Plasmid 69 (2013) 24–35

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Cointegrate-resolution of toluene-catabolic transposon Tn4651:Determination of crossover site and the segment requiredfor full resolution activity

Hirokazu Yano a,b,⇑, Hiroyuki Genka a, Yoshiyuki Ohtsubo a, Yuji Nagata a, Eva M. Top b,Masataka Tsuda a

a Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Sendai 980-8577, Japanb Institute for Bioinformatics and Evolutionary Studies, Biological Sciences, University of Idaho, Moscow, ID 83844, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 9 March 2012Accepted 23 July 2012Available online 1 August 2012Communicated by Prof. R. Chalmers

Keywords:PseudomonasTransposonTyrosine recombinaseCointegrate-resolutionTn4652Tn3

0147-619X/$ - see front matter � 2012 Elsevier Inchttp://dx.doi.org/10.1016/j.plasmid.2012.07.004

⇑ Corresponding author. Present address: Universfor Bioinformatics and Evolutionary Studies, Moscow+1 208 885 7905.

E-mail address: hyano@uidaho.edu (H. Yano).

Tn3-family transposon Tn4651 from Pseudomonas putida mt-2 plasmid pWW0 carries twodivergently transcribed genes, tnpS and tnpT, for cointegrate-resolution. While tnpSencodes a tyrosine recombinase, tnpT encodes a protein that shows no homology to anyother characterized protein. The Tn4651 resolution site was previously mapped withinthe 203-bp fragment that covered the tnpS and tnpT promoter region. To better understandthe molecular mechanisms underlying the Tn4651 cointegrate-resolution, we determinedthe extent of the functional resolution site (designated the rst site) of Tn4651 and the loca-tion of the crossover site for the cointegrate-resolution. Deletion analysis of the rst regionlocalized the fully functional rst site to a 136-bp segment. The analysis of the site-specificrecombination between Tn4651 rst and a rst variant from the Tn4651-related transposon,Tn4661, indicated that the crossover occurs in the 33-bp inverted repeat region, which sep-arates the 136-bp functional rst site into the tnpS- and tnpT-proximal segments. Electro-phoretic mobility shift assays demonstrated specific binding of TnpT to the 20-bpinverted repeat region in the tnpT-proximal segment. The requirement for accessorysequences on both sides of the crossover site and the involvement of the unique DNA-bind-ing protein TnpT suggest that the Tn4651-specified resolution system uses a differentmechanism than other known resolution systems. Furthermore, comparative sequenceanalysis for Tn4651-related transposons revealed the occurrence of DNA exchange at therst site among different transposons, suggesting an additional role of the TnpS-TnpT-rstsystem in the evolution of Tn4651-related transposons.

� 2012 Elsevier Inc. All rights reserved.

1. Introduction

Bacterial replicative transposons play an important rolein dissemination of antibiotic resistance, heavy-metal resis-tance, and degradation of recalcitrant chemicals, becausethey move and duplicate genes upon transposition between

. All rights reserved.

ity of Idaho, Institute, ID 83844, USA. Fax:

horizontally transferrable DNA molecules such as plasmids(Schmitt et al., 1979; Liebert et al., 1999; Yano et al., 2007).Replicative transposons also act as intracellular mutagensthat can induce beneficial mutations and thereby allowbacteria to adapt to new conditions (Kasak et al., 1997;Devers et al., 2008). Thus, the replicative transposons playsignificant roles in bacterial evolution. Since the spreadand persistence of antibiotic resistance in bacterial commu-nities are a great concern to human health, it is important toimprove our understanding of these transposition mecha-nisms (Levy and Marshall, 2004; Willems et al., 2011).

Fig. 1. (a) Schematic representation of the genetic structure of Tn4651-related transposons. Only transposition- and site-specific recombination-associated genes are shown. Amino acid identity between Tn4651homolog and its counterpart in other transposons is shown below thegene indicated by pentagons. Abbreviations used are as follows: chr.,chromosome; xyl, toluene-catabolic genes; car, carbazole-catabolic genes;and mer, mercury-resistance genes. (b) Similarity of the 46-bp terminalinverted repeats of Tn4651-related transposons. The dots indicate thenucleotides that are identical to those in the Tn4651 sequence. FIS and IHFindicate their respective binding regions that overlap with 46-bp IRs ofTn4651 (Teras et al., 2009). IRL and IRR correspond to those shown in (a).(c) Predicted secondary structures of Tn4651 TnpT and Tn5041 OrfQ. Darkgray denotes an alpha-helix. The region with a P-value <0.03 in Paricoil2(McDonnell et al., 2006) was considered to form a coiled-coil structure,and is indicated by horizontal line.

H. Yano et al. / Plasmid 69 (2013) 24–35 25

Tn3-family transposons are composed of genes respon-sible for transposition [tnpA and terminal inverted repeats(IRs)], site-specific recombination [tnpR and a resolutionsite (res)], and genes encoding phenotypic traits or un-known functions (Kleckner, 1981). This gene organizationallows for transposition in a two-step replicative mode(Grindley, 2002). In the first step, the tnpA product (trans-posase) mediates strand-exchange between IRs and a targetmolecule, and the following DNA replication generates acointegrate intermediate in which the donor and a targetmolecule are connected by two directly repeated copies ofthe transposon. In the next step, the tnpR product (resol-vase) mediates strand-exchange between the two directlyrepeated res sites to separate the cointegrate into the targetand donor molecule, each of which carries a single copy ofthe transposon. Most Tn3-family transposons carry thetnpR gene encoding a small (<200 aa) serine recombinase,while transposons Tn4430 and Tn5401 from genus Bacillusencode a tyrosine recombinase (TnpI) for resolution (Mah-illon and Lereclus, 1988; Baum, 1994). Moreover, the inser-tion sequence IS1071, which has been predominantly foundin Betaproteobacterial hosts, has no resolution gene despitethe fact that its transposition generates a cointegrate (Sotaet al., 2006). It is still unknown how diversity within theresolution systems affects the spread and persistence ofeach transposon type in bacterial communities.

The toluene-catabolic transposon, Tn4651, discoveredin plasmid pWW0 from Pseudomonas putida mt-2 (Tsudaand Iino, 1987), has a unique genetic organization.Tn4651 carries two genes, tnpA and tnpC, for the cointe-grate-formation process, and two other genes, tnpS andtnpT, for the cointegrate-resolution process (Fig. 1a). TnpC(120 aa) regulates expression of TnpA at the post-tran-scriptional level (Horak and Kivisaar, 1999). TnpS (323aa) is a tyrosine recombinase differing from phage integ-rases in that TnpS does not have the N-terminal DNA-bind-ing domain of phage integrases. TnpS is phylogeneticallymore closely related to the Cre resolvase of bacteriophageP1 than to phage integrases (Hallet et al., 2004). TnpT(332 aa) is a hypothetical protein postulated to enhancethe TnpS-mediated recombination reaction (Genka et al.,2002). To date, several transposons carrying a set of tnpA,tnpC, tnpS and tnpT homologs have been reported. Theseare (i) Tn4652, a deletion derivative of Tn4651 lacking alltoluene-catabolic genes (Tsuda and Iino, 1987), (ii) mer-cury resistance transposon Tn5041 from Pseudomonas sp.(Kholodii et al., 1997), (iii) carbazole-catabolic transposonTn4676 from Pseudomonas resinovorans plasmid pCAR1(Maeda et al., 2003; Shintani et al., 2005), and (iv) a 13-kb cryptic transposon, designated Tn4661 in this study,from a Pseudomonas aeruginosa plasmid and chromosomes(Fig. 1a). It has been shown that transposition of Tn4652 isregulated not only by the transposon itself but also by thehost chromosomal gene products (Ilves et al., 2001; Horaket al., 2004). The IHF proteins positively regulate both thetranscription of Tn4652 tnpA and the binding of TnpA tothe terminal IRs (Horak and Kivisaar, 1998; Ilves et al.,2004), while FIS negatively regulates the transposition byoutcompeting IHF on both of the terminal regions ofTn4652 (Horak et al., 2004; Ilves et al., 2004; Teras et al.,2009). Furthermore, the CorR/ColS two-component signal

transduction system positively regulates transposition ofTn4652 (Horak et al., 2004).

