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Journal of Bacteriology, January 2004, p. 212-225, Vol. 186, No. 1
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.1.212-225.2004

Characterization of the 101-Kilobase-Pair Megaplasmid pKB1, Isolated from the Rubber-Degrading Bacterium Gordonia westfalica Kb1

Daniel Bröker,1 Matthias Arenskötter,1 Antje Legatzki,2 Dietrich H. Nies,2 and Alexander Steinbüchel1*

Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, D-48149 Münster,1 Institut für Mikrobiologie, Martin-Luther-Universität Halle-Wittenberg, D-06099 Halle, Germany2

Received 16 July 2003/ Accepted 29 September 2003


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ABSTRACT
 
The complete sequence of the circular 101,016-bp megaplasmid pKB1 from the cis-1,4-polyisoprene-degrading bacterium Gordonia westfalica Kb1, which represents the first described extrachromosomal DNA of a member of this genus, was determined. Plasmid pKB1 harbors 105 open reading frames. The predicted products of 46 of these are significantly related to proteins of known function. Plasmid pKB1 is organized into three functional regions that are flanked by insertion sequence (IS) elements: (i) a replication and putative partitioning region, (ii) a putative metabolic region, and (iii) a large putative conjugative transfer region, which is interrupted by an additional IS element. Southern hybridization experiments revealed the presence of another copy of this conjugational transfer region on the bacterial chromosome. The origin of replication (oriV) of pKB1 was identified and used for construction of Escherichia coli-Gordonia shuttle vectors, which was also suitable for several other Gordonia species and related genera. The metabolic region included the heavy-metal resistance gene cadA, encoding a P-type ATPase. Expression of cadA in E. coli mediated resistance to cadmium, but not to zinc, and decreased the cellular content of cadmium in this host. When G. westfalica strain Kb1 was cured of plasmid pKB1, the resulting derivative strains exhibited slightly decreased cadmium resistance. Furthermore, they had lost the ability to use isoprene rubber as a sole source of carbon and energy, suggesting that genes essential for rubber degradation are encoded by pKB1.


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INTRODUCTION
 
The genus Gordonia was proposed by Tsukamura for coryneform bacteria isolated from sputa of patients with pulmonary disease or from soil (65-67). Gordonia belongs to the so-called CMN group (Corynebacterium, Mycobacterium, and Nocardia) of actinomycetes, which synthesize mycolic acids (13, 59). Gordonia strains also play an important role in bioremediation and biodegradation of pollutants and have attracted much interest in recent years due to their unusual and diverse metabolic capabilities (23, 29, 30, 70). Three strains of Gordonia polyisoprenivorans (2, 37) and the novel species G. westfalica Kb1 (39) were described as bacteria able to degrade natural rubber and synthetic cis-1,4-polyisoprene, which allows species of this genus to serve as model organisms for the investigation of the hitherto unknown biochemical and molecular mechanisms of rubber biodegradation (36). So far, no native plasmids of the genus Gordonia have been detected. Since factors encoded by linear or circular plasmids are often involved in degradation of complex xenobiotics (62), rubber-degrading bacterial strains were screened for the occurrence of extrachromosomal DNA. This publication gives the first example and the complete DNA sequence of a native Gordonia plasmid, which also provides the basis for the development of shuttle vectors that may serve as a novel genetic tool for the study of Gordonia and related genera.


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MATERIALS AND METHODS
 
Bacterial strains, plasmids, and cultivation conditions. Bacteria and plasmids used in this study are listed in Table 1. All strains of the genera Gordonia and Rhodococcus were grown at 30°C in standard I complex nutrient broth (St-I; E. Merck AG, Darmstadt, Germany), mycobacterial strains were grown at 30°C in Luria-Bertani broth (LB) (52) containing Tween 80 (50 ml/liter of broth), and strains of Streptomyces were cultivated at 30°C in yeast extract-malt extract medium (24). For growth experiments with cis-1,4-polyisoprene as the sole carbon source, G. westfalica Kb1, G. westfalica Kb1-K38, and G. westfalica Kb1-K43 were cultivated at 30°C on mineral salts medium (54). Cells of Escherichia coli were cultivated at 37°C in LB broth. Antibiotics were applied according to the method of Sambrook et al. (52) and as indicated in the text. Carbon sources were added as indicated in the text. Liquid cultures were made in Erlenmeyer flasks and incubated on a horizontal rotary shaker. Solid media were prepared by the addition of agar (15 g/liter).


