Differential Single-stranded DNA Binding Properties of the Paralogous SsbA and SsbB Proteins from Streptococcus pneumoniae *

The naturally transformable Gram-positive bacterium Streptococcus pneumoniae has two single-stranded DNA-binding (SSB) proteins, designated SsbA and SsbB. The SsbA protein is similar in size to the well character-ized SSB protein from Escherichia coli (SsbEc). The SsbB protein, in contrast, is a smaller protein that is specifically induced during natural transformation and has no counterpart in E. coli . In this report, the single-stranded DNA (ssDNA) binding properties of the SsbA and SsbB proteins were examined and compared with those of the SsbEc protein. The ssDNA binding charac-teristics of the SsbA protein were similar to those of the SsbEc protein in every ssDNA binding assay used in this study. The SsbB protein differed from the SsbA and SsbEc proteins, however, both in its binding to short homopolymeric dT n oligomers (as judged by polyacryl- amide gel-shift assays) and in its binding to the longer naturally occurring (cid:1) X and M13 ssDNAs (as judged by agarose gel-shift assays and electron microscopic anal-ysis). The results indicate that an individual SsbB protein binds to ssDNA with an affinity that is similar or higher than that of the SsbA and SsbEc proteins. How-ever, the manner in which multiple SsbB proteins as-semble onto a ssDNA molecule differs from that observed with the SsbA

P-end-labeled dT n oligomers were prepared using [␥-32 P]ATP and polynucleotide kinase (Amersham Biosciences). Circular X ssDNA(ϩ strand) was from New England Biolabs. GelStar® nucleic acid gel stain was from Cambrex. G250 BioSafe Coomassie protein gel stain was from Bio-Rad. X ssDNA concentrations were determined by absorbance at 260 nm using the conversion factor 36 g ml Ϫ1 A 260 Ϫ1 . dT n concentrations were determined by absorbance at 260 nm using the extinction coefficient 8.4 mM Ϫ1 cm Ϫ1 (2). All ssDNA concentrations are expressed as total nucleotides.
Gel-exclusion Chromatography-Gel-exclusion chromatography analysis of the SSB proteins was carried out using a Superose 12-gelexclusion column (24 ml, Amersham Biosciences) with 20 mM Tris-Cl (pH 7.5), 5% glycerol, 100 mM NaCl, 1 mM EDTA, and 1 mM dithiothreitol. The SSB proteins were detected by absorbance at 280 nm, and the apparent molecular masses were determined by comparison with a standard curve that was developed using the following gel-exclusion calibration proteins: albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and ribonuclease A (13.7 kDa) (Amersham Biosciences).
Polyacrylamide Gel Electrophoresis Mobility Shift Assays-The reaction solutions contained 25 mM Tris acetate (pH 7.5), 10 mM Mg(acetate) 2 , 5% glycerol, 1 mM dithiothreitol, and the concentrations of dT n and SSB protein given in the figure legends. Reaction solutions were incubated at 37°C, as indicated in the figure legends. Aliquots (20 l) were removed from each reaction solution and added to 2 l of gelloading solution (0.25% bromphenol blue, 40% sucrose). The aliquots were analyzed by electrophoresis on a 5% native polyacrylamide gel using a Tris borate/EDTA buffer system (45 mM Tris borate (pH 8.5)/1 mM EDTA). Bands corresponding to unbound and SSB-bound dT n oligomers were visualized by autoradiography (these reactions contained 32 P-end-labeled dT n oligomers), and bands corresponding to unbound and dT n -bound SSB protein were visualized by G250 BioSafe Coomassie staining (these reactions contained only unlabeled dT n oligomers).
Agarose Gel Electrophoresis Mobility Shift Assays-The reaction solutions contained 25 mM Tris acetate (pH 7.5), 10 mM Mg(acetate) 2 , 5% glycerol, 1 mM dithiothreitol, and the concentrations of X ssDNA and SSB protein given in the figure legends. Reaction solutions were incubated at 37°C as indicated in the figure legends. Aliquots (20 l) were removed from each reaction solution and added to 2 l of gel-loading solution (0.25% bromphenol blue, 40% sucrose). The aliquots were analyzed by electrophoresis on a 0.8% agarose gel using a Tris borate/ EDTA buffer system (45 mM Tris borate (pH 8.5)/1 mM EDTA). Bands corresponding to unbound X ssDNA and the various SSB-X ssDNA complexes were visualized by GelStar® nucleic acid staining.
