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Journal of Bacteriology, November 2004, p. 7161-7174, Vol. 186, No. 21
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.21.7161-7174.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

The Completely Sequenced Plasmid pEST4011 Contains a Novel IncP1 Backbone and a Catabolic Transposon Harboring tfd Genes for 2,4-Dichlorophenoxyacetic Acid Degradation

Eve Vedler,* Merle Vahter, and Ain Heinaru

Department of Genetics, Institute of Molecular and Cell Biology, Tartu University, Tartu, Estonia

Received 7 July 2004/ Accepted 27 July 2004


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ABSTRACT
 
The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterium Achromobacter xylosoxidans subsp. denitrificans strain EST4002 contains plasmid pEST4011. This plasmid ensures its host a stable 2,4-D+ phenotype. We determined the complete 76,958-bp nucleotide sequence of pEST4011. This plasmid is a deletion and duplication derivative of pD2M4, the 95-kb highly unstable laboratory ancestor of pEST4011, and was self-generated during different laboratory manipulations performed to increase the stability of the 2,4-D+ phenotype of the original strain, strain D2M4(pD2M4). The 47,935-bp catabolic region of pEST4011 forms a transposon-like structure with identical copies of the hybrid insertion element IS1071::IS1471 at the two ends. The catabolic regions of pEST4011 and pJP4, the best-studied 2,4-D-degradative plasmid, both contain homologous, tfd-like genes for complete 2,4-D degradation, but they have little sequence similarity other than that. The backbone genes of pEST4011 are most similar to the corresponding genes of broad-host-range self-transmissible IncP1 plasmids. The backbones of the other three IncP1 catabolic plasmids that have been sequenced (the 2,4-D-degradative plasmid pJP4, the haloacetate-catabolic plasmid pUO1, and the atrazine-catabolic plasmid pADP-1) are nearly identical to the backbone of R751, the archetype plasmid of the IncP1 ß subgroup. We show that despite the overall similarity in plasmid organization, the pEST4011 backbone is sufficiently different (51 to 86% amino acid sequence identity between individual backbone genes) from the backbones of members of the three IncP1 subgroups (the {alpha}, ß, and {gamma} subgroups) that it belongs to a new IncP1subgroup, the {delta} subgroup. This conclusion was also supported by a phylogenetic analysis of the trfA2, korA, and traG gene products of different IncP1 plasmids.


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INTRODUCTION
 
Microbial degradation of 2,4-dichlorophenoxyacetic acid (2,4-D), a xenobiotic herbicide used worldwide for almost 60 years, is a well-studied process. Various soil bacteria can use 2,4-D as a carbon and energy source. Therefore, this compound has become a model for studying the evolution and distribution of genes for the degradation of chloroaromatic compounds. A number of bacterial strains belonging to different phylogenetic groups able to mineralize this compound have been found to possess genetically and enzymatically different 2,4-D-catabolic pathways (9, 10, 16-18). The best-studied 2,4-D degradation genes (located in a chromosome or a plasmid) are tfd-like (pJP4-like). The very recently sequenced 87,688-bp plasmid pJP4 (48) from Wautersia eutropha JMP134 (formerly Ralstonia eutropha) was originally isolated in Australia (8), and its tfd genes and the corresponding enzymes responsible for converting 2,4-D to 3-oxoadipate are well characterized (22, 23, 25, 26, 35, 58). Besides pJP4, there are only two cases in which the DNA regions containing tfd genes for the whole 2,4-D degradation pathway have been sequenced, a chromosomal transposon-like structure (about 30 kb) from Delftia acidovorans P4a (15) and Tn5530 (41 kb) located in plasmid pIJB1 from Burkholderia cepacia 2a (36, 56). The tfd genes of these two elements are homologous to the pJP4 tfd genes but are organized in a different way.

Bacterial catabolic genes are often encoded by mobile genetic elements, including transposons and conjugative plasmids. Genes that encode degradation of man-made compounds are often found in IncP1 plasmids which are divided into three subgroups, the {alpha}, ß, and {gamma} subgroups (1). These plasmids are the most promiscuous self-transmissible plasmids that have been characterized to date (46) and thus could be responsible for efficient dissemination of genes encoding degradation of recently introduced xenobiotic compounds. The (almost) complete nucleotide sequences of four IncP1 catabolic plasmids, pUO1 (39), pADP-1 (30), pJP4 (48), and pTSA (47) coding for haloacetate, atrazine, 2,4-D, and p-toluenesulfonate degradation, respectively, have been determined, and they have all been shown to be members of the IncP1 ß subgroup. Although only the catabolic region of the 2,4-D-degradative plasmid pIJB1 has been sequenced, it has been shown that this plasmid belongs to the same subgroup (36).

Nearly 20 years ago, several 2,4-D-metabolizing bacterial strains were isolated from different soil samples from Estonian agricultural enterprises. They all contained 2,4-D-degradative plasmids that were the same size (2; V. Kõiv, unpublished data). One strain, D2M4, containing plasmid pD2M4 (about 95 kb), was selected for further investigation as it showed the best growth characteristics on 2,4-D as a sole source of carbon and energy. However, the 2,4-D+ phenotype of this strain was very unstable. Different laboratory manipulations were performed in order to obtain a more stable phenotype. As a result, strain EST4002 containing plasmid pEST4011 (approximately 70 kb, as estimated by restriction analysis) was isolated. This strain was determined to be a strain of Achromobacter xylosoxidans subsp. denitrificans. According to hybridization experiments, the tfd-like genes for 2,4-D degradation were all located in pEST4011. 2,4-D– strain EST4003 (obtained when EST4002 was grown in Luria-Bertani medium) contained plasmid pEST4012 (about 30 kb), from which the whole catabolic region had been deleted (28, 53).

