Journal of Bacteriology, September 2003, p . 5269-5274, Vol . 185, No . 17

Complete Nucleotide Sequence and Genetic Organization of the 210-Kilobase Linear Plasmid of Rhodococcus erythropolis BD2

Christiane Stecker,¹ Andre Johann,² Christina Herzberg,³ Beate Averhoff,^1,3* and Gerhard Gottschalk^2,3

Department Biologie I, Bereich Genetik und Mikrobiologie, Ludwig-Maximilians-Universität, D-80638 Munich,¹ Laboratorium für Genomanalyse,² Abteilung für Allgemeine und Angewandte Mikrobiologie, Institut für Mikrobiologie und Genetik, Georg-August-Universität, D-37077 Göttingen, Germany³

Received 3 February 2003/ Accepted 19 May 2003

Rhodococcus erythropolis strain BD2 is a gram-positive isopropylbenzene (IPB) degrader and was found to cometabolize trichloroethene with dependence on IPB as an inducing substrate . Analysis of the IPB degradation pathway led to the finding that strain BD2 harbors a linear transmissible plasmid, pBD2, of approximately 210 kb carrying the ipb genes for IPB and trichloroethene oxidation and mediating arsenite and mercury resistance (4, 5) . Linear plasmid pBD2 was sequenced to further characterize its genetic organization .

General features of pBD2. To perform a complete sequence analysis, pBD2 linear DNA was isolated from R . erythropolis BD2 as described recently (4) and a cosmid library comprising 350 clones was generated by use of pWE15 (17) . To map the cosmids on pBD2, hybridization studies with digoxigenin-labeled probes were performed under high-stringency conditions as described by Anderson and Young (1) . The first cosmid was mapped with a probe of pMK34 carrying the ipb genes (8) . Further rounds of hybridization with labeled end fragments of mapped cosmids led to the identification of seven cosmid clones covering 96% of pBD2 . The cosmids were analyzed with respect to the presence of chromosomal contaminations and chimeric DNA, respectively . After assembly of the sequence generated from a small insert library of the selected cosmid DNAs in pTZr19 (12) and from pBD2 end fragments (cloning is described below), two gaps remained . One gap of 931 bp was closed by sequencing clones from a pDA71 small insert plasmid library (8) . The other gap of 212 bp was closed by PCR using primers deduced from the sequence flanking the gap (P1, 5'-GAGCCACACAAACACCAG-3', and P2, 5'-GCACGAAGTATGGCGAAC-3') .

Sequence data were analyzed with the software package (version 10.0) of the Genetics Computer Group (University of Wisconsin Biotechnology Center), and similarity searches were done by BLAST and FASTA .

The average GC content of the 210,205-bp plasmid pBD2 is 62.2%, which is lower than the average GC content of rhodococcal genomes (64 to 72%) . Interestingly, the GC content was found to increase from the left to the right end . The region from the left end to 91 kb comprises 60.7% GC on average (region A), and the region from 91 kb to the right end contains 63.4% GC (region B) .

The coding region comprises 87.6% of the plasmid, and a total of 212 open reading frames (ORFs) were identified (Table 1), and 97 of them could be assigned to six functional groups: 7 code for plasmid maintenance and replication, 16 code for transport and metalloresistance, 23 code for catabolism, 32 code for transposition, 14 code for regulation, and 5 code for protein modification . Forty-seven ORFs code for conserved proteins, and 68 ORFs are hypothetical . Their locations on the plasmid are depicted in Fig . 1 .

 
The deduced proteins of 20 of the pBD2 ORFs, are similar to proteins encoded by other linear plasmids; for example, PBD2.001 shows significant similarities of 73 and 54%, respectively, to 201L1 of the linear plasmid pHG201 from Rhodococcus opacus MR11 (7) and to FIR1 of linear plasmid pFiD188 from Rhodococcus fascians D188 (11) . PBD2.002 exhibits a similarity of 54% to FIR2 of pFiD188 . Eighteen ORFs (PBD2.014 to PBD2.018, PBD2.020 to PBD2.025, PBD2.027, PBD2.052, PBD2.054 to PBD2.056, PBD2.064, and PBD2.065) show significant similarities to ORFs of the 350-kb linear plasmid SCP1 from S . coelicolor A3(2) (20) .