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  		TABLE 1. Bacterial strains and plasmids used for this study

Determination of metal tolerance. For the gram-positive bacteria of the genera Gordonia, Rhodococcus, and Mycobacterium, St-I agar plates containing 100 to 800 µM CdCl2 were prepared in a 0.1 M stock solution of CdCl2 H2O (E. Merck AG) which was sterilized by autoclaving. The plates were inoculated with the bacterial strains and tolerance for cadmium was evaluated after incubation for 3 days at 30°C. Resistance of E. coli strains was determined by use of dose-response curves generated by growth in Lennox medium (Becton-Dickinson, Sparks, Md.). The medium contained 50 µg of anhydrotetracycline per liter to induce expression of the plasmid-encoded genes in these strains. Overnight cultures of E. coli strains were used to inoculate parallel cultures with increasing metal concentrations. Cells were cultivated for 16 h with shaking at 37°C, and the optical density was determined at 600 nm.

Cadmium uptake experiments. Cadmium uptake experiments were performed with E. coli cells in Tris buffer (10 mM; pH 7.0) by filtration, as published previously (44). The cells were cultivated in Tris-buffered mineral salts medium in the presence of 2 g of glucose per liter and 1 g of yeast extract per liter, up to 100 Klett units, when 200 µg of anhydrotetracycline per liter was added, and incubation was continued with shaking for 3 h at 30°C. Cells were harvested by centrifugation, washed, and suspended in 10 mM Tris-HCl buffer (pH 7.0). Radioactive 109Cd2+ (87.4 GBq/g) was added at a concentration of 10 µM, incubation was continued with shaking at 30°C, and the metal content in washed cells (dry weight) was determined at various time points by use of an equilibration curve. Background binding was not subtracted.

Isolation, analysis, and manipulation of DNA. Plasmid DNA was prepared from crude lysates by the alkaline extraction method (8). Before lysis, cells of Gordonia, Rhodococcus, and Mycobacterium were incubated in the presence of lysozyme (2 g/liter) for 2 h at 37°C. Total DNA of Gordonia was prepared as described by Ausubel et al. (4), with modifications as follows: cells of 50-ml cultures were harvested by centrifugation and suspended in 8.5 ml of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and 1 ml of lysozyme solution (10 g of TE per liter). After incubation at 37°C for 2 h, 500 µl of a sodium dodecyl sulfate solution (100 g/liter) and 50 µl of a proteinase K solution (20 g of TE per liter) were added and mixed gently. After additional incubation at 37°C for 1 h, 5 ml of 5 M NaCl and 1.5 ml of a CTAB solution (100 g of cetyltrimethylammonium bromide per liter of 0.7 M NaCl) were added and the solution was incubated at 65°C for 20 min. DNA was digested with various restriction endonucleases (Gibco/BRL, Gaithersburg, Md.) under the conditions recommended by the manufacturer. All other genetic procedures and manipulations were conducted as described by Sambrook et al. (52).

Extrachromosomal DNA was detected by pulsed-field gel electrophoresis (PFGE). Preparation of total DNA embedded in low-melting-temperature agarose and linearization of circular plasmid DNA were done according to the methods of Barton et al. (5). Electrophoresis was performed in the CHEF-DR III system (Bio-Rad GmbH, Munich, Germany).

Plasmid curing. For generation of plasmid-free mutants of G. westfalica Kb1, heat curing was performed (43). Fifty milliliters of St-I medium in 300-ml Erlenmeyer flasks was inoculated with cells of G. westfalica Kb1 and incubated at 42°C. Every 2 days, Erlenmeyer flasks with fresh St-I medium were inoculated with 1 ml of the grown culture and subsequently cultivated at 42°C. After 20 passages, the cells were diluted and spread on St-I agar plates. The colonies obtained were screened for the absence of plasmid pKB1 as described previously (27).

Cloning procedures. The cadA gene from G. westfalica Kb1 was amplified by PCR from plasmid pKB1 with the primers P1 and P2 (listed in Table 2) and was cloned downstream of the tet promoter in plasmid pASK3 (IBA GmbH, Göttingen, Germany), leading to plasmid pECA34. The two insertion sequence (IS) elements, IS1 and IS3 (comprising ORF1/ORF2 and ORF53), were amplified from total DNA of wild-type G. westfalica Kb1 and the pKB1-free mutants with the primer sets P3 plus P4 and P5 plus P6 (listed in Table 2). The two IS elements, IS1 and IS3, with their contiguous regions, were then amplified with the two primer sets P3 plus P7 and P8 plus P6 (Table 2).


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  		TABLE 2. PCR and sequencing primers used in this study

Gene transfer. Hybrid plasmids containing oriV of pKB1 were transferred to species of the genera Gordonia, Mycobacterium, and Rhodococcus by electroporation in a Model 2510 electroporator (Eppendorf-Netheler-Hinz, Hamburg, Germany). Preparation of the electrocompetent cells and the execution of electroporation were done as described recently (3, 28). For transformation of Streptomyces coelicolor strain A3(2) and Streptomyces lividans strain TK23, protoplasts of these strains were prepared as described by Okanishi et al. (47), Bibb et al. (7), and Thompson et al. (63). Transformations were done according to the rapid small procedure, as described by Bibb et al. (6), Thompson et al. (63), and Okanishi et al. (46). Conjugational plasmid transfer was carried out, applying a previously described protocol (18) employing E. coli S17-1 as donor and G. polyisoprenivorans as recipient.

