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Originally published In Press as doi:10.1074/jbc.M414057200 on January 12, 2005

J. Biol. Chem., Vol. 280, Issue 12, 11067-11073, March 25, 2005
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Differential Single-stranded DNA Binding Properties of the Paralogous SsbA and SsbB Proteins from Streptococcus pneumoniae*

Diane E. Grove{ddagger}, Smaranda Willcox§, Jack D. Griffith§, and Floyd R. Bryant{ddagger}

From the {ddagger}Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205 and the §Lineberger Comprehensive Cancer Center and Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, North Carolina 27599

Received for publication, December 14, 2004 , and in revised form, January 10, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 characterized 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 characteristics 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 dTn oligomers (as judged by polyacrylamide gel-shift assays) and in its binding to the longer naturally occurring {phi}X and M13 ssDNAs (as judged by agarose gel-shift assays and electron microscopic analysis). 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. However, the manner in which multiple SsbB proteins assemble onto a ssDNA molecule differs from that observed with the SsbA and SsbEc proteins. These results represent the first analysis of paralogous SSB proteins from any bacterial species and provide a foundation for further investigations into the biological roles of these proteins.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Streptococcus pneumoniae is a naturally transformable Gram-positive bacterium that is able to take up DNA from its environment and incorporate this exogenous DNA into its chromosome (1). This process, known as transformational recombination, serves as a general mutational mechanism that allows S. pneumoniae to change its genetic composition in response to environmental changes and stresses (1). The transformational recombination reaction involves the assimilation of a single-stranded form of the exogenous DNA into a homologous region of the double-stranded S. pneumoniae chromosome (1). An inspection of the genome sequence reveals that S. pneumoniae has two single-stranded DNA-binding (SSB) proteins, designated SsbA and SsbB (see Fig. 1A). The SsbA protein (17,350 Da, 156 amino acids) is similar in size and sequence to the extensively studied SSB protein from Escherichia coli (18,874 Da, 178 amino acids), a non-sequence-specific ssDNA1-binding protein that is involved in many aspects of DNA metabolism in E. coli (2). The SsbB protein (14,926 Da, 131 amino acids), in contrast, is a smaller protein that is specifically induced during natural transformation in S. pneumoniae (3). These results suggest that the SsbA protein may be a general SSB protein involved in routine DNA functions (analogous to the E. coli SSB protein), and that the SsbB protein may be a specialized SSB protein used primarily during transformational recombination. This idea is consistent with a recent analysis of the genome sequences of 69 different bacterial species, which revealed that those naturally transformable Gram-positive bacteria that are closely related to S. pneumoniae (e.g. Bacillus subtilis) contain two ssb-like genes, whereas the non-naturally transformable Gram-negative bacteria related to E. coli (e.g. Salmonella typhimurium) contain only a single ssb gene (4).



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  		FIG. 1.
S. pneumoniae SsbA and SsbB proteins. A, the amino acid sequence of the S. pneumoniae SsbA and SsbB proteins are aligned with that of the E. coli SSB protein (SsbEc). Identical residues are highlighted in black. B, SDS-polyacrylamide gel electrophoresis of purified S. pneumoniae SsbA and SsbB protein. The gel lanes contain purified S. pneumoniae SsbA protein, S. pneumoniae SsbB protein, E. coli SSB protein, and molecular mass standards (M), as indicated. The acrylamide concentration was 5% in the stacking gel and 13% in the separating gel. The gel was stained with 0.1% Coomassie Brilliant Blue R-250.

 

