Figures
Abstract
Background
Streptococcus pneumoniae has two paralogous, homotetrameric, single-stranded DNA binding (SSB) proteins, designated SsbA and SsbB. Previous studies demonstrated that SsbA and SsbB have different solution-dependent binding mode preferences with variable DNA binding capacities. The impact of these different binding properties on the assembly of multiple SsbAs and SsbBs onto single-stranded DNA was investigated.
Methodology/Principal Findings
The complexes that were formed by the SsbA and SsbB proteins on dTn oligomers of defined lengths were examined by polyacrylamide gel electrophoresis. Complexes containing either two SsbAs or two SsbBs, or mixed complexes containing one SsbA and one SsbB, could be formed readily, provided the dTn oligomer was long enough to satisfy the full binding mode capacities of each of the bound proteins under the particular solution conditions. Complexes containing two SsbAs or two SsbBs could also be formed on shorter dTn oligomers via a “shared-strand binding” mechanism in which one or both proteins were bound using only a portion of their potential binding capacity. Mixed complexes were not formed on these shorter oligomers, however, indicating that SsbA and SsbB were incompatible for shared-strand binding. Additional experiments suggested that this shared-strand binding incompatibility may be due in part to differences in the structure of a loop region on the outer surface of the subunits of the SsbA and SsbB proteins.
Conclusion/Significance
These results indicate that the SsbA and SsbB proteins may co-assemble on longer DNA segments where independent binding is possible, but not on shorter DNA segments where coordinated interactions between adjacent SSBs are required. The apparent compatibility requirement for shared-strand binding could conceivably serve as a self-recognition mechanism that regulates the manner in which SsbA and SsbB interact in S. pneumoniae.
Citation: Salerno B, Anne G, Bryant FR (2011) DNA Binding Compatibility of the Streptococcus pneumoniae SsbA and SsbB Proteins. PLoS ONE 6(9): e24305. https://doi.org/10.1371/journal.pone.0024305
Editor: Vladimir N. Uversky, University of South Florida College of Medicine, United States of America
Received: July 15, 2011; Accepted: August 4, 2011; Published: September 7, 2011
Copyright: © 2011 Salerno et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by funds from the Johns Hopkins University Bloomberg School of Public Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
The naturally transformable Gram-positive bacterium Streptococcus pneumoniae has two paralogous, homotetrameric, single-stranded DNA binding (SSB) proteins, designated SsbA and SsbB (Figure 1) [1]–[3]. The SsbA protein (156 amino acids/17,350 Da per monomer) is expressed constitutively whereas the SsbB protein (131 amino acids/14,926 Da per monomer) is induced specifically during transformational competence. These expression patterns suggest that the SsbA protein may serve as a general SSB protein for routine DNA functions, and that the SsbB protein may be a specialized SSB protein used primarily during natural transformation [1].
The amino acid sequences 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. The division between the N-terminal and C-terminal domains is indicated by an arrow, and the putative Loop 1 region is denoted with a box.
The N-terminal domains of the SsbA and SsbB proteins (amino acids 1–105/106) are similar in sequence. The C-terminal domain of the SsbB protein (amino acids 106–131), however, is significantly shorter than that of the SsbA protein (amino acids 107–156) (Figure 1). Structural studies of the corresponding regions of the homotetrameric SSB protein from Escherichia coli (SsbEc) and other bacterial SSB proteins have shown that the N-terminal domain contains the DNA binding and subunit tetramerization sites, whereas the C-terminal domain may serve as a binding site for other proteins involved in various DNA functions [4].
Bacterial SSB proteins bind single-stranded DNA in a non-sequence-specific manner. The DNA binding properties of the SsbEc protein have been the most extensively characterized. Two major binding modes have been identified: the SSB35 mode and the SSB65 mode. In the SSB35 mode (favored at lower salt concentrations), two subunits of the SsbEc tetramer interact with the single-stranded DNA (occluding ∼35 nucleotides per tetramer), whereas in the SSB65 mode (favored at higher salt concentrations), all four subunits of the SsbEc tetramer interact with the single-stranded DNA (occluding ∼65 nucleotides per tetramer) [5].
We previously carried out a comparative analysis of the DNA binding mode properties of the SsbEc, SsbA, and SsbB proteins. In that study, the various SSB proteins were incubated with the oligomer, dT35, under different solution conditions and the resulting complexes were examined by polyacrylamide gel electrophoresis. In standard reaction solution (25 mM Tris acetate (pH 7.5)), the SsbEc protein was able to bind a single dT35 molecule, consistent with the SSB35 mode of binding. When Mg2+ (10 mM) was included in the reaction solution, however, the SsbEc protein was able to bind two dT35 molecules, consistent with the SSB65 mode of binding. The SsbA protein behaved similarly to the SsbEc protein under all reaction conditions, indicating that it interacted with dT35 in SSB35 and SSB65 modes that were analogous to those of the SsbEc protein. The SsbB protein, in contrast, appeared to bind two dT35 molecules in an SSB65-like mode in the absence of Mg2+, and in an enhanced SSB65-like mode (with positive intersubunit cooperativity) in the presence of Mg2+ [2].
