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Characterization of HdnoR, the Transcriptional Repressor of the 6-Hydroxy-D-Nicotine
Oxidase Gene of Arthrobacter nicotinovorans pAO1, and its DNA-binding Activity in
Response to L- and D-Nicotine Derivatives *
Cristinel Sandu2, Calin B. Chiribau and Roderich Brandsch#
Institute of Biochemistry and Molecular Biology, University of Freiburg, Freiburg, Germany
# Correspondence to
Dr. Roderich Brandsch
Institute of Biochemistry and Molecular Biology
Hermann-Herder-Str. 7
79104 Freiburg
Germany
Telephon: ++49-761-2035231
Telefax: ++49-761-2035253
e-mail: [email protected]
Running title: HdnoR
* This work was supported by a grant of the Deutsche Forschungsgemeinschaft to R. B. The
costs of publication were defrayed in part by the payment of page charges. This article must
therefore be hereby marked “advertisement “ in accordance with 18 U.S.C. Section 1734,
solely to indicate this fact.
Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on October 8, 2003 as Manuscript M307797200 by guest on A
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SUMMARY
Utilization of L-nicotine as growth substrate by Arthrobacter nicotinovorans pAO1 starts with
hydroxylation of the pyridine ring at C6. Next, the pyrrolidine ring is oxidized by 6-hydroxy-
L-nicotine oxidase which acts strictly stereo specific on the L-enantiomer. Surprisingly, L-
nicotine induces in the bacteria also the synthesis of a 6-hydroxy-D-nicotine specific oxidase.
Genes of nicotine degrading enzymes are located on the catabolic plasmid pAO1. The pAO1
sequence revealed that the 6-hydroxy-D-nicotine oxidase gene is flanked by two ORFs with
similarity to amino acid permeases and a divergently transcribed ORF with similarity to
proteins of the tetracycline repressor TetR family. Reverse transcription PCR and primer
extension analysis of RNA transcripts isolated from A. nicotinovorans pAO1 indicated that
the 6-hydroxy-D-nicotine oxidase gene represents a transcriptional unit. DNA electromobility
shift assays established that the purified TetR similar protein represents the 6-hydroxy-D-
nicotine oxidase gene repressor HdnoR and binds to the 6-hydroxy-D-nicotine oxidase gene
operator with a Kd of 21 nM. The enantiomers 6-hydroxy-D- and 6-hydroxy-L-nicotine acted
in vitro as inducers. In vivo analysis of 6-hydroxy-D-nicotine oxidase gene transcripts from
bacteria grown with L- and D-nicotine confirmed this conclusion. The poor discrimination by
HdnoR between the 6-hydroxy-L- and 6-hydroxy-D-nicotine enantiomers explains the
presence of the 6-hydroxy-D-nicotine specific enzyme in bacteria grown on L-nicotine.
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INTRODUCTION
The Gram + soil bacterium Arthrobacter nicotinovorans pAO1, formerly known as A.
oxidans (1), has the metabolic ability to grow on the tobacco alkaloid nicotine (2). The main
alkaloid produced by the tobacco plant is L- nicotine and in the presence of this compound the
bacteria produce a nicotine dehydrogenase, which hydroxylates C6 of the pyridine ring of
nicotine (3, 4). A stereo specific 6-hydroxy-L-nicotine oxidase (6HLNO)1 leads to the
formation of N-methylaminopropyl-(6-hydroxypyridyl-3)-ketone (5). Surprisingly, L-nicotine
also induces the synthesis of a 6-hydroxy-D-nicotine specific oxidase (6HDNO) (6,7, Fig. 1).
When chemically synthesised D-nicotine was added to A. nicotinovorans pAO1 cultures the
same enzyme activities were induced. The induction of both stereo specific enzymes 6HLNO
and 6HDNO by either L- or D-nicotine was a long standing puzzle (3). It could be explained
by the presence of a L-nicotine racemase which produces the D-enantiomer. However, there
was no evidence found for the presence of a L-nicotine racemase (3, 5, 8).
Genes of nicotine degrading enzymes are situated on the catabolic plasmid pAO1 (9).
It has been shown before that a protein present in A. nicotinovorans pAO1 extracts binds to an
operator site consisting of two inverted repeats: IR1, covering the 6hdno promoter region, and
IR2, situated upstream from the 6hdno promoter (10). However, the protein of the
transcriptional regulator remained elusive and had not been identified. Recently the position
on pAO1 of genes of enzymes involved in nicotine catabolism by A. nicotinovorans has been
determined (11). The gene of 6HDNO was not part of this gene cluster. The sequence of
pAO1 (12) revealed, that 6hdno is positioned in close proximity to two open reading frames,
ORF111 and ORF113, with high similarity to amino acid permeases, and to ORF114, with
similarity to transcriptional regulators of the TetR family (PROSITE PS01081; 13, 14).
In this work we performed a transcriptional analysis of the 6hdno gene cluster and
present evidence, that 6hdno represents a transcriptional unit. We cloned and expressed the
DNA carrying ORF114, purified the TetR similar transcriptional regulator and show that it
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represents the 6hdno repressor. Evidence is presented, that 6-hydroxy-D-nicotine and 6-
hydroxy-L-nicotine act as inducers of 6hdno expression. Induction of 6hdno expression by
both, L- and D-nicotine enantiomers, can be explained by the poor discrimination of HdnoR
between 6-hydroxy-L- and 6-hydroxy-D-nicotine.
