical to the corresponding segment in strain NCIMB13064, except for three nucleotide substitutions in dhaA and a 12-nucleotide deletion in the
Rhodococcus regulatory gene dhaR. No further
similarity was found between the two sequences.
The haloalkane dehalogenase gene regions of the three phylogenetically
different bacteria R. rhodochrous NCIMB13064, P. pavonaceae 170, and Mycobacterium sp. strain GP1 thus
appear to have a common genetic origin. The globally distributed
1-chlorobutane catabolic gene cluster on plasmid pRTL1 (G. J. Poelarends, M. Zandstra, T. Bosma, L. A. Kulakov, M. J. Larkin, J. R. Marchesi, A. J. Weightman, and D. B. Janssen, submitted for publication) seems to be ancestral to the DNA
fragments harboring the dhaA gene in strains 170 and GP1.
These dhaA gene fragments probably originated from a
catabolic gene cluster similar to the one present on pRTL1 in strain
NCIMB13064 and were horizontally transferred to the present hosts.
Since the structural genes are identical, we conclude that horizontal transfer of the dhaA gene fragment to strain 170 has
occurred naturally and recently, resulting in the formation of a newly evolved pathway for 1,3-dichloropropene degradation (Fig. 1). The
hydrolytic product of 1,3-dichloropropene, 3-chloroallyl alcohol, can
be used as a carbon source by several gram-negative bacteria (5,
43). We also propose that the 1,2-dibromoethane-degrading pathway
of strain GP1 arose by horizontal transfer of a DNA fragment harboring
dhaA from a donor strain present in the original mixed enrichment culture to a 2-bromoethanol-degrading host. The host that
obtained a complete degradation pathway for 1,2-dibromoethane in this
way gained a selective advantage and became the predominant organism in
the mixed culture.
The role of the dhaR gene in the regulation of
dhaA expression.
The sequence analysis indicated that
expression of the dhaA gene in R. rhodochrous
NCIMB13064 may be regulated by the dhaR gene product. If
dhaR encodes a regulatory protein, its inactivation should
lead to either constitutive expression or noninducibility of the
dhaA gene, depending on the negative or positive nature of
the regulation, respectively. E. coli HB101 harboring
recombinant cosmid pLTL1k, which carried intact dhaR and
dhaA genes (Fig. 5),
constitutively expressed dhaA (Table
2), suggesting that expression of
dhaA is not regulated in E. coli. Introduction of pLTL1k into Pseudomonas sp. strain GJ1 did not lead to
constitutive expression of dhaA. We therefore used strain
GJ1 to analyze the role of the dhaR gene in the regulation
of dhaA expression.
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FIG. 5.
Schematic overview of the 1-chlorobutane-degradative
gene cluster in recombinant cosmid pLTL1k and of the two subclones used
for dhaA expression studies in Pseudomonas sp.
strain GJ1. Genes are indicated by hatched boxes, and arrows indicate
the direction of transcription. Cosmid pLTL1k was cut at the indicated
restriction sites, and the corresponding dhaA gene fragments
were isolated and inserted into the BamHI or SalI
site of pDSK519, yielding plasmids pDSKB1 and pDSKS4, respectively. The
direction of the pDSK519-localized lac promoter is
indicated. BamHI and SalI restriction sites are
indicated by B and S, respectively.
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TABLE 2.
Dehalogenase activities in crude extracts of E. coli HB101 and Pseudomonas sp. strain GJ1 harboring
different constructs
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Plasmids that carried either dhaA and an intact
dhaR gene (pDSKB1) or dhaA and the
dhaR gene with a deletion in its proximal part (pDSKS4) were
constructed (Fig. 5). In contrast to cell extracts prepared from
strains GJ1(pLTL1k) and GJ1(pDSKB1), cell extract from strain
GJ1(pDSKS4) displayed haloalkane dehalogenase activity (Table 2). Cell
extract from strain GJ1 carrying pDSKS5, in which the SalI
insert that is present in pDSKS4 was placed in the direction opposite
of that of the lac promoter of pDSK519, also displayed dehalogenase activity (Table 2), indicating that the constitutive expression of dhaA was controlled by its own promoter and
not by the lac promoter of pDSK519. These results show that
inactivation of dhaR leads to constitutive expression of the
dhaA gene, indicating that the dhaR gene product
putatively acts as a repressor of dhaA expression.
