On Linear Plasmids in Phylogenetically Different Bacteria

Since chloroethenes, which include known or suspected carcinogens, are widely distributed at many contaminated sites, their microbial degradation has been investigated intensely, and aerobic as well as anaerobic transformation pathways have been described (for a review, see Fetzner 1998a). Linear plasmids have been implicated in alkene and chloroethene metabolism by phylogenetically very different bacteria, such as Mycobacterium strains, Gor-donia rubripertincta B-276 (formerly Rhodococcus corallinus, Nocardia coral-lina), Nocardioides sp. JS614, Xanthobacter sp. Py2, Pseudomonas putida AJ, and Ochrobactrum sp. TD (Saeki et al. 1999; Krum and Ensign 2001; Danko et al. 2004, 2006; Mattes et al. 2005).

The Gram-negative bacterium Xanthobacter sp. Py2 oxidizes propene via epoxidation and carboxylation to form acetoacetate (Fig. 1A), which can be easily converted to central metabolites (for a review, see Ensign and Allen 2003). Loss of the 320-kb megaplasmid pEK1, proposed to be linear, was accompanied by loss of alkene monooxygenase and carboxylase activity, and loss of coenzyme M biosynthesis. The alkene monooxygenase genes xamoABCDEF, as well as the xecABCDEFG cluster that codes for the four enzymes of epoxide metabolism (XecA, -D, -E, -C), two proteins of unknown function (XecB, -F), and a putative (2R)-phospho-3-sulfolactate synthase

(XecG = ComA) involved in biosynthesis of coenzyme M, are located on pEK1 (Krum and Ensign 2001) (Table 1).

Gordonia rubripertincta B-276, which utilizes propene and oxidizes trichloroethene (TCE), contains four linear plasmids (Table 1). A three-component alkene monooxygenase (Saeki and Furuhashi 1994) catalyzes the oxidation of propene, trichloroethene, and probably dichloroethenes and

A Fig. 1 Alkene degradation via the coenzyme M pathway. (A): Propene degradation by Xanthobacter sp. strain Py2 (Ensign and Allen 2003). CoM, coenzyme M (2-mer-captoethanesulfonate); 1, propene; 2a, (R)-epoxypropane; 2b, (S)-epoxypropane; 3a, 2-(R )-hydroxypropyl-CoM; 3b, 2-(S)-hydroxypropyl-CoM; 4, 2-ketopropyl-CoM; 5, ace-toacetate. Xamo, alkene monooxygenase; XecA, epoxyalkane:CoM transferase; XecD, 2-(R)-hydroxypropyl-CoM dehydrogenase; XecE, 2-(S)-hydroxypropyl-CoM dehydrogenase; XecC, NADPH:2-ketopropyl-CoM carboxylase/oxidoreductase. (B): Hypothetical pathway of vinyl chloride degradation by Nocardioides sp. JS614 (Mattes et al. 2005). CoM, coenzyme M (2-mercaptoethanesulfonate); 1, vinyl chloride; 2, chloroepoxyethane; 3, 2-chloro-2-hydroxyethyl-CoM; 4, 2-ketoethyl-CoM; 5, carboxymethyl-CoM; 6, CoM-acetyl-coenzyme A; 7, acetyl-coenzyme A. EtnABCD, alkene monooxygenase; EtnE, epoxyalkane:CoM transferase. For proteins of open reading frames (ORFs) 7-9, see text dichloropropenes to the corresponding epoxides. The alkene monooxygenase genes amoABCD, as well as genes involved in the downstream catabolism of epoxides, are encoded by the 185-kb linear plasmid pNC30 (Saeki et al. 1999).