EXPERIMENTAL PROCEDURES
Growth of A. nicotinovorans pAO1 and preparation of bacterial extracts. A.
nicotinovorans pAO1 was grown at 30 °C on citrate medium supplemented with trace
elements and vitamin solution (2). For enzyme assays and Western blots the cultures were
induced with 0.05% of different nicotine derivatives for 3 hours at 30 °C. Bacterial pellets
harvested from 100 ml cultures were re-suspended in 3 ml of 0.1 M phosphate buffer, pH 7.4,
1mM phenylmethylsulfonyl fluoride and 5 mg/ml lysosyme. After 1h incubation on ice, the
suspensions were passed 3 times through a French pressure cell at 132 Mpa and the lysate was
centrifuged for 30 min at 12,000 g.
For total RNA isolation, a 5 ml over night A. nicotinovorans pAO1 culture grown in
citrate medium at 30 °C, was induced with 0.05% of L-nicotine, D-nicotine, 6-hydroxy- L-
nicotine, or 6-hydroxy-D-nicotine and growth was continued for 3h. The cultures were then
frozen in liquid nitrogen to stabilize the RNA, melted again and the bacteria were harvested
by centrifugation at 4,000 g for 10 min, re-suspended in 100 µl of 14 mg/ml lysosyme and
incubated at 28 °C for 3 hours. The suspension was used for RNA isolation following the
protocol of the supplier of the RNA isolation kit.
Total RNA isolation. RNA was isolated from bacteria pre-treated as described above
with the RNeasy Kit (Qiagen, Hilden, Germany). Contaminating DNA was digested by “on
column” DNAse I treatment, as described by the supplier. A second round of DNAse I
digestion was performed to remove traces of DNA as follows: 3 µg RNA was incubated in a
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15 µl assay with 3U of RNase free-DNaseI in buffer supplied with the kit for 1h at 30 °C. The
DNAse I was inactivated by the addition of 1mM EDTA and incubation at 65 °C for 15 min.
Reverse transcription (RT)-PCR. cDNA was prepared from 1 µg of total RNA with
avian myeloblastoma virus reverse transcriptase (20 U/µg of RNA; Amersham Pharmacia
Biotech, Freiburg, Germany) and a mixture of random hexanucleotides in the presence of 1U
of RNAsin (Amersham Pharmacia Biotech, Freiburg, Germany) in a total volume of 10 µl.
Reverse transcription was started by a cycle of 10 min at 25 °C, followed by a second cycle of
1h at 42 °C and by the inactivation of the enzyme at 70 °C for 15 min. 1 µl (1:10) of the
cDNA was used as template in PCR reactions with primers specific for different transcripts
(Table I). 6hdno transcripts were amplified by RT-PCR from total RNA prepared from A.
nicotinovorans pAO1 cultures induced with L- and D-nicotine enantiomers. The PCR
program was as follows: 50 °C 31min; 95 °C 15 min; [94 °C 1 min; 55 °C 1min; 72 °C 1min
20 sec] x35; 72 °C 10 min.
Primer extension analysis. A. nicotinovorans pAO1 total RNA was extracted as
described above. 10 picomoles of primer 14 were 5' [32P] labeled using γ-[32P]ATP
(Amersham Pharmacia Biotech, Freiburg, Germany) and T4 Polynucleotide kinase (Promega,
Madison, USA) for 10 minutes at 37 °C. The kinase was heat inactivated at 95 °C for 2
minutes. The concentration of the labelled primer was brought to 100 fmoles/µl and 1 µl of
primer was hybridised with 5-10 µg total RNA for 20 minutes at 58 °C. The annealed primer
was extended for 30 minutes at 42 °C using the AMV reverse transcriptase (Promega,
Madison, USA). The extension products were ethanol precipitated, resuspended in formamide
dye (Promega, Madison, USA) and loaded onto a 6% polyacrylamide, 7M urea sequencing
gel. DNA fragment VII was amplified using primers 15 (Table I) and sequenced with the
primer employed in the primer extension reaction. The gel was dried and exposed to X-
OMAT type Kodak film.
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Recombinant DNA work. The DNA carrying ORF114 was amplified by PCR with
primers 12 (Table I) carrying a BamHI and a XhoI restriction site, respectively. The restricted
fragments were ligated into the BamHI-XhoI digested pH6EX3 (15) with the aid of the Rapid
DNA Ligation Kit (Roche Diagnostics, Mannheim, Germany) and the DNA was transformed
into transformation competent E.coli XL-1 blue prepared with the Roti-Transform Kit
according to the instructions of the supplier (Roth, Bruchsal, Germany).
Overexpression and purification of HdnoR. An overnight culture of E. coli XL-1 blue
harbouring the pH6EX3-hdnoR was diluted 10 times in LB medium, supplemented with 50
µg/µl ampicillin and induced for 3 h at 37 °C with 1 mM IPTG. The bacteria were harvested
by centrifugation, re-suspended in 40 mM Hepes, pH 7.4, 0.5M NaCl and lysed by sonication
at 4 °C in a Branson sonifier J17V (scale adjustment 1). The lysate was centrifuged for 15 min
at 12,000 g and the supernatant was used to purify the His6-HdnoR protein on Talon
Sepharose (Clontech, USA). The protein was eluted at 0.5 M imidazol in 40 mM Hepes pH
7.4, 0.5M NaCl and revealed a single band in SDS-PAGE. The HdnoR protein fractions were
concentrated by 50 % ammonium sulphate precipitation and centrifugation at 4,000 g for 5
min. The protein pellet was re-suspended in 10 mM Tris pH 8.0, 1 mM EDTA, 10 mM DDT
and 10 % glycerol and the concentration of the protein was adjusted to 0.1 µg/µl in the same
buffer. Aliquots of protein were frozen in liquid nitrogen and kept until use at –70 °C. For
EMSA the protein was diluted at the required concentration as indicated in the Legend to
Figures.
Protein cross-linking. 2 µg HdnoR protein in a final volume of 25 µl of 89 mM Tris-
borate pH 8.0, 2 mM EDTA (TBE) was cross-linked with 1% formaldehyde (16) for 10
minutes at room temperature. The reaction was stopped by acetone precipitation and the
samples were analysed by SDS-PAGE.