Electron Microscopy-The SsbA and SsbB proteins were incubated with X ssDNA in a buffer containing 20 mM Hepes (pH 7.6)/50 mM NaCl, at a protein to DNA ratio (g:g) of 20:1 in a total volume of 40 l, for 15 min at 37°C. The samples were loaded onto a 2-ml column of Bio-Gel A-50m (Agarose Bead Technologies, Tampa, FL) previously equilibrated in 10 mM Tris-Cl (pH 7.5)/0.5 mM EDTA. The same buffer was then used to elute the sample from the column, and 250-l fractions were collected. Aliquots of the protein-DNA-containing fractions were mixed with a buffer containing spermidine (6) for 3 s and quickly applied to a mesh copper grid coated with a thin carbon film, glowcharged shortly before sample application. Following adsorption of the samples to the electron microscopy support for 2-3 min, the grids were subjected to a dehydration procedure in which the water content of the washes was gently replaced by a serial increase in ethanol concentration to 100% and then air-dried. The samples were then rotary shadowcast with tungsten at 10 Ϫ7 torr and examined in a Tecnai G 2 TEM instrument at 40 kV. Images of the SsbA-X ssDNA and SsbB-X ssDNA complexes were captured using a Gatan ultrascan US4000SP digital camera, and panels were arranged using Adobe Photoshop. The estimates of the relative areas of the SsbA-X ssDNA and SsbB-X ssDNA complexes were derived from the electron microscopy images using the NIH Image software.

RESULTS
Oligomerization State of the SsbA and SsbB Proteins-The E. coli SSB protein (SsbEc) forms a stable tetramer in solution, and it is the tetrameric form of the protein that binds to ssDNA (2). Therefore, before examining the ssDNA binding properties, the oligomerization states of the S. pneumoniae SsbA and SsbB proteins were determined by gel-exclusion chromatography. In the gel-exclusion analysis, the SsbEc protein eluted as a single peak at a position corresponding to a protein with an apparent molecular mass of ϳ72,400 Da (chromatograph not shown). This is in agreement with the calculated value of 75,496 Da for the SsbEc tetramer (2). Under the same conditions, the SsbA and SsbB proteins also eluted as single peaks at positions corresponding to proteins with apparent molecular masses of ϳ63,300 and 54,100 Da, respectively (chromatographs not shown). These values are close to the calculated values of 69,400 and 59,704 Da for the SsbA and SsbB tetramers. These results indicate that the SsbA and SsbB proteins also form stable tetramers in solution, and it will therefore be presumed in the discussion below that it is the tetrameric form of these proteins that binds to ssDNA as well.
Binding of SSB Proteins to dT n Oligomers-The ssDNA binding activities of the SsbA and SsbB proteins were compared with those of the SsbEc protein using a series of dT n oligomers ranging in size from n ϭ 35-100. In these experiments, a fixed concentration of dT n was incubated with various concentrations of SSB protein, and the resulting complexes were analyzed by polyacrylamide gel electrophoresis.
dT 50 -The binding of the various SSB proteins to dT 50 is shown in Fig. 2. The amount of dT 50 that was bound to SsbEc protein increased with increasing protein concentration until all of the dT 50 was incorporated into an SsbEc-dT 50 complex. Increasing the SsbEc concentration further did not result in the formation of any additional complexes. A similar pattern of binding was observed when the SsbA and SsbB proteins were added to dT 50 (Fig. 2). Although the mobilities of the complexes that were formed with the various SSB proteins were different (presumably because of the difference in the sizes of the proteins), the dependence of complex formation on protein concentration was similar for the three proteins. These results indicated that each of the SSB proteins binds to dT 50 to form a complex in which a single SSB tetramer is bound to dT 50 . Similar results were obtained for all three SSB proteins with the shorter oligomer, dT 35 (data not shown).
