Dual pathogenicity island transfer by piggybacking lateral transduction.

Lateral transduction (LT) is the process by which temperate phages mobilize large sections of bacterial genomes. Despite its importance, LT has only been observed during prophage induction. Here, we report that superantigen-carrying staphylococcal pathogenicity islands (SaPIs) employ a related but more versatile and complex mechanism of gene transfer to drive chromosomal hypermobility while self-transferring with additional virulence genes from the host. We found that after phage infection or prophage induction, activated SaPIs form concatamers in the bacterial chromosome by switching between parallel genomic tracks in replication bubbles. This dynamic life cycle enables SaPIbov1 to piggyback its LT of staphylococcal pathogenicity island vSaα, which encodes an array of genes involved in host-pathogen interactions, allowing both islands to be mobilized intact and transferred in a single infective particle. Our findings highlight previously unknown roles of pathogenicity islands in bacterial virulence and show that their evolutionary impact extends beyond the genes they carry.

Control of resistance against bacteriophage killing by a metabolic regulator in meningitis-associated Escherichia coli.

Ecologically beneficial traits in bacteria are encoded by intrinsic and horizontally acquired genes. However, such traits are not universal, and the highly mosaic nature of bacterial genomes requires control at the transcriptional level to drive these processes. It has emerged that regulatory flexibility is widespread in the Escherichia coli species, whereby preexisting transcription factors can acquire new and unrelated roles in regulating beneficial traits. DsdC is the regulator of D-serine tolerance in E. coli, is essential for D-serine catabolism, and is often encoded by two copies in neonatal meningitis-associated E. coli (NMEC). Here, we reveal that DsdC is a global regulator of transcription in NMEC and does not require D-serine for the control of novel beneficial traits. We show that DsdC binds the chromosome in an unusual manner, with many binding sites arranged in clusters spanning entire operons and within gene coding sequences, such as neuO. Importantly, we identify neuO as the most significantly down-regulated gene in a strain deleted for both dsdC copies, in both the presence and absence of D-serine. NeuO is prophage encoded in several NMEC K1 isolates and mediates capsule O-acetylation but has no effect on attachment to or invasion of human brain endothelial cells. Instead, we demonstrate that NeuO provides resistance against K1 bacteriophage attack and that this critical function is regulated by DsdC. This work highlights how a horizontally acquired enzyme that functions in cell-surface modulation can be controlled by an intrinsic regulator to provide a key ecological benefit to an E. coli pathotype.

The RecD2 helicase balances RecA activities.

DNA helicases of the RecD2 family are ubiquitous. Bacillus subtilis RecD2 in association with the single-stranded binding protein SsbA may contribute to replication fork progression, but its detailed action remains unknown. In this work, we explore the role of RecD2 during DNA replication and its interaction with the RecA recombinase. RecD2 inhibits replication restart, but this effect is not observed in the absence of SsbA. RecD2 slightly affects replication elongation. RecA inhibits leading and lagging strand synthesis, and RecD2, which physically interacts with RecA, counteracts this negative effect. In vivo results show that recD2 inactivation promotes RecA-ssDNA accumulation at low mitomycin C levels, and that RecA threads persist for a longer time after induction of DNA damage. In vitro, RecD2 modulates RecA-mediated DNA strand-exchange and catalyzes branch migration. These findings contribute to our understanding of how RecD2 may contribute to overcome a replicative stress, removing RecA from the ssDNA and, thus, it may act as a negative modulator of RecA filament growth.

Nucleoid-associated Rok differentially affects chromosomal transformation on Bacillus subtilis recombination-deficient cells.

Rok, a Bacillus subtilis nucleoid-associated protein (NAP), negatively regulates competence development and silences xenogeneic genes. We show that rok inactivation increases rpoB482 natural intraspecies chromosomal transformation (CT) and plasmid transformation to a different extent. In ΔaddAB, ΔrecO, recF15, ΔrecU, ΔruvAB or rec+ cells intraspecies CT significantly increases, but the ΔrecD2 mutation reduces, and the ΔrecX, ΔradA or ΔdprA mutation further decreases CT in the Δrok context when compared to rok+ cells. These observations support the idea that rok inactivation, by altering the topology of the recipient DNA, differentially affects the integration of homologous DNA in rec-deficient strains, and in minor extent the competent subpopulation size. The impairment of other NAP (Hbsu or LrpC) also increased intra- and interspecies CT (nonself-DNA, ~8% nucleotide sequence divergence) in rec+ cells, but differentially reduced both types of CTs in certain rec-deficient strains. We describe that rok inactivation significantly stimulates intra and interspecies CT but differentially reduces them in transformation-deficient cells, perhaps by altering the nucleoid architecture. We extend the observation to other NAPs (Hbsu, LrpC).