Electromobility Shift Assay (EMSA). Protein DNA-binding assays were performed
according to (17). DNA fragments employed in EMSA were amplified by PCR using the
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primers listed in Table I and labelled with γ[P32]-ATP using the Ready-to-go T4
polynucleotide kinase Kit (Amersham Pharmacia Biotech, Freiburg, Germany). Binding of
HdnoR to DNA was carried out in 25 µl of a reaction mixture containing 0.3 ng DNA and
various amounts of HdnoR in 10 mM Tris, pH 8.0, 50mM KCl, 0.1 mM DDT, 0.1 mg/ml
BSA and 5% glycerol. After incubation at room temperature for 20 minutes, loading buffer
was added to a final concentration of 10 % glycerol and 0.05 % bromphenol blue and the
mixture was immediately applied to a 4 % native polyacrylamide gel. Electrophoresis was
carried out in TBE buffer at a constant current of 30 mA for 1.5 h. After drying, the gel was
developed by incubation with a phosphoimaging plate. The effect of potential inducers on
HdnoR DNA-binding was tested by pre-incubation for 2 minutes of the protein with nicotine
derivatives dissolved in H2O, prior to adding the radio-labelled DNA. Incubation continued
for 20 min at room temperature before the sample was loaded onto a polyacrylamide gel as
described above.
Western- blotting. Cell extracts of A. nicotinovorans pAO1 induced with L- and D-
nicotine enantiomers were separated by SDS-PAGE on a 12 % polyacrylamide gel and blotted
on nitrocellulose membrane (Millipore, Bedford, Germany). Polyclonal antibodies raised in
rabbit against A. nicotinovorans pAO1 6HDNO were used to detect the presence of the
protein in a second antibody bound peroxidase mediated colour reaction.
Enzyme assays. Extracts of un-induced, or L- or D-nicotine induced A. nicotinovorans
pAO1 grown on citrate medium were prepared as described above and 6HDNO activity was
determined in the cleared lysates as outlined in (18).
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RESULTS
Organization and transcriptional analysis of the 6HDNO gene region of pAO1. The
6HDNO gene of pAO1 is flanked by two ORFs, ORF111 and ORF113 (Ac. Nr: AJ507836,
12) both with similarity to amino acid permeases. ORF111 is positioned 550 bp upstream of
and divergently oriented to 6hdno, ORF113 is positioned 505 bp downstream of 6hdno and
oriented into the same direction (Fig. 2A). 81 bp downstream of ORF113 and in opposite
orientation there is ORF114 with similarity to transcriptional repressors of the TetR family.
An analysis of transcripts generated from this DNA region was performed to establish
whether 6hdno and the hypothetical gene of ORF113 form a transcriptional unit. RNA was
isolated from A. nicotinovorans pAO1 grown in the presence of L-nicotine, DNase I treated
and reverse transcribed into cDNA with the aid of a random hexanucleotide. The cDNA was
then employed in PCR with specific primers (Table I) derived from regions I to VI, as
indicated in Fig. 2A. Fig. 2B shows the results. Lanes marked M show a 1kb DNA ladder as
molecular weight marker. Lanes 1, 4, 7, 10, 13, and 16 show the PCR products obtained with
pAO1 DNA as template and primers amplifying fragments I, II, III, IV, V and VI,
respectively. This PCR control was positive with all primer pairs. Lanes 2, 5, 8, 11, 14 and 17
show the results of the negative control PCR with RNA as template, which were all negative
and proof that the RNA samples did not contain DNA. Lanes 3, 6, 9, 12, 15 and 18 show the
PCR results with cDNA as template. Only primer pairs derived from coding regions of the
ORFs amplified in the PCR the expected DNA fragments. No amplification product was
obtained with primers devised to amplify region II of Fig. 2A, as expected for an intergenic
region. The absence of a PCR product with cDNA as template and primers devised to amplify
region IV of Fig. 2A, supports the conclusion that 6hdno and the gene carrying ORF113 do
not form a transcriptional unit.
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The transcriptional analysis also indicated that the genes of this pAO1 region are
expressed in the presence of L-nicotine. PCR assays performed with cDNA derived from
RNA prepared from A. nicotinovorans pAO1 grown without nicotine gave no amplification
products (not shown). Transcripts corresponding to ORF114 could be detected in the absence
of nicotine, at a level corresponding to that shown in lane 18 of Fig. 2B (not shown), an
indication that the gene of this hypothetical transcriptional regulator was constitutively
expressed.
Expression and purification of the ORF114 protein. The hypothetical protein of
ORF114, with a predicted MW of 21,934, typical of TetR family repressors (PROSITE,
PS01081), contains a predicted helix-turn-helix (HTH) motif at its N-terminal amino acid
sequence (Fig. 3A) and shows highest similarity to hypothetical repressor proteins from
Streptomyces coelicolor (21.1 kDa protein EbrA, 26% identity in 167 aa, AN Q9X9V5),
Actinosynnema pretiosum (transcriptional regulator Asm29, 24% identity in 197 aa, AN
Q8KUH9), and the regulatory protein AcrR from Proteus mirabilis (21% identity in 195 aa,
AN Q8VPB0).
The DNA carrying ORF114 was inserted into pH6EX3 and expressed in E. coli as a
6His-tagged protein. Thus a fusion protein was generated with the N-terminal amino acid
sequence MSPIHHHHHHLVPRGSKL, L corresponding to the UUG translation start of
ORF114. The protein was purified and formation of dimers was determined by cross linking
with formaldehyde. Fig. 3B shows the purified protein (lane 2) and the cross link product
migrating at the size of a homo-dimer (lane 3). The purified protein was tested for DNA-
binding activity in EMSA with DNA fragments a, b and c shown in Fig. 2A. Only fragment b,
which caries the 6hdno promoter gave a band shift in the presence of the protein (Fig. 3C).
