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The acquisition of multidrug resistance by pathogenic bacteria is a potentially incipient pandemic. Horizontal transfer of DNA from mobile integrative conjugative elements (ICEs) provides an important way to introduce genes that confer antibiotic (Ab)-resistance in recipient cells. Sizable numbers of SXT/R391 ICEs encode a hypermutagenic Rum DNA polymerase (Rum pol), which has significant homology with Escherichia coli pol V. Here, we show that even under tight transcriptional and post-transcriptional regulation imposed by host bacteria and the R391 ICE itself, Rum pol rapidly accelerates development of multidrug resistance (CIPR, RifR, AmpR) in E. coli in response to SOS-inducing Ab and non-Ab external stressors bleomycin (BLM), ciprofloxacin (CIP) and UV radiation. The impact of Rum pol on the rate of acquisition of drug resistance appears to surpass potential contributions from other cellular processes. We have shown that RecA protein plays a central role in controlling the ability of Rum pol to accelerate antibiotic resistance. A single amino acid substitution in RecA, M197D, acts as a 'Master Regulator' that effectively eliminates the Rum pol-induced Ab resistance. We suggest that Rum pol should be considered as one of the major factors driving development of de novo Ab resistance in pathogens carrying SXT/R391 ICEs., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

A total of 75 bacterial isolates were obtained from nodules of beans cultivated across 10 sites in six agro-ecological zones in Uganda. Using recA gene sequence analysis, 66 isolates were identified as members of the genus Rhizobium, while 9 were related to Agrobacterium species. In the recA gene tree, most Rhizobium strains were classified into five recognized species. Phylogenetic analysis based on six concatenated sequences (recA-rpoB-dnaK-glnII-gyrB-atpD) placed 32 representative strains into five distinct Rhizobium species, consistent with the species groups observed in the recA gene tree: R. phaseoli, R. etli, R. hidalgonense, R. ecuadorense, and R. sophoriradicis, with the first three being the predominant. The rhizobial strains grouped into three nodC subclades within the symbiovar phaseoli clade, encompassing strains from distinct phylogenetic groups. This pattern reflects the conservation of symbiotic genes, likely acquired through horizontal gene transfer among diverse rhizobial species. The 32 representative strains formed symbiotic relationships with host beans, while the Agrobacterium strains did not form nodules and lacked symbiotic genes. Multivariate analysis revealed that species distribution was influenced by the environmental factors of the sampling sites, emphasizing the need to consider these factors in future effectiveness studies to identify effective nitrogen-fixing strains for specific locations., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

The replicative helicase, DnaB, is a central component of the replisome and unwinds duplex DNA coupled with immediate template-dependent DNA synthesis by the polymerase, Pol III. The rate of helicase unwinding is dynamically regulated through structural transitions in the DnaB hexamer between dilated and constricted states. Site-specific mutations in DnaB enforce a faster more constricted conformation that dysregulates unwinding dynamics, causing replisome decoupling that generates excess ssDNA and induces severe cellular stress. This surplus ssDNA can stimulate RecA recruitment to initiate recombinational repair, restart, or activation of the transcriptional SOS response. To better understand the consequences of dysregulated unwinding, we combined targeted genomic dnaB mutations with an inducible RecA filament inhibition strategy to examine the dependencies on RecA in mitigating replisome decoupling phenotypes. Without RecA filamentation, dnaB:mut strains had reduced growth rates, decreased mutagenesis, but a greater burden from endogenous damage. Interestingly, disruption of RecA filamentation in these dnaB:mut strains also reduced cellular filamentation but increased markers of double strand breaks and ssDNA gaps as detected by in situ fluorescence microscopy and FACS assays, TUNEL and PLUG, respectively. Overall, RecA plays a critical role in strain survival by protecting and processing ssDNA gaps caused by dysregulated helicase activity in vivo., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Aim: Ohmic heating (OH) (i.e. heating by electric field) more effectively kills bacterial spores than traditional wet heating, yet its mechanism remains poorly understood. This study investigates the accelerated spore inactivation mechanism using genetically modified spores., Methods and Results: We investigated the effects of OH and conventional heating (CH) on various genetically modified strains of Bacillus subtilis: isogenic PS533 (wild type_1), PS578 [lacking spores' α/β-type small