Recombination proteins differently control the acquisition of homeologous DNA during Bacillus subtilis natural chromosomal transformation.

Natural chromosomal transformation (CT) plays a major role in prokaryote evolution, yet factors that govern the integration of DNA from related species remain poorly understood. We show that in naturally competent Bacillus subtilis cells the acquisition of homeologous sequences is governed by sequence divergence (SD). Integration initiates in a minimal efficient processing segment via homology-directed CT, and its frequency decreases log-linearly with increased SD up to 15%. Beyond this and up to 23% SD the interspecies boundaries prevail, the CT frequency marginally decreases, and short (<10-nucleotides) segments are integrated via homology-facilitated micro-homologous integration. Both mechanisms are RecA dependent. We identify the other recombination proteins required for the acquisition of homeologous DNA. The absence of AddAB, RecF, RecO, RuvAB or RecU, crucial for repair-by-recombination, did not affect CT. However, dprA, radA, recJ, recX or recD2 inactivation strongly decreased intraspecies and interspecies CT. Interspecies CT was not detected beyond ~8% SD in ΔdprA, ~10% in ΔrecJ, ΔradA, ΔrecX and ~14% in ΔrecD2 cells. We propose that DprA, RecX, RadA/Sms, RecJ and RecD2 accessory proteins are important for the generation of genetic diversity. Together with RecA, they facilitate gene acquisition from bacteria of related species.

Reversible regulation of conjugation of Bacillus subtilis plasmid pLS20 by the quorum sensing peptide responsive anti-repressor RappLS20.

Quorum sensing plays crucial roles in bacterial communication including in the process of conjugation, which has large economical and health-related impacts by spreading antibiotic resistance. The conjugative Bacillus subtilis plasmid pLS20 uses quorum sensing to determine when to activate the conjugation genes. The main conjugation promoter, Pc, is by default repressed by a regulator RcopLS20 involving DNA looping. A plasmid-encoded signalling peptide, Phr*pLS20, inactivates the anti-repressor of RcopLS20, named RappLS20, which belongs to the large group of RRNPP family of regulatory proteins. Here we show that DNA looping occurs through interactions between two RcopLS20 tetramers, each bound to an operator site. We determined the relative promoter strengths for all the promoters involved in synthesizing the regulatory proteins of the conjugation genes, and constructed an in vivo system uncoupling these regulatory genes to show that RappLS20 is sufficient for activating conjugation in vivo. We also show that RappLS20 actively detaches RcopLS20 from DNA by preferentially acting on the RcopLS20 molecules involved in DNA looping, resulting in sequestration but not inactivation of RcopLS20. Finally, results presented here in combination with our previous results show that activation of conjugation inhibits competence and competence development inhibits conjugation, indicating that both processes are mutually exclusive.

Bacillus subtilis RarA Acts as a Positive RecA Accessory Protein.

Ubiquitous RarA AAA+ ATPases play crucial roles in the cellular response to blocked replication forks in pro- and eukaryotes. Here, we provide evidence that absence of RarA reduced the viability of ΔrecA, ΔrecO, and recF15 cells during unperturbed growth. The rarA gene was epistatic to recO and recF genes in response to H2O2- or MMS-induced DNA damage. Conversely, the inactivation of rarA partially suppressed the HR defect of mutants lacking end-resection (ΔaddAB, ΔrecJ, ΔrecQ, ΔrecS) or branch migration (ΔruvAB, ΔrecG, ΔradA) activity. RarA contributes to RecA thread formation, that are thought to be the active forms of RecA during homology search. The absence of RarA reduced RecA accumulation, and the formation of visible RecA threads in vivo upon DNA damage. When ΔrarA was combined with mutations in genuine RecA accessory genes, RecA accumulation was further reduced in ΔrarA ΔrecU and ΔrarA ΔrecX double mutant cells, and was blocked in ΔrarA recF15 cells. These results suggest that RarA contributes to the assembly of RecA nucleoprotein filaments onto single-stranded DNA, and possibly antagonizes RecA filament disassembly.