The specificity of the protein-DNA complex formed was evaluated in EMSA in the presence
of competing unlabelled fragment b and salmon sperm DNA, respectively (Fig. 3D). Only the
unlabelled fragment b competed with the labelled fragment for DNA binding, but not the
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unspecific salmon sperm DNA. Thus ORF114 represents the gene of the transcriptional
regulator of the 6HDNO gene and the protein was named accordingly 6hdno repressor
(HdnoR).
Interaction of HdnoR with IR1 and IR2 of the 6hdno operator DNA. A schematic
representation of the 5´-DNA region of 6hdno is presented in Fig. 4A. The transcriptional
start site (+1), situated 51 nucleotides upstream of the translation start codon UUG, and the –
10 and –35 elements of the 6hdno promoter were established previously (20). The two
inverted repeats IR1 and IR2 were shown by DNAase I protection assays to be the recognition
sites of a DNA-binding protein present in crude extracts and (NH4)2SO4 fractions of A.
nicotinovorans (10). This DNA-binding protein was present in nicotine un-induced and
induced bacterial extracts, but did not react to the presence of L-nicotine when tested in vitro
in EMSA. When a DNA-fragment of 191 bp (Table I) carrying the two inverted repeats was
employed in EMSA with purified HdnoR, the protein did bind concentration dependent, first
to one site (middle band in Fig. 4B, 10 nM) and then to both sites (Fig. 4B, 20 nM), an
indication that binding of HdnoR to one site may stimulate binding of the protein to the
second site and that binding of HdnoR to both sites was co-operative (10). Titration of the 105
bp DNA encompassing IR1 and the 107 bp DNA fragment encompassing IR2, respectively,
with HdnoR (Fig. 4C and 4D) allowed the determination of a Kd (50% binding of HdnoR to
its recognition sequence) of approximately 20 nM for the protein-DNA interaction at IR1 and
IR2, respectively. When a 41 bp double stranded oligonucleotide (Table 1, number 13), with
the 37 bp sequence of IR1 at its centre was tested in EMSA for HdnoR-binding, the same
results as with the 105 bp fragment (Fig. 4C) were obtained (not shown).
Determination of the transcriptional start site of the gene carrying ORF111. 6hdno
and the divergently transcribed permease similar gene could form a regulatory unit controlled
by HdnoR, with two promoters positioned each on one of the complementary DNA strands.
The 6hdno promoter is part of IR1, with the –35 region TTGACA followed 16 nucleotides
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downstream by the –10 region TATCAAT (Fig. 4A, 19). The first 6 nucleotides of IR2 (Fig.
4A) read on the complementary strand TTGTCA, which represents a reasonable –35 region,
and are followed 17 nucleotides downstream by the sequence AATGAT, a possible –10
region. Therefore the transcriptional start site of the gene carrying ORF111 was determined
by primer extension analysis. It revealed one strong signal as potential transcriptional start
site (Fig. 5) 85 bp upstream of the proposed translation start site of ORF111 (12). Weaker
signals corresponding to shorter primer extension products may represent cDNAs generated
from processed RNA molecules, or premature termination products of the reverse
transcriptase reaction. The strong termination signal of the primer extension reaction may
represent a genuine transcriptional start site, since secondary structure predictions of a
hypothetical RNA transcript upstream and downstream from the proposed transcriptional start
site showed no hairpin loop structures which may act as potential stop sites for the reverse
transcriptase, or an unusual high GC content of the sequence. 6 bp upstream of the proposed –
35 promoter region, the palindrome sequence 5´CTCCCCGGGAG (Fig. 5, panel A) is
positioned, which may represent a potential binding site for a transcriptional regulator.
The primer extension analysis was corroborated by the results of RT-PCR reactions
performed with primer pairs covering the proposed 5´end of the RNA transcript (Fig. 5, panel
A) and primer pairs with one of the primers downstream of the +1 transcriptional start site
(Fig. 5, panel A). Only primer 14 in combination with primer 16 gave in RT-PCR an
amplification product (Fig. 5, panel C).
The effect of nicotine derivatives on HdnoR-binding to IR1. The interaction of the
protein with IR1, which covers the 6hdno promoter, was tested in EMSA in the presence of
various nicotine-derived compounds (Fig. 6A). Only 6-hydroxy-D-and 6-hydroxy-L-nicotine
prevented HdnoR from binding to the IR1 DNA (Fig. 6B).
The effect of 6-hydroxy-D-, 6-hydroxy-L- and L-nicotine on HdnoR/DNA complex
formation was analysed into greater detail at 20 nM HdnoR, which gave half-maximal
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binding to IR1 (Fig. 7, panel A, B and C). Both 6-hydroxy-nicotine enantiomers prevented
DNA-protein complex formation at µM concentrations, with complete inhibition at 50 µM 6-
hydroxy-D-nicotine and at 100 µM 6-hydroxy-L-nicotine, respectively. A thousand fold
higher L-nicotine concentration was required to elicit a similar effect. Thus, 6-hydroxy-
nicotine may be regarded as the compound active in 6hdno induction, with the D-enantiomer
twice as potent as the L-enantiomer.
The effect of L-nicotine, D-nicotine and 6-hydroxy-D-nicotine in vivo on 6HDNO
activity, protein level and 6hdno transcripts. A. nicotinovorans pAO1 cultures were grown in
the presence of 0.05% L-nicotine, D-nicotine or 6-Hydroxy-D-nicotine, respectively, and
6HDNO activity in the bacterial extracts was determined. Highest specific activity was found
in cultures grown with 6-hydroxy-D-nicotine and lowest in cultures grown in the presence of
L-nicotine (Fig. 8A). The enzyme activity levels correlated with the observed 6hdno transcript
levels, which were lower in L-nicotine grown bacteria and higher in 6-hydroxy-D-nicotine
and D-nicotine grown bacteria (Fig. 8B) and with the 6HDNO protein levels on Western blots
(Fig. 8C).