To evaluate the relative binding efficiencies of the SsbA and SsbB proteins, a set of reactions was carried out in which dT 50 was incubated with a saturating concentration of either SsbA or SsbB protein alone or with a saturating concentration of each protein simultaneously. As shown in Fig. 3, either SSB protein alone was able to complex all of the dT 50 in the reaction solution. When dT 50 was incubated with both SsbA protein and SsbB protein simultaneously, bands corresponding to both the SsbA-dT 50 and the SsbB-dT 50 complex were observed. The in- tensity of the band corresponding to the SsbB-dT 50 complex appeared to be somewhat more intense than that for the SsbA-dT 50 complex, suggesting that the binding of the SsbB protein was more favorable under these reaction conditions. Furthermore, a similar distribution of the SsbA-dT 50 and SsbB-dT 50 complex bands was observed when dT 50 was incubated with a saturating concentration of SsbA protein prior to the addition of SsbB protein or when dT 50 was incubated with a saturating concentration of SsbB protein prior to the addition of SsbA protein (Fig. 3). These results indicated that even if dT 50 was fully complexed by one of the SSB proteins prior to the addition of the second SSB protein, the SsbA and SsbB proteins were able to exchange on dT 50 to generate a mixture containing both the SsbA-dT 50 and SsbB-dT 50 complexes.
An additional set of reactions was carried out to assess the binding efficiencies of the SsbA and SsbB proteins, relative to that of the SsbEc protein. As shown in Fig. 3, when a saturating concentration of both SsbA and SsbEc protein was added to dT 50 , bands corresponding to both the SsbA-dT 50 and SsbEc-dT 50 complex were observed. The intensity of the SsbA-dT 50 complex band was greater than that of the SsbEc-dT 50 complex band regardless of the order of addition of the two proteins, suggesting that the binding of the SsbA protein was more favorable under these reaction conditions. The difference was more pronounced when a saturating concentration of both SsbB and SsbEc protein was added to dT 50 . In this case, the intensity of the SsbB-dT 50 complex band was clearly greater than that of the SsbEc-dT 50 band regardless of the order of addition of the two proteins, indicating that the binding of the SsbB protein was more favorable under these reaction conditions (Fig. 3).
dT 75 -The binding of the various SSB proteins to dT 75 is shown in Fig. 4. The amount of dT 75 that was bound to SsbEc protein increased with increasing protein concentration until all of the dT 75 was incorporated into an SsbEc-dT 75 complex. In contrast to the results that were obtained with dT 50 , however, a further increase in the SsbEc protein concentration resulted in the disappearance of the initial complex and the appearance of a new complex of even lower gel mobility (Fig. 4). A similar pattern of binding was observed when SsbA protein was added to dT 75 (Fig. 4). These results indicated that a second tetramer of SsbEc or SsbA protein was able to bind to dT 75 at the higher protein concentrations. This conclusion is consistent with recent biophysical studies, which showed that the SsbEc protein forms a complex with one SsbEc tetramer bound to dT 70 at lower binding densities and a complex with two SsbEc tetramers bound to dT 70 at higher binding densities (7). By comparison, when increasing concentrations of SsbB protein were added to dT 75 , an initial complex was formed at the same protein concentration that was observed for the formation of the first complex with the SsbEc and SsbA proteins (Fig. 4). In contrast to the results with the SsbEc and SsbA proteins, however, increasing the SsbB protein concentration further did not result in the formation of any additional complexes (Fig. 4). These results indicated that only a single tetramer of the SsbB protein was able to bind to dT 75 , even at the highest protein concentrations examined. Similar results were obtained for all three SSB proteins with the shorter oligomer, dT 65 (data not shown).
To further evaluate the binding of the SSB proteins to dT 75 , an additional set of experiments was carried out in which the polyacrylamide gels were treated with Coomassie stain to indicate the relative amounts of SSB protein present in various complexes. As shown in Fig. 5, the staining intensities of the bands that were formed by the SsbEc and SsbA proteins were consistent with there being approximately twice as much protein in the second complex (complex II) as in the first complex (complex I). Moreover, SsbEc or SsbA protein concentrations above that necessary to convert all of the dT 75 into the second complex led to the appearance of a new staining band, which corresponded to excess unbound protein (Fig. 5). By comparison, concentrations of SsbB protein above that necessary to convert all of the dT 75 into the first complex (complex I) did not result in the appearance of a new complex band but did lead to the appearance of a new staining band that corresponded to excess unbound SsbB protein (Fig. 5). These results provide further support for the conclusion that two tetramers of either SsbA or SsbEc protein, but only one tetramer of SsbB protein, were able to bind to dT 75 .