In this paper we report the complete nucleotide sequence of a 2,4-D degradative plasmid, the 76,958-bp pEST4011 plasmid, and we describe and analyze all open reading frames (ORFs) and other features detected in pEST4011. The restriction patterns of pEST4011 and its ancestor, pD2M4, which is unstable in the laboratory, are compared. On the basis of multiple alignments of all available trfA2, korA, and traG gene products of different IncP1 plasmids, we show that the backbone of pEST4011 is quite different from that of other IncP1 plasmids. Consequently, we suggest that pEST4011 belongs to a new IncP1subgroup, the {delta} subgroup.


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MATERIALS AND METHODS
 
Isolation of pEST4011 DNA. A. xylosoxidans subsp. denitrificans EST4002 cells harboring pEST4011 were maintained on minimal salts agar plates containing 5 mM 2,4-D. Plasmid pEST4011 was isolated from EST4002 cells that were grown overnight at 30°C in minimal salts medium supplemented with 5 mM 2,4-D. The culture obtained was inoculated into minimal salts medium supplemented with 20 mM sodium citrate and 0.2% Casamino Acids and grown overnight at 30°C. Plasmid DNA was isolated by using a method for isolation of large bacterial plasmids described by Hansen and Olsen (13), followed by CsCl buoyant density ultracentrifugation (38).

Construction of pEST4011 library. For construction of a pEST4011 library, partial digestion with Bsp143I was used to produce overlapping fragments that were approximately 1 kb long. These fragments were cloned into the BamHI site of the vector plasmid pBluescript II (SK) (Stratagene) and transferred into Escherichia coli DH5{alpha} cells. This and all other cloning procedures in this study were performed by using standard methods (38).

DNA sequencing. Templates for DNA sequencing were prepared by PCR amplification of inserted DNA from randomly selected insert-containing clones with the vector-specific primers T3 and T7. The PCR products obtained were directly sequenced by using the same primers (subsequent primer walking was used if necessary), a DYEnamic ET terminator cycle sequencing kit (Amersham Pharmacia Biotech), and an ABI Prism 377 DNA sequencer (Applied Biosystems).

pEST4011 sequence assembly. All sequences were edited and analyzed and the complete sequence of pEST4011 was assembled by using the BioEdit sequence alignment editor, version 5.0.9 (12). Vector sequences were removed, and sequences were assembled into contigs manually according to alignment results with the most similar complete sequences obtained from the GenBank, including those of Birmingham IncP-alpha plasmids (R18, R68, RK2, RP1, RP4) (accession number L27758), Enterobacter aerogenes plasmid R751 (NC_001735), and B. cepacia 2a plasmid pIJB1 (AF029344). In order to close gaps, custom primers were designed by using the ends of each contig. These primers were used for PCR amplification of DNA fragments containing the necessary sequences; purified pEST4011 DNA was used as a template. Again, the PCR products obtained were directly sequenced by using the same primers. Finally, assembly of the pEST4011 nucleotide sequence was verified by PCR analysis by producing overlapping products covering the whole plasmid and by restriction analysis with BamHI, ClaI, EcoRI, HindIII, KpnI, MfeI, MluI, NdeI, StuI, and XmnI.

Resequencing the identical regions of pEST4011. In order to separately resequence the identical copies of IS1071::IS1471 and the 6,991-bp duplications, two strategies were used. First, pEST4011 regions were cloned into the vector plasmid pBluescript II (SK) and sequenced by using vector-specific and custom primers. Second, two overlapping PCR products were generated by using one primer annealing outside the duplicated area and another primer annealing inside the duplicated area, and they were sequenced by using the same primers and subsequent primer walking.

Analysis of pEST4011 ORFs. The presence of ORFs in pEST4011 was analyzed by using the National Center for Biotechnology Information ORF Finder program (http://www.ncbi.nlm.nih.gov/gorf.html). All predicted ORFs that were at least 150 bp long were analyzed further with the BLASTN and BLASTP programs (http://www.ncbi.nlm.nih.gov/BLAST), and only the ORFs with reasonable homology to some other known sequence were selected. Translation start codon positions were determined manually on the basis of the presence of a potential Shine-Dalgarno sequence upstream of a start codon.

Phenotypic analysis with Biolog GN2 microplates. One day before inoculation of Biolog GN2 plates (Biolog Inc., Hayward, Calif.), strain EST4002 harboring plasmid pEST4011 and strain EST4003 harboring plasmid pEST4012 were streaked on BUGM (Biolog Inc.) agar plates. The wells of the Biolog GN2 plates were inoculated with 150 µl of a bacterial suspension adjusted to an optical density at 580 nm of 0.150 for both strains. The plates were incubated at 30°C for 72 h. Development of color was automatically recorded by using a microplate reader with a 620-nm-wavelength filter.

Multiple alignment and phylogenetic tree construction. The amino acid sequences of different IncP1 trfA2, korA, and traG gene products were aligned, and bootstrapped neighbor-joining trees were derived by using the program CLUSTALX, version 1.8 (43), with default parameters. The PHYLIP 3.6 software package (http://evolution.genetics.washington.edu/phylip.html) programs SEQBOOT, PROTDIST, NEIGHBOR, CONSENSE, and PROML were used to calculate protein distances and to derive the corresponding phylogenetic trees.

Nucleotide sequence accession number. The complete nucleotide sequence of pEST4011 from A. xylosoxidans subsp. denitrificans strain EST4002 has been deposited in the GenBank database under accession number AY540995.


