From the Department of Pharmacology and Toxicology, School of Medicine, Philipps University of Marburg, Karl-von-Frisch-Strasse 1, D-35033 Marburg, Germany
Received for publication, December 5, 2000
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ABSTRACT |
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The Comamonas testosteroni
3 Comamonas (formerly Pseudomonas)
testosteroni is a Gram-negative bacterium found in soil,
mud, and water, but it has also been isolated from the gastrointestinal
tract in humans (1, 2). Interestingly, C. testosteroni can
use a variety of steroid substrates as a sole carbon source through the
reaction of a set of steroid-inducible enzymes (3, 4). Although the
metabolic intermediates of the steroid substrates have been identified, and the basic catabolic pathway is known, only limited information is
available on the molecular structure and function of the participating enzymes (5-11). Moreover, nothing was known about the mechanism of
induction of steroid-metabolizing enzymes or about the basis of their
transcriptional regulation.
The occurrence of several hydroxysteroid dehydrogenases
(HSDs)1 in microbial
organisms raises the question of the physiological function of these
enzymes in procaryotes. Because steroids simultaneously serve both as
signal molecules and as a carbon source, they play a particularly
important role in certain procaryotes. The same applies to procaryotic
HSDs. On the one hand, procaryotic HSDs may play a regulatory role in
the steroid-inducible gene expression. On the other hand, it is
established that they participate in steroid degradation.
Enzymatic C3-dehydrogenation of ring A, which occurs at the initial
stage of steroid catabolism in C. testosteroni, is mediated by 3 In previous investigations, we isolated and characterized the 3 The present paper provides the first data on the molecular mechanism of
steroid signaling in procaryotes. It reports on the regulation of the
steroid-inducible gene (hsdA) of 3 Bacterial Strains and Plasmids--
Host strains
Escherichia coli HB101 (Promega) and C. testosteroni ATCC 11996 (Deutsche Sammlung für
Mikroorganismen) were used for gene cloning and expression. Subcloning
of fragments was carried out in plasmids pUC18 and pUC19 (Life
Technologies, Inc.), pKK232-8 (Amersham Pharmacia Biotech)
containing the cat gene, and pBBR1MCS-2 (27). Of special
importance was the broad host range cloning vector pBBR1MCS-2, which
contains the kanamycin resistance gene and which was found in our
investigations to be able to replicate not only in E. coli
but also in C. testosteroni ATCC 11996. pK18, which contains
the kanamycin resistance gene, was a gift obtained from CIBA-Geigy AG,
Department of Biotechnology, Basel. The plasmid copy numbers determined
were as follows: 80 copies per cell of pUC18 and pUC19 in E. coli, 10 copies per cell of pKK232-8 in E. coli, 6 copies per cell of pBBR1MCS-2 in both E. coli and C. testosteroni, 80 copies per cell of pK18 in E. coli.
Copy numbers remained unchanged, notwithstanding the size of the
inserted fragments.
Growth Media and Growth Conditions--
Bacterial cells were
grown in standard I nutrient broth medium (Merck) or standard LB
medium at 37 °C (E. coli) or at 30 °C (C. testosteroni).
Restriction Enzymes and Other Reagents--
Restriction enzymes,
ligase, shrimp alkaline phosphatase, and S1 nuclease were
obtained from Roche Molecular Biochemicals, Biolabs, MBI
Fermentas, and Amersham Pharmacia Biotech, and used according to
the manufacturers' instructions. Ampicillin and kanamycin antibiotics
were from AGS (Heidelberg, Germany).
DNA Manipulations--
Recombinant DNA work was carried out
following standard techniques according to Sambrook et al.
(28).
Subcloning of the C. testosteroni hsdA Regulatory
Region--
Subcloning of the C. testosteroni hsdA
regulatory region and the preparation of respective plasmids are shown
in Fig. 1. The 5.257-kb EcoRI fragment, which has been
isolated from C. testosteroni chromosomal DNA in previous
investigations (25), was cloned into pUC18 to get p6. p6 was digested
with HincII and XmaI, and the resulting 2.979- and 2.937-kb fragments were ligated again into pUC18 to give pH2 and
pX12, respectively. AvrII (restriction site on the p6
C. testosteroni insert upstream of hsdA) and
XbaI (restriction site in pUC18) were used for double
digestion of p6 and ligated to yield pAX1. For transforming C. testosteroni with the same fragments, HincII,
XmaI, AvrII, and XbaI restriction enzymes were used again to subclone respective inserts from p6 into
pBBR1MCS-2, to obtain pBB21B, pBBH31, pBBX1, and pBBAX7. The
AvrII/XbaI-EcoRI fragment was also
cloned into pK18 to get pKAX1. The 2.708-kb
EcoRI-ClaI fragment from p6 was cloned into pBBR1MCS-2, and a corresponding 2.741-kb
SalI-ClaI fragment was cloned into
pKK232-8, to generate pBB7 and pBKB12, respectively. The same cloning
strategies as described above were performed to generate pBKH1, pBKX8,
and pBKAX1, which represent descendants from pKK232-8 containing the
HincII-ClaI (0.430 kb),
XmaI-ClaI (0.388 kb), and
AvrII/XbaI-ClaI (0.185 kb) fragments,
respectively, from p6 upstream of the cat gene. For getting
deleted pBBR1MCS-2/CAT reporter constructs, the cat gene
fragment from pKK232-8 was first subcloned with BamHI and
AviII into pBBR1MCS-2 to yield pBBK1 (data not
shown). A 2.735-kb XbaI-ClaI fragment from p6 was
then ligated to pBBK1 upstream of cat to receive pBBK4.
