M. pneumoniae strain MAC (ATCC^® no. 15492™) was cultured in Mycoplasma medium containing 1.4% Difco™ PPLO broth (Becton Dickinson), 0.15% Difco™ TC Yeastolate, UF (Becton Dickinson), 1.4% glucose, 20% horse serum, 1,000 U/ml Penicillin G, 500 U/ml Polymyxine B, 3 μg/ml Fungizone, 5 μg/ml Voriconazol and 0.02 mg/ml phenol red. The pH of the medium was adjusted to 7.8–8.0 using a solution of 2 N NaOH. Finally, the medium was filter-sterilized. Cells were harvested upon color change of the medium (from red/orange to yellow).

DNA was purified from cultures of M. pneumoniae as follows. Cultures (3 ml) were harvested by centrifugation for 10 min in a microcentrifuge at full speed. The cell pellet was resuspended in 1 ml 0.9% NaCl. After centrifugation, the cell pellet was resuspended in 400 μl of TE buffer (10 mM Tris-HCl, 1 mM EDTA pH 8.0). Then, 70 μl of 10% sodium dodecyl sulfate (SDS) and 5 μl of Proteinase K (10 mg/ml) was added. After incubation for 10 min at 65°C, 100 μl of 5 M NaCl was added, followed by the addition of 100 μl of a solution of 10% N-cetyl-N,N,N-trimethylammonium bromide (CTAB) in 0.7 M NaCl. The solution was incubated for 10 min at 65°C. The DNA was subsequently extracted from the solution using 500 μl of chloroform/isoamyl alcohol (24:1, vol:vol). The DNA in the upper, aqueous phase was transferred to a fresh tube and precipitated by the addition of 360 μl of isopropanol. Following incubation at -20°C for 30 min, the DNA was pelleted by centrifugation for 10 min at full speed in a microcentrifuge. The DNA pellet was washed with 70% ethanol, dried, and resuspended in 10 μl of H₂O. The MPN229 (or G07_orf166 3) ORF was amplified by PCR from the M. pneumoniae MAC genomic DNA. The PCR mixture (50 μl) contained 0.3 μM of oligonucleotide primer SSB_fw (5'-GGTCGTCATATGAACCGCGTTTTTTTATTTG-3'; the sequence in bold indicates a unique NdeI restriction endonuclease recognition site, which overlaps with the translation initiation codon [in italics] of MPN229), 0.3 μM of primer SSB_rv (5'-GCAGCCGGATCCTTATTCATCATCACTCTCCTC-3'; the sequence in bold indicates a unique BamHI restriction site, which is adjacent to the translation termination codon [anticodon in italics] of MPN229), 10 ng of M. pneumoniae genomic DNA, 0.2 mM of each dNTP, 1 unit of Pfu DNA polymerase (Fermentas), and 1 × Pfu buffer containing MgSO₄ (20 mM Tris-HCl [pH 8.8], 10 mM (NH₄)₂SO₄, 10 mM KCl, 0.1% Triton X-100, 0.1 mg/ml BSA, and 2 mM MgSO₄). PCR was performed using the following conditions: 3 min at 95°C, followed by 30 cycles of 30 sec at 95°C, 30 sec at 50°C, and 2 min at 72°C. The resulting 522-base pairs (bp) PCR fragment was cloned into the pJET1/blunt cloning vector (Fermentas) using the GeneJET™ PCR cloning kit (Fermentas). From the generated plasmid, pJET1-MpnSSB, the MPN229 ORF was excised by digestion with NdeI and BamHI, and cloned into NdeI- and BamHI-digested expression vectors pET-11c and pET-16b (Novagen), resulting in plasmid pET-11c-MpnSSB and pET-16b-MpnSSB, respectively. In plasmid pET-11c-MpnSSB, the MPN229 ORF is cloned such as to express Mpn SSB in its natural, non-tagged form. In plasmid pET-16b-MpnSSB, the MPN229 ORF is fused at its 5' end to a sequence encoding a polyhistidine tag (MG(H)₁₀SSGHIEGRH). This polyhistidine sequence allows one-step affinity purification of the MPN229-encoded protein by Ni²⁺-affinity chromatography (see below). For the generation of a glutathione S-transferase (GST)-Mpn SSB fusion protein expression construct, the NdeI-BamHI fragment containing MPN229 was first made blunt-ended by incubation with dNTPs and 'Klenow fragment' (Fermentas) and then cloned into SmaI-digested and Shrimp alkaline phosphatase (Promega)-treated vector pRP265 (NCCB Plasmids database, element No. PC-V3271; http://www.cbs.knaw.nl/scripts/Plasmids.dll/ShowPlasmid?Nr=3271). Plasmid pRP265 is a derivative of the expression plasmid pGEX-2T in which the polylinker 5'-GGATCCCCGGGAATTC-3' has been replaced by the sequence 5'-GGATCCCCATGGTACCCGGGTCGACTAGTATGCATAAGCTTGAATTC-3'. The resulting plasmid was termed pRP265-MpnSSB. Vector pRP265 was used for the production of native GST. The integrity of all DNA constructs used in this study was verified by DNA sequencing.