The linear plasmid pNoc614 of Nocardioides sp. JS614 was discussed to encode the complete pathway of vinyl chloride and ethene oxidation to central metabolites (Mattes et al. 2005; see Fig. 1B for vinyl chloride oxidation). In the first degradation step, alkene monooxygenase (EtnABCD) catalyzes the formation of epoxyethane and chlorooxirane from ethene and vinyl chloride, respectively. Subsequent coenzyme M transfer to these epoxyalka-nes, catalyzed by EtnE, yields 2-hydroxyethyl-CoM from epoxyethane, and 2-chloro-2-hydroxyethyl-CoM from chlorooxirane. Whereas the latter may eliminate HCl to form 2-ketoethyl-CoM (Fig. 1B), formation of 2-ketoethyl-CoM from 2-hydroxyethyl-CoM would require the action of a dehydroge-nase. Further steps of the hypothetical pathway may involve oxidation to carboxymethyl-CoM, activation to CoM-acetyl-CoA, and reductive cleavage of the CoM-acetyl-CoA thioether to acetyl-CoA and CoM-SH (Mattes et al. 2005). Hybridization studies and sequencing revealed that pNoc614 carries the etnEABCD locus, putative acyl-CoA transferase and acyl-CoA synthetase genes (ORFs 9 and 10), and two putative oxidoreductase genes (ORFs 7 and 8) (Table 1; Fig. 2). The deduced gene products of ORF9, 0RF10, and etnEABCD of strain JS614 show significant similarity to the corresponding proteins of Mycobacterium rhodesiae JS60 (36-83% amino acid (aa) identity), which are also encoded on a linear plasmid (Coleman and Spain 2003b). The EtnABCD proteins of strain JS60 also share 41-59% sequence identity with the corresponding gene products of amoABCD of G. rubripertincta B-276, suggesting a common ancestry (Fig. 2). Remarkably, the ORF7 and ORF8 proteins of pNoc614 of Nocardioides sp. JS614 also show some similarity to NADPH:2-ketopropyl-CoM carboxylase/oxidoreductase (XecC; 24% aa identity) and 2-(S)-hydroxypropyl-CoM dehydrogenase (XecE; 47% aa identity), respectively, of the Gram-negative strain Xanthobacter sp. Py2. A gene presumed to code for (2R)-phospho-3-sulfolactate synthase, which catalyzes the first step in CoM biosynthesis, likewise is localized on the plasmids of both

Table 1 Overview of linear catabolic plasmids

Organism

Plasmid"

Plasmid size (kb)

Significance in catabolism; catabolic genes identified [accession number]

Refs.

Xanthobacter sp. Py2

pEKl

320

(Chloro)alkene metabolism via coenzyme M pathway: xamoABCDEF encoding alkene monooxygenase [AJ012090]; xecABCDEFG involved in epoxide metabolism and coenzyme M synthesis [X79863, AY024334],

Swaving et al. 1995 Zhou et al. 1999 Krum and Ensign 2001

Gordonia rubripertincta

B-276

pNCIO pNC20 pNC30

70 85 185

Alkene metabolism and co-oxidation of trichloroethene: amoABCD encoding alkene monooxygenase [D37875],

Saeki et al. 1999

pNC40

235

Nocardioides sp. JS614

pNoc614

290

(Chloro)alkene metabolism via coenzyme M pathway: etnEABCD (ORFs 11-15) encoding alkene monooxygenase (EtnABCD) and epoxyalkane: coenzyme M transferase (EtnE), genes for putative CoA transferase (ORF9), putative acyl-CoA synthetase (ORFIO), two putative oxidoreductases (ORF7, xecC-like, and ORF8, xecE-like) [AY772007].

Mattes et al. 2005

Xanthobacter autotrophicus GJ10

pXAUl

225

1,2-Dichloroethane metabolism: haloalkane dehalogenase gene dhlA [M26950]; aldA encoding 2-chloroacetaldehyde dehydrogenase [AF029733],

Tardif et al. 1991 van der Ploeg et al. 1994 Bergeron et al. 1998

Rhodococcus erythropolis PR4

pRELl pRECla pREC2a

271.577 104.014 3.637

Alkane oxidation to acyl-CoA (putative): ORFs pRELl_0256-pRELl_0261 and pRELl_0282-pRELl_0286 [AP008931], Complete set of genes for (5-oxidation of fatty acids (ORFs pRECl_0064-pRECl_0074) [AP008932], [AP008933],

Sekine et al. 2006

Organism Plasmida

Plasmid Significance in catabolism; catabolic genes identified size (kb) [accession number]

Refs.

