1 Characterization of HdnoR, the Transcriptional Repressor of the 6

1

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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234-239

2. Eberwein, H., F.A. Gries, and K. Decker (1961) Hoppe-Seyler´s Z. Physiol. Chem. 323,

236-248

3. Gloger, M. and Decker, K. (1969) Z. Naturforschg. 24b, 1016-1025

4. Hochstein, L. I., Rittenberg, S. C. (1959) J. Biol. Chem. 234, 156-160

5. Decker, K., and H. Bleeg. 1965. Biochim. Biophys. Acta. 105, 313-334.

6. Decker, K., and Dai, V. D. (1967) Eur. J. Biochem. 3, 132-138

7. Decker, K., Dai, D., Möhler, H., and Brühmüller, M. (1972) Z. Naturforsch. 27, 1072-1073

8. Mesnard, F., Girard, S., Fliniaux, O., Bhogal, R. K., Gillet, F., Lebreton, J., Fliniaux, M.-

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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online

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.

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Documents

1 Characterization of HdnoR, the Transcriptional Repressor of the 6