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Journal of Bacteriology, October 2000, p. 5683-5691, Vol. 182, No. 20
Laboratory of Microbiology, Wageningen
University, NL-6703 CT Wageningen, The Netherlands
Received 24 May 2000/Accepted 15 July 2000
To characterize the expression and possible regulation of reductive
dehalogenation in halorespiring bacteria, a 11.5-kb genomic fragment
containing the o-chlorophenol reductive
dehalogenase-encoding cprBA genes of the gram-positive
bacterium Desulfitobacterium dehalogenans was subjected to
detailed molecular characterization. Sequence analysis revealed the
presence of eight designated genes with the order
cprTKZEBACD and with the same polarity except for cprT. The deduced cprC and cprK
gene products belong to the NirI/NosR and CRP-FNR families of
transcription regulatory proteins, respectively. CprD and CprE are
predicted to be molecular chaperones of the GroEL type, whereas
cprT may encode a homologue of the trigger factor folding
catalysts. Northern blot analysis, reverse transcriptase PCR, and
primer extension analysis were used to elucidate the transcriptional
organization and regulation of the cpr gene cluster. Results indicated halorespiration-specific transcriptional induction of
the monocistronic cprT gene and the biscistronic
cprBA and cprZE genes. Occasional read-through
at cprC gives rise to a tetracistronic cprBACD
transcript. Transcription of cprBA was induced 15-fold upon
addition of the o-chlorophenolic substrate
3-chloro-4-hydroxyphenylacetic acid within 30 min with concomitant
induction of dehalogenation activity. Putative regulatory protein
binding motifs that to some extent resemble the FNR box were identified
in the cprT-cprK and cprK-cprZ intergenic
regions and the promoter at cprB, suggesting a role for
FNR-like CprK in the control of expression of the
cprTKZEBACD genes.
Halorespiring bacteria have received
increasing attention during the last decade, as they are able to couple
the reductive dehalogenation of a large variety of halogenated aromatic
and aliphatic hydrocarbons to energy conservation and hence to
microbial growth. These compounds are present in the environment as a
consequence of their past and present application in industry and
agriculture and because of natural production, compromising
environmental integrity and health (14, 15).
Halorespiring bacteria are believed to play an important role in
the in situ bioremediation of soil and groundwater polluted with
halogenated hydrocarbons. The ability to perform halorespiration
appears to be widespread throughout the Bacteria, as
halorespiring bacteria have been found in the groups of
low-G+C-content gram-positive bacteria, green nonsulfur
bacteria, and In order to understand the molecular basis of this novel respiratory
system, efforts have focused not only on the reductive dehalogenases as
the central enzymes in halorespiration (16, 22, 40) but also
on the identification of additional structural and regulatory
components of the halorespiratory electron transport chain. An
efficient conjugation system has been used for the integration of
conjugative transposon Tn916 into the chromosome of
D. dehalogenans, leading to the isolation of a number
of halorespiration-deficient mutants, which were characterized at the
physiological, biochemical, and genetic levels
(31).
It is known from physiological experiments that halorespiration is
induced by the presence of a halogenated substrate in most halorespiring bacteria described to date. For two
halorespiring strains, Desulfomonile tiedjei and
Desulfitobacterium frappieri TCE1, the influence of
alternative electron acceptors on the activity of the dehalogenating
system has been described, indicating that particularly sulfur
oxyanions are potential inhibitors of halorespiration (12,
35). In contrast, expression of halorespiration by
3-chloro-4-hydroxyphenylacetic acid (Cl-OHPA) in nonacclimated cultures
of D. dehalogenans was not affected by the presence of
equimolar amounts of sulfite (20). However, the level at
which regulation takes place, the control mechanisms involved, and the
inducing signal remain to be elucidated.
This study addresses the molecular analysis of the regulation of
reductive dehalogenation in a halorespiring bacterium.