Constructs pET-11c-MpnSSB, pET-16b-MpnSSB, pRP265-MpnSSB and pRP265 were introduced into E. coli BL21(DE3) and the resulting strains were grown overnight at 37°C in LB medium containing 100 μg/ml ampicillin. The cultures were diluted 1:100 in 100–150 ml LB medium with ampicillin and grown at 37°C to an optical density at 600 nm of 0.6. Protein expression was then induced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.3 mM. After 3 hr, the bacteria were harvested by centrifugation and stored at -20°C.

Non-tagged Mpn SSB was purifed as follows. Frozen, bacterial pellets corresponding to 100 ml of culture were resuspended in 5 ml of buffer A (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT). The suspension was sonicated on ice and clarified by centrifugation for 20 min at 12,000 × g (4°C). To the supernatant, 5 ml of saturated ammonium sulphate was added, followed by 15 min of incubation on ice. After centrifugation for 10 min at 10,000 × g (4°C), the protein pellet, which contained the Mpn SSB protein, was dissolved in 10 ml of buffer A. Subsequently, 1 ml of a 50% suspension of DNA-cellulose (Worthington biochemical corp., Lakewood NJ), equilibrated in buffer A, was added, and the suspension was left on a rotating mixer for 1 hour at 4°C. The DNA-cellulose was poured into a column, and protein was eluted stepwise by the addition of 0.5 ml of a buffer (20 mM Tris-HCl pH 7.5, 1 mM DTT) containing increasing concentrations of NaCl (from 200 to 1500 mM). The fractions containing Mpn SSB were pooled and dialyzed against 20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1% Tween 20, 50% glycerol. The protein was stored at -20°C.

The polyhistidine-tagged Mpn SSB protein (Mpn H₁₀-SSB) was purified as follows. Bacterial pellets corresponding to 150 ml of culture were resuspended in 10 ml of buffer B (20 mM Tris-HCl pH 8.0, 0.3 M NaCl) plus 5 mM imidazole. The suspension was sonicated on ice and clarified by centrifugation for 20 min at 12,000 × g (4°C). To the supernatant, 1 ml was added of a 50% slurry of Ni²⁺-nitroloacetic acid (Ni-NTA)-agarose (Qiagen), equilibrated previously in buffer B containing 5 mM imidazole. The suspension was stirred for 1 hr at 4°C and subsequently poured into a column. Non-specifically bound proteins were washed from the Ni-NTA agarose by two subsequent washes (of 4 ml each) with buffer B containing 5 mM, 10 mM and 20 mM imidazole, respectively. The bound Mpn SSB protein was eluted from the column with 2.5 ml of buffer B containing 250 mM imidazole. Fractions of 0.5 ml were collected, analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), pooled, and dialyzed against buffer B plus 1 mM DTT and 50% glycerol. Aliquots of purified protein were stored at -20°C.

Both GST and GST-tagged Mpn SSB protein (GST-SSB) were purified as follows. Bacterial pellets corresponding to 100 ml of culture were resuspended on ice in 5 ml of PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.3) and sonicated. Then, Triton X-100 was added to a final concentration of 1%, and the suspension was left on a rotating mixer for 30 min at 4°C. Subsequently, the suspension was clarified by centrifugation for 20 min at 10,000 × g (4°C). To the supernatant, 0.8 ml was added of a 50% slurry of Glutathione-Agarose (Sigma, Saint Louis, MO, USA), equilibrated previously in PBS. The suspension was incubated on a rotating mixer for 30 min at 4°C and subsequently poured into a column. The column was washed consecutively with 3 ml of PBS containing 1% Triton X-100 and with 5 ml of PBS. Specifically bound protein was eluted from the column by addition of 3 ml of 50 mM Tris-HCl pH 8.0 containing 10 mM reduced glutathione (Sigma). Fractions of 0.5 ml were collected, analyzed by SDS-PAGE, pooled, and dialyzed against 20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1% Tween 20, 50% glycerol. Aliquots of purified protein were stored at -20°C. Protein concentrations were determined using the BC Assay protein quantitation kit (Interchim) as well as by SDS-PAGE analysis in conjunction with bovine serum albumin standards. All purified proteins were obtained at concentrations of at least 1 mg/ml, having an estimated homogeneity of 95% or greater. The concentrations of all proteins used in this study refer to monomeric protein concentrations throughout the manuscript.

