Research in Microbiology 159 (2008) 118e127www.elsevier.com/locate/resmic

Nucleotide sequence, organization and characterizationof the (halo)aromatic acid catabolic plasmid pA81

from Achromobacter xylosoxidans A8

Vera Jencova a, Hynek Strnad b, Zdenek Chodora b, Pavel Ulbrich b,Cestmir Vlcek b, W.J. Hickey c,*, Vaclav Paces a,b

a Department of Biochemistry and Microbiology, Institute of Chemical Technology in Prague, Prague, Czech Republicb Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic

c Department of Soil Science, University of Wisconsin-Madison, Madison, WI 53706, USA

Received 22 August 2007; accepted 29 November 2007

Available online 8 December 2007

Abstract

The complete 98,192 bp nucleotide sequence was determined for plasmid pA81, which is harbored by the haloaromatic acid-degradingbacterium Achromobacter xylosoxidans A8. The majority of the 103 open reading frames identified on pA81 could be categorized as either‘‘backbone’’ genes, genes encoding (halo)aromatic compound degradation, or heavy metal resistance determinants. The backbone genescontrolled conjugative transfer, replication and plasmid stability, and were well conserved with other IncP1-b plasmids. Genes encoding (halo)aromatic degradation were clustered within a type I transposon, TnAxI, and included two ring-hydroxylating oxygenases (ortho-halobenzoateoxygenase, salicylate 5-hydroxylase) and a modified ortho-cleavage pathway for chlorocatechol degradation. The cluster of heavy metal resis-tance determinants was contained within a Type II transposon TnAxII, and included a predicted P-type ATPase and cation diffusion facilitatorsystem. Genes identical to those carried by TnAxI and TnAxII were identified on other biodegradative/resistance plasmids and genomic islands,indicating an evolutionary relationship between these elements. Collectively, these insights further our understanding of how mobile elements,and interactions between mobile elements affect the fate of organic and inorganic toxicants in the environment.� 2007 Elsevier Masson SAS. All rights reserved.

Keywords: Achromobacter xylosoxidans A8; Biodegradation; Chlorobenzoic acids; Chlorocatechols; Modified ortho-cleavage pathway; Salicylate 5-hydroxylase;

Ortho-halobenzoate oxygenase; Horizontal gene transfer; Plasmid; incP1-b; DNA sequencing; Heavy metal resistance; Mobile genetic elements

1. Introduction

Chlorobenzoates are intermediates produced during bacte-rial degradation of polychlorinated biphenyls. Microbialgrowth on ortho-substituted chlorobenzoates (CBs) such as2- and 2,5-(di)chlorobenzoic acid (2-CB; 2,5-DCB) is initiatedby an ortho-halobenzoate oxygenase, which transforms 2-CBand 2,5-DCB into catechol and 4-chlorocatechol, respectively[6,30]. Chlorocatechols are subsequently transformed by the

* Corresponding author. Tel.: þ1 608 262 9018; fax: þ1 608 265 2595.

E-mail address: wjhickey@wisc.edu (W.J. Hickey).

0923-2508/$ - see front matter � 2007 Elsevier Masson SAS. All rights reserved.

doi:10.1016/j.resmic.2007.11.018

enzymes of the so-called ‘‘modified ortho-metabolic path-way’’ [3,8,10,13,16,31].

Bacterial biodegradation genes are often carried on mobilegenetic elements (MGEs), including transposons, phage-related elements (genomic islands), conjugative plasmids andcombinations/derivatives thereof [26]. Self-transmissible andhighly promiscuous plasmids of the IncP1 group are emergingas a group of MGEs important in xenobiotic degradation. Thecomplete nucleotide sequence of several IncP1-b catabolicplasmids was reported recently; these plasmids carry genesencoding for catabolism of atrazine (pADP-1; [12]), p-toluenesulfonate (pTSA; [27]), 2,4-dichlorophenoxyacetic acid (pJP4and pEST4011; [28,32]) or haloacetate (pUO1; [21]).

119V. Jencova et al. / Research in Microbiology 159 (2008) 118e127

Achromobacter xylosoxidans A8 was isolated in the CzechRepublic from PCB-contaminated soil [15], and is able to use2-CB and 2,5-DCB as sole carbon and energy source. StrainA8 harbors two plasmids, pA81 (98.2 kbp) and pA82 (ca.250 kbp), the former of which encodes for CB degradation[8]. Here we report the complete 98,192 bp nucleotide se-quence of pA81, in silico analysis of the core plasmid ‘‘back-bone’’ as well as two resident transposons, and physiologicaltesting to determine if functions predicted to be encoded bythe transposons are phenotypes likely to be conferred bypA81.

2. Materials and methods

2.1. Plasmid DNA isolation and sequencing

trf

trb

TnAxII

81 kb

98,2 kb

16,3 kb

met

kor

oriV

A. xylosoxidans was grown for 36 h at 28 �C in 1 l of ABCmineral medium [19] containing 750 mg/l 2,5-DCB. PlasmidDNA was isolated by an alkali lysis method [19], purified byseparation in low-melting agarose gel electrophoresis, digestedwith SacI and HindIII and then cloned into pUC19. The SacIand HindIII clones and the entire pA81 DNA were partially di-gested with HinPI, and fragments (1e2 kbp) were insertedinto the AccI site of pUC19.

DNA sequencing was done on ss- or dsDNA templates us-ing a Thermo Sequenase cycle sequencing kit with 7-deaza-dGTP (Pharmacia). Fragments were analyzed using an ALFexpress sequencing machine (Pharmacia). Sequences were as-sembled with Staden software [22] and gap closing was doneby primer-walking with multiplex PCR. Sequence assemblywas verified by PCR analysis.