Chromosomal fragments flanking o-chlorophenol reductive
dehalogenase-encoding gene cprA in D. dehalogenans were cloned and characterized, revealing the
presence of open reading frames that encode polypeptides possibly involved in regulation and maturation of the dehalogenating
system. The expression of the different genes identified in the
cpr gene cluster was studied under various growth conditions
and was found to be tightly controlled at the transcriptional level.
Materials.
Cl-OHPA was purchased from Sigma-Aldrich
Chemie (Zwijndrecht, The Netherlands) and filtered prior to
use. All gases were obtained from Hoek Loos (Schiedam, The
Netherlands). When appropriate, experiments were carried out in an
anaerobic glove box (Coy Laboratories Products, Grass Lake, Mich.)
under an atmosphere of 96% N2 and 4% H2. The
oxygen concentration was kept low with the palladium catalyst RO-20,
provided by BASF (Arnhem, The Netherlands).
Bacterial strains, plasmids, and growth and induction conditions.
D. dehalogenans strain JW/IU-DC1 (DSM 9161) (38)
was routinely grown under anaerobic conditions (100% N2
gas phase) at 37°C in rubber-stoppered serum bottles containing basal
mineral medium, as described by Neumann et al. (21),
supplemented with 0.1% peptone, 30 mM NaHCO3, and trace
elements and vitamin solution as recommended by the German Collection
of Microorganisms and Cell Cultures (Braunschweig, Germany). An
electron donor and acceptor were added to a concentration of 20 mM from
anaerobic stock solutions. Growth was monitored spectrophotometrically
by determining the optical density at 600 nm
(A600). The concentrations of Cl-OHPA and OHPA
during growth in the presence of Cl-OHPA as the electron acceptor were
determined by high-performance liquid chromatography analysis on a
SpectraSystem high-performance liquid chromatograph, with a
SpectraSystem P2000 pump, an AS3000 autosampler, and a UV1000 UV
detector (ThermoQuest, Austin, Tex.). The sample (20 µl) was injected
into a pesticide reversed-phase column (Chrompack, Middelburg, The
Netherlands). The mobile phase was acetonitrile-0.01 M
H3PO4 (10:90 [vol/vol]). A flow rate of 1 ml
min
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Transcriptional Regulation of the cpr Gene Cluster in
ortho-Chlorophenol-Respiring Desulfitobacterium
dehalogenans
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
and 
proteobacteria (9). Among
these, the gram-positive genus
Desulfitobacterium comprises a major group of
isolates. The versatile Desulfitobacterium
dehalogenans has been isolated because of its ability to use
o-halogenated phenolic compounds as terminal electron
acceptors in an anaerobic respiratory chain with lactate, pyruvate,
formate, and molecular hydrogen as electron donors (38).
Recently, the reductive dehalogenation of tetrachloroethene and
hydroxylated polychlorinated biphenyls by the halorespirational system
of D. dehalogenans has been reported (42).
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
1 was applied, and Cl-OHPA and OHPA were quantified by
their absorption at 206 nm. For the induction with Cl-OHPA, cells were
grown with pyruvate to an A600 of 0.1 and
supplemented with 5 mM Cl-OHPA. Samples were taken before and after
induction and stored on ice prior to further processing.
DNA isolation and manipulation.
Chromosomal DNA of D. dehalogenans was isolated as described previously (40).
Plasmid DNA was isolated from E. coli by using the alkaline
lysis method, and standard DNA manipulations were performed according
to established procedures (28) and manufacturers' instructions. Enzymes were purchased from Life Technologies B.V. (Breda, The Netherlands), Roche Molecular Biochemicals (Mannheim, Germany), and New England Biolabs (Beverly, Mass.). Oligonucleotides were obtained from Eurogentec (Seraing, Belgium), Life Technologies B.V., and MWG Biotech (Ebersberg, Germany). PCR products were purified
prior to subsequent manipulation using the QIAquick PCR purification
kit (Qiagen GmbH, Hilden, Germany). A Hybond-N+ nylon transfer membrane
(Amersham Pharmacia Biotech) was used for Southern blot analysis.
