Genetic recombination in Bacillus subtilis: a division of labor between two single-strand DNA-binding proteins

We have investigated the structural, biochemical and cellular roles of the two single-stranded (ss) DNA-binding proteins from Bacillus subtilis, SsbA and SsbB. During transformation, SsbB localizes at the DNA entry pole where it binds and protects internalized ssDNA. The 2.8-Å resolution structure of SsbB bound to ssDNA reveals a similar overall protein architecture and ssDNA-binding surface to that of Escherichia coli SSB. SsbA, which binds ssDNA with higher affinity than SsbB, co-assembles onto SsbB-coated ssDNA and the two proteins inhibit ssDNA binding by the recombinase RecA. During chromosomal transformation, the RecA mediators RecO and DprA provide RecA access to ssDNA. Interestingly, RecO interaction with ssDNA-bound SsbA helps to dislodge both SsbA and SsbB from the DNA more efficiently than if the DNA is coated only with SsbA. Once RecA is nucleated onto the ssDNA, RecA filament elongation displaces SsbA and SsbB and enables RecA-mediated DNA strand exchange. During plasmid transformation, RecO localizes to the entry pole and catalyzes annealing of SsbA- or SsbA/SsbB-coated complementary ssDNAs to form duplex DNA with ssDNA tails. Our results provide a mechanistic framework for rationalizing the coordinated events modulated by SsbA, SsbB and RecO that are crucial for RecA-dependent chromosomal transformation and RecA-independent plasmid transformation.     

Roles of Bacillus subtilis DprA and SsbA in RecA-mediated Genetic Recombination

Bacillus subtilis competence-induced RecA, SsbA, SsbB, and DprA are required to internalize and to recombine single-stranded (ss) DNA with homologous resident duplex. RecA, in the ATP·Mg²⁺-bound form (RecA·ATP), can nucleate and form filament onto ssDNA but is inactive to catalyze DNA recombination. We report that SsbA or SsbB bound to ssDNA blocks the RecA filament formation and fails to activate recombination. DprA facilitates RecA filamentation; however, the filaments cannot engage in DNA recombination. When ssDNA was preincubated with SsbA, but not SsbB, DprA was able to activate DNA strand exchange dependent on RecA·ATP. This work demonstrates that RecA·ATP, in concert with SsbA and DprA, catalyzes DNA strand exchange, and SsbB is an accessory factor in the reaction. In contrast, RecA·dATP efficiently catalyzes strand exchange even in the absence of single-stranded binding proteins or DprA, and addition of the accessory factors marginally improved it. We proposed that the RecA-bound nucleotide (ATP and to a lesser extent dATP) might dictate the requirement for accessory factors.     

RecO-mediated DNA homology search and annealing is facilitated by SsbA

Bacillus subtilis RecO plays a central role in recombinational repair and genetic recombination by (i) stimulating RecA filamentation onto SsbA-coated single-stranded (ss) DNA, (ii) modulating the extent of RecA-mediated DNA strand exchange and (iii) promoting annealing of complementary DNA strands. Here, we report that RecO-mediated strand annealing is facilitated by cognate SsbA, but not by a heterologous one. Analysis of non-productive intermediates reveals that RecO interacts with SsbA-coated ssDNA, resulting in transient ternary complexes. The self-interaction of ternary complexes via RecO led to the formation of large nucleoprotein complexes. In the presence of homology, SsbA, at the nucleoprotein, removes DNA secondary structures, inhibits spontaneous strand annealing and facilitates RecO loading onto SsbA–ssDNA complex. RecO relieves SsbA inhibition of strand annealing and facilitates transient and random interactions between homologous naked ssDNA molecules. Finally, both proteins lose affinity for duplex DNA. Our results provide a mechanistic framework for rationalizing protein release and dsDNA zippering as coordinated events that are crucial for RecA-independent plasmid transformation.     

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 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 dT(n) oligomers (as judged by polyacrylamide gel-shift assays) and in its binding to the longer naturally occurring 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.     

Chromosomal transformation in Bacillus subtilis is a non-polar recombination reaction

Natural chromosomal transformation is one of the primary driving forces of bacterial evolution. This reaction involves the recombination of the internalized linear single-stranded (ss) DNA with the homologous resident duplex via RecA-mediated integration in concert with SsbA and DprA or RecO. We show that sequence divergence prevents Bacillus subtilis chromosomal transformation in a log-linear fashion, but it exerts a minor effect when the divergence is localized at a discrete end. In the nucleotide bound form, RecA shows no apparent preference to initiate recombination at the 3′- or 5′-complementary end of the linear duplex with circular ssDNA, but nucleotide hydrolysis is required when heterology is present at both ends. RecA·dATP initiates pairing of the linear 5′ and 3′ complementary ends, but only initiation at the 5′-end remains stably paired in the absence of SsbA. Our results suggest that during gene transfer RecA·ATP, in concert with SsbA and DprA or RecO, shows a moderate preference for the 3′-end of the duplex. We show that RecA-mediated recombination initiated at the 3′- or 5′-complementary end might have significant implication on the ecological diversification of bacterial species with natural transformation.     