The pronounced difference in binding mode preferences raises the question of whether SsbA and SsbB would be able to interact together on single-stranded DNA. To address this issue, we have now examined the assembly of multiple SsbAs or SsbBs on dTn oligomers of various defined lengths. Polyacrylamide gel electrophoresis was particularly well suited for this analysis because the various SSB·dTn complexes were readily resolvable and remarkably stable during electrophoresis, and the effect of solution conditions on complex formation could be assessed by varying the composition of the electrophoresis running buffer. The results indicate that: i) different mechanisms of assembly are available to the SsbA and SsbB proteins, depending on the length of the DNA and the specific solution conditions, and ii) SsbA and SsbB may co-assemble on longer DNA segments where independent binding is possible, but not on shorter DNA segments where coordinated interactions between adjacent SSBs are required.
Results
Experimental Design
The binding of the Streptococcus pneumoniae SsbA and SsbB proteins to a set of dTn oligomers ranging in length from dT50 to dT130 was examined. Particular attention was placed on determining the shortest dTn oligomer that was able to accommodate the binding of two SsbAs, two SsbBs, or one SsbA and one SsbB, in either the absence or presence of Mg2+. The expectation with this approach was that two SSBs would have to interact in a coordinated manner to form a complex on a minimal length dTn, whereas the SSBs would be able to bind independently to isolated sites on longer dTn oligomers. All binding reactions were carried out in solutions containing 25 mM Tris acetate (pH 7.5) and either 0 or 10 mM magnesium acetate, and the resulting complexes were analyzed by polyacrylamide gel electrophoresis using a running buffer identical in composition to that of the individual reaction solutions.
SsbA protein assembly
The complexes that were formed by the SsbA protein with the various dTn oligomers in the absence and presence of Mg2+ are shown in Figures 2 and 3, respectively (note: in these experiments, the electrophoretic mobilities of the free dTn oligomers exhibit a greater inverse-dependence on length than do the corresponding SsbA·dTn complexes, leading to a progressive decrease in the separation between the free dTn oligomers and the SsbA·dTn complexes with increasing dTn length).
The reaction solutions contained 25 mM Tris acetate (pH 7.5), 5 µM dTn (nucleotide concentration), and the indicated concentrations of SsbA protein (tetramer concentrations). The reactions were analyzed by polyacrylamide gel electrophoresis using a gel running buffer consisting of Tris acetate (pH 7.5). The bands corresponding to the unbound dTn oligomers, and the A1, A2, and A3 complexes, were visualized by phosphorimaging.
The reaction solutions contained 25 mM Tris acetate (pH 7.5), 10 mM magnesium acetate, 5 µM dTn (nucleotide concentration), and the indicated concentrations of SsbA protein (tetramer concentrations). The reactions were analyzed by polyacrylamide gel electrophoresis using a gel running buffer consisting of Tris acetate (pH 7.5) and 10 mM magnesium acetate. The bands corresponding to the unbound dTn oligomers, and the A1 and A2 complexes, were visualized by phosphorimaging.
Absence of Mg2+.
When increasing concentrations of SsbA were added to dT50 in the absence of Mg2+, a single complex with a gel mobility lower than that of the unbound dT50 was formed (A1 complex). All of the dT50 was converted to this complex at an SsbA concentration that corresponded to approximately one SsbA tetramer per dT50 molecule, and there was no indication of the formation of a second complex at higher SsbA concentrations (Figure 2 and additional data not shown). A similar pattern of binding was obtained with the longer oligomer, dT65 (gel not shown). These results indicated that a single SsbA was able to bind to dT50 and dT65 under these reaction conditions.
When the oligomer length was increased to dT75, an A1 complex was formed with increasing SsbA concentrations in a manner similar to that observed with the shorter oligomers. As the concentration of SsbA was increased further, however, the A1 complex disappeared and a new complex with an even lower gel mobility was formed (A2 complex). This result indicated that a second SsbA was able to bind to dT75 under these conditions. A similar pattern of binding was observed with the longer oligomers, dT85, dT90, and dT100, indicating that two SsbAs were able to bind to each of these oligomers as well (Figure 2).
When the oligomer length was increased to dT130, A1 and A2 complexes were formed with increasing SsbA concentrations in a manner similar to that observed with dT100. At higher SsbA concentrations, however, a third complex with a mobility lower than either the A1 and A2 complexes was formed (A3 complex). This result indicated that three SsbAs were able to bind to dT130 under these conditions (Figure 2).
The results in Figure 2 indicated that the shortest dTn oligomer in the set that was able to bind two SsbAs in the absence of Mg2+ was dT75, and the shortest dTn that was able to bind three SsbAs was dT130. These results are summarized in Table 1.
Presence of Mg2+.
When increasing concentrations of SsbA were added to dT50 in the presence of Mg2+, a single complex with a gel mobility lower than that of the unbound dT50 was formed (A1 complex), with no indication of the formation of a second complex at higher SsbA concentrations (Figure 3). A similar pattern of binding was obtained with dT65 (gel not shown). These results were similar to those that were obtained in the absence of Mg2+ and indicated that a single SsbA was able to bind to dT50 and dT65 in the presence of Mg2+. A1 complexes were also formed when increasing concentrations of SsbA were added to the longer oligomers, dT75 and dT85, but in contrast to the results that were obtained in the absence of Mg2+, A2 complexes were not detected with these oligomers (Figure 3).
