INTRODUCTION

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Pseudomonas sp. strain B13 is a sewage isolate capable of utilizing 3-chlorobenzoate (3CBA) as its sole carbon and energy source (14). The degradation of 3CBA involves an initial oxidation to chlorocatechols, which are subsequently converted to 3-oxoadipate by the action of four enzymes of the modified ortho cleavage pathway, encoded by the clcABDE genes (15). The clc genes have been transferred from strain B13 to different Pseudomonas recipient bacteria, thereby enabling the recipients to degrade chlorocatechols as well (22, 26, 27, 40, 41). We have recently demonstrated that the clc genes are located on a 105-kb mobile element (named the clc element) which is the transfer determinant and is capable of integrating in the chromosome (25, 34). The original host Pseudomonas sp. strain B13 also carries two nonadjacent chromosomal copies of the clc element, although the isolation of small amounts of a 110-kb plasmid (pB13) carrying the clc genes in strain B13 has been reported elsewhere (10). The EcoRI restriction patterns of pB13 and the integrated clc element were basically identical, and the apparent 5-kb size difference was due only to inaccurate sizing of the largest EcoRI fragments (25). This suggested that pB13 and the integrating clc element exist in two different forms of the same entity, i.e., an integron and a free "plasmid."

The chromosomal location of the clc element was demonstrated by Southern hybridization on digested chromosomal DNAs separated by pulsed-field gel electrophoresis for transconjugants of Pseudomonas putida F1, P. putida BN10, Burkholderia cepacia WR401, Alcaligenes eutrophus CH34, and Ralstonia spp. (34). Some transconjugants carried only one chromosomal copy of the clc element, others carried two, and the F1 transconjugants carried up to eight copies (25, 34). Interestingly, chromosomal integrations in the F1 transconjugants occurred in two loci, with tandem amplification mainly in one locus. Integration of the clc element was shown to be RecA independent and site specific and should therefore have been mediated by functions encoded on the element itself (25). The integration sites in F1 were both identified as glycine tRNA structural genes, and the integrations appeared to take place at the 3' end of the tRNA gene. A wide variety of genetic elements are known to integrate into the host chromosome by means of site-specific recombinases which use tRNA genes as their target sites. Such elements include the bacteriophages R73 (17, 37), P4 and P22 (24), and T12 (21); insertional actinomycete plasmids (11); virulence determinants of Dichelobacter nodosus (11, 12) and of Vibrio cholerae (18); and the Bacteroides NBU1 element (33).

In this paper, we present the characterization of a novel, unusually long recombinase gene (int-B13) of the phage P4 integrase family and demonstrate its function in site-specific integration of the clc element. To our knowledge, this is the first time that a bacteriophage-related integrase has been shown to be associated with horizontal transfer of genes involved in degradation of aromatic substances, further demonstrating the importance of this class of genetic elements in bacterial evolution.

	MATERIALS AND METHODS

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Bacterial strains, media, and growth conditions. Escherichia coli DH5 (31) was used for routine cloning experiments with plasmids. E. coli BL21(DE3)(pLysS) (35) was used as a host strain for inducible protein overexpression of pET3c-derived plasmids (29). E. coli cultures were grown at 37°C on Luria-Bertani medium (31), which was supplemented with the following antibiotics when appropriate: ampicillin, 100 µg/ml; chloramphenicol, 10 µg/ml; and tetracycline, 20 µg/ml. For induction of int-B13 in E. coli BL21, isopropyl--D-thiogalactopyranoside (IPTG) was added to the medium at a 1 mM concentration.

Plasmids. The plasmid constructs used in this study are listed in Table 1. The plasmids pUC18Not (16), pUC28 (4), and pACYC184 (New England Biolabs, Beverly, Mass.) were used as general cloning vehicles. Plasmid pET3c (29) is an ATG vector derived from pBR322 which contains the ₁₀ promoter, ribosome binding site, and terminator optimized for T7-directed protein expression. The linearized vector pGEM-T Easy (Promega Corporation, Madison, Wis.), containing single 3'-thymidine overhangs, was used for cloning of DNAs amplified by PCR.