DISCUSSION
The experimental data presented in this paper demonstrate that the protein encoded by
ORF114 of pAO1 represents the transcriptional repressor of 6hdno. The HdnoR protein shows
all characteristics of a repressor of the TetR family. Its predicted molecular weight of 21 kDa,
the predicted HTH motif at the initial third of the protein and the formation of dimers are
typical for these regulatory proteins. The DNA sequence of IR1 and IR2 protected by the
protein was determined by S1-mapping before, with a (NH4)2SO4 fraction prepared from A.
nicotinovorans pAO1 (10). Here we identified the gene of this protein and purified the
repressor. HdnoR binds to the same IR1 and IR2 DNA sequences as the protein shown to be
present in extracts of A. nicotinovorans pAO1 and thus we conclude that HdnoR is identical
with this protein. The absence of an effect of L-nicotine in EMSA with IR1 and IR2 in the
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presence of A. nicotinovorans pAO1 (NH4)2SO4 fraction containing the transcriptional
regulator, which has been reported previously (10), may be explained by the finding that in
EMSA solely the 6-hydroxy-nicotine derivatives acted as inducers.
Expression of hdnoR seemed to be constitutive, since specific transcripts could be
detected both, in un-induced and nicotine induced bacteria. This finding supports the
conclusion, that the gene codes for a repressor. There are no HdnoR binding sites present on
the DNA upstream of the repressor gene. Therefore the repressor seems not to auto-regulate
the transcription of its gene. Inspection of the 5´-regions of the ndh, kdh and dhph (11) genes
revealed no HdnoR binding site. A search for the core palindrome sequence of IR1 and IR2
revealed no additional consensus sequences on the pAO1 DNA. Apparently there is a
complex regulation of genes involved in nicotine utilization and one may assume additional
transcription factors in charge of regulating expression of the ndh-6hlno, kdh and dhph
operons.
HdnoR is the first nicotine-responsive transcriptional regulator of genes belonging to
the pAO1 encoded nicotine regulon which has been characterized. The repressor
discriminates poorly between 6-hydroxy-D-nicotine and 6-hydroxy-L-nicotine as inducers,
which explains the surprising finding made many years ago, that a strictly stereo specific 6-
hydroxy-D-nicotine oxidase is induced by L-nicotine (3,5). From the effect of various
nicotine derivatives on the formation of the HdnoR-DNA complex, one may conclude that the
L- or D-position of the pyrrolidine ring of nicotine is not that important for binding to the
repressor as is the presence of the hydroxyl group at C6 of the pyridine ring. L- and D-
nicotine, N-methyl-myosmine, 6-and 2-amino-L-nicotine or 6-hydroxy-pyridine did not
prevent HdnoR DNA-binding. Since 6-hydroxy-pyridine had no effect, the pyrrolidine ring of
nicotine seems to be required for its interaction with HdnoR.
6hdno is not co-transcribed with the permease similar gene located downstream of it,
but appears to represent a transcriptional unit by itself. However, the genes of the two
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permeases appear to belong to a nicotine regulon, since transcripts of both of these genes can
be detected only in nicotine-induced bacteria. 6hdno and the divergently transcribed permease
gene may form a functional unit, with the permease responsible for uptake of the compound
which serves as substrate for the enzyme 6HDNO. However, as suggested by the primer
extension analysis and the RT-PCR results, they seem not to form a transcriptional unit
regulated by HdnoR. There are no hints yet available as to the function of these hypothetical
permeases. It is tempting to assume a role in transport of nicotine or nicotine derivatives, in
and/or out of the bacterial cell. Efforts to inactivate in A. nicotinovorans pAO1 the permease
genes by homologous recombination with an antibiotic cassette failed so far as did the
heterologous expression of the permease genes in E. coli. .
A. nicotinovorans pAO1 contains promoters resembling in principle σ70 E. coli
promoters. The 6hdno promoter situated at IR1 exhibits a consensus –35 TTGACA sequence
separated by 16 bp from the sequence TATCAAT, very similar to the consensus –10 sequence
TATAAT, with one C residue inserted (Fig. 4, panel A). Whether the proposed promoter (Fig.
5, panel A) which resembles the 6hdno promoter, is indeed the promoter of the permease
gene, has to be proven in functional tests, by fusion with an indicator gene. The accumulation
of C residues at promoter sites, as suggested here for pAO1 promoters, has been observed in
the case of other bacteria with a high GC content, like Mycobacterium tuberculosis (20).
Consensus sequences typical for promoters regulated by alternative sigma factors have not
been detected by inspection of the intergenic regions of the 6hdno and permease genes.
The expression pattern of 6hdno in vivo in the presence of L-, D- or 6-hydroxy-D-
nicotine may be explained by the interaction of the HdnoR protein with these nicotine
derivatives. L-nicotine induces the expression of both, ndh and 6hlno, and thus L-nicotine is
turned over into N-methlyaminopropyl(6-hydroxy-pyridil-3)ketone, giving no time to 6-
hydroxy-L-nicotine to accumulate and to interact with HdnoR. This results in a low level of
6hdno expression. When turnover of L-nicotine slows down, possibly because of feedback
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inhibition of the pathway enzymes by end products, more 6-hydroxy-L-nicotine may
accumulate, resulting in a delayed increase of 6hdno expression, as has been observed in A.
nicotinovorans pAO1 cultures grown with L-nicotine (3, 5).
