Completing the specificity swap: Single-stranded DNA recognition by F and R100 TraI relaxase domains

Plasmid 80 (2015) 1–7

Contents lists available at ScienceDirect

Plasmid j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y p l a s

Completing the specificity swap: Single-stranded DNA recognition by F and R100 TraI relaxase domains Kip E. Guja 1, Joel F. Schildbach * Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218, USA

A R T I C L E

I N F O

Article history: Available online 1 April 2015 Communicated by Julian Rood Keywords: Relaxase Nickase Plasmid Conjugation Specificity

A B S T R A C T

During conjugative plasmid transfer, one plasmid strand is cleaved and transported to the recipient bacterium. For F and related plasmids, TraI contains the relaxase or nickase activity that cleaves the plasmid DNA strand. F TraI36, the F TraI relaxase domain, binds a single-stranded origin of transfer (oriT) DNA sequence with high affinity and sequence specificity. The TraI36 domain from plasmid R100 shares 91% amino acid sequence identity with F TraI36, but its oriT DNA binding site differs by two of eleven bases. Both proteins readily distinguish between F and R100 binding sites. In earlier work, two amino acid substitutions in the DNA binding cleft were shown to be sufficient to change the R100 TraI36 DNAbinding specificity to that of F TraI36. In contrast, three substitutions could make F TraI36 more “R100-like”, but failed to completely alter the specificity. Here we identify one additional amino acid substitution that completes the specificity swap from F to R100. To our surprise, adding further substitutions from R100 to the F background were detrimental to binding instead of being neutral, indicating that their effects were influenced by their structural context. These results underscore the complex and subtle nature of DNA recognition by relaxases and have implications for the evolution of relaxase binding sites and oriT sequences. © 2015 Elsevier Inc. All rights reserved.

1. Introduction During bacterial conjugation, a single conjugative plasmid strand is transferred in a unidirectional manner between bacteria (Cascales and Christie, 2003; Lawley et al., 2003). Conjugation occurs within and between bacterial species, and cross-kingdom transfer has also been described (Christie et al., 2005; Lessl and Lanka, 1994; Waters, 2001). Conjugation contributes to the diversification of prokaryotic genomes as well as the dissemination of antibiotic resistance and the increased virulence of some pathogens

Abbreviations: oriT, origin of transfer. * Corresponding author. Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218, USA. Fax: 410-516-5213. E-mail address: [email protected] (J.F. Schildbach). 1 Current address: Department of Pharmacological Sciences, Stony Brook University, Stony Brook, New York 11794, USA. http://dx.doi.org/10.1016/j.plasmid.2015.03.006 0147-619X/© 2015 Elsevier Inc. All rights reserved.

(Gogarten and Townsend, 2005; Goldenfield and Woese, 2007; Reisner et al., 2006; Sorensen et al., 2005). Clinically significant factors such as toxins, hemolysins, and antibiotic resistance proteins can be encoded by conjugative plasmids and spread throughout bacterial populations in medical settings. Conjugative plasmids also have been linked to accelerated rates of evolution seen in pathogenic Escherichia coli strains (Wirth et al., 2006). Although conjugative plasmids are extremely diverse, many elements involved in the transfer process are conserved, including relaxase proteins (Zechner et al., 2000). Relaxases, also called nickases, are responsible for binding and cleaving one plasmid strand at the plasmid origin of transfer, or oriT (Inamoto et al., 1991; Lang et al., 2010; Matson and Morton, 1991; Pansegrau et al., 1993; Reygers et al., 1991; Traxler and Minkley, 1988). Relaxases form covalent phosphotyrosyl linkages between an active site Tyr and a DNA backbone phosphate, and the attached relaxase acts as a pilot protein, helping deliver the DNA to the