In contrast to the increasing insights into the regulationof the transposition initiation process, the molecularmechanisms underlying the cointegrate-resolution processof Tn4651 have remained unclear. A previous study onTn4651 localized the cis-acting element required for coin-tegrate-resolution within the 203-bp AatII-SacII segment

26 H. Yano et al. / Plasmid 69 (2013) 24–35

covering the tnpS and tnpT promoter region (heredesignated the rst region: resolution associated with TnpSand TnpT, Fig. 2a) (Tsuda and Iino, 1987). Our subsequentstudy indicated that (i) the 20-bp inverted repeat in the203-bp segment (Acc. Site in Fig. 2a) is indispensable forthe full resolution activity of Tn4651, (ii) cointegrate-reso-lution requires both TnpS and TnpT, and (iii) TnpS canmediate intermolecular site-specific recombination (Genkaet al., 2002). Comparative analysis of Tn5041 and its vari-ants identified in mercury-resistant soil bacterial strainssuggested that a 34-bp inverted repeat region (att5041) be-tween the orfI (tnpS homolog) and orfQ (tnpT homolog)might serve as an active crossover site (Kholodii et al.,2002). However, the recombination at att5041 has not

Fig. 2. Effect of rst deletions on resolution efficiency. (a) Nucleotide sequence ofcorresponding sequences in its related transposons. The dots indicate the nucleorst variant is indicated by a bar below the sequence. rstTn4661v1 and rstTn4661v2 aare those from rstTn4651. (b) Effect of rst deletions in the tnpS-proximal end. ‘+S4040-bp upstream sequence. All data points and error bars indicate means and SEMEffect of rst deletions in the tnpT-proximal end. ‘+T40’ indicates the rst variansequence. Significance was determined by comparison to the 180-bp rst region

experimentally been demonstrated. Furthermore, due tothe sequence dissimilarity between the tnpS-tnpT andorfI-orfQ intergenic regions, it was unclear if Tn4651 andTn5041 carry each of the homologs that correspond toatt5041 and the 20-bp inverted repeat, respectively.

In this work, we first described the features of unchar-acterized transposon Tn4661. Then, we determined theextremities of the functional resolution site in Tn4651,the location of the crossover site upon its cointegrate-res-olution, and the biochemical function(s) of the Tn4651TnpT protein, using genes and sites from Tn4651 andTn4661. The results strongly suggest that the cointegrate-resolution of Tn4651 is distinct from known resolution sys-tems in terms of regulation of the recombination reaction.

the rst region from positions �12 (the AatII site) to 191 in Tn4651 and thetide identical to those in the Tn4651 sequence. The deleted region in eachre the derivatives of rstTn4661, in which the nucleotides marked by triangle’ indicates the rst variant containing the entire rst region and an additional

from triplicate experiments. The Y-axis is shown in logarithmic scale. (c)t containing the entire rst region and an additional 40-bp downstream(Dunnett’s test for the last data point): ⁄P < 0.05; ⁄⁄⁄P < 0.001.

H. Yano et al. / Plasmid 69 (2013) 24–35 27

2. Materials and methods

2.1. Bacterial strains, plasmids, and media

Escherichia coli strain DH5a [F�, /80lacZDM15, D(lac-ZYA-argF) U169, recA1, endA1, deoR, hsdR17(rK

- mK+), phoA,

supE44, k-, thi-1, gryA96, relA1] was used for DNA cloning(Sambrook and Russell, 2001). E. coli Rosetta 2 (DE3) [F-,ompT, hsdSB(rB

� mB�), gal, dcm, lacY1, pRARE2] (Takara Bio,

Shiga, Japan) was used for overexpression and purificationof TnpT. P. putida F1 (Zylstra et al., 1988), a strain free ofTn4651-related transposons, was used as a host for in vivorecombination assays. The E. coli strains were incubated at37 �C in Luria–Bertani (LB) broth, while the P. putida strainswere incubated at 30 �C in LB broth. Solid medium was pre-pared by the addition of 1.5% agar. Antibiotics were addedto the media at the following concentrations: chloramphen-icol (Cm), 50 lg/ml; tetracycline (Tc), 40 lg/ml for P. putidaand 20 lg/ml for E. coli; gentamicin (Gm), 10 lg/ml; kana-mycin (Km), 50 lg/ml. 3-Methylbenzotate (3MB) wasadded to the media at the concentration of 0.1–1 mM,depending on the experimental conditions. The plasmids

Table 1Plasmids used in this study.

Plasmids Genotypes and relevant charact

pWW0 IncP-9 replicon, Tol+, Tn4651Rms148 IncP-7 replicon, Smr, Tra+, Tn46pHSG396 Cmr, pMB1 replicon, cloning vecpHSG398 Cmr, pMB1 replicon, cloning vecpSTV29 Cmr, p15A replicon, cloning vecpBBR1MCS-5 Gmr, pBBR1 replicon, cloning vepSOV5 Gmr, pBBR1-MCS5DmobpIROE1 Gm, pSOV5::Tn4661pUC4K Apr, Kmr, pMB1 repliconpJB866 Tcr, RK2 replicon, expression vepColdIV Apr, pMB1 replicon, expressionpCold51T Apr, pColdIV::tnpTpHY444 Cmr, pHSG396::rstpHY470 Cmr, pHSG396::rstTn4661

pHY445 Cmr, pSTV29::(rst-tnpS)pHY452 Cmr, pSTV29::(rst-tnpSY293F)pHY453 Tcr, pJB866::tnpT, Mob+

pRST449 Gmr Kmr, pSOV5::(rst-kan-rst-tnpRST449YF Gmr Kmr, pSOV5::(rst-kan-rst-tnpRSTDS13 Gmr Kmr, pSOV5::(rstDS13-kan-pRSTDS18 Gmr Kmr, pSOV5::(rstDS18-kan-pRSTDS25 Gmr Kmr, pSOV5::(rstDS25-kan-pRSTDS31 Gmr Kmr, pSOV5::(rstDS31-kan-pRST+S40 Gmr Kmr, pSOV5::(rst+S40-kan-pRSTDT0 Gmr Kmr, pSOV5::(rstDT0-kan-rpRSTDT9 Gmr Kmr, pSOV5::(rstDT9-kan-rpRSTDT14 Gmr Kmr, pSOV5::(rstDT14-kan-pRSTDT20 Gmr Kmr, pSOV5::(rstDT20-kan-pRSTDT26 Gmr Kmr, pSOV5::(rstDT26-kan-pRSTDT30 Gmr Kmr, pSOV5::(rstDT30-kan-pRSTDT32 Gmr Kmr, pSOV5::(rstDT32-kan-pRSTDT34 Gmr Kmr, pSOV5::(rstDT34-kan-pRSTDT37 Gmr Kmr, pSOV5::(rstDT37-kan-pRST+T40 Gmr Kmr, pSOV5::(rst+T40-kan-pRST5161 Gmr Kmr, pSOV5::(rstTn4661-kan-pRST5161v1 Gmr Kmr, pSOV5::(rstTn4661v1-kapRST5161v2 Gmr Kmr, pSOV5::(rstTn4661v2-kapRST5161v1YF Gmr Kmr, pSOV5::(rstTn4661v1-ka

a Abbreviations used to describe phenotypes are as follows: Tol+, tolmissible; and Mob+, mobilizable.

used are listed in Table 1. Cloning vector pSOV5 wasconstructed from pBBR1-MCS5 (Kovach et al., 1995) byremoving its Eco52I-flanked fragment in the mob region,followed by the blunt-end and self-ligation reaction of thepBBR-1MCS5 replicon. Plasmid pIROE1, which carries aTn4651-related transposon Tn4661, was obtained in thisstudy (see Supplementary materials for details).