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.

Surface Exclusion Revisited: Function Related to Differential Expression of the Surface Exclusion System of Bacillus subtilis Plasmid pLS20.

During conjugation a genetic element is transferred from a bacterial donor to a recipient cell via a connecting channel. It is the major route responsible for the spread of antibiotic resistance. Conjugative elements can contain exclusion system(s) that inhibit its transfer to a cell already harboring the element. Our limited knowledge on exclusion systems is mainly based on plasmids of Gram-negative bacteria. Here we studied the conjugative plasmid pLS20 of the Gram-positive Bacillus subtilis. We demonstrate that pLS20 contains an exclusion system and identified the single gene responsible for exclusion, named sespLS20 , which is embedded in the conjugation operon. SespLS20 is the founding member of a novel family of surface exclusion proteins encoded by conjugative elements of Gram-positive origin. We show that the extent of surface exclusion correlates with the level of sespLS20 expression, and that sespLS20 is expressed at basal low-levels in all donor cells but becomes highly expressed in conjugating cells. Accordingly, the transfer of pLS20 from a conjugation-primed donor cell to an un-primed or conjugation-primed donor is inhibited moderately and very efficiently, respectively. The consequences of this differential regulation, which appears to be a conserved feature of surface exclusion systems of Gram-positive and Gram-negative origin, are discussed.

Bacillus subtilis RecA interacts with and loads RadA/Sms to unwind recombination intermediates during natural chromosomal transformation.

During natural transformation Bacillus subtilis RecA, polymerized onto the incoming single-stranded (ss) DNA, catalyses DNA strand invasion resulting in a displacement loop (D-loop) intermediate. A null radA mutation impairs chromosomal transformation, and RadA/Sms unwinds forked DNA in the 5'→3' direction. We show that in the absence of RadA/Sms competent cells require the RecG translocase for natural chromosomal transformation. RadA/Sms tetracysteine motif (C13A and C13R) variants, which fail to interact with RecA, are also deficient in plasmid transformation, but this defect is suppressed by inactivating recA. The RadA/Sms C13A and C13R variants bind ssDNA, and this interaction stimulates their ATPase activity. Wild-type (wt) RadA/Sms interacts with and inhibits the ATPase activity of RecA, but RadA/Sms C13A fails to do it. RadA/Sms and its variants, C13A and C13R, bound to the 5'-tail of a DNA substrate, unwind DNA in the 5'→3' direction. RecA interacts with and loads wt RadA/Sms to promote unwinding of a non-cognate 3'-tailed or 5'-fork DNA substrate, but RadA/Sms C13A or C13R fail to do it. We propose that wt RadA/Sms interaction with RecA is crucial to recruit the former onto D-loop DNA, and both proteins in concert catalyse D-loop extension to favour integration of ssDNA during chromosomal transformation.

Bacillus subtilis RadA/Sms contributes to chromosomal transformation and DNA repair in concert with RecA and circumvents replicative stress in concert with DisA.

Bacillus subtilis radA is epistatic to disA and recA genes in response to methyl methane sulfonate- and 4-nitroquinoline-1-oxide-induced DNA damage. We show that ΔradA cells were sensitive to mitomycin C- and H2O2-induced damage and impaired in natural chromosomal transformation, whereas cells lacking DisA were not. RadA/Sms mutants in the conserved H1 (K104A and K104R) or KNRFG (K255A and K255R) motifs fail to rescue the sensitivity of ΔradA in response to the four different DNA damaging agents. A RadA/Sms H1 or KNRFG mutation impairs both chromosomal and plasmid transformation, but the latter defect was suppressed by inactivating RecA. RadA/Sms K255A, K255R and wild type RadA/Sms reduced the diadenylate cyclase activity of DisA, whereas RadA/Sms K104A and K104R blocked it. Single-stranded and Holliday junction DNA are preferentially bound over double-stranded DNA by RadA/Sms and its variants. Moreover, RadA/Sms ATPase activity was neither stimulated by a variety of DNA substrates nor by DisA. RadA/Sms possesses a 5´â†’3´ DNA helicase activity. The RadA/Sms mutants neither hydrolyze ATP nor unwind DNA. Thus, we propose that RadA/Sms has two activities: to modulate DisA and to promote RecA-mediated DNA strand exchange. Both activities are required to coordinate responses to replicative stress and genetic recombination.