View larger version (61K): [in this window] [in a new window]   FIG. 1.   Physical map of the right end of the integrated clc element in P. putida F1 harboring the integrase gene int-B13. (A) Restriction map of the insert DNA of pRR108 (Table 1), showing some of the important restriction sites. Grey shading indicates DNA which is part of the clc element. The hatched box below corresponds to the sequence depicted in panel B (opposite orientation). (B) Nucleotide sequence of the region containing glyV and int-B13. The sequence of glyV is boxed, and the identity segment of 18 bp is underlined. Arrows indicate inverted repeats (IR) within glyV and one between the tRNA gene and int-B13. The proposed amino acid sequence of Int-B13 and the ribosomal binding sites of putative ORFs are shown.

Expression of int-B13 in E. coli. E. coli BL21(DE3)(pLysS) harboring plasmid pRR169 or pRR169Not was grown in Luria-Bertani medium to an optical density at 540 nm of 0.45 to 0.55. Subsequently, cells were induced by the addition of 1 mM IPTG, and the culture was grown for another 90 min. Bacterial cells (1 ml) were harvested by centrifugation, resuspended in 50 µl of protein loading buffer (19), and boiled for 5 min. After 1 min of centrifugation (at 15,000 × g), samples of 5 to 10 µl were used directly for analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which was performed according to the method of Laemmli (19).

DNA isolations and manipulations. Plasmid DNA isolations (from E. coli DH5), transformations, and other DNA manipulations were carried out according to established procedures (31). Total DNA was isolated with the Easy-DNA kit (Invitrogen, Carlsbad, Calif.) or by the method of Marmur (20). Restriction enzymes and other DNA-modifying enzymes were purchased from Amersham Life Science (Little Chalfont, Buckinghamshire, United Kingdom) and used according to the specifications of the manufacturer.

DNA amplification by PCR. PCRs were performed with Taq polymerase according to the descriptions of the supplier (Life Technologies, Basel, Switzerland). PCR primers used in this study (Table 2) were purchased from MWG Biotech (Ebersberg, Germany) or Microsynth (Balgach, Switzerland). The method referred to as colony PCR was performed as follows. One bacterial colony from an agar plate was transferred with a sterile toothpick into a 0.5-ml PCR tube containing 100 µl of distilled sterile water. The sample was heated to 98°C for 6 min in order to lyse the bacterial cells and release their DNA. For DNA amplification by PCR, 1 µl of this solution was added to a PCR mixture with a total volume of 50 µl.

DNA sequencing and sequence analysis. Double-stranded template sequencing was performed on plasmids with the Thermo Sequenase fluorescently labelled primer cycle kit with 7-deaza-dGTP (Amersham Life Science). Primers labelled with the fluorescent dye IRD-800 at the 5' end were purchased from MWG Biotech. An automated DNA sequencer, model 4000L (LI-COR Inc., Lincoln, Nebr.), was used for sequencing. Computer analysis of the DNA and amino acid sequences was done with DNASTAR software (DNASTAR Inc., Madison, Wis.). Comparisons of our own sequence data with published sequences in GenBank were performed with the BLAST software via the Internet (http://www.ncbi.nlm.nih.gov/BLAST/ [2]).

Nucleotide sequence accession number. The nucleotide sequence presented in this article (Fig. 1) has been deposited in GenBank under accession no. AJ004950.