acid-soluble proteins (SASP)], PS2318 (lacking recA, encoding a DNA repair protein), isogenic PS4461 (wild type_2), and PS4462 (having the 2Duf protein in spores, which increases spore wet heat resistance and decreases spore inner membrane fluidity). Removal of SASP brought the inactivation profiles of OH and CH closer, suggesting the interaction of these proteins with the field. However, the reemergence of a difference between CH and OH killing for SASP-deficient spores at the highest tested field strength suggested there is also interaction of the field with another spore core component. Additionally, RecA-deficient spores yielded results like those with the wild-type spores for CH, while the OH resistance of this mutant increased at the lower tested temperatures, implying that RecA or DNA are a possible additional target for the electric field. Addition of the 2Duf protein markedly increased spore resistance both to CH and OH, although some acceleration of killing was observed with OH at 50 V/cm., Conclusions: In summary, both membrane fluidity and interaction of the spore core proteins with electric field are key factors in enhanced spore killing with electric field-heat combinations., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Aim: Recombinase RecA and its homologs play a key role in homologous recombination DNA repair and revive stalled replication fork DNA synthesis. RecA plays an essential role in the evolution of antibiotic-resistant strains via stress-induced DNA repair mechanisms during the SOS response. Accordingly, RecA has become an attractive target to slow down antibiotic resistance rates and prevent mutations in pathogenic bacterial species., Methods and Results: We employed RecA conserved activities: DNA binding, displacement loop formation, strand exchange, ATP hydrolysis, and LexA cleavage, to elucidate the inhibitory role of gallic acid on Staphylococcus aureus RecA functions. Gallic acid inhibition of the SOS response by western blot analysis and its antibacterial activity were measured. The gallic acid inhibited all the canonical activities of S. aureus RecA protein., Conclusion: The natural phenolic compound gallic acid interferes with RecA protein DNA complex formation and inhibits activities such as displacementloop formation, strand exchange reaction, ATP hydrolysis, and coprotease activity of S. aureus. Additionally, gallic acid can obstruct ciprofloxacin-induced RecA expression and eventually confer the inhibitory role of gallic acid in the SOS survival mechanism in S. aureus., (© The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International.)

Bacterial defence systems are tightly regulated to avoid autoimmunity. In Type I restriction-modification (R-M) systems, a specific mechanism called restriction alleviation (RA) controls the activity of the restriction module. In the case of the Escherichia coli Type I R-M system EcoKI, RA proceeds through ClpXP-mediated proteolysis of restriction complexes bound to non-methylated sites that appear after replication or reparation of host DNA. Here, we show that RA is also induced in the presence of plasmids carrying EcoKI recognition sites, a phenomenon we refer to as plasmid-induced RA. Further, we show that the anti-restriction behavior of plasmid-borne non-conjugative transposons such as Tn5053, previously attributed to their ardD loci, is due to plasmid-induced RA. Plasmids carrying both EcoKI and Chi sites induce RA in RecA- and RecBCD-dependent manner. However, inactivation of both RecA and RecBCD restores RA, indicating that there exists an alternative, RecA-independent, homologous recombination pathway that is blocked in the presence of RecBCD. Indeed, plasmid-induced RA in a RecBCD-deficient background does not depend on the presence of Chi sites. We propose that processing of random dsDNA breaks in plasmid DNA via homologous recombination generates non-methylated EcoKI sites, which attract EcoKI restriction complexes channeling them for ClpXP-mediated proteolysis., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Homologous recombination (HR) is essential for genome stability and for maintaining genetic diversity. In eubacteria, RecA protein plays a key role during DNA repair, transcription, and HR. RecA is regulated at multiple levels, but majorly by RecX protein. Moreover, studies have shown RecX is a potent inhibitor of RecA and thus acts as an antirecombinase. Staphylococcus aureus is a major food-borne pathogen that causes skin, bone joint, and bloodstream infections. To date, RecX's role in S. aureus has remained enigmatic. Here, we show that S. aureus RecX (SaRecX) is expressed during exposure to DNA-damaging agents, and purified RecX protein directly interacts physically with RecA protein. The SaRecX is competent to bind with single-stranded DNA preferentially and double-stranded DNA feebly. Significantly, SaRecX impedes the RecA-driven displacement loop and inhibits formation of the