When the oligomer length was increased to dT90, an A1 complex was formed at lower SsbA concentrations in a manner similar to that observed with the shorter oligomers. As the concentration of SsbA was increased further, however, the A1 complex disappeared and a new complex with an even lower gel mobility was formed (A2 complex). This result indicated that a second SsbA was able to bind to dT90 under these conditions. A similar pattern of binding was obtained with dT100 and dT130, indicating that two SsbAs were able to bind to each of these oligomers as well. In contrast to the results that were obtained in the absence of Mg2+, however, there was no indication of the formation of a third complex with dT130, even at the highest concentrations of SsbA that were examined (Figure 3).
The results in Figure 3 indicated that the shortest dTn oligomer in the set that was able to bind two SsbAs in the presence of Mg2+ was dT90, and that only two SsbAs were able to bind even when the oligomer length was increased to dT130. These results are summarized in Table 1.
SsbB protein assembly
The complexes that were formed by the SsbB protein with the various dTn oligomers in the absence and presence of Mg2+ are shown in Figures 4 and 5, respectively (note: in these experiments, the separation between the free dTn oligomers and the various SsbB·dTn complexes is less than that observed with the SsbA protein, owing to the smaller molecular size of the SsbB protein and the increased electrophoretic mobility of the SsbB·dTn complexes).
The reaction solutions contained 25 mM Tris acetate (pH 7.5), 5 µM dTn (nucleotide concentration), and the indicated concentrations of SsbB protein (tetramer concentrations). The reactions were analyzed by polyacrylamide gel electrophoresis using a gel running buffer consisting of Tris acetate (pH 7.5). The bands corresponding to the unbound dTn oligomers, and the B1 and B2 complexes, were visualized by phosphorimaging.
The reaction solutions contained 25 mM Tris acetate (pH 7.5), 10 mM magnesium acetate, 5 µM dTn (nucleotide concentration), and the indicated concentrations of SsbB protein (tetramer concentrations). The reactions were analyzed by polyacrylamide gel electrophoresis using a gel running buffer consisting of Tris acetate (pH 7.5) and 10 mM magnesium acetate. The bands corresponding to the unbound dTn oligomers, and the B1 and B2 complexes, were visualized by phosphorimaging.
Absence of Mg2+.
When increasing concentrations of SsbB were added to dT50 in the absence of Mg2+, a single complex with a gel mobility lower than that of the unbound dT50 was formed (B1 complex). All of the dT50 was converted to this complex at an SsbB concentration that corresponded to approximately one SsbB tetramer per dT50 molecule, and there was no indication of the formation of a second complex at higher SsbB concentrations (Figure 4 and additional data not shown). A similar pattern of binding was obtained with the longer oligomers, dT65 (gel not shown) and dT75 (Figure 4). These results indicated that only a single SsbB was able to bind to these oligomers under these conditions.
When the oligomer length was increased to dT85, a B1 complex was formed at lower SsbB concentrations in a manner similar to that observed with the shorter oligomers. As the concentration of SsbB was increased further, however, the B1 complex disappeared and a new complex with an even lower gel mobility was formed (B2 complex). This result indicated that a second SsbB was able to bind to dT85 under these conditions (Figure 4). A similar pattern of binding was observed with the longer oligomers, dT90, dT100, and dT130, indicating that two SsbBs were able to bind to each of these oligomers as well. There was no indication of the formation of a third complex with these oligomers, however, even at the highest concentrations of SsbB that were examined (Figure 4).
The results in Figure 4 indicated that the shortest dTn oligomer in the set that was able to bind two SsbBs in the absence of Mg2+ was dT85, and that only two SsbBs were able to bind even when the oligomer length was increased to dT130. These results are summarized in Table 1.
Presence of Mg2+.
When increasing concentrations of SsbB were added to dT50 in the presence of Mg2+, a single complex with a gel mobility lower than that of the unbound dT50 was formed (B1 complex), with no indication of the formation of a second complex at higher SsbB concentrations (Figure 5). A similar pattern of binding was obtained with the longer oligomers, dT65 (gel not shown) and dT75 (Figure 5). These results were similar to those that were obtained in the absence of Mg2+ and indicated that only a single SsbB was able to bind to these oligomers under these conditions. B1 complexes were also formed when increasing concentrations of SsbB were added to the longer oligomers, dT85 and dT90, but in contrast to the results that were obtained in the absence of Mg2+, B2 complexes were not detected with these oligomers (Figure 5).
When the oligomer length was increased to dT100, a B1 complex was formed at lower SsbB concentrations in a manner similar to that observed with the shorter oligomers. When the concentration of SsbB was increased further, however, the B1 complex disappeared and a new complex of even lower gel mobility was formed (B2 complex). This result indicated that a second SsbB was able to bind to dT100 under these conditions (Figure 5). A similar pattern of binding was obtained with dT130, indicating that two SsbBs were able to bind to this oligomer as well. There was no indication of the formation of a third complex with these oligomers, however, even at the highest concentrations of SsbB that were examined (Figure 5).
The results in Figure 5 indicated that the shortest dTn oligomer in the set that was able to bind two SsbBs in the presence of Mg2+ was dT100, and that only two SsbBs were able to bind even when the oligomer length was increased to dT130. These results are summarized in Table 1.