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RESULTS AND DISCUSSION
 
Nucleotide sequence and organization of pEST4011. The 2,4-D-degradative plasmid pEST4011 is circular and 76,958 bp long and has an overall G+C content of 62.03%. Thus, pEST4011 is 7 kb larger than we previously calculated on the basis of restriction analysis. A circular physical map of pEST4011 is shown in Fig. 1. The first nucleotide of the HindIII restriction site between the upf54.4 and upf54.8 genes corresponds to position 1. The recognition site positions of 10 different restriction endonucleases are also shown in Fig. 1. Interestingly, 101 of 115 of these sites are located in the catabolic transposon of pEST4011. These restriction enzymes together with the PCR analysis were used to verify the assembly of pEST4011 (data not shown). Plasmid pEST4011 consists of the 29-kb IncP1 backbone loaded with the 48-kb catabolic transposon containing, among other genes, the tfd-like genes for complete 2,4-D degradation, bordered by identical IS1071::IS1471 hybrid insertion elements. The nucleotide sequence of this catabolic transposon is 99% identical to the sequence of Tn5530 (41 kb) of pIJB1, except for duplication of a 6,991-bp region in the case of pEST4011. As a result, the pEST4011 region from position 27334 to position 34324 is identical to the adjacent region from position 34325 to position 41315, with only one mismatch. In addition, the pEST4011 region from position 20101 to position 31555 is 99% identical to the sequenced region of Variovorax paradoxus TV1 2,4-D-degradative plasmid pTV1 (49, 50). The IncP1 backbone of pEST4011 consists of the standard elements (Fig. 1), including the origin for the theta mode of replication (oriV) regulated by iterons; the region necessary for autonomous replication initiation, copy number control, and stable maintenance in the host cell; and two regions involved in plasmid conjugation, Tra1 (tra genes) with an origin of transfer (oriT) and Tra2 (trb genes). However, seven trb genes (trbE to trbL) essential for mating pair formation during conjugation are not present in pEST4011.



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  		FIG. 1. Circular physical map of 76,958-bp plasmid pEST4011. The identified ORFs, the insertion elements, and the vegetative (oriV) and transfer (oriT) origins are shown inside the circle. The blue genes are necessary for plasmid autonomous replication initiation, copy number control, and stable maintenance in the host cell; the yellow genes are involved in plasmid conjugation; all genes in the pEST4011 catabolic transposon are purple. The 7-kb duplicated regions are indicated; the site from which the essential transfer genes trbE to trbL are missing is indicated by a line with a triangle at the end. R indicates the righthand copy of IS1071::IS1471, and L indicates the lefthand copy of IS1071::IS1471. The recognition site positions of 10 restriction endonucleases, which were used to verify the assembly of pEST4011, are indicated outside the circle. The colored circle indicates the G+C contents in 192-bp blocks starting from the position 1 (the first nucleotide in the corresponding HindIII recognition site). The products of the genes in quotation marks are truncated and thus probably not functional.

By using the National Center for Biotechnology Information ORF Finder program and BLAST similarity searches, 68 ORFs were identified in the pEST4011 sequence (Fig. 1; Table 1); 58 of these ORFs were named after the closest relative found in the GenBank database. Twenty-two ORFs are orientated in the same direction, whereas 46 are orientated in the opposite direction. Similarly, 34 ORFs are located both in the backbone and in the catabolic transposon of pEST4011 (9 of these ORFs are the tfd genes for 2,4-D catabolism). Six ORFs in the catabolic region (including one tfd gene) have identical copies due to duplication of a 7-kb DNA segment. Five ORFs code for putative transposases, and two of them are identical tnp1471 genes of IS1071::IS1471. Ten ORFs encode putative transcriptional regulators. Four ORFs start with GTG, and the rest start with an ATG translation start codon. The translation stop codons are TGA (42 ORFs), TAA (15 ORFs), and TAG (12 ORFs). A ribosome-binding site with low similarity (two to four matches) to the Shine-Dalgarno consensus sequence (5'-TAAGGAGGT-3') precedes 24 ORFs, while for 44 ORFs there are five to seven matches with the consensus sequence.


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  		TABLE 1. Locations and predicted functions of pEST4011 coding sequences

Genes involved in 2,4-D degradation. In A. xylosoxidans subsp. denitrificans EST4002, all tfd genes necessary for 2,4-D degradation (the pathway is shown in Fig. 2) are located on plasmid pEST4011 (Table 1; Fig. 1). Figure 3 shows the portion of the pEST4011 catabolic region containing the tfd genes, which are organized as follows. There is a cluster of genes comprising tfdC (which codes for 3,5-dichlorocatechol 1,2-dioxygenase) (21), tfdE (which codes for 2-chlorodienelactone hydrolase), tfdB (which codes for 2,4-dichlorophenol hydroxylase), tfdK (which codes for 2,4-D transporter), and tfdA (which codes for 2,4-D/{alpha}-ketoglutarate dioxygenase). tfdR, which encodes a LysR-type transcriptional activator (54), is located immediately upstream and is transcribed divergently from this cluster; almost 2 kb downstream of this cluster lies a gene for (2-chloro)maleylacetate reductase (tfdF); finally, 2,4-dichloro-cis,cis-muconate cycloisomerase is encoded by two identical copies of tfdD which are located 2.6 and 9.6 kb upstream of tfdR.



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  		FIG. 2. 2,4-D degradation pathway encoded by tfd genes. TCA, tricarboxylic acid.