ORFs were predicted with GeneMark (opal.biology.gatech.edu/GeneMark), Generation (compbio.ornl.gov/tools/pipeline)and Glimmer (compbio.ornl.gov/tools/pipeline) using Pseudo-monas aeruginosa PAO, Ralstonia solanacearum and Escher-ichia coli as models. We also used DicodonUse [14] withappropriate template sequences (e.g., from Achromobacter,Alcaligenes and various IncP-b plasmids) to assess the proba-bility that candidate ORFs were bona fide genes. Output fromthese programs was compared in Artemis (sanger.ac.uk/Software/Artemis/) and all predicted ORFs were analyzed us-ing BLAST (www.ncbi.nlm.nih.gov/BLAST/) and FASTA(www.ebi.ac.uk/fasta33/).

pA81

98192 bp

mocpkleklc

2.2. Heterologous expression

ohb

hyb

tra

tra

51.9 kb

TnAxI

upforiT

Fig. 1. Circular map of pA81. The ORFs are colored as follows: degradative

genes and genes for resistance to heavy metals, dark gray; plasmid backbone,

light gray; recombinases, black; other functions, white.

Genes encoding ohbAB and hybAD were cloned and ex-pressed in E. coli as described elsewhere [6]. Enzymatic activ-ity was tested in lysates from cells expressing OhbAB orHybAD, or non-transformed E. coli. Lysates were added toan assay cocktail that contained substrate (2,5-DCB;0.5 mM) and NADH (1 mM) in phosphate buffer (20 mM,pH 7). The mixtures were incubated at 25 �C for 2 h and sam-ples were taken periodically for analysis of 2,5-DCB and theexpected transformation product (4-chlorocatechol, 4-CC) byusing HPLC.

2.3. Nucleotide sequence accession number

The sequence of pA81 was deposited in EMBL underaccession number AJ515144 and the 16S rRNA gene ofAchromobacter xylosoxidans A8 under accession numberAJ875020.

3. Results and discussion

3.1. General features

Plasmid pA81 is a circular element (Fig. 1) that is predictedto encompass 103 ORFs. Function was predicted for the puta-tive products of 77 ORFs (Table 1); the remainder were eitheraligned to conserved hypothetical proteins (7 ORFs), or couldnot be assigned a function (19 ORFs). The plasmid consists ofan IncP1-b ‘‘backbone’’ (41.6 kbp, 58 ORFs) into which twotransposons, TnAxI and TnAxII, are inserted (Fig. 1, Table1). TnAxI is a type I transposon (39.2 kbp, 39 ORFs) that isinserted between the trb and tra gene clusters and carriesgenes for (halo)aromatic acid degradation (Figs. 1e3, Table1). TnAxII is a type II transposon (17.4 kbp, 16 ORFs) con-taining heavy metal resistance genes, and is inserted betweenoriV and trfA (Figs. 1e3, Table 1).

The junctions between the trb-tra cluster and oriV-trfA arecommon insertion points for transposons in IncP1-b plasmids.However, the structure of pA81 differs from other IncP plas-mids encoding biodegradative functions (including the newlydetermined IncP1-b plasmids) in that, in the latter group,biodegradation transposons are integrated between the oriVand trfA regions [1]).

120 V. Jencova et al. / Research in Microbiology 159 (2008) 118e127

3.2. (Halo)aromatic acid degradation functions encodedon TnAxI

The (halo)aromatic acid transposon TnAxI carries operonsfor catabolism of ortho-substituted CB (ohbRAB, mocpRABCD),and also includes an operon predicted to function in salicylatedegradation (hybRABCD). A similar combination of genes wasdescribed in P. aeruginosa JB2, where it is believed to be partof a clc element-like genomic island (see below). Of the knownIncP1-b degradative plasmids, only pJP4 has genes encodinghaloaromatic compound degradation [28]. The rest of theIncP1-b degradative plasmids encode for the catabolism ofhaloacetate (pUO1, [21]), atrazine (pADP, [12]) or toluenesulfonate (pTSA, [27]).

Catabolism of o-CB is initiated by an o-halobenzoate, aro-matic ring-hydroxylating oxygenase, which transforms o-CBand polychlorinated CB into catechol and chlorocatechols, re-spectively [6,30]. The o-halobenzoate oxygenase is composedof a small subunit iron sulfur protein (ISPb) and a large sub-unit iron sulfur protein (ISPa) that are encoded by ohbB andohbA, respectively (Table 1). OhbB of TnAxI is 100% identi-cal to an OhbB homolog in Burkholderia xenovorans LB400[4], and has 99% identity to OhbB homologs of P. aeruginosa142 [30] and P. aeruginosa JB2 [6]. The OhbA homologs fromall four of these sources are identical.

Proteins having the greatest identity in amino acid sequenceto OhbB are AhdA1c (51%) from Sphingomonas sp. P2,PhnA1b (49%) of Sphingomonas sp. CHY-1 and BphA1c(49%) of pNL1 in Novosphingobium aromaticivorans F199.Both AhdA1c and PhnA1b have been demonstrated to be sub-units of salicylate 1-hydroxylase, which transforms salicylateto catechol. While, to the best of our knowledge, activityhas not been determined for BphA1c, this protein is 96%identical to AhdA1c and thus is also likely to function as a sa-licylate 1-hydroxylase. For OhbB, the most similar proteins(50e51% identity) are the small subunits of the three salicy-late 1-hydroxylases listed above. The arrangement of genesencoding the large and small subunits of the terminal oxygen-ase is similar in the o-halobenzoate oxygenase and salicylate1-hydroxylase gene clusters (Fig. 4A). Thus, the o-haloben-zoate oxygenase and salicylate 1-hydroxylases may havediverged from a common ancestor.