Co-assembly of SsbA and SsbB proteins
The ability of the SsbA and SsbB proteins to co-assemble on dTn oligomers was also investigated. For these experiments, dTn oligomers were selected that were long enough to accommodate the binding of either two SsbAs or two SsbBs, under various solution conditions (see Table 1).
Absence of Mg2.
The first set of co-assembly experiments in the absence of Mg2+ was carried out with dT90 (Figure 6). When an excess concentration of SsbA alone was added to dT90, an A2 complex with two SsbAs bound to the dT90 was formed. Similarly, when an excess concentration of SsbB alone was added to dT90, a B2 complex with two SsbBs bound to the dT90 was formed. When SsbA and SsbB were added together to dT90, however, the A2 and B2 complexes were again formed, but little if any mixed complexes with one SsbA and one SsbB bound to the same dT90 molecule were detected (as judged by the absence of a new band with a mobility intermediate between that of the A2 and B2 complexes). These results indicated that although either two SsbAs or two SsbBs could bind to dT90 in the absence of Mg2+, the binding of one SsbA and one SsbB to dT90 was unfavorable under these conditions.
The reaction solutions contained 25 mM Tris acetate (pH 7.5), 5 µM dT90 (left) or dT100 (right) (total nucleotide concentration), and the indicated concentrations of SsbA and SsbB protein (tetramer concentrations). A, The reactions were analyzed by polyacrylamide gel electrophoresis using a gel running buffer consisting of Tris acetate (pH 7.5). The bands corresponding to the unbound dTn oligomers, and the A2, B2, and A·B complexes, were visualized by phosphorimaging. B, The lanes for the reactions that contained 0.15 µM SsbA, 0.15 µM SsbB, and either dT90 (left) or dT100 (right) were scanned to show the relative intensities of the bands for the indicated complexes.
A second set of co-assembly experiments was carried out in the absence of Mg2+ with the longer oligomer, dT100 (Figure 6). When an excess concentration of either SsbA or SsbB alone was added to dT100, the corresponding A2 or B2 complexes were formed, as with dT90. In contrast to the results that were obtained with dT90, however, the A2 and B2 complexes, and a third complex with an intermediate mobility were formed when SsbA and SsbB were added together to dT100. The intermediate band was excised from the gel, analyzed by SDS-polyacrylamide gel electrophoresis, and found to contain approximately equal amounts of SsbA and SsbB protein (gel not shown). These results indicated that the intermediate band corresponded to a mixed complex in which one SsbA and one SsbB were bound to the dT100 (A·B complex).
The results in Figure 6 indicated that although the simultaneous binding of SsbA and SsbB to dT90 was unfavorable in the absence of Mg2+, SsbA and SsbB were able to bind together on dT100 under these conditions.
Presence of Mg2+.
The first set of co-assembly experiments in the presence Mg2+ (10 mM) was carried out with dT100 (Figure 7). When an excess concentration of either SsbA or SsbB alone was added to dT100, the corresponding A2 and B2 complexes were formed as expected. When SsbA and SsbB were added together to dT100, however, the A2 and B2 complexes were again formed, but no mixed complexes with one SsbA and one SsbB bound to the same dT100 molecule were detected (as judged by the absence of a new band with a mobility intermediate between that of the A2 and B2 complexes). These results indicated that although two SsbAs or two SsbBs could bind to dT100 in the presence of Mg2+, the binding of one SsbA and one SsbB to dT100 was unfavorable under these conditions.
The reaction solutions contained 25 mM Tris acetate (pH 7.5), 10 mM magnesium acetate, 5 µM dT100 (left) or dT130 (right) (total nucleotide concentration), and the indicated concentrations of SsbA and SsbB protein (tetramer concentrations). A, The reactions were analyzed by polyacrylamide gel electrophoresis using a gel running buffer consisting of Tris acetate (pH 7.5) and 10 mM magnesium acetate. The bands corresponding to the unbound dTn oligomers, and the A2, B2, and A·B complexes, were visualized by phosphorimaging. B, The lanes for the reactions that contained 0.15 µM SsbA, 0.15 µM SsbB, and either dT100 (left) or dT130 (right) were scanned to show the relative intensities of the bands for the indicated complexes.
A second set of co-assembly experiments was carried out in the presence of Mg2+ with the longer oligomer, dT130 (Figure 7). When an excess concentration of either SsbA or SsbB alone was added to dT130, the corresponding A2 or B2 complexes were formed, as with dT100. In contrast to the results that were obtained with dT100, however, the A2 and B2 complexes, and a third complex with an intermediate mobility were formed when SsbA and SsbB were added together to dT100. The appearance of the intermediate band was consistent with the formation of a mixed complex in which one SsbA and one SsbB were bound to the dT130 (A·B complex).
The results in Figure 7 indicated that although the simultaneous binding of SsbA and SsbB to dT100 was unfavorable in the presence of Mg2+, SsbA and SsbB were able to bind together on dT130 under these conditions.