2.2. General DNA manipulations

PCR was carried out using KOD-plus high-fidelity DNApolymerase (Toyobo, Osaka, Japan). Extraction of DNA frag-ments from agarose gels was performed using a QiaEX IIbead kit (Qiagen, Valencia, CA, USA). Alkaline lysis withphenol–chloroform purification (Sambrook and Russell,2001) was used to extract plasmids from P. aeruginosaand P. putida strains. A LaboPass minikit (Hokkaido SystemSciences, Sapporo, Japan) was used to purify plasmids fromE. coli strains. T4 Polynucleotide kinase (Takara Bio, Shiga,Japan) was used for the site-directed mutagenesisexperiments. DNA sequences were determined usingBigDye terminator ver. 3.1 (Applied Biosystems, Foster

eristicsa References

Greated et al. (2002)61 Sagai et al. (1975)tor Takeshita et al. (1987)tor Takeshita et al. (1987)

tor Takara Bio, Shiga, Japan.ctor, Mob+ Kovach et al. (1995)

This studyThis studyTaylor and Rose (1988)

ctor Blatny et al. (1997)vector Qing et al. (2004)

This studyThis studyThis studyThis studyThis studyThis study

pS) This studypSY293F) This studyrst-tnpS) This studyrst-tnpS) This studyrst-tnpS) This studyrst-tnpS) This study

rst-tnpS) This studyst-tnpS) This studyst-tnpS) This studyrst-tnpS) This studyrst-tnpS) This studyrst-tnpS) This studyrst-tnpS) This studyrst-tnpS) This studyrst-tnpS) This studyrst-tnpS) This study

rst-tnpS) This studyrst-tnpS) This studyn-rst-tnpS) This studyn-rst-tnpS) This studyn-rst-tnpSY293F) This study

uene utilization; Nah+, naphthalene utilization; Tra+, self-trans-

28 H. Yano et al. / Plasmid 69 (2013) 24–35

City, CA, USA), and ABI prism 310 (Applied Biosystems,Foster City, CA, USA). The oligonucleotides used in thisstudy are listed in the Supplementary materials.

2.3. Construction of cointegrate analogs and TnpT expressionplasmids

Cointegrate analog pRST449 (Fig. 3a) was constructed asfollows. The 180-bp rst region (base positions from 1 to 180in Fig. 2a) was PCR-amplified using pWW0 as a template,and the PCR product was cloned into the BamHI-XbaI regionof pHSG398 to construct pHY444. The rst-tnpS region (fromthe stop codon of tnpS to position 180 in Fig. 2a) was alsoPCR-amplified, and the PCR product was cloned into theKpnI-EcoRI region of pSTV29 to construct pHY445. The rstregion of pHY444 and the Km-resistance (Kmr) gene (kan)from pUC4K (Taylor and Rose, 1988) were cloned into theBamHI-SphI region of pHY445. The pHY445 derivative car-rying the tnpS and kan genes with opposite transcriptiondirections was selected, and the rst-kan-rst-tnpS region of

Fig. 3. Experimental design for the cointegrate-resolution analysis. (a)Structure of cointegrate analog pRST449 and resolution of the cointegrateanalog. Depending on the purpose of the experiment, one of the two rstregions on pRST449 was replaced with a rst deletion mutant. Alterna-tively, tnpS was replaced with an inactive tnpS mutant, tnpSY293F.Transcription of tnpS was designed to start from its authentic promoterin the rst region. (b) TnpS- and TnpT-dependent cointegrate-resolution.Each symbol indicates the concentration of inducer (3MB) for the TnpTexpression. Y293F denotes the cointegrate analog encoding an inactiveTnpS mutant. All data points and error bars indicate means and SEM fromtriplicate experiments. Note that the Y-axis is shown in logarithmic scale.

this pHY445 derivative was cloned into the SphI-EcoRI re-gion of pSOV5, giving rise to pRST449.

pRST449YF differs from pHY449 in that it carries thetnpS mutant gene (tnpSY293F) which codes for phenylala-nine instead of the tyrosine residue at the 293rd positionof TnpS; the codon was modified from UUC to UAC. Uponconstruction of pRST449YF, a point mutation was intro-duced into the pHY445-loaded tnpS gene by PCR-amplifi-cation of pHY445 using the primer pair of Y293FF andY293FR (Table S1 in the Supplementary materials). Subse-quent phosphorylation and self-ligation of the PCR productgenerated pHY452, which carries the rst-tnpSY293F frag-ment. The rst region and the kan gene were next insertedinto pHY452, and the resulting rst-kan-rst-tnpSY293F regionof the pHY452 derivative was cloned into pSOV5 to obtainpRST449YF.

pRST449 derivatives carrying a series of rst deletionvariants in either the tnpS- or tnpT-proximal end of the180-bp rst region were constructed as follows. Deletionderivatives of the 180-bp rst region were PCR-amplified,and the PCR products were cloned into pHSG396 orpHSG398 (Takeshita et al., 1987) to confirm the sequenceof the insert. The rst variant lacking the tnpS-proximalend was designated rstDS13, rstDS18, rstDS25, or rstDS31(Fig. 2a), while the rst variant lacking the tnpT-proximalend was designated rstDT9, rstDT14, rstDT20, rstDT26,rstDT30, rstDT32, rstDT34, or rstDT37 (Fig. 2b). The rstvariants, rst + S40 and rst + T40, contain an additional 40-bp sequence of the 50-region of tnpS and tnpT, respectively.A series of the rst variants in the pHSG396 or pHSG398derivatives in conjunction with the kan gene was clonedinto the BamHI-SphI region of pHY445 as described above.Subsequent transfer of the rst-kan-rst-tnpS derivatives topSVO5 yielded pRSTDS13, pRSTDS18, pRSTDS25,pRSTDS31, pRST + S40, pRSTDT9, pRSTDT14, pRSTDT20,pRSTDT26, pRSTDT30, pRSTDT32, pRSTDT34, pRSTDT37,and pRST + T40. Cointegrate analog pRSTDT0, which wasused as a control in the rst deletion analysis for the tnpT-proximal end, is identical to pRST449 except that pRSTDT0carries the SphI site instead of the XbaI site at the borderbetween the rst region and the vector backbone.

To construct pRST5161 (Fig. 4a), a cointegrate analogcarrying the rst region of Tn4661 (rstTn4661), the 180-bprstTn4661 region (Fig. 2a) was first PCR-amplified using plas-mid pIROE1 as a template, and the PCR product was clonedinto pHSG398. The rstTn4661 region of the resulting pHSG398derivative (pHY470) and the kan gene were cloned intopHY445 to obtain pRST5161. The two rstTn4661 variants,rstTn4661v1 and rstTn4661v2, were constructed by site-direc-ted mutagenesis using pHY470 as a PCR template. Thesevariants, in conjunction with the kan gene, were moved topHY445 or pHY452 to obtain the pRST5161 derivatives car-rying rstTn4661v1 (pRST5161v1), rstTn4661v2 (pRST5161v2),or both rstTn4661v1 and tnpSY293F (pRST5161v1YF).

TnpT expression plasmids, pCold51T and pHY453, wereconstructed by cloning the PCR-amplified Tn4651 tnpTfragment into the NdeI-XhoI sites of pColdIV (Qing et al.,2004), and the SacI-HindII sites of a broad-host-range pro-tein expression plasmid pJB866 (Blatny et al., 1997),respectively. Transcription of tnpT in pHY453 is controlled

Fig. 4. Intramolecular recombination between rstTn4651 and rstTn4661. (a)Structure of cointegrate analog pRST5161 carrying rstTn4651 and rstTn4661

and resolution of the cointegrate analog. (b) Plasmids extracted from P.putida F1 clones carrying different types of cointegrate analogs. Plasmidswere linearized by digestion with EcoRI prior to electrophoresis. The tnpS-distal rst region in pRST449 was replaced with rstTn4661, rstTn4661v1, orrstTn4661v2 (from left). One of the cointegrate analogs carries bothrstTn4661v1 and tnpSY293F. In rstTn4661v1, the original 50-TCATCGG-30

sequence in the rst core site spacer region was replaced with 50-CACTCCA-30 . In rstTn4661v2, the original 50-ATGGTA-30 in the accessorysite IR2 was replaced with 50-ATACTA-30 (See Fig. 2). (c) Nucleotidesequence in the 33-bp inverted repeat region of the hybrid rst site on theresolution product. Dots indicate nucleotide sequences identical to thoseon Tn4651.

H. Yano et al. / Plasmid 69 (2013) 24–35 29

by the Pm promoter, and is activated by the vector-en-coded regulator protein XylS in the presence of 3MB.