Similarly, constructs of the HincII-ClaI,
XmaI-ClaI, and
AvrII/XbaI-ClaI fragments plus the
cat gene yielded pBBKH13, pBBKX7, and pBBKAX1.
Subcloning of the hsdA Repressor Gene--
Subcloning of the
C. testosteroni hsdA repressor gene and the preparation of
respective plasmids are also shown in Fig. 1. A 2.451-kb
KpnI-NdeI fragment of p6 was cloned into either
pUC18 or pUC19 to yield pKpN6 or pKpN7, respectively. Plasmids pKAN10 and pKAN12 (containing a 1.030-kb insert) were obtained by excising a
1.421-kb KpnI-AvrII fragment from pKpN6 or pKpN7, respectively.
Transformation of Bacteria--
All constructs were verified by
restriction enzyme digestions of the isolated plasmids. Plasmids were
purified with the Qiagen Tip-100 kit (Qiagen, Hilden, Germany).
Ligations were transferred into E. coli or C. testosteroni. Competent cells were prepared by the
CaCl2 or electroporation methods. Double plasmid
transformations were performed by exploiting the kanamycin resistance
gene of pK18 and pBBR1MCS-2 and the ampicillin resistance gene
of pUC18, pUC19, and pKK232-8. In these cases both antibiotics were
added to the culture medium. Plasmid isolation and agarose gel
electrophoresis were performed to prove successful double transformations.
Construction of Mutant Promoters (pM) by Polymerase Chain
Reactions--
The mutant promoters (pMx, where x indicates the
plasmid number) were generated by overlap extension polymerase chain
reaction mutagenesis (29). The following set of primers was used (see Table I): pL1 and pR2 for hsdA promoter mutations, pL1 and
pR1 for wild-type hsdA promoter-cat constructs,
pML and pMR for the mutated promoter-cat constructs, and pM0
and pR2 for promoterless hsdA constructs. Mutant plasmids
were then transferred into E. coli HB101, and 3 Primer Extension--
Total RNA was prepared from E. coli HB101, previously transformed with plasmids p6, pX12, or
pAX1, after growth for 6, 9, 12, or 16 h. The 15-mer
oligonucleotide primer pRse (complementary to a sequence within
hsdA and later used as the sequencing primer; see Table
I) was end-labeled with [32P]ATP (Roche Molecular
Biochemicals kit). Primer extension was performed by hybridizing 20,000 cpm-labeled pRse for 20 min at 65 °C with 5 µg of total RNA of
each preparation, followed by 10 min at room temperature. Annealed
primer was extended with avian myeloblastosis virus reverse
transcriptase (Promega) for 1 h at 41 °C. The resulting mixture
was separated on a sequencing gel. As a reference, DNA sequence
reactions of pX12 and pAX1 were performed with the same primer (pRse).
S1 Nuclease Analysis--
A 204-mer oligonucleotide was prepared
from plasmid p6 by polymerase chain reaction with primers pL1 and pR2
(see Table I), agarose gel electrophoresis, and ClaI
digestion. The oligonucleotide was end-labeled with
[32P]ATP and subsequently digested with XbaI
to yield a single-stranded 32P-labeled 185-mer. The
oligonucleotide (50,000 cpm) was mixed in hybridization buffer with 10 µg of total RNA from E. coli HB101, previously transformed
with plasmids p6, pX12, or pAX1, after growth for 6, 9, 12 or 16 h. The mixture was denatured at 85 °C for 10 min and then incubated
at 37 °C for 6 h. The annealed oligonucleotide/mRNA hybrid
was treated with 200 units of S1 nuclease (Roche Molecular Biochemicals) at 37 °C for 1 h, and the resulting products were separated on a sequencing gel. As a reference, DNA sequence reactions of pX12 and pAX1 were performed with the sequencing primer pRse.
Point and Frameshift Mutations of repA--
Point mutations of
both possible ATG start codons (resulting in pRm1 and pRm3) and shift
mutations by insertion of an additional base within the repA
sequence (resulting in pRm2 and pRm4) were generated by two-step
polymerase chain reaction mutagenesis (29) according to standard
procedures. The resulting plasmids were used in cotransformation
experiments together with pAX1, and 3 Specific Binding of Testosterone to RepA--
E. coli
HB101 cells, carrying pKpN6, were grown for 18 h, and total
protein was isolated. Ten µg of protein (10 µl) was mixed with 0.1 µCi of [3H]testosterone at room temperature for 20 min.
Nonlabeled testosterone was used in increasing concentrations as a
specific binding competitor. The samples were washed on nitrocellulose
filters with 0.2 × TEN (10 mM Tris-HCl, 1 mM EDTA, 0.1 M NaCl, pH 8.0) and 0.5% Tween 20 buffer and mixed with a scintillator, and the cpm were determined.
Preparation of 5-Nucleotide and 10-Nucleotide Deleted Operator
Constructs--
Two-step polymerase chain reaction mutagenesis was
used to prepare 5-nucleotide and 10-nucleotide deleted mutants (29). All primers and oligonucleotides were prepared by MWG Biotec. The polymerase chain reaction fragments were cloned into p6 for the
preparation of pL10n (deletion at 0.935 kb) and pL5n (TGGGC deleted on
location 2.568 kb). All clones prepared were sequenced by MWG Biotec.