Role of the single-stranded DNA-binding protein SsbB in pneumococcal transformation: maintenance of a reservoir for genetic plasticity

Bacteria encode a single-stranded DNA (ssDNA) binding protein (SSB) crucial for genome maintenance. In Bacillus subtilis and Streptococcus pneumoniae, an alternative SSB, SsbB, is expressed uniquely during competence for genetic transformation, but its precise role has been disappointingly obscure. Here, we report our investigations involving comparison of a null mutant (ssbB(-)) and a C-ter truncation (ssbBΔ7) of SsbB of S. pneumoniae, the latter constructed because SSBs' acidic tail has emerged as a key site for interactions with partner proteins. We provide evidence that SsbB directly protects internalized ssDNA. We show that SsbB is highly abundant, potentially allowing the binding of ~1.15 Mb ssDNA (half a genome equivalent); that it participates in the processing of ssDNA into recombinants; and that, at high DNA concentration, it is of crucial importance for chromosomal transformation whilst antagonizing plasmid transformation. While the latter observation explains a long-standing observation that plasmid transformation is very inefficient in S. pneumoniae (compared to chromosomal transformation), the former supports our previous suggestion that SsbB creates a reservoir of ssDNA, allowing successive recombination cycles. SsbBΔ7 fulfils the reservoir function, suggesting that SsbB C-ter is not necessary for processing protein(s) to access stored ssDNA. We propose that the evolutionary raison d'être of SsbB and its abundance is maintenance of this reservoir, which contributes to the genetic plasticity of S. pneumoniae by increasing the likelihood of multiple transformation events in the same cell.     

Dynamic structures of Bacillus subtilis RecN-DNA complexes

Genetic and cytological evidences suggest that Bacillus subtilis RecN acts prior to and after end-processing of DNA double-strand ends via homologous recombination, appears to participate in the assembly of a DNA repair centre and interacts with incoming single-stranded (ss) DNA during natural transformation. We have determined the architecture of RecN–ssDNA complexes by atomic force microscopy (AFM). ATP induces changes in the architecture of the RecN–ssDNA complexes and stimulates inter-complex assembly, thereby increasing the local concentration of DNA ends. The large CII and CIII complexes formed are insensitive to SsbA (counterpart of Escherichia coli SSB or eukaryotic RPA protein) addition, but RecA induces dislodging of RecN from the overhangs of duplex DNA molecules. Reciprocally, in the presence of RecN, RecA does not form large RecA–DNA networks. Based on these results, we hypothesize that in the presence of ATP, RecN tethers the 3′-ssDNA ends, and facilitates the access of RecA to the high local concentration of DNA ends. Then, the resulting RecA nucleoprotein filaments, on different ssDNA segments, might promote the simultaneous genome-wide homology search.     

Bacillus subtilis RecO and SsbA are crucial for RecA-mediated recombinational DNA repair

Genetic data have revealed that the absence of Bacillus subtilis RecO and one of the end-processing avenues (AddAB or RecJ) renders cells as sensitive to DNA damaging agents as the null recA, suggesting that both end-resection pathways require RecO for recombination. RecA, in the rATP·Mg²⁺ bound form (RecA·ATP), is inactive to catalyze DNA recombination between linear double-stranded (ds) DNA and naked complementary circular single-stranded (ss) DNA. We showed that RecA·ATP could not nucleate and/or polymerize on SsbA·ssDNA or SsbB·ssDNA complexes. RecA·ATP nucleates and polymerizes on RecO·ssDNA·SsbA complexes more efficiently than on RecO·ssDNA·SsbB complexes. Limiting SsbA concentrations were sufficient to stimulate RecA·ATP assembly on the RecO·ssDNA·SsbB complexes. RecO and SsbA are necessary and sufficient to ‘activate’ RecA·ATP to catalyze DNA strand exchange, whereas the AddAB complex, RecO alone or in concert with SsbB was not sufficient. In presence of AddAB, RecO and SsbA are still necessary for efficient RecA·ATP-mediated three-strand exchange recombination. Based on genetic and biochemical data, we proposed that SsbA and RecO (or SsbA, RecO and RecR in vivo) are crucial for RecA activation for both, AddAB and RecJ–RecQ (RecS) recombinational repair pathways.     