DNA sequence analysis. To obtain the complete sequence of megaplasmid pKB1, we constructed a shotgun library. Plasmid DNA was fragmented by hydro-shearing, cloned into pGEM-T vector DNA (Promega, Madison, Wis.), and sequenced by MWG Biotech (Ebersberg, Germany), resulting in fivefold sequence coverage. A few regions for which only uncertain sequences were obtained were sequenced by employing individual primers. Hybrid plasmids containing oriV-comprising DNA fragments were sequenced with IRD800-labeled universal and reverse primers, using the SequiTherm EXCEL II Long-Read L-C kit and a LI-COR 4200 sequencer (LI-COR Biosciences, Lincoln, Nebr.).

Open reading frames (ORFs) were identified by use of the program GeneMark (http://opal.biology.gatech.edu/GeneMark/) to indicate start codons, stop codons, and codon usage statistics for each reading frame (41). Database searches of the predicted protein sequences were performed with the BLAST program provided by EMBL/Heidelberg (1). Multiple sequence alignments were carried out with the program BioEdit (21). Protein sequences were also analyzed for functionally important motifs by use of SMART software (http://smart.embl-heidelberg.de/) (35, 55).

Nucleotide sequence accession number. The DNA sequence of pKB1 has been deposited in the EMBL database under accession number AJ576039.


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RESULTS AND DISCUSSION
 
Detection and characterization of the megaplasmid pKB1. Since metabolic pathways involved in degradation of unusual compounds may be encoded by bacterial plasmids, the rubber-degrading bacteria G. polyisoprenivorans strains Kd2T, VH2, and Y2K and G. westfalica strain Kb1 were screened for the occurrence of plasmids. Cells were embedded in low-melting-temperature agarose and lysed, and the immobilized total DNA was treated with Aspergillus oryzae S1 nuclease to linearize possible circular plasmids. When the DNA samples were separated by PFGE, only those from G. westfalica Kb1 displayed a distinct band, which was visible only after S1 treatment and corresponded to a size of about 100 kbp (data not shown). Consequently, this strain contained a circular megaplasmid that we named pKB1. Plasmid pKB1 represents the first extrachromosomal DNA identified for the genus Gordonia.

To investigate the possible metabolic function of pKB1, we completely sequenced it (Fig. 1). Plasmid pKB1 is a circular DNA molecule with a size of 101,016 bp, which confirmed the PFGE results. The G+C content was 66 mol%, as is expected for DNA from a bacterium of the CMN group. The plasmid carried 105 ORFs, 47 on one DNA strand and 58 on the other. The predicted products of 67 ORFs were related to proteins in the current databases, as indicated by a BLAST analysis, of which 46 were homologous to proteins with putative functions (Table 3). According to the predicted functions of their products, the genes located on plasmid pKB1 were clustered into three major regions that were all flanked by putative IS elements: a replication and partitioning region, a metabolic region, and a conjugational transfer region (Fig. 1). Investigations were carried out with ORF8 within the replication and partitioning region, which encodes a replication gene and mediates autonomous replication, and with ORF34, which encodes cadA, a cadmium P-type ATPase.



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  		FIG. 1. Schematic representation of the ORFs of the 101,016-bp plasmid pKB1. The predicted ORFs located on plasmid pKB1 are shown as arrows. The direction of the arrow indicates the DNA strand on which the ORF is located. The colors of the arrows group the assigned functions of the putative ORF products as follows: orange, ORFs related to replication and partitioning; yellow, ORFs with putative metabolic functions; blue, ORFs related to heavy-metal resistance; dark green, ORFs related to conjugation; red, ORFs related to IS elements and transposons; green, putative regulator proteins; pink, putative peptidases; light blue, putative membrane proteins; dark gray, conserved hypothetical ORFs; light gray, ORFs with no known function. The small black arrows indicate binding sites and orientations of pKB1-specific primers (Table 2).


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  		TABLE 3. Summary of ORFs identified by significant homology (BLAST search) or GENEMARK prediction

Identification of oriV of plasmid pKB1. The replication and partitioning region of plasmid pKB1 was further characterized, with the intention of constructing E. coli-Gordonia shuttle vectors. For cloning and identification of the origin of replication, oriV, of plasmid pKB1, a plasmid library of pKB1 was generated by use of the mobilizable vector pBBR1MCS-2, which is not able to replicate in Gordonia strains (unpublished data). The DNA of pKB1 was partially digested with XhoI and ligated to XhoI-linearized pBBR1MCS-2 plasmid DNA. Since plasmid transfer into G. westfalica yielded no transformants, the DNA mixture was electroporated into G. polyisoprenivorans strain VH2. The cells were screened for 4 days at 30°C for kanamycin resistance, which is encoded by plasmid pBBR1MCS-2.