Proteins were separated on SDS-polyacrylamide gels, essentially as described by Laemmli 13. Following electrophoresis, gels were stained with Coomassie brilliant blue (CBB), destained in 40% methanol/10% acetic acid, and photographed using a GelDoc XR system (Bio-Rad). Digital images were processed using Quantity One^® 1-D Analysis Software (Bio-Rad).

Gel filtration chromatography was carried out by applying a sample of 500 μl of Mpn SSB (at 0.3 mg/ml in 50 mM Tris-HCl (pH 7.5), 135 mM NaCl) to a Sephadex G-150 column with a length of 1 m and an inner diameter of 1.0 cm. The column was eluted with 50 mM Tris-HCl (pH 7.5), 135 mM NaCl at an elution rate of 4 ml/h. The column was calibrated with bovine serum albumin (BSA, 66.4 kDa), ovalbumin (42.9 kDa), and cytochrome C (12.3 kDa). Void volume was determined with blue dextran (2,000 kDa). Fractions of 1.0 ml were collected and analyzed by measuring the optical density at 280 nm (OD₂₈₀). Since a relatively low concentration of Mpn SSB was loaded on the column, the eluted fractions were precipitated with trichloroacetic acid, and separated on 14% SDS-PAGE gels. Gels were stained with CBB and recorded using the GelDoc XR system (Bio-Rad). In fractions that contained Mpn SSB, the amount of protein was determined semi-quantitatively using BioNumerics Version 3.0 software (Applied Maths).

The sequences of the oligonucleotide substrates that were used in this study are listed in Table 1. Oligonucleotides were purchased from Eurogentec and were used either unlabeled or 5'-end labeled with ³²P. Bacteriophage M13mp18 circular, single-stranded DNA was purified from cultures of infected E. coli XL-Blue cells (Stratagene), essentially as described previously 14. Bacteriophage φX174 RF1 DNA was purchased from Fermentas. The DNA was linearized by digestion with PstI (Invitrogen), followed by inactivation of the restriction enzyme by incubation for 20 min at 80°C. Viron DNA from φX174 was purchased from New England Biolabs. The concentration of bacteriophage-derived DNAs was determined using the NanoDrop 1000 (Thermo Scientific). The DNA concentrations are indicated for DNA molecules and not for nucleotides for all substrates throughout the manuscript.

DNA-binding experiments were carried out with either oligonucleotide substrates (Table 1), bacteriophage M13mp18 DNA, or bacteriophage φX174 DNA (both single- and double-stranded). Reactions were performed in volumes of 20 μl with various concentrations of purified protein in 25 mM Tris-OAc pH 7.5, 10 mM Mg(OAc)₂, 1 mM DTT, 5% glycerol, and either a single-stranded (ss) unlabeled oligonucleotide (5 μM), 5'-³²P-labeled oligonucleotide (1 μM), double-stranded (ds) supercoiled bacteriophage DNA (M13mp18 or φX174 DNA) (1 nM), or ss bacteriophage DNA (M13mp18 or φX174 DNA) (2 nM). After incubation for 15 min at 37°C, 2 μl was added of a solution containing 40% glycerol and 0.25% bromophenol blue. Subsequently, the samples were electrophoresed in 0.5 × TBE buffer, either through 1.0% agarose gels (when using unlabeled oligonucleotides), 0.6% agarose gels (when using bacteriophage DNA) or 5% polyacrylamide gels (when using ³²P-labeled oligonucleotides). Agarose gels were stained with ethidium bromide and photographed. The polyacrylamide gels were subjected to autoradiography.