The four known ohb-encoded o-halobenzoate oxygenasesare alike in that genes encoding the ferredoxin and ferredoxinreductase components, which are required for electron transferand enzyme activity, are not clustered with the oxygenase genes(Fig. 4A). In A. xylosoxidans A8, hybD and hybA (encoding fer-redoxin reductase and ferredoxin components, respectively, ofsalicylate 5-hydroxylase; see below) are located on TnAxI 4 kbdownstream from ohbAB, and could fulfill this role. Heterolo-gous expression was done to verify activity predicted for oh-bAB and to test the hypothesis that ohbAB-mediatedtransformation of o-halobenzoates might be supported or en-hanced by interaction with hybAD. In assay mixtures contain-ing lysates from cells expressing OhbAB and HybAD, about20% of the 2,5-DCB was degraded with stoichiometricproduction of 4-CC. In contrast, there was no detectable

degradation of 2,5-DCB or 4-CC formation in assays con-taining lysates from non-transformed cells, cells expressingOhbAB alone or HybAD alone. These results support theabove-stated hypothesis.

Expression of ohbAB is presumably controlled by an IclR-type transcriptional regulator (encoded by ohbR) that is imme-diately upstream of these genes (Table 1, Fig. 4A). The IclRproteins possess an N-terminal helix-turn-helix (HTH) motiffor DNA binding; domains in the IclR C-terminal are believedto be involved in effector binding and protein multimerization[29]. B. xenovorans LB400, P. aeruginosa 142 andP. aeruginosa JB2 all possess an ohbR homolog upstream ofthe ohbAB genes (Fig. 4A). OhbR of P. aeruginosa strain142 possesses the N-terminal HTH motif, and its size is withinthe range expected for IclR proteins (238e280 amino acids[29]. However, the predicted OhbR of pA81, as well as thatof P. aeruginosa JB2 and B. xenovorans LB400, are appar-ently truncated (70e108 aa). Compared to OhbR of P. aerugi-nosa 142, all that remains in the truncated forms of OhbR isthe N-terminal HTH motif, a region in which the OhbR homo-logs are essentially identical. Given that the truncated OhbRforms apparently lack the C-terminal domains that may influ-ence OhbR functionality, it remains to be determined how (orif) these proteins effect regulation of ohbAB.

The o-CB degradation pathway is completed by the moc-pRABCD, which encodes the modified ortho-cleavage path-way for assimilatory catabolism of chlorocatechols [8]. Theproducts of the TnAxI mocpRABCD cluster have 95e100%sequence identity with homologs carried on pRW71 (tet-RABCD, [17]), pP51 (tcbRABCD, [31]) and pENH91(cbnRABCD, [13]). Of the three, mocpRABCD is most similarto the cbnRABCD cluster, which is encompassed by a compos-ite transposon Tn5707 [13]. Furthermore, TnAxI and Tn5707are delineated by similar insertion sequence elements (seebelow), and the congruity between these transposons suggestthat they are descendants of a common ancestral element.

Upstream of the mocp cluster are genes predicted to encodea salicylate 5-hydroxylase (hybRABCD). The predicted prod-ucts of hybRABCD were 99% identical to those from genesin P. aeruginosa JB2 which encode a salicylate 5-hydroxylase(hybACBD; [6]) and a LysR-type regulatory element (hybR).HybRABCD also had ca. 83% identity to NdsRABCD of Pig-mentiphaga NDS-2 and ca. 79% identity to NagRAaGAbH ofPolaromonas napthalenivorans CJ2, Polaromonas sp. JS666and Ralstonia sp. U2 (pWWU2). In the hyb, nds and nag clus-ters, the arrangement of genes encoding different componentsof salicylate 5-hydroxylase is well conserved (Fig. 4B) andincludes a ferredoxin reductase, large subunit of terminaloxygenase, a small subunit of terminal oxygenase and a ferre-doxin, the exception being that, in the case of TnAxI, hybC(encoding the oxygenase small subunit) is disrupted by a trans-posase and the resultant polypeptide is predicted to be trun-cated from its full length of 160 amino acids to 68 aminoacids. While the effects of HybC truncation on salicylate 5-hydroxylase activity are unknown, tests with whole cells ofA. xylosoxidans A8 show that it can degrade salicylate and5-chlorosalycilate.

Table 1

Summary of ORFs annotated on pA81

Genea Position aab dirc Function of closest relative (source) IDd E value

TrfA 1286e60 408 � Replication initiation protein (pB4) Q8RSK0 4.63e-141

ssb 1673e1335 112 � Single-strand DNA binding protein (pB4) Q8RSJ8 3.4e-47

trbA 1783e2142 119 þ Transcriptional regulator (pB4) Q8RSJ7 3.2e-45

trbB 2457e3422 321 þ Mating pair formation protein TrbB (pB4) Q8RSJ6 1.3e-121

trbC 3452e3916 154 þ Mating pair formation protein TrbC-prepilin (pB4) Q8RSJ5 1e-56

trbD 3920e4231 103 þ Mating pair formation protein TrbD (pB4) Q8RSJ4 2.2e-43

trbE 4228e6786 852 þ Mating pair formation protein TrbE (pB4) Q8RSJ3 0

trbF 6783e7571 262 þ Mating pair formation protein TrbF (pB4) Q8RSJ2 1.3e-106

trbG 7568e8482 304 þ Mating pair formation protein TrbG (pB4) Q8RSJ1 63.6e-126

trbH 8485e8946 153 þ Mating pair formation protein TrbH (pB4) Q8RSJ0 8.1e-59

trbI 8950e10,329 459 þ Mating pair formation protein TrbI (pB4) Q8RSI9 7.6e-150

trbJ 10,346e11,119 257 þ Mating pair formation protein TrbJ (Sphingomonas sp. pA1) BAE19737 3.9e-90