In contrast to the negatively regulated expression of dhaA
in R. rhodochrous NCIMB13064, the haloalkane dehalogenase
genes in P. pavonaceae 170 and Mycobacterium sp.
strain GP1 are constitutively expressed (35, 36). The
constitutive expression of dhaA in strain 170 may be caused
by the absence of a dhaR gene. Although strain GP1 possesses
the dhaR gene in front of dhaAf, the
12-nucleotide deletion present in dhaR may inactivate the
regulatory protein (DhaR), leading to constitutive expression. To
confirm that this deletion in dhaR inactivated its gene
product, recombinant cosmid pGP1-4B5, which carried the
dhaAf gene region of strain GP1 (Fig. 2), was
introduced into Pseudomonas sp. strain GJ1. In contrast to
cell extract prepared from strain GJ1 harboring cosmid pLTL1k, which
carried the dhaA gene region of strain NCIMB13064 (Fig. 5),
cell extract of strain GJ1(pGP1-4B5) displayed haloalkane dehalogenase
activity (Table 2), indicating that the deletion in dhaR had
inactivated its gene product. This further emphasizes the negative
regulatory role of DhaR.
In strains 170 and GP1, a putative DNA integrase gene is present
next to the conserved dhaA gene fragment.
The sequence
comparisons indicated that novel catabolic pathways for
1,3-dichloropropene and 1,2-dibromoethane were built by adding existing
DNA fragments harboring dhaA to the genomes of P. pavonaceae 170 and Mycobacterium sp. strain GP1,
respectively. This suggests the involvement of a mechanism for the
acquisition of distinct DNA fragments. Gene acquisition by bacterial
genomes could occur either by excisive-integrative recombination events mediated by insertion elements or by site-specific integration events
mediated by DNA integrases (40, 42, 47).
Interestingly, an ORF encoding a putative DNA integrase
(intP) was found upstream of the conserved dhaA
gene fragment in strain 170 (Fig. 2 and Table 1). IntP shares
significant similarity with proteins belonging to the integrase (Int)
family of site-specific recombinases (Fig.
6A). Although the members of the Int
family exhibit a large diversity in their sequences, they all harbor two regions of marked sequence similarity, called box I and box II
(27). IntP shows 55% identity to its nearest neighbor, the integrase-like protein of Selenomonas ruminantium, in these
two conserved domains (Fig. 6A). The tetrad R-H-R-Y, which includes the
active-site tyrosine (31), is conserved in almost all
members of the Int family (1, 4, 27) and is present as
Arg-183, His-327, Arg-330, and Tyr-362 in IntP.
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FIG. 6.
(A) Amino acid sequence alignment of IntP with the
conserved segments of the recombinases Cre of bacteriophage P1
(accession no. P06956) and XerD of E. coli (M54884) and the
integrase-like proteins of S. ruminantium (AB011029) and
Tn5041 (X98999). Asterisks represent bases identical to
those in the upper sequence. Invariant amino acid residues which are
believed to be involved in catalysis are numbered (IntP numbering) and
shown in boldface. (B) Amino acid sequence alignment of IntM with the C
termini of the recombinases Sss of P. aeruginosa (S61402),
XerC of E. coli (P22885), and CodV of Bacillus
subtilis (P39776) and the putative phage type integrase of
Sphingomonas aromaticivorans (AF079317). Asterisks represent
bases identical to those in the upper sequence. Dashes represent bases
absent in other sequences. The conserved residues His-352, Arg-355, and
Tyr-415 are numbered (IntM numbering) and shown in boldface.
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An ORF encoding a putative DNA integrase (intM) was also
found upstream of the conserved dhaA gene fragment in strain
GP1 (Fig. 2 and Table 1). The C-terminal region of IntM shares
considerable similarity with proteins of the Int family (Fig. 6B). The
conserved tetrad R-H-R-Y of the Int family (1, 4, 27) is
present as Arg-260, His-352, Arg-355, and Tyr-415 in IntM (Fig. 6B).
The presence of an integrase gene in the vicinity of the conserved
dhaA gene fragment both in strain 170 and in strain GP1 suggests acquisition of these DNA fragments by site-specific
integration. Assuming that no additional recombinations have taken
place after the primary insertion event, comparisons of the boundaries
between the conserved dhaA gene fragments and the sequences
unique for strain 170 or GP1 define the insertion site. No short
duplication of bases on either side of the presumed point of insertion
or regions of dyad symmetry that could serve as integrase binding sites
were found when the flanking sequences were compared. The absence of
these features could be due to an adjacent deletion removing the border
segment on one side. Such a deletion could also explain the fusion of
the dhaA gene to the 3' end of the haloalcohol dehalogenase
gene hheB (Fig. 2).
The presence of insertion elements in the vicinity of the conserved
dhaA gene fragments.