Rhodo-coccus sp. RHA1

pRHLl 1123.075 (Polychlorinated) biphenyl and ethylbenzene metabolism: etbDl, bphAaAbAcAdClBl, bphSl, bphTl, bphF3G3E3, bphRGlFlEl. Putative extradiol dioxygenases (ro08079, ro09005), catechol 1,2-dioxygenases (ro08069, ro08511), C-C hydrolases (ro08081, ro09014), maleylacetate reductase (ro08510), phenol monooxygenase (ro08076-08078). Terephthalate and phthalate metabolism: tpaKBCAbAa, tpaR, patDABCE, padR, padAaAbBAcAdC. Possible carveol dehydrogenases limCl and limC2 (ro08210, ro08632) and limonene monooxygenase (ro09085). Putative ring hydroxylating oxygenase (ro08637-08639); genes for cytochrome P450 systems (ro08067-08068, ro08984-08986); genes involved in steroid catabolism (ro09001-09005) [CP000432], pRHL2 442.536 (Polychlorinated) biphenyl and ethylbenzene metabolism: bphF4, bphG4E4, bphT2, bphS2, etbAd, bphB2, etbAalAblC, bphDlE2F2, etbAa2Ab2AcD2 (formerly ebdAlA2A3D2); extradiol dioxygenase gene (rol0315). Terephthalate and phthalate metabolism: tpaKBCAbAa, tpaR, patDABCE, padR, padAaAbBAcAdC. Putative 2,3-dichlorophenol 6-monooxygenase (rol0313) [CP000433], pRHL3 332.361 Putative carveol dehydrogenase gene (RHL3.41, limC3) and limonene monooxygenase gene (RHL3.42, limB), genes for cytochrome P450 proteins (RHL3.62, 3.246, 3.287). Metabolism of aromatic compounds: putative maleylacetate reductase, intradiol dioxygenase, (indole) dioxygenase genes (RHL3.276-3.280) [CP000434],

Alkylbenzene metabolism: akbTS, (ORFU)akbA4B, (ORFsl5-20), akbAlbA2bCDEF, (ORF21), akbAlaA2aA3; putative genes for 4-hydroxy-2-oxovalerate aldolase, acetaldehyde dehydrogenase, 2-hydroxypenta-2,4-dienoate hydratase, enoyl-CoA hydratase, acyl-CoA ligase. Phthalate and terephthalate metabolism: ophAlA2, (ORFO), ophBA3A4C, ophR, pehA, ptrDABC, tphR, tphAlA2BA4 [AY502075],

Masai et al. 1997 Shimizu et al. 2001 Warren et al. 2004 Patrauchan et al. 2005

McLeod et al. 2006 www.rhodococcus.ca

Kim et al. 2002, 2004 Choi et al. 2005

Organism

Plasmid"

Plasmid size (kb)

Significance in catabolism; catabolic genes identified [accession number]

Refs.

pDK3 (?)

750

Phthalate and terephthalate metabolism: ophAlA2, (ORFO), ophBA3A4C, ophR, pehA, ptrDABC, tphR, tphAlA2BA4 [AY502076].

R. erythropolis TA421

pTA421

560

Biphenyl metabolism: bphC2 [D88014]; bphA 1A2A3A4BC3STD [D88020, D88015, AB014348]; bphAlA2A3BA4C4D [D88021, D88016],

Kosono et al. 1997 Arai et al. 1998

R. globerulus P6

pLP6 pSP6

650 360

Biphenyl metabolism: bphC2 (extradiol dioxygenase).

Biphenyl metabolism: bphC2, bphC3, bphC4 (extradiol dioxygenases).

Kosono et al. 1997

R. rhodochrous K37

270 200

Biphenyl degradation: bphC6 [AB117724]; bphC8 [AB117726] (extradiol dioxygenases).

Taguchi et al. 2004

R. erythropolis BD2

pBD2

210.250

Isopropylbenzene metabolism and co-oxidation of trichloroethene: ipbAlA2A3A4CB (PBD2.153-158), ipbST (PBD2.159-169), ipbD (PBD2.174) [AY223810],

Dabrock et al. 1994 Kesseler et al. 1996 Stecker et al. 2003

Rhodococcus sp. 124

(?) (?)