trbK 11,129e11,350 73 þ Entry exclusion protein TrbK (Sphingomonas sp. pA1) BAE19738 9.8e-28

trbL 11,362e13,080 572 þ Mating pair formation protein TrbL (pB4) Q8RSI6 6e-162

trbM 13,099e13,677 192 þ TrbM protein (pB4) Q8RSI5 5.5e-92

trbN 13,679e14,314 211 þ Muramidase TrbN (Sphingomonas sp. pA1) BAE19741 2e-91

trbO 14,344e14,613 89 þ TrbO protein (pB4) Q8RSI3 1.7e-33

trbP 14,610e15,308 232 þ TrbP protein (Sphingomonas sp. pA1) BAE19743 1e-98

upf 30.5 15,324e15,767 147 þ Outer membrane protein (pB4) Q8RSI1 4.7e-54

orf20 15,864e16,286 140 þ Putative major outer membrane lipoprotein precursor (pKJK5) QOVUV3 6e-44

IstA21 16,386e17,942 518 þ Transposase IstA protein (Alcaligenes eutrophus pENH91) BAA33969 0

IstB22 17,932e18,726 264 þ Transposase IstB protein (Alcaligenes eutrophus pENH91) BAA33970 5e-141

orf23 18,859e18,996 45 þ Hypothetical protein fr. (P. chlororaphis RW71) Q9L3W3 2.8e-17

orf24 20,012e18,993 339 � Hypothetical protein (Delftia acidovorans P4a) AAL86584 0

mocpD 21,164e20,106 352 � Maleylacetate reductase (Pseudomonas sp. PP51) P27101 0

mocpC 21,877e21,161 238 � Dienelactone hydrolase (Alcaligenes eutrophus pENH91) BAA74533 1.1e-135

orfX 22,909e21,899 336 � Hypothetical protein (Delftia acidovorans P4a) AAL86583 2e-170

mocpB 24,023e22,911 370 � Chloromuconate cycloisomerase (Delftia acidovorans P4a) Q9RNZ9 0

mocpA 24,775e24,020 251 � Chlorocatechol-1,2-dioxygenase (Alcaligenes eutrophus pENH91) Q9WXC8 5e-142

mocpR 24,925e25,809 294 þ Lys-R type regulatory protein (Alcaligenes eutrophus pENH91) Q9WXC7 5e-144

orf31 26,937e26,563 124 � Fumarylacetoacetate hydrolase (Burkholderia xenovorans LB400) YP_559887 7e-66

orf32 27,379e26,963 126 � Hypothetical protein (Burkholderia xenovorans LB400) YP_559886 8e-63

orf33 28,274e27,345 309 � Putative dioxygenase (Burkholderia xenovorans LB400) YP_559885 0

orf34 28,600e28,271 109 � Hypothetical protein (B. xenovorans LB400) YP_559884 3e-49

orf35 29,518e28,691 275 � Putative dehydrogenase (B. xenovorans LB400) YP_559883 3e-131

orf36 30,747e29,755 330 � ABC transporter periplasmic protein (B. xenovorans LB400) YP_559882 6e-179

orf37 31,968e31,060 302 � Putative LysR-type transcriptional regulator (P. aeruginosa JB2) AAC69490 9e-114

orf38 32,492e32,007 161 � Putative substrate binding protein (P. aeruginosa JB2) AAC69489 6e-84

Tnp39 33,617e32,649 322 � Transposase (P. aeruginosa JES) AAD38927 9e-112

orf40 34,224e33,937 95 � ABC transporter ATP binding protein (P. aeruginosa JB2) AAC69488 4.2e-44

orf41 34,692e34,366 108 � ABC transporter ATP binding protein (Polaromonas sp. JS666) YP_547841 1e-31

orf42 35,687e34,689 332 � ABC transporter permease protein (Polaromonas naphtalenivorans CJ2) ZP01019783 1e-72

orf43 36,625e35,690 311 � Putative membrane spanning protein (P. aeruginosa JB2) AAC69487 2e-135

hybD 36,977e36,663 104 � Ferredoxin component of salicylate 5-hydroxylase (P. aeruginosa JB2) AAC69486 1.7e-49

tnpA 37,342e38,310 322 þ Transposase (P. aeruginosa JES) AAD38932 9e-112

hybC 38,660e38,442 72 � 2-hydroxybenzoate 5-hydroxylase beta subunit (P. aeruginosa JB2) AAC69485 2e-31

hybB 39,925e38,663 420 � 2-hydroxybenzoate 5-hydroxylase alpha subunit (P. aeruginosa JB2) AAC69484 0

hybA 40,939e39,953 328 � 2-hydroxybenzoate 5-hydroxylase alpha subunit reductase (P. aeruginosa JB2) AAC69483 2e-178

hybR 41,047e41,979 310 þ Putative LysR-type transcriptional regulator (P. aeruginosa JB2) AAC69482 1e-173

orf50 43,192e42,038 384 � Extracellular ligand binding receptor (Polaromonas sp. JS666) YP_547846 2.8e-162

ohbR 43,605e43,330 71 � Probable transcriptional regulator (P. aeruginosa 142) AAD20004 1e-25

ohbA 43,967e44,497 176 þ Ortho-halobenzoate oxygenase small subunit (B. xenovorans LB400) YP_559890 5e-94

ohbB 44,538e45,824 428 þ Ortho-halobenzoate oxygenase large subunit (B. xenovorans LB400) YP_559891 0

Tnp54 46,576e46,061 171 � Transposase fr.(Idiomarina loihiensis L2TR) AAV81475 2.1e-93

orf55 46,590e46,760 56 þ Putative MerR family regulator fr. (Shewanella sp. W3-18-1) ZP00905340 8e-18

Tnp56 46,648e47,673 318 þ Transposase (Burkholderia cenocepacia PC184) ZP00978227 1e-106