2

K.E. Guja, J.F. Schildbach / Plasmid 80 (2015) 1–7

recipient cell (Draper et al., 2005; Inamoto et al., 1991; Llosa et al., 1995; Matson et al., 1993; Pansegrau et al., 1990; Parker and Meyer, 2007). Early studies established that a relaxase specifically targets the oriT of the plasmid encoding it (Thompson et al., 1984; Traxler and Minkley, 1988; Willetts and Maule, 1979, 1986). The TraI protein of conjugative plasmid F factor is the F relaxase, and F TraI also possesses a helicase activity (Abdel-Monem et al., 1983). Biochemical studies of F TraI36, the N-terminal 36-kDa relaxase domain of TraI, have shown that F TraI36 is highly specific for its cognate oriT sequence (Harley and Schildbach, 2003; Hekman et al., 2008; Stern and Schildbach, 2001; Williams and Schildbach, 2006). In vivo, this specificity is reflected in the poor efficiency with which F TraI mobilizes plasmids containing the TraI binding site for the highly homologous R100 plasmid, despite a difference of only two nucleotide bases between the F oriT and the R100 oriT sequences (Fekete and Frost, 2000; Stern and Schildbach, 2001). In fact, the in vitro affinity of F TraI36 for the R100 oriT sequence relative to its cognate oriT is reduced by over three orders of magnitude (Harley and Schildbach, 2003). While R100 TraI36 shows a similar high level of in vitro specificity, R100 TraI appears less specific in vivo, showing some activity against both R100 and F oriT sequences, permitting transfer of chimeric plasmids, albeit with reduced efficiency (Fekete and Frost, 2000). Despite the degree of sequence specificity shown by F and R100 TraI, their relaxase domains share 91% sequence identity (301/330 amino acids; Fig. 1). Previous work from this laboratory demonstrated that substituting two amino acids from the F TraI36 sequence into the R100 TraI36 background (variant R100–R193Q/ Q201R, where “R100” indicates the background) generates a protein that possesses wild-type (wt) F-specificity and affinity for the F oriT sequence (Harley and Schildbach, 2003). The converse substitutions in F TraI36 (F-Q193R/R201Q), however, do not confer the wt R100 specificity. Although F-Q193R/R201Q has an “R100-like” specificity, binding the R100 oriT site with 10-fold greater affinity than the F site, the protein binds the R100 oriT site with an affinity that is reduced more than 100-fold relative to wt R100 TraI36. Adding another substitution, E153D, to F-Q193R/R201Q increased affinity for the R100 site approximately 10-fold. The resulting F TraI36 variant E153D/Q193R/R201Q, which we will refer to as the F-triple mutant, still falls short of the R100 specificity and shows only a 60-fold preference for the R100 site, as opposed to the 1000-fold preference shown by wt R100 TraI36. In the structures of F TraI36, unbound and in complex with a single-stranded F oriT oligonucleotide (Datta et al., 2003; Larkin et al., 2005), Gln193 and Arg201 line either side of a pocket within the protein’s binding surface (Fig. 2; Datta et al., 2003; Harley and Schildbach, 2003; Larkin et al., 2005). The guanine (Gua) base that docks into that pocket in F TraI36 (Gua145′ using the numbering of Frost and colleagues (Frost et al., 1994), where the prime designates the “bottom” or transfer strand) is a Thy in the R100 oriT sequence. Gua147′ of the F oriT sequence, which also contacts Gln193 in the complex, is replaced by Ade in the R100 oriT sequence. Examining the structure, we identified four amino acids that both differ between F and R100 TraI36 and are within close

proximity of the two bases that differ between F and R100 TraI binding sites (Gua145′ and Gua147′): Met2, Ile4, Ala5 and Arg198 (Fig. 2). We engineered several additional F TraI36 variants using the F-triple mutant (F-E153D/Q193R/ R201Q) as a template, with substitutions made at these positions. These variant proteins were expressed and purified, and their binding was assayed. 2. Materials and methods 2.1. Protein engineering and purification Primer sequences are shown in Supplementary Table S1. All cloned gene segments and mutations were confirmed by DNA sequencing (DNA Analysis Facility, Johns Hopkins Medical Institutions). The wt F TraI36, F-triple mutant, and wt R100 TraI36 expression constructs were engineered previously (Harley and Schildbach, 2003; Street et al., 2003). Plasmids were purified from Escherichia coli strain XL-1 Blue (Stratagene) or TB1 using the PureYield Plasmid Midiprep System (Promega). Single and double mutations were PCR generated using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). Most mutants were generated in a step-wise fashion starting with the F-triple mutant. Expression plasmids were transformed into BL21(DE3)pLysS cells (Stratagene). All proteins were expressed and purified as described for wt F TraI36 (Street et al., 2003). 2.2. DNA binding affinity measurements Binding affinities of TraI36 and F TraI36 variants for a single-stranded oriT DNA sequence were measured by following changes in fluorescence intensity upon binding of a 3′ carboxy-tetramethylrhodamine (TAMRA)-labeled F-TAM (5′-TTTGCGTGGGGTGT^GGTGCTTT-3′) or R100TAM (5′-TTTGCGTAGTGTGT^GGTGCTTT-3′) oriT binding site oligonucleotide (sequence differences between the two oligonucleotide are underlined and the caret indicates the nic site.) Oligonucleotides were purchased from Integrated DNA Technologies. F-TAM or R100-TAM oligonucleotides were diluted to 4 nM in binding buffer (100 mM NaCl, 50 mM Tris– HCl [pH 7.5], and 0.1 mM EDTA). Changes in fluorescence emission intensity of the F-TAM and R100-TAM oriT oligonucleotides were measured in an AVIV Biomedical Automated Titrating Differential/Ratio Spectrofluorometer Model ATF-105. Typically, for wt or near wt F binding affinity, varying volumes of 250 nM protein in binding buffer were injected into 4 nM TAMRA-labeled oligonucleotide. Lower affinity interactions were measured using a stock protein solution of higher concentration. An initial volume of 1.7 mL of the oligonucleotide solution was used. Bandwidths were set at 4 nM excitation and 6 nM emission. Excitation and emission wavelengths were set at 520 nM and 580 nM, respectively. Measurements were averaged for 10 s. Samples were stirred continuously, and stir speed was set to 10. A 50 μL or 100 μL syringe was used to inject increasing amounts of protein. Experiments were performed at 25 °C with a stir time of 3–4 minutes between injections.