strand exchange. Notably, SaRecX also abrogates adenosine triphosphate hydrolysis and abolishes the LexA coprotease activity. These findings highlight the role of the RecX protein as an antirecombinase during HR and play a pivotal role in regulation of RecA during the DNA transactions., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The recombinase RecA/Rad51 ATPase family proteins catalyze paramount DNA strand exchange reactions that are critically involved in maintaining genome integrity. However, it remains unclear how DNA strand exchange proceeds when encountering RecA-free defects in recombinase nucleoprotein filaments. Herein, by designing a series of unique substrates (e.g. truncated or conjugated incoming single-stranded DNA, and extended donor double-stranded DNA) and developing a two-color alternating excitation-modified single-molecule real-time fluorescence imaging assay, we resolve the two key steps (donor strand separation and new base-pair formation) that are usually inseparable during the reaction, revealing a novel long-range flanking strand separation activity of synaptic RecA nucleoprotein filaments. We further evaluate the kinetics and free energetics of strand exchange reactions mediated by various substrates, and elucidate the mechanism of flanking strand separation. Based on these findings, we propose a potential fundamental molecular model involved in flanking strand separation, which provides new insights into strand exchange mechanism and homologous recombination., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In rapidly growing cells, with recombinational DNA repair required often and a new replication fork passing every 20 min, the pace of RecA-mediated DNA strand exchange is potentially much too slow for bacterial DNA metabolism. The enigmatic RadD protein, a putative SF2 family helicase, exhibits no independent helicase activity on branched DNAs. Instead, RadD greatly accelerates RecA-mediated DNA strand exchange, functioning only when RecA protein is present. The RadD reaction requires the RadD ATPase activity, does not require an interaction with SSB, and may disassemble RecA filaments as it functions. We present RadD as a new class of enzyme, an accessory protein that accelerates DNA strand exchange, possibly with a helicase-like action, in a reaction that is entirely RecA-dependent. RadD is thus a DNA strand exchange (recombination) synergist whose primary function is to coordinate closely with and accelerate the DNA strand exchange reactions promoted by the RecA recombinase. Multiple observations indicate a uniquely close coordination of RadD with RecA function., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Meiosis is a fundamental process for sexual reproduction in most eukaryotes and the evolutionarily conserved recombinases RADiation sensitive51 (RAD51) and Disrupted Meiotic cDNA1 (DMC1) are essential for meiosis and thus fertility. The mitotic function of RAD51 is clear, but the meiotic function of RAD51 remains largely unknown. Here we show that RAD51 functions as an interacting protein to restrain the Structural Maintenance of Chromosomes5/6 (SMC5/6) complex from inhibiting DMC1. We unexpectedly found that loss of the SMC5/6 partially suppresses the rad51 knockout mutant in terms of sterility, pollen inviability, and meiotic chromosome fragmentation in a DMC1-dependent manner in Arabidopsis thaliana. Biochemical and cytological studies revealed that the DMC1 localization in meiotic chromosomes is inhibited by the SMC5/6 complex, which is attenuated by RAD51 through physical interactions. This study not only identified the long-sought-after function of RAD51 in meiosis but also discovered the inhibition of SMC5/6 on DMC1 as a control mechanism during meiotic recombination., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Integrons confer a rapid adaptation capability to bacteria. Integron integrases are able to capture and shuffle novel functions embedded in cassettes. Here, we investigated cassette recruitment in the Vibrio cholerae chromosomal integron during horizontal transfer. We demonstrated that the endogenous integrase expression is sufficiently triggered, after SOS response induction mediated by the entry of cassettes during conjugation and natural transformation, to mediate significant cassette insertions. These insertions preferentially occur at the attIA site, despite the presence of about 180 attC sites in the integron array. Thanks to the presence of a promoter in the attIA site vicinity, all these newly inserted cassettes are expressed and prone to selection. We also showed that the RecA protein is critical for cassette recruitment in the V. cholerae chromosomal integron but not in mobile integrons. Moreover, unlike the mobile integron integrases, that of V. cholerae is not active in other bacteria. Mobile integrons might have evolved from the chromosomal ones by overcoming host factors, explaining their large dissemination in bacteria and their role in antibioresistance expansion., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