	RESULTS

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Identification of the clc element's putative integrase gene. Previously, we have reported chromosomal integration of a 105-kb genetic element (named the clc element) at two sites in P. putida F1 (25). The two integration sites in F1 were both identified as glycine tRNA structural genes, and each integration appeared to occur at the 3' end of the target gly-tRNA. This observation suggested that a site-specific recombinase was responsible for the chromosomal integrations. Near the right end of the clc element (insert of plasmid pRR108 [Table 1]), an ORF was identified by sequencing and its predicted amino acid sequence was homologous to those of site-specific recombinases of the bacteriophage P4 integrase subfamily. The nucleotide sequence of this ORF (tentatively named int-B13) had a length of 1,971 bp, corresponding to a coding capacity of 657 amino acids (aa) and a molecular mass of 74 kDa. To confirm the presence and actual size of the int-B13-encoded polypeptide, the gene was cloned into pET3c and overexpressed in E. coli BL21(DE3)(pLysS). The DNA sequence of the int-B13 ORF predicted two possible translational starts close to another (Fig. 1, nt 262 and 304). Plasmid pRR169 contained the int-B13 gene under the transcriptional control of the T7 promoter and carried a translational fusion from the ATG start codon at nt 262. When an E. coli BL21 culture with pRR169 was induced with IPTG and the crude extract was separated by SDS-PAGE, a protein band of the expected size (74 kDa) for Int-B13 was obtained (Fig. 2). However, at this resolution it was not possible to discern whether the ATG at nt 262 or the second ATG codon at nt 304 was actually used (or whether both were used). To confirm the origin of this protein band from the int-B13 gene, a frameshift mutation was introduced in the unique NotI restriction site at nt 1158 in the sequence of int-B13 (Fig. 1B). Induction of E. coli BL21 carrying this plasmid (pRR169Not) produced a protein of 56 kDa on an SDS-PAGE gel (Fig. 2), which was the expected size for the truncated Int-B13. This confirmed the coding capacity of the int-B13 ORF.

View larger version (83K): [in this window] [in a new window]   FIG. 2.   Overexpression of the Int-B13 polypeptide in E. coli. Shown is an SDS-PAGE gel of crude extracts from E. coli BL21(DE3)(pLysS) cultures harboring either pRR169 (intact int-B13) or pRR169Not (frameshift mutation in int-B13), with or without induction with IPTG. The sizes of protein standards are listed. Arrows indicate the positions of full-length Int-B13 (74 kDa) and truncated Int-B13 (56 kDa).

Int-B13 is a member of the integrase family of site-specific recombinases. A clustal alignment (MegAlign, DNASTAR software) of Int-B13's amino acid sequence with the seven most similar integrases is shown in Fig. 3A. The protein sequence with the highest similarity to Int-B13 is the integrase of retronphage R73 (37) with 37% identity and 57% homology in an alignment covering 407 aa. The conserved residues His-396, Arg-399, and Tyr-433 (integrase family positions) found in all site-specific recombinases of the integrase (Int) family, with the exception of pSAM2 Int (3, 8), were also present in the Int-B13 sequence (His-365, Arg-368, and Tyr-401 [Fig. 3A]). The conserved Tyr-342 of  Int (corresponding to Tyr-401 for Int-B13) has been shown to be the residue forming a covalent bond between the Int protein and the DNA at the attachment site(s) during recombination (23). In accordance with the findings of Abremski and Hoess (1), a second conserved arginine residue was also found in Int-B13, Arg-261 (Fig. 3A). The Int-B13 protein, however, was considerably longer than the phage P4-related integrases, which are all between 385 and 440 aa in length. The C-terminal 220 aa of Int-B13 had high similarity to only one other polypeptide in GenBank, encoded by an ORF present near the pah gene cluster for naphthalene degradation found in Pseudomonas aeruginosa PaK1 (Fig. 3B). No function has been assigned to the predicted protein from this ORF. Interestingly, the P. aeruginosa ORF could be part of a larger ORF, since it is located at the end of the DNA fragment sequenced. The translated sequence upstream of the ORF also matched the Int-B13 protein sequence very well (Fig. 3B).