K.E. Guja, J.F. Schildbach / Plasmid 80 (2015) 1–7

3

Fig. 1. Alignments of F and F-like relaxase and oriT sequences. A. An alignment of the amino acid sequences of the relaxase domains (amino acids 1–330) of the TraI proteins from F, R100 (NCBI Reference Sequence NC_002134.1), and P307 (this paper; Frost et al., 1994) The alignment was generated using ClustalW2 (www.ebi.ac.uk/clustalw/) and rendered with ESPript (http://espript.ibcp.fr/). Sequence identity is denoted with blue shading, and substitutions considered conservative are shaded gray. The secondary structure of F TraI36 is depicted above the alignment, and positions of substitutions described in this study are marked with a star. Key specificity determinants or beneficial substitutions are denoted with a red star, while neutral or detrimental substitutions are marked with a yellow star (see Section 3 for details). B. An alignment of the DNA sequences of the origin of transfer for plasmids F, R100 (NCBI Reference Sequence NC_002134.1), and P307 (Frost et al., 1994; Goldner et al., 1987). The triangles mark nic, the site at which TraI cleaves. Differences from the F sequence are denoted in red. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Binding data were fit to a single binding site model using KaleidaGraph (Synergy Software) using the equation:

2.3. Sequences

((BLU − BLL ) (2 × [ DT ])) × ([ DT ] + [ PT ] + K D )

R100-like relaxases are defined as TraI from plasmids R100 [Swiss-Prot:Q9WTB0], p1ESCUM [Swiss-Prot:B7LJ26], pUT189 [Swiss-Prot:Q1R1Q0] and pO86A1 [Swiss-Prot: Q08JE3]. F-like relaxases are defined as TraI from plasmids F [Swiss-Prot:P14565], R1 [Swiss-Prot:Q6TDU5], P1658/ 97 [Swiss-Prot:Q84A21], pSMS35_130 [Swiss-Prot:B1LRJ1], pIP1206 [Swiss-Prot:B1VCB3], p53638_226 [Swiss-Prot: B2U5N8], pSS_046 [Swiss-Prot:Q3YTF0], pAPEC-O2-ColV

±

(([D ] + [P ] + K T

T

D

)2 − (4 ∗ [ DT ][ PT ])) + BLL

where BLU is the upper baseline, BLL is the lower baseline, DT is the total amount of labeled ssDNA, PT is the total amount of protein and KD is the dissociation constant. Representative binding curves are shown in Fig. 3.

4

K.E. Guja, J.F. Schildbach / Plasmid 80 (2015) 1–7

Fig. 2. The X-ray crystallographic structure of F TraI36 bound F oriT oligonucleotide. The protein is shown in blue cartoon representation, with residues selected for mutagenesis shown as space-filling models (Met2 in red, Ile4 in purple, Ala5 in yellow, Tyr190 in orange, and Arg198 in brown). The bound oriT oligonucleotide is shown as beige stick models, while bases Gua145′ and Gua147′ (the two base differences between F and R100 oriT sites) are shown as green and cyan stick models, respectively. The top inset depicts the relative positions of Tyr190, Ile4 and base Gua147′ of the oriT DNA. Experiments have shown a substitution at the 4 position requires a compensatory mutation at the 190 position. The bottom inset depicts the F TraI36 protein surface in gold, with specificity determining residues highlighted (Met2 in red, Gln193 in blue, and Arg201 in purple). Base Gua145′ is shown as a stick model in green. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