The resulting kanamycin-resistant colonies of G. polyisoprenivorans strain VH2 contained hybrid plasmids that were composed of at least three XhoI fragments: plasmid pBBR1MCS-2 DNA plus two additional fragments, of 2,331 and 4,927 bp (data not shown). Neither fragment alone was able to mediate replication in G. polyisoprenivorans VH2 (data not shown), indicating that essential elements were located on both fragments, which together correspond to the 7,258-bp region from nucleotides 3,743 to 11,000 on the sequence map of plasmid pKB1. This region contains nine ORFs (ORF 6 to ORF 14) (Fig. 2A) and five tandem repeats, with a period size of 16 bp, downstream of ORF8 (nucleotides 7,558 to 7,637) (Fig. 2B).



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  		FIG. 2. Mapping of the oriV region of plasmid pKB1. (A) The ability of DNA fragments of plasmid pKB1 to confer autonomous replication in Gordonia to a suicide plasmid was tested in G. polyisoprenivorans. Successfully replicating hybrid plasmids conferred kanamycin resistance to the host, as indicated on the right. Initially, a 7-kbp dual XhoI fragment of plasmid pKB1 was identified in a gene bank of this plasmid. A hybrid plasmid containing this 7-kbp XhoI region (pBBRKB1:XhoI7) and the ORFs carried by it is diagrammed at the top. Below, the sizes and locations of derivatives of the 7-kbp DNA fragment are given. The numbers at the right and left margins of the fragments indicate the exact positions of these fragments in the map of plasmid pKB1. (B) To identify the smallest DNA region of plasmid pKB1 that is essential to confer stable replication, three fragments were amplified by PCR: pBBRKB1:XhoI2,6 (top), pBBRKB1:XhoI2,3 (middle), and pBBRKB1:ORF82,0 (bottom). The gray arrows indicate the size and position of ORF8, which encodes a protein with unknown function. The ORF8 product may be translated by two possible start codons, ATG (at position 5,323 on the pKB1 map) and GTG (at position 5,344). The stop codon, TAA, of ORF8 is at position 7,441. Moreover, the positions of two possible cis-acting sites are given, one a 39-bp sequence upstream of ORF8 with 92% identity to a region of plasmid pSOX from Rhodococcus sp. strain X309 (nucleotides 5,327 to 5,365) and the other downstream tandem repeats (nucleotides 7,558 to 7,637). Single restriction sites for BclI (5,549), BglII (5,799), and KpnI (7,034) are also shown.

To identify the genes and cis-acting elements essential for replication and partitioning, we deleted the 7,258-bp gene region from both ends by exonuclease III and endonuclease treatment. The autonomous replication ability of the resulting fragments was tested (Fig. 2A). The smallest of the fragments able to mediate stable plasmid replication in G. polyisoprenivorans strain VH2 had a size of 2,332 bp and harbored only the predicted ORF8 plus a sequence of 39 bp (nucleotides 5,327 to 5,365) (Fig. 2B). Further reduction of the 2,332-bp fragment by use of restriction enzymes (BclI, BglII, and KpnI) (Fig. 2B) or by amplification of ORF8 by PCR did not generate fragments that were able to maintain stable plasmid replication. The ORF8 product had a predicted size of 680 amino acid (aa) residues. Neither the sequence of this fragment nor the predicted amino acid sequence of ORF8 showed similarities to DNA elements or proteins known to be involved in plasmid replication. The only element associated with replication function was the 39-bp sequence. Thus, the 39-bp sequence was also required for replication. This sequence was 92% identical at the nucleotide level to a region of plasmid pSOX from Rhodococcus sp. which is located between the replication genes of that plasmid (14). The tandem repeats downstream of ORF8 were not essential for autonomous replication.

ORF9 may encode an ATPase (238 aa) involved in chromosome partitioning. The predicted protein, containing a parA domain, was 62% identical to a hypothetical protein of a virulence plasmid from Rhodococcus equi (60). ORF10 encodes a predicted protein (109 aa) comprising a helix-turn-helix-like motif of the CopG family, which is involved in dimerization of RepA proteins but not in DNA binding. Homologies were obtained to hypothetical proteins of the virulence plasmids p33701 and p103 of R. equi strains ATCC 33701 and 103, respectively. These are plasmids with an origin of replication (oriV) that may belong to a novel type (60). The predicted ORF11 product (293 aa) was 42% identical to a putative prophage excisionase, which again is harbored by the virulence plasmids p33701 and p103 as part of a cluster of replication genes (60). The functions of the other ORFs in this region remained unknown or could be not related to replication.