Bacillus subtilis MutS Modulates RecA-Mediated DNA Strand Exchange Between Divergent DNA Sequences.

The efficiency of horizontal gene transfer, which contributes to acquisition and spread of antibiotic resistance and pathogenicity traits, depends on nucleotide sequence and different mismatch-repair (MMR) proteins participate in this process. To study how MutL and MutS MMR proteins regulate recombination across species boundaries, we have studied natural chromosomal transformation with DNA up to ∼23% sequence divergence. We show that Bacillus subtilis natural chromosomal transformation decreased logarithmically with increased sequence divergence up to 15% in wild type (wt) cells or in cells lacking MutS2 or mismatch repair proteins (MutL, MutS or both). Beyond 15% sequence divergence, the chromosomal transformation efficiency is ∼100-fold higher in ΔmutS and ΔmutSL than in ΔmutS2 or wt cells. In the first phase of the biphasic curve (up to 15% sequence divergence), RecA-catalyzed DNA strand exchange contributes to the delineation of species, and in the second phase, homology-facilitated illegitimate recombination might aid in the restoration of inactivated genes. To understand how MutS modulates the integration process, we monitored DNA strand exchange reactions using a circular single-stranded DNA and a linear double-stranded DNA substrate with an internal 77-bp region with ∼16% or ∼54% sequence divergence in an otherwise homologous substrate. The former substrate delayed, whereas the latter halted RecA-mediated strand exchange. Interestingly, MutS addition overcame the heterologous barrier. We propose that MutS assists DNA strand exchange by facilitating RecA disassembly, and indirectly re-engagement with the homologous 5'-end of the linear duplex. Our data supports the idea that MutS modulates bidirectional RecA-mediated integration of divergent sequences and this is important for speciation.

Measurement of the Length of the Integrated Donor DNA during Bacillus subtilis Natural Chromosomal Transformation.

For natural transformation to occur, bacterial cells must first develop a programmed physiological state called competence. Competence in Bacillus subtilis, which occurs only in a fraction of cells, is a transient stress response that allows cells to take up DNA from the environment. During natural chromosomal transformation, the internalized linear single-stranded (ss) DNA recombines with the identical (homologous) or partially identical (homeologous) sequence of the resident duplex. The length of the integrated DNA, which can be measured, depends on the percentage of sequence divergence between the donor (internalized) and the recipient (chromosomal) DNAs. The following protocol describes how to induce the development of competence in B. subtilis cells, how to transform them with donor DNAs representing different percentages of sequence divergence compared with the recipient chromosomal DNA, how to calculate the chromosomal transformation efficiency for each of them, and how to amplify the chromosomal DNA from the transformants in order to measure the length in base pairs (bp) of the integrated donor DNA.

Novel regulatory mechanism of establishment genes of conjugative plasmids.

The principal route for dissemination of antibiotic resistance genes is conjugation by which a conjugative DNA element is transferred from a donor to a recipient cell. Conjugative elements contain genes that are important for their establishment in the new host, for instance by counteracting the host defense mechanisms acting against incoming foreign DNA. Little is known about these establishment genes and how they are regulated. Here, we deciphered the regulation mechanism of possible establishment genes of plasmid p576 from the Gram-positive bacterium Bacillus pumilus. Unlike the ssDNA promoters described for some conjugative plasmids, the four promoters of these p576 genes are repressed by a repressor protein, which we named Reg576. Reg576 also regulates its own expression. After transfer of the DNA, these genes are de-repressed for a period of time until sufficient Reg576 is synthesized to repress the promoters again. Complementary in vivo and in vitro analyses showed that different operator configurations in the promoter regions of these genes lead to different responses to Reg576. Each operator is bound with extreme cooperativity by two Reg576-dimers. The X-ray structure revealed that Reg576 has a Ribbon-Helix-Helix core and provided important insights into the high cooperativity of DNA recognition.

RecA Regulation by RecU and DprA During Bacillus subtilis Natural Plasmid Transformation.