[Swiss-Prot:Q2TU09], pAPEC-O2-R [Swiss-Prot:Q5JBP8], pVM01 [Swiss-Prot:B1P7Q9], pECOS88 [Swiss-Prot:B7LI91], pCVM29188_146 [Swiss-Prot:B4TLB7], pAPEC-O1-ColBM [Swiss-Prot:Q27TG4], pMAR7 [Swiss-Prot:Q0H0A7], pMAR2 [Swiss-Prot:B7UTI8]; W0095 from plasmid pSFO157 [SwissProt:Q93QL7]; and TraI from E. coli strains 101-1 [SwissProt:B3XEJ8], B7A [Swiss-Prot:B3HH87], E22 [Swiss-Prot: B3ID91], E110019 [Swiss-Prot:B3IND3]. Multiple sequence alignments were performed using Clustal W using the ClustalW2 server at the European Bioinformatics Institute [http://www.ebi.ac.uk/Tools/clustalw2/]. P307 plasmid was generously supplied by Prof. Ellen Zechner (Univ. of Graz). The DNA sequence of the region encoding the TraI relaxase domain was determined by sequencing PCR product. PCR amplification used primers based on sequences conserved among F-like plasmids. Primer sequences are available upon request. DNA sequencing was performed by Eurofins MWG Operon using supplied primers. 3. Results and discussion

Fig. 3. Variant TraI36 proteins demonstrate altered specificity. Emission intensity data of a 3′ 22-base TAMRA-labeled oligonucleotide encoding the R100 oriT were followed during titration of wt R100 (blue circles), wt F (red squares), F-triple mutant (purple triangles), or F-triple mutant + M2L (green diamonds) TraI36 protein. Intensity data were adjusted for fluorophore dilution and normalized for initial intensity (arbitrary units) relative to each other. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.1. Binding of wild-type F and R100 TraI36, the F-triple mutant, and other F variants Binding affinities were measured by recording changes in total emission intensity of 3′-TAMRA-labeled oriT binding site oligonucleotides upon titration of protein; representative binding curves are shown in Fig. 3. Experimentally

determined dissociation constants (KD) of the wt and variant TraI36 proteins are listed in Table 1. Binding experiments that yielded μM KD values did not reach saturation, and therefore these KD values are approximations. Consistent with previous measurements, both F TraI36 (F oriT: 0.48 nM

K.E. Guja, J.F. Schildbach / Plasmid 80 (2015) 1–7

5

Table 1 Binding affinities of wt and variant TraI36 proteins for F and R100 oriT sequences. Dissociation constants for the binding of TraI36 proteins to ssDNA oligonucleotides including the F or R100 oriT sequence around nic. Listed values are averages and standard deviations of a minimum of 3 measurements, except the values for F-Triple mutant and the R100 affinity for F oriT, which were measured twice. An amino acid matrix illustrates the positions that differ from the F TraI36 sequence. Values preceded by a tilde are approximations based on binding curves that lack well-developed upper baselines. TraI36 protein

F factor (wt) R100 (wt) F-Triple mutant F-Triple + M2L F-Triple + I4F F-Triple + R198K F-Triple + Y190L F-Triple + M2L/I4F F-Triple + I4F/Y190L F-Triple + M2L/I4F/R198K F-Triple + M2L/I4F/Y190L F-Triple + M2L/I4F/A5S/R198K F-Triple + M2L/I4F/Y190L/R198K

KD ± SD (nM) for:

Amino acid at position: 2

4

5

153

190

193

198

201

R100 oriT

F oriT

M L

I F

A S

E D D D D D D D D D D D D

Y L

Q R R R R R R R R R R R R

R K

R Q Q Q Q Q Q Q Q Q Q Q Q

~3200 2.1 ± 0.1 75 ± 2.5 3.2 ± 0.21 ~1600 22 ± 5.5 560 ± 74 32 ± 2.6 150 ± 17 54 ± 13 26 ± 2.4 30 ± 4.1 18 ± 2.1

0.48 ± 0.02 ~3200 ~4700 ~3500 ~4400 ~2000 ~4300 ~4800 ~6600 ~3500 ~6600 ~2200 ~3400