dT 100 -The binding of the various SSB proteins to dT 100 is shown in Fig. 6. The amount of dT 100 that was bound to SsbB protein increased with increasing protein concentration until all of the dT 100 was incorporated into an SsbB-dT 100 complex. In contrast to the results with dT 50 and dT 75 , however, a further increase in the SsbB protein concentration resulted in the disappearance of the initial complex and the appearance of a new complex of even lower mobility (Fig. 6). This result indicated that a second tetramer of SsbB protein was able to bind to dT 100 at higher protein concentrations. By comparison, when increasing concentrations of SsbA protein were added to dT 100 , an initial complex and a second complex were formed at protein concentrations similar to those observed for the formation of the first and second complexes with the SsbB protein.
However, a further increase in SsbA protein concentration resulted in the disappearance of the second complex and the appearance of a third complex of even lower mobility (Fig. 6). A similar pattern of binding was observed when SsbEc protein was added to dT 100 (Fig. 6). These results suggested that three tetramers of SsbA or SsbEc protein, but only two tetramers of SsbB protein, were able to bind to dT 100 .
Binding of SSB Proteins to X ssDNA-The binding of the various SSB proteins to the long naturally occurring circular X ssDNA (5386 nucleotides) was also examined. In these experiments, a fixed concentration of X ssDNA was incubated with increasing concentrations of SSB protein, and the resulting complexes were analyzed by agarose gel electrophoresis.
When increasing concentrations of SsbEc protein were added to X ssDNA, there was a progressive decrease in the mobility of the X ssDNA until a discrete slower moving complex was observed at higher SsbEc protein concentrations (Fig. 7). A similar pattern of binding was observed when SsbA protein was added to X ssDNA (Fig. 7). These results are similar to agarose gel shift results that have been reported previously for the SsbEc protein (8) and reflect the binding of increasing amounts of SSB protein until the X ssDNA is saturated with SSB protein. In contrast, when increasing concentrations of SsbB protein were added to X ssDNA, there was an increase in the mobility of the ssDNA until a discrete faster moving complex was observed at higher SsbB protein concentrations (Fig. 7). In a parallel experiment, X ssDNA was first incubated with SsbB protein (to form the faster moving complex) and then treated with SDS (to remove the SsbB protein from the ssDNA) before loading onto the agarose gel. In this case, intact circular X ssDNA was recovered, demonstrating that the formation of the faster moving complex was not because of degradation of the X ssDNA (data not shown). In addition, when the agarose gels were treated with Coomassie stain, unbound SsbB protein was found to migrate more slowly than unbound SsbA protein (consistent with the calculated pI values of the SsbA protein (pI ϭ 5.2) and SsbB protein (pI ϭ 6.0)). This indicated that the rapid migration of the SsbB-X ssDNA complexes was not due simply to an increased mobility of the SsbB protein relative to that of the SsbA protein (data not shown). Furthermore, a similar pattern of results was obtained for all three SSB proteins when the agarose gels were run using a Tris acetate buffer system in place of the Tris borate buffer system, showing that the difference in binding patterns was not specific to the gel-running buffer (data not shown). Finally, a similar pattern of results was obtained for all three SSB proteins when circular M13 mp18 ssDNA (7249 nucleotides) was used in place of circular X ssDNA, demonstrating that the formation of the faster moving complex by the SsbB protein was not specific to X ssDNA (data not shown). Taken together, these results indicated that the complexes that were formed with X ssDNA by the SsbB protein were different from those formed by the SsbA and SsbEc proteins.
The complexes that were formed by the SsbA and SsbB proteins with X ssDNA were also examined by electron microscopy (Fig. 8). The results (obtained with saturating concentrations of the SSB proteins) showed that the SsbA protein formed open circular complexes in which the X ssDNA was almost completely covered by extended tracts of SsbA protein.