Probes for hybridization experiments were labeled by nick translation
in the presence of [
-32P]dATP (Amersham Pharmacia Biotech).
Sequence analysis of the cpr gene cluster.
In
order to extend the sequence downstream of the cprA locus,
as it was determined previously from pLUW910 and pLUW913
(40) (Fig. 1), a 0.9-kb
HincII-EcoRI restriction fragment of pLUW910 was
subcloned in pUC19, yielding pLUW910EH2, and
sequenced. Subsequently, Southern blot analysis of
HincII-HindIII-digested chromosomal DNA of
D. dehalogenans revealed a 1.9-kb fragment that strongly hybridized with the aforementioned radiolabeled 0.9-kb fragment. The
1.9-kb fragment was cloned in E. coli using
HincII-HindIII-digested pUC19, resulting in
pLUW911. pLUW916 was obtained by inverse PCR (36), which was
performed as described previously (40) from NcoI-digested and self-ligated chromosomal DNA of
D. dehalogenans with the divergent primer pair BG580
and BG581 (BG580, positions 9023 to 9002, and BG581, positions 9671 to
9692, of the cpr gene cluster) (see below; Fig. 1). The
resulting 1.8-kb PCR product was cloned in E. coli using
XcmI-digested pMON38201. Subsequently, PCR was performed
with chromosomal DNA of D. dehalogenans with primers BG581
and HS22 (positions 10260 to 10240) and HS23 and HS27 (HS23, positions
10237 to 10257, and HS27, positions 11117 to 11097). Both PCR
products were cloned in E. coli using
XcmI-digested pMON38201, yielding pLUW921 and pLUW922,
respectively.
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Amplification, cloning, and sequencing of rRNA genes. The 16S rRNA-encoding gene was amplified from chromosomal DNA of D. dehalogenans with the universal primer pair 7f-1510r (19). Primers 1492f (19) and 23InsR (24) were used for the amplification of the 3' and 5' ends of the 16S and 23S rRNA genes, respectively, and the 16S-23S intergenic spacer. PCR products were cloned in the pGEM-T vector, yielding pLUW900 (16S) and pLUW901 (16S-23S), and their authenticity was verified by nucleotide sequence analysis.
DNA sequencing and DNA and protein sequence analysis. DNA sequencing was performed using DNA sequencer 4000L (LiCor, Lincoln, Nebr.). Plasmid DNA used for sequencing reactions was purified with the QIAprep Spin Miniprep kit (Qiagen GmbH). Reactions were performed using the Thermo Sequenase fluorescently labeled primer cycle sequencing kit (Amersham Pharmacia Biotech). Fluorescently (IRD 800) labeled universal sequencing primers were purchased from MWG Biotech. Sequence similarity searches and alignments were performed using the BLAST, version 2.0, program (1) (National Center for Biotechnology Information, Bethesda, Md.) and the programs Clustal X, GeneDoc (34; K. B. Nicholas and H. B. J. Nicholas, GeneDoc: a tool for editing multiple sequence alignments, 1997), and the DNAstar package (DNASTAR Inc., Madison, Wis.). Protein secondary structure and helical transmembrane region predictions were made with the profile network systems PHDsec and PHDhtm (25-27), respectively. Prediction of helix-turn-helix (H-T-H) DNA-binding motifs was made using the method of Dodd and Egan (8).
Isolation of total RNA, Northern analysis, RT-PCR, and primer extension. Total RNA was isolated from exponentially growing cultures of D. dehalogenans by the Macaloid method described by Kuipers et al. (18). For Northern blot analysis, 7 µg of RNA was separated on a formaldehyde-1% agarose gel, transferred to a Hybond-N+ nylon transfer membrane (Amersham Pharmacia Biotech) by downwards capillary transfer as described by Chomczynski (5) with 5× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-10 mM NaOH as the transfer liquid, and immobilized by UV cross-linking. Prehybridization and hybridization were performed in ULTRAhyb hybridization buffer (Ambion, Austin, Tex.) as recommended by the manufacturer.