We have recently amplified the ssbA and ssbB genes from S. pneumoniae genomic DNA, developed efficient expression systems, and purified the S. pneumoniae SsbA and SsbB proteins to near homogeneity (Fig. 1B). In this report, the ssDNA binding properties of the S. pneumoniae SsbA and SsbB proteins are examined and compared with those of the E. coli SSB protein. The results represent the first analysis of paralogous SSB proteins from any bacterial species.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—S. pneumoniae SsbA protein was prepared as described (5). The S. pneumoniae SsbB protein was prepared by a modification of the method used to prepare the S. pneumoniae SsbA protein.2 E. coli SSB protein was from Promega. dTn oligomers were from Invitrogen. 32P-end-labeled dTn oligomers were prepared using [{gamma}-32P]ATP and polynucleotide kinase (Amersham Biosciences). Circular {phi}X ssDNA(+ strand) was from New England Biolabs. GelStar® nucleic acid gel stain was from Cambrex. G250 BioSafe Coomassie protein gel stain was from Bio-Rad. {phi}X ssDNA concentrations were determined by absorbance at 260 nm using the conversion factor 36 µg ml–1 A260–1. dTn concentrations were determined by absorbance at 260 nm using the extinction coefficient 8.4 mM–1 cm–1 (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-gel-exclusion 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 dTn and SSB protein given in the figure legends. Reaction solutions were incubated at 37 °C, as indicated in the figure legends. Aliquots (20 µl) were removed from each reaction solution and added to 2 µl of gel-loading 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 dTn oligomers were visualized by autoradiography (these reactions contained 32P-end-labeled dTn oligomers), and bands corresponding to unbound and dTn-bound SSB protein were visualized by G250 BioSafe Coomassie staining (these reactions contained only unlabeled dTn 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 {phi}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 µl) were removed from each reaction solution and added to 2 µl 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 {phi}X ssDNA and the various SSB-{phi}X ssDNA complexes were visualized by GelStar® nucleic acid staining.

Electron Microscopy—The SsbA and SsbB proteins were incubated with {phi}X ssDNA in a buffer containing 20 mM Hepes (pH 7.6)/50 mM NaCl, at a protein to DNA ratio (µg:µg) of 20:1 in a total volume of 40 µl, 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-µl 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, glow-charged 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 10–7 torr and examined in a Tecnai G2 TEM instrument at 40 kV. Images of the SsbA-{phi}X ssDNA and SsbB-{phi}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-{phi}X ssDNA and SsbB-{phi}X ssDNA complexes were derived from the electron microscopy images using the NIH Image software.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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 ~72,400 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 ~63,300 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 dTn Oligomers—The ssDNA binding activities of the SsbA and SsbB proteins were compared with those of the SsbEc protein using a series of dTn oligomers ranging in size from n = 35–100. In these experiments, a fixed concentration of dTn was incubated with various concentrations of SSB protein, and the resulting complexes were analyzed by polyacrylamide gel electrophoresis.

dT50—The binding of the various SSB proteins to dT50 is shown in Fig. 2. The amount of dT50 that was bound to SsbEc protein increased with increasing protein concentration until all of the dT50 was incorporated into an SsbEc-dT50 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 dT50 (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 dT50 to form a complex in which a single SSB tetramer is bound to dT50. Similar results were obtained for all three SSB proteins with the shorter oligomer, dT35 (data not shown).



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  		FIG. 2.
Binding of SSB proteins to dT50. The reaction solutions contained 5.3 µM dT50 (total nucleotide concentration) and the indicated concentrations of SsbEc, SsbA, or SsbB protein (0–1.0 µM tetramer). The reaction solutions were incubated at 37 °C for 15 min and then analyzed by polyacrylamide gel electrophoresis. The bands corresponding to unbound dT50 (ssDNA) and the various SSB-dT50 complexes were visualized by autoradiography.

 
To evaluate the relative binding efficiencies of the SsbA and SsbB proteins, a set of reactions was carried out in which dT50 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 dT50 in the reaction solution. When dT50 was incubated with both SsbA protein and SsbB protein simultaneously, bands corresponding to both the SsbA-dT50 and the SsbB-dT50 complex were observed. The intensity of the band corresponding to the SsbB-dT50 complex appeared to be somewhat more intense than that for the SsbA-dT50 complex, suggesting that the binding of the SsbB protein was more favorable under these reaction conditions. Furthermore, a similar distribution of the SsbA-dT50 and SsbB-dT50 complex bands was observed when dT50 was incubated with a saturating concentration of SsbA protein prior to the addition of SsbB protein or when dT50 was incubated with a saturating concentration of SsbB protein prior to the addition of SsbA protein (Fig. 3). These results indicated that even if dT50 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 dT50 to generate a mixture containing both the SsbA-dT50 and SsbB-dT50 complexes.