Host range of pKB1 replicon. The broad-host-range cloning vectors pBBR1MCS-2 (Kmr) and pBBR1MCS-5 (Gmr), mediating resistance to kanamycin and gentamicin, respectively, were chosen for construction of E. coli-Gordonia shuttle vectors. Both plasmids and the 2,332-bp oriV-containing fragment of plasmid pKB1 were amplified by PCR (primer pairs P9-P10 and P11-P12, respectively) (Table 2), ligated, and tested for replication in E. coli and Gordonia, leading to the shuttle vectors pDBMCS-2 and pDBMCS-5 (Fig. 3). Both new vector plasmids possess an extended multiple cloning site (MCS), allow for blue-white selection in E. coli via alpha complementation, are compatible with IncP, IncQ, IncW, ColE1, and P15a group plasmids, and can be mobilized if the RK2 transfer functions are provided in trans. We also constructed derivatives of plasmids pBBR1MCS (Cmr), pBBR1MCS-3 (Tcr), and pBBR1MCS-4 (Apr), containing the 2,332-bp fragment comprising oriV cloned into the MCS, which did not mediate resistance to the respective antibiotics in G. polyisoprenivorans.



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  		FIG. 3. Physical maps of two E. coli-Gordonia shuttle vectors. The 2,332-bp fragment of plasmid pKB1 that is the smallest essential part necessary to confer stable replication in Gordonia was amplified by PCR (open white bar) and cloned into suicide plasmids pBBR1MCS-2 and pBBR1MCS-5 (31), leading to the new vector plasmids pDBMCS-2 and pDBMCS-5, respectively. The genes located on the suicide plasmids are indicated by black arrows. Unique restriction sites present in the MCS of each plasmid are shown.

To test the host range of oriV from plasmid pKB1, the oriV-carrying hybrid plasmid pBBRKB1:XhoI7 was transferred to different species of the genera Gordonia, Rhodococcus, and Mycobacterium by electroporation as well as to two species of the genus Streptomyces by transformation of protoplasts (Table 4). Kanamycin-resistant derivatives of Mycobacterium smegmatis mc2155, Rhodococcus opacus PD630, G. polyisoprenivorans VH2, and G. polyisoprenivorans Y2K were obtained, indicating functional expression of the pDBMCS-2-encoded kanamycin resistance gene and stable propagation of the vector plasmid. For all other strains listed in Table 4, no kanamycin-resistant colonies could be obtained. This indicates a rather narrow host range of the oriV from plasmid pKB1 that is limited to Gordonia strains and closely related bacteria. In addition to the E. coli-Rhodococcus shuttle vector systems based on pNC903, which were previously described as functional replicons for G. polyisoprenivorans Y2K and VH2 (3), the two E. coli-Gordonia shuttle vectors constructed in this study represent a second functional replication system.


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  		TABLE 4. Transfer of pKb1 oria

Generation of a plasmid-free mutant strain of G. westfalica Kb1. Thirty-four ORFs were localized within the metabolic region of pKB1. Three ORFs represented putative regulators, four represented putative membrane proteins, four represented putative peptidases, eight represented putative metabolic functions, and four were putatively involved in heavy-metal resistance. No function could be assigned to 11 ORFs. For identification of the functions encoded by plasmid pKB1, a plasmid-cured derivative strain of G. westfalica Kb1 was generated by heat curing (43). This method was used because the bacterium is very sensitive to mitomycin C. The curing procedure was successful, and 14 of 50 tested mutant strains did not contain plasmid pKB1 DNA (data not shown). The plasmid-free G. westfalica strains Kb1-K38 and Kb1-K43 were further characterized.

Since plasmid pKB1 contains many putative IS elements, total DNA isolated from the plasmid-free strain Kb1-K43 was analyzed for possible chromosomal insertions of parts of pKB1. Southern hybridization experiments were performed with the complete plasmid pKB1 as probe and total DNA from the plasmid-containing wild-type strain G. westfalica Kb1 serving as a positive control, and all 11 EcoRI fragments of plasmid pKB1 were visible (Fig. 4). An additional fragment (8,000 bp) for the positive control was observed which could not be derived from plasmid pKB1. When total DNA from the plasmid-free derivative strain Kb1-K43 was used instead of wild-type DNA, signals corresponding to the two largest EcoRI fragments (16,247 and 24,349 bp) of plasmid pKB1 and the signal corresponding to the 8,000-bp fragment were still obtained. This indicated the presence of DNA that was highly homologous to parts of plasmid pKB1 in the chromosome of the cured derivative strain Kb1-K43.



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  		FIG. 4. Southern hybridization analysis for detection of pKB1 in total DNA of pKB1-deficient mutant Kb1-K43 and G. westfalica Kb1. The isolated total DNA of the G. westfalica Kb1 wild type and of the pKB1-deficient mutant Kb1-K43 was digested with EcoRI, separated by agarose gel electrophoresis, and stained with ethidium bromide (A), and the fragments were transferred to a nylon membrane for Southern blotting (B). The DNA of complete plasmid pKB1 was used as a digoxigenin-labeled probe. {lambda}:PstI, {lambda} DNA digested with PstI; WT, total DNA of G. westfalica Kb1 wild type; K43, total DNA of G. westfalica K43.