Mobility Shift Assays--
Three 34-bp double-stranded DNA
fragments (Op1, Op2, and Co) for gel shift experiments with RepA were
prepared by the annealing of six oligonucleotides synthesized by MWG
Biotec. Oligonucleotides (100 pmol) were incubated pairwise in TEN
buffer at 95 °C for 10 min and allowed to cool down slowly to
25 °C in 1.5 h. Finally, 4 pmol of DNA was labeled with
digoxigenin-11-ddUTP. Electrophoresis was performed according to
the instructions in the kit from Roche Molecular Biochemicals. 8%
polyacrylamide was used in gel shift assays. Op1 contained the 10-bp
palindromic motif TCAAAGCCCA at 0.935 kb, Op2 contained the 10-bp
palindromic motif TGGGCTTTGA at 2.568 kb, and Co represented a region
outside the operator motifs and served as a control.
ELISA of 3 Assay of 3 Protein Determination--
Protein was measured by the method of
Lowry et al. (30).
Statistical Analysis--
The results of enzymatic activities
(HPLC) and protein expressions (ELISA) were analyzed by the Student's
t test. Four independent plates of cells were transformed,
and each extract was assayed in duplicate.
Analysis of the Upstream Regulatory Region of hsdA--
As shown
in Fig. 1, plasmids p6, pH2, pX12, and
pAX1, which are derived from pUC18, were transferred into E. coli strain HB101. Plasmids pBB21B, pBBH31, pBBX1, and pBBAX7,
which represent descendants of pBBR1MCS-2, were successfully
transferred into C. testosteroni. Subsequent expression of
3
Fig. 2 also shows that, compared with transformations with pH2, pBBH31,
pX12, pBBX1, pAX1, and pBBAX7, 3 Mapping of the Transcription Start Site--
The transcription
start site of hsdA was determined by primer extension
analysis and confirmed by S1 nuclease protection assay (Fig.
3). Total RNA from E. coli
HB101 transformed with p6, pX12, or pAX1 was obtained after growth for
6, 9, 12, or 16 h. For the primer extension reaction (Fig.
3A) the sequencing primer pRse (Table
I), complementary to a region within the
hsdA coding region (positions +93 to +78), was used, and a
single band representing the 5'-end was determined. For the S1 nuclease
protection assay (Fig. 3A), a 32P-labeled
antisense DNA probe that extended from the AvrII site (at
2.532 kb) to the ClaI site (at 2.708 kb) on the entire
EcoRI C. testosteroni fragment of plasmid p6 was
used together with total RNA as described above. The 5'-end was thus
localized 28 bp upstream of the ATG codon. The 5'-end (marked by an
arrow in Fig. 3B) is preceded by a promoter
sequence ( Point Mutations for Determination of the Promoter
Sequence--
Site-specific mutations in the
As controls, two mutations located 2 and 3 bp upstream of the
These experiments identified the promoter region of hsdA and
demonstrate that the Identification of a Gene Coding for a Negative Regulator of hsdA
Expression--
As is the case with many negatively regulated enzymes,
the coding region of a putative repressor protein was suspected to lie
adjacent to hsdA. To test this hypothesis, the upstream and downstream regions of hsdA were searched for potential
repressor genes in separate experiments. First, the 2.708-kb
EcoRI-ClaI upstream fragment of hsdA
was subcloned into pBBR1MCS-2 to generate pBB7 (Fig. 1). Levels of
3
To search for the coding region of the repressor protein
downstream of hsdA, we fused the
EcoRI-ClaI fragment from p6 (lacking hsdA) to the cat gene in pKK232-8 (Fig. 1).
pKK232-8 (control) and the resulting plasmid descendants pBKB12,
pBKH1, pBKX8, and pBKAX1 were each double-transformed into E. coli with the second plasmids pBBR1MCS-2 (control), pBB7, pBBAX7,
and pBB21B in medium containing ampicillin and kanamycin. As has
already been found with 3
To define the exact coding region of the repressor protein, several
plasmids containing deleted constructs of the EcoRI fragment downstream of the ClaI site were prepared. Cotransformation
experiments with pKpN6 and pAX1 suggest that the repressor gene is
present between the KpnI (at 1.111 kb) and NdeI
(at 3.562 kb) restriction sites on the EcoRI fragment (Fig.
4). The presence of an open reading frame
(orf 4) between 2.065 kb and 3.328 kb gave rise to the
possibility that orf 4 is the coding gene for the repressor protein, which we named RepA. This was confirmed by cotransformation experiments with pKpN7, which contains the lacZ promoter
upstream of orf 4 (Fig. 1) and which did not affect
hsdA expression (Fig. 4). To specify the active region of
RepA, plasmids pKAN10 and pKAN12 were generated; both plasmids code for
a C-terminal truncated form of RepA but differ in their lacZ
orientation (Fig. 1). The resulting hsdA expression data
(Fig. 4) proved that RepA is encoded by orf 4, which has an
opposite orientation to hsdA and which, interestingly
enough, overlaps with the hsdA sequence. Obviously, the
N-terminal half of RepA contains the repressor active region.
RepA Acts as Repressor of hsdA--
Further verification for RepA
acting as negative regulator of hsdA was achieved by point
and frameshift mutations generated within the RepA gene
(repA) (Fig. 5). Two possible
translation start sites are found within the repA sequence
at 2.929 and 3.328 kb. Point mutations of both ATGs to GTGs (pRm1 and
pRm3) decreased the inhibitory effect of RepA on hsdA
expression compared with respective controls (pKpN6) with full RepA
activity and low hsdA expression (Fig. 5). The generation of
a frameshift mutation by insertion of an additional base after the
putative start codons of repA (pRm2 and pRm4) led, in both
cases, to a complete loss of RepA activity and full hsdA
expression. These results indicate the translation start point of
repA to be located at 3.328 kb. Moreover, the fact that a
frameshift mutation within repA provides full
hsdA expression proves that RepA acts as a repressor protein and excludes the possibility of an antisense RNA effect.