RecA is essential for double-strand-break repair (DSBR) and the SOS response in Escherichia coli K-12. RecN is an SOS protein and a member of the Structural Maintenance of Chromosomes family of proteins thought to play a role in sister chromatid cohesion/interactions during DSBR. Previous studies have shown that a plasmid-encoded recA4190 (Q300R) mutant had a phenotype similar to ∆ recN (mitomycin C sensitive and UV resistant). It was hypothesized that RecN and RecA physically interact, and that recA4190 specifically eliminated this interaction. To test this model, an epistasis analysis between recA4190 and ∆ recN was performed in wild-type and recBC sbcBC cells. To do this, recA4190 was first transferred to the chromosome. As single mutants, recA4190 and ∆ recN were Rec + as measured by transductional recombination, but were 3-fold and 10-fold decreased in their ability to do I-SceI-induced DSBR, respectively. In both cases, the double mutant had an additive phenotype relative to either single mutant. In the recBC sbcBC background, recA4190 and ∆ recN cells were very UV S (sensitive), Rec - , had high basal levels of SOS expression and an altered distribution of RecA-GFP structures. In all cases, the double mutant had additive phenotypes. These data suggest that recA4190 (Q300R) and ∆ recN remove functions in genetically distinct pathways important for DNA repair, and that RecA Q300 was not important for an interaction between RecN and RecA in vivo recA4190 (Q300R) revealed modest phenotypes in a wild-type background and dramatic phenotypes in a recBC sbcBC strain, reflecting greater stringency of RecA's role in that background., (Copyright © 2020 by the Genetics Society of America.)

Several functions have been proposed for the Escherichia coli DNA polymerase IV (pol IV). Although much research has focused on a potential role for pol IV in assisting pol III replisomes in the bypass of lesions, pol IV is rarely found at the replication fork in vivo. Pol IV is expressed at increased levels in E. coli cells exposed to exogenous DNA damaging agents, including many commonly used antibiotics. Here we present live-cell single-molecule microscopy measurements indicating that double-strand breaks induced by antibiotics strongly stimulate pol IV activity. Exposure to the antibiotics ciprofloxacin and trimethoprim leads to the formation of double strand breaks in E. coli cells. RecA and pol IV foci increase after treatment and exhibit strong colocalization. The induction of the SOS response, the appearance of RecA foci, the appearance of pol IV foci and RecA-pol IV colocalization are all dependent on RecB function. The positioning of pol IV foci likely reflects a physical interaction with the RecA* nucleoprotein filaments that has been detected previously in vitro. Our observations provide an in vivo substantiation of a direct role for pol IV in double strand break repair in cells treated with double strand break-inducing antibiotics., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Awa-bancha is a post-fermented tea produced in Naka and Kamikatsu, Tokushima, Japan. We investigated the lactic acid bacteria in each stage of production of Awa-bancha and evaluated the relationships with the components. Lactic acid bacteria were isolated from tea leaves cultured with de Man, Rogosa, and Sharpe (MRS) agar plates, and the species were identified by homology of the 16 S rRNA gene and multiplex polymerase chain reaction (PCR) of the recA gene to distinguish the Lactobacillus plantarum group. As a result, a variety of species were isolated from the raw tea leaves, and Lactobacillus pentosus was isolated most frequently after anaerobic fermentation. Regarding the tea leaf components, organic acids, such as lactic acid, increased, free amino acids decreased, and catechins changed owing to anaerobic fermentation. Our results suggest that the microbial flora mainly composed of L. pentosus is important in the anaerobic fermentation process for flavor formation of Awa-bancha.

G-quadruplex (G4) DNA structures can form physical barriers within the genome that must be unwound to ensure cellular genomic integrity. Here, we report unanticipated roles for the Escherichia coli Rep helicase and RecA recombinase in tolerating toxicity induced by G4-stabilizing ligands in vivo. We demonstrate that Rep and Rep-X (an enhanced version of Rep) display G4 unwinding activities in vitro that are significantly higher than the closely related UvrD helicase. G4 unwinding mediated by Rep involves repetitive cycles of G4 unfolding and refolding fueled by ATP hydrolysis. Rep-X and Rep also dislodge G4-stabilizing ligands, in agreement with our in vivo G4-ligand sensitivity result. We further demonstrate that RecA filaments disrupt G4 structures and remove G4 ligands in vitro, consistent with its role