In the presence of D-nicotine, D-nicotine is turned over into 6-hydroxy-D-nicotine by
NDH, which does not discriminate between the L- and D-nicotine enantiomers (3, 5). The 6-
hydroxy-D-nicotine formed, however, is no substrate for 6HLNO. Therefore 6-hydroxy-D-
nicotine accumulates, leading to a higher expression of 6hdno.
From the results presented in this study one may assume, that 6-hydroxy-D-nicotine
represents the natural inducer of 6hdno expression. However, the question remains: is D-
nicotine found in nature? There are reports that L-nornicotine, a side product of nicotine
biosynthesis by the tobacco plant, was racemized into the D-enantiomer in leaves of the plant
(21). It has also been shown that during cigarette smoking L-nicotine is racemized into its
enantiomer (22). Possibly D-nicotine or D-nornicotine is formed during the decay of the
tobacco plant in the soil. In this case, 6HDNO would be an essential enzyme, required for the
biodegradation of these compounds, and regulation of the expression of its gene by HdnoR
and 6-hydroxy-D-nicotine may reflect this fact. However, the possibility can not be excluded
that the natural inducer of 6hdno and the natural substrate of 6HDNO is a molecule which just
resembles 6-hydroxy-D-nicotine.
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REFERENCES
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5. Decker, K., and H. Bleeg. 1965. Biochim. Biophys. Acta. 105, 313-334.
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A., and Robins, R. (2001) Plant Science 161, 1011-1018
9. Brandsch, R., Decker, K. (1984) Arch. Microbiol. 138, 15-17
10. Bernauer, H, Mauch, L. and Brandsch, R. (1992) Mol. Microbiol. 6, 1809-1820
11. Baitsch, D., C. Sandu, R. Brandsch, and G. L. Igloi. 2001. J. Bacteriol. 183, 5262-5267
12. Igloi, G. L. and Brandsch, R. (2003) J. Bacteriol. 185, 1976-1986
13. Orth, P., Schnappinger, D., Hillen, W., Saenger, W. and Hinrichs, W. (2000) Nature
Struct. Biol. 7, 215-219
14. Hinrichs, W., Kisker, C., Duvel, M., Muller, A., Tovar, K., Hillen, W. and Saenger, W.
(1994) Science 264, 418-20
15. Berthold, H., Scanarini, M., Abney, C.C., Frorath, B., Northemann, W. (1992). Protein.
Expr. Purif. 3, 50-6
16. Grkovic, S., M.H. Brown, M.A. Schumacher, R.G. Brennan, and R.A. Skurray. (2001) J.
Bacteriol. 183, 7102-7109
17. Fried M and Crothers, D.M. (1981). Nucleic Acids Res. 9, 6505-25
18. Brühmüller, M., H. Möhler and K. Decker (1972) Eur. J. Biochem. 29, 143-151
19. Mauch, L., Bichler, V. and Brandsch, R. (1990) Mol. Gen. Genet. 221, 427-434
20. Recchi. C., Sclavi, B., Rauzier, J., Gicquel, B. and Reyrat, J.-M. (2003) J. Biol. Chem.
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21. Kisaki, T., and Tamaki, E. (1961) Arch. Biochem. Biophys. 92, 351-355
22. Nwosu, C. G., and Crooks, P. A. (1988) Xenobiotica 18, 1361-1372
Footnote1. The abbreviations used are: 6HDNO, 6-hydroxy-D-nicotine oxidase; 6HLNO, 6-
hydroxy-L-nicotine oxidase; KDH, ketone dehydrogenase; DHPH, dihydroxypyridine
hydroxylase; TBE, Tris-borate EDTA buffer; EDTA, ethylenediamine tetraacetic acid;
EMSA, electromobility shift assay; bp, base pair; RT, reverse transcription; DDT,
dithiotreitol; Tris, Tris(hydroxymethyl)aminoethane.
Footnote2. Present address: Molecular Biophysics, Rockefeller University, NY, USA
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LEGEND TO FIGURES
Fig 1. First enzymatic steps in nicotine degradation by Arthrobacter nicotinovorans
pAO1. Nicotine dehydrogenase is a complex heterotrimeric enzyme with FAD, molybdenum
cofactor (MoCo) and iron-sulphur clusters as prosthetic groups. 6-Hydroxy-L-nicotine
oxidase consists of a homodimer with one FAD molecule per subunit. 6-Hydroxy-D-nicotine
oxidase is a monomeric protein with FAD covalently bound to a histidine residue.
Fig. 2. Transcriptional analysis of the 6hdno region of pAO1. Panel A, schematic
representation of the position of 6hdno and flanking ORFs on the 165,137 bp pAO1 sequence
(Accession number AJ507836). a, b, c, DNA-fragments analysed by EMSA. I, II, III, IV, V,
VI PCR amplified DNA fragments. Panel B, lanes 1, 4, 7, 10, 13 and 16, PCR products
obtained with pAO1 DNA as template and primer pairs (see Table I) amplifying fragments I,
II, III, IV, V and VI, respectively; lanes 2, 5, 8, 11, 14 and 17, as before, but with RNA as
template; lanes 3, 6, 9, 12, 15, and 18, as before but with cDNA as template. M, 1 kb DNA
ladder.