Natural plasmid transformation plays an important role in the dissemination of antibiotic resistance genes in bacteria. During this process, Bacillus subtilis RecA physically interacts with RecU, RecX, and DprA. These three proteins are required for plasmid transformation, but RecA is not. In vitro, DprA recruits RecA onto SsbA-coated single-stranded (ss) DNA, whereas RecX inhibits RecA filament formation, leading to net filament disassembly. We show that a null recA (ΔrecA) mutation suppresses the plasmid transformation defect of competent ΔrecU cells, and that RecU is essential for both chromosomal and plasmid transformation in the ΔrecX context. RecU inhibits RecA filament growth and facilitates RecA disassembly from preformed filaments. Increasing SsbA concentrations additively contributes to RecU-mediated inhibition of RecA filament extension. DprA is necessary and sufficient to counteract the negative effect of both RecU and SsbA on RecA filament growth onto ssDNA. DprA-SsbA activates RecA to catalyze DNA strand exchange in the presence of RecU, but this effect was not observed if RecU was added prior to RecA. We propose that DprA contributes to RecA filament growth onto any internalized SsbA-coated ssDNA. When the ssDNA is homologous to the recipient, DprA antagonizes the inhibitory effect of RecU on RecA filament growth and helps RecA to catalyze chromosomal transformation. On the contrary, RecU promotes RecA filament disassembly from a heterologous (plasmid) ssDNA, overcoming an unsuccessful homology search and favoring plasmid transformation. The DprA-DprA interaction may promote strand annealing upon binding to the complementary plasmid strands and facilitating thereby plasmid transformation rather than through a mediation of RecA filament growth.

Bacillus subtilis RecA with DprA-SsbA antagonizes RecX function during natural transformation.

Bacillus subtilis DprA and RecX proteins, which interact with RecA, are crucial for efficient chromosomal and plasmid transformation. We showed that RecA, in the rATP·Mg2+ bound form (RecA·ATP), could not compete with RecX, SsbA or SsbB for assembly onto single-stranded (ss)DNA, but RecA·dATP partially displaced these proteins from ssDNA. RecX promoted reversible depolymerization of preformed RecA·ATP filaments. The two-component DprA-SsbA mediator reversed the RecX negative effect on RecA filament extension, but not DprA or DprA and SsbB. In the presence of DprA-SsbA, RecX added prior to RecA·ATP inhibited DNA strand exchange, but this inhibition was reversed when RecX was added after RecA. We propose that RecA nucleation is more sensitive to RecX action than is RecA filament growth. DprA-SsbA facilitates formation of an active RecA filament that directly antagonizes the inhibitory effects of RecX. RecX and DprA enable chromosomal transformation by altering RecA filament dynamics. DprA-SsbA and RecX proteins constitute a new regulatory network of RecA function. DprA-SsbA contributes to the formation of an active RecA filament and directly antagonizes the inhibitory effects of RecX during natural transformation.

Activity and in vivo dynamics of Bacillus subtilis DisA are affected by RadA/Sms and by Holliday junction-processing proteins.

Bacillus subtilis c-di-AMP synthase DisA and RecA-related RadA/Sms are involved in the repair of DNA damage in exponentially growing cells. We provide genetic evidence that DisA or RadA/Sms is epistatic to the branch migration translocase (BMT) RecG and the Holliday junction (HJ) resolvase RecU in response to DNA damage. We provide genetic evidence damage. Functional DisA-YFP formed dynamic foci in exponentially growing cells, which moved through the nucleoids at a speed compatible with a DNA-scanning mode. DisA formed more static structures in the absence of RecU or RecG than in wild type cells, while dynamic foci were still observed in cells lacking the BMT RuvAB. Purified DisA synthesizes c-di-AMP, but interaction with RadA/Sms or with HJ DNA decreases DisA-mediated c-di-AMP synthesis. RadA/Sms-YFP also formed dynamic foci in growing cells, but the foci moved throughout the cells rather than just on the nucleoids, and co-localized rarely with DisA-YFP foci, suggesting that RadA/Sms and DisA interact only transiently in unperturbed conditions. Our data suggest a model in which DisA moving along dsDNA indicates absence of DNA damage/replication stress via normal c-di-AMP levels, while interaction with HJ DNA/halted forks leads to reduced c-di-AMP levels and an ensuing block in cell proliferation. RadA/Sms may be involved in modulating DisA activities.

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.

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·Mg(2+) 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.

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 · Mg(2+)-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.