Tnp57 47,670e48,530 286 þ DNA replication protein (Burkholderia cenocepacia PC184) ZP00978228 5e-100

Tnp58 48,591e49,409 272 þ Putative transposase A (P. stutzeri M1) CAB42636 4e-141

IstA59 49,516e51,072 518 þ Transposase IstA protein (Alcaligenes eutrophus pENH91) BAA33969 0

IstB60 51,062e51,856 264 þ Transposase IstB protein (Alcaligenes eutrophus pENH91) BAA33970 5e-141

Tnp61 51,775e52,800 289 þ Transposase (Ralstonia metallidurans CH34) YP_145631 2e-160

Tnp62 55,639e52,769 956 � Transposase (Delftia acidovorans P4a) AAM76780 0

traC 60,765e56,032 1577 � DNA replication primase (Sphingomonas sp. PA1) YP_302610 0

traD 61,167e60,769 132 � Conjugal transfer protein TraD (Sphingomonas sp. pA1) YP_302611 2e-40

(continued on next page)

121V. Jencova et al. / Research in Microbiology 159 (2008) 118e127

Table 1 (continued)

Genea Position aab dirc Function of closest relative (source) IDd E value

traE 63,246e61,186 686 � DNA topoisomerase (Sphingomonas sp. pA1) YP_302612 0

traF 63,797e63,261 178 � Plasmid transfer protein traF (pB4) Q8RSH3 2e-72

traG 65,698e63,794 634 � Conjugal transfer protein TraG (pB4) Q8RSH2 0

traH 66,407e65,997 752 � Conjugal transfer protein TraH (pB4) Q8RSH0 1.1e-35

traI 67,953e65,695 136 � DNA relaxase (Sphingomonas sp. pA1) YP_302615 0

traJ 68,359e67,988 123 � Conjugal transfer protein TraJ (pB4) Q8RSG8 9e-44

traK 68,949e69,368 139 þ Conjugal transfer protein TraK (Sphingomonas sp. pA1) YP_302618 4e-34

traL 69,368e70,093 241 þ Conjugal transfer protein TraL (Sphingomonas sp. pA1) YP_302619 2e-133

traM 70,093e70,533 146 þ Conjugal transfer TraM protein (pB4) Q8RSG5 1e-35

traN 71,363e70,728 211 � TraN protein (Acidovorax sp.JS42) ZP01384249 3e-46

traO 71,740e71,393 115 � TraO protein (Sphingomonas sp. pA1) YP_302622 9e-50

kfrA 72,936e71,914 340 � KfrA protein (Acidovorax sp.JS42) ZP01384247 4e-33

korB 74,198e73,137 353 � Transcriptional repressor protein KorB (pB4) Q8RSN4 2.9e-133

incC 75,277e74,195 360 � Inclusion membrane protein (pB10) Q7X3C2 4e-170

korA 75,264e74,956 102 � Transcriptional repressor protein KorA (pB10) Q7X3C3 7e-41

kleF 75,795e75,385 136 � KleF protein (pB3) Q5W3F3 9.9e-37

kleE 76,123e75,797 108 � KleE protein (pJP4) YP_025374 7e-28

kleA 76,569e76,333 78 � KleA protein (Acidovorax sp.JS42) ZP01384242 6e-26

korC 76,984e76,727 85 � Transcriptional repressor protein KorC (pB10) Q7X3B8 3e-29

klcB 78,210e76,981 409 � KlcB protein (pB10) Q7X3B7 3e-165

klcA 78,913e78,485 142 � Anti-restriction protein KlcA (R751) NP_044227 3e-60

dinJ 79,086e79,349 87 þ Putative DNA-damage-inducible protein (pB4) Q8RSM4 3e-27

orf87 79,336e79,617 93 þ Addiction module toxin RelE/StbE (Acidovorax sp.JS42) ZP01384236 4.6e-40

orf88 80,762e81,037 91 þ Hypothetical protein (Mycobacterium sp. JLS) Q1U2E9 5.5

orf89 81,761e82,123 120 � Hypothetical protein (Verminephrobacter eiseniae EF01-2) ZP01400433 3e-16

orf90 82,039e82,356 105 þ Hypothetical protein KpsF/GutQ (Psychrobacter sp. PRwf-1) ZP01272066 2.9

orf91 82,393e82,740 115 � Hypothetical protein (Oryza sativa) Q94E85 0.076

orf92 82,867e83,376 169 � hypothetical protein (Acidovorax sp.JS42) ZP01383248 7e-18

int 83,581e84,753 390 þ Phage integrase/recombinase (Acidovorax sp.JS42) ZP01383249 7e-118

orf94 84,662e85,990 442 � Putative glycerate kinase (Paracoccus denitrificans PD1222) ZP00633121 2e-84

orf95 85,995e86,498 167 � Putative lipoprotein signal peptidase (Ralstonia metallidurans CH34) YP_584452 7e-50

orf96 86,586e87,122 178 � Lipoprotein signal peptidase (Shewanella frigidimarina NCIMB400) YP_752133 2e-20

metA 87,208e90,117 969 � Metal-transporting P-type ATPase (Ralstonia metallidurans CH34) YP_584451 0

metR 90,207e90,599 130 þ Transcriptional regulator, MerR family (Ralstonia metallidurans CH34) YP_584450 1.7e-47

orf99 90,767e91,963 398 � Sterol desaturase (Rubrivivax gelatinosus PM1) ZP00242269 5e-63

orf100 92,038e92,673 211 þ Cation efflux protein (Ralstonia metallidurans CH34)) YP_584447 8e-63

pbrT 92,704e94,596 630 � Cytochrome c, Iron permease FTR1(Acidovorax sp. JS42) ZP01382706 3e-170

tnpA 94,797e97,823 1008 þ Transposase (Acidovorax sp. JS42) ZP01382707 0

tnpR 97,820e98,170 116 þ Putative tnpA repressor protein (Acidovorax sp. JS42) ZP01382612 3e-36

a Coding sequence designations were assigned from the closest relative as determined by BLAST searches.b Number of amino acids.c Direction of transcription.d GenBank accession number of the closest relative.