View larger version (107K): [in this window] [in a new window]   FIG. 3.   Amino acid sequence alignment between the Int-B13 protein and homologous sequences identified in the databases. The ruler shown above the sequences is that for Int-B13. (A) Alignment of the N-terminal 460 aa of Int-B13 with the seven most similar integrases. The four conserved amino acid residues presumed to be responsible for catalytic function of integrases are indicated with asterisks. The GenBank accession numbers for the integrase sequences shown are as follows: clc element of Pseudomonas sp. strain B13, AJ004950; retronphage R73, A42465; D. nodosus vap region, L31763; M. loti symbiosis island, AF049242; bacteriophage Sf6, P37317; satellite phage P4, RSBPP4; prophage CP4-57, P32053; and V. cholerae pathogenicity island, U02372. (B) Alignment between the C-terminal 247 aa of the Int-B13 protein and a translated nucleotide sequence from P. aeruginosa PaK1 (GenBank accession no. D84146). The predicted amino acid sequence from strain PaK1 is translated starting from nt 3 of the published sequence and is in frame with the proposed ORF1, which starts at Met-24.

Attachment sites of Int-B13. Site-specific recombinases like the integrases of bacteriophages P4 and R73 mediate recombination between a phage attachment site, attP, and a bacterial chromosomal attachment site, attB (9). Likewise, the junction sequences between a prophage (or another type of integrated element) and chromosomal DNA are termed attL (left-end junction) and attR (right-end junction). During integration, the actual recombination event involving strand exchange occurs within short sequences identical to both attP and attB, the att core or identity segment. Since the putative chromosomal attachment sites attB1-F1 of P. putida F1 (formerly INT1) and attB2-F1 (INT2) had been identified previously (25), we now determined the attP site of the clc element itself. The precise sequence of the attP site would be evident only from a circular form with a direct junction between the left and right ends, which in the integrated form are 105 kb apart. This junction could be amplified from total DNA of Pseudomonas sp. strain B13 by using PCR with primers RR316a (left end) and RR319 (right end) (Table 2). As expected, the DNA sequence (insert of pRR146) of the amplified fragment contained the left- and right-end sequences and an 18-bp segment identical to the 3' end of glyV (Fig. 4). The 18-bp segment seemed to be the core sequence of the clc element's attP, since only this segment was 100% identical to part of the chromosomal attB sites.

View larger version (51K): [in this window] [in a new window]   FIG. 4.   Nucleotide sequences for Int-B13 attachment sites. The positions of relevant restriction sites, of large inverted repeats (IR), the Gly-tRNA gene or part of it, and putative other tRNA structural genes are shown. Grey shading indicates DNA that is part of the clc element. (A) attL from B. cepacia JH230. A sequence segment homologous to cysT from E. coli K-12 (GenBank accession no. X52789) is indicated. (B) attL1 from Pseudomonas sp. strain B13. (C) attP from Pseudomonas sp. strain B13. The exact border between the clc element's left and right ends is indicated, and the proposed ATG start codon for int-B13 is underlined. (D) attL from Ralstonia sp. strain S11. A sequence segment homologous to cysT from S. lividans (GenBank accession no. X52072) is indicated. (E) Summarized overview of the features of the Int-B13 attachment sites in the different hosts. For an explanation, see the text. Vertical arrows point to the insertion sites in P. putida F1. Loop structures (not necessarily the exact same sequence) are indicated. Diagram is not drawn to scale.

Sequence specificity of Int-B13-mediated integrations in different bacteria. The PCR was used to amplify the junctions between chromosomal DNA and the left end of the integrated clc element (attL) in different recipients. The aim was to analyze whether glyV would be used as the target for Int-B13-mediated integration in hosts other than the previously analyzed P. putida F1 (25). The exact junction would be evident only from the nucleotide sequence at the left end, where the remainder of the glyV gene from attB is found. The bacteria analyzed for this purpose were Pseudomonas sp. strain B13 itself, Ralstonia sp. strain S11 (26), and B. cepacia JH230 (34). First, we determined by Southern hybridizations of NaeI- and SphI-digested total DNAs what fragment sizes were to be expected from inverse PCR (iPCR) and what the copy number of the element in each strain was. From these hybridizations it was evident that the B13 genome contained two separate copies of the clc element, whereas Ralstonia sp. strain S11 and B. cepacia JH230 both contained only one integrated element (results not shown). The left junction of one of the integration sites of the clc element in strain B13 was amplified by iPCR. For this purpose, total DNA was digested with NaeI, religated, and subjected to PCR with the primers RR315 and RR316. Cloning and sequencing revealed the 3' end of a glycine tRNA gene directly adjacent to the left end of the clc element, as in P. putida F1 (Fig. 4). A second, complete Gly-tRNA gene was detected 99 bp to the left of the integration site in strain B13.