The two IS elements that are putatively responsible for integration, comprising ORF1/ORF2 and ORF53 (only weak homologies to tnpA from Arthrobacter nicotinivorans, GenBank CAA65743), respectively, coding for putative transposases, were identified by PCR (Fig. 1). The PCR products generated with the primer sets P3-P4 and P5-P6 (Table 2; Fig. 1), containing only the sequences of the putative IS elements (ORF1/ORF2 and ORF53), could be amplified from both the wild type and the mutant Kb1-K43, whereas the PCR products generated with the primer sets P3-P7 and P8-P6 (Table 2; Fig. 1), containing the complete sequences of the two putative IS elements and parts of the adjacent regions, which were expected to be absent from the mutant Kb1-K43 (Fig. 4), could indeed only be amplified from the genome of the wild type and not from that of the mutant Kb1-K43. Therefore, an insertion into the chromosome of the two IS elements harboring ORF1/ORF2 and ORF53 and of the 51,527-bp region between these two IS elements must have already occurred in the wild-type strain Kb1 of G. westfalica. The insertion was then maintained in the chromosomes of the pKB1-free mutant; however, this insertion contained the complete conjugation region only. This demonstrates that the metabolic and heavy-metal resistance genes were eliminated during plasmid curing and that insertion of the conjugation region may have occurred early in the history of G. westfalica.

In conclusion, the metabolic region may constitute a catabolic transposon, which is a widespread occurrence among eubacteria and is found in gram-negative as well as gram-positive bacteria. Composite transposons are flanked by related, but not necessarily identical, IS elements and may be very large, exceeding 50 kbp (62).

The plasmid-free mutant strain of G. westfalica was not able to grow on cis-1,4-polyisoprene as sole carbon source. Since several examples for plasmid-encoded degradation pathways are known (62), the possible involvement of plasmid pKB1 in rubber degradation was tested. The G. westfalica strain Kb1 wild type and plasmid-free mutant derivatives were cultivated for 40 days in the presence of cis-1,4-polyisoprene as the sole carbon source. While the turbidity at 600 nm of the wild-type culture increased from 0.15 to 0.75 during incubation, that of the mutant strains did not. This indicated that the mutant strains were not able to use cis-1,4-polyisoprene as a sole carbon source and that, therefore, at least some genes of plasmid pKB1 of G. westfalica must be involved in rubber degradation.

Genes essential for rubber biodegradation are probably encoded by the 49,489-bp region that is definitively missing from the cured mutants (Fig. 4). Since little is known about the biodegradation of rubber (25, 36) and proteins involved in this biochemical process have not been identified, it is not surprising that many ORFs with a hitherto unknown function are located in this metabolic region. These are strong candidates for rubber degradation genes. Electron transport proteins are often involved in catabolic and anabolic reactions and may also be involved in the initiation of biodegradation of rubber. Three ORFs (ORF27, ORF39, and ORF42) encode putative proteins that may be involved in cytochrome c biosynthesis. ORF27 encodes a protein of 520 aa, exhibiting 52% identity to a conserved hypothetical protein from Mycobacterium tuberculosis, which is putatively involved in cytochrome c biosynthesis. ORF39 encodes a protein of 271 aa, whose predicted protein comprises a DsbD (cytochrome c biogenesis) domain, and exhibits 51% identity to CCDA from Mycobacterium bovis. ORF42 encodes a protein of 597 aa which contains a COX1 domain and exhibits 77% identity to the cytochrome c oxidase subunit I isolated from M. tuberculosis. Cytochromes are involved in various electron transport systems and do not function only in aerobic or anaerobic respiration (17), e.g., cytochrome c catalyses peroxidase-like reactions in the presence of an electron acceptor like hydrogen peroxide (68). During rubber biodegradation, cytochrome c may catalyze epoxidation of the cis-1,4-polyisoprene molecule.

A putative epoxide hydrolase encoded by ORF6, which is localized about 10 kbp downstream of the metabolic region, may subsequently catalyze hydrolysis of the epoxide to the corresponding diol as has been shown for other epoxide hydrolases (69). A similar initiation of isoprene degradation was described by Johan et al. (26) for Rhodococcus sp. strain AD45. Such a sequence of reactions would in principal be consistent with the occurrence of cleavage products identified during biodegradation of natural rubber or related compounds (9, 10, 16, 53, 64). In addition, ORF28 and ORF40 encode proteins which are putatively necessary for the transport of electrons. The predicted ORF40 product (209 aa) contains a thioredoxin domain and exhibits 52% identity to a hypothetical protein isolated from Mycobacterium leprae.