122 V. Jencova et al. / Research in Microbiology 159 (2008) 118e127

3.3. Heavy metal resistance functions on TnAxII

A. xylosoxidans A8 is resistant to both Pb(II) and Cd(II),and six ORFs within TnAxII are predicted to encode proteinsrelated to heavy metal resistance. Five of these ORFs have thegreatest similarity to genes identified on chromosome 1 of Ral-stonia metallidurans CH34, and the overall arrangement ofthis gene cluster in R. metallidurans CH34 is similar to thatof TnAxII. The cluster includes three genes predicted to func-tion in metal translocation (Table 1): metA encodes a protein90% identical to a metal efflux P-type ATPase, pbrT is pre-dicted to encode a protein with 60% identity to the Pb(II) up-take protein PbrT, and the product predicted for orf100 has themotif of a Co/Zn/Cd efflux system component and was 87%identical to CzcD (Rmet_2299) of R. metallidurans CH34.

The substrate spectrum of efflux systems operative in bac-terial resistance to heavy metal ions vary from broad to highly

specific for a particular metal ion. The P-type ATPase (MetA)encoded on TnAxII could serve as an efflux system eitheritself or as a part of a larger complex similar to those of R. met-allidurans CH34. Overall, the similarities in gene arrangementand protein sequences suggest an evolutionary relationshipbetween the clusters on TnAxII and R. metallidurans CH34.

3.4. Plasmid backbone structure

The structure of the pA81 backbone is similar to that ofother IncP1-b plasmids including: pA1 [5], pJP4 [28], pB4[23], pADP-1 [12], pUO1 [21], pTSA [27] and R751 [25].The pA81 backbone contained 44 highly conserved IncP1-b genes, including orf87, which is unique to IncP1-b back-bones [24].

The backbone of IncP1-b plasmids contains gene clustersresponsible for conjugative transfer, initiation of vegetative

5 kbp

mocpRABCD

ISAx1a

ohbR

ABhybRABCDABC transporter

ISAx2a ISAx2b ISAx1b

tnp62

tnp6

1 (f

r)

tnp5

4 (f

r)tn

p56

tnp5

7tn

p58

(fr)

istA

istB

istA

istB

2 kbp

OriV heavy metal resistance

tnpAR

in t

16,3 kbp55,7 kbp

istA

istB

orf2

3

orf2

4moc

pD

mocpC

orfX

mocpB

mocpA

mocpR

orf3

1or

f32

orf3

3

orf3

4

orf3

5or

f36

tnp3

9tn

p45

orf3

8or

f37

orf4

0

orf4

1

orf4

2or

f43hy

bDhy

bChy

bBhy

bAhy

bRor

f50

orf5

5oh

bRoh

bAoh

bB

tnp5

4tn

p56

tnp5

7tn

p58ist

Aist

Btn

p61

tnp6

2

trf trb TnAxI

TnAxI

b

b

a

a

TnAxII

TnAxII

80,7 kbp

traCDEFG

oriT

oriVtra

JIH

traKLM kfr ko

r kle klc

98,2 kbp

tnpAmetAint pbrT tnpRor

f100

orf9

9or

f88or

f89

orf9

0

orf9

1or

f92

orf9

4or

f95or

f96

metR

Fig. 2. Structural characteristics of TnAxI and TnAxII. Features are coded as follows: black arrows, recombinases; gray dots, direct repeats; white rectangles, LTR

sequences. Coloration of ORFs is as indicated in Fig. 1.

123V. Jencova et al. / Research in Microbiology 159 (2008) 118e127

replication, stable inheritance and regulatory networks forthese functions. The conjugative transfer modules (trb andtra regions) and replication module of pA81 are closely relatedto corresponding segments of multiresistance plasmid pB4[23], whereas the stable inheritance modules klc and kle aremore closely related to the IncP1-b multi-resistance plasmidpB10 [20]. The latter has been proposed to be a recombinant,since its transfer region is closest to pADP-1 and R751, but itsklc and kle regions are more similar to pB4: the structure ofpA81 appears to be the reciprocal of pB4.

The IncP1 plasmids described to date carry either catabolicgenes or multiresistance determinants. The fact that thebackbone of catabolic plasmid pA81 is most similar to

B

C

trf trb TnAxI

backbone

62.2 %

42 % 56 % 62 %

0 kbp 20

40

A

Fig. 3. Physical characteristics of pA81. Panel A, % G þ C profile (1 kb window).

EcoRV, HindIII, KpnI, NdeI, SacI, SmaI, SpeI, XbaI). Panel C, genetic map (whi

indicate direction of transcription of individual gene clusters).

multiresistance plasmids is consistent with the hypothesisthat IncP1 plasmids represent a single evolutionary line andvary their composition depending on the environment, ratherthan representing multiple evolutionary lines [26]. As withother IncP1-b plasmids, the pA81 backbone has a mosaicstructure [48] that is indicative of recombination with otherplasmids encountered in the environment.