The repA gene is 1.263 kb long, and the deduced amino
acid sequence comprises 420 amino acid residues with a calculated
molecular mass of 45.4 kDa. A homology search of the predicted primary
structure of RepA against protein data bases failed to detect
significant identities to any other known protein.
Binding of Testosterone to RepA--
The binding of testosterone
to RepA was tested by mixing protein extracts from E. coli
HB101 cells, carrying pKpN6 (repA) or pK18 (control), with
[3H]testosterone (Fig. 6).
Nonlabeled testosterone served as a specific competitor and decreased
[3H]testosterone binding to RepA in a
concentration-dependent manner, finally leading to control
values (pK18) at an excess of 100-fold nonlabeled steroid
(Fig. 6).
Two 10-bp Palindromic Sequences as Cis-acting Elements for hsdA
Regulation--
Two palindromic 10-bp motifs were localized at 0.935 kb (TCAAAGCCCA = Op1) and at 2.568 kb (TGGGCTTTGA = Op2)
upstream of hsdA and were tested as cis-acting
operator elements for hsdA regulation (Figs.
7 and 8).
The amount of 3 Gel Mobility Shift Experiments--
The specific interaction
between RepA, testosterone, and the operator sequences was proved by
gel mobility assays (Fig. 8). Three 34-bp double-stranded DNA
fragments, Op1 and Op2 (containing the two palindromic 10-bp operator
sequences on locations 0.935 kb and 2.568 kb, respectively) as well as
Co (a sequence 17 bp upstream of Op2 that served as a control), were
used. As shown in Fig. 8B (lanes 1 and
6), both Op1- and Op2-containing DNA fragments gave band
shifts upon the presence of RepA, indicating the binding of RepA to the
operator sequences. The specificity of this protein-DNA interaction was
confirmed by mixing a 100-fold excess of nonlabeled Op1- or
Op2-containing fragments to the assays. The band shift of both Op1 and
Op2 disappeared with nonlabeled Op1 and Op2 (and vice versa)
as specific competitors (lanes 2, 3,
7, and 8). Fragment Co, which did not contain the
palindromic operator motif, could not compete for specific RepA binding
(Fig. 8B, lanes 4 and 9). Finally,
protein extracts from E. coli carrying pK18 (lacking repA) did not result in band shifts, either with Op1 or with
Op2 (Fig. 8B, lanes 5 and 10). The
interaction between RepA and Op2 was then investigated in the presence
or absence of testosterone (Fig. 8C). The reversal of RepA
binding to Op2 by testosterone was clearly demonstrated.
A Model on the Regulation of hsdA Expression--
The factors
controlling the expression of hsdA in C. testosteroni are summarized in Fig.
9. A negative regulator of
hsdA is encoded by a gene, repA, which has an
opposite orientation to hsdA and which overlaps the
hsdA sequence. In the absence of "inducing" steroids,
the RepA protein binds to operator sequences (Op1 and Op2) and blocks
hsdA transcription. In the presence of appropriate steroids,
however, these bind to RepA, thereby reducing its ability to bind to
the operator region. Hence, induction of hsdA
expression by steroids in fact is a derepression, where steroid
"inducers" prevent the binding of a repressor protein to the
operator. Upon dissociation of the repressor from the operator, the
polymerase binds to the promoter, and transcription of 3 Microbial metabolism of steroids is of considerable interest
academically and because of the potential applications of this process
in the pharmaceutical and food industries as well as in human medicine
and environmental biotechnology. Because biotransformation of steroids
in procaryotes is so important, there is considerable interest in the
mechanism of its regulation. This is especially true because the
expression of steroid-transforming enzymes is controlled by steroids
themselves. Here, the question still remains whether
steroid-dependent gene induction in procaryotes resembles that found in eucaryotes, where steroid hormones act by binding to
specific nuclear receptors, which leads to transcriptional regulation
of different gene products and the desired physiological response. So
far nothing is known on the steroid-dependent
transcriptional regulation in procaryotes, either on hsdA or
on the complete steroid degradative pathway.
To follow up our previous work on the molecular biology and structure
of 3 Analysis of the C. testosteroni hsdA regulatory region led
to the identification of the operator and promoter region upstream of
hsdA. Interestingly, we could identify a gene
(repA) that codes for a negative regulator protein for
hsdA expression. The repA gene has an opposite
orientation to hsdA and overlaps the hsdA sequence. The repressor protein (RepA) obviously binds to the operator
region under noninduced conditions, thus preventing binding of the RNA
polymerase to the hsdA promoter. It is suggested that in the
presence of appropriate steroidal inducers, these bind to RepA, thereby
altering its conformation and reducing its affinity to the
cis-regulating operator sequences. Upon dissociating from the operator, the polymerase binds to the promoter, and transcription of 3 Interestingly, downstream of hsdA is another gene encoding a
steroid-metabolizing enzyme, the gene for From an examination of the proposed catabolic pathway (36), it can be
estimated that the complete degradation of the steroid nucleus requires
more than 20 enzymatic reactions. With a pathway of this size, it is
conceivable that the encoding genes are clustered into operons or
belong to a steroid-dependent regulon. In this respect, the
close proximity of the two genes, hsdA and
It is meanwhile well established that HSDs act as pre-receptor control
devices by promoting either the synthesis or degradation of steroid
hormones, thereby regulating their physiological action. Therefore,
like other short-chain dehydrogenase/reductase proteins, 3 Steroid hormones were originally assumed to be exclusively the products
of vertebrate endocrine organs, implying a recent evolutionary origin.