2.4. Site-specific recombination in P. putida

The resolution activities of rst and its variants were ana-lyzed in P. putida F1. This is because this strain does notcarry a Tn4651-related transposon in the chromosome,and can provide Pseudomonas species-specific host factorspotentially involved in the TnpS-mediated recombination.The resolution activities of cointegrate analog pRST449and its variants were analyzed by counting the frequencyof Km-sensitive (Kms) clones in the F1 population, and aga-rose-gel electrophoresis of plasmids extracted from the li-quid cultures. The frequency of Kms clones was monitoredas follows. P. putida F1 harboring pHY453 was transformedwith a series of cointegrate analogs. Then, the transfor-mants (three replicates for each cointegrate analog) wereinoculated in 2 ml of LB containing Tc, Km and Gm. Afterovernight cultivation, a 4.9-ll aliquot of each culture wastransferred to 5 ml of fresh media supplemented with3MB and Tc. Every 24 h, the 4.9 ll aliquot of culture wastransferred to fresh media, while a small fraction of theculture was serially diluted, and plated on LB agar

containing Tc and Gm and LB agar containing Tc and Km.The frequency of cointegrate-carrying cells were repre-sented as the number of Km resistant colonies over thenumber of Gm resistant colonies. The serial batch culturetransfer and the measurement of the frequency of cointe-grate in the population was repeated twice. To analyzethe effect of deletions in the rst region, we cultured strainF1 in the presence of 0.1 mM 3MB. In statistical analysis,we compared the fractions of cointegrate-carrying cells atthe last time point (generation 30) using ANOVA and Dun-nett’s test considering the 180-bp rst as a control.

2.5. Purification of TnpT

TnpT was overexpressed from pCold51T in E. coli Roset-ta 2 (DE3) and was purified from the cell extracts usingammonium sulfate fractionation as described in theSupplementary materials. TnpT was stored at �20 �C in abuffer containing 50 mM Tris–HCl (pH 8.0), 250 mM NaCl,50% glycerol, 2 mM DTT and 1.0 mg/ml TnpT (>80% pure inSDS–PAGE).

2.6. Electrophoretic mobility shift assays (EMSAs)

The 32P-labeled 259-bp fragment containing the rst re-gion was obtained by 50-end labeling of the gel-purifiedPCR product, followed by purification using Bio-Rad micro-bio-pin P-6 columns. The binding reaction in a final volumeof 10 ll was performed in a buffer containing 10 mMTris–HCl (pH 8.0), 50 mM NaCl, 10% glycerol and 0.8 mMDTT, 0.1 nM of labeled rst fragment, and the TnpT protein(0–50 nM). When required, sheared salmon sperm DNAor the unlabeled 259-bp fragment was added as aDNA-binding competitor to the binding reaction. The41-bp competitor DNA containing either one of the 35-bpsequences (segments A–F in Fig. 5) in the rst region wasprepared by annealing two complementary 41-mer nucle-otides listed in Table S1 of the Supplementary materials.The reaction mixtures were electrophoresed in 5% non-denaturing polyacrylamide gels in 0.5 � TBE for 2 h at50 V. After electrophoresis, gels were dried and exposedto a phosphor imaging plate overnight. The plate imagewas analyzed using the LAS-2000 system (Fujifilm, Tokyo,Japan).

2.7. Bioinformatic analysis

Protein secondary structures were estimated based onthe consensus secondary structure prediction programJpred3 (Cole et al., 2008) and the coiled-coil motif predic-tion program Paircoil2 (McDonnell et al., 2006). Genome-Matcher (Ohtsubo et al., 2008) was used to comparenucleotide sequences.

2.8. Nucleotide accession number

Complete nucleotide sequence of Tn4661 was depositedin the database under GenBank ID: AB375440.

Fig. 5. Electromobility shift assays to analyze the interaction of rst and TnpT. (a) Schematic representation of the location of the DNA segments used in theassays. The bars designated A to F are the segments used in the experiments in panel (d). (b) TnpT-binding to the 259-bp fragment containing the full-lengthfunctional rst site. (c) Competition of the labeled 259-bp fragment with non-labeled DNA. The data indicate means and SEM from three binding assays. (d)Competition of the labeled 259-bp fragment with non-labeled 41-bp fragment containing the 35-bp segment from the rst region.

30 H. Yano et al. / Plasmid 69 (2013) 24–35

3. Results

3.1. Transposon Tn4661 in P. aeruginosa plasmid Rms148

Recent complete sequence determinations of P. aerugin-osa strains revealed the presence of Tn4651-like elementsin various strains (Klockgether et al., 2004; Wurdemannand Tummler, 2007). Furthermore, Southern hybridizationanalysis against the plasmid collection from our laboratoryusing the probes for the Tn4651 tnpS-tnpT or tnpA-tnpC re-gions suggested the presence of a Tn4651-related transpo-son in P. aeruginosa plasmid Rms148 (Sagai et al., 1975).We isolated the Rms148-loaded transposon, designatedTn4661, by examining its transposition from Rms148 intoa non-mobilizable cloning vector pSOV5 (see the Supple-mentary materials). Nucleotide sequence determinationof Tn4661 in the pSOV5 derivative revealed that Tn4661is identical to the 12.7-kb transposon in the genomic island

PAGI-4 of P. aeruginosa strain C (Klockgether et al., 2004).With respect to the transposition and resolution genes,Tn4661 showed higher similarity to Tn4676 on pCAR1 fromP. resinovorans than to Tn4651 on pWW0 from P. putida(Table S2 of the Supplementary materials, Fig. 1b, andFig. 2a). The rst region (rstTn4661) of Tn4661 was used inthe recombination assays described below.

3.2. Reconstitution of an inducible cointegrate-resolutionsystem of Tn4651

To address the efficiency of cointegrate-resolution ofTn4651, we first attempted to reconstitute an induciblecointegrate-resolution system in the Tn4652-free P. putidastrain F1. Cointegrate analog pRST449 carries the kan andtnpS genes, and two directly repeated copies of the 180-bp rst region (Fig. 3a). The authentic promoter of tnpS inthe rst region controls its transcription on pRST449.

H. Yano et al. / Plasmid 69 (2013) 24–35 31

pRST449 was introduced into strain F1 carrying pHY453, inwhich the transcription of tnpT is induced in the presenceof 3MB. Since the resolution reaction excises a segmentcarrying the kan gene, the resolution reaction was ex-pected to generate Kms clones.

The P. putida F1 cells harboring both pRST449 andpHY453 were cultured for 30 generations in the presenceof different concentrations of 3MB to induce expressionof TnpT. Incubation with 3MB led to the appearance ofKms clones, and higher 3MB concentrations resulted inthe detection of Kms clones at earlier time points(Fig. 3b). When pHY453 was replaced by its parental vectorplasmid (pJB866), the resolution product of pRST449 wasnot detected at any time points during the 30 generations.This indicates that cointegrate-resolution is dependent onthe expression of TnpT. No resolution was detected whentnpS on pRST449 was replaced by tnpSY293F, which codesfor an inactive TnpS mutant (Fig. 3b). These results indicatethat both TnpT and the catalytic function of TnpS are re-quired for cointegrate-resolution of Tn4651 in P. putida.

3.3. The extent of the functional rst site

Our previous study showed that the 203-bp segmentfrom the AatII to SacII sites in Fig. 2a contains a cis-actingsite required for cointegrate-resolution (Tsuda and Iino,1987). A subsequent deletion analysis of this segment indi-cated that the outermost ends of the segment required forfull resolution activity (the functional rst site) are situatedbetween the AatII site and position 53, and between posi-tion 121 and the SacII site (Fig. 2a), respectively (Genkaet al., 2002). To more precisely define the segment in-volved in the resolution reaction, one of the two copiesof the rst region on pRST449 was replaced with a seriesof deletion variants in the 180-bp rst region (Fig. 2), andthe effect of the deletions on resolution efficiency wasinvestigated.