A cprA-specific probe was generated by PCR amplification from chromosomal DNA of D. dehalogenans with the primer pair BG475 and BG476 (BG475, positions 6778 to 6797; BG476, positions 7213 to 7192). As probes specific for cprC, cprD, and cprE, PCR products that were obtained after PCR amplification with primer pairs BG619 and BG620 (BG619, positions 7632 to 7658, and BG620, positions 8282 to 8255), BG581 and HS22, and BG624 and BG577 (BG624, positions 4147 to 4171), respectively, were used. For the detection of transcription products of ORFU, cprT, cprK, and cprZE, probes were generated by PCR with primer pairs HS29 and HS30, HS24 and HS28, HS31 and HS32 (HS31, positions 2906 to 2927, and HS32, positions 3440 to 3419), and HS25 and BG577, respectively. Reverse transcriptase PCR (RT-PCR) was performed to analyze the transcriptional organization of the genes in the cpr gene cluster. Five hundred nanograms of DNase-treated RNA (RQ1 RNase-free DNase; Promega) was used in a 25-µl reaction mixture containing the following: 25 pmol of each primer, 200 µM dATP, dCTP, dGTP, and dTTP, 1.7 mM MgSO4, 5 µl of avian myeloblastosis virus (AMV)-Tfl 5× reaction buffer, and 2.5 U of AMV RT and Tfl polymerase (Access RT-PCR system; Promega). In negative controls, AMV RT was omitted, and chromosomal DNA of D. dehalogenans was added to positive-control reaction mixtures. cDNA synthesis and subsequent PCR amplification were performed using the GeneAmp PCR system 9700 (Perkin-Elmer Cetus, Norwalk, Conn.). The reaction mixture was incubated at 48°C for 45 min. After the mixture was preheated to 94°C for 2 min, 40 amplification cycles, consisting of denaturation at 94°C for 20 s, primer annealing at 50°C for 30 s, and elongation at 68°C for 1 min 30 s, were performed. A final extension of 7 min at 68°C was performed. Primer extension analysis was performed to determine the transcription start sites of the cprBA and cprCD transcripts. For this purpose, 10 or 30 µg of RNA and 4 pmol of the fluorescently (IRD 800) labeled oligonucleotides BG600 (positions 5670 to 5648) and HS26 (positions 7476 to 7456), respectively, were dissolved in 10 µl of 1× RT buffer (50 mM Tris-HCl [pH 8.3], 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol), incubated at 70°C for 5 min, and slowly cooled to room temperature. After addition of 10 µl of 1× RT buffer containing 2 mM dATP, dCTP, dGTP, and dTTP, 10 U of RNasin, and 200 U of Superscript II RT (Life Technologies), the sample was incubated at 48°C for 30 min. RNase (0.2 mg/ml) was added, and the sample was precipitated with ethanol and washed once with 70% ethanol. The pellet was dried, dissolved in 2 µl of formamide loading buffer, and separated on a Li-Cor 4000L DNA sequencer.Nucleotide sequence accession number. The nucleotide sequence of the cpr gene cluster described here has been deposited in the GenBank database under accession no. AF115542.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Genetic organization of the cpr gene cluster.
Previously we analyzed the nucleotide sequence of the cprBA
genes that encode the catalytic subunit and putative membrane anchor of
the o-chlorophenol reductive dehalogenase in D. dehalogenans (40). Here we report the transcriptional
organization of the cprBA genes. The chromosomal regions
flanking the cluster were characterized by sequence and transcriptional
analysis, revealing the presence of six additional transcribed genes,
tentatively designated cprC, cprD,
cprE, cprK, cprT, and cprZ,
and one untranscribed open reading frame, ORFU (Fig. 1; see below).