These complexes are similar to those that have been reported previously for the SsbEc protein (9, 10) and are consistent with the similar DNA binding patterns that were obtained for the SsbA and SsbEc proteins in the agarose gel-shift assays (Fig.  7). In contrast, the complexes that were formed by the SsbB protein were highly condensed in appearance and contained numerous stem-like projections that were not prominent in the SsbA complexes (Fig. 8). Furthermore, an analysis of the electron microscopy images revealed that the projected surface area of the SsbB-X ssDNA complexes was 30% smaller than that of the SsbA-X ssDNA complexes, indicating that there was less protein bound in the SsbB complexes than in the SsbA complexes. The condensed appearance of the SsbB-X ssDNA complexes is presumably related to the anomalous mobilities that were observed for these complexes in the agarose gel-shift assays (Fig. 7).
Because the SsbA and SsbB proteins appeared to form different types of complexes with X ssDNA, an additional set of experiments was carried out to examine the complexes that would be formed if SsbA and SsbB protein were added together to X ssDNA. In these experiments, X ssDNA was incubated with a saturating concentration of either SsbA protein or SsbB protein alone, or with a saturating concentration of each protein simultaneously. As shown in Fig. 9A, the SsbA and SsbB proteins alone were able to convert all of the X ssDNA in the reaction solution to the discrete slower or faster moving complexes, respectively. When X ssDNA was incubated with SsbA and SsbB protein simultaneously, however, a single discrete slower moving complex was observed with a mobility that was identical to that of the complex formed with the SsbA protein alone (Fig. 9A). This single slower moving complex was observed even when the X ssDNA was incubated with a saturating concentration of SsbA protein prior to the addition of SsbB protein, or when the X ssDNA was incubated with a saturating concentration of SsbB protein prior to the addition of SsbA protein (Fig. 9A). These results indicated that either the SsbA protein was binding preferentially over the SsbB protein to form a complex with the X ssDNA that contained only SsbA protein or that the SsbA and SsbB proteins were binding together to form a mixed SsbA/SsbB complex with the X ssDNA that had a mobility indistinguishable from that of the complex formed with SsbA protein alone.
To distinguish between these possibilities, X ssDNA was incubated with a saturating concentration of either SsbA protein or SsbB protein alone, or with a saturating concentration of each protein simultaneously. The resulting complexes were then isolated by preparative agarose gel electrophoresis and analyzed by SDS-polyacrylamide gel electrophoresis. As shown in Fig. 9B, the slower moving complex that was formed in the presence of a mixture of SsbA and SsbB protein contained approximately equal amounts of the two proteins. Furthermore, the intensities of the bands corresponding to the SsbA and SsbB proteins were approximately one-half of the intensities of the SsbA and SsbB bands that were obtained from the complexes formed with SsbA or SsbB protein alone (Fig. 9B). These results indicated that although the complex that was formed in the presence of a mixture of SsbA and SsbB protein had a gel mobility that was indistinguishable from the complex formed with SsbA protein alone, the complex did contain approximately equal amounts of SsbA and SsbB protein bound to the X ssDNA.

DISCUSSION
The polyacrylamide gel shift results indicated that the Ss-bEc, SsbA, and SsbB proteins each bind to the shorter oligomer, dT 50 , to form a complex in which a single SSB tetramer is bound to the ssDNA. Furthermore, the competition experiments indicated that the affinity of the SsbB protein for dT 50 is higher than that of either the SsbA or SsbEc protein. However, the gel shift results indicated that two SsbEc or SsbA tetramers are able to bind to the intermediate length oligomer, dT 75 , whereas only a single SsbB tetramer is able to bind to this ssDNA. Similarly, the gel shift results indicated that three tetramers of SsbEc or SsbA protein are able to bind to the longer oligomer, dT 100 , but only two SsbB tetramers are able to bind to this ssDNA. These results indicated that the SsbB protein interacts with ssDNA in a manner that is different from that of the SsbEc and SsbA proteins.