E. coli RecA-promoted DNA strand transfer reactions were carried out using φX174 DNA, in a similar fashion as described previously 1516. Reactions (30 μl) contained 70 mM Tris-HCl pH 7.6, 10 mM MgCl₂, 5 mM DTT, 5 mM ATP, 0.08 U/μl Pyruvate Kinase Preparation (Type VII, from rabbit muscle [Sigma]), 3.2 mM phospho(enol)pyruvic acid (Sigma), 1 nM ss circular φX174 DNA, 2 nM PstI-digested, ds φX174 DNA, 0.2 μg/μl E. coli Rec A (New England Biolabs), and 3 ng/μl of either E. coli SSB (Epicentre Biotechnologies), or 6.5 ng/μl of either Mpn SSB, GST-SSB or GST. Samples of 10 μl were taken at 0, 30 and 60 min of incubation at 37°C, and reactions were terminated by the addition of 1 μl of a solution of 5% SDS and 50 mM EDTA pH 8.0, followed by incubation for 10 min at 55°C. After the addition of loading dye, the samples were separated on 0.6% agarose gels in 0.5 × TBE. The gels were stained with ethidium bromide and photographed.

The M. pneumoniae MPN229 ORF was recognized as an ORF putatively encoding a homolog of single-stranded DNA binding proteins after determination of the complete genome sequence of M. pneumoniae strain M129 3. MPN229 has the capacity to encode a 166-amino acid protein (termed Mpn SSB) with a theoretical molecular mass of 18.4 kDa. The predicted amino acid sequence of Mpn SSB displays only limited similarity with the protein encoded by the SSB gene from E. coli (17% identity). A significantly higher similarity was seen with the protein that is putatively encoded by the MG091 gene 17 from Mycoplasma genitalium (61% identity). The residues that are shared among the predicted amino acid sequences of the (putative) SSB proteins from M. pneumoniae, M. genitalium, Ureaplasma parvum (all belonging to the family of Mycoplasmataceae) as well as from E. coli, are depicted in a multiple sequence alignment in Fig. 1. Most bacterial SSB proteins that have been studied to date possess a so-called oligonucleotide/oligosaccharide-binding (OB) fold and function as homotetramers 1819. By contrast, the SSB proteins from Thermus aquaticus, Thermus thermophilus 20 and Deinococcus radiodurans 21, which have a significantly higher molecular weight than the E. coli (-like) SSB protein(s), were reported to contain two OB folds per monomer and function as homodimers. As shown in Fig. 1, Mpn SSB, which is 12 amino acids smaller than the 178-amino acid E. coli SSB, may contain a single OB fold. The amino acid motifs that are typical of the OB fold are indicated by the open boxes below the aligned sequences in Fig. 1.

The C-terminus of the Mpn SSB sequence contains a high proportion of acidic amino acid residues: 13 of the 24 C-terminal amino acids are aspartic acid (D) or glutamic acid (E) residues. Albeit less extensive, acidic residues are also concentrated near the C-terminus of other SSB proteins. In E. coli, these residues have been demonstrated to be involved in the interaction of SSB with other proteins, such as exonuclease I, uracil DNA glycosylase, and DNA polymerase III 222324252627282930. Several residues of the E. coli SSB protein have been demonstrated to play a role in DNA-binding. These include three tryptophan (W) residues at position 41 (adjacent to OB box '2' in Fig. 1), 55 (within box '3') and 89 (adjacent to box '45₁') 31. However, none of these residues appear to be conserved with the SSB proteins from the Mycoplasmataceae. Only W55 seems to have a clear counterpart in the mycoplasma proteins, i.e. a tyrosine (Y) residue in M. pneumoniae (Y44) and a phenylalanine (F) in both M. genitalium (F44) and U. parvum (F43). Other residues that were found to be implicated in E. coli SSB DNA-binding, however, do have a counterpart in the mycoplasma SSBs. The arginine (R) and lysine (K) residues at positions 57 and 63, respectively, of E. coli SSB, which were shown to be oriented toward the DNA binding cleft 32, are conserved in the M. pneumoniae and M. genitalium SSB proteins (R46 and K52, respectively, in both proteins). These residues are located in the predicted OB fold motifs '3' and 'α' (Fig. 1). Interestingly, the histidine (H) residue that was described to be important for tetramerization of E. coli SSB (H56, central to OB fold box '3') 33 is not conserved among the proteins listed in Fig. 1; an isoleucine (I) is present at a position congruent to that of H56 in the mycoplasma proteins (residue I45 in M. pneumoniae SSB). The lack of conservation of this apparently crucial amino acid residue, however, can be reconciled with the finding that substitution of residue H56 of E. coli SSB for either an F or I residue, did not change the properties of the protein measurably 33.