RecFOR Proteins Target RecA Protein to a DNA Gap with Either DNA or RNA at the 5′ Terminus: IMPLICATION FOR REPAIR OF STALLED REPLICATION FORKS*

The repair of single-stranded gaps in duplex DNA by homologous recombination requires the proteins of the RecF pathway. The assembly of RecA protein onto gapped DNA (gDNA) that is complexed with the single-stranded DNA-binding protein is accelerated by the RecF, RecO, and RecR (RecFOR) proteins. Here, we show the RecFOR proteins specifically target RecA protein to gDNA even in the presence of a thousand-fold excess of single-stranded DNA (ssDNA). The binding constant of RecF protein, in the presence of the RecOR proteins, to the junction of ssDNA and dsDNA within a gap is 1–2 nm, suggesting that a few RecF molecules in the cell are sufficient to recognize gDNA. We also found that the nucleation of a RecA filament on gDNA in the presence of the RecFOR proteins occurs at a faster rate than filament elongation, resulting in a RecA nucleoprotein filament on ssDNA for 1000–2000 nucleotides downstream (5′ → 3′) of the junction with duplex DNA. Thus, RecA loading by RecFOR is localized to a region close to a junction. RecFOR proteins also recognize RNA at the 5′-end of an RNA-DNA junction within an ssDNA gap, which is compatible with their role in the repair of lagging strand gaps at stalled replication forks.     

Bacillus subtilis RecA and its accessory factors,RecF, RecO,RecR and RecX,are required for spore resistance to DNA double-strand break

Bacillus subtilis RecA is important for spore resistance to DNA damage, even though spores contain a single non-replicating genome. We report that inactivation of RecA or its accessory factors, RecF, RecO, RecR and RecX, drastically reduce survival of mature dormant spores to ultrahigh vacuum desiccation and ionizing radiation that induce single strand (ss) DNA nicks and double-strand breaks (DSBs). The presence of non-cleavable LexA renders spores less sensitive to DSBs, and spores impaired in DSB recognition or end-processing show sensitivities to X-rays similar to wild-type. In vitro RecA cannot compete with SsbA for nucleation onto ssDNA in the presence of ATP. RecO is sufficient, at least in vitro, to overcome SsbA inhibition and stimulate RecA polymerization on SsbA-coated ssDNA. In the presence of SsbA, RecA slightly affects DNA replication in vitro, but addition of RecO facilitates RecA-mediated inhibition of DNA synthesis. We propose that repairing of the DNA lesions generates a replication stress to germinating spores, and the RecA·ssDNA filament might act by preventing potentially dangerous forms of DNA repair occurring during replication. RecA might stabilize a stalled fork or prevent or promote dissolution of reversed forks rather than its cleavage that should require end-processing.     

Transformation of Streptococcus pneumoniae relies on DprA- and RecA-dependent protection of incoming DNA single strands

Seventy-five years after the discovery of transformation with Streptococcus pneumoniae, it is remarkable how little we know of the proteins that interact with incoming single strands in the early processing of transforming DNA. In this work, we used as donor DNA in transformation a radioactively labelled homologous fragment to examine the fate of the single-stranded (ssDNA) products of uptake in cells mutant for DprA or RecA, two proteins essential for transformation. Fifteen minutes after uptake, the labelling of specific chromosomal restriction fragments that demonstrated homologous integration in the wild type was not detected in dprA or recA cells, indicating that in the mutants incoming ssDNA could not be processed into recombinants. Investigation of the fate of donor label 1 min after uptake revealed that incoming ssDNA was immediately degraded in the absence of DprA or RecA. Our results demonstrate that incoming ssDNA requires active protection prior to the RecA-driven search for homology and that both DprA and RecA are needed for this protection.     

SSB protein limits RecOR binding onto single-stranded DNA

The RecO and RecR proteins form a complex that promotes the nucleation of RecA protein filaments onto SSB protein-coated single-stranded DNA (ssDNA). However, even when RecO and RecR proteins are provided at optimal concentrations, the loading of RecA protein is surprisingly slow, typically proceeding with a lag of 10 min or more. The rate-limiting step in RecOR-promoted RecA nucleation is the binding of RecOR protein to ssDNA, which is inhibited by SSB protein despite the documented interaction between RecO and SSB. Full activity of RecOR is seen only when RecOR is preincubated with ssDNA prior to the addition of SSB. The slow binding of RecOR to SSB-coated ssDNA involves the C terminus of SSB. When an SSB variant that lacks the C-terminal 8 amino acids is used, the capacity of RecOR to facilitate RecA loading onto the ssDNA is largely abolished. The results are used in an expanded model for RecOR action.     