However, several steroid hormones have been isolated from plant
sources, where they are considered to play a prominent part in plant
growth, development, and flowering (42, 43). Interestingly, recent
findings provide evidence for the existence in microbial cells of fully
developed functional signal transduction pathways that are capable of
recognizing and responding to the corresponding mammalian hormones
(44). Detailed analysis of steroid-protein interactions in bacteria
might therefore provide insight into the evolution of the hormonal
systems found in more complex life forms. Recognition may also lead to
the discovery of new systems for the microbial transformations of
steroids and identification of novel ligands, which, through genetic
manipulation, can be effectively utilized in health care or biotechnology.
Hence, steroids play a particularly important role in certain
procaryotes, because they may simultaneously serve both as signal molecules and as a carbon source. The same applies for procaryotic HSDs, such as 3 In conclusion, induction of hsdA in C. testosteroni actually appears to be a derepression of gene
transcription by specific binding of the steroid inducer molecule to a
repressor protein. Under noninduced conditions the repressor protein
(encoded by repA) prevents transcription by binding as a
trans-acting negative regulator to operator sequences
upstream of hsdA. The present paper is the first to give
insights into the genetics of steroid-dependent gene
regulation in procaryotes and provides a firm foundation for further
investigations into the genetics of steroid catabolism in bacteria.
-hydroxysteroid dehydrogenase/carbonyl reductase gene
(hsdA) codes for an adaptive enzyme in the degradation of
steroid compounds. However, no information was available on the
molecular regulation of steroid-inducible genes nor on the mechanism of
steroid signaling in procaryotes. We, therefore, investigated the
cis- and trans-acting elements of
hsdA expression to infer the mechanism of its molecular
regulation by steroids. The gene was localized on a
5.257-kilobase EcoRI fragment of C. testosteroni chromosomal DNA. The promoter was characterized, and
the transcriptional start site was identified. Two palindromic operator
domains were found upstream of hsdA. A new gene coding for
a trans-acting negative regulator (repressor A, RepA) of
hsdA expression was characterized. The specific interaction between RepA, testosterone, and the operator domain is demonstrated. From our results we conclude that hsdA is under negative
transcriptional control by an adjacent gene product (RepA).
Accordingly, induction of hsdA by steroids in fact is a
derepression, where steroidal inducers bind to the repressor, thereby
preventing its binding to the hsdA operator.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-HSD, the production of which has been shown to be inducible by
testosterone, progesterone, and lanosterol (12). Since the pioneering
work of Marcus and Talalay (13), it is well known that 3
-HSD is one
of the first enzymes of the steroid catabolic pathway and that,
therefore, it plays a central role in steroid metabolism. 3
-HSD,
which was first identified by its activity in converting
dihydrocortisone to 3
-tetrahydrocortisone (14), has been found in
mammalian cells (15) and in procaryotes such as Clostridium
perfringens (16), Eubacterium lentum (17), Pseudomonas putida (18), and C. testosteroni (10,
19, 20). Despite similar substrate specificities, eucaryotic and
procaryotic 3
-HSDs belong to two different protein superfamilies.
Whereas eucaryotic 3
-HSD (EC 1.1.1.213) belongs to the aldo-keto
reductase superfamily (21), the procaryotic 3
-HSD (EC 1.1.1.50) is a
member of the short chain dehydrogenase/reductase superfamily (22-24).
-HSD
enzyme from C. testosteroni and determined the primary structure by the cloning and sequencing of its gene (10, 24, 25).
3
-HSD has been shown to mediate the oxidoreduction at position 3 of
the steroid nucleus of a great variety of C19 to C27 steroids (10). Surprisingly, this enzyme is also
capable of catalyzing the carbonyl reduction of nonsteroidal xenobiotic aldehydes and ketones and has therefore been named 3
-HSD/CR (10). Further studies revealed that the substrate pluripotency of
3
-HSD/CR, as well as its inducibility, not only increase the
resistance of C. testosteroni to the steroid antibiotic
fusidic acid but also enhance the metabolic capacity of insecticide
degradation in this organism (26). However, no information is available on the transcriptional regulation of 3
-HSD/CR itself or on the molecular regulation of the complete steroid degradation pathway.
-HSD/CR, one of the
first enzymes in steroid degradation in C. testosteroni. The
results suggest that hsdA in C. testosteroni is
under negative transcriptional control by a repressor protein, the gene
of which has an opposite orientation to that of hsdA and
which overlaps the hsdA sequence.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-HSD/CR
activity was assayed by HPLC analysis as described previously (10). For
determining the mutant promoter activity with the CAT reporter assay,
mutant promoter-cat constructs were transformed into
C. testosteroni, and the CAT activities were assayed as
described below. All mutant promoters generated in this study were
confirmed by DNA sequencing (MWG Biotec, Ebersberg, Germany).
-HSD/CR protein production was
measured by ELISA.
-HSD/CR--
For the quantification of 3
-HSD/CR
protein expression, an ELISA was established, and respective antibodies
were generated. Rabbit antibodies directed against 3
-HSD/CR from
C. testosteroni were prepared according to standard methods.
ELISA plates were coated with protein extracts containing 3
-HSD/CR
in coating buffer. After washing, antibodies against 3
-HSD/CR were
added in 1:10,000 dilution. The further procedure corresponded to that
of the CAT ELISA kit from Roche Molecular Biochemicals.