340 80

Toluene-inducible dioxygenase, indene oxidation: tidABCD [AF452376], Naphthalene metabolism and indene oxidation: nidABCD [AF121905]; nimR (transcriptional regulator), nimA (naphthalene inducible monooxygenase), nimB (extradiol dioxygenase) [AF452375.2],

Priefert et al. 2004

R. opacus 1CP

plCP

740

4-Chloro-/3,5-dichlorocatechol metabolism (clcBRAD) [AF003948]; 3-chloro-catechol metabolism (clcA2D2B2F) [AJ439407]; macA encoding maleylacetate reductase [AF030176],

Moiseeva et al. 2002 K├Ânig et al. 2004

Rhodococcus sp. NCIMB12038

p2SLl

380

Naphthalene metabolism: rubl, narRl, narR2, rub2, (ORF7), narAaAbB, AnarC; [AF082663.3]

Kulakov et al. 2005

Organism Plasmid" Plasmid Significance in catabolism; catabolic genes identified size (kb) [accession number]

Refs.

Rhodococcus P40L1 420 sp. P400 P40C1a 180

P40L2 45

P40L3 20

Kulakov et al. 2005

Naphthalene metabolism: rubl, narRl, narR2, (ORF7), narAaAbBC [AY392423.2],

R. opacus SA0101

pWK301 1100

pWK302 1000 pWK303 700

Naphthalene, dibenzofuran, and dibenzo-p-dioxin metabolism: rubl, narRl, narR2, ORF7, narAaAbBC (ORF6, dodRl, dodR2, ORF7, dodA, do dB, dodC, ORF8 [ABl 10633]).

Kitagawa et al. 2004 Kimura et al. 2006

R. opacus M213

pNUOl 750 pNU02 (?) 200-300

Metabolism of aromatic compounds: edoD encoding catechol 2,3-dioxygenase. Uz et al. 2000

Terrabacter sp. DBF63

pDBFl pDBF2

Angular dioxygenation of dibenzofuran to 2,2',3-trihydroxybiphenyl (dbfAlA2). Fluorene oxidation to phthalate: flnR, finB, dbfAlA2,flnEDl, ORF16,flnC; phthalate conversion to protocatechuate: phtAlA2, (ORF11), BphtA3A4CR; protocatechuate branch of (5-ketoadipate pathway: pcaR, pcaHGBDCFIJ; putative extradiol dioxygenase gene (ORF62) [AP008980], Derivative of pDFl (dbf-fln and pht genes)

Nojiri et al. 2002 Habe et al. 2004 Habe et al. 2005

Arthrobacter pALl 112.992 Quinaldine conversion to anthranilate: xdhCAike gene; qoxS, qoxM, qoxL, nitro- moq, hod, amq (ORF1, qoxS, qoxM, qoxL, ORF2, hod, ORF4 [AJ537472]);

guajacolicus putative anthranilate metabolism via anthranoyl-CoA and 2-amino-5-oxo-

Ru61a cyclohex-l-ene-carbonyl-CoA.

Overhage et al. 2005 Parschat et al. 2007

a: Indicates circularity; (?): Denotes unclear topology

Fig. 2 Organization of genes involved in alkene degradation. JS614, etn locus on the linear plasmid pNoc614 of Nocardioides sp. strain JS614 (Mattes et al. 2005; GenBank accession number AY772007); JS60, homologues on a linear plasmid of Mycobacterium rhodesiae JS60 (Coleman et al. 2003a; AY243034); B-276, amo genes located on pNC30 of Gordonia rubripertincta B-276 (Saeki and Furuhashi 1994; D37875). The amoABCD and etnABCD genes code for multicomponent alkene monooxygenases; etnE encodes epoxyalkane:CoM transferase. For the other ORFs, see text. The arrows indicate the size, location, and direction of transcription of the genes and ORFs; homologous ORFs feature the same pattern. Truncated regions are marked with the symbol S