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  		FIG. 3. Comparison of the arrangement of the tfd genes in plasmid pEST4011 and the homologous genes in D. lusatiensis (accession number AJ536297), D. acidovorans P4a (AY078159), and W. eutropha JMP134 plasmid pJP4 (AY365053). A pEST4011 tfd gene and its most similar counterpart are indicated by the same color. The red numbers below the genes indicate percentage of amino acid identity/percentage of amino acid similarity compared with the corresponding pEST4011 tfd analogue. The boxes around the genes indicate duplicated regions.

The A. xylosoxidans subsp. denitrificans EST4002 tfdE, tfdB, tfdK, and tfdA genes are almost identical to the same genes of the D. acidovorans P4a chromosomal transposon for 2,4-D degradation and are also organized in the same order (Table 1; Fig. 3). The tfdC and tfdR genes are most similar to the corresponding genes of W. eutropha JMP134 plasmid pJP4. The pEST4011 tfdD gene and orf5, which are immediately downstream of tfdD, are most similar to D. acidovorans P4a tfdD and orfI. The function of the product encoded by orfI is unknown, but this ORF has been found in many operons determining ortho cleavage pathways of chlorocatechols. The strongly conserved amino acid sequences encoded by these ORFs suggest that the products might play an essential role in the function of these operons (31). The chloromaleylacetate reductase encoded by pEST4011 tfdF is quite different from all other known chloromaleylacetate reductases; only 52% of its amino acids are identical to the amino acids of the closest relatives, the {alpha}-proteobacterial ClpF involved in chlorophenol degradation by Defluvibacter lusatiensis and Bradyrhizobium japonicum USDA 110 MacA (52% amino acid identity and 77% amino acid similarity).

All tfd gene products except TfdD and TfdF are very similar to pJP4 tfd gene products, including TfdA (84% amino acid identity and 94% amino acid similarity), TfdBII (91% amino acid identity and 97% amino acid similarity), TfdCII (89% amino acid identity and 97% amino acid similarity), TfdEII (79% amino acid identity and 93% amino acid similarity), and TfdK (74% amino acid identity and 90% amino acid similarity) (Fig. 3). The ancestors of the pEST4011 tfdRCEBKA gene cluster and the pJP4 tfdRDIICIIEIIFIIBIIK gene cluster probably had a common origin. In the case of pEST4011, the regions containing the tfdD and tfdF genes have been deleted. In the chromosomal transposon of D. acidovorans P4a only a portion of the tfdRCEBKA cluster is present in pEST4011 (Fig. 3). As tfdF and tfdD are not pJP4-like genes and are separated from the tfdRCEBKA gene cluster, they were probably individually recruited in the precursor of the pEST4011 catabolic transposon to replace the genes lost from the putative ancestor the tfdRDCEFBK(A) cluster during evolution. Vallaeys et al. have also shown that diverse gene cassettes were independently recruited during assembly of 2,4-D-catabolic pathways having different origins (51).

At a position 1.2 kb upstream of tfdD there is a gene (ccp) whose predicted product is most similar to Burkholderia fungorum EriC (41% amino acid identity and 65% amino acid similarity) and Xanthomonas axonopodis pv. citri str.306 (accession number NC_00125171) YadQ (37% amino acid identity and 65% amino acid similarity), which are the chloride-channeling proteins (37). As chloride ions are liberated during 2,4-D dissimilation, this protein may have a role in removal of these ions from cells.

The proposed functions and the levels of amino acid identity and similarity of other predicted gene products encoded by the pEST4011 catabolic transposon are shown in Table 1. In this transposon only 11 of 34 ORFs (nine tfd genes plus two copies of ccp coding for a chloride-channeling protein) are necessary for 2,4-D degradation. In addition to these ORFs, the presence of two copies of orf5 could be bound to the same function (31). Five ORFs code for (putative) transposases, and four ORFs are translated into (putative) transcriptional regulators; the functions of two ORFs are unknown. The remaining 10 ORFs potentially code for different catabolic functions, including indole acetamide hydrolase (two copies of iaaH), proline dehydrogenase/pyrroline-5-carboxylate dehydrogenase bifunctional protein (putA), and malonate decarboxylase (mdc operon of seven genes). As shown in Table 1, the closest relatives of these catabolic genes are found in bacteria known to be plant pathogens (X. axonopodis and Pseudomonas syringae) and in a nitrogen-fixing symbiontic bacterium (B. japonicum). We analyzed strains EST4002 (containing pEST4011) and EST4003 (containing the deletant plasmid pEST4012) by a microtiter plate-bound substrate utilization assay (Biolog Inc.). The scatter plot of substrate utilization activities for the two strains studied showed a high correlation (R2 = 97.8%, P < 0.0001), which means that all 95 carbon substrates from Biolog GN2 microplate wells were utilized by these strains at approximately the same rate (Fig. 4).



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  		FIG. 4. Scatter plot of substrate utilization activities for strain EST4002 containing plasmid pEST4011 and strain EST4003 containing deletant plasmid pEST4012, with a regression line. All 95 carbon substrates from Biolog GN2 microplate wells are represented by dots, and the locations of L-proline and malonic acid are indicated by arrows. OD620nm, optical density at 620 nm.

Indole acetamide hydrolase (IaaH) performs the second reaction in the two-step pathway for synthesis of indole acetic acid (a plant hormone auxin) from tryptophan, which is found in associated plant-growth-promoting rhizobacteria (41). Thus, even if the iaaH gene codes for a functional enzyme, plasmid pEST4011 itself does not enable its host to synthesize auxin. The PutA bifunctional protein converts L-proline to glutamate for use as a carbon and nitrogen source; in addition, it acts as a repressor of its own expression in response to the proline supply (27). The Biolog analysis showed that strains EST4002 and EST4003 both used L-proline at physiological concentrations as a growth substrate (Fig. 4). Thus, this ability must rely on chromosomal genes.