-HSD/CR Activity and CAT Reporter Gene
Expression--
3
-HSD/CR enzyme activity was assessed by HPLC as
described previously (10). CAT expression from pKK232-8 and its
derivatives was measured by the ELISA kit (Roche Molecular
Biochemicals) according to the manufacturer's instructions. Because
nontransformed E. coli and C. testosteroni cells
exhibited some background CAT activity, fresh competent cells were
transformed and then cultivated for 16 h until used. In these
cases CAT activity showed very low background levels, thus facilitating
the procedure.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-HSD/CR in E. coli and C. testosteroni resulted in comparable enzyme activities in both bacteria, as determined by HPLC (Fig. 2), revealing
the promoter region of hsdA to be recognized by the E. coli transcription machinery. Importantly, nontransformed E. coli cells had no detectable 3
-HSD/CR background activity.
Accordingly, E. coli represents a suitable system for
investigating the hsdA regulatory domains.
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Fig. 1.
Schematic illustration of the various
deletion and reporter constructs. Deleted fragments are
represented by lines, and the corresponding restriction
sites are given as one-letter abbreviations above the lines
(A, AvrII; C, ClaI;
E, EcoRI; H, HincII;
K, KpnI; N, NdeI;
X, XmaI. CAT reporter fusions appear as
boxes downstream of the ClaI site.
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Fig. 2.
Deletion analysis of the upstream regulatory
region of hsdA. 3 -HSD activity (nmol × mg
1 × min
1) was
assayed in E. coli and C. testosteroni after
transformation with pUC18 and pBBR1MCS-2, respectively, each carrying
deleted inserts of the 5.257-kb EcoRI fragment (as shown in
Fig. 1). Nontransformed E. coli cells and those containing
pUC18 had no detectable 3
-HSD/ CR background activity. Similarly,
C. testosteroni cells transformed with pBBR1MCS-2
expressed no 3
-HSD/CR activity.
-HSD/CR activities in strains
carrying p6 and pBB21B are very low. This low level of enzyme
production with p6 and pBB21B in E. coli and C. testosteroni, respectively, not only reveals a pattern of negative
regulation of hsdA transcription, but also suggests that a
repressor protein might be encoded by a gene that is also located on
the 5.257-kb EcoRI fragment of C. testosteroni
chromosomal DNA. In addition, because deletions downstream of the
AvrII site (plasmids pAX1 and pBBAX7) lead to high enzyme
activity, the promoter region of hsdA must be located within
this 93-bp domain. The higher enzyme activity in strains carrying pAX1
and pBBAX7, relative to that with pH2, pBBH31, pX12, and pBBX1, may be
explained by the action of two operator domains identified at 0.935 and
2.568 kb on the EcoRI fragment (see below). The same
promoter regions of hsdA fused to the cat
reporter gene (plasmids pBBK4, pBBKH13, pBBKX7, and pBBKAX1) and
transferred into C. testosteroni yielded a similar increase
in CAT expression (data not shown).
35, TAGCCT;
10, TTTGAT; with a spacing of 17 bp) that has
been verified by point mutation analysis (see below).
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Fig. 3.
Primer extension analysis, S1 nuclease
mapping, and nucleotide sequence of the hsdA promoter
region. A, the primer-extended products using total RNA
obtained from E. coli HB101 were electrophoresed with a
sequencing ladder (lanes A, C, G, and
T) generated by using the same template and primer. The S1
nuclease protection assay (cf. "Experimental Procedures") confirms
the transcription start site, which has been identified by primer
extension analysis. A portion of the nucleotide sequence deduced from
the sequencing lanes is shown on the right. The most intense
extended product is indicated by an arrow (on the
left), and the corresponding transcriptional start
site (at 28 upstream from the AUG codon) is indicated by an
asterisk (on the right). Lanes 1,
4, 7, 10, plasmid p6; lanes
2, 5, 8, 11, plasmid pX12;
lanes 3, 6, 9, 12, plasmid
pAX1. Lanes 1-3, after 6 h of growth; lanes
4-6, after 9 h of growth; lanes 7-9, after
12 h of growth; lanes 10-12, after 16 h of
growth. B, the upstream region of hsdA containing
the promoter sequence is shown. The transcriptional start site is
marked by an arrow. Upstream of this position,
the AvrII restriction site and the
35 and
10 DNA
sequences are underlined. In addition, the ribosome binding
site (Shine-Dalgarno sequence) is indicated by SD.
Primers used in this study
35 (TAGCCT) and
10
(TTTGAT) motifs were introduced by polymerase chain reaction and are
shown in Table II. Mutant promoter
fragments were inserted into pAX1 descendants and transferred
into E. coli HB101 to determine 3
-HSD/CR activity,
or pMx-cat constructs (in plasmid pBBKAX1) were
transferred into C. testosteroni to test for CAT expression.
As compared with respective control experiments, all point mutations
except one resulted in a decrease of 3
-HSD/CR activities or CAT
reporter expressions (Table II). For example, a T to G transition of
the first T in the
10 motif resulted in a residual 3
-HSD/CR
activity of only 1.8%, and a T to G transition of the first T in the
35 motif gave a residual CAT expression of only 13%. Interestingly, the C to A transition in the
35 motif yielded an enhanced activity of
up to 135% (3
-HSD/CR) and 123% (CAT).
Point and deletion mutations in the hsdA promoter region and the
effects thereof on the transcriptional activity
-HSD activity or CAT expression as
described under "Experimental Procedures." Single point mutations
are underlined and indicated in italics. 3
-HSD activities (nmol × min
1 × mg
1) and CAT expression (ng × mg
1 of protein) are given as percentages relative to control
values obtained with plasmids pAX1 and pBBKAX1. The data correspond to
averages ± S.D. of at least four independent determinations.
