1 Interdisziplinäres Ökologisches Zentrum, Technische Universität Bergakademie Freiberg, Leipziger Str. 29, D-09599 Freiberg, Germany
2 Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany
Correspondence
Michael Schlömann
michael.schloemann{at}ioez.tu-freiberg.de
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ABSTRACT |
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Dedicated to the memory of Olga Maltseva, who started the Rhodococcus work in our lab and persuaded us to follow up on it.
The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this article are AY569453 and AY569454.
Present address: NOXXON Pharma AG, Max-Dohrn-Str. 810, D-10589 Berlin, Germany.
Present address: Europroteome AG, Neuendorfstraße 24a, D-16761 Hennigsdorf, Germany.
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INTRODUCTION |
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The enzymes of chlorocatechol pathways of proteobacteria are often encoded by circular catabolic plasmids such as pJP4 of Ralstonia eutropha JMP134 (Don & Pemberton, 1985), pP51 of Pseudomonas sp. P51 (van der Meer et al., 1991a
, b
), pAC27 of Pseudomonas putida AC866 (Ghosal et al., 1985
), and pEST4011 of P. putida EST4021 (Mae et al., 1993
), all of which have been characterized in detail.
Plasmids of rhodococci, which, like their Streptomyces counterparts often possess linear topology, frequently encode catabolic genes, enabling their hosts either to grow with unusual carbon and energy sources such as CO2/hydrogen (Kalkus et al., 1990), (chloro)biphenyl (Kosono et al., 1997
; Masai et al., 1997
), and isopropylbenzene (Dabrock et al., 1994
; Stecker et al., 2003
) or to just convert such compounds as was shown for trichloroethene (Dabrock et al., 1994
; Saeki et al., 1999
). In addition, linear plasmids of rhodococci were shown to harbour genes for resistance to thallium (Kalkus et al., 1990
) and arsenic (Dabrock et al., 1994
; Stecker et al., 2003
), as well as genes for phytopathogenicity (Maes et al., 2001
).
The high similarities between some genes and gene clusters, located on linear plasmids of different Rhodococcus species, suggest that they may have been distributed in this genus via horizontal gene transfer. Thus, similar bphC genes, expressing 2,3-dihydroxybiphenyl dioxygenase activity, were found to be plasmid encoded in Rhodococcus erythropolis TA421 and in Rhodococcus globerulus P6 (Kosono et al., 1997). Extremely high ratios of identical positions (96100 %) were found between the deduced catabolic proteins for isopropylbenzene degradation (ipbA1A2A3BC) encoded on the 210 kb transmissible linear plasmid of R. erythropolis BD2 (Dabrock et al., 1994
; Stecker et al., 2003
) and the analogous proteins of a linear-plasmid-encoded biphenyl degradation pathway in Rhodococcus sp. strain RHA1 (Masai et al., 1997
; Shimizu et al., 2001
) and of an isopropylbenzene degradation pathway in Rhodococcus sp. strain I1 (GenBank accession no. AJ006127). Linear plasmids, therefore, seem likely to play a key role in the propagation of genes for the catabolism of aromatic compounds comparable to that of circular plasmids. Moreover, compared to circular plasmids and chromosomes, linear replicons have been presumed, in addition, to enhance the genomic plasticity and fluidity (Redenbach & Altenbuchner, 2002
).
At present the number of identified linear plasmids in rhodococci is still limited and characterization has usually been restricted to the determination of their termini and of special phenotypic properties. The only and very recently available complete sequence is that of pBD2 (210 kb) of the isopropylbenzene degrader R. erythropolis strain BD2 (Stecker et al., 2003). On plasmids of rhodococci or on other linear plasmids of actinobacteria, chlorocatechol catabolic genes have so far not been identified.
The investigation reported in the present paper aimed at elucidating whether the chlorophenol-degrading R. opacus 1CP carries a linear plasmid and if so, to investigate the localization of chlorocatechol catabolic and other degradative genes on it and to determine the basic properties of the plasmid. Some of the results have been reported previously in a preliminary communication (Eulberg et al., 1998c).
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METHODS |
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Escherichia coli DH5 was obtained from Gibco-BRL and was grown aerobically with constant shaking (120 r.p.m.) at 37 °C in baffled Erlenmeyer flasks with dYT medium (1·6 % tryptone, 1 % yeast extract, 0·5 % NaCl). When appropriate for selection, ampicillin was added to a final concentration of 100 µg ml1.
The plasmids used in this study are listed in Table 1.
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Determination of linearity.
In order to determine whether the presumed extrachromosomal element was in the closed-circular supercoiled form, DNA-containing agarose plugs were additionally treated with S1 nuclease (Barton et al., 1995). The gel plugs were washed twice (15 min; room temperature) with freshly prepared 1 mM PMSF in TE10.1 (pH 7·5) to inactivate the proteinase K, and were then soaked twice (15 min; 37 °C) in 1 ml 10 mM Tris/HCl (pH 7·6). Single agarose plugs were incubated (30 min; 37 °C) with S1 nuclease in 200 µl of a solution containing 1 U Aspergillus oryzae S1 nuclease (Gibco), 50 mM NaCl, 30 mM sodium acetate (pH 4·6), 5 mM ZnSO4 and 5 % (v/v) glycerol. The reaction was stopped by transferring the slices to 100 µl ES buffer on ice.