With the exception of cprT, all genes are transcribed in the
same direction as cprBA. In front of each of the genes
potential Shine-Dalgarno sequences are present; these sequences are
complementary to the 3' end of the D. dehalogenans 16S rRNA
(3'-AGAAUCUUUCCUCCA-5'; see below). The cprC
gene, previously designated orf1 (40), is located downstream of the structural gene for the o-chlorophenol reductive
dehalogenase (cprA) and is predicted to encode a polypeptide
of 395 amino acids with a molecular mass of 43,867 Da. Secondary
structure prediction suggests the presence of six transmembrane helices
(Fig. 2). Within the C-terminal
cytoplasmic domains, two cysteine-rich signatures of the type
CXXXCP were identified. For the C-terminal part of the
predicted protein, CprC, significant similarity was observed with
membrane-bound regulators of the NosR/NirI type, which have been shown
to play a role in a signal transduction pathway that eventually
controls the transcription of the nitrous oxide (nos) and
nitrite reductase (nir) gene clusters of Pseudomonas
stutzeri and Paracoccus denitrificans, respectively
(7, 30). This similarity was most pronounced in the vicinity
of the cysteine-rich motifs (Fig. 2). However, CprC is significantly
smaller than known proteins of the NosR/NirI family, due to a much
shorter N-terminal extracytoplasmic loop (approximately 170 amino acids
compared to 350) and the lack of a C-terminal cytoplasmic domain
containing two additional ferredoxin-like motifs binding either
[2Fe-2S] or [4Fe-4S] clusters in NosR and NirI (4).
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Transcriptional analysis of the cpr gene cluster.
The observation that the cprBA genes for the
o-chlorophenol reductive dehalogenase are flanked by five
genes that could encode proteins which can be expected to play a role
in regulation, maturation, or action of CprA prompted us to investigate
the transcription of these genes under different conditions. Northern
blot analysis was performed on total RNA isolated from cells grown with
pyruvate as the electron donor and either Cl-OHPA, fumarate, nitrate,
or pyruvate as the electron acceptor. The sizes of the transcripts were
estimated by comparison with RNA molecular weight markers. Hybridization with a 32P-labeled cprA-specific
probe revealed the presence of two transcripts of approximately 1.7 and
5.2 kb, which were solely detectable in RNA isolated from cells grown
by halorespiration (Fig. 4). The major
hybridizing transcript of approximately 1.7 kb indicated cotranscription of the structural gene cprA with
cprB, as was anticipated because of the fact that both genes
are only 12 nucleotides apart (40) (Fig. 4). Hybridization
with probes specific for genes downstream of cprA unveiled
transcripts of approximately 2.4 and 5.2 kb for cprC and
0.7, 2.4, and 5.2 kb for cprD. All transcripts were observed
only in RNA obtained from cells grown with Cl-OHPA as the electron
acceptor (Fig. 4). The presence of a large transcript of about 5.2 kb
hybridizing with probes specific for cprA, cprC,
and cprD and its concentration relative to that of the major
1.7-kb cprBA transcript, indicate occasional read-through after cprA. This would imply that the cprBACD
genes are cotranscribed from the promoter preceding cprB
(see below). The smaller transcripts (2.4 and 0.7 kb) detected with the
cprC- and cprD-specific probes could be products
of posttranscriptional processing of either the large 5.2-kb
polycistronic cprBACD mRNA or a 3-kb biscistronic cprCD transcript transcribed from a promoter preceding
cprC. Hybridization with probes specific for
cprE, cprZE, and cprT indicated a
bicistronic transcript of cprE and cprZ and
monocistronic transcription of cprT specifically induced
under halorespiring conditions (Fig. 4). A small 0.5-kb
transcript that was detected with the cprZE-specific probe
in RNA from cells grown on pyruvate, fumarate, or nitrate as the
electron acceptor was absent from halorespiring
cells, indicating that halorespiration induced read-through
between cprZ and cprE. A transcript of
approximately 2.6 kb could be detected with a probe specific for
cprK, which was constitutively produced at very low
levels. The same transcript was also detected with probes specific for
cprE and cprZE, indicating constitutive
transcription of a tricistronic cprKZE transcript (Fig. 4).