The 13- and 18-bp deletions in the tnpS-proximal end ofthe rst region (using pRSTDS13 and pRSTDS18, respec-tively) did not affect the resolution efficiency (Fig. 2b). Bycontrast, the 25- and 31-bp deletions (pRSTDS25 andpRSTDS31) resulted in a significant reduction in resolutionefficiency. The use of an rst fragment containing both theentire 180-bp rst region and an additional 40-bp regionof tnpS (pRST + S40) did not increase the resolution effi-ciency. These results suggest that the tnpS-proximalboundary of the functional rst site is situated betweenpositions 19 and 25 in Fig. 2a. The effect of deletions inthe tnpT-proximal end on resolution efficiency was ana-lyzed in the same manner. The 9-, 14-, 20-, and 26-bp dele-tions (pRSTDT9, pRSTD14, pRSTDT20, and pRSTDT26) didnot result in a significant reduction in resolution efficiency.However, deletions longer than 26 bp in size (pRSTDT30,pRSTDT32, pRSTDT34, and pRSTDT37) exhibited step-wisereductions of resolution efficiency (Fig. 2c). The use of anrst fragment containing both the entire 180-bp rst regionand an additional 40-bp region of tnpT (pRST + T40) didnot result in an increase in resolution efficiency. These re-sults suggest that the tnpT-proximal boundary of the func-tional rst site is situated between positions 151 and 154 inFig. 2a. These two types of experiments indicate that the

fully functional rst site is located within the 136-bp seg-ment from positions 19 to 154 in Fig. 2a.

3.4. Strand-exchange takes place in the 33-bp inverted repeatregion

It was unclear at what location in the rst site the strand-exchange takes place during the resolution of the Tn4651-mediated cointegrate. The approximate crossover site canbe determined by analyzing the hybrid recombination sitethat is generated by a site-specific recombination betweentwo highly similar but not identical sites (Reed, 1981;Summers et al., 1985; Canosa et al., 1996). In accordancewith the high amino acid sequence identities in TnpS andTnpT between Tn4651 and Tn4661 (Fig. 1), the rst regionof Tn4651 also showed similarity to the corresponding re-gion of Tn4661 (Fig. 2a). To determine the Tn4651 cross-over site, we constructed the cointegrate analogpRST5161, where one of the two rst regions of pRST449was replaced with the rstTn4661 region (Fig. 2a). We thenexamined whether resolution would occur dependentupon TnpSTn4651 and TnpTTn4651. Contrary to our expecta-tions, the resolution product of pRST5161 was not detectedeven after 30 generations. This indicated that the presenceof nucleotide sequences unique to rstTn4661 inhibited thecointegrate-resolution mediated by the Tn4651 resolutionproteins.

The regions involved in site-specific recombination of-ten contain sequence motifs such as inverted or direct re-peats that serve as the binding sites for site-specificrecombinases and their auxiliary proteins participating inthe recombination process (e.g., Xis for phage DNA excisionfrom the chromosome and ArgR for resolution of plasmid-dimers) (Colloms et al., 1996; Van Duyne, 2005). Withinthe functional rst site, two distinguishable inverted repeatpairs were found. One was a 13-bp repeat pair within a 33-bp inverted repeat region, and each 13-bp repeat was des-ignated IRL or IRR (Fig. 2a). The other was a 9-bp repeatpair within a 20-bp inverted repeat region, and each 9-bprepeat was designated IR1 or IR2 (Fig. 2a). In the former re-gion, rstTn4651 and rstTn4661 showed six mismatches, five ofwhich were located in the spacer region between IRL andIRR. In the latter IR2 region, two mismatches were foundbetween Tn4651 and Tn4661. When the recombinationprocess involves the Holliday junction formation betweentwo recombination sites, a mismatch in the spacer se-quence between the nicking points reduces the recombina-tion efficiency (Weisberg et al., 1983; Hoess et al., 1986;Lee and Saito, 1998). Thus, we considered that Tn4661-spe-cific sequences within these motifs might have preventedthe potential binding of TnpS or TnpT to the rst region orthe strand-exchange after DNA cleavage by TnpS.

To investigate whether the nucleotide differences in thetwo inverted repeated regions between rstTn4651 andrstTn4661 inhibited the recombination, we constructed tworstTn4661 variants. One was rstTn4661v1, in which the se-quence of the spacer region between IRL and IRR inrstTn4661 was replaced with that from rstTn4651. The otherwas rstTn4661v2, in which the sequence of IR2 was replacedwith that from rstTn4651 (Fig. 4a). The cointegrate analogs,pRST5161v1 and pRST5161v2 carrying rstTn4661v1 and

32 H. Yano et al. / Plasmid 69 (2013) 24–35

rstTn4661v2, respectively, were introduced into P. putida F1containing pHY453. The transformants were cultivatedfor 40 generations of growth in the presence of expressionof TnpT, and the sizes of the residing plasmids were ana-lyzed by agarose gel electrophoresis (Fig. 4b). The resolu-tion product of pRST5161v1 with a size equivalent to theresolution product of pRST449 was detected, while the res-olution product of pRST5161v2 was not detected. The res-olution product of pRST5161v1YF, a pRST5161v1derivative encoding an inactive TnpS mutant, was alsonot detected. These results indicate that TnpS mediatedrecombination between rstTn4651 and rstTn4661v1, and themismatches in the spacer region between IRL and IRRinhibited the resolution reaction between rstTn4651 andrstTn4661.

To obtain further information about the crossover site,the resolution product of pRST5161v1 was purified, andthe hybrid rst region was sequenced. The region consistedof rstTn4651 from positions 1 to 91, and rstTn4661v1 frompositions 62 to 180 (Fig. 4c) period. The 30-bp sequencefrom positions 62 to 91 was shared among the three rst re-gions, rstTn4651, rstTn4661v1 and their hybrid rst region. Theseresults indicate that the TnpS-mediated strand-exchangetakes place within the 30-bp segment. Since an invertedrepeat pair flanking a crossover site often serves as arecombinase-binding site (Hallet et al., 2004), it is plausi-ble that the 33-bp inverted region, designated the core site,is a TnpS-binding sequence.

3.5. TnpT specifically binds to the rst region

Phage DNA excision from the chromosome and resolu-tion of plasmid-dimers mediated by tyrosine recombinasesoften require the binding of auxiliary proteins to therecombination site in order to introduce recombinationdirectionality by modulating the shape of the synapticcomplex (Hallet et al., 2004; Grindley et al., 2006). Wehypothesized that TnpT also contributes to the resolutionreaction by binding to the rst region. To investigate the bio-chemical function of TnpT and the role of DNA segmentsother than the core site, TnpT was purified and its bindingto the rst region was analyzed using EMSAs. In this assay,the 259-bp DNA containing the entire rst region (Fig. 2aand 5a) was used as the target DNA, since electrophoresisof a labeled rst fragment with a shorter size yielded severalretarded bands in the absence of TnpT. In the absence ofthe DNA-binding competitor, the addition of TnpT reducedthe free probe signal, and the shifted band (TnpT-rst com-plex) was observed only at the well position of polyacryl-amide gels (Fig. 5d). The addition of increasing amountsof the unlabeled 259-bp DNA increased the free probe sig-nal, while the addition of equivalent amounts of salmonsperm DNA hardly generated any free probe signal(Fig. 5c). This indicates specific binding of TnpT to theprobe containing the entire rst region. To obtain furtherinformation about the TnpT-binding site(s), we analyzedthe competition between the labeled DNA and one of sixdifferent unlabeled 41-bp DNA fragments, each containinga particular 35-bp segment from the rst region (segments Ato F in Fig. 5a), in the presence of a constant amount ofTnpT and labeled DNA. The addition of increasing amounts

of the 41-bp fragment containing segment E (from posi-tions 118 to 152 covering IR1 and IR2) increased the freeprobe signal (Fig. 5d), while the labeled DNA competedmore effectively with the other 41-bp fragments with re-spect to binding to TnpT. This indicates that TnpT bindsto segment E with a much higher affinity than the othersegments in the rst region. These results suggest that TnpTspecifically binds to the DNA segment from positions 118to 152 (Fig. 2a and Fig. 5a) or to a larger fragment that in-cludes this segment.

4. Discussion

There is increasing evidence indicating that Tn4651-re-lated transposons have spread across strains of the genusPseudomonas (Fig. 1a). This research was intended to clar-ify the mechanisms underlying cointegrate-resolution ofthese transposons. Previous genetic studies on Tn4651and comparative sequence analysis of Tn5041 and its re-lated elements revealed potential cis-acting sites [20-bprepeat region (IR1 and IR2) for Tn4651 and att5041 forTn5041] involved in the cointegrate-resolution of thesetransposons (Genka et al., 2002; Kholodii et al., 2002).However, these studies were not successful in providingsolid evidence for the role of the two DNA segments inthe resolution.