For the isolation of native megaplasmid and to detect covalently linked proteins, which are a general property of actinobacterial linear plasmids (Sakaguchi, 1990), a plasmid preparation was made as described above but omitting the proteinase K treatment. Genomic DNA in which covalently linked proteins remained attached to plasmid ends was obtained by classical phenol/chloroform extraction without proteinase K.
Electrophoresis.
Conventional electrophoresis was performed by standard methods (Sambrook et al., 2001). PFGE was performed by contour-clamped homogeneous field electrophoresis (Chu et al., 1986
), initially using a CHEF-DRII system from Bio-Rad and at a later point a 2015 Pulsaphor system from Pharmacia LKB. Slab gels with 0·8 % or 1 % (w/v) agarose in TBE buffer [44·5 mM Tris-base, 44·5 mM boric acid, 1 mM EDTA (pH 8·5)] were used at 14 °C for the separations. In general, a constant pulse time duration of 80 s was maintained during the total run time (24 h; 6 V cm1). For restriction analysis, a linear increasing pulse time from 1 to 25 s during the total run time (24 h; 6 V cm1) was used. At a later point an additional step of increasing pulse time from 25 to 30 s over 6 h was performed. Lambda Ladder PFG Marker, MidRange I PFG Marker obtained from New England BioLabs and chromosomes of Saccharomyces cerevisiae obtained from Pharmacia were used as high-molecular-mass DNA standards. Gels were stained by ethidium bromide or SYBR Gold. To visualize the mobility of the native plasmid, and of restriction fragments not treated with proteinase K, 0·2 % SDS was added to slab gels and electrophoresis buffer.
Localization of genes by PCR.
For experiments on the localization of catabolic genes, genomic DNA of R. opacus 1CP was obtained as described previously (Eulberg et al., 1997). Plasmid DNA was prepared in sufficient amounts by excision of the plasmid band, which was visible after PFGE of lysed, agarose-embedded cells of R. opacus 1CP, and elution from the gel with an Easy Pure kit (Biozym). Specific and degenerate primers, which were used for PCR experiments are listed in Table 2
.
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Localization of genes by hybridization.
In hybridization experiments, all labelling, dot blot, hybridization and detection procedures were done using a DIG DNA Labelling and Detection Kit Nonradioactive (Roche) and positively charged nylon membranes from Qiagen or Roth. DIG-labelled probes were either prepared by PCR or generated by randomly primed labelling with Klenow polymerase using genomic DNA or the inserts of plasmids pRER1, pRER3, pRER7 and pMARRE0 (Table 1) as template. All probes were hybridized under high-stringency conditions as described in the Boehringer manual either with dot blots of genomic DNA or p1CP DNA, or with Southern blots of p1CP or its deletion mutants p1CP.01 and p1CP.02 from PFGE agarose gels. To enhance the transfer efficiency of the Southern blots, the PFGE gels were exposed to UV radiation for a few minutes to nick the DNA, followed by a downward alkaline transfer (Sambrook et al., 2001
).
Initial restriction analysis and exonuclease digestion.
After equilibration of agarose plugs in TE buffer (pH 8) and then in restriction buffer, incubation in the presence of 30 units of the restriction enzyme took place for at least 16 h. The reaction was terminated by incubation for 1 h in 1 ml 20 mM Tris/HCl (pH 8)/50 mM EDTA (pH 8). Plugs were then equilibrated with 0·5x TBE, and analysed for restriction fragments formed by PFGE as well as (for fragments <20 kb) by standard agarose gel electrophoresis. The size estimation was performed with the software TotalLab version 1.1 (Nonlinear Dynamics). To determine the localization of genes or plasmid ends on restriction fragments, Southern blots were performed as described above, followed by hybridization with probes specific for macA, clcA, clcA2, clcF, or the ends of p1CP, respectively.
For exonuclease digestion, agarose plugs containing a visible amount p1CP DNA after equilibration for 1 h with the appropriate exonuclease buffer were incubated for 4 h at 37 °C in 250 µl buffer with 30 units of exonuclease III (MBI Fermentas) or exonuclease (New England BioLabs). After stopping the reaction by addition of 5 µl 0·5 M EDTA, plugs equilibrated in 0·5x TBE were analysed on a PFGE gel. For digestion experiments with native p1CP, plugs of an SDS-PFGE gel were used after having been washed in 10 mM Tris/HCl, 1 mM EDTA (pH 8) to eliminate the SDS.
DNA sequence analysis.
Sequencing was done on an ALFexpress Sequencer (Pharmacia) or a LI-COR 4200 IR Sequencer using the Thermo Sequenase fluorescent-labelled primer cycle sequencing kit with 7-deaza-dGTP (Amersham Pharmacia; both sequencers) or the Cycle Reader Auto DNA Sequencing kit (MBI Fermentas; LI-COR only) with labelled T3 or T7 primer. Extensive secondary structures hampered sequencing of the outer 100 bp of p1CP-J. Therefore, the respective subclone was commercially sequenced by SEQLAB. Sequence analysis was done using the Lasergene 99 program package v4.05 (DNASTAR) and BLAST provided by the National Center of Biotechnology Information (NCBI) (Altschul et al., 1990, 1997
).