L F

L L L L L

F F F F F F

S

KD; R100 oriT: ~3.2 μM KD) and R100 TraI36 (F oriT: ~3.2 μM KD; R100 oriT: 2.1 nM KD) bind their cognate oriT DNA sequences highly specifically (Harley and Schildbach, 2003; Harley et al., 2002; Stern and Schildbach, 2001). As shown previously, the F triple mutant has a clear binding preference for the R100 sequence, but its affinity falls short of the wt R100 protein (F oriT: ~4.7 μM KD; R100 oriT: 75 nM KD). We generated several F-background mutants having substitutions in addition to those in the F-triple mutant intending to improve affinity for the R100 oriT oligonucleotide sequence. The substitutions M2L, I4F, A5S and R198K were chosen because these residues are within 5 Å of the two bases that differ in the F and R100 oriT sequences, Gua145′ and Gua147′ (Fig. 2; Larkin et al., 2005). We expected A5S to be least likely to have a significant effect given its greater relative distance from the binding cleft. These substitutions (except A5S) were added singly to the F-triple mutant background. We found that two single substitutions increased affinity for the R100 site, and one substitution drastically reduced it. The more dramatic improvement, caused by adding the M2L substitution to the F-triple mutant, increased affinity for the R100 oriT binding site by over 20-fold, to approximately wt R100 affinity levels (3.2 nm K D ), while having little effect on affinity for the F oriT (~3.5 μM KD). This M2L substitution effectively completes the F to R100 specificity swap. The Met2 residue therefore plays a key role in determining the specificity difference observed between F and R100 TraI36. Met2 may directly interact with DNA bases because it is one of several residues that form a pocket into which Gua4 of the oriT DNA docks, as shown in Fig. 2 (Larkin et al., 2005). The Met2 residue may also affect specificity indirectly, through its proximity to Gln193, a residue that has been shown to contribute significantly to binding specificity (Harley and Schildbach, 2003). Like the M2L substitution, R198K in the F-triple mutant background increased affinity for the R100 site, although to a lesser extent (~3-fold). The variant F-triple + R198K bound to the R100 and F sites with KD values of 22 nM and ~2 μM, respectively. Although Arg198 is not a contact residue

L L L L

K

K K K

in the F TraI36:ssDNA crystal structure, it is proximal to residues Arg201 and His221, both of which contribute significantly to binding, suggesting that residues at 198 affect binding affinity indirectly (Harley and Schildbach, 2003; Larkin et al., 2005). In contrast to R198K, I4F in the F-triple mutant background decreased affinity for the R100 site ~20fold (to ~1.6 μM), while having no discernible effect on affinity for the F oriT sequence (KD ~ 4.4 μM). Next, several substitutions were combined and added to the F-triple mutant protein. The M2L/I4F combination increased affinity for the R100 oriT sequence more than 2-fold relative to the F-triple mutant (to KD = 32 nM) without substantially affecting affinity for the F oriT (KD ~ 4.8 μM). The combination of M2L/I4F/R198K in the F-triple mutant background caused only a slight increase in affinity for the R100 oriT sequence (to KD = 54 nM) and no apparent change in the affinity for the F oriT sequence (KD ~ 3.5 μM), relative to the F-triple mutant. Finally, the quadruple substitution M2L/I4F/A5S/R198K in the F-triple mutant background caused an increase in affinity for the R100 oriT of ~2-fold (KD = 30 nM), while the affinity for the F oriT sequence was unchanged. The I4F substitution in either the F-triple mutant background or the F-triple + M2L variant dramatically reduces binding. In the F TraI36 structure with bound oriT ssDNA, Ile4 side chain atoms are located within 4 Å of the Gua147′ base and DNA contact residue Q193 (Larkin et al., 2005). An I4F substitution might therefore reduce binding by disrupting the shape complementarity between protein and oriT DNA. In the F TraI36 structure, Ile4 also packs against DNA contact Y190 (Supplementary Fig. S1). Because R100 has a Leu190, we reasoned that the I4F substitution might be less disruptive in the context of a Y190L substitution. To test this hypothesis, we also engineered the Y190L substitution into the F-triple mutant + M2L/I4F background and other mutant backgrounds as well. The F-triple + Y190L variant showed more than 7-fold decreased affinity for the R100 site (KD = 560 nM), relative to the F-triple. However, when Y190L was added to the F-triple + I4F, binding affinity increased more than 10-fold relative