The MPN229 gene was cloned into different expression vectors in order to express Mpn SSB either in its native form (Mpn SSB), as an N-terminal polyhistidine-tagged polypeptide (Mpn H₁₀-SSB) or as a glutathione S-transferase (GST)-tagged fusion protein (GST-SSB). Each of these proteins was expressed to high levels in E. coli and was purified to near homogeneity (Fig. 2A). The estimated molecular weights of the purified proteins corresponded to their theoretical molecular weights of 18.4 kDa, 20.9 kD and 45.0 kDa for Mpn SSB, Mpn H₁₀-SSB, and GST-SSB, respectively. As a control, we also purified non-fused GST (Fig. 2A, lane 5). While most experiments were performed using Mpn H₁₀-SSB, the activities of the three different SSB variants were found to be indistinguishable.

In order to determine the oligomeric state of Mpn SSB in solution, the untagged, purified protein was subjected to size-exclusion chromatography. As shown in Fig. 2B, Mpn SSB eluted from the column as a single, major protein species with an estimated molecular weight of ~75 kDa. This molecular weight corresponds to the theoretical molecular weight of the tetrameric form of Mpn SSB (73.6 kDa). These data therefore suggests that Mpn SSB exists predominantly as a homo-tetramer in solution. A similar oligomeric composition has previously also been found for SSB proteins from other bacterial species, such as E. coli 32.

To investigate the potential of Mpn H₁₀-SSB to bind DNA, the protein was incubated at various concentrations with three different single-stranded oligonucleotide (ssDNA) substrates. The resulting complexes were analyzed by non-denaturing agarose gel electrophoresis, followed by staining with ethidium bromide. As shown in Fig. 3A, an increasing amount of product with a lower mobility than that of the free oligonucleotides was generated with increasing Mpn H₁₀-SSB concentrations. At the highest protein concentration tested (9.4 μM; lanes 5, 9 and 13), virtually all of the ssDNA formed part of the lower mobility complex, which was assumed to represent Mpn H₁₀-SSB-ssDNA complexes. DNA-binding by Mpn H₁₀-SSB was found to be rapid, as the DNA-protein complexes shown in Fig. 3A were already produced in less than 1 min of incubation at 37°C (data not shown). Similar results were obtained with the other recombinant SSB proteins (data not shown).

Because protein-DNA complexes were generated with each of the three different ssDNA substrates tested, we hypothesized that Mpn SSB may bind ssDNA in a sequence-independent manner. To further investigate this, we tested the ability of Mpn H₁₀-SSB to bind ³²P-labeled homooligomers (50-mers) of dA, dT, dC and dG (Table 1). Although the (T)₅₀ substrate seemed to be bound most efficiently, each of the homooligomers was bound by the protein (Fig. 3B). The (C)₅₀ substrate, despite being labeled inefficiently, most likely due to the formation of inaccessible secondary structures, was also bound by Mpn H₁₀-SSB. These data show that the protein indeed binds ssDNA irrespective of the DNA substrate sequence. It is interesting to note that binding of Mpn H₁₀-SSB to the 50-nucleotide homooligomer substrates resulted in the formation of two different DNA-protein complexes (Fig. 3B). As the formation of the larger of the two complexes was favored at higher protein concentrations, it is likely that this larger species represents DNA-protein complexes in which a higher number of SSB DNA-binding units (eg., dimers or tetramers) are present than in the smaller species. Thus, a 50-mer DNA substrate may be bound by a single binding unit at relatively low protein concentrations, whereas two or more binding units may occupy the substrate at higher protein concentrations. To study this further, we tested binding of Mpn H₁₀-SSB to a series of oligonucleotides with lengths ranging from 15 to 50 nucleotides, and each containing the same 15-nucleotide 'core sequence', which was randomly chosen (Table 1). As shown in Fig. 3C, only the 15-nucleotide substrate was bound inefficiently by the protein, whereas the other substrates were bound efficiently, each being completely complexed at higher protein concentrations. Interestingly, a second, larger DNA-protein complex was seen exclusively with substrates with a length equal to or larger than 30 nucleotides. The efficiency of formation of these larger complexes was even higher with the 40- or 50-mer substrates (lanes 14–20). Thus, whereas a 20-mer substrate may accommodate binding of a single SSB DNA-binding unit, longer substrates may allow binding of two or more units, generating protein-DNA complexes with a lower electrophoretic mobility than do single unit-bound complexes.