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RESULTS |
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In contrast to p1CP obtained from cell lysis with proteinase K treatment, the native plasmid (obtained from proteinase K-free cell lysis) did not migrate into the agarose gel (Fig. 2a), indicating the presence of a covalently linked protein. During PFGE under denaturing conditions (0·2 % SDS), mobility of native (i.e. not protease treated) p1CP was restored (Fig. 2b
).
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Identification of genes for (chloro)aromatic catabolism on p1CP
PCR and hybridization experiments with PFGE-separated p1CP DNA were performed for a number of genes involved in catabolism of catechol, 3-chlorocatechol, 4-chloro-/3,5-dichlorocatechol, protocatechuate, and benzoate, in order to find out whether they are localized on p1CP or on the chromosome.
For clcA (encoding 4-chloro-/3,5-dichlorocatechol 1,2-dioxygenase), clcA2 (encoding 3-chlorocatechol 1,2-dioxygenase), clcB (encoding 3-chloro-/2,4-dichloro-cis,cis-muconate cycloisomerase), clcF (encoding 5-chloromuconolactone dehalogenase), and macA (encoding maleylacetate reductase), p1CP DNA gave PCR products of expected size with the respective specific primers (Table 2). Because of the possible existence of more than one maleylacetate reductase gene in strain 1CP (Seibert et al., 1998
), the PCR product obtained was ligated into pBluescript II SK(+) and after transformation eight clones were sequenced. All gave sequences that were identical to that of macA (Seibert et al., 1998
), indicating that the detected gene is in fact the one already described.
The positive hybridizations of PFGE-separated p1CP DNA with DIG-labelled probes for clcA, clcB and macA (Fig. 3) clearly support the PCR results. In addition, positive hybridizations were obtained for clcA2 and clcF, indicating that not only the 4-chloro-/3,5-dichlorocatechol pathway and at least one maleylacetate reductase gene but also the 3-chlorocatechol pathway is located on the megaplasmid.
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Cloning and sequence analysis of terminal fragments of p1CP
Terminal sequences of linear replicons in actinomycetes are characterized by the occurrence of differently sized inverted repeats and palindromic motifs important for replication and propagation of linearity (Chen, 1996; Sakaguchi, 1990
). In order to characterize p1CP in this respect, both ends of p1CP were cloned and sequenced.
One terminal fragment was identified by hybridization of SstII-digested purified p1CP DNA using a DIG-labelled 2·3 kb SalI fragment of the right end of the linear Aut plasmid pHG201 from R. opacus MR11 (Kalkus et al., 1998). Ligation into SstII/EcoRV-digested pBluescript II KS(+) and transformation into E. coli DH5
, followed by colony hybridization, led to the identification of one positive clone, p1CP-S, containing a 1·56 kb SstII/blunt-end insert. Its sequence was determined after restriction analysis (see Fig. 5b
) and subcloning.
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Sequence alignment of the 1·56 kb insert of p1CP-S and the 4·08 kb insert of p1CP-J, designated the left and right end of p1CP, respectively, showed identity of the terminal 13 nucleotides (Fig. 7). In addition to this short perfect inverted repeat, a similarity of 89 % identical positions between the terminal 587 bp of the left end and the terminal 583 bp of the right end indicated the presence of a larger imperfect TIR. The homologous regions are sharply separated from the residual sequences which do not show significant similarities to each other.
BLASTN screening of NCBI and EBI databases with the inserts of p1CP-S and p1CP-J showed high similarities to the ends of other linear plasmids of rhodococci (Table 3). Highest scores were obtained for the right end of pHG204 (AF001834), which showed 92 % identical positions over 710 and 331 bp to p1CP-S, and the 3' end of pHG207 (L14442), which shared 95 %, 91 % and 87 % identical positions over 239, 992 and 339 bp, respectively, with p1CP-J. As indicated by these data, regions of high similarity are often interrupted, obviously by deletions or insertions. The fact that only one of the two ends of plasmids pHG201, pRHL2 and pBD2 is detected by BLASTN searches reflects the fact that linear plasmids of rhodococci, like their counterparts from streptomycetes (Chen et al., 1993
, 1996
; Lin et al., 1993
; Pandza et al., 1998
), may exchange their ends (Kalkus et al., 1993
). Even plasmids with very similar ends may be the product of a recombination process, as was shown for pHG207. The two ends of pHG207 share 95 and 91 % identical base pairs over two regions of 237 and 329 positions, respectively; however, restriction analysis confirmed that the left and right end in fact originate from pHG204 and pHG205, respectively (Kalkus et al., 1993
).
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Both terminal sequences p1CP-S and p1CP-J were screened with BLASTX against the February 2004 version of the non-redundant NCBI and EBI databases for open reading frames which would encode polypeptides. However, no ORFs were found to show significant similarity either to proteins of known function or to hypothetical proteins.