Transcription of ORFU could not be detected by Northern blot analysis.
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Transcription initiation from putative cprB and
cprC promoters.
The results obtained by Northern blot
analysis and RT-PCR indicated transcription initiation under
halorespiring conditions from a promoter preceding
cprB. The start site of Cl-OHPA-specific transcription from
the cprB promoter could be identified 43 nucleotides upstream of the translation start site by primer extension using total
RNA extracted from cells of D. dehalogenans grown by
halorespiration with pyruvate as the electron donor and Cl-OHPA as the
electron acceptor (Fig. 5A). No primer
extension product was obtained with RNA isolated from cells grown by
pyruvate fermentation. Upstream of the transcription initiation site,
the consensus sequence for a 
10 region could be detected, but no
consensus 35 region was observed (Fig.
6A). Northern blot analysis also
suggested posttranscriptional processing of a polycistronic
mRNA or another transcription initiation at a site preceding
cprC. A halorespiration-specific primer extension product
indicated that this site is localized 58 nucleotides upstream of the
translation start of cprC (Fig. 5B).
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Kinetics of induction of cprBA operon expression.
To investigate the induction kinetics of cprBA expression
under halorespiring conditions, cells grown with pyruvate
as the sole carbon and energy source were amended with Cl-OHPA during exponential growth and cprBA transcription and
dechlorination of Cl-OHPA to OHPA were determined. Normalized by
comparison to the 16S rRNA levels, transcription of cprBA
was already induced 15-fold 30 min after induction, whereas significant
amounts of the dechlorination product, OHPA (5.8% of Cl-OHPA
added), were detected after 2 h (Fig.
7). Considering a specific growth rate of
approximately 0.2 h1 (generation time
[tD] 
3 h), the massive induction of
halorespiration-specific transcription within 0.15 × tD is remarkably fast. Maximal induction of 18-fold was observed 3 h after addition of Cl-OHPA.
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). The unusual ratio obtained
after 2 h of induction may be due to degradation of the
cprBA transcript compared to the 16S rRNA. Concentrations of
Cl-OHPA and OHPA were determined by reverse-phase high-performance
liquid chromatography analysis. The degree of conversion of Cl-OHPA to
OHPA was calculated as [OHPA] × 100%/([Cl-OHPA] + [OHPA])
(
).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Halorespiring bacteria have been demonstrated to be actively involved in the reductive dehalogenation of chlorinated aliphatic and aromatic compounds of natural and anthropogenic origin. Hence they contribute significantly to the detoxification of these contaminants in the environment (10). The reductively dehalogenating enzymes from a limited number of halorespiring bacteria have been characterized at the biochemical and genetic levels, indicating similar modes of catalytic action (16). However, insight into the regulatory circuits involved in the induction and repression of the halorespiration process is still very limited. For the first time, we here present a molecular analysis of the control of halorespiration-specific gene expression.
We have cloned and sequenced extended chromosomal fragments up- and downstream of the ortho-chlorophenol reductive dehalogenase-encoding cprBA gene cluster in halorespiring D. dehalogenans. Previously, we showed that cprA codes for a preprotein containing a twin-arginine (RR) signal sequence (40). This signal peptide is cleaved off in the mature protein and is thought to play a major role in the maturation and translocation of mainly periplasmic proteins binding different redox cofactors by the recently described twin-arginine translocation system (2). Putative functions of the newly detected open reading frames could in most cases be assigned by the similarity of the products to proteins present in the databases. CprC and CprK are potentially involved in regulation of transcription at different levels, whereas CprD, CprE, and CprT have significant similarity to molecular chaperones involved in the correct folding, processing, and assembly of proteins. However, no function could be assigned to the predicted gene products of ORFU and cprZ.