Decarboxylation of malonate to acetate and CO2 is a key reaction as it initiates decomposition of this compound in both aerobic and anaerobic bacteria (7, 20). The predominant portion of malonate in the environment originates from industrial production; therefore, bacterial degradation of this compound is now of great interest. In the case of B. cepacia both strain 2a (which contains pIJB1) and mutant strain 2a-1 (which contains plasmid pIJB2, in which the whole catabolic region has been deleted) (56) were able to grow on malonate as a sole source of carbon and energy. Xia et al. (56) concluded that the ability to grow on malonate must reside on the chromosome, although the plasmid-borne analogues of mdc genes could also be active. According to the substrate utilization assay, strains EST4002 and EST4003 do not use malonic acid as a growth substrate (Fig. 4). Therefore, as the nucleotide sequences of pEST4011 and pIJB1 mdc operons are 100% identical, this gene cluster alone is not adequate for malonate dissimilation in either host. Consequently, it seems that the only detectable catabolic property of EST4002 provided by plasmid pEST4011 is the ability to degrade 2,4-D. However, it is possible that in other hosts this plasmid contributes to additional properties (for example, auxin biosynthesis or malonate dissimilation).

The (putative) regulatory proteins encoded by pEST4011 (for example, Lrp) may alter the expression pattern of both plasmid and chromosomal genes. Genome analyses have revealed that members of the Lrp family of transcriptional regulators are widely distributed among prokaryotes. The archetype leucine-responsive regulatory protein from E. coli is a global regulator involved in modulating a variety of metabolic functions, including catabolism and anabolism of amino acids, as well as pilus synthesis (4). Lrp has also been shown to be a positive modulator of conjugal transfer of F-like plasmids (5, 40). However, the metabolic patterns of strains EST4002 and EST4003 with the carbon substrates available on Biolog GN2 microplates are not significantly different (Fig. 4).

Insertion elements in pEST4011. The 48-kb catabolic transposon of pEST4011 is bordered by identical (two mismatches) copies of the hybrid insertion element IS1071::IS1471 (positions 9700 to 14019 and 53315 to 57634) (Fig. 3). This element consists of the 1.1-kb IS1471 element (which belongs to the IS630 family described by Mahillon and Chandler [29]) inserted into the 3.2-kb class II transposable element IS1071 (57). While IS1071 flanks a variety of catabolic genes and operons (6), IS1471 has been detected only in the hybrid IS1071::IS1471 insertion elements present in pEST4011 and pIJB1. In the case of pIJB1, both copies of IS1071::IS1471 flanking Tn5530 have been sequenced only partially, but Poh et al. (36) have suggested that these copies are not identical.

At a position 1.5 kb upstream of the lefthand copy ofIS1071::IS1471 there is another insertion element in pEST4011 (Fig. 1), an ISBPH-like structure whose transposase gene is most similar to the Achromobacter georgiopolitanum KKS102 transposase gene tnpBPH of insertion element ISBPH (Table 1). In A. georgiopolitanum KKS102, ISBPH is located between bphS and bphE, the genes involved in biphenyl degradation (32).

Comparison of the pEST4011 backbone with other IncP1 backbones. A 29-kb region of pEST4011 (positions 1 to 9699 and 57635 to 76958) contains genes for plasmid replication initiation, stable maintenance in the host cell, and the conjugation machinery for plasmid transfer into new hosts (Fig. 1). The overall structure of the pEST4011 backbone is similar to that of the archetype plasmids belonging to the IncP1 {alpha} subgroup (RK2, etc.) (33) and IncP1 ß subgroup (R751) (44) (Table 2 briefly describes all plasmids discussed in this paper). The predicted gene products of the backbones of the IncP1 plasmids (completely) sequenced by May 2004, including pUO1 (39), pADP-1 (30), pJP4 (48), pTSA (47), and pB4 (42), are 65 to 100% identical to those of R751. However, the corresponding gene products of the pEST4011 backbone show only 51 to 86% identity to either R751 or RK2 backbone gene products. The proposed functions and levels of amino acid identity and similarity of all predicted pEST4011 backbone gene products are shown in Table 1.


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  		TABLE 2. Plasmids discussed in this paper

A comparison of the six IncP1 plasmids mentioned above (not including pJP4) (1, 39) showed that various resistance and catabolic determinants are inserted mostly into two regions in the plasmid backbones, between oriV and the trfA gene and between the Tra1 and Tra2 regions. When we compared plasmids pEST4011 and R751, the archetype plasmid of the IncP1 ß subgroup, we found that pEST4011 contains only one catabolic transposon inserted between the core of oriV and the trfA gene (between iterons 2 and 3) (Fig. 5).



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  		FIG. 5. Comparison of the linear genetic maps of plasmids pEST4011 and R751 (accession number NC_001735). Different types of boxes represent common sets of backbone genes, and the corresponding functions are indicated between the two maps. The arrows with boxes indicate insertions not found in the two plasmids; the pEST4011 sites from which the kleG and kleB genes are missing (compared with R751) are indicated by triangles. Some of the pEST4011 and R751 backbone gene positions are also indicated for orientation.

In the pEST4011 backbone two ORFs were found between the upf54.8 and kfrA genes, namely, orf1 and orf2, whose predicted amino acid sequences are most similar to the sequences of the Xylella fastidiosa 9a5c cell filamentation protein and to an unknown protein, respectively (Table 1). These two ORFs are not present in any of the plasmids mentioned above. In addition, two small genes, kleB and kleG, which are devoted to stable inheritance, as well as two genes having unknown functions, kluA and kluB, located in the R751 backbone were not identified in pEST4011.