Since biodegradation of rubber must occur outside of the cell or at the cell surface, membrane and transport proteins most probably perform a crucial function in rubber biodegradation. Furthermore, special proteins or other biopolymers may be required to establish a tight contact of the cells with the rather hydrophobic rubber molecules, in particular for those bacteria exhibiting adhesive growth as a biofilm on natural rubber, such as all species of the genus Gordonia (36). The protein encoded by ORF4, which is located 10 kbp downstream of the metabolic region, exhibited weak homology to TmtpC from M. smegmatis, which is involved in the transport of glycopeptidolipids through the cytoplasm membrane. Glycopeptidolipids are especially necessary for the formation of biofilms on polyvinylchloride (48). Finally, the probably complex biochemical process of rubber biodegradation and of other functions encoded by pKB1 will most probably be strongly regulated, thus explaining the occurrence of several genes for putative regulator proteins on pKB1. Thus, plasmid pKB1 encodes numerous candidate proteins that may be involved in the initial attack on natural rubber and/or in facilitating physical contact between the degrading bacterium and its substrate.

Furthermore, the metabolic region harbors a putative methyltransferase (ORF31, isolated from Shewanella oneidensis; 37% identity), a putative polyprenol-phosphate-mannose synthase 2 (ORF35, isolated from M. smegmatis; 42% identity), and a putative oxidoreductase (ORF36, isolated from Bacillus cereus; 22% identity).

Cadmium sensitivity of the wild type and the plasmid-free mutant. In addition to genes encoding putative degradation pathways, plasmid pKB1 contains several genes that might be involved in heavy-metal homeostasis (ORFs 27, 29, 32, 33, 37, and 45) (Table 3). Moreover, G. westfalica was able to grow on St-I agar plates in the presence of 800 µM Cd(II) (Table 5). The bacterial species G. amicalis, G. bronchialis 43341, and G. nitida exhibited similar cadmium resistance, while the tolerance of cadmium under the same conditions of other related bacteria, G. alkanivorans 44369 (growth up to 600 µM cadmium), G. desulfuricans (growth up to 300 µM cadmium), G. polyisoprenivorans, G. sputi, M. smegmatis mc155, R. opacus PD630, Rhodococcus rhodochrous (growth up to 200 µM cadmium), G. terrae (growth up to 100 µM cadmium), was significantly lower. Thus, G. westfalica was more resistant to cadmium than 8 of 12 bacteria tested.


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  		TABLE 5. Determination of cadmium tolerance for different strains of Gordonia, Rhodococcus, and Mycobacteriuma

To investigate whether plasmid pKB1 mediates cadmium resistance in this bacterium, we compared growth of G. westfalica and the plasmid-free mutant strain Kb1-K43 in liquid medium in the presence of increasing cadmium concentrations (Fig. 5). A concentration of about 300 µM cadmium was required to decrease growth of the G. westfalica wild type to 50%, while about 40 µM cadmium was sufficient to reduce growth of the plasmid-free strain to half. Thus, the absence of plasmid pKB1 diminished cadmium resistance of G. westfalica by a factor of about 7, indicating that plasmid pKB1 is involved in cadmium resistance.



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  		FIG. 5. Effect of Cd2+ on growth of G. westfalica. Dose-response curves for cadmium were done with the G. westfalica Kb1(pKB1) wild type (•) and the plasmid-cured derivative strain K43 ({circ}).

The ORF34 gene product CadA may contribute to pKB1-mediated cadmium resistance. ORF34 from G. westfalica Kb1 plasmid pKB1 encodes a putative Cd2+/Zn2+-transporting P-type ATPase (Table 3). The predicted protein exhibits the closest similarity (58% identity) to a cation-transporting P-type ATPase G (CtpG; NP_216508) of M. tuberculosis, another gram-positive bacterium with high GC content, and to other P-type ATPases, e.g., CadA from Staphylococcus aureus, the first described example of a heavy-metal effluxing P-type or CPx-type ATPase (45).

ORF34 was amplified by PCR from plasmid DNA of G. westfalica Kb1 and the gene was cloned into plasmid pASK3. The resulting plasmid, pECA34, was transferred into E. coli strain RW3110 ({Delta}zntA), a metal-sensitive strain with a deletion in the native Zn2+/Cd2+-effluxing P-type ATPase ZntA of this bacterium (50). The G. westfalica Kb1 gene corresponding to ORF34 conferred cadmium resistance to E. coli RW3110, but not zinc resistance (Fig. 6). It was therefore designated cadA and its product was designated GoCadA.



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  		FIG. 6. Cadmium and zinc resistance of E. coli strains expressing a P-type ATPase from G. westfalica Kb1, GoCadA. Dose-response curves for cadmium (A) and zinc (B) are shown for E. coli strain RW3110 ({Delta}zntA) complemented in trans with the cadA gene of G. westfalica Kb1 cloned into plasmid pASK3 ({blacksquare}). The negative control strain is RW3110(pASK3) ({circ}), and the positive control is the wild-type strain W3110(pASK3) (•). The mean results of three (cadmium) or two (zinc) independent experiments are shown, with standard deviation bars.