Co-assembly of SsbA protein with SsbA/B and SsbBRYTP proteins
Additional co-assembly experiments were carried out with the SsbA protein and two SSB variants: the SsbA/B protein and the SsbBRYTP protein. The SsbA/B protein is a chimeric protein in which the C-terminal domain of the SsbA protein (amino acids 106–156) has been replaced with the C-terminal domain from the SsbB protein (amino acids 105–131) [2]. The SsbBRYTP protein is a modified SsbB protein in which a four-amino acid sequence from the N-terminal domain of the SsbB protein (18HKTN21) has been replaced with the corresponding sequence from the SsbA protein (18RYTP21) (Figure 1, Material and Methods). The SsbA/B protein (15,039 Da per monomer) and the SsbBRYTP protein (14,963 Da per monomer) are similar in size to the SsbB protein (14,926 Da per monomer), and form complexes on dT90 that are clearly resolvable by gel electrophoresis from the complexes formed by the SsbA protein (17,350 Da per monomer). Experiments analogous to those carried out for the SsbA and SsbB proteins indicated that either two SsbA/Bs ((A/B)2 complex) or two SsbBRYTPs (BRYTP2 complex) were able to bind to dT90 in the absence of Mg2+.
SsbA/B protein.
The initial set of SsbA and SsbA/B co-assembly experiments was carried out with dT90 in the absence of Mg2+ (Figure 8). When an excess concentration of either SsbA or SsbA/B alone was added to dT90, the corresponding A2 and (A/B)2 complexes were formed, as expected. When SsbA and SsbA/B were added together to dT90, however, the A2 and (A/B)2 complexes, and a third complex with an intermediate mobility, were formed. The appearance of the intermediate band was consistent with the formation of a mixed complex in which one SsbA and one SsbA/B were bound to the dT90 (A·A/B complex).
The reaction solutions contained 25 mM Tris acetate (pH 7.5), 5 µM dT90 (total nucleotide concentration), and the indicated concentrations of SsbA and SsbA/B protein (tetramer concentrations). A, The reactions were analyzed by polyacrylamide gel electrophoresis using a gel running buffer consisting of Tris acetate (pH 7.5). The bands corresponding to the unbound dT90 oligomer, and the A2, (A/B)2, and A·A/B complexes, were visualized by phosphorimaging. B, The lane for the reaction that contained 0.15 µM SsbA and 0.10 µM SsbA/B was scanned to show the relative intensities of the bands for the indicated complexes.
These results indicated that although the simultaneous binding of SsbA and SsbB was unfavorable (Figure 6), SsbA was able to form a mixed complex with SsbA/B on dT90 in the absence of Mg2+ (Figure 8). Additional experiments indicated that SsbA/B also differed from SsbB it that it was able to form a mixed complex with SsbA on dT100 in the presence of Mg2+ (gel not shown).
SsbBRYTP protein.
The initial set of SsbA and SsbBRYTP co-assembly experiments was also carried out with dT90 in the absence of Mg2+ (Figure 9). When an excess concentration of either SsbA or SsbBRYTP alone was added to dT90, the corresponding A2 and BRYTP2 complexes were formed, as expected. When SsbA and SsbBRYTP were added together to dT90, however, the A2 and BRYTP2 complexes, and a third complex with an intermediate mobility were formed. The appearance of the intermediate band was consistent with the formation of a mixed complex in which one SsbA and one SsbBRYTP were bound to the dT90 (A·BRYTP complex).
The reaction solutions contained 25 mM Tris acetate (pH 7.5), 5 µM dT90 (total nucleotide concentration), and the indicated concentrations of SsbA and SsbBRYTP protein (tetramer concentrations). A, The reactions were analyzed by polyacrylamide gel electrophoresis using a gel running buffer consisting of Tris acetate (pH 7.5). The bands corresponding to the unbound dT90 oligomer, and the A2, BRYTP2, and A•BRYTP complexes, were visualized by phosphorimaging. B, The lane for the reaction that contained 0.15 µM SsbA and 0.15 µM SsbBRYTP was scanned to show the relative intensities of the bands for the indicated complexes.
These results indicated that although the simultaneous binding of SsbA and SsbB was unfavorable (Figure 6), SsbA was able to form a mixed complex with SsbBRYTP on dT90 in the absence of Mg2+ (Figure 9). Additional experiments, however, indicated that SsbBRYTP, like SsbB, was unable to form a mixed complex with SsbA on dT100 in the presence of Mg2+ (gel not shown).
Discussion
The results presented here indicate that the shortest dTn oligomer that is able to accommodate the binding of two SsbAs or two SsbBs is strictly defined, and depends on whether Mg2+ is included in the reaction solution (Table 1). This finding suggests that the minimal oligomer length may be determined by the preferred binding modes and potential binding capacities of the individual SSB proteins under the particular solution conditions.
SsbA protein assembly
The shortest dTn oligomer that was able to bind two SsbAs in the absence of Mg2+ was dT75 (Table 1). Assuming that SsbA interacts with dTn oligomers as a tetramer in an SSB35-like mode in the absence of Mg2+ (see Introduction), two SsbAs may assemble on dT75 under these conditions in a manner in which each of the SsbAs interacts with a ∼35-nucleotide segment of the oligomer. The observation that only two SsbAs were able to bind even when the oligomer length was increased to dT100, whereas three SsbAs were able to bind to dT130 indicates that at least a ∼35 nucleotide segment of dTn was required for the stable binding of each SsbA (Table 1). In all cases, as the concentration of SsbA was increased, the dTn complex with a lesser number of SsbAs bound was replaced completely by the complex with the greater number of SsbAs bound. These results indicate that under SSB35-like binding mode conditions, individual SsbAs can organize themselves so as to maximize the number of SsbAs bound to a dTn oligomer while satisfying the ∼35-nucleotide binding requirement of each bound SsbA.