Initial restriction analysis of p1CP
In order to get a first view of p1CP, a rough restriction map of p1CP was generated by means of the AT-specific 6 bp cutters VspI and DraI (Fig. 5a, c). DIG-labelled gene probes for macA, clcA, clcA2 and clcF as well as for both ends were used as additional markers. While clcA and macA were shown to be both localized on a 160 kb large DraI fragment, clcA2 and clcF were found together on the adjoining 190 kb large DraI fragment (Fig. 5a
), which also hybridized with a probe for the right end. More detailed analysis led to the detection of the clcA2 and clcF genes on an internal 110 kb VspI fragment. However, because of three additional VspI fragments of unknown order, its exact position could not be determined.
Deletion of the clc and clc2 gene cluster in mutants 1CP.01 and 1CP.02
Attempts to cure strain 1CP from p1CP were performed by prolonged cultivation under non-selective conditions in LB medium. After 130 generations, appropriate dilutions were plated on LB agar plates and incubated for 3 days at 30 °C. Five hundred colonies were screened for their ability to convert 4-chlorocatechol, a property which is encoded by the p1CP-located clc gene cluster. For this, material from the colonies was transferred to 200 µl agarized mineral medium in microtitre plates containing 10 mM succinate plus 0·5 mM 4-chlorocatechol. While incubation of such sterile medium for several days at 30 °C led to autoxidation of the 4-chlorocatechol, resulting in a brownish colour, wells inoculated with wild-type R. opacus 1CP did not turn dark, due to 4-chlorocatechol degradation prior to autoxidation. Of the 500 picked colonies only two mutants occurred that had lost the ability to degrade 4-chlorocatechol, as indicated by the brown colour of the respective wells after 57 days at 30 °C. PFGE analysis of the two mutants, designated 1CP.01 and 1CP.02, showed that in both strains a megaplasmid was still present which obviously had undergone a large deletion (Fig. 8
).
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That the deletions within the shortened plasmids p1CP.01 (500 kb) and p1CP.02 (400 kb) in fact include the clc gene region was verified by Southern blot hybridization with a clcB-specific probe as well as by dot blot hybridization and PCR with a clcA-specific probe and primer, respectively, which were all negative. In addition, clcB could not be detected in genomic DNA of the mutants, excluding the possibility that the clc-containing region of p1CP had integrated into the chromosome by some recombination process.
Because of the relative proximity of clc and clc2 genes in p1CP, the deletion mutants 1CP.01 and 1CP.02 were also investigated for the presence of a functional 3-chlorocatechol pathway. The additional absence of the clc2 gene cluster in both mutants was proven by PCR as well as by Southern blot hybridization of digested genomic DNA with clcA2-specific primers and probes (data not shown).
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DISCUSSION |
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Despite the obvious metabolic versatility of rhodococci, relatively little is known about the genetics of their degradative pathways for chloroaromatic compounds. In this respect, the strain that has so far received most attention is R. opacus 1CP. It was originally isolated due to its ability to utilize 4-chloro- and 2,4-dichlorophenol (Gorlatov et al., 1989), but it also grows with phenol, styrene, benzoate and 4-hydroxybenzoate. Prolonged cultivation in the presence of 2-chlorophenol or 3-chlorobenzoate led to the isolation of a mutant also able to utilize these compounds as sole carbon source (Moiseeva et al., 1999
). Gene clusters for the metabolism of catechol (catRABC), protocatechuate (pcaHGBLR), 4-chloro-/3,5-dichlorocatechol (clcBRAD), 3-chlorocatechol (clcA2D2B2F) and benzoate (benABCDK) were identified and their specific function has already been shown (Bauer, 2002
; Eulberg et al., 1997
, 1998a
, b
; Moiseeva et al., 2001
, 2002
). Available biochemical and sequence evidence suggests that chlorocatechol catabolism of strain 1CP evolved independently of that in proteobacteria (Eulberg et al., 1998a
; Moiseeva et al., 2002
; Solyanikova et al., 1995
). Neither of the chlorocatechol gene clusters of R. opacus 1CP includes a maleylacetate reductase gene. A separate gene for this activity, macA, was cloned and sequenced, but turned out not to be involved in 4-chlorophenol degradation (Seibert et al., 1998
).
In proteobacteria the genes of chlorocatechol pathways usually reside on plasmids (Frantz & Chakrabarty, 1987; Köiv et al., 1996
; Perkins et al., 1990
; van der Meer et al., 1991a
) or other transferable genetic elements (van der Meer et al., 2001
). In contrast, the genes for the degradation of numerous non-chlorinated aromatic compounds via the catechol and protocatechuate pathways in most strains investigated appear to be located on the chromosome (Doten et al., 1987
; Holloway et al., 1994
; Sauret-Ignazi et al., 1996
; Zylstra et al., 1989
). These facts raised the question whether the situation in the Gram-positive R. opacus 1CP is similar or different.
In the present paper it is shown that also in R. opacus 1CP the chlorocatechol genes are located on a plasmid, p1CP. This is true for both the clc cluster for 4-chlorocatechol and 3,5-dichlorocatechol degradation and the clc2 cluster for 3-chlorocatechol degradation as well as for the macA gene. In contrast, the genes for benzoate, catechol and protocatechuate catabolism, as in most proteobacteria, are obviously not located on the plasmid. Plasmid p1CP was shown to be a huge megaplasmid of 740 kb and to have linear topology. Thus, chlorocatechol genes of R. opacus 1CP are the first such genes known to be located on a linear plasmid.