Fig. 3. Purification and DNA-binding activity of the ORF 114 protein. Panel A, alignment
of the N-terminal amino acid sequence of ORF 114 with the amino acid sequence of TetR
family repressors. Underlined is the amino acid sequence predicted to form a HTH domain of
the protein. Panel B, lane 1, protein molecular weight standard; lane 2, purified ORF 114
protein and, lane 3, dimers of the protein following formaldehyde cross linking. Panel C,
EMS assays with [32P]-labelled DNA-fragments a, b and c (see Fig. 2A and Table 1); - , no
protein, +, with protein added to the assays. Panel D, the specificity of interaction of HdnoR
(0.5 µM) with [32P]-labelled DNA fragment b (30 ng) was tested by competition with 1 µg
(lane 2), 500 ng (lane 3) and 100 ng (lane 4) of unlabelled DNA fragment b, and with 2.5 µg
(lane 7), 1.2 µg (lane 8), 0.25 µg (lane 9) salmon sperm DNA. Lanes 1 an 6, 30 ng [32P]-
labelled fragment only; lanes 5 and 10, EMSA in the presence of HdnoR only.
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Fig. 4. The ORF 114 protein is the transcriptional regulator of the 6HDNO gene
(HdnoR). Panel A, schematic representation of the 5´6hdno region. Indicated are the two
inverted repeats IR1 and IR2 and the 6hdno transcriptional start site +1. Also shown is the
sequence of the inverted repeats and the –35 and –10 sequences of the 6hdno promoter. Panel
B, EMSA showing the concentration dependent binding of HdnoR to the [32P]-labelled 191 bp
PCR amplified fragment carrying IR1 and IR2. Panel C and panel D, titration of [32P]-
labelled 105 bp PCR amplified fragment carrying IR1 and 107 bp fragment carrying IR2 with
HdnoR, respectively.
Fig. 5. Determination of the transcriptional start site of the permease gene carrying
ORF111 by primer extension and RT-PCR. Panel A shows a schematic representation of
the divergently transcribed permease and 6hdno genes, indicates fragment VII (see Table 1)
amplified for the sequencing reaction performed for the identification of the transcriptional
start site of the permease gene, the +1 nucleotide of the transcript, the putative -10 and -35
regions of a proposed promoter of the permease gene, and a putative operator site. ORF111
starts with TTG. Also indicated is the transcriptional start of 6hdno. Panel B gives the result
of the primer extension analysis. Lanes GATC, sequencing reaction of fragment VII; lanes 1,
2, 3, three independent primer extension reactions; lane 4, control reaction in the absence of
RNA. The arrow indicates the fragment obtained by primer extension and the asterisk
indicates the first nucleotide of the transcript. Panel C. Lanes 1, 3, 5, PCR reactions with
primers 16, 17 and 18 and pAO1 DNA as template amplifying fragments VIII, IX and X,
respectively (see Table I); lanes 2, 4, 6, RT-PCR with the primers 16, 17, 18, respectively;
lane 7, control RT-PCR in the absence of RNA; M, 100 bp DNA ladder. The amount of
template and primers was the same in all reactions.
Fig 6. HdnoR behaves like a repressor. Panel A, compounds tested in EMSA on their effect
on HdnoR binding to IR1 DNA. Panel B, EMSA performed with 30 nM [32P]-labelled 105 bp
DNA fragment carrying IR1 and 50 nM HdnoR in the presence of: lane 1, [32P]-labelled DNA
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control; lane 2, HdnoR protein only; lane 3, 100 µM L-nicotine, lane 4, 100 µM D-nicotine;
lane 5, 100 µM 6-OH-L-nicotine; lane 6, 100 µM 6-hydroxy-D-nicotine; lane 7, 100 µM N-
methyl-myosmine; lane 8, 100 µM 6-amino-L-nicotine; lane 9, 100 µM 2-amino-L-nicotine;
lane 10, 100 µM 6-hydroxy-pyridine.
Fig. 7. Inducers levels required to prevent HdnoR DNA-binding. EMSA were performed
with [32P]-labelled 105 bp IR1 DNA fragment in the presence of increasing amounts of : panel
A, 6-hydroxy-D-nicotine; panel B, 6-hydroxy-L-nicotine, and panel C, L-nicotine. First lanes,
without protein, lanes 0, protein only, additional lanes in the presence of protein and
increasing inducers concentrations as indicated.
Fig. 8. In vivo analysis of 6hdno expression in the presence of nicotine enantioners. 6hdno
expression was assayed as 6HDNO activity in extracts of A. nicotinovorans pAO1 (panel A),
as 6hdno transcripts obtained by RT-PCR from RNA extracted from A. nicotinovorans pAO1
(panel B), and as 6HDNO protein levels revealed on Western Blots of A. nicotinovorans
pAO1 extracts decorated with 6HDNO-specific antiserum. A. nicotinovorans pAO1 extracts
were prepared from n.i., non induced, L, L-nicotine induced, D-OH, 6-hydroxy-D-nicotine
induced, and D, D-nicotine induced bacteria.