Half-maximal inhibition of E. coli strain RW3110 ({Delta}zntA) and wild-type strain W3110 occurred at about 5 and 650 µM Cd2+, respectively (Fig. 6). Complementation in trans with cadA from G. westfalica Kb1 led to half-maximal inhibition at about 250 µM Cd2+. Thus, GoCadA was able to protect E. coli cells against cadmium about half as efficiently as the native efflux system ZntA.

The metal cation uptake into cells of E. coli strain RW3110 expressing GoCadA was examined (Fig. 7). Cells of metal-sensitive E. coli strain RW3110 accumulated 6.6 µmol of 109Cd2+ per g of cell dry weight within 20 min in an assay buffer containing 10 µM cadmium. In contrast, cells containing GoCadA accumulated only 1.4 µmol of cadmium per g of cell dry weight, only 20% of the amount bound to metal-sensitive control cells. Thus, GoCadA was functionally expressed in E. coli and decreased the accumulation of cadmium by E. coli cells, probably by cadmium efflux. These data indicate that GoCadA may contribute to plasmid pKB1-mediated cadmium resistance in this bacterium.



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  		FIG. 7. Presence of P-type ATPase GoCadA from G. westfalica Kb1 diminishes accumulation of Cd2+ in E. coli strain RW3110. E. coli strain RW3110 ({Delta}zntA) was complemented in trans with the gene encoding GoCadA ({blacksquare}). The negative control ({circ}) contained only the vector plasmid, pASK3. The mean values of two independent experiments with 10 µM 109Cd2+ are shown. d.w., dry weight.

Plasmid pKB1 harbored putative heavy-metal resistance genes in addition to cadA, which is a notable situation. ORF28 encodes a protein of 415 aa containing a domain of the Nramp family and exhibiting 59% identity to a divalent cation transporter integral membrane protein isolated from M. bovis. ORF38 encodes a predicted protein (314 aa) comprising a domain of the cation efflux family, and 35% identity was observed to a cobalt-zinc-cadmium resistance protein from Xanthomonas axonopodis. ORF46 encodes a protein of 730 aa and exhibits highest identity (46%) to a cation-transporting ATPase of M. leprae.

Thus, plasmid pKB1 seems to harbor two genes for P-type ATPases. Bacteria that contain more than one CPx-type ATPase of the Zn/Cd/Pb group of proteins may exhibit differentiation of the functions of these proteins, while in bacteria that contain only one of these proteins, the substrate specificity of the single transporter should be broader. In E. coli, ZntA is responsible for detoxification of Zn2+, Cd2+, and Pb2+ (49-51). In contrast, in the gram-negative bacterium Ralstonia metallidurans, the three CPx-type ATPases of the Zn/Cd/Pb group mainly concentrate on Zn2+ (ZntA), Cd2+ (CadA), or Pb2+ (PbrA) with respect to the regulation of their expression and their substrate specificities (11, 34). CadA from Bacillus subtilis confers resistance to cadmium, zinc, and cobalt (19), and Bxa1 from Oscillatoria brevis confers resistance to zinc and cadmium (40) in E. coli (34).

Conjugation region. The third region of the plasmid appears to be concerned with conjugation, a complex process which involves many genes (39). This region is divided into two parts by the presence of an IS element comprising ORF76 and ORF77. This conjugation region contains 49 detected ORFs and the putative transcriptional products of 6 ORFs exhibiting homologies to proteins putatively involved in conjugational processes. ORF67 presumably encodes a TraA-like protein with 33% identity to a protein from R. equi that contains an ATP/GTP binding motive. The transcriptional product predicted for ORF75 represents a protein of 1,631 aa, comprising a DEAD-like helicase superfamily domain, and 37% identity was observed to a putative methylase from R. equi. Further similarities were observed in particular to the plasmid-encoded conjugative transfer gene complexes (trs) of Lactococcus lactis DPC3147 (15) and S. aureus (42). This provides evidence of possible pKB1-encoded conjugative transfer and should be further investigated.


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ACKNOWLEDGMENTS
 
We are grateful for financial support provided by the Deutsche Bundesstiftung Umwelt (Osnabrück, Germany) to A.S. (AZ. 13072 within the ICBIO project), by the Deutsche Forschungsgemeinschaft to D.H.N. (Ni262/3-3), and by the Fonds der Chemischen Industrie to A.S. and D.H.N.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstrasse 3, 48149 Münster, Germany. Phone: 49-251-8339821. Fax: 49-251-8338388. E-mail: steinbu{at}uni-muenster.de. Back


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Journal of Bacteriology, January 2004, p. 212-225, Vol. 186, No. 1
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.1.212-225.2004




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