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  		FIG. 3.
Relative dT50 binding efficiencies of SSB proteins. The reaction solutions contained 5.3 µM dT5050 (total nucleotide concentration) and no SSB protein (lane 1), 0.3 µM SsbEc protein (lane 2), 0.3 µM SsbA protein (lane 3), 0.3 µM SsbB protein (lane 4), 0.3 µM SsbEc protein and 0.3 µM SsbA protein added simultaneously (lane 5), 0.3 µM SsbEc protein added at 0 min followed by 0.3 µM SsbA protein at 15 min (lane 6), 0.3 µM SsbA protein added at 0 min followed by 0.3 µM SsbEc protein at 15 min (lane 7), 0.3 µM SsbEc protein and 0.3 µM SsbB protein added simultaneously (lane 8), 0.3 µM SsbEc protein added at 0 min followed by 0.3 µM SsbB protein at 15 min (lane 9), 0.3 µM SsbB protein added at 0 min followed by 0.3 µM SsbEc protein at 15 min (lane 10), 0.3 µM SsbA protein and 0.3 µM SsbB protein added simultaneously (lane 11), 0.3 µM SsbA protein added at 0 min followed by 0.3 µM SsbB protein at 15 min (lane 12), or 0.3 µM SsbB protein added at 0 min followed by 0.3 µM SsbA protein at 15 min (lane 13). After a total incubation period of 30 min at 37 °C, the reactions were analyzed by polyacrylamide gel electrophoresis. The bands corresponding to unbound dT50 (ssDNA) and the various SSB-dT50 complexes were visualized by autoradiography.

 
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 dT50, bands corresponding to both the SsbA-dT50 and SsbEc-dT50 complex were observed. The intensity of the SsbA-dT50 complex band was greater than that of the SsbEc-dT50 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 dT50. In this case, the intensity of the SsbB-dT50 complex band was clearly greater than that of the SsbEc-dT50 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).

dT75—The binding of the various SSB proteins to dT75 is shown in Fig. 4. The amount of dT75 that was bound to SsbEc protein increased with increasing protein concentration until all of the dT75 was incorporated into an SsbEc-dT75 complex. In contrast to the results that were obtained with dT50, 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 dT75 (Fig. 4). These results indicated that a second tetramer of SsbEc or SsbA protein was able to bind to dT75 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 dT70 at lower binding densities and a complex with two SsbEc tetramers bound to dT70 at higher binding densities (7). By comparison, when increasing concentrations of SsbB protein were added to dT75, 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 dT75, even at the highest protein concentrations examined. Similar results were obtained for all three SSB proteins with the shorter oligomer, dT65 (data not shown).



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  		FIG. 4.
Binding of SSB proteins to dT75. The reaction solutions contained 5.3 µM dT75 (total nucleotide concentration) and the indicated concentrations of SsbEc, SsbA, or SsbB protein (0–1.0 µM tetramer). The reaction solutions were incubated at 37 °C for 15 min and then analyzed by polyacrylamide gel electrophoresis. The bands corresponding to unbound dT75 (ssDNA) and the various SSB-dT75 complexes were visualized by autoradiography.

 
To further evaluate the binding of the SSB proteins to dT75, 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 dT75 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 dT75 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 dT75.



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  		FIG. 5.
Visualization of unbound and dT75-bound SSB proteins. The reaction solutions contained 30 µM dT75 (total nucleotide concentration) and the indicated concentrations of SsbEc, SsbA, or SsbB protein (0–1.8 µM tetramer). The reaction solutions were incubated at 37 °C for 15 min and then analyzed by polyacrylamide gel electrophoresis. The bands corresponding to unbound SSB protein and the various SSB-dT75 complexes were visualized by Coomassie protein staining (the lane designated M corresponds to the SSB proteins in the absence of dT75). Note that the concentrations of dT75 and SSB protein were increased 6-fold relative to those in Fig. 4 so that the unbound and bound SSB proteins would be clearly visible by Coomassie staining.

 
dT100—The binding of the various SSB proteins to dT100 is shown in Fig. 6. The amount of dT100 that was bound to SsbB protein increased with increasing protein concentration until all of the dT100 was incorporated into an SsbB-dT100 complex. In contrast to the results with dT50 and dT75, 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 dT100 at higher protein concentrations. By comparison, when increasing concentrations of SsbA protein were added to dT100, 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 dT100 (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 dT100.