Site-specific integration mediated by the int-B13 gene product in E. coli. To demonstrate that the int-B13 gene product was sufficient to promote site-specific recombination between attP and attB, we looked for integrase activity in E. coli DH5, a strain which by itself is deficient in recombination. We cloned the int-B13 gene plus the attP sequence on one plasmid (as on the circular form of the clc element) and looked at integration into a cloned attB site, present on a second plasmid. Surprisingly, the attP region could not be combined with the int-B13 gene on a high-copy ColE1-based replicon, but only on the low-copy vector pACYC184 (i.e., pRR172 [Fig. 5]). Even then, E. coli DH5 cells harboring plasmid pRR172 had a reduced growth rate. These observations indicated that some kind of detrimental activity was associated with the construct. E. coli DH5 cotransformed with the plasmids pRR123 (containing attB1) and pRR172 (attP plus int-B13) showed the formation of a chimeric plasmid in some instances. Restriction enzyme analysis of this chimeric plasmid indicated an integrative recombination between the attP site of pRR172 and the attB1 site of pRR123 (results not shown). DNA sequencing of the chimeric plasmid revealed that the sites of recombination were in fact identical to those previously characterized in the F1 transconjugant RR221 (25). The chimeric plasmid contained one attR site with a complete copy of glyV and one attL site with only the 18-bp 3' end of glyV (Fig. 5).

View larger version (17K): [in this window] [in a new window]   FIG. 5.   Circular maps of the plasmids pRR123 and pRR172 (Table 1) and the chimeric plasmid resulting from Int-B13-mediated recombination between these two plasmids in E. coli DH5. The relevant genetic markers, restriction sites, and PCR primer binding sites are shown. The arrows on the attachment sites (attP, attB1-F1, attL, and attR) indicate the direction of the glycine tRNA gene, or part of it. Inactivated genes are depicted within parentheses.

View this table: [in this window] [in a new window]   TABLE 3.   Colony PCR detection of a chimeric plasmid after cotransformations into E. coli DH5

	DISCUSSION

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To the right end of the mobile clc element from Pseudomonas sp. strain B13, we localized functions involved in site-specific chromosomal integration. An ORF (int-B13) coding for an integrase of the bacteriophage P4 subfamily started approximately 200 bp from the junction between the element's right end and the chromosomal target, a glycine tRNA structural gene (Fig. 1). The sequence similarity of the 657-aa product of the int-B13 gene to P4-related integrases and a demonstration of the gene's functionality gave evidence that Int-B13 was responsible for site-specific integrative recombination between the clc element's attachment site (attP) and chromosomal attachment sites (attB sites). Based on these results, we speculate that the int-B13 gene is also responsible for site-specific chromosomal integration of the complete clc element.

The clc element's integrase showed significant amino acid sequence homology to integrases from bacteriophages like R73, P4, and Sf6 (13, 17, 24, 37) (Fig. 3B). A high degree of amino acid sequence homology was also found between Int-B13 and the integrase IntS from the 500-kb symbiosis island of Mesorhizobium loti (36) and between Int-B13 and the integrase from the vap region of D. nodosus (11, 12). The majority of these P4-type integrases mediate site-specific integrative recombination involving tRNA structural genes. For instance, retronphage R73 integrates into a sel-tRNA gene (37), satellite phage P4 integrates into a leu-tRNA (24), the symbiosis island from M. loti uses a phe-tRNA as a target site (36), and the vap region from D. nodosus seems to integrate into a ser-tRNA gene (11, 12). Similar to the observations for the clc element, these integrases mediate insertions into the 3' ends of their target tRNA genes. Upon integration, the 3' portion of the tRNA gene at attB is replaced by an identical segment carried on attP. For the clc element, this identity segment had a length of 18 bp. The exact reconstruction of the gene sequence of the target tRNA's 3' end is an important feature in maintaining its essential function (9). Reiter et al. (28) pointed out that the identity segments of many elements inserting into tRNA genes extend from the anticodon loop through the 3' end. However, the identity segments of the att sites from the clc element (18 bp) (Fig. 1B), phage P4 (20 bp), the vap region of D. nodosus (19 bp), and the M. loti symbiosis island (17 bp) are shorter, extending from the TC loop through the 3' end. Regions of dyad symmetry characteristic of tRNA genes are supposed to serve as integrase binding sites (28). However, this can be true only for attB or attL DNA containing the complete tRNA gene and not for the corresponding attP site which contains only the 3' portion of the sequence.