Over the last few years, several linear plasmids from rhodococci have been described. The R. opacus wild-type strains MR11 and MR22 were each shown to contain three linear plasmids, pHG201 (270 kb), pHG202 (400 kb) and pHG203 (420 kb), as well as pHG204 (190 kb), pHG205 (280 kb) and pHG206 (500 kb), respectively (Kalkus et al., 1990). Two of these plasmids, pHG201 and pHG205, as well as pHG207, obtained from the transconjugant strain MR2253 (Kalkus et al., 1993
), enable their hosts to grow chemolithoautotrophically on gaseous hydrogen and carbon dioxide. Furthermore, pHG204 correlates with thallium resistance. Again three linear plasmids, pRHL1 (1100 kb), pRHL2 (450 kb) and pRHL3 (330 kb), were identified in the polychlorinated biphenyl degrader Rhodococcus sp. strain RHA1, and it was shown that gene clusters encoding (chloro)biphenyl conversion are localized on the two largest plasmids (Masai et al., 1997
). Rhodococcus corallinus B-276 was even found to harbour four linear plasmids, pNC10 (70 kb), pNC20 (85 kb), pNC30 (185 kb) and pNC40 (235 kb). The gene for an alkene monooxygenase, which allows this organism to grow on propene and to co-oxidize trichloroethene, was shown to be located on pNC30 (Saeki et al., 1999
). In contrast, only single linear plasmids, pBD2 (210 kb) and pTA421 (500 kb), were detected in the isopropylbenzene-degrading strain R. erythropolis BD2 (Kesseler et al., 1996
; Stecker et al., 2003
) and the (chloro)biphenyl degrader R. erythropolis TA421 (Kosono et al., 1997
), respectively. However, both plasmids contain at least some of the genes responsible for aromatic catabolism.
Taking into account the 1100 kb pRHL1, p1CP at 740 kb is presently the second largest linear megaplasmid identified from rhodococci.
The propagation of all linear replicons, because of the 5'3' polarity of DNA replication, requires a solution to the end replication problem. Linear plasmids as well as linear chromosomes of actinomycetes have overcome this problem by invertrons consisting of perfect or imperfect TIRs and terminal bound proteins (TPs) (Sakaguchi, 1990). Like other linear plasmids of rhodococci, p1CP was shown to have TPs as well as TIRs. The ends of p1CP have a short perfect TIR of 13 bp, which belongs to a larger imperfect TIR of 583/587 bp, in which the two parts share 89 % identical positions with each other. Sequence comparison of both p1CP ends with the database revealed significant similarities to the right ends of pHG204, pHG201, pRHL2, the 3' end of pBD2, and both ends of pHG207, whereas the corresponding left ends of pHG201 and pRHL2, as well as the 5' end of pBD2 were not revealed by BLASTN searches with the ends of p1CP. However, manual alignment of the terminal 100117 nucleotides of all hitherto available ends of linear Rhodococcus plasmids clearly confirmed the presence of strongly conserved palindromic motifs. Two copies of the motif GCTXCGC were found in each terminus and it was already assumed earlier that these structures are essential for replication (Kalkus et al., 1998
; Shimizu et al., 2001
). Essentially the same central motif with the potential to form a stable single C-residue loop close to the sheared T : C pairing was found in the linear Streptomyces plasmids pSCL1 (Wu & Roy, 1993
), pSLA2 (Hirochika et al., 1984
) and SLP2 (Chen et al., 1993
), as well as in the terminal segments of several linear Streptomyces chromosomes (Huang et al., 1998
). In pSLA2 these structures were shown to be essential for replication and propagation of linearity (Qin & Cohen, 1998
). The specific binding of TPs (Huang et al., 1998
) and the patching of the single-stranded gaps at the 3' termini during replication (Chen, 1996
) are two possible functions of palindromes. Additionally, a conserved CCXXCGG motif with an unknown, but probably important function, was found at the very end of all termini, including those of p1CP.
Attempts to cure the megaplasmid present in strain 1CP, in order to determine the phenotypic properties encoded on p1CP, have up to the present been unsuccessful. However, prolonged cultivation under non-selective conditions led to the identification of two mutants 1CP.01 and 1CP.02, harbouring shortened megaplasmids p1CP.01 (500 kb) and p1CP.02 (400 kb) which must have undergone large deletions, both covering the clc as well as the clc2 gene cluster, and macA. Electrophoretic immobility of the native mutant plasmids indicated the presence of protein-linked termini. The deletions must, therefore, have taken place within the replicons. It will be interesting to elucidate the plasmid regions and mechanisms involved in the occurrence of these deletions.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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---|
Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 33893402.
Barton, B. M., Harding, G. P. & Zuccarelli, A. J. (1995). A general method for detecting and sizing large plasmids. Anal Biochem 226, 235240.[CrossRef][Medline]
Bauer, J. (2002). Klonierung und Sequenzierung der peripheren Benzoat-Abbaugene aus Rhodococcus opacus 1CP und Untersuchungen zu ihrer Substratspezifität. Senior project thesis, Technische Universität Bergakademie Freiberg.
