Plasmids for Nicotine-Dependent and -Independent Gene Expression in Arthrobacter nicotinovorans and Other Arthrobacter Species

The first inducible Arthrobacter overexpression system, basedon the promoter/operator and the repressor of the 6-D-hydroxynicotineoxidase gene of Arthrobacter nicotinovorans, is described here.Nicotine-dependent overproduction and affinity purificationof recombinant proteins are presented. The system will allowthe production of complex enzymes and genetic complementationstudies in Arthrobacter species.

INTRODUCTION

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Introduction

The genus Arthrobacter of gram-positive soil bacteria playsan important role in the mineralization of natural and man-madeorganic matter in the environment. In spite of the enormousecological and potential industrial importance of these bacteria,recombinant DNA work with these bacteria has been seriouslyhampered because until now there were no suitable plasmid vectorsavailable for gene cloning and expression purposes for Arthrobacterspecies.

Most of the Arthrobacter genes characterized so far have beencloned and expressed in Escherichia coli (10, 15-17) and otherbacterial hosts (16, 18, 19), or alternatively, the proteinshave been purified directly from crude lysates of Arthrobacter.Heterologous expression, however, can lead to inactive proteinsdue to misfolding, a lack of appropriate enzyme cofactors andproper insertion of these cofactors into apoenzymes, or posttranslationalmodifications. The development of an Arthrobacter expressionsystem would be beneficial for the production of active Arthrobacterenzymes, for use in genetic complementation assays, or for theengineering of Arthrobacter strains with new, biotechnologicallysignificant catabolic features.

Our laboratory has investigated protein biochemistry and nicotinedegradation in Arthrobacter nicotinovorans for over 2 decades.A large catabolic plasmid, pAO1, confers on Arthrobacter nicotinovoransthe ability to degrade nicotine (3). Various enzymes of thenicotine degradation pathway in A. nicotinovorans (1, 3, 4)have been identified and characterized, and recently the sequencingof the 165-kb pAO1 plasmid was completed (8). We have previouslycloned and biochemically characterized the 6-D-hydroxynicotineoxidase gene (hdnO) from pAO1 (12, 13) and characterized itspromoter region (hdnOp) (2). Recently, we also cloned the hdnOrepressor gene (hnoR), biochemically characterized its geneproduct, and identified the inducer molecule for hdnO expression(20).

It has been reported previously that shuttle vectors, developedfrom cryptic plasmids of phylogenetically related species suchas Corynebacterium glutamicum (22) and Brevibacterium lactofermentum(9), can also replicate in some Arthrobacter species, and somereports have previously described the cloning of genes in Arthrobacterspecies (10, 14, 21) using such plasmids.

Shuttle plasmids for inducible gene expression in Arthrobacter species.

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Shuttle plasmids for inducible...

We have created two vectors, pART2 and pART3, for the expressionof genes in A. nicotinovorans as a host. The plasmids are madeof DNA modules derived from A. nicotinovorans, C. glutamicum,and E. coli and required a number of cloning steps, which aredescribed in Table 1.

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TABLE 1. Construction of pART2 and pART3 vectors and their derivatives

pART plasmids contain a 1.9-kb fragment from the cryptic plasmidpCG100 from C. glutamicum ATCC 13058 and can replicate autonomouslyin A. nicotinovorans and Arthrobacter globiformis. pART plasmidDNA can be reisolated from these species, and DNA restrictiongives a pattern of digestion identical to the one expected forpART vectors (data not shown). These plasmids contain the ColE1ori from E. coli and also the Kan^r gene from Tn903, which confersresistance to kanamycin in all tested species. The expressionof genes is driven by the promoter/operator of the 6-D-hydroxynicotineoxidase gene from A. nicotinovorans plasmid pAO1. pART2 andpART3 maps as well as details about the start codon, the multiplecloning sites (MCS), and the polyhistidine stretch sequenceare presented in Fig. 1.