Fig. 2 Organization of genes involved in alkene degradation. JS614, etn locus on the linear plasmid pNoc614 of Nocardioides sp. strain JS614 (Mattes et al. 2005; GenBank accession number AY772007); JS60, homologues on a linear plasmid of Mycobacterium rhodesiae JS60 (Coleman et al. 2003a; AY243034); B-276, amo genes located on pNC30 of Gordonia rubripertincta B-276 (Saeki and Furuhashi 1994; D37875). The amoABCD and etnABCD genes code for multicomponent alkene monooxygenases; etnE encodes epoxyalkane:CoM transferase. For the other ORFs, see text. The arrows indicate the size, location, and direction of transcription of the genes and ORFs; homologous ORFs feature the same pattern. Truncated regions are marked with the symbol S

strains JS614 and Py2 (comA = xecG, ORF6 on the pNoc614 segment; Mattes et al. 2005).

When six vinyl chloride degrading mycobacteria, which all possess homologous epoxyalkane:CoM transferase genes (etnE), were screened for plas-mids, large linear DNA elements (110-330 kb) were found in all strains, namely, Mycobacterium sp. strains JS61, JS616, JS617, JS619, JS621, and M. rhodesiae JS60. In fact, almost identical etnE sequences were found in a total of ten ethene/vinyl chloride degrading isolates that were obtained from geographically distant locations. The phylogeny of the 16SrDNA of the strains significantly differed from the phylogeny of the etnE gene sequences (Coleman and Spain 2003a,b), indicating horizontal gene transfer among these mycobacteria. Notably, a recent report suggests that gene transfer may have occurred even between Gram-positive and Gram-negative bacteria. Plasmid DNA of Pseudomonas putida AJ and Ochrobactrum sp. TD was found to contain etnE genes almost identical to each other and to those of Mycobacterium strains JS60 and JS621 (Danko et al. 2006). Based on their behavior in pulsed-field gel electrophoresis, the plasmids of P. putida AJ and Ochrobactrum sp. TD, which are essential for the ability to degrade vinyl chloride and ethene, were reported to have a linear topology. These plasmids, however, appear to readily undergo deletions and rearrangements, depending on the carbon source used for growth; in complex medium, they were cured rapidly (Danko et al. 2004, 2006).

Genes Encoding 1,2-Dichloroethane Degradation by Xanthobacter autotrophicus GJ10 Are Segregated Between the Chromosome and pXAU1

Xanthobacter autotrophicus GJ10 is able to metabolize 1,2-dichloroethane (Janssen et al. 1985, 1989), a compound produced by the chemical industry primarily for use in the synthesis of vinyl chloride. Degradation (Fig. 3) is initiated by haloalkane dehalogenase DhlA, which catalyzes a hydrolytic dehalo-genation reaction to form 2-chloroethanol (Janssen et al. 1989; Verschueren et al. 1993). Subsequent dehydrogenation by an alcohol dehydrogenase (Mox)

Fig. 3 Degradation of 1,2-dichloroethane by Xanthobacter autotrophicus GJ10 (Janssen et al. 1985). 1, 1,2-Dichloroethane; 2, 2-chloroethanol; 3, chloroacetaldehyde; 4, chloroac-etate; 5, glycolate. DhlA, haloalkane dehalogenase; Mox, (pyrroloquinoline quinone-dependent) alcohol dehydrogenase; Ald, chloroacetaldehyde dehydrogenase; DhlB, 2-haloacid dehalogenase results in formation of the toxic compound 2-chloroacetaldehyde, which subsequently is oxidized to 2-chloroacetate (van der Ploeg et al. 1994). Strain GJ10 harbors a second hydrolytic dehalogenase, namely, haloacid dehaloge-nase DhlB (Ridder et al. 1997), which converts 2-chloroacetate to glycolate (van der Ploeg et al. 1991). Two chloroacetaldehyde dehydrogenases have been identified: one is encoded by the chromosomal aldB gene, the other by aldA located on the 225-kb plasmid pXAU1 which was shown to be linear by pulsed-field gel electrophoresis (Bergeron et al. 1998). Haloalkane dehalogenase DhlA likewise is encoded by pXAU1, whereas the mox and dhlb genes are located on the chromosome (Tardif et al. 1991; van der Ploeg et al. 1994).

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