Viral SPP1 DNA is infectious in naturally competent Bacillus subtilis cells: inter- and intramolecular recombination pathways

A proteolyzed bacteriophage (phage) might release its DNA into the environment. Here, we define the recombination functions required to resurrect an infective lytic phage from inactive environmental viral DNA in naturally competent Bacillus subtilis cells. Using phage SPP1 DNA, a model that accounts for the obtained data is proposed (i) the DNA uptake apparatus takes up environmental SPP1 DNA, fragments it, and incorporates into the cytosol different linear single-stranded (ss) DNA molecules shorter than genome-length; (ii) the SsbA-DprA mediator loads RecA onto any fragmented linear SPP1 ssDNA, but negative modulators (RecX and RecU) promote a net RecA disassembly from these ssDNAs not homologous to the host genome; (iii) single strand annealing (SSA) proteins, DprA and RecO, anneal the SsbA- or SsbB-coated complementary strands, yielding tailed SPP1 duplex intermediates; (iv) RecA polymerized on these tailed intermediates invades a homologous region in another incomplete molecule, and in concert with RecD2 helicase, reconstitutes a complete linear phage genome with redundant regions at the ends of the molecule; and (v) DprA, RecO or viral G35P SSA, may catalyze the annealing of these terminally redundant regions, alone or with the help of an exonuclease, to produce a circular unit-length duplex viral genome ready to initiate replication.     

A key presynaptic role in transformation for a widespread bacterial protein: DprA conveys incoming ssDNA to RecA

Natural transformation is a mechanism for genetic exchange in many bacterial genera. It proceeds through the uptake of exogenous DNA and subsequent homology-dependent integration into the genome. In Streptococcus pneumoniae, this integration requires the ubiquitous recombinase, RecA, and DprA, a protein of unknown function widely conserved in bacteria. To unravel the role of DprA, we have studied the properties of the purified S. pneumoniae protein and its Bacillus subtilis ortholog (Smf). We report that DprA and Smf bind cooperatively to single-stranded DNA (ssDNA) and that these proteins both self-interact and interact with RecA. We demonstrate that DprA-RecA-ssDNA filaments are produced and that these filaments catalyze the homology-dependent formation of joint molecules. Finally, we show that while the Escherichia coli ssDNA-binding protein SSB limits access of RecA to ssDNA, DprA lowers this barrier. We propose that DprA is a new member of the recombination-mediator protein family, dedicated to natural bacterial transformation.     

Multiple interactions among the competence proteins of Bacillus subtilis

Proteins required for transformation of Bacillus subtilis and other competent bacteria are associated with the membrane or reside in the cytosol. Previous work has shown that RecA, ComGA, ComFA and SsbB are directed to the cell poles in competent cells, and that the uptake of transforming DNA occurs preferentially at the poles. We show that ComGA, ComFA, DprA (Smf), SsbB (YwpH), RecA and YjbF (CoiA) are located at the cell poles, where they appear to colocalize. Using fluorescence resonance energy transfer, we have shown that these six competent (Com) proteins reside in close proximity to one another. This conclusion was supported by the effects of com gene knockouts on the stabilities of Com proteins. Data obtained from the com gene knockout studies, as well as information from other sources, extend the list of proteins in the transformation complex to include ComEC and ComEA. Because ComGA and ComFA are membrane-associated, while DprA, SsbB, RecA and YjbF are soluble, a picture emerges of a large multiprotein polar complex, involving both cytosolic and membrane proteins. This complex mediates the binding and uptake of single-stranded DNA, the protection of this DNA from cellular nucleases and its recombination with the recipient chromosome.     

DNA binding compatibility of the Streptococcus pneumoniae SsbA and SsbB proteins

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 dT_n 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 dT_n 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 dT_n 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.     