Bey, S. J., Tsou, M. F., Huang, C. H., Yang, C. C. & Chen, C. W. (2000). The homologous terminal sequence of the Streptomyces lividans chromosome and SLP2 plasmid. Microbiology 146, 911922.[Medline]
Cain, R. B. (1981). Regulation of aromatic and hydroaromatic catabolic pathways in nocardioform actinomycetes. Zentbl Bakteriol Mikrobiol Hyg Abt 1 Suppl 11, 335354.
Chen, C. W. (1996). Complications and implications of linear bacterial chromosomes. Trends Genet 12, 192196.[CrossRef][Medline]
Chen, C. W., Yu, T. W., Lin, Y. S., Kieser, H. M. & Hopwood, D. A. (1993). The conjugative plasmid SLP2 of Streptomyces lividans is a 50 kb linear molecule. Mol Microbiol 7, 925932.[Medline]
Chu, G., Vollrath, D. & Davis, R. W. (1986). Separation of large DNA molecules by contour-clamped homogeneous electric fields. Science 234, 15821585.[Medline]
Dabrock, B., Kesseler, M., Averhoff, B. & Gottschalk, G. (1994). Identification and characterization of a transmissible linear plasmid from Rhodococcus erythropolis BD2 that encodes isopropylbenzene and trichloroethene catabolism. Appl Environ Microbiol 60, 853860.[Abstract]
Don, R. H. & Pemberton, J. M. (1985). Genetic and physical map of the 2,4-dichlorophenoxyacetic acid-degradative plasmid pJP4. J Bacteriol 161, 466468.[Medline]
Dorn, E., Hellwig, M., Reineke, W. & Knackmuss, H. J. (1974). Isolation and characterization of a 3-chlorobenzoate degrading pseudomonad. Arch Microbiol 99, 6170.[Medline]
Doten, R. C., Ngai, K. L., Mitchell, D. J. & Ornston, L. N. (1987). Cloning and genetic organization of the pca gene cluster from Acinetobacter calcoaceticus. J Bacteriol 169, 31683174.[Medline]
Dugan, I. N. & Golovlev, E. L. (1983). Induction of dioxygenases for aromatic substrates in rhodococci under nutrient limitation. Microbiology (English translation of Mikrobiologiya) 52, 751755.
Eulberg, D. (1997). Genetik von Wegen des Aromatenabbaus durch Rhodococcus opacus 1CP Konvergente Evolution des Chlorbrenzkatechin-Katabolismus. PhD thesis, Universität Stuttgart.
Eulberg, D., Golovleva, L. A. & Schlömann, M. (1997). Characterization of catechol catabolic genes from Rhodococcus erythropolis 1CP. J Bacteriol 179, 370381.[Abstract]
Eulberg, D., Kourbatova, E. M., Golovleva, L. A. & Schlömann, M. (1998a). Evolutionary relationship between chlorocatechol catabolic enzymes from Rhodococcus opacus 1CP and their counterparts in Proteobacteria: sequence divergence and functional convergence. J Bacteriol 180, 10821094.
Eulberg, D., Lakner, S., Golovleva, L. A. & Schlömann, M. (1998b). Characterization of a protocatechuate catabolic gene cluster from Rhodococcus opacus 1CP: evidence for a merged enzyme with 4-carboxymuconolactone-decarboxylating and 3-oxoadipate enol-lactone-hydrolyzing activity. J Bacteriol 180, 10721081.
Eulberg, D., Seibert, V. & Schlömann, M. (1998c). A linear megaplasmid, p1CP, carrying the chlorocatechol catabolic genes from Rhodococcus opacus 1CP. Biospektrum special edn, p. 125, poster PF214.
Finnerty, W. R. (1992). The biology and genetics of the genus Rhodococcus. Annu Rev Microbiol 46, 193218.[CrossRef][Medline]
Frantz, B. & Chakrabarty, A. M. (1987). Organization and nucleotide sequence determination of a gene cluster involved in 3-chlorocatechol degradation. Proc Natl Acad Sci U S A 84, 44604464.[Abstract]
Ghosal, D., You, I. S., Chatterjee, D. K. & Chakrabarty, A. M. (1985). Genes specifying degradation of 3-chlorobenzoic acid in plasmids pAC27 and pJP4. Proc Natl Acad Sci U S A 82, 16381642.[Abstract]
Golovlev, E. L. (1995). Ecological physiology of rhodococci. In Abstract Book of European Environmental Research Organization Workshop: Enzymatic and Genetic Aspects of Environmental Biotechnology, p. 18. Pushchino, Russia: European Environmental Research Organization.
Gorlatov, S. N., Maltseva, O. V., Shevchenko, V. I. & Golovleva, L. A. (1989). Degradation of chlorophenols by Rhodococcus erythropolis. Microbiology (English translation of Mikrobiologiya) 58, 647651.