A longer dTn oligomer was required for the binding of two SsbAs when Mg2+ was included in the reaction solution (Table 1). Assuming that SsbA interacts with dTn oligomers as a tetramer in an SSB65-like binding mode in the presence of Mg2+ (see Introduction), it is likely that the increased length requirement is due to the higher binding capacity of SsbA under these conditions. The complexes containing two SsbAs that were observed with dT130 are consistent with an SSB65-like mode of binding in that dT130 is long enough to satisfy the full capacity of ∼65 nucleotides expected for each of the bound SsbAs (∼130 nucleotides total) (Figure 10). However, stable complexes containing two SsbAs could also be formed on oligomers as short as dT90 under these conditions (Table 1). With these shorter oligomers, one or both of the SsbAs were presumably bound using only a portion of their potential binding capacity. These results suggest that under SSB65-like binding mode conditions, two SsbAs are able to assemble onto shorter segments of single-stranded DNA via a coordinated sharing of the DNA strand between the bound proteins.
The first complex represents an independent binding complex in which two SSB tetramers are each bound to a 65-nucleotide segment of a dT130 molecule. The second complex represents a shared-strand binding complex in which one SSB tetramer is bound to a 65-nucleotide segment and the second SSB tetramer is bound to the remaining 25-nucleotide segment of a dT90 molecule (unequal sharing). The third complex represents a shared-strand binding complex in which two SSB tetramers are each bound to a 45-nucleotide segment of a dT90 molecule (equal sharing). The paths shown for the dTn strands in these complexes are speculative and are based on a structural model of the SsbEc protein bound to single-stranded DNA in the SSB65 binding mode [7].
Various arrangements can be envisioned for the DNA strand in a “shared-strand binding” mechanism (Figure 10). With dT90 for example, one SsbA could be bound to a ∼65-nucleotide segment, with the second SsbA bound to the remaining ∼25-nucleotide segment of the oligomer. Alternatively, the dT90 may be more equally shared between the two SsbAs, with each binding to a ∼45-nucleotide segment of the oligomer. These possibilities are not necessarily mutually exclusive and a combination of different binding arrangements may also occur. In any case, the observation that complexes with two SsbAs were not formed on dTn oligomers shorter than dT90 when Mg2+ was included in the reaction solution, but could be formed on oligomers as short as dT75 in absence of Mg2+ (Table 1) suggests that the shared-strand arrangement that is adopted when two SsbAs bind in the SSB65-like mode may be different from the binding arrangement that is used when two SsbAs bind in the SSB35-like mode.
SsbB protein assembly
Longer dTn oligomers were required for the binding of two SsbBs than were needed for two SsbAs, both in the absence and presence of Mg2+ (Table 1). Assuming that SsbB interacts with dTn oligomers as a tetramer in an SSB65-like mode in the absence of Mg2+ and in an enhanced SSB65-like mode in the presence of Mg2+ (see Introduction), the longer length requirement may reflect the higher binding capacity of SsbB, relative to that of SsbA, under these reaction conditions. The complexes containing two SsbBs that were observed with dT130 in both the absence and presence of Mg2+ are consistent with either the SSB65-like mode or the enhanced SSB65-like mode of binding in that dT130 is long enough to satisfy the full ∼65 nucleotide binding capacity expected for each of the bound SsbBs in either case (∼130 nucleotides total) (Figure 10). However, stable complexes containing two SsbBs could also be formed on oligomers as short as dT85 (in the absence of Mg2+) or dT100 (in the presence of Mg2+). These results suggest that two SsbBs can bind to the shorter oligomers using a shared-strand binding mechanism analogous to that proposed for SsbA in the presence of Mg2+ (SSB65-like binding mode conditions) (Figure 10). In the case of SsbB, the observation that dT85 was able to accommodate the binding of two SsbBs in absence of Mg2+, whereas at least dT100 was required for the binding of two SsbBs in the presence of Mg2+ may reflect a difference in the arrangement of the shared strand between the two SsbBs under normal SSB65-like mode versus enhanced SSB65-like mode binding conditions.
SsbA and SsbB protein co-assembly
The shortest dTn oligomer that was able to accommodate the simultaneous binding of one SsbA and one SsbB was also strictly defined and depended on the solution conditions.
In the absence of Mg2+, complexes containing either two SsbAs or two SsbBs were readily formed on dT90. However, little or no mixed complexes with one SsbA and one SsbB were detected when both proteins were added together to dT90 (Figure 6). If the binding capacity of SsbA under these conditions is assumed to be ∼35 nucleotides (SSB35-like mode) and the binding capacity of SsbB is assumed to be ∼65 nucleotides (SSB65-like mode), a dT90 molecule would not be long enough to satisfy the full binding capacities of one SsbA and one SsbB (∼100 nucleotides total). Therefore, the simultaneous binding of SsbA and SsbB to dT90 would presumably require one or both of these proteins to bind using only a portion of their potential binding capacity. The absence of mixed complex formation on dT90 thus suggests that SsbA and SsbB are not able to engage in shared-strand binding in the absence of Mg2+.