In R751, RK2, pJP4, and pB4 there are two forms of the replication initiation protein TrfA encoded by the trfA1 and trfA2 genes, the larger TrfA-44 protein (382 to 407 amino acids) and the smaller TrfA-33 protein (284 to 289 amino acids). These proteins are translated from the same reading frame, and the translational start sites are 97 to 122 amino acids apart. It has been shown that the smaller protein is sufficient for plasmid replication in many hosts (59). In pUO1, pTSA, and pEMT3 only the gene coding for TrfA-44 (406 to 411 amino acids) has been annotated, while in pADP-1 and pEST4011 only TrfA-33 proteins are encoded (303 and 289 amino acids, respectively). In pADP-1, the region corresponding to the N-terminal part of TrfA-44 is missing, and in pEST4011 the deletion also encompasses the ssb gene, which codes for a single-stranded DNA-binding protein involved in DNA replication, recombination, and repair and is present in all of the other plasmids mentioned above just upstream of trfA. In pEST4011 an additional 2,360 bp of DNA (positions 57635 to 59995) is inserted between the catabolic transposon and iteron 2 (positions 60146 to 60166) of oriV. The nucleotide sequence of this region is not similar to any sequence in the GenBank database.

In pEST4011, a large amount of DNA coding for the C-terminal half of TrbE and genes trbF to trbN, as well as the region coding for most of the C-terminal region of TraC2 and TraC3 and all of the traC4 gene, which are present in R751, RK2, pJP4, pADP-1, pUO1, and pB4, are missing. In the latter plasmids, this region may also contain additional genes, but the important fact is that it includes seven trb genes (trbE to trbL) essential for mating pair formation during conjugation (1).

As in other IncP1 plasmids, five transcriptional regulators are encoded in the pEST4011 backbone. KorA, KorB, and KorC are three global repressors that regulate transcription of the genes necessary for plasmid replication, stable inheritance, and conjugation; TrbA is a transcriptional repressor of conjugation genes; and KfrA has been shown to regulate only its own transcription (1). Consensus sequences for the binding of these regulators have been determined for KorA, KorB, KorC, and TrbA (3, 33, 44). Most of these binding sites are also present in pEST4011; the only exception is one TrbA binding site present in the RK2 traJ/traK promoter-operator region (Table 3). Additionally, one KorB binding site upstream of traJ and one KorC binding site upstream of klcA, which are not present in R751 or RK2, were found in pEST4011. As shown in Table 3, the binding sites of KorA and KorB are well conserved in the three plasmids, while the KorC binding sites of pEST4011 look like hybrids of the corresponding R751 and RK2 sites. Interestingly, the TrbA operator sequences are more heterogeneous and contain more mismatches with the proposed consensus binding site (WWCGATATATCGWW) (3). In the case of RK2, the individual TrbA operators contain one to three mismatches, while the corresponding sequences of pEST4011 and R751 have up to six mismatches. Only one TrbA binding site shown in Table 3, the site in the promoter region of the R751 trbB gene, is an exact match with the consensus sequence. Bingle and Zatyka (3) assumed that such suboptimal operators play an important role in the balanced regulation of plasmid transfer. Finally, the TraJ binding sites of pEST4011 and R751 are identical. This is in agreement with the fact that the amino acid sequence of the pEST4011 TraJ protein is more similar to the corresponding sequence of R751 than to the corresponding sequence of RK2, although the level of identity is only 55%.


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  		TABLE 3. DNA binding proteins encoded by the pEST4011 backbone

The pEST4011 origin of replication (oriV) consists of the same components as the origins of replication described for other IncP1 plasmids (1, 24, 33): 10 binding sites (called iterons) for the TrfA protein (which initiates replication at oriV), including iterons 2 to 11 plus one additional iteron (iteron 1) not present in R751 or RK2 (Table 3; Fig. 6A); four binding sites for the host protein DnaA; three palindromic sequences which are also proposed to bind TrfA; an A-T-rich region; and a G-C-rich region. Table 3 shows that the conserved TGACA motif of the TrfA iterons of R751 and RK2 is much more frequently AGACA in pEST4011. Also, the second part of the pEST4011 iterons is more frequently CTTGAG than it is in R751 and RK2. In RK2 this sequence quite strictly alternates between CTTGAG and GATGAG.



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  		FIG. 6. (A) oriV region of pEST4011. The binding sites for replication initiation protein TrfA (iterons i1 to i11) together with the directions are indicated by triangles. The gray boxes represent palindromic sequences (P); the black boxes are DnaA binding sites (D). The G-C- and A-T-rich regions, the direction of replication, the site of additional DNA insertion, and the trfA gene (with the direction of transcription) are also indicated. (B) oriT region of pEST4011. The arrows indicate the binding sites for the TraI, TraJ, and TraK proteins and the nic site. The gray box represents the region that is highly conserved in different IncP1 plasmids. The translation start codons together with the directions are indicated for the traJ and traK genes. The relaxation region and the bent region are indicated by dotted lines; the directions of transcription from the promoters of traJ (PtraJ) and traK (PtraK) and the putative direction of DNA transfer during conjugation are indicated by horizontal arrows.

The structure of the origin of transfer (oriT) is also similar to that of other IncP1 plasmids (1, 33). It consists of a relaxation region containing TraJ and TraI binding sites (Table 3; Fig. 6B) and of the nic site GC (positions 74414 and 74415 in pEST4011), in which one strand of DNA is nicked during conjugation (34).