Hirochika, H., Nakamura, K. & Sakaguchi, K. (1984). A linear DNA plasmid from Streptomyces rochei with an inverted terminal repetition of 614 base pairs. EMBO J 3, 761766.[Abstract]
Holloway, B. W., Römling, U. & Tümmler, B. (1994). Genomic mapping of Pseudomonas aeruginosa PAO. Microbiology 140, 29072929.[Medline]
Huang, C.-H., Lin, Y.-S., Yang, Y.-L., Huang, S.-W. & Chen, C. W. (1998). The telomeres of Streptomyces chromosomes contain conserved palindromic sequences with potential to form complex secondary structures. Mol Microbiol 28, 905916.[CrossRef][Medline]
Kalkus, J., Reh, M. & Schlegel, H. G. (1990). Hydrogen autotrophy of Nocardia opaca strains is encoded by linear megaplasmids. J Gen Microbiol 136, 11451151.[Medline]
Kalkus, J., Dörrie, C., Fischer, D., Reh, M. & Schlegel, H. G. (1993). The giant linear plasmid pHG207 from Rhodococcus sp. encoding hydrogen autotrophy: characterization of the plasmid and its termini. J Gen Microbiol 139, 20552065.[Medline]
Kalkus, J., Menne, R., Reh, M. & Schlegel, H. G. (1998). The terminal structures of linear plasmids from Rhodococcus opacus. Microbiology 144, 12711279.[Medline]
Kesseler, M., Dabbs, E. R., Averhoff, B. & Gottschalk, G. (1996). Studies on the isopropylbenzene 2,3-dioxygenase and the 3-isopropylcatechol 2,3-dioxygenase genes encoded by the linear plasmid of Rhodococcus erythropolis BD2. Microbiology 142, 32413251.[Medline]
Köiv, V., Marits, R. & Heinaru, A. (1996). Sequence analysis of the 2,4-dichlorophenol hydroxylase gene tfdB and 3,5-dichlorocatechol 1,2-dioxygenase gene tfdC of 2,4-dichlorophenoxyacetic acid degrading plasmid pEST4011. Gene 174, 293297.[CrossRef][Medline]
Koronelli, T. V., Dermicheva, S. G. & Korotaeva, E. V. (1988). Survival of hydrocarbon-oxidizing bacteria under complete starvation conditions. Mikrobiologiya 57, 298304.
Kosono, S., Maeda, M., Fuji, F., Arai, H. & Kudo, T. (1997). Three of the seven bphC genes of Rhodococcus erythropolis TA421, isolated from a termite ecosystem, are located on an indigenous plasmid associated with biphenyl degradation. Appl Environ Microbiol 63, 32823285.[Abstract]
Krauß, U. (2002). Überexpression der 5-Chlormuconolacton-Dehalogenase. Ein Enzym eines neuen modifizierten ortho-Abbauweges von 2-Chlorphenol des Stammes Rhodococcus opacus 1CP. Senior project thesis, Technische Universität Bergakademie Freiberg.
Lin, Y. S., Kieser, H. M., Hopwood, D. A. & Chen, C. W. (1993). The chromosomal DNA of Streptomyces lividans 66 is linear. Mol Microbiol 10, 923933.[Medline]
Mae, A. A., Marits, R. O., Ausmees, N. R., Koiv, V. M. & Heinaru, A. L. (1993). Characterization of a new 2,4-dichlorophenoxyacetic acid degrading plasmid pEST4011: physical map and localization of catabolic genes. J Gen Microbiol 139, 31653170.
Maes, T., Vereecke, D., Ritsema, T., Cornelis, K., Thu, H. N., Van Montagu, M., Holsters, M. & Goethals, K. (2001). The att locus of Rhodococcus fascians strain D188 is essential for full virulence on tobacco through the production of an autoregulatory compound. Mol Microbiol 42, 1328.[CrossRef][Medline]
Masai, E., Sugiyama, K., Iwashita, N., Shimizu, S., Hauschild, J. E., Hatta, T., Kimbara, K., Yano, K. & Fukuda, M. (1997). The bphDEF meta-cleavage pathway genes involved in biphenyl/polychlorinated biphenyl degradation are located on a linear plasmid and separated from the initial bphACB genes in Rhodococcus sp. strain RHA1. Gene 187, 141149.[CrossRef][Medline]
Moiseeva, O. V., Lin'ko, E. V., Baskunov, B. P. & Golovleva, L. A. (1999). Degradation of 2-chlorophenol and 3-chlorobenzoate by Rhodococcus opacus 1CP. Microbiology (English translation of Mikrobiologiya) 68, 400405.
Moiseeva, O. V., Belova, O. V., Solyanikova, I. P., Schlömann, M. & Golovleva, L. A. (2001). Enzymes of a new modified ortho-pathway utilizing 2-chlorophenol in Rhodococcus opacus 1CP. Biochemistry (English translation of Biokhimiya) 66, 548555.[Medline]
Moiseeva, O. V., Solyanikova, I. P., Kaschabek, S. R., Gröning, J., Thiel, M., Golovleva, L. A. & Schlömann, M. (2002). A new modified ortho-cleavage pathway of 3-chlorocatechol degradation by Rhodococcus opacus 1CP: genetic and biochemical evidence. J Bacteriol 184, 52825292.