The apparent incompatibility in shared-strand binding does not appear to preclude SsbA and SsbB from binding independently on longer dTn oligomers where strand sharing would not be required. Although they were unable to co-assemble on dT90, SsbA and SsbB were able to form a mixed complex on dT100 in the absence of Mg2+ (Figure 6). In this case, a dT100 molecule could potentially provide a ∼35-nucleotide segment for the SsbA and a ∼65-nucleotide segment for the SsbB, and thereby satisfy the full binding capacities of both proteins under the reaction conditions.
DNA binding compatibility also appears to govern the co-assembly of SsbA and SsbB on dTn oligomers when Mg2+ is included in the reaction solution. Although complexes with two SsbAs or two SsbBs were readily formed on either dT100 or dT130 in the presence of Mg2+, mixed complexes with one SsbA and one SsbB were detected only with dT130 (Figure 7). If the binding capacity of SsbA under these conditions is assumed to be ∼65 nucleotides (SSB65-like mode) and the binding capacity of SsbB is also assumed to be ∼65 nucleotides (enhanced SSB65-like mode), a dT130 molecule would be able to satisfy the full binding capacities of one SsbA and one SsbB (∼130 nucleotides total), whereas a dT100 molecule would only be able to partially satisfy the binding capacities of the two proteins. Thus, the formation of mixed complexes on dT130, but not on dT100, indicates that SsbA and SsbB are able to bind independently, but are not able to engage in shared-strand binding, in the presence of Mg2+.
SsbA/B and SsbBRYTP proteins
Although SsbA and SsbB appeared to be incompatible for shared-strand binding, SsbA was able to form mixed complexes under some shared-strand binding conditions with two SSB protein variants: the SsbA/B protein and the SsbBRYTP protein.
The SsbA/B protein, in which the C-terminal domain of the SsbA protein has been replaced with the C-terminal domain from the SsbB protein, was prepared previously to assess the contribution of the C-terminal domains to the DNA binding properties of the SsbA and SsbB proteins [2]. The DNA binding mode preferences of the SsbA/B protein were found to be similar to those of the SsbA protein, suggesting that the primary structural determinants of DNA binding may be contained within the N-terminal domains of the various SSB proteins [2]. Moreover, the SsbA/B protein was able to form a mixed complex with SsbA on dT90 in the absence of Mg2+ and on dT100 in the presence of Mg2+ (Figure 8, and gel not shown). These findings suggest that the inability of SsbA to engage in shared-strand binding with SsbB may not be due to the dissimilar C-terminal domain of the SsbB protein, inasmuch as the C-terminal domain of the SsbA/B protein is identical to that of the SsbB protein. It is possible, however, that the C-terminal domain functions differently in the SsbB protein than in the chimeric SsbA/B protein, and contributes to the incompatibility of SsbA and SsbB in shared-strand binding.
The SsbBRYTP protein, in which the 18HKTN21 sequence of the SsbB protein has been replaced with the corresponding 18RYTP21 sequence from the SsbA protein, was also prepared in an effort to determine the structural basis for the differential DNA binding properties of the SsbA and SsbB proteins. The DNA binding properties of the SsbBRYTP protein were found to be similar to those of the SsbB protein, indicating that the 18HKTP21 sequence may not be responsible for the distinctive DNA binding mode preferences of the SsbB protein (see Experimental Procedures). The SsbBRYTP protein differed from the SsbB protein, however, in that it was able to form a mixed complex with SsbA on dT90 in the absence of Mg2+ (Figure 9). These results suggest that the shared-strand binding incompatibility that was observed with the SsbA and SsbB proteins in absence of Mg2+ was not due to the difference in their preferred DNA binding modes, but may be attributable to the divergent 18RYTP21 and 18HKTN21 sequences of these proteins. The observation that SsbBRYTP was unable to form a mixed complex with SsbA on dT100 in the presence of Mg2+, however, indicates that this sequence difference is not sufficient to account for the shared-strand binding incompatibility that was observed with SsbA and SsbB in the presence of Mg2+. Thus, the introduction of the 18RYTP21 sequence into the SsbB protein has the effect of uncoupling the Mg2+-independent shared-strand binding incompatibility from the Mg2+-dependent incompatibility.
The 18RYTP21 sequence of the SsbA protein is at least partially conserved in the SsbEc protein and in a number of other homotetrameric bacterial SSB proteins whose x-ray crystal structures have been determined (Figure 11). An inspection of the various structures shows that the tertiary folds of the N-terminal domains are similar in all cases, and that the 18RYTP21 sequence of the SsbA protein, and the divergent 18HKTN21 sequence of the SsbB protein, correspond to the Loop 1 region on the outer surface of the subunits of the SSB tetramers (Figure 11) [6]–[7]. The differences in the composition of the Loop 1 region may account for the inability of the SsbA and SsbB proteins to engage in shared-strand binding in the absence of Mg2+. For example, the Loop 1 variations could conceivably affect the precise orientation of the DNA strand as it winds around the individual SSB tetramers, or influence the manner in which two SSB tetramers interact when bound in close proximity on a DNA strand. Other molecular determinants are apparently required, however, for shared-strand binding compatibility in the presence of Mg2+. A definitive determination of the molecular basis for shared-strand binding and DNA binding compatibility will require further structural analysis of the various SSB-dTn complexes. These studies are currently underway in our laboratory.