The binding of the SsbEc protein to ssDNA has been analyzed extensively (2). In general, at low DNA binding densities the SsbEc protein binds in the "(SSB) 65 mode" in which all four subunits of the tetramer interact with ssDNA (occluding ϳ65 nucleotides of ssDNA/tetramer). At high DNA binding densities, however, the SsbEc protein binds in the "(SSB) 35 mode " in which only two subunits of the tetramer interact with ssDNA (occluding ϳ35 nucleotides of ssDNA/tetramer). It cannot be determined from the gel-shift assays reported here whether the SsbEc and SsbA proteins are in the (SSB) 35 or (SSB) 65 mode when only a single SsbEc or SsbA tetramer is bound to dT 75 . However, the SsbEc and SsbA proteins presumably must be in the (SSB) 35 mode when two tetramers are bound to dT 75 (7). Similarly, the SsbEc and SsbA proteins presumably must be in the (SSB) 35 mode when three SsbEc or SsbA tetramers are bound to dT 100 . The finding that only one SsbB tetramer was able to bind to dT 75 and only two SsbB tetramers were able to bind to dT 100 suggests that either the SsbB protein is unable to bind to the dT n oligomers in an (SSB) 35 -like mode or that FIG. 6. Binding of SSB proteins to dT 100 . The reaction solutions contained 5.3 M dT 100 (total nucleotide concentration) and the indicated concentrations of SsbEc, SsbA, or SsbB protein (0 -1.0 M tetramer). The reaction solutions were incubated at 37°C for 15 min and then analyzed by polyacrylamide gel electrophoresis. The bands corresponding to unbound dT 100 (ssDNA) and the various SSB-dT 100 complexes were visualized by autoradiography. The minor bands that are visible below the bands for the first complex correspond to 20% of the total DNA and are apparently because of lower molecular weight contaminants in the dT 100 preparation.
multiple SsbB tetramers are unable to bind in close proximity on the same dT n molecule.
The agarose gel shift results indicated that the SsbB protein also forms complexes with circular X ssDNA that are different from those formed by the SsbA and SsbEc proteins. At saturating protein concentrations, the SsbA and SsbEc proteins each formed a discrete complex with X ssDNA that migrated more slowly than unbound X ssDNA during agarose gel electrophoresis. Electron microscopy showed that these slower moving SsbA complexes corresponded to open circular complexes in which the X ssDNA molecules were almost completely covered by SsbA protein. These open circular complexes were essentially identical to those that were reported previously for the SsbEc protein (8,9). In contrast, the SsbB protein formed complexes with X ssDNA that migrated more rapidly than unbound X ssDNA during agarose gel electrophoresis. Electron microscopy indicated that these faster moving complexes were more condensed than the open circular complexes that were formed when X ssDNA was covered with SsbEc or SsbA protein.
Unbound circular X ssDNA appears as a condensed bushlike structure when visualized by electron microscopy because of intrastrand DNA pairing interactions (9). The open circular appearance of the complexes that were formed by the SsbA and SsbEc proteins indicates that these proteins are largely able to destabilize and bind to the regions of the secondary structure in the X ssDNA. The condensed appearance of the complexes that were formed by the SsbB protein, in contrast, suggests that it may be less effective than the SsbA and SsbEc proteins in destabilizing and binding to regions of DNA secondary structure. It is not clear, however, whether this possibility would completely account for the anomalous mobilities of the SsbB-X ssDNA complexes that were observed by agarose gel electrophoresis; the complexes that were formed with X ssDNA at subsaturating concentrations of SsbEc and SsbA protein still migrated more slowly than unbound X ssDNA, whereas the complexes that were formed by the SsbB protein migrated more rapidly than unbound X ssDNA across the entire range of SsbB protein concentrations examined. An alternate possibility is that the SsbB protein may differ from the SsbA and SsbEc proteins by being able to interact with two spatially separated sites on the X ssDNA and this is responsible for the condensed appearance and anomalous electrophoretic mobility of the SsbB-X ssDNA complexes. Either of these possibilities would be consistent with the electron micrographs, which indicated that there was 30% less protein bound in the SsbB-X ssDNA complexes than in the SsbA-X ssDNA complexes, and with the results that demonstrated that the SsbA and SsbB proteins are able to bind together to X ssDNA to form a mixed SsbA/SsbB-X ssDNA complex. The determination of the physical basis for the unusual electrophoretic behavior of the SsbB-X ssDNA complexes will require further investigation.