6

K.E. Guja, J.F. Schildbach / Plasmid 80 (2015) 1–7

to the F-triple + I4F, to a KD of 150 nM. Thus, Y190L largely compensates for the detrimental effects of I4F, suggesting an interaction between those two residues. Results from additional variants suggest the effect can be modulated by other interactions. When the Y190L substitution is added to the F-triple + M2L/I4F variant, binding affinity for the R100 oriT sequence remains essentially unchanged (32 nM vs. 25 nM KD). When, however, Y190L was added to the F-triple + M2L/I4F/R198K variant, binding affinity increased more than 2-fold (from KD =54 nM to KD = 18 nM). In all variant proteins studied, the M2L substitution improves binding to the R100 oriT sequence. In F TraI36, M2 is one of several residues that form a pocket into which Gua147′ of the F oriT sequence docks. This pocket participates in one of two knob-into-hole interactions that play a central role in determining the specificity of F TraI36 (Datta et al., 2003; Harley and Schildbach, 2003; Larkin et al., 2005). The role of the M2L substitution in improving binding to the R100 sequence is therefore not surprising. We also identified other substitutions that improved binding to the R100 oriT sequence in certain contexts (for example, R198K added to the F-triple variant and Y190L added to the F-triple + I4F variant). Interestingly, though, no combination tested produced a protein with a higher binding affinity than the F-triple + M2L. Thus, combinations of substitutions that made the variant more “R100-like” in amino acid sequence actually made the resulting proteins less “R100-like” in binding affinity. This result surprised us somewhat. Given earlier work, in which dramatic shifts in specificity between R100 and F relaxases could be caused by few amino acid changes, we naively assumed that amino acid substitutions would either contribute to the switch or would be neutral. 3.2. Evolutionary implications Earlier studies on the DNA binding function of the MATa1 homeodomain provide another example of myriad compensatory forces combining to direct DNA recognition events (Hart et al., 2002). Much like that of the MATa1 homeodomain, the evolution of TraI relaxases likely included several compensatory mutations outside of the binding cleft that relieved unfavorable interactions with residues that directly contact DNA bases. Prior work on Cre recombinase variants also underscores the importance of distal mutations that can disrupt shape complementarity with DNA bases and side-chain interactions with the phosphate backbone (Baldwin et al., 2003). The differing specificities of Cre recombinase variants may have evolved through a relaxation of specificity from intermediates that can bind either of two different LoxP sites (Santoro and Schultz, 2002). Over the past few years, several genomes and plasmids containing genes that encode F-like and R100-like relaxases have been sequenced. These sequences, however, do not provide a great deal of insight into how F-like and R100like relaxases may have diverged from a common precursor. Most oriT sequences of these plasmids include either the F sequence or the R100 sequence around nic. Relative to F and F-like relaxases, the R100-like relaxases all contain Asp153, Val160, Ser235, and several substitutions between residues 2 and 9 and between residues 186 and 207.

The oriT of P307 around nic differs from F and R100 each by one base, having the G147′A but not the G145′T substitution of R100 relative to F (Fig. 1; Goldner et al., 1987). In an effort to gain further insight into relaxase specificity, we sequenced the portion of the P307 traI gene that corresponds to the relaxase domain (Fig. 1). We found that despite the oriT sequence difference, the P307 TraI relaxase closely resembles F TraI. While P307 and R100 TraI share S235, P307 and F TraI are identical in key specificity-determining regions, including residues 2–9, 153, and 186–207. We demonstrated previously that the wt F TraI36 binds the P307 oriT sequence with >100-fold reduced affinity, and given its similar sequence, it is possible that P307 TraI binds a singlestranded oligonucleotide with its cognate oriT sequence with a KD ≥ 100 nM (Harley and Schildbach, 2003). We more recently have shown that while in general a reduced affinity of F TraI for an oriT sequence translates into reduced transfer efficiency for a plasmid carrying that sequence, the plasmid may still be transferred with >10% of the efficiency of the wt plasmid (Hekman et al., 2008). This level of transfer efficiency may be sufficient for the plasmid to be maintained long-term within, or distributed through, a bacterial population. Furthermore, based on the results presented here, it is quite possible that some of the observed sequence differences between F and P307 TraI contribute to the specificity and affinity of P307 TraI for its cognate oriT sequence. 4. Conclusions The relaxase domains of F and R100 plasmids share significant sequence homology yet show an exquisite level of binding specificity for the oriT of their cognate plasmids. The TraI36 domain from plasmid R100 has more than 90% amino acid sequence identity with F TraI36, and its oriT DNA binding site differs by only two of eleven bases. Despite this extensive sequence similarity, the F and R100 relaxase domains bind to their cognate oriT site three orders of magnitude more tightly than to the oriT site of the non-cognate plasmid. Previously, we demonstrated that the R100 TraI36 DNA-binding specificity could be swapped by making two amino acid substitutions in the DNA binding cleft. In contrast, three substitutions could make F TraI36 more “R100like”, but the specificity swap was incomplete. We have identified one additional amino acid substitution that completes the specificity swap from R100 to F. Interestingly, adding further substitutions from R100 to the F background was detrimental to binding instead of being neutral, indicating that their effects were influenced by their structural context. These results suggest that DNA binding by relaxases can be strongly influenced by amino acids that do not contact the DNA directly, and emphasize the complexity of evolution of relaxase binding sites and oriT sequences. Authors’ contributions KEG participated in the design of the study, carried out the experiments, analyzed the data and drafted the manuscript. JFS conceived of the study, participated in the design of the study, assisted in data analysis, and helped draft the