Many catabolic genes are
associated with transposons (42, 47), and transposition is
clearly a major mechanism for the acquisition of catabolic genes by
bacterial genomes. Sequence analysis of the regions (>4 kb) adjacent
to the conserved dhaA gene fragment in
Mycobacterium sp. strain GP1 showed that these regions do
not encode any proteins related to known transposases. Distal to the
dhaA gene fragment in P. pavonaceae 170, however, a large ORF (tnpA) was found (Fig. 2) of which the deduced
amino acid sequence showed extensive similarity with the putative
transposase of Pseudomonas pseudoalcaligenes JS45 (Table 1).
The tnpA ORF, however, appeared to be interrupted by an
insertion element, identified as IS1071 (Fig. 2), causing a
truncation of its product. Nucleotide sequencing of a 1.7-kb fragment
of IS1071 revealed no differences with the known sequence of
IS1071, which is involved in transposition of the
chlorobenzoate genes (26). PCR amplification, using a primer
specific for both ends of IS1071 (due to the inverted
repeats at its termini) and recombinant cosmid pPC33 as template DNA, showed the formation of a 3.2-kb PCR product corresponding to the size
of IS1071 (26). Thus, a complete copy of
IS1071 was present as an insertion in the tnpA
gene approximately 1.6 kb downstream of dhaA.
Southern hybridization analysis revealed five copies of
IS1071 in strain 170 but only two copies in strain 170M4, a
spontaneous mutant of strain 170 that has lost the dhaA gene
(35) (results not shown). The 60-kb plasmid pPC170, that was
previously identified in strain 170 (44), is still present
in strain 170M4, showing that the dhaA gene is not plasmid
localized. Assuming that the mutation in strain 170M4 was caused by a
single deletion event, this could suggest that the dhaA gene
region in strain 170 is flanked by several copies of IS1071,
forming a composite class I element, and that a homologous
recombination event between two insertion sequences was responsible for
the loss of the dhaA gene and three copies of
IS1071. Strain 170M4 also lacked the integrase gene
intP, which is consistent with such a deletion event.
Many antibiotic resistance genes found on transposons in gram-negative
bacteria are located within a conserved DNA sequence (15, 25,
40). These conserved elements, called integrons (16,
40), are formally distinct from other genetic elements in that
they determine site-specific integration functions, a DNA integrase and
a recombination site, and are thus able to acquire resistance genes at
the specific site without the need for the presence of insertion
sequences or integrase genes in the DNA segments that are acquired. If
the integrase gene intP in P. pavonaceae 170 is
flanked by IS1071 sequences and confers on this transposon a
capability of taking up individual and unrelated catabolic genes by
integrase-mediated recombination, strain 170 may possess a mobile DNA
element similar to the previously identified integrons which are
capable of taking up antibiotic resistance genes (16, 40).
No sequences similar to IS1071 were found in strains
NCIMB13064 and GP1, indicating that this insertion element was not
involved in distribution of the dhaA gene among these
strains. Similarly, insertion sequence IS2112, which is
located approximately 6 kb upstream of the dhaA gene in
strain NCIMB13064 (Fig. 2) (21), is not present in strains
170 and GP1. These results are consistent with the hypothesis that
site-specific integration events mediated by IntP and IntM were
responsible for the acquisition of the dhaA gene fragments,
rather than excisive-integrative recombination events mediated by
insertion elements. However, the presence of multiple copies of an
active mobile element (IS1071) around the dhaA
gene in strain 170, combined with the fact that strain 170 was recently
isolated from a 1,3-dichloropropene-contaminated environment in which
natural genetic exchange is likely to be important (44),
suggests that transposition has played a part in the mobilization of
the dhaA gene among members of the microbial community
present in this environment.
Concluding remarks.
Our data provide further support for
previous studies suggesting that horizontal transfer of genes involved
in pollutant biodegradation may play an important role in the evolution
of catabolic pathways and the adaptation of microbial communities to
different environmental contaminants. Up to now, catabolic genes were
thought to be transferred mainly by means of conjugative plasmids and
transposons (42, 47). The results presented here suggest
that genes specifying adaptation to xenobiotics can also spread as
integrons, as has been proposed in the case of genes specifying
antibiotic resistance (16, 40). The widespread use of
antibiotics and the introduction of xenobiotics into the environment
seem to lead to adaptation by similar molecular mechanisms.
This study was supported by the Life Sciences Foundation (SLW),
which is subsidized by the Netherlands Organization for Scientific Research (NWO), and by the EC Environment and Climate Research Program
contract ENV4-CT95-0086.
We thank P. Terpstra (BioMedical Technology Centre, University of
Groningen, Groningen, The Netherlands) for his assistance in DNA
sequencing and analysis.
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Abstract
Introduction