CprD and CprE belong to the family of GroEL chaperonins, which are found in prokaryotes, chloroplasts, and mitochondria. GroEL chaperonins are tetradecameric proteins that are involved in preventing protein aggregation and facilitating protein folding and assembly (11). In addition, it has been suggested that accessory proteins such as GroEL might play a role in correct assembly and cofactor insertion during the maturation of RR signal peptide-containing proteins, such as the reductive dehalogenases from halorespiring bacteria (2, 29). CprT has significant similarity to the trigger factor, a prolyl peptidyl isomerase that catalyzes proline cis-trans isomerization, a potential rate-limiting step in protein folding (11). Studies on the trigger factor from E. coli showed its association with nascent polypeptide chains and the 50S ribosome, suggesting a role in protein folding (33, 39). Interestingly, complexes between trigger factor and GroEL formed in vivo have been reported to have a much higher affinity for partially folded polypeptides than GroEL alone (17). Moreover, overexpression of trigger factor together with GroEL and GroES significantly improved the solubility of recombinant proteins, normally prone to aggregation into inclusion bodies (23). Unlike known trigger factors, CprT lacks part of the N-terminal domain, which is noncatalytic but which appears to be required for full activity in protein folding (43). Nonetheless, the specific coordinated expression of cprD, cprE, and cprT under halorespiring conditions suggests a synergistic role of these molecular chaperones in the maturation of the dehalogenating complex.
Northern blot analysis and RT-PCR revealed that the transcription of almost all genes identified in the cpr gene cluster is induced under halorespiring growth conditions, whereas no or significantly less-abundant transcription was observed under pyruvate-fermenting and fumarate- or nitrate-respiring conditions. We could reveal the transcriptional organization of the locus, i.e., two biscistronic units, cprZE and cprBA, with occasional read-through at cprC, yielding expression of the polycistronic cprBACD genes. Possibly, transcription of a third biscistronic unit, cprCD, might be initiated at a promoter preceding cprC. cprT, encoding a trigger factor, is obviously transcribed into a monocistronic mRNA (Fig. 4). Low-level constitutive transcription under all tested conditions was solely observed for tricistronic transcript cprKZE. Transcription from the cprB promoter was strongly induced within 30 min upon the addition of Cl-OHPA to cells growing by fermentation of pyruvate with concomitant dehalogenation of Cl-OHPA to OHPA, indicating o-chlorophenol reductive dehalogenase activity. This is in agreement with the earlier result that dehalogenation cannot be induced in the presence of chloramphenicol, showing that activation of dehalogenation requires de novo protein synthesis (37).
Sequence analysis revealed the presence of gene cprK,
constitutively expressed at a low level, encoding a potential
transcription regulatory protein. The observed tight control of the
expression of the structural and putative accessory cpr
genes might imply a direct involvement of CprK in the
functionality of the D. dehalogenans halorespirational
system. CprK has significant similarity to FNR- and FixK-like
regulators, which are important trans-acting factors in
regulatory networks of anaerobic assimilation and dissimilation. Like
FixK, CprK lacks the N-terminal cysteine cluster, a characteristic of
FNR, which is involved in the binding of an Fe/S center, and as such in
redox sensing (44). However, CprK does show an unusually high content of five cysteine residues, among which is the conserved internal cysteine residue Cys105. In the E. coli
FNR protein, the corresponding Cys122 has been shown to be
essential for Fe binding, disulfide bond formation, and covalent
modification (13). The FNR- and FixK-like regulatory
proteins share a common conserved DNA-binding motif in their C-terminal
recognition helices, which is complementary to a palindromic
recognition motif, the so-called FNR or anaerobox, in the promoter of
target genes (TTGAT-N4-ATCAA) (32).