3.5. Structure and evolution of TnAxI

TnAxI encodes CB degradation and its integration in thetrb-tra region is unique as the mobile elements containing deg-radation clusters are in the IncP plasmids normally integrated

backbone

TnAxIItraCDEFG

oriT oriV

traJIH

traKLM kfr ko

r kle klc

60 80 98,2

Panel B, localization of restriction endonuclease sites (BamHI, BglII, EcoRI,

te boxes, plasmid backbone clusters; gray boxes, TnAxI and TnAxII; arrows

idX1 nagR nagAa nagG nagH nagA

bnagAc nagAd nagB nagF nagC nagG

R

R

hybDhybChybBhybAhybR hybE

orf41hybDtnphybChybBhybAhybRorf48

R

R

nagR nagAa nagG nagH nagAb nagAc nagAd nagB

idX973 idX972 idX971 idX970 idX969

ndsR ndsA ndsB ndsC ndsD orf6

R

B

phnC phnA3 phnA2b phnA1b phnD

ohbC

ohbAohbRorf ohbB tnp tnp -

top - ohbR ohbA ohbB tnpA

ohbC

xylY xylX bphCbp

hA3

bphA

2cbphA1c nahD

bxeA1110 bxeA1106ohbR ohbA ohbB

nahDahdA1cbphcxylXxylY ahdA

2c

ahdA

3

ohbR ohbA ohbB tnp tnporf50A

Fig. 4. Comparison of gene arrangements of the ring-hydroxylating oxygenase clusters. Panel A, ortho-halobenzoate oxygenases and salicylate 1-hydroxylase gene

clusters. Panel B, salicylate 5-hydroxylase clusters. Genes illustrated are as follows: ,, ferredoxin; B, small subunit of oxygenase (ISPb); #, large subunit of

oxygenase (ISPa); R, ferredoxin-reductase; recombinase, black; regulatory genes, dark gray; degradation genes (ring-hydroxylating oxygenase subunits), light

gray; other genes, white. Sequences illustrated are from (GenBank accession number): 1, Achromobacter xylosoxidans A8 pA81 (AJ515144); 2, Burkholderia xen-

ovorans LB400 (CP000272); 3, Pseudomonas aeruginosa 142 (AF121970); 4, Pseudomonas aeruginosa JB2 (AF087482); 5, Sphingomonas sp. P2 (AB091692); 6,

Sphingomonas sp. CHY-1 (AJ633552); 7, Sphingomonas aromaticivorans pNL1 (AF079317); 8, Achromobacter xylosoxidans A8 pA81 (AJ515144); 9, Pseudo-monas aeruginosa JB2 (AF087482); 10, Pigmentiphaga sp. NDS-2 (AB098505); 11, Polaromonas naphthalenivorans CJ2 (NZ_AANM01000003); 12, Polaromo-

nas sp. JS666 (NC_007948); 13, Ralstonia sp. U2 pWWU2 (AF036940).

124 V. Jencova et al. / Research in Microbiology 159 (2008) 118e127

into the oriV-trfA region. TnAxI is flanked by two identical in-sertion sequences, ISAxIa and ISAxIb, oriented in the samedirection (Fig. 2), an arrangement characteristic of type Itransposons [2]. There are 3 bp (TGT) direct repeats (DR) atboth termini of TnAxI. These IS elements belong to theIS21 family [11], which is characterized by terminal invertedrepeats (right/left; IRR/L) of 11e50 bp, usually in two or threecopies. In the case of ISAx1, there are duplicated 30 bp IRLand 25 bp IRR repeats. The IS21-type recombinase is com-posed of two subunits, IstA and IstB, and the IstABISAx1

subunits are 99.8% identical to IstAB of Tn5707 identifiedon pENH91 of R. eutropha NH9 [13]. As mentioned above,TnAxI and Tn5707 contain identical genes encoding a modi-fied ortho-cleavage pathway, and the collective similaritiesbetween TnAxI and Tn5707 indicate that they descendedfrom a common ancestral element.

Alignment of TnAxI and Tn5707 indicates that there hasbeen at least one additional insertion into the ancestral

transposon (Fig. 5). Variations in average % G þ C contentof the DNA inserted into TnAxI indicate that there are threedistinct regions originating from different sources. The first re-gion (25.8e32 kbp; orf31e36, Table 1, Fig. 3) has an average% G þ C of 42 and contains a number of hypothetical pro-teins. The second region (32e43.3 kbp; orf38e50, Table 1,Fig. 3) has an average G þ C content of 56% and encompasseshybRABCD and ORFs of an ABC transport system. The thirdarea (43.3e46 kbp; ohbR-tnp54) has average G þ C content of62% and spans the genes encoding the o-halobenzoateoxygenase.

The extensive similarities between TnAxI, B. xenvoransLB400 and P. aeruginosa JB2 suggest that a common elementwas involved in dissemination of the three regions describedabove. In B. xenvorans LB400 and P. aeruginosa JB2, theseareas are carried on a genomic island similar to the clc-element [4], and alignments of TnAxI, clc-JB2 and clc-LB400illustrate areas of shared DNA sequences (Fig. 5). All three

tnppA81 TnAxI

P4a

pENH91Tn5707

clc JB2

clc LB400

tnpmocp

ABC hyb

ohbclc glyV

IS 1600 IS 1600cbn

tfd IS 1071IS 1071(fr)

ABC ohb

clc

Fig. 5. Alignment between TnAxI and evolutionarily related mobile elements. Areas of identical nucleotide sequence are joined by gray shading. White boxes

indicate degradation clusters, black boxes indicate recombinase genes and gene clusters, arrows indicate direction of transcription. Sequences illustrated are

from (abbreviation, GenBank accession number): Delftia acidovorans P4a (P4a, AY078159); Ralstonia eutropha NH91 (pENH91 Tn5707, AB019032); Achromo-bacter xylosoxidans A8 (pA81 TnAxI, AJ515144), Pseudomonas aeruginosa JB2 (clc JB2, AF087482), Burkholderia xenovorans LB400 (clc LB400, CP000272).