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  		FIG. 6.
Binding of SSB proteins to dT100. The reaction solutions contained 5.3 µM dT100 (total nucleotide concentration) and the indicated concentrations of SsbEc, SsbA, or SsbB protein (0–1.0 µM tetramer). The reaction solutions were incubated at 37 °C for 15 min and then analyzed by polyacrylamide gel electrophoresis. The bands corresponding to unbound dT100 (ssDNA) and the various SSB-dT100 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 dT100 preparation.

 
Binding of SSB Proteins to {phi}X ssDNA—The binding of the various SSB proteins to the long naturally occurring circular {phi}X ssDNA (5386 nucleotides) was also examined. In these experiments, a fixed concentration of {phi}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 {phi}X ssDNA, there was a progressive decrease in the mobility of the {phi}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 {phi}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 {phi}X ssDNA is saturated with SSB protein. In contrast, when increasing concentrations of SsbB protein were added to {phi}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, {phi}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 {phi}X ssDNA was recovered, demonstrating that the formation of the faster moving complex was not because of degradation of the {phi}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 = 5.2) and SsbB protein (pI = 6.0)). This indicated that the rapid migration of the SsbB-{phi}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 {phi}X ssDNA, demonstrating that the formation of the faster moving complex by the SsbB protein was not specific to {phi}X ssDNA (data not shown). Taken together, these results indicated that the complexes that were formed with {phi}X ssDNA by the SsbB protein were different from those formed by the SsbA and SsbEc proteins.



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  		FIG. 7.
Binding of SSB proteins to {phi}X ssDNA. The reaction solutions contained 5.0 µM circular {phi}X ssDNA (total nucleotide concentration) and the indicated concentrations of SsbEc, SsbA, or SsbB protein (0–0.3 µM 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 {phi}X ssDNA (ssDNA) and the various SSB-{phi}X ssDNA complexes were visualized by GelStar® staining. The minor band that is visible in the 0 µM SSB protein lanes is because of a small amount of linear {phi}X ssDNA that is present in the circular {phi}X ssDNA preparation.

 
The complexes that were formed by the SsbA and SsbB proteins with {phi}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 {phi}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-{phi}X ssDNA complexes was 30% smaller than that of the SsbA-{phi}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-{phi}X ssDNA complexes is presumably related to the anomalous mobilities that were observed for these complexes in the agarose gel-shift assays (Fig. 7).



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  		FIG. 8.
Electron microscopic analysis of SSB-{phi}X ssDNA complexes. Electron micrographs showing the complexes formed with circular {phi}X ssDNA by the SsbA protein (A) and SsbB protein (B). The bar represents 50 nm.

 
Because the SsbA and SsbB proteins appeared to form different types of complexes with {phi}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 {phi}X ssDNA. In these experiments, {phi}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 {phi}X ssDNA in the reaction solution to the discrete slower or faster moving complexes, respectively. When {phi}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 {phi}X ssDNA was incubated with a saturating concentration of SsbA protein prior to the addition of SsbB protein, or when the {phi}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 {phi}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 {phi}X ssDNA that had a mobility indistinguishable from that of the complex formed with SsbA protein alone.