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TABLE I Oligonucleotides used in this study
Primer
Sequence
Length of DNA (bp)
Fragment
1 forward 5’-cgtttgcgacccctcccg-3’ reverse 5’-cctcggtggtggcattcacc-3’
228
Fragment I
2 forward 5’-ctcgaggagccggtttggcc-3’ reverse 5’-cgtcttcgagagagtgatcacc-3’
535 Fragment II
3 forward 5’-cgtcaaactggagatcgagg-3’ reverse 5’-gtccaaacccagaagtcgtc-3’
194 Fragment III
4 forward 5’-cctcggtggtggcattcacc-3’ reverse 5’-gcaaagaagccagagacagag-3’
1193 Fragment IV
5 forward 5’-gtgcattgtgcttgccgtggt-3’ reverse 5’-gcaaagaagccagagacagag-3’
197 Fragment V
6 forward 5’-cgtctgaaaccatctggg-3’ reverse 5’-cctaagaacgatagccagcg-3’
208 Fragment VI
7 forward 5’-ctcgaggagccgtttggcca-3’ reverse 5’-gctgcaaagggcgggcgatct-3’
205 Fragment 5’ to ORF 111
8 forward 5’-gacaaagagcgatgtgttccg-3’ reverse 5’-cgtcttcgagagagtgatcacc-3’
105 Fragment 5’to 6hdno carrying IR1
9 forward 5’-gcaaggaatcgccatagacgg-3’ reverse 5’-cgtcttcgagagagtgatcacc-3’
191 Fragment 5’ to 6hdno carrying IR1 + IR2
10 forward 5’-gcaaggaatcgccatagacgg-3’ reverse 5’-cggaacacatcgctctttgtc-3’
107 Fragment carrying IR2
11 forward 5’-cgagggatcttgaaacagc-3’ reverse 5’-ggactcagacataggtatcacc-3’
531 Fragment 5’ to ORF 113
12 forward 5’-gggcaaggatccaagttgcg-3’ reverse 5’-cccaatagtctcgagcgaagaaagacg-3’
662 hdnoR (ORF114)
13 5’-cccccattgacatggacagctgtccatgtatcaatagggtg-3’ 5’-gggggtaactgtacctgtcgacaggtacatagttatcccac-3’
41 Double stranded oligo carrying IR1
14 5’-gctggctctcagaagaagaaacttg-3’ 25 Primer extension analysis
15 forward 5’-gctggctctcagaagaagaaacttg-3’ reverse 5’-gtagcgaatgctgcagttatagag-3’
307 Fragment VII
16 forward 5’-gctggctctcagaagaagaaacttg-3’ reverse 5’-tctcgcagtcgatcaccatct-3’
112 Fragment VIII
17 forward 5’-gctggctctcagaagaagaaacttg-3’ reverse 5’-gtagacgaaaaggcacttt-3’
137 Fragment IX
18 forward 5’-gctggctctcagaagaagaaacttg-3’ reverse 5’-ccttcgggatgctaatgagtc-3’
189 Fragment X
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L-Nicotine
* NCH3N
1/2 O2 Nicotine DehydrogenaseFAD;[2Fe-2S];MoCo
D-Nicotine
NCH3N
*
6-Hydroxy-L-nicotine
* NCH3NOH
* NCH3NOH
6-Hydroxy-D-nicotine
O2 6-Hydroxy-D-nicotine-oxidase6-Hydroxy-L-nicotine-oxidaseO2
O
NHCH3O
NH
N-Methylaminopropyl-(6-hydroxypyridyl-3)-ketone
FAD covalent FAD
Fig. 1. Sandu et al.
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ORF 111 6hdno ORF 113 ORF 114
112000 113000 114000 115000 116000 117000 118000
228bp 535bpI II
194bp
1193bp
197bpIII V
IV208bp
VI
bppAO1a b c
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 M
I II III VIV VI
A
B
Fig. 2 Sandu et al.
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Fig. 3 Sandu et al.
HdnoR 7DRRQQLIDAAIRV-IRDGVESASLRTIASEAKASLAAVHVCFTNKDELMQAAAEbrA 8VRRQDFIEAAVKVIAEYGVANATTRRIAAAANSPLASLHYVFHTKDELFDAVYASM29 5VRREQLVAAALRVMKRDGIAAATTRAICAEADMPHGAFHYCFRSKQELYTALLAcrR 11ETRQQIIDAALRLFTVQGVSATSLSDIATEAGVTRGAIYWHFKNKVDLFTEAC
rRqq.idAAlrv....Gv..as.r.IaaeA..p.ga.hy.F..K.eLf.a..
A
a cb
+-+-+-
CB
16
2024
3645556684
116205Mr
1X
2X
1 2 3
D
1 2 3 4 5 6 7 8 9 10
free DNA
DNA-proteincomplex
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Fig. 4 Sandu et al.
0 5040302010 60 70 80 90 100 150 nM HdnoR
IR2/HdnoRD
IR1/HdnoR
C
IR1+IR2/HdnoR
B IR1/IR2/HdnoR
free fragment
free fragment
free fragment
50 bp
TGACAAGGACAAGTGTCCATGTCA CCCATTGACATGGACAGCTGTCCATGTATCAATAGGGTGA
24 bp 37 bp
IR2 IR1 6hdno
TTGACA TATCAAT-35 -10 +1
A
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Fig. 6 Sandu et al.
L-nicotine
* NCH3N
6-hydroxy-L-nicotine
* NCH3NOH
N OH
2-hydroxypyridine
6-NH2-L-nicotine
* NCH3NHN2
D-nicotine
NCH3N
*
* NCH3NOH
6-hydroxy-D-nicotine
N
NCH3
* NCH3N HN2
2-NH2-L-nicotine
N-CH3-myosmine
L -ni
c
D-n
ic
L-O
H-n
ic
D-O
H-n
ic
MM
SM
2-N
H2-
nic
2 -O
H-p
yrid
in
6-N
H2-
nic
A
B
free DNA
DNA-Proteincomplex
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Fig. 7 Sandu et al.
C
0 10 20 30 50 75 100 mM L-nicotine
B
0 10 20 30 50 75 100 µµµµM 6-OH-L-nicotine
0 10 20 30 50 75 100
A
µµµµM 6-OH-D-nicotinefree DNA
free DNA
free DNA
DNA-proteincomplex
DNA-proteincomplex
DNA-proteincomplex
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01234567
mU
6H
DN
O /
mg
prot
.
RT-PCR
W-Blot
n.i. L D-OH D
A
B
C
Fig. 8 Sandu et al.
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Cristinel Sandu, Calin B. Chiribau and Roderich BrandschDNA-binding activity in response to L- and D-nicotine derivatives
6-hydroxy-D-nicotine oxidase gene of arthrobacter nicotinovorans pAO1, and its Characterization of HdnoR, the transcriptional repressor of the
published online October 8, 2003J. Biol. Chem.
10.1074/jbc.M307797200Access the most updated version of this article at doi:
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