K.E. Guja, J.F. Schildbach / Plasmid 80 (2015) 1–7

manuscript. Both authors read and approved the final manuscript. Acknowledgments We thank Dr. Christopher Larkin and Dr. Lubomir Dostal for insightful discussions and helpful advice regarding the binding assays, as well as Dr. Miguel Garcia-Diaz for critical evaluation of the manuscript. This work was supported by National Institutes of Health grant GM61017 to J.F.S. with a National Institutes of Health research supplement award to K.E.G. Accession codes The sequence of the TraI relaxase domain from plasmid P307 has been deposited in GenBank under accession code KP990665. Conflict of interest The authors declare that they have no competing interests. Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.plasmid.2015.03.006. References Abdel-Monem, M., et al., 1983. Identification of Escherichia coli DNA helicase I as the traI gene product of the F sex factor. Proc. Natl. Acad. Sci. U.S.A. 80, 4659–4663. Baldwin, E.P., et al., 2003. A specificity switch in selected Cre recombinase variants is mediated by macromolecular plasticity and water. Chem. Biol. 10, 1085–1094. Cascales, E., Christie, P.J., 2003. The versatile bacterial type IV secretion systems. Nat. Rev. Microbiol. 1, 137–149. Christie, P.J., et al., 2005. Biogenesis, architecture, and function of bacterial type IV secretion systems. Annu. Rev. Microbiol. 59, 451–485. Datta, S., et al., 2003. Structural insights into single-stranded DNA binding and cleavage by F factor TraI. Structure (Camb.) 11, 1369–1379. Draper, O., et al., 2005. Site-specific recombinase and integrase activities of a conjugative relaxase in recipient cells. Proc. Natl. Acad. Sci. U.S.A. 102, 16385–16390. Fekete, R.A., Frost, L.S., 2000. Mobilization of chimeric oriT plasmids by F and R100-1: role of relaxosome formation in defining plasmid specificity. J. Bacteriol. 182, 4022–4027. Frost, L.S., et al., 1994. Analysis of the sequence and gene products of the transfer region of the F sex factor. Microbiol. Rev. 58, 162–210. Gogarten, J.P., Townsend, J.P., 2005. Horizontal gene transfer, genome innovation and evolution. Nat. Rev. Microbiol. 3, 679–687. Goldenfield, N., Woese, C., 2007. Biology’s next revolution. Nature 445, 369. Goldner, A., et al., 1987. The origin of transfer of P307. Plasmid 18, 76–83. Harley, M.J., Schildbach, J.F., 2003. Swapping single-stranded DNA sequence specificities of relaxases from conjugative plasmids F and R100. Proc. Natl. Acad. Sci. U.S.A. 100, 11243–11248. Harley, M.J., et al., 2002. R150A mutant of F TraI relaxase domain: reduced affinity and specificity for single-stranded DNA and altered fluorescence anisotropy of a bound labeled oligonucleotide. Biochemistry 41, 6460–6468. Hart, B., et al., 2002. Engineered improvements in DNA-binding function of the MATa1 homeodomain reveal structural changes involved in combinatorial control. J. Mol. Biol. 316, 247–256.