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FIG. 1. DNA maps of plasmids pART2 and pART3. The following plasmid features are presented: pCG100 and ColE1 origins of replication; Kan^r, kanamycin resistance gene; P, promoter/operator of the hdnO gene (hdnOp); MCS; His₈, eight-histidine tag coding sequence; and plasmid size in bp. Relevant restriction sites, including unique restriction sites, and bp positioning are represented. pART3 additionally carries the hdnO repressor gene (hnoR). The bottom sequence shows the following features: rbs, inferred ribosomal binding site; ATG, the translation start codon; MCS with nine unique restriction sites; coding sequence for a His₈ tag; and TAA, translation stop codon. Note that the SalI* site is present twice in the pART3 vector. DNA analysis and map displays were prepared with BioEdit (7).

pART2 is a vector for constitutive gene expression, whereaspART3 also carries the hnoR gene and therefore is a vector forcontrolled gene expression in A. nicotinovorans. The HnoR proteinbinds to the hdnO operators IR1 and IR2 and blocks the expressionof genes from this promoter (20). pART3 allows nicotine- and6-hydroxynicotine-dependent gene expression in A. nicotinovoransand A. globiformis.

Overproduction of GFP in Arthrobacter sp.

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Overproduction of gfp in...

A simple experiment to test the functionality of the pART plasmidsin A. nicotinovorans is to express the green fluorescent protein(GFP) gene (gfp) from these plasmids (Table 1) and to monitorthe fluorescence of bacteria by fluorescence microscopy.

As shown in Fig. 2, A. nicotinovorans produces bright fluorescencewhen transformed with pART2-gfp (panel B).A. nicotinovoranscontaining plasmid pAO1 (A. nicotinovorans/pAO1) transformedwith pART2-gfp produces slightly reduced fluorescence (panelC). This is explained by the presence of HnoR, produced fromthe pAO1 plasmid, which leads to the repression of hdnOp-drivengfp expression. The pART2 vector is designed for constitutivegene expression in Arthrobacter species, independent of thegrowth conditions. However, the expression of nicotine uptakeand nicotine catabolic genes in A. nicotinovorans/pAO1 is poorin rich medium. Therefore, nicotine-inducible gene expressionfrom the pART3 plasmid was carried out on citrate minimal medium.When A. nicotinovorans/pAO1 was transformed with pART3-gfp,no fluorescent bacteria were detectable under the fluorescencemicroscope (panel D). The presence of HnoR, produced from pART3and additionally from pAO1, leads to efficient repression ofhdnOp-driven gfp expression. The addition of nicotine to thecultures resulted in the induction of gfp expression, as demonstratedby the appearance in the cultures of fluorescent bacteria following60 min of growth in the presence of nicotine (panel E) and anincreased fluorescence of bacteria in cultures grown with nicotineovernight (panel F). The expression of gfp from the pART3 vectorwas also tested in A. globiformis, and we observed the appearanceof nicotine- and 6-hydroxy-nicotine-dependent GFP fluorescence(data not shown). Cells induced with 6-hydroxynicotine expressedbrighter fluorescence than those induced with nicotine, whichis in agreement with the different induction efficiencies ofthe two compounds. These experiments demonstrate the functionalityof pART vectors in A. nicotinovorans and other Arthrobacterspp. Nevertheless, not every species can be used as a host fornicotine-inducible expression from pART3, unless it has a nicotineuptake system. A. globiformis was described previously to degradenicotine, and thereby it should have a nicotine uptake system(11).

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FIG. 2. Fluorescent A. nicotinovorans cells upon gfp expression. (A) Light microscopy image of A. nicotinovorans bacteria. Inset, magnified individual cell field. (B) Fluorescence microscopy image of A. nicotinovorans transformed with pART2-gfp; (C) A. nicotinovorans/pAO1 transformed with pART2-gfp; (D to F) A. nicotinovorans/pAO1 transformed with pART3-gfp and grown in the absence of nicotine (D), in the presence of nicotine for 60 min (E), and in the presence of nicotine overnight (F). Bacteria shown in panels A to C were grown in LB medium, and bacteria shown in panels D to F were grown in citrate medium supplemented with 0.5% yeast extract, mineral salts (5), and, as required, 0.05% L-nicotine. Pictures of A. nicotinovorans bacteria were taken using an Zeiss Axioskop 50 epifluorescence microscope equipped with a Plan-Nofluar 100x (1.3-numerical-aperture) objective and a triple-pass filter set. Digital images were recorded with a Nikon D100 camera.