Pandza, S., Biukovic, G., Paravic, A., Dadbin, A., Cullum, J. & Hranueli, D. (1998). Recombination between the linear plasmid pPZG101 and the linear chromosome of Streptomyces rimosus can lead to exchange of ends. Mol Microbiol 28, 11651176.[CrossRef][Medline]
Perkins, E. J., Gordon, M. P., Caceres, O. & Lurquin, P. F. (1990). Organization and sequence analysis of the 2,4-dichlorophenol hydroxylase and dichlorocatechol oxidative operons of plasmid pJP4. J Bacteriol 172, 23512359.[Medline]
Picardeau, M. & Vincent, V. (1998). Mycobacterial linear plasmids have an invertron-like structure related to other linear replicons in actinomycetes. Microbiology 144, 19811988.[Medline]
Qin, Z. & Cohen, S. N. (1998). Replication at the telomeres of the Streptomyces linear plasmid pSLA2. Mol Microbiol 28, 893903.[CrossRef][Medline]
Redenbach, M. & Altenbuchner, J. (2002). Why do some bacteria have linear chromosomes and plasmids? Biospektrum 8, 158163.
Robertson, J. G. & Batt, R. D. (1973). Survival of Nocardia corallina and degradation of constituents during starvation. J Gen Microbiol 78, 109117.
Saeki, H., Akira, M., Furuhashi, K., Averhoff, B. & Gottschalk, G. (1999). Degradation of trichloroethene by a linear-plasmid-encoded alkene monooxygenase in Rhodococcus corallinus (Nocardia corallina) B-276. Microbiology 145, 17211730.[Medline]
Sakaguchi, K. (1990). Invertrons, a class of structurally and functionally related genetic elements that includes linear DNA plasmids, transposable elements, and genomes of adeno-type viruses. Microbiol Rev 54, 6674.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (2001). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sauret-Ignazi, G., Gagnon, J., Beguin, C., Barrelle, M., Markowicz, Y., Pelmont, J. & Toussaint, A. (1996). Characterisation of a chromosomally encoded catechol 1,2-dioxygenase (E.C. 1.13.11.1) from Alcaligenes eutrophus CH34. Arch Microbiol 166, 4250.[CrossRef][Medline]
Seibert, V., Kourbatova, E. M., Golovleva, L. A. & Schlömann, M. (1998). Characterization of the maleylacetate reductase MacA of Rhodococcus opacus 1CP and evidence for the presence of an isofunctional enzyme. J Bacteriol 180, 35033508.
Shimizu, S., Kobayashi, H., Masai, E. & Fukuda, M. (2001). Characterization of the 450-kb linear plasmid in a polychlorinated biphenyl degrader, Rhodococcus sp. strain RHA1. Appl Environ Microbiol 67, 20212028.
Solyanikova, I. P., Maltseva, O. V., Vollmer, M. D., Golovleva, L. A. & Schlömann, M. (1995). Characterization of muconate and chloromuconate cycloisomerase from Rhodococcus erythropolis 1CP: indications for functionally convergent evolution among bacterial cycloisomerases. J Bacteriol 177, 28212826.[Abstract]
Stecker, C., Johann, A., Herzberg, C., Averhoff, B. & Gottschalk, G. (2003). Complete nucleotide sequence and genetic organization of the 210-kilobase linear plasmid of Rhodococcus erythropolis BD2. J Bacteriol 185, 52695274.
van der Meer, J. R., Eggen, R. I., Zehnder, A. J. & de Vos, W. M. (1991a). Sequence analysis of the Pseudomonas sp. strain P51 tcb gene cluster, which encodes metabolism of chlorinated catechols: evidence for specialization of catechol 1,2-dioxygenases for chlorinated substrates. J Bacteriol 173, 24252434.[Medline]
van der Meer, J. R., van Neerven, A. R. W., De Vries, E. J., De Vos, W. M. & Zehnder, A. J. B. (1991b). Cloning and characterization of plasmid-encoded genes for the degradation of 1,2-dichloro-, 1,4-dichloro-, and 1,2,4-trichlorobenzene of Pseudomonas sp. strain P51. J Bacteriol 173, 615.[Medline]
van der Meer, J. R., Ravatn, R. & Sentchilo, V. (2001). The clc element of Pseudomonas sp. strain B13 and other mobile degradative elements employing phage-like integrases. Arch Microbiol 175, 7985.[CrossRef][Medline]
Warhurst, A. M. & Fewson, C. A. (1994). Biotransformations catalyzed by the genus Rhodococcus. Crit Rev Biotechnol 14, 2973.[Medline]
Wu, X. & Roy, K. L. (1993). Complete nucleotide sequence of a linear plasmid from Streptomyces clavuligerus and characterization of its RNA transcripts. J Bacteriol 175, 3752.[Abstract]
Zylstra, G. J., Olsen, R. H. & Ballou, D. P. (1989). Genetic organization and sequence of the Pseudomonas cepacia genes for the alpha and beta subunits of protocatechuate 3,4-dioxygenase. J Bacteriol 171, 59155921.[Medline]
Received 6 April 2004;
revised 15 June 2004;
accepted 17 June 2004.
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