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  		FIG. 9.
Binding of SsbA and SsbB proteins to {phi}X ssDNA. A, the reaction solutions contained 30 µM circular {phi}X ssDNA (total nucleotide concentration) and no SSB protein (lane 1), 1.8 µM SsbA protein (lane 2), 1.8 µM SsbB protein (lane 3), 1.8 µM SsbA protein and 1.8 µM SsbB protein added simultaneously (lane 4), 1.8 µM SsbA protein added at 0 min followed by 1.8 µM SsbB protein at 8 min (lane 5), or 1.8 µM SsbB protein added at 0 min followed by 1.8 µM SsbA protein at 8 min (lane 6). After an incubation period of 16 min (total) at 37 °C, the reactions were analyzed by agarose gel electrophoresis and the bands corresponding to unbound {phi}X ssDNA (ssDNA) and the SsbA-{phi}X ssDNA and SsbB-{phi}X ssDNA complexes were visualized by GelStar® staining. The minor band that is visible in the various lanes is because of a small amount of linear {phi}X ssDNA that is present in the circular {phi}X ssDNA preparation. Note that the concentrations of {phi}X ssDNA and SSB protein were increased 6-fold relative to those in Fig. 7 so that there would be sufficient material for the subsequent analysis of the composition of the various SSB-{phi}X ssDNA complexes. B, the bands corresponding to unbound {phi}X ssDNA and the complexes that were formed with {phi}X ssDNA by SsbA protein, SsbB protein, or SsbA and SsbB proteins together were excised from an agarose gel analogous to the one shown in A. SDS-loading dye was added to each gel slice, and the samples were heated at 100 °C until the agarose melted. The samples were then analyzed by SDS-polyacrylamide gel electrophoresis. The bands corresponding to the various SSB proteins were visualized by Coomassie staining.

 
To distinguish between these possibilities, {phi}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 {phi}X ssDNA.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The polyacrylamide gel shift results indicated that the SsbEc, SsbA, and SsbB proteins each bind to the shorter oligomer, dT50, 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 dT50 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, dT75, 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, dT100, 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 ~65 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 ~35 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 dT75. However, the SsbEc and SsbA proteins presumably must be in the (SSB)35 mode when two tetramers are bound to dT75 (7). Similarly, the SsbEc and SsbA proteins presumably must be in the (SSB)35 mode when three SsbEc or SsbA tetramers are bound to dT100. The finding that only one SsbB tetramer was able to bind to dT75 and only two SsbB tetramers were able to bind to dT100 suggests that either the SsbB protein is unable to bind to the dTn oligomers in an (SSB)35-like mode or that multiple SsbB tetramers are unable to bind in close proximity on the same dTn molecule.

The agarose gel shift results indicated that the SsbB protein also forms complexes with circular {phi}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 {phi}X ssDNA that migrated more slowly than unbound {phi}X ssDNA during agarose gel electrophoresis. Electron microscopy showed that these slower moving SsbA complexes corresponded to open circular complexes in which the {phi}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 {phi}X ssDNA that migrated more rapidly than unbound {phi}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 {phi}X ssDNA was covered with SsbEc or SsbA protein.

Unbound circular {phi}X ssDNA appears as a condensed bush-like 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 {phi}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-{phi}X ssDNA complexes that were observed by agarose gel electrophoresis; the complexes that were formed with {phi}X ssDNA at subsaturating concentrations of SsbEc and SsbA protein still migrated more slowly than unbound {phi}X ssDNA, whereas the complexes that were formed by the SsbB protein migrated more rapidly than unbound {phi}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 {phi}X ssDNA and this is responsible for the condensed appearance and anomalous electrophoretic mobility of the SsbB-{phi}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-{phi}X ssDNA complexes than in the SsbA-{phi}X ssDNA complexes, and with the results that demonstrated that the SsbA and SsbB proteins are able to bind together to {phi}X ssDNA to form a mixed SsbA/SsbB-{phi}X ssDNA complex. The determination of the physical basis for the unusual electrophoretic behavior of the SsbB-{phi}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 (dT35 and dT50) 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 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 ability 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.


   FOOTNOTES
 
* This work was supported by NIEHS, National Institutes of Health Training Grant ES07141 (to D. E. G. and F. R. B.) and National Institutes of Health Grant GM31819 (to J. D. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, 615 N. Wolfe St., Baltimore, MD 21205. Tel.: 410-955-3895; E-mail: fbryant{at}jhsph.edu.

1 The abbreviations used are: ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; {phi}X, bacteriophage {phi}X174. Back

2 M. A. Hedayati, D. E. Grove, S. E. Steffen, and F. R. Bryant, manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank Mohammad Hedayati and Ray Enke for their assistance with some of the preliminary experimental work that led to the results described in this paper.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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