Cointegrate-resolution of Tn3-family transposons isgenerally achieved by a single species of site-specificrecombinases, such as TnpR of Tn3 (Benjamin et al.,1985) and TnpI of Tn4430 (Vanhooff et al., 2006). In con-trast, cointegrate-resolution of Tn4651 was highly depen-dent on TnpT as well as TnpS (Fig. 3b). Tyrosinerecombinase itself, including TnpS, mediates both inter-and intra-molecular recombination (Genka et al., 2002;Vanhooff et al., 2006). Therefore, it is theoretically possiblethat TnpS mediates the integration of excised resolutionproduct circles into the substrate plasmid, thus generatinga plasmid carrying more than three copies of rst regions, ashas been observed for XerC/D-mediated resolution of achromosome-dimer analog carrying two dif sites (Recchiaet al., 1999). However, such plasmid derivatives were notobserved in this study, and only substrate plasmids andresolution products were detected in P. putida lysates inthe presence of TnpT. This observation suggests that TnpTcoordinates the directionality of recombination to yieldonly resolution products.

We here reported for the first time the binding of TnpTto a specific DNA sequence. The secondary structure pre-diction suggested a unique structure of TnpT (Fig. 1c),when compared with the structures of other knownrecombination directionality factors such as ArgR, PepA,and Xis (Ni et al., 1999; Strater et al., 1999; Abbani et al.,2007). The C-terminal 267 amino-acid region consists ofalpha helices, and forms a coiled-coil structure. A similarsecondary structure was predicted for the Tn5041 OrfQprotein, despite the fact that TnpT and OrfQ show only29% identity. A coiled-coil motif is generally responsiblefor the formation of protein dimers or multimers (Woolf-son, 2005). It is thus possible that TnpT forms a quaternarystructure upon its binding to DNA. The potential target for

H. Yano et al. / Plasmid 69 (2013) 24–35 33

TnpT is the 20-bp inverted repeat region (Fig. 4) that is lo-cated 35 bp upstream of the transcriptional start point oftnpS (Fig. 2a) (Genka et al., 2002). This suggests that TnpTplays a role in transcriptional control of tnpS.

Deletions extending to the rst position 151 in segment E(Fig. 2a and Fig. 5a) resulted in a significant reduction inthe resolution efficiencies of cointegrate analogs(pRSTDT30, pRSTDT32, pRSTDT34 and pRSTDT37 inFig. 2c). Although the results from EMSA suggested thatTnpT does not efficiently bind to fragment F (positions148–182), deletion of positions 151–154 reduced resolu-tion efficiency. This could be mainly due to an impairedinteraction of TnpT with DNA. It is currently unclear howTnpT binds to the fragment E region, but we speculate thatseveral TnpT molecules cooperatively bind to DNA andform oligomers to introduce DNA-bending as proposedfor PepA-binding to cer of ColE1 (Strater et al., 1999) andXis-binding to attR (Abbani et al., 2007). The oligomeriza-tion may depend on a weak affinity of TnpT to positions151–154. Progressive elimination of the tnpT-proximalend might have resulted in a stepwise reduction in thenumber of TnpT molecules for the oligomerization on thefragment E region. This may explain a gradual decreasein resolution efficiency. Our foot-printing analysis to deter-mine the TnpT-binding site was unsuccessful so far. Fur-thermore, we could not purify TnpS in the solublefraction. Future biochemical analysis under optimized con-ditions will provide a clear picture for the DNA-bindingmechanisms of TnpT and TnpS.

The tnpS-proximal end of the functional rst site did notshow any homology to the 20-bp inverted repeat regionin the tnpT-proximal side or the 33-bp inverted repeats atthe core site. Therefore, the requirement for the segmentat the tnpS-proximal side for efficient resolution could beunrelated to the binding of TnpT or TnpS. In the case ofres of Tn1000 (cd), six of pSM19035, and cer of ColE1, theoutermost ends of the functional resolution sites are almostidentical to those of the protein-binding regions (Wells andGrindley, 1984; Summers and Sherratt, 1988; Rojo andAlonso, 1995; Alen et al., 1997). Extending deletions inthe accessory regions resulted in a gradual decrease in res-olution efficiency for Tn1000 and ColE1 (Wells and Grind-ley, 1984; Summers and Sherratt, 1988). Our preliminaryin vitro resolution assays using partially purified TnpSTn4661

and TnpTTn4661 as well as P. putida crude extract suggestthat an unknown host protein(s) other than IHF is necessaryfor the resolution of the Tn4661-mediated cointegrate(Wakase and Tsuda, unpublished data). It is possible thatthe requirement of both outermost ends in the functionalrst site is related to the binding of a host protein or unchar-acterized gene products encoded by the transposon.

The rst deletion analysis indicated that the fully func-tional rst site is 136 bp in length (position 19–154 inFig. 2a), and the crossover site was within the 33-bp invertedrepeat region (core site: position 59–91 in Fig. 2a). Thus, thecore site splits the 136-bp functional rst site into the 40-bptnpS-proximal and 63-bp tnpT-proximal segments. Thisrequirement for the DNA segments seems to be unusualamong the resolution sites analyzed so far. In most resolu-tion systems, the accessory sites required for the resolutionreaction (e.g., cer, six, res of Tn1000, and irs of Tn4430) are

situated on only one side of the core site (Wells and Grindley,1984; Summers and Sherratt, 1988; Canosa et al., 1996; Van-hooff et al., 2006). This is true even for the phage excisionsites (attL and attR) (Van Duyne, 2005). Therefore, availableevidence suggests that the molecular mechanisms underly-ing cointegrate-resolution of Tn4651-related transposonsare significantly different from those of known intramolecu-lar recombination systems.

The resolution site in Tn3-family transposons often of-fers a recombination hot spot for gene exchange (shuffling)among transposons (Kholodii, 2001; Yano et al., 2007).Gene exchange at the resolution site can provide variousphenotypic genes to cryptic transposons, and can also offeropportunities for inactivated transposons to regain func-tional transposition genes and sites (Yano et al., 2007,2010). To investigate whether the rst site also serves as arecombination hot spot, we searched the nucleotide se-quences of Tn4651-related transposons in the database. ATn4661-like element was found in the P. aeruginosa PA7and PA14 chromosomes (Fig. S1 in Supplementary materi-als, Fig. 6a). The Tn4661-like element in the PA14 chromo-some shares an identical structure with Tn4661 in the10.8-kb segment from the tnpA-proximal IR to the rst re-gion (Fig. 6a), while the remaining 2.8-kb rst-tnpT-IR re-gion of the Tn4661-like element does not show similarityto the equivalent part of Tn4661. Instead the 2.8-kb regionshows a relatively high similarity with the equivalent partof Tn4651/Tn4652 (Fig. 6a). This suggests that recombina-tion in the rst site between Tn4661 and another relatedtransposon had occurred in the past. A closer look at therst sequences revealed that the Tn4661-like element andTn4661 share identical sequences from rst positions 1 to72, but that the remaining sequences are different(Fig. 6b, only positions 50–100 are shown). This stronglysuggests that the strand-exchange in the rst core site, pos-sibly mediated by TnpS, generated a chimeric transposonin the PA14 chromosome or other genomes.

It should be noted that the sequences in the core site andthe 20-bp inverted repeat region in the Tn4651 rst site showlittle similarity to those in the corresponding region ofTn5041. In contrast, Tn4651, Tn4661, and Tn4676 sharehighly similar sequences in the inverted repeat motif inthe rst core site (IRL and IRR in Fig. 2a). Interestingly, thesethree transposons do not share the same sequence in theirrespective spacer region (Fig. 2a). As shown in this study,sequence mismatch in the spacer region prevents the intra-molecular recombination between the rst sites (Fig. 4).Thus, the recombinations between those three transposonsare unlikely to ocurr. However, our results from compara-tive sequence analysis (Fig. 6) seem to suggest that thestrand exchange within the rst core site can happen evenin the presence of mismatches in the spacer regions. Thisinconsistency should be addressed in future research.