Affinity purification of overproduced proteins in A. nicotinovorans.

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Affinity purification of...

To assess whether the pART vectors can be used for protein overproductionand purification in A. nicotinovorans, we have chosen as a proteinmodel the enzyme 2,6-dihydroxypyridine 3-hydroxylase (DhpH;see Table 1 for dhpH cloning details). DhpH-His₈ is a yellowprotein, a characteristic given by the flavin adenine dinucleotidecofactor, which makes it easy to monitor binding and elutionof the protein from the column. Additionally, the hydroxylationof 2,6-dihydroxypyridine (2,6-DHP) to 2,3,6-trihydroxypyridine(2,3,6-THP) is accompanied by the formation of a blue pigmentin the enzyme assay (1). In Fig. 3A, a gel is presented fora typical purification of DhpH-His₈ from extracts of A. nicotinovoranstransformed with pART2-dhpH. The yield of DhpH-His₈ producedin A. nicotinovorans transformed with pART2 and grown in LBmedium was up to 5% of the total protein present in the bacteriallysates. The specific activity of DhpH-His₈ was ${approx}$ 18 U per mgprotein, which corresponds to the previously reported specificactivity of the enzyme (1). DhpH-His₈ was also produced fromA. nicotinovorans/pAO1 transformed with pART3-dhpH, grown incitrate minimal medium, and induced with nicotine (Fig. 3B).The yield of DhpH-His₈ from A. nicotinovorans/pAO1 lysates wasapproximately 3%.

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FIG. 3. Purification of DhpH-His₈ from A. nicotinovorans extracts. (A) Ni²⁺-chelating Sepharose affinity chromatography purification of DhpH-His₈ from crude extracts of A. nicotinovorans transformed with pART2-dhpH and grown in LB medium without nicotine induction. Lane 1, crude extract; lane 2, purified protein fraction, eluted with 200 mM imidazole. (B) Ni²⁺-affinity purification of DhpH-His₈ from extracts of A. nicotinovorans/pAO1 transformed with pART3-dhpH and grown in citrate minimal medium in the presence of 0.05% L-nicotine. Lane 1, crude extract; lane 2, purified protein fraction, eluted with 200 mM imidazole. The transformation of A. nicotinovorans with pART plasmids was achieved by electroporation with competent cells as described previously (6), with selection on kanamycin (140 µg ml^–1). E. coli was selected with 20 µg ml^–1 kanamycin.

Since pART2 and pART3 are shuttle vectors, they may also beused in E. coli for the production and purification of proteins.Genes expressed from pART2 lead to constitutive expression fromhdnOp, whereas expression from pART3 is reduced due to the presenceof the hnoR gene.

We have proven in this work the functionality and usefulnessof the pART2 and pART3 vectors for protein overproduction inArthrobacter spp. These vectors are particularly valuable forthe production of proteins, which cannot be produced in theiractive form in heterologous systems. These plasmids will beused to overproduce active nicotine dehydrogenase and ketonedehydrogenase for biochemical studies with their natural host,A. nicotinovorans. These enzymes are inactive when overproducedin E. coli (P. Sachelaru and R. Brandsch, unpublished) becausethe molybdopterin cytosine dinucleotide cofactor required fortheir activity is not made in E. coli, which instead synthesizesthe molybdopterin guanine dinucleotide cofactor. In addition,these plasmids will be used as essential tools in genetic complementationstudies with Arthrobacter spp.

Nucleotide sequence accession numbers.

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Nucleotide sequence accession...

The DNA sequences of the two plasmids described in this studyhave been deposited in GenBank under accession numbers DQ191047(pART2) and DQ191048 (pART3).

ACKNOWLEDGMENTS

This work was supported by a grant from the Deutsche Forschungsgemeinschaftto R.B.

We thank K. H. Gartemann, Bielefeld, Germany, for providingplasmid p25435.

FOOTNOTES

* Corresponding author. Present address: Laboratory of Molecular Biophysics, The Rockefeller University, 1230 York Avenue, Box 42, New York, NY 10021. Phone: (212) 327-7220. Fax: (219) 327-7222. E-mail: sanduc{at}rockefeller.edu.

REFERENCES

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