A, Comparison of sequences from the Loop 1 region of the SSB proteins from Escherichia coli (PDB code 1eyg), Mycobacterium tuberculosis (PDB code 1ue1), Mycobacterium smegmatis (PDB code 1x3e), Helicobacter pylori (PDB code 2vw9), and Streptomyces coelicolor (PDB code 3eiv) with the corresponding sequences from the Streptococcus pneumoniae SsbA and SsbB proteins. B, Structural model for an SsbEc tetramer (green)-single-stranded DNA (blue) complex (based on PDB code 1eyg) [7]. The RYMP sequence in the Loop 1 region of each SsbEc subunit is highlighted (red).
SsbA and SsbB binding compatibility
SsbA is a constitutively-expressed protein, and presumably functions as the primary SSB protein during the routine replication and maintenance of chromosomal DNA in S. pneumoniae (analogous to the SsbEc protein in E. coli). SsbB, in contrast, is induced specifically during natural transformation, and associates transiently with a single-stranded form of the exogenous DNA before the DNA is incorporated into a homologous region of the S. pneumoniae chromosome (there is no analog of the SsbB protein in E.coli) [1], [8].
The extent to which the SsbA and SsbB proteins are functionally interchangeable in these various activities is not clear. Our results, however, indicate that the SsbA and SsbB proteins will be able to bind together on longer single-stranded DNA segments where independent binding is possible, but suggest that they may not co-assemble on shorter single-stranded DNA segments where coordinated interactions between adjacent SSBs are required. The compatibility requirement for shared-strand binding could conceivably serve as a self-recognition mechanism that regulates the manner in which SsbA and SsbB interact in S. pneumoniae.
Materials and Methods
Materials
S. pneumoniae SsbA protein [9], SsbB protein [10], and SsbA/B protein [2] were prepared as previously described. Gel-purified dTn oligomers were from Invitrogen. 32P-end-labeled dTn oligomers were prepared using [γ-32P]ATP (PerkinElmer) and T4 polynucleotide kinase (New England Biolabs).
Preparation and characterization of the SsbBRYTP protein
The SsbBRYTP protein coding sequence, in which the nucleotide sequence of the SsbB protein corresponding to amino acids 18HKTN21 was replaced with a sequence coding for the amino acids 18RYTP21, was generated by overlap-extension PCR mutagenesis. The initial mutagenesis template was our previously described pETssbB construct, which contains the wild type SsbB sequence cloned into the NdeI/HindIII site of the pET21a expression vector (Novagen) [10]. Primer a (5′-CGGATAACAATTCCCCTCTAG-3′) and primer d (5′-TTAGCAGCCGGATCTCAGTGG-3′) flanked the ssbB gene, and primer b (5′-CTTGTCATTTGGAGTGTAACGCAATTCTGGTGTAGAC-3′) and primer c (5′-GAATTGCGTTACACTCCAAATGACAAGTCGGTAGC-3′) were the internal overlapping mutagenic primers (mutagenic bases are underlined). The final SsbBRYTP-coding PCR product was digested with NdeI and HindIII and then cloned into the NdeI/HindIII site of a pET21a expression vector to yield the construct pETssbBRYTP. The insert was sequenced and found to be identical to the expected SsbBRYTP protein coding sequence.
The pETssbBRYTP expression plasmid was introduced into E. coli strain Rosetta(DE3)pLysS (Novagen), and the SsbBRYTP protein was purified from the resulting Rosetta(DE3)pLysS/pETssbBRYTP cells using a procedure analogous to that described previously for the wild type SsbB protein [10]. The final fraction of SsbBRYTP protein was greater than 95% pure as judged by SDS-polyacrylamide gel electrophoresis.
The purified SsbBRYTP protein was characterized using the dT35 binding assay that was described previously for the SsbA and SsbB proteins [2]. The results were similar to those that were obtained with the wild type SsbB protein, and indicated that the SsbBRYTP protein was able to bind two dT35 molecules in the absence of Mg2+, and two dT35 molecules (with positive intersubunit cooperativity) in the presence of 10 mM Mg2+.
Polyacrylamide gel electrophoresis assays
The dTn binding reaction solutions (30 µl) contained 25 mM Tris acetate (pH 7.5), 5% glycerol, 1 mM dithiothreitol, and the concentrations of magnesium acetate, dTn (32P-end-labeled), and SSB protein given in the figure legends. The reactions solutions were incubated at 25°C for 15 min, and then 3 µl of gel loading solution (0.25% bromophenol blue, 40% sucrose) was added. An aliquot (20 µl) of the final solution was analyzed by electrophoresis on 5% native polyacrylamide gels using a buffer system consisting of 25 mM Tris acetate (pH 7.5) and the same concentration of magnesium acetate as in the reaction solutions. The bands corresponding to unbound and SSB-bound dTn oligomers were visualized using a Fuji FLA-7000 imager. The specific protein concentrations for the individual gels in Figures 2–9 were selected to illustrate the concentration-dependent formation of the various SSB-dTn complexes.
Author Contributions
Conceived and designed the experiments: FRB BS. Performed the experiments: BS GA. Analyzed the data: FRB BS. Contributed reagents/materials/analysis tools: FRB BS GA. Wrote the paper: FRB BS.
References
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