The complete functional Int-B13 protein had a considerably higher molecular mass than other known P4-type integrases. Even so, several smaller ORFs were found in frame with the largest coding region (Fig. 1B). Although not investigated, the translational start of Int-B13 may be ATG at nt 304 rather than that at nt 262, due to a better ribosome binding site. Other downstream translational starts may result in truncated forms of Int-B13 lacking part of the N-terminal domain. For the conjugative transposon Tn916, truncated integrase proteins are thought to be involved in regulating recombinational activity (32) by interacting with the full-length integrase protein or the attachment sites. Since Int-B13 is so much longer than other P4-related integrases and the C-terminal region is not homologous to the site-specific recombinases, the ORF starting at Val-453 could have a different role. Perhaps this part codes for the excionase function, which is typically clustered together with an integrase. The excionase stimulates excisive recombination and is usually a small protein of 60 to 120 aa (5, 30). The C-terminal region of Int-B13 did not show homology with known excionases, but this type of protein normally has very little homology (7, 30). The only sequence with significant homology to the C-terminal domain of Int-B13 was an ORF originating in P. aeruginosa PaK1 (38). The ORF is located upstream of an NAH7-like pah gene cluster, putatively encoding naphthalene degradation. Interestingly, a translation of the (published) nucleotide sequence upstream of the ORF also revealed the RRMMQDWADRLDL residue motif, which forms the last conserved region in the C termini of the P4-related integrases (Fig. 3). Therefore, we suspect that the proposed ORF represents the C-terminal domain of a larger ORF, similar to Int-B13. No function has yet been assigned to the ORF flanking the pah cluster, but the entire gene cluster is thought to be part of a mobile element (39).

The previously isolated and characterized plasmid pB13 carrying the clc genes in Pseudomonas sp. strain B13 (10) seems to be identical to our integrated clc element (25). The fact that other research groups have been unable to isolate plasmid DNA from strain B13 (22, 41) was probably due to the clc element's integration into the chromosome. Our observations at the moment indicate that the extrachromosomal circular form of the element is abundant only in strain B13 in stationary phase during growth on 3CBA (results not shown). Interestingly, in most other transconjugants analyzed so far, none or very little of the circular form can be detected by PCR amplification (results not shown). How excision and transfer of the clc element are regulated will therefore have to be studied more extensively.

It is becoming clear that a new form of mobile genetic elements exists, which we propose to call "gene islands" (after the use of the terms pathogenicity island and symbiosis island). Such elements harbor integrases related to those of bacteriophages and may have been evolutionarily derived from those. Examples include insertional plasmids from actinomycetes (5-8), some of the pathogenicity islands from gram-negative pathogens (11, 12, 18), and a recently discovered 500-kb transferable region (symbiosis island) from M. loti (36). This symbiosis island seems to carry all the genetic information required for nodule formation, symbiotic nitrogen fixation, and synthesis of three vitamins. For the first time, our work demonstrated that a bacteriophage-related integrase was associated with horizontal transfer of genes coding for degradation of xenobiotics, and the clc element could therefore be considered a "degradation island." Apparently, bacteriophage-related integrases using tRNA structural genes as (chromosomal) insertion sites are involved in horizontal transfer of very diverse genetic determinants, not only those of bacterial virulence (11). This class of integrating elements may have been underestimated and could have greater evolutionary importance than previously thought.