125V. Jencova et al. / Research in Microbiology 159 (2008) 118e127

elements are alike in possession of the ohb cluster, as well as aregion upstream of ohb that corresponds to orf31e36 on TnAxI.The clc-element in P. aeruginosa JB2 and TnAxI differs fromclc-LB400 in that the hyb and ABC regions are inserted betweenthe ohb cluster and the orf31e36 region (Fig. 5). Compared toclc-JB2, the hyb and ABC transporter regions on TnAxI haveundergone extensive rearrangement and deletion. The clc genesand mocp/cbn genes represent two different evolutionarybranches within the family of genes encoding the modified or-tho-cleavage pathway for chlorocatechol metabolism [8]. Thus,the pairing of ohb with clc genes in clc-JB2 and clc-LB400, andwith mocp in TnAxI indicates a pattern of modular assembly forthe o-CB degradation pathway.

The alignment between Tn5707, TnAxI, clc-JB2 and clc-LB400 (Fig. 5) clearly indicates that these elements havecrossed evolutionary paths. It is likely that the ohb-hyb-ABCtransporter cassette assembled once and was then disseminatedinto the environment, rather than assembling multiple times indifferent organisms. All known ohb clusters are alike in thatthey have the same transposase (tnpA) immediately down-stream and this recombinase was possibly involved in transferof the ohb cluster to the cassette. As mentioned above, com-pared to clc-JB2, the hyb and ABC transporter regions onTnAxI have undergone extensive rearrangement and deletion,and thus the ohb-hyb-ABC transporter cassette of TnAxI ap-pears to represent an evolutionary progression from that inclc-JB2. However, the evolutionary relationship between theclc elements in P. aeruginosa JB2 and B. xenovorans LB400is unclear: the presence or absence of the hyb-ABC transporterregion could represent either acquisition of this DNA in thecase of clc-JB2 or deletion of it in the case of clc-LB400. Itis also unclear whether the ohb-hyb-ABC transporter cassettefirst assembled on a clc-like element and subsequently inte-grated into a plasmid-borne transposon or vice versa.

Next to TnAx1 (region 51.9e56 kbp), orf62 and orf61(fragment) are identical to the transposase of IS1071. IS1071transposases flank chlorocatechol degradation genes in p4a

[7] and other biodegradative genes in pJP4 [28], pUO1 [21],pTSA [27], pEST4011 [32] and pADP1 [12]. The only exam-ple of an IS1071 homolog not associated with degradationgenes is on the IncP1-b antibiotic resistance plasmid pB10[20]. In pA81, it is possible that the transposition event(s) di-rected by orf62-encoded recombinase played a role in inser-tion of TnAxI into pA81. However, we did not identify anyLTR sequences characteristic of the Tn3 family of transpo-sases that would indicate borders of this TnAxI. Alternatively,the IS1071 transposases may have been associated witha different transposon that was inserted into pA81, and whichhas since been largely deleted.

3.6. Structure and evolution of TnAxII

The boundaries of TnAxII are delimited by tnpAR (orf102and orf103) and the corresponding LTR sequences, and ithas structural features characteristic of the type II transposons[18]. Transcription of Tn21-type transposases is regulated byrepressor TnpR encoded by an ORF localized next to thetransposase gene [9]. TnpATnAxII had 65% identity to a transpo-sase identified on Tn5041 from Pseudomonas sp. KHP41 [9],which carries the heavy metal resistance mer genes. TnpAbelongs to the Tn21 transposase family, one characteristic ofwhich is a 38 bp LTR that usually begins with GGGG andends in TAAG [23]; similar LTR sequences border TnAxII.The ends of TnAxII are bordered by 5 bp (TCATC) directrepeats.

TnpATnAxII also has homology to other transposases identi-fied on elements carrying genes responsible for heavy metalresistance or catabolism of xenobiotics. These include TnpAof s-triazine-degrading P. huttiensis NRRLB-12228 (GenBankQ9ALU6), TnpAc from the carbazol/dioxine catabolic trans-poson Tn4676 in pCAR1 of P. resinovorans CA10 andTnpA in the pWWO plasmid of P. putida mt-2, which ispart of the Tn4651 transposon carrying xyl genes for xyleneand toluene catabolism.

126 V. Jencova et al. / Research in Microbiology 159 (2008) 118e127

TnAxII also carries an int gene (orf93), which is predictedto encode a phage integrase similar to that of Acidovorax sp.JS42. This integrase could have driven a transposition eventthat affected the composition of TnAxII. However, nucleotidesequence markers that would be indicative of the event werenot identified.

3.7. Concluding remarks

DNA sequence analysis of pA81 has provided insight intothe basic biology of the plasmid, specific metabolic functionsthe plasmid may confer on a host organism, the evolutionaryrelationships of the inserted transposons to other mobile ge-netic elements, and the evolutionary relationship of pA81 toother IncP1-b plasmids. The finding that the backbone of thebiodegradation/heavy metal resistance plasmid pA81 is mostsimilar to multiresistance plasmids is consistent with the hy-pothesis that IncP1 plasmids comprise a single lineage and al-ter their gene content depending on the environment inhabitedby the host organism. Analysis of the two transposons TnAxIand TnAxII carried by pA81 reveals specialization for biodeg-radation and heavy metal resistance, and also establishes thatthese transposons form a linkage between pA81, other biode-gradative/resistance plasmids and genomic islands. Collec-tively, these insights further our understanding of mobileelements and interactions between elements that affect thefate of organic and inorganic toxicants in the environment.

Acknowledgements

This work was supported by the project of the Czech Minis-try of Education, Youth and Sports No. 1M6837805002 (Centerfor Applied Genomics), by the project of the Academy of Sci-ences of the Czech Republic No. AV0Z50520514, the UnitedStates Department of Agriculture. (9501422 to WJH) and theUnited States National Science Foundation (9722620 to WJH).

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