To investigate whether Mpn SSB binds with similar affinities to 20- and 50-mer substrates, a DNA-binding competition experiment was performed in which Mpn H₁₀-SSB was incubated with a constant amount of radiolabeled oligonucleotide and increasing amounts of the other, unlabeled oligonucleotide (Fig. 3D). Complexes with 20-mer substrates were no longer observed when a 5-fold molar excess of the 50-mer substrate was present in the DNA-binding reaction (compare lanes 2 and 3). By contrast, the formation of complexes with the 50-mer substrate was only partially inhibited in the presence of a 20-100-fold excess of unlabeled 20-mer oligonucleotide (Fig. 3D, lanes 11–13). These results indicate that Mpn H₁₀-SSB binds with a higher affinity to the 50-mer substrate than to the 20-mer substrate.

To study the ability of Mpn SSB to also bind dsDNA, the protein was incubated with either single- or double-stranded φX174 DNA (Fig. 4A). While almost all ssDNA was shifted towards a lower mobility form at the lowest concentration of Mpn SSB tested (1.3 μM; lane 10), significant binding to dsDNA could not be observed at any protein concentration used (lanes 14–16). These results demonstrate that Mpn SSB preferentially binds ssDNA. Fig. 4A further shows that the gel mobility of Mpn SSB-ssDNA complexes becomes lower when Mpn SSB concentrations are increased (lanes 10–12). Similarly as described above for the oligonucleotide substrates, it is likely that increasing numbers of Mpn SSB molecules accumulate on the ssDNA substrates when higher concentrations of protein are present. Consequently, the size of the DNA-protein complexes will increase when the protein concentration becomes higher, resulting in lowering of the gel mobility of these complexes. Similar DNA-binding results were obtained for each of the purified, recombinant Mpn SSB proteins, including GST-SSB (Fig. 4B, lanes 7–11), as well as for E. coli SSB (Fig. 4A, lanes 2–4). Interestingly, the E. coli SSB protein may assemble on DNA substrates in a different fashion than does Mpn SSB, as more heterogeneously sized protein-DNA complexes were formed by the E. coli protein than by the M. pneumoniae protein, in particular at relatively low protein concentrations (compare lanes 2 and 10 in Fig. 4A).

To determine whether Mpn SSB is dependent upon divalent cations for its activity, a DNA-binding experiment was conducted in either the presence or absence of Mg²⁺. Fig. 4C shows that similar DNA-protein complexes are generated irrespective of the presence of Mg²⁺ in the binding reaction (compare lanes 3–5 to lanes 7–9), which indicates that the protein does not require divalent cations for DNA-binding. Similarly, the SSB proteins from E. coli and S. pneumoniae were previously also shown to bind DNA irrespective of the presence of Mg²⁺ in the binding reaction 2234. However, these proteins do display different binding modes in either the presence or absence of divalent cations 2234.

Single-stranded DNA-binding proteins from several micro-organisms have previously been shown to stimulate in vitro DNA strand exchange reactions catalyzed by (homologs of) RecA. Also, the SSB proteins from S. pneumoniae and D. radiodurans were found to stimulate the activity of RecA from E. coli 1621. To study the potential of Mpn SSB to stimulate E. coli RecA-catalyzed DNA recombination, so-called three-strand exchange reactions were performed. In these reactions, RecA promotes the transfer of one of the strands of a linear, dsDNA molecule (donor) to a complementary, circular ssDNA molecule (acceptor), resulting in a double-stranded, circular product and a linear, single-stranded product (Fig. 5A).

Either alone (data not shown) or in the presence of GST (Fig. 5B, lanes 7–9), E. coli RecA did not detectably catalyze φX174 DNA strand exchange. However, in the presence of E. coli SSB, strand transfer was readily detectable after 60 min of incubation at 37°C (lane 6), while a faint DNA species at the position of the double-stranded, open circular recombination product was already visible after 30 min (lane 5). Similar results were obtained when E. coli SSB was replaced by either Mpn SSB (lanes 13–15), Mpn H₁₀-SSB (data not shown) or GST-SSB (lanes 10–12), albeit that the latter protein was somewhat less efficient than the other recombinant proteins in stimulating RecA activity.

Taken together, these data show that Mpn SSB, despite having a relatively low level of sequence similarity with E. coli SSB, is capable of stimulating DNA strand transfer activity of E. coli RecA. Consequently, Mpn SSB may play a similar, crucial role as E. coli SSB in DNA recombination processes. The elucidation of the activities of Mpn SSB can therefore be considered an important step in unraveling the recombinatorial events that may occur in the genome of M. pneumoniae, and which may play a pivotal role in antigenic variation of this bacterium.