Multiple alignments of trfA2, korA, and traG gene products of IncP1 plasmids. As described above, the backbone gene products of pEST4011 are homologous but still quite different from the corresponding products of both RK2 and R751, the archetype plasmids of the IncP1 {alpha} and IncP1 ß subgroups, respectively. In order to examine to which IncP1 subgroup pEST4011 belongs, we constructed multiple alignments of the available full-length amino acid sequences of the short form of TrfA (TrfA-33), KorA, and TraG of IncP1 plasmids (Table 2). In cases in which the coding sequences for TrfA-33 were not annotated, we generated the necessary sequences from TrfA-44 by deleting the N-terminal 122 amino acids in the case of pUO1 and pTSA and 123 amino acids in the case of pEMT3 (based on similarities with RK2 and R751). The bootstrapped neighbor-joining trees derived from these alignments (obtained by using the program CLUSTALX) (Fig. 7) showed that pEST4011 TrfA, KorA, and TraG are distinct from all other corresponding sequences and that they occupy individual branches of each tree. Similar results were obtained with the different PHYLIP programs used (data not shown). As C. M. Thomas and coworkers have assigned plasmid pQKH54 (14) to the {gamma} subgroup (personal communication) and the amino acid sequence of TrfA of pQKH54 appeared to be different from the pEST4011 TrfA sequence (unpublished data), we placed pEST4011 in a new IncP1 subgroup, the {delta} subgroup. The other four IncP1 catabolic plasmids sequenced, pJP4, pADP-1, pUO1, and pTSA, together with pIJB1, possess the well-conserved IncP1 subgroup ß backbone. The TrfA2 phylogenetic tree (Fig. 7A) shows that the 2,4-D-degradative plasmid pEMT3 is also different from all other IncP1 plasmids and could also form a separate subgroup. Unfortunately, the incompatibility group of the 2,4-D-degradative plasmid pTV1 is unknown (T. Vallaeys, personal communication).



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  		FIG. 7. Bootstrapped neighbor-joining trees derived from multiple alignments of trfA2 (A), korA (B), and traG (C) gene products of different IncP1 plasmids, constructed with the CLUSTALX program. The GenBank accession numbers of the proteins are indicated in parentheses. The numbers at the nodes of the trees represent the bootstrap values (1,000 replicates) for each node.

Comparison of pEST4011 and its laboratory ancestor, pD2M4. The original strain D2M4, containing plasmid pD2M4 (about 95 kb), had a very unstable 2,4-D+ phenotype; 99% of the cells lost this phenotype when they were grown in Luria-Bertani medium, and all of the 2,4-D– clones studied contained no plasmid. Subsequently, a series of selections were performed (V. Kõiv, unpublished data); in each step D2M4 cells were cultivated in nonselective medium, and this was followed by selection on medium containing 2,4-D as a sole source of carbon and energy. The resulting 2,4-D+ clone with the best growth on 2,4-D was selected for the next step. Finally, a strain with improved stability of the 2,4-D+ phenotype was obtained; about 50% of the cells lost the 2,4-D+ phenotype when they were grown in nonselective medium. The selected strain was designated EST4002, and it contained a smaller plasmid, pEST4002 (estimated size, 78 kb). In order to increase the stability of the plasmid and to overcome the difficulties with DNA extraction, pEST4002 was transferred by conjugation into a plasmid-free recipient strain, Pseudomonas putida PaW340 (28). The resulting 2,4-D+ transconjugants contained plasmid pEST4011 (70 kb, as determined by restriction analysis). After maintenance of strain EST4002 on 2,4-D minimal medium agar plates for years, the strain acquired a very stable 2,4-D+ phenotype; only 1 to 2% of the cells lost the phenotype when they were grown in Luria-Bertani medium. According to a restriction analysis this strain contained exactly the same pEST4011 plasmid as the 2,4-D+ transconjugants of PaW340 (53).

Due to problems arising from instability of the 2,4-D+ phenotype and the difficulties with DNA extraction, we were not able to maintain either the original strain D2M4 containing pD2M4 or plasmid pEST4002. As Mäe et al. have shown (28), plasmid pEST4002 had one additional 7.8-kb EcoRI restriction fragment compared with pEST4011. However, the exact location and size of the deletion were not determined. During the present study we compared the EcoRI and HindIII restriction patterns of pD2M4 (Kõiv, unpublished) and pEST4011 (Fig. 8A and B). We found that pEST4011 had lost the approximately 23.5- and 20.5-kb HindIII restriction fragments and the approximately 26-, 9.5-, and 4.6-kb EcoRI fragments compared with pD2M4. In addition, the 19,609-bp HindIII fragment and the 6,991- and 10,872-bp EcoRI fragments of pEST4011 are not present in pD2M4. Instead of the 21,849-bp pEST4011 HindIII fragment, pD2M4 had a corresponding band at about 15 kb.