35
sequence were introduced to give plasmids pM-2 and
pM-3, which were transferred into E. coli and
C. testosteroni. No changes in the promoter activity could
be observed, as revealed by both 3
-HSD/CR and CAT determinations
(Table II). In addition, we prepared mutant plasmids (pM0 and pBBK1) in
which the entire promoter sequence (downstream of the AvrII
restriction site) was deleted. Cells transformed with these plasmids
showed no 3
-HSD/CR activity or very low CAT activities, respectively
(Table II).
35 and
10 regions are critical for full activity. These sequences are separated by 17 bp, the optimal distance
(17 ± 2 bp) for promoters recognized by the
70 RNA polymerase holoenzyme.
-HSD/CR expression of p6, pH2, pX12, and pAX1 in E. coli
were compared after double plasmid transformation with either
pBBR1MCS-2 (control) or pBB7 in medium containing ampicillin and
kanamycin. From the fact that pBB7 did not affect 3
-HSD/CR
expression, it was obvious that no repressor gene is located on the
2.708-kb upstream fragment of hsdA (data not shown).
-HSD/CR expression, the
EcoRI-ClaI upstream fragment of hsdA
(pBB7) failed to inhibit CAT expression after double plasmid transformation with pBKH1, pBKX8, and pBKAX1. In contrast, strong inhibition of CAT expression was obtained when either the downstream fragment of hsdA (pBBAX7) or the complete 5.257-kb
EcoRI fragment (pBB21B) were double-transformed with the
cat plasmids (data not shown). These results indicate that
the coding region of the repressor protein must be located downstream
of the AvrII site (at 2.532 kb) on the EcoRI fragment.
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Fig. 4.
Repression of hsdA by
RepA. 3 -HSD/CR (µg/mg of protein) was assayed by ELISA in
E. coli after pAX1 cotransformations with pK18, pKpN6,
pKpN7, pKAN10, and pKAN12. Cotransformation of p6 and pK18 served as a
control. Four independent plates were transformed, and each extract was
assayed in duplicate. Repression of hsdA by RepA (gene
product of orf 4 in Fig. 1) is observed if the
lacZ promoter is at the NdeI site (pKpN6 and
pKAN10) but not at the KpnI (pKpN6) or AvrII
(pKAN12) sites. Obviously, the repressor active region is located on
the N-terminal half of RepA, due to hsdA repression in cells
carrying pKAN10.
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Fig. 5.
Point and frameshift mutations of repA.
Point mutations of both possible ATG start codons (pRm1 and pRm3) and
shift mutations within the repA sequence (pRm2 and
pRm4) were generated, and the effect on hsdA expression was
tested in cotransformation experiments with pAX1. 3 -HSD/CR protein
production was measured by ELISA. In contrast to the point mutations,
the frameshifts led, in both cases, to a complete loss of RepA activity
and full hsdA expression, as was the case with pK18 as a control.
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Fig. 6.
Specific binding of testosterone to
RepA. Ten µg of protein extracts from E. coli,
carrying pKpN6 (repA) or pK18 (control), was mixed with 0.1 µCi of [3H]testosterone, and steroid binding was
measured. The concentration-dependent competition of
nonlabeled testosterone for RepA binding is clearly shown.
-HSD/CR produced in E. coli cells
transformed with plasmids carrying deletions in these sequences is
shown in Fig. 7. Deletion of Op1 (pL10n) resulted in an increase in
hsdA expression compared with p6. Upon addition of the
steroid testosterone (Fig. 7, + ster.),
hsdA expression increased 8-fold in strains carrying either
p6 or pL10n, compared with "noninduced" conditions, i.e.
without steroid. Because Op2 overlaps the
10 binding site of the
70 RNA polymerase by 5 bp, only half of Op2 could be deleted (pL5n)
without affecting the hsdA promoter structure. Here, no
alterations in hsdA expression could be observed, even in
the presence of testosterone. However, the effect of testosterone
"induction" of hsdA is seen in E. coli strains cotransformed with pAX1 and pKpN6 (the latter coding for RepA)
as well as in wild-type C. testosteroni cells (Fig. 7). In
these cases, testosterone led to a reversal of hsdA
repression. Together, these results point to a specific interaction
between RepA, testosterone, and the operator sequences.
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Fig. 7.
Characterization of two operator
elements. Two palindromic 10-bp motifs (Op1 and Op2 in Fig. 8)
were tested as cis-acting operator elements for
hsdA regulation. The amount of 3 -HSD/CR produced in
E. coli cells transformed with plasmids carrying deletions
in these sequences was determined by ELISA. Deletion of Op1 (pL10n)
resulted in an increase in hsdA expression compared with p6 (control).
Deletion of Op2 by 5 bp (pL5n) increased hsdA expression compared with
pAX1. However, hsdA expression was unchanged with pL5n + steroid (ster.) compared with pL5n. Complete
reversal of hsdA repression by testosterone (+ ster.) led to
an 8-fold increase in 3
-HSD/CR production in strains carrying either
p6 or pL10n. The same induction was observed in E. coli
strains cotransformed with pAX1 and pKpN6 (repA), as well as
in wild-type C. testosteroni cells.
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Fig. 8.