In positively regulated promoters, this FNR binding motif is
preferentially centered at a distance of 41.5 nucleotides upstream of
the transcription start (32). Inspection of the mapped
halorespiration-inducible cprB promoter showed that it lacks
the 35 consensus motif of strong constitutive promoters but does
contain an anaerobox-like palindromic structure (I-1;
TTAAT-N4-ACTAA). This putative regulatory protein binding motif is centered 41.5 nucleotides upstream of the
apparent transcription start, suggesting positive regulation of
transcription by an FNR-like factor (Fig. 6A). Another interesting feature of the cprB promoter is the presence and position of
an additional long inverted repeat (I-3) that overlaps with the
transcription start site, suggesting a function in control of
transcription initiation. Northern blot analysis revealed that
expression from putative promoters preceding cprT and
cprZ was also stimulated under halorespiring
conditions. FNR box-like motifs centered 87.5 and 77.5 bp upstream of
cprT and cprZ, respectively (I-7 and I-9; Fig. 6B
and C), could be identified. Moreover, in both cases conserved 10
consensus motifs were found at the same distance (19 bp) downstream of
the FNR box-like motifs as in the mapped cprB promoter (Fig. 6B, C, and E). Both the lower degree of conservation of the proposed FNR box-like motif at cprC (Fig. 6D and E) and the small
difference in spacing to the transcription start (45.5 bp instead of
41.5 bp) and a less well conserved 10 motif favor the idea that the mapped start site of the cprCD mRNA is the site of
processing of the larger tetracistronic unit cprBACD rather
than of transcription initiation.
In conclusion, it is tempting to speculate that the FNR homologue CprK is the factor binding to the different anaerobox-like motifs preceding at least the three halorespiration-inducible promoters present in the cpr gene cluster. An alignment of the identified motifs revealed a consensus sequence for all analyzed promoters that only slightly differs from that of the FNR box (Fig. 6E), which could reflect corresponding differences in the recognition helix of CprK (Fig. 3). CprK might be activated by the addition of a halogenated substrate, either directly or via an o-chlorophenol sensor, such as the two-component regulatory system that was previously detected from the detailed analysis of halorespiration-deficient mutants (31). If so, active CprK then induces transcription from the apparent cprB promoter and the putative cprT and cprZ promoters, generating the set of polypeptides required to obtain a functional dehalogenating complex. Such a model suggests a regulatory loop similar to that which has been proposed recently for nitrite reductase- and nitrous oxide reductase-encoding gene clusters from denitrifying bacteria. There, the expression of both the structural genes, nirS and nosZ, and of the membrane-bound regulator, encoded by nirI and nosR, is under the control of FNR-like regulatory proteins NNR and FnrD, respectively (6, 30). However, the exact function and mode of action of the NirI, NosR, and CprC regulatory proteins remain unknown. Interestingly, the analysis of the partially available genome sequence of the halorespiring Dehalococcoides ethenogenes (preliminary sequence data were obtained from The Institute for Genomic Research website at http://www.tigr.org) has revealed the occasionally close linkage of reductive dehalogenase-encoding genes with cprC and cprK homologues, as well as with genes potentially coding for two-component regulatory systems. This might serve as an additional, although only indirect, indication for the involvement of such regulatory proteins in the regulation of expression of reductive dehalogenases.
The molecular analysis of the cpr gene cluster reported here for the first time provides insight into the molecular bases of regulation and maturation of the halorespiratory system and suggests regulatory circuits which are similar to those proposed for respiratory complexes present in denitrifying bacteria. The remarkably fast induction of halorespiration-specific gene expression and its relative insensitivity to the presence of alternative electron acceptors (20; Smidt et al., unpublished results) indicate the potential of D. dehalogenans as a dedicated degrader in contaminated environments.
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ACKNOWLEDGMENTS |
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This work was partly supported by a grant of the Studienstiftung des Deutschen Volkes and contract BIO4-98-0303 of the European Union.
Sequencing of Dehalococcoides ethenogenes was accomplished with support from the DOE Microbial Genome Program.
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FOOTNOTES |
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* Corresponding author. Mailing address: Laboratory of Microbiology, Wageningen University, Hesselink van Suchtelenweg 4, NL-6703 CT Wageningen, The Netherlands. Phone: 31-(0)-317483118. Fax: 31-(0)-317483829. E-mail: hauke.smidt{at}algemeen.micr.wag-ur.nl.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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