The direction of RecA protein assembly onto single strand DNA is the same as the direction of strand assimilation during strand exchange

The RecA protein of Escherichia coli optimally promotes DNA strand exchange reactions in the presence of the single strand DNA-binding protein of E. coli (SSB protein). Under these conditions, assembly of RecA protein onto single-stranded DNA (ssDNA) occurs in three steps. First, the ssDNA is rapidly covered by SSB protein. The binding of RecA protein is then initiated by nucleation of a short tract of RecA protein onto the ssDNA. Finally, cooperative polymerization of additional RecA protein accompanied by displacement of SSB protein results in a ssDNA-RecA protein filament (Griffith, J. D., Harris, L. D., and Register, J. C. (1984) Cold Spring Harbor Symp. Quant. Biol. 49, 553-559). We report here that RecA protein assembly onto circular ssDNA yields RecA protein-covered circles in which greater than 85% are completely covered by RecA protein with no remaining SSB protein-covered segments (as detected by electron microscopy). However, when linear ssDNA is used, 90% of the filaments contain a short segment at one end complexed with SSB protein. This suggests that RecA protein assembly is unidirectional. Visualization of the assembly of RecA protein onto either long ssDNA tails (containing either 5' or 3' termini) or ssDNA gaps generated in double strand DNA allowed us to determine that the RecA protein polymerizes in the 5' to 3' direction on ssDNA and preferentially nucleates at ssDNA-double strand DNA junctions containing 5' termini.     

Molecular determinants of the DprA?RecA interaction for nucleation on ssDNA

Natural transformation is a major mechanism of horizontal gene transfer in bacteria that depends on DNA recombination. RecA is central to the homologous recombination pathway, catalyzing DNA strand invasion and homology search. DprA was shown to be a key binding partner of RecA acting as a specific mediator for its loading on the incoming exogenous ssDNA. Although the 3D structures of both RecA and DprA have been solved, the mechanisms underlying their cross-talk remained elusive. By combining molecular docking simulations and experimental validation, we identified a region on RecA, buried at its self-assembly interface and involving three basic residues that contact an acidic triad of DprA previously shown to be crucial for the interaction. At the core of these patches, ^DprAM238 and ^RecAF230 are involved in the interaction. The other DprA binding regions of RecA could involve the N-terminal α-helix and a DNA-binding region. Our data favor a model of DprA acting as a cap of the RecA filament, involving a DprA−RecA interplay at two levels: their own oligomeric states and their respective interaction with DNA. Our model forms the basis for a mechanistic explanation of how DprA can act as a mediator for the loading of RecA on ssDNA.     

Assembly of presynaptic filaments. Factors affecting the assembly of RecA protein onto single-stranded DNA

We have previously shown that the assembly of RecA protein onto single-stranded DNA (ssDNA) facilitated by SSB protein occurs in three steps: (1) rapid binding of SSB protein to the ssDNA; (2) nucleation of RecA protein onto this template; and (3) co-operative polymerization of additional RecA protein to yield presynaptic filaments. Here, electron microscopy has been used to further explore the parameters of this assembly process. The optimal extent of presynaptic filament formation required at least one RecA protein monomer per three nucleotides, high concentrations of ATP (greater than 3 mM in the presence of 12 mM-Mg2+), and relatively low concentrations of SSB protein (1 monomer per 18 nucleotides). Assembly was depressed threefold when SSB protein was added to one monomer per nine nucleotides. These effects appeared to be exerted at the nucleation step. Following nucleation, RecA protein assembled onto ssDNA at net rates that varied from 250 to 900 RecA protein monomers per minute, with the rate inversely related to the concentration of SSB protein. Combined sucrose sedimentation and electron microscope analysis established that SSB protein was displaced from the ssDNA during RecA protein assembly.     

Expression and purification of the SsbB protein from Streptococcus pneumoniae

The Gram positive bacterium, Streptococcus pneumoniae, has two genes, designated ssbA and ssbB, which are predicted to encode single-stranded DNA binding proteins (SSB proteins). We have shown previously that the SsbA protein is similar in size and in biochemical properties to the well-characterized SSB protein from Escherichia coli. The SsbB protein, in contrast, is a smaller protein and has no counterpart in E. coli. This report describes the development of an expression system and purification procedure for the SsbB protein. The ssbB gene was amplified from genomic S. pneumoniae DNA and cloned into the E. coli expression vector, pET21a. Although, we had shown previously that the SsbA protein is strongly expressed from pET21a in the E. coli strain BL21(DE3)pLysS, no expression of the SsbB protein was detected in these cells. However, the SsbB protein was strongly expressed from pET21a in the Rosetta(DE3)pLysS strain, a derivative of BL21(DE3)pLysS which supplies the tRNAs for six codons that are used infrequently in E. coli. The differential expression of the two SSB proteins in the parent BL21(DE3)pLysS strain was apparently due to the presence of two rare codons in the ssbB gene sequence that are not present in the ssbA sequence. Using the Rosetta(DE3)pLysS/pETssbB expression system, a protocol was developed in which the SsbB protein was purified to apparent homogeneity. DNA binding assays confirmed that the purified SsbB protein had single-stranded DNA binding activity. The expression and purification procedures reported here will facilitate further investigations into the biological role of the SsbB protein.