Gel mobility shift experiments. The
specific interaction between RepA, testosterone, and the two operator
sequences, at 0.935 kb (Op1, TCAAAGCCCA) and at 2.568 kb (Op2,
TGGGCTTTGA), was proved by gel mobility assays. A, three
34-bp double-stranded DNA fragments (containing Op1 and Op2, as well as
a sequence 17 bp upstream of Op2 that served as a control (Co)) were
used. B, both Op1- and Op2-containing DNA fragments gave
band shifts upon the presence of RepA (lanes 1 and
6). Upon addition of a 100-fold excess of nonlabeled Op1 or
Op2, the band shifts disappeared (lanes 2, 3,
7, 8). Fragment Co could not compete for specific
RepA binding (lanes 4 and 9). Extracts from
E. coli carrying pK18 (lacking repA) did not
result in band shifts (lanes 5 and 10).
C, the interaction between RepA and Op2 was tested in the
presence or absence of testosterone. The reversal of RepA binding to
Op2 by testosterone is clearly shown
-HSD/CR
mRNA is initiated.
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Fig. 9.
Scheme of the transcriptional regulation of
hsdA in C. testosteroni. In the
absence of inducing steroids, the RepA protein binds to operator
sequences (Op1 and Op2) and blocks hsdA transcription. In
the presence of appropriate steroids, however, these bind to RepA,
which is then released from the operator region. Upon dissociation of
the repressor from the operator, the polymerase binds to the promoter,
and transcription of 3 -HSD mRNA is initiated. Hence, induction
of hsdA expression by steroids in fact is a derepression.
E, EcoRI.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-HSD/CR (10, 19, 24, 25, 31), we investigated the
transcriptional regulation of this important enzyme in C. testosteroni. In this study, we present the first data on the molecular mechanism of steroid induction in procaryotes. Southern blot
experiments revealed the 3
-HSD/CR gene (hsdA) to be
located on a 5.257-kb EcoRI genomic fragment of C. testosteroni ATCC 11996 (25). Immediately downstream of
hsdA, the gene of another steroid-metabolizing enzyme was
found,
5-3-ksi, which has been cloned and
sequenced previously (32).
-HSD/CR mRNA is initiated. Hence, induction of 3
-HSD/CR expression in C. testosteroni by steroids actually appears
to be a derepression by preventing the binding of a repressor protein to regulatory DNA regions (Fig. 9), like that of the well known lac operon in E. coli. Corresponding to this
idea, Arai et al. (33) suggested that the expression of the
genes for phenol degradation in C. testosteroni TA 441 are
under negative control by AphS, which belongs to the GntR family
of transcriptional regulators (34).
5-3-ketosteroid
isomerase (32, 35).
5-3-ketosteroid isomerase (steroid
-isomerase, EC 5.3.3.1) catalyzes the isomerization of the double
bond of, e.g. 5-androstene-3,17-dione to
4-androstene-3,17-dione by a direct intramolecular and stereospecific
transfer of the C-4
proton to the C-6
position. The resulting
double bond is then in conjugation with the 3-keto group, a fact that
is important prior to ring A aromatization and ring B opening.
5-3-ksi, is intriguing. Interestingly, parts
of a bile acid-inducible operon have been identified in
Eubacterium sp. strain VPI 12708. In this procaryote, at
least six new polypeptides of different sizes have been identified by
one- and two-dimensional gel electrophoresis, and their encoding genes
have been cloned, sequenced, and localized to overlapping fragments of
chromosomal DNA (37). From these and some other observations, the
authors concluded that all six open reading frames are transcribed as a
polycistronic message from a single bile acid-induced operon. In
addition, evidence was provided that the genes of some
steroid-metabolizing enzymes, 3-oxo-steroid
1-dehydrogenase and
3-oxo-steroid
4-5
-dehydrogenase, in Arthrobacter
simplex and C. testosteroni (38) are clustered to one operon.
-HSD/CR from C. testosteroni may also have an important
function in the conversion of signaling molecules (steroids) to either the active or inactive state. Moreover, the analysis of short-chain dehydrogenase/reductase protein sequences revealed an unexpected relationship between steroid dehydrogenases and NodG and FixR proteins, signal molecules that mediate nodulation of legume roots by
nitrogen-fixing (22, 23, 25). The nodulation process is initiated by
the secretion from the plant of a flavonoid, which stimulates the
transcription of nodulation (nod) genes in the target
rhizobia. The remarkable similarities between the actions of flavonoids
in the communication between plants and rhizobia to that of
steroid-mediated actions in vertebrates (39), together with the
occurrence of steroid-binding proteins and HSDs in bacteria (40, 41),
are relevant to the question whether the elements for transcriptional
activation or inactivation of genes by steroid-like molecules are also
present in procaryotes.
-HSD/CR from C. testosteroni. These
enzymes, on the one hand, have their role in procaryotic steroid
hormone signal transduction, and, on the other hand, participate in
steroid degradation.
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FOOTNOTES |
---|
* This work was supported by grants (to E. M.) from the European Community and by the Deutsche Forschungsgemeinschaft (SFB 395).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number AF092031.
To whom correspondence should be addressed. Tel.:
49-6421-28-65465; Fax: 49-6421-28-65600; E-mail:
maser@mailer.uni-marburg.de.
Published, JBC Papers in Press, January 3, 2001, DOI 10.1074/jbc.M010962200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
HSD, hydroxysteroid dehydrogenase;
3-HSD/CR, 3
-HSD/carbonyl
reductase;
kb, kilobase(s);
CAT, chloramphenicol acetyltransferase;
pM, mutant promoter;
HPLC, high pressure liquid chromatography;
RepA, repressor A;
ELISA, enzyme-linked immunosorbent assay;
bp, base pair(s);
Co, control.
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REFERENCES |
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