The tnpS and tnpT homologs have been found not onlyin transposons but also in plasmids and chromosomeswithout their associated transposition genes [e.g., plasmidspND6–1 (Li et al., 2004) and pADP-1 (Martinez et al., 2001),and the chromosomes of strains within genera Pseudomo-nas and Xanthomonas (H. Yano, and M. Tsuda, unpublisheddata)]. Although it is unclear whether these genes aresimply remnants of transposons, this finding suggests an

Fig. 6. Evidence for recombination at the rst core site between Tn4651-related transposons in extant genomes. (a) Structural similarity between theTn4661-like element in the PA14 chromosome (GenBank ID: CP000438) and Tn4651 (GenBank ID: AJ344068), or Tn4661 (GenBank ID: AB375440).Pentagons indicate coding sequences. Amino-acid sequence identities (%) between Tn4661 products and their homologs are indicated between thetransposable elements. (b) Alignment of rst regions from the three elements shown in (a). Dots indicate nucleotides identical to those in the Tn4651sequence. Positions 50–100 of rst are shown.

34 H. Yano et al. / Plasmid 69 (2013) 24–35

additional role of the TnpS/TnpT/rst-related resolution sys-tems in the maintenance of plasmids and chromosomes ofPseudomonas-related genera.

In conclusion, we demonstrated that the crossover sitein the Tn4651-specified resolution system is within the33-bp inverted repeat region in the 136-bp functional rstsite, and that TnpT is a novel type of DNA-binding proteinthat determines recombination directionality to yield onlyresolution products. Deletion analysis of the rst regionstrongly suggested that accessory sites are present on bothsides of the crossover site. This implies that cointegrate-resolution of the Tn4651-related transposon is mechani-cally different from that of the well-characterized resolu-tion systems. Comparative analysis for Tn4651-relatedtransposons suggests that recombination at the rst coresite plays a role in the evolution of this family of transpo-sons as well as in cointegrate-resolution.

Acknowledgments

We thank Dr. Christopher M .Thomas at University ofBirmingham for providing us unpublished sequence infor-mation of plasmid Rms148. We thank Shiho Wakase andDr. Yukari Sato at Tohoku University for technical adviceand meaningful discussion. We also thank Gail E. Deckertat University of Idaho for improving the manuscript. Thisresearch was supported by a Grant-in-Aid from the Minis-try of Education, Culture, Sports, Science and Technology,Japan.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.plasmid.2012.07.004.

References

Abbani, M.A., Papagiannis, C.V., Sam, M.D., Cascio, D., Johnson, R.C., Clubb,R.T., 2007. Structure of the cooperative Xis–DNA complex reveals amicronucleoprotein filament that regulates phage lambda intasomeassembly. Proc. Natl. Acad. Sci. USA 104, 2109–2114.

Alen, C., Sherratt, D.J., Colloms, S.D., 1997. Direct interaction ofaminopeptidase A with recombination site DNA in Xer site-specificrecombination. EMBO Journal 16, 5188–5197.

Baum, J.A., 1994. Tn5401, a new class II transposable element fromBacillus thuringiensis. Journal of Bacteriology 176, 2835–2845.

Benjamin, H.W., Matzuk, M.M., Krasnow, M.A., Cozzarelli, N.R., 1985.Recombination site selection by Tn3 resolvase: topological tests of atracking mechanism. Cell 40, 147–158.

Blatny, J.M., Brautaset, T., Winther-Larsen, H.C., Karunakaran, P., Valla, S.,1997. Improved broad-host-range RK2 vectors useful for high and lowregulated gene expression levels in gram-negative bacteria. Plasmid38, 35–51.

Canosa, I., Rojo, F., Alonso, J.C., 1996. Site-specific recombination by the bprotein from the streptococcal plasmid pSM19035: minimalrecombination sequences and crossing over site. Nucleic AcidsResearch 24, 2712–2717.

Cole, C., Barber, J.D., Barton, G.J., 2008. The Jpred 3 secondary structureprediction server. Nucleic Acids Research 36, W197–201.

Colloms, S.D., McCulloch, R., Grant, K., Neilson, L., Sherratt, D.J., 1996. Xer-mediated site-specific recombination in vitro. EMBO Journal 15,1172–1181.

Devers, M., Rouard, N., Martin-Laurent, F., 2008. Fitness drift of anatrazine-degrading population under atrazine selection pressure.Environmental Microbiology 10, 676–684.

Genka, H., Nagata, Y., Tsuda, M., 2002. Site-specific recombination systemencoded by toluene catabolic transposon Tn4651. Journal ofBacteriology 184, 4757–4766.

Greated, A., Lambertsen, L., Williams, P.A., Thomas, C.M., 2002. Completesequence of the IncP-9 TOL plasmid pWW0 from Pseudomonas putida.Environmental Microbiology 4, 856–871.

Grindley, N.D., Whiteson, K.L., Rice, P.A., 2006. Mechanisms of site-specificrecombination. Annual Review of Biochemistry 75, 567–605.

Grindley, N.D.F. 2002. The movement of Tn3-like elements; transpositionand cointegrate resolution. In: Craig, N.L., Craigie, R., Lambowitz, A.M.(Eds.), Mobile DNA II. ASM press, Washington DC., pp. 272–302.

Hallet, B., Vanhooff, V., Cornet, F., 2004. DNA site-specific resolutionsystems. In: Funnell, B.E., Phillips, G.J. (Eds.), Plasmid biology. ASMpress, Washington D.C., pp. 145–180.

Hoess, R.H., Wierzbicki, A., Abremski, K., 1986. The role of the loxP spacerregion in P1 site-specific recombination. Nucleic Acids Research 14,2287–2300.

H. Yano et al. / Plasmid 69 (2013) 24–35 35

Horak, R., Ilves, H., Pruunsild, P., Kuljus, M., Kivisaar, M., 2004. The ColR-ColS two-component signal transduction system is involved inregulation of Tn4652 transposition in Pseudomonas putida understarvation conditions. Molecular Microbiology 54, 795–807.

Horak, R., Kivisaar, M., 1998. Expression of the transposase gene tnpA ofTn4652 is positively affected by integration host factor. Journal ofBacteriology 180, 2822–2829.

Horak, R., Kivisaar, M., 1999. Regulation of the transposase of Tn4652 bythe transposon-encoded protein TnpC. Journal of Bacteriology 181,6312–6318.

Ilves, H., Horak, R., Kivisaar, M., 2001. Involvement of sigma(S) instarvation-induced transposition of Pseudomonas putida transposonTn4652. Journal of Bacteriology 183, 5445–5448.

Ilves, H., Horak, R., Teras, R., Kivisaar, M., 2004. IHF is the limiting hostfactor in transposition of Pseudomonas putida transposon Tn4652 instationary phase. Molecular Microbiology 51, 1773–1785.

Kasak, L., Horak, R., Kivisaar, M., 1997. Promoter-creating mutations inPseudomonas putida: a model system for the study of mutation instarving bacteria. Proc. Natl. Acad. Sci. USA 94, 3134–3139.

Kholodii, G., 2001. The shuffling function of resolvases. Gene 269, 121–130.

Kholodii, G., Gorlenko, Z., Mindlin, S., Hobman, J., Nikiforov, V., 2002.Tn5041-like transposons: molecular diversity, evolutionaryrelationships and distribution of distinct variants in environmentalbacteria. Microbiology 148, 3569–3582.

Kholodii, G.Y., Yurieva, O.V., Gorlenko, Z., Mindlin, S.Z., Bass, I.A.,Lomovskaya, O.L., Kopteva, A.V., Nikiforov, V.G., 1997. Tn5041: achimeric mercury resistance transposon closely related to thetoluene degradative transposon Tn4651. Microbiology 143, 2549–2556.

Kleckner, N., 1981. Transposable elements in prokaryotes. Annual Reviewof Genetics 15, 341–404.

Klockgether, J., Reva, O., Larbig, K., Tummler, B., 2004. Sequence analysisof the mobile genome island pKLC102 of Pseudomonas aeruginosa C.Journal of Bacteriology 186, 518–534.

Kovach, M.E., Elzer, P.H., Hill, D.S., Robertson, G.T., Farris, M.A., Roop,R.M.n., Peterson, K.M., 1995. Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166, 175–176.