7

Hekman, K., et al., 2008. An intrastrand three-DNA-base interaction is a key specificity determinant of F transfer initiation and of F TraI relaxase DNA recognition and cleavage. Nucleic Acids Res. 36, 4565–4572. Inamoto, S., et al., 1991. Site- and strand-specific nicking in vitro at oriT by the traY-traI endonuclease of plasmid R100. J. Biol. Chem. 266, 10086–10092. Lang, S., et al., 2010. Molecular recognition determinants for type IV secretion of diverse families of conjugative relaxases. Mol. Microbiol. 78, 1539–1555. Larkin, C., et al., 2005. Inter- and intramolecular determinants of the specificity of single-stranded DNA binding and cleavage by the F factor relaxase. Structure (Camb.) 13, 1533–1544. Lawley, T.D., et al., 2003. F factor conjugation is a true type IV secretion system. FEMS Microbiol. Lett. 224, 1–15. Lessl, M., Lanka, E., 1994. Common mechanisms in bacterial conjugation and Ti-mediated T-DNA transfer to plant cells. Cell 77, 321–324. Llosa, M., et al., 1995. Nicking activity of TrwC directed against the origin of transfer of the IncW plasmid R388. J. Mol. Biol. 246, 54–62. Matson, S.W., Morton, B.S., 1991. Escherichia coli DNA helicase I catalyzes a site- and strand-specific nicking reaction at the F plasmid oriT. J. Biol. Chem. 266, 16232–16237. Matson, S.W., et al., 1993. Characterization of the reaction product of the oriT nicking reaction catalyzed by Escherichia coli DNA helicase I. J. Bacteriol. 175, 2599–2606. Pansegrau, W., et al., 1990. Covalent association of the traI gene product of plasmid RP4 with the 5′-terminal nucleotide at the relaxation nick site. J. Biol. Chem. 265, 10637–10644. Pansegrau, W., et al., 1993. Relaxase (TraI) of IncP alpha plasmid RP4 catalyzes a site-specific cleaving-joining reaction of single-stranded DNA. Proc. Natl. Acad. Sci. U.S.A. 90, 2925–2929. Parker, C., Meyer, R.J., 2007. The R1162 relaxase/primase contains two, type IV transport signals that require the small plasmid protein MobB. Mol. Microbiol. 66, 252–261. Reisner, A., et al., 2006. Synergistic effects in mixed Escherichia coli biofilms: conjugative plasmid transfer drives biofilm expansion. J. Bacteriol. 188, 3582–3588. Reygers, U., Wessel, R., Muller, H., Hoffman-Berling, H., 1991. Endonuclease activity of Escherichia coli DNA helicase I directed against the transfer origin of the F factor. EMBO J. 10, 2689–2694. Santoro, S.W., Schultz, P.G., 2002. Directed evolution of the site specificity of Cre recombinase. PNAS 99, 4185–4190. Sorensen, S.J., Bailey, M., Hansen, L.H., Kroer, N., Wuertz, S., 2005. Studying plasmid transfer in in situ: a critical review. Nat. Rev. Microbiol. 3, 700–710. Stern, J.C., Schildbach, J.F., 2001. DNA recognition by F Factor TraI36: highly sequence-specific binding of single-stranded DNA. Biochemistry 40, 11586–11595. Street, L.M., et al., 2003. Subdomain organization and catalytic residues of the F factor TraI relaxase domain. Biochim. Biophys. Acta 1646, 86–99. Thompson, R., et al., 1984. The F plasmid origin of transfer: DNA sequence of wild-type and mutant origins and location of origin-specific nicks. EMBO J. 3, 1175–1180. Traxler, B.A., Minkley, E.G., Jr., 1988. Evidence that DNA helicase I and oriT site-specific nicking are both functions of the F TraI protein. J. Mol. Biol. 204, 205–209. Waters, V.L., 2001. Conjugation between bacterial and mammalian cells. Nat. Genet. 29, 375–376. Willetts, N., Maule, J., 1979. Investigations of the F conjugation gene traI:traI mutants and lambdatraI transducing phages. Mol. Gen. Genet. 169, 325–336. Willetts, N., Maule, J., 1986. Specificities of IncF plasmid conjugation genes. Genet. Res. 47, 1–11. Williams, S.L., Schildbach, J.F., 2006. Examination of an inverted repeat within the F factor origin of transfer: context dependence of F TraI relaxase DNA specificity. Nucleic Acids Res. 34, 426–435. Wirth, T., et al., 2006. Sex and virulence in Escherichia coli: an evolutionary perspective. Mol. Microbiol. 60, 1136–1151. Zechner, E.L., et al., 2000. Conjugative-DNA transfer processes. In: Thomas, C.M. (Ed.), The Horizontal Gene Pool. Harwood Academic Publishers, Amsterdam.