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  		FIG. 8. (A) Restriction analysis of plasmid pD2M4 digested with EcoRI and HindIII. Plasmid pWW0 (accession number NC_003350) was digested with the same restriction endonucleases to provide size markers. The DNA fragments were separated in a 0.7% agarose gel. The numbers and arrows on the right indicate the approximate positions of the corresponding fragments (in base pairs). The arrows with the open arrowheads indicate the pD2M4 restriction fragments not present in pEST4011. (B) Restriction analysis of plasmid pEST4011 digested with EcoRI and HindIII. The DNA fragments were separated in a 0.8% agarose gel. The numbers and arrows on the right indicate the approximate positions of the corresponding fragments (in base pairs). The arrows with the open arrowheads indicate the pEST4011 restriction fragments not present in pD2M4. (C) Aligned circular restriction maps of plasmids pEST4011 and pD2M4. The outer circle with recognition positions of the corresponding restriction enzymes represents pEST4011. The inner discontinuous circle represents pD2M4, and the boxes indicate the corresponding digestion products. The restriction fragments of pD2M4 not present in pEST4011 are shown outside the circles, and the arrows indicate the sites in pD2M4 where these fragments are probably embedded in an unknown order. The line with the open triangle at the end indicates the site in pEST4011 where the essential transfer genes trbE to trbL are missing. The duplicated regions of pEST4011 are indicated by dotted lines.

These results show that (i) pEST4011 lost approximately 25 to 29 kb of DNA from somewhere between position 57349 and position 66873 (i.e., between the righthand copy of IS1071::IS1471 and the traD gene) and (ii) pD2M4 did not contain the 6,991-bp duplication present in pEST4011 containing genes lrp to iaaH (Fig. 1 and 8C). Indeed, when pEST4011 was compared with R751, we noticed that in case of pEST4011 about 14 kb of backbone DNA containing (among other genes) a set of trb genes essential for conjugation (trbE to trbL) (Fig. 5) was missing. The remaining 11 to 15 kb could comprise additional DNA inserted between the Tra1 and Tra2 regions, as reported for all other IncP1 plasmids discussed in this study except pJP4 (1, 39, 48). Thus, on the one hand, this deletion somehow ensured its host a very stable 2,4-D+ phenotype, and on the other hand, pEST4011 lost the ability to self-transmit.

The 6,991-bp duplication in the pEST4011 catabolic region is not present either in pD2M4 or in catabolic transposon Tn5530 of pIJB1. At least two of six ORFs in this region can be directly related to 2,4-D degradation; namely, tfdD is responsible for the fourth step in the corresponding pathway (Fig. 2), and the ccp product may function as a channeling protein for the removal of chloride liberated during 2,4-D dissimilation from cells. The predicted amino acid sequence of the pEST4011 TfdD protein is far more similar to the sequence of TcbD of Pseudomonas sp. strain P51 plasmid pP51 (P27099) than to the sequence of pJP4 TfdDI or TfdDII; the levels of amino acid identity are 83, 64, and 32%, respectively, and the levels of amino acid similarity are 96, 87, and 57%, respectively. The tcb genes of pP51 are responsible for degradation of 1,2,4-trichlorobenzene by its host strain (52), and the substrate for TcbD is 2,3,5-trichloro-cis,cis-muconate. The corresponding 2,4-D degradation intermediate and substrate for the pEST4011 and pJP4 tfdD gene products is 2,4-dichloro-cis,cis-muconate. Vollmer et al. (55) showed that although they could not measure enzyme kinetics with 2,3,5-trichloro-cis,cis-muconate as the substrate, 2,4-dichloro-cis,cis-muconate was the best substrate for pP51 TcbD and pJP4 TfdDI. However, the catalytic efficiency of TcbD was somewhat lower than that of TfdD (kcat/Km, 53 and 120 µM–1 min–1, respectively). Therefore, it is tempting to speculate that the 7-kb pEST4011 region was duplicated in order to increase the tfdD copy number and consequently the amount of the corresponding enzyme in a cell in order to degrade 2,4-D more efficiently.

Mercury resistance is a common heavy metal resistance in bacterial isolates from soil and water environments (19). As shown in Table 2, all catabolic IncP1 plasmids except the least-studied plasmid pEMT3 bear mercury resistance genes. Although plasmid pEST4011 does not carry any Hgr determinants, it is probable that the precursor plasmid pD2M4 did. In pADP-1 and pUO1 the mer genes are located between the Tra1 and Tra2 regions; if this was the case for pD2M4 as well, the corresponding genes were lost during laboratory evolution. However, mercury resistance has not been determined for either strain EST4002 or EST4003.

Our first conclusion is that it seems that despite similarities in overall organization, the backbones of IncP1 plasmids are far more heterogeneous than previously thought. Also, as shown in Table 2, very similar IncP1 backbones, although loaded with different additionalfunctions, can be isolated from geographically distinct regions of the world. Second, diverse genes and gene cassettes are assembled into (2,4-D) catabolic pathways having different origins, but once a working set of necessary genes has been brought together (for example, in a composite catabolic transposon), these genes may spread horizontally as one unit and this unit may insert itself into different vehicles, like conjugative (broad-host-range) plasmids. Other catabolic and/or regulatory genes encoded by these composite transposons may be just entrapped passengers with no significance in one host, while they can probably confer an altered phenotype to another host in certain conditions. Finally, serious attention should be paid to the fact that the occurrence of laboratory evolution caused by continuous enrichment procedures during the isolation of natural catabolic plasmids may hinder studies and distort our understanding of evolution in nature.


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ACKNOWLEDGMENTS
 
This work was supported by Estonian Science Foundation grant 5682.

We gratefully thank Christopher Thomas for fruitful discussions and sending us the amino acid sequence of TrfA of pQKH54. We also thank Tatiana Vallaeys for the information concerning plasmid pTV1 and Jaak Truu and Viia Kõiv for their substantial contributions to this paper.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Genetics, Institute of Molecular and Cell Biology, 23 Riia Street, Tartu 51010, Estonia. Phone: 372-7375014. Fax: 372-7420286. E-mail: eve.vedler{at}ut.ee. Back


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Journal of Bacteriology, November 2004, p. 7161-7174, Vol. 186, No. 21
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.21.7161-7174.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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