Lee, G., Saito, I., 1998. Role of nucleotide sequences of loxP spacer regionin Cre-mediated recombination. Gene 216, 55–65.

Levy, S.B., Marshall, B., 2004. Antibacterial resistance worldwide: causes,challenges and responses. Nature Medicine 10, S122–9.

Li, W., Shi, J., Wang, X., Han, Y., Tong, W., Ma, L., Liu, B., Cai, B., 2004.Complete nucleotide sequence and organization of the naphthalenecatabolic plasmid pND6-1 from Pseudomonas sp. strain ND6. Gene336, 231–240.

Liebert, C.A., Hall, R.M., Summers, A.O., 1999. Transposon Tn21, flagship ofthe floating genome. Microbiology and Molecular Biology Reviews 63,507–522.

Maeda, K., Nojiri, H., Shintani, M., Yoshida, T., Habe, H., Omori, T., 2003.Complete nucleotide sequence of carbazole/dioxin-degradingplasmid pCAR1 in Pseudomonas resinovorans strain CA10 indicatesits mosaicity and the presence of large catabolic transposon Tn4676.Journal of Molecular Biology 326, 21–33.

Mahillon, J., Lereclus, D., 1988. Structural and functional analysis ofTn4430: identification of an integrase-like protein involved in the co-integrate-resolution process. EMBO Journal 7, 1515–1526.

Martinez, B., Tomkins, J., Wackett, L.P., Wing, R., Sadowsky, M.J., 2001.Complete nucleotide sequence and organization of the atrazinecatabolic plasmid pADP-1 from Pseudomonas sp. strain ADP. Journalof Bacteriology 183, 5684–5697.

McDonnell, A.V., Jiang, T., Keating, A.E., Berger, B., 2006. Paircoil2:improved prediction of coiled coils from sequence. Bioinformatics22, 356–358.

Ni, J., Sakanyan, V., Charlier, D., Glansdorff, N., Van Duyne, G.D., 1999.Structure of the arginine repressor from Bacillus stearothermophilus.Natural Structural Biology 6, 427–432.

Ohtsubo, Y., Ikeda-Ohtsubo, W., Nagata, Y., Tsuda, M., 2008.GenomeMatcher: a graphical user interface for DNA sequencecomparison. BMC Bioinformatics 9, 376.

Qing, G., Ma, L.C., Khorchid, A., Swapna, G.V., Mal, T.K., Takayama, M.M.,Xia, B., Phadtare, S., Ke, H., Acton, T., Montelione, G.T., Ikura, M.,Inouye, M., 2004. Cold-shock induced high-yield protein productionin Escherichia coli. Nature Biotechnology 22, 877–882.

Recchia, G.D., Aroyo, M., Wolf, D., Blakely, G., Sherratt, D.J., 1999. FtsK-dependent and -independent pathways of Xer site-specificrecombination. EMBO Journal 18, 5724–5734.

Reed, R.R., 1981. Resolution of cointegrates between transposons cd andTn3 defines the recombination site. Proc. Natl. Acad. Sci. USA 78,3428–3432.

Rojo, F., Alonso, J.C., 1995. The b recombinase of plasmid pSM19035 bindsto two adjacent sites, making different contacts at each of them.Nucleic Acids Research 23, 3181–3188.

Sagai, H., Krcmery, V., Hasuda, K., Iyobe, S., Knothe, H., 1975. R factor-mediated resistance to aminoglycoside antibiotics in Pseudomonasaeruginosa. Jpn J Microbiol. 19, 427–432.

Sambrook, J., Russell, D.W., 2001. Molecular Cloning. Cold Spring HarborLaboratory Press, Cold Spring Harbor, New York.

Schmitt, R., Bernhard, E., Mattes, R., 1979. Characterisation of Tn1721, anew transposon containing tetracycline resistance genes capable ofamplification. Molecular and General Genetics 172, 53–65.

Shintani, M., Yoshida, T., Habe, H., Omori, T., Nojiri, H., 2005. Largeplasmid pCAR2 and class II transposon Tn4676 are functional mobilegenetic elements to distribute the carbazole/dioxin-degradative cargene cluster in different bacteria. Applied Microbiology andBiotechnology 67, 370–382.

Sota, M., Yano, H., Nagata, Y., Ohtsubo, Y., Genka, H., Anbutsu, H.,Kawasaki, H., Tsuda, M., 2006. Functional analysis of unique class IIinsertion sequence IS1071. Applied and Environment Microbiology72, 291–297.

Strater, N., Sherratt, D.J., Colloms, S.D., 1999. X-ray structure ofaminopeptidase A from Escherichia coli and a model for thenucleoprotein complex in Xer site-specific recombination. EMBOJournal 18, 4513–4522.

Summers, D., Yaish, S., Archer, J., Sherratt, D., 1985. Multimer resolutionsystems of ColE1 and ColK: localisation of the crossover site.Molecular and General Genetics 201, 334–338.

Summers, D.K., Sherratt, D.J., 1988. Resolution of ColE1 dimers requires aDNA sequence implicated in the three-dimensional organization ofthe cer site. EMBO Journal 7, 851–858.

Takeshita, S., Sato, M., Toba, M., Masahashi, W., Hashimoto-Gotoh, T.,1987. High-copy-number and low-copy-number plasmid vectors forlacZa-complementation and chloramphenicol- or kanamycin-resistance selection. Gene 61, 63–74.

Taylor, L.A., Rose, R.E., 1988. A correction in the nucleotide sequence ofthe Tn903 kanamycin resistance determinant in pUC4K. Nucleic AcidsResearch 16, 358.

Teras, R., Jakovleva, J., Kivisaar, M., 2009. Fis negatively affects binding ofTn4652 transposase by out-competing IHF from the left end ofTn4652. Microbiology 155, 1203–1214.

Tsuda, M., Iino, T., 1987. Genetic analysis of a transposon carrying toluenedegrading genes on a TOL plasmid pWW0. Molecular and GeneralGenetics 210, 270–276.

Van Duyne, G.D., 2005. Lambda integrase: armed for recombination.Current Biology 15, R658–60.

Vanhooff, V., Galloy, C., Agaisse, H., Lereclus, D., Revet, B., Hallet, B., 2006.Self-control in DNA site-specific recombination mediated by thetyrosine recombinase TnpI. Molecular Microbiology 60, 617–629.

Weisberg, R.A., Enquist, L.W., Foeller, C., Landy, A., 1983. Role for DNAhomology in site-specific recombination. The isolation andcharacterization of a site affinity mutant of coliphage lambda.Journal of Molecular Biology 170, 319–342.

Wells, R.G., Grindley, N.D., 1984. Analysis of the cd res site. Sites requiredfor site-specific recombination and gene expression. Journal ofMolecular Biology 179, 667–687.

Willems, R.J., Hanage, W.P., Bessen, D.E., Feil, E.J., 2011. Population biologyof Gram-positive pathogens: high-risk clones for dissemination ofantibiotic resistance. FEMS Microbiology Reviews 35, 872–900.

Woolfson, D.N., 2005. The design of coiled-coil structures and assemblies.Advances in Protein Chemistry 70, 79–112.

Wurdemann, D., Tummler, B., 2007. In silico comparison of pKLC102-likegenomic islands of Pseudomonas aeruginosa. FEMS MicrobiologyLetters 275, 244–249.

Yano, H., Garruto, C.E., Sota, M., Ohtsubo, Y., Nagata, Y., Zylstra, G.J., Williams,P.A., Tsuda, M., 2007. Complete sequence determination combined withanalysis of transposition/site-specific recombination events to explaingenetic organization of IncP-7 TOL plasmid pWW53 and related mobilegenetic elements. Journal of Molecular Biology 369, 11–26.

Yano, H., Miyakoshi, M., Ohshima, K., Tabata, M., Nagata, Y., Hattori, M.,Tsuda, M., 2010. Complete nucleotide sequence of TOL plasmid pDK1provides evidence for evolutionary history of IncP-7 catabolicplasmids. Journal of Bacteriology 192, 4337–4347.

Zylstra, G.J., McCombie, W.R., Gibson, D.T., Finette, B.A., 1988. Toluenedegradation by Pseudomonas putida F1: genetic organization of thetod operon. Applied and Environment Microbiology 54, 1498–1503.