The protein products of six ORFs within region A—PBD2.002, PBD2.010, PBD2.027, PBD2.059, PBD2.072, PBD2.086, and the ORF located close to the right terminus, PBD2.200—show similarities to proteins implicated in plasmid maintenance and DNA processing (Table 1) . In addition, all of the ORFs conserved on linear plasmids and nearly none of the potential transposon functions were detected within these regions . It is tempting to speculate that several of these hypothetical proteins are implicated in plasmid maintenance, especially those which are conserved and widely distributed among linear plasmids . Taken together, these findings suggest that region A and the right pBD2 terminus are essential for plasmid maintenance and DNA processing .

Plasmid dynamics in the region encoding resistance and catabolic functions. Within the high-GC region, region B, a cluster of conserved genes mediating pBD2-encoded arsenite resistance was detected . Ten kilobases downstream of the resistance functions, the ipb gene cluster, encoding the three subunits of the IPB dioxygenase, the 3-isopropylcatechol dioxygenase, and the IPB dihydrodiol dehydrogenase, was found (ORFs PBD2.153 to PBD2.158) (Table 1) . The deduced proteins are 94 to 100% similar to the analogous proteins of a linear-plasmid-encoded biphenyl (BPH) degradation pathway in Rhodococcus sp . strain RHA1 and the BPH degradation pathway in Rhodococcus sp . strain I1 (16) (accession no . CAA06877) . The deduced proteins of two ORFs downstream of the ipb genes, PBD2.159 and PBD2.160, show 62 to 80% similarities to two-component signal transduction systems of the BPH degradation pathways in Rhodococcus sp . strain M5 and R . erythropolis TA421 (9) (accession no . AB014348) . The close association together with the very high similarities suggests that this potential two-component regulatory system represents the IPB pathway regulatory system . The product of ORF PBD2.174, located 10 kb downstream of the ipb cluster, is identical to the 2-hydroxy-6-oxohepta-2,4-dienoate hydrolase (EtbD1) in Rhodococcus sp . strain RHA1 (19) . Therefore, PBD2.174 is designated ipbD .

Taken together, the similarities of the key enzymes and the regulators of the IPB degradative pathway genes in R . erythropolis BD2 and the linear-plasmid-encoded functions of BPH degradation pathways indicate that the ipb and bph operons have been distributed among gram-positive soil bacteria via linear-plasmid-mediated horizontal gene transfer .

The ipb structural and regulatory genes are flanked by a total of 22 ORFs showing significant similarities to insertion sequences, integrases, and transposases (Fig . 1; Table 1) . This high number of transposon-related ORFs in the close vicinity of the ipb genes indicates that the ipb genes could have been acquired via transposition events . Furthermore, this suggests that this part of the plasmid has undergone a high frequency of dynamic rearrangements .

Structural characteristics of the pBD2 termini. The presence of terminal proteins bound to the 5' ends of pBD2 was demonstrated by exonuclease, exonuclease III, and mung bean nuclease treatment and gel retardation analyses under proteolytic and nonproteolytic conditions according to the protocol of Kalkus et al . (6) . The two terminal fragments, a 1.5-kb KpnI and a 3.8-kb BamHI fragment, were cloned from pBD2 DNA isolated under proteolytic conditions . Additionally, a 4.9-kb EcoRI/NotI fragment with a 1-kb overlap of the KpnI fragment was cloned from pBD2 .

It is apparent from sequence analyses of the pBD2 termini that pBD2 does not contain long TIRs and that the similarity of the terminal sequences at both ends is reduced to two inverted repeats which share a central motif, GCTXCGC . This motif is characteristic of linear rhodococcal plasmids and is suggested to be involved in extending the 5' lagging strand after each replication round (3, 7) . In addition to this conserved central motif, the linear plasmids pBD2 and pHG201 from R . opacus MR11 and pRHL2 from Rhodococcus sp . strain RHA1 (16) show significant similarities over a range of 1,000 bp at the left termini and of 130 bp of the right termini . The short rhodococcal TIRs are in contrast to the long TIRs of the Streptomyces linear plasmids, which comprise many palindromes with the potential to form very stable complex secondary structures at the 3' ends of linear replicons . Although the significance is not clear, the conservation of these structures suggests an important biological role, which obviously is not conserved with respect to linear rhodococcal plasmids .

Nucleotide sequence accession number. The sequence of the linear plasmid pBD2 from R . erythropolis BD2 is available in GenBank under the accession number AY223810 . The graphical representation and a detailed annotation are available at the Laboratorium für Genomanalyse, Göttingen, Germany, at http://www.g2l.bio.uni-goettingen.de .

We thank Maria Kesseler and Jutta Kalkus for providing the ends of the linear plasmid . We are grateful to Arnim Wiezer and Heiko Liesegang for assistance in analyzing the sequence information and for submission of the sequence information to the databanks .

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