A comparison of the primary sequences reveals that the N-terminal regions of the SsbA and SsbB proteins (amino acids 1-106) are highly similar in sequence to the corresponding region of the SsbEc protein (amino acids 1-115) (Fig. 1A). Structural studies have shown that this region of the SsbEc protein contains the subunit tetramerization and ssDNA binding sites (2). Thus, the observations that the SsbA and SsbB proteins form stable tetramers in solution and bind to shorter ssDNAs (dT 35 and dT 50 ) with efficiencies that are at least as high as that of the SsbEc protein are consistent with the high sequence similarity of the N-terminal domains of the three proteins. In addition to the core N-terminal DNA binding/ tetramerization domain, the SsbEc protein also has a C-terminal acidic tail (amino acids 166 -177), which is connected to the N-terminal core domain by a proline/glycine-rich spacer region (amino acids 116 -165). Although the SsbA and SsbB proteins also have C-terminal acidic tails, the SsbB protein differs from the SsbA and SsbEc proteins in having a much shorter spacer region between the acidic tail and the N-terminal core domain (Fig. 1A). A recent crystal structure shows that the entire C-terminal region (spacer plus tail) of the SsbEc protein is highly disordered and extends out laterally from the N-terminal core DNA binding domain of the protein (11). It is conceivable that the shorter spacer region of the SsbB protein may bring the acidic tail closer to the core DNA binding domain, and this may modulate the DNA binding properties of the SsbB protein, relative to those of the SsbEc and SsbA proteins. It is also possible that some other structural feature is responsible for the differences between the ssDNA binding properties of the SsbB protein and those of the SsbEc and SsbA proteins.
The close similarity of the ssDNA binding properties of the SsbA and SsbEc proteins is consistent with the SsbA protein being the S. pneumoniae analog of the SsbEc protein, a general purpose SSB protein involved in routine DNA functions. The recent reports that the expression of the SsbB protein is strongly induced during natural transformation, on the other hand, suggest that the SsbB protein may be a specialized SSB protein that plays a direct role in this process (3). During natural transformation, exogenous dsDNA binds to a DNA uptake site on the surface of the S. pneumoniae cell. One of the strands of the exogenous dsDNA is degraded by a cell surface nuclease, whereas the remaining complementary linear ssDNA is transported into the cell interior. The linear ssDNA is then incorporated into a homologous region of the double-stranded S. pneumoniae chromosome in a RecA-dependent process (1). It is conceivable that the role of the SsbB protein is to bind to the FIG. 7. Binding of SSB proteins to X ssDNA. The reaction solutions contained 5.0 M circular X ssDNA (total nucleotide concentration) and the indicated concentrations of SsbEc, SsbA, or SsbB protein (0 -0.3 M tetramer). The reaction solutions were incubated at 37°C for 15 min and then analyzed by agarose gel electrophoresis. The bands corresponding to the unbound X ssDNA (ssDNA) and the various SSB-X ssDNA complexes were visualized by GelStar® staining. The minor band that is visible in the 0 M SSB protein lanes is because of a small amount of linear X ssDNA that is present in the circular X ssDNA preparation. exogenous linear ssDNA (perhaps to protect it from cellular nucleases) as it is being transported into the cell interior. If this is the case, the particular ssDNA binding properties of the SsbB protein may reflect adaptations, which optimize the abil-ity of the SsbB protein to carry out this function. It is also possible that the SsbB protein acts as an accessory protein for various recombination proteins during the assimilation of the exogenous ssDNA into the S. pneumoniae chromosome. In this regard, several studies have indicated that the C-terminal region of the SsbEc protein may be involved in the interaction of the SsbEc protein with a host of other proteins that are involved in various aspects of DNA metabolism in E. coli. The similar length of the C-terminal region of the SsbA protein is consistent with the idea that the SsbA protein plays a role in S. pneumoniae that is analogous to that of the SsbEc protein in E. coli. The shorter C-terminal region of the SsbB protein, on the other hand, may not only modify the ssDNA binding properties, but may also alter the spectrum of protein-protein interactions that are available to the SsbB protein and enable it to function more effectively in conjunction with various recombination proteins during natural transformation. The ssDNA binding studies that are presented in this report, together with the recent isolation and characterization of other proteins that have been implicated in transformational recombination (12,13), will provide a foundation for further investigations into the biological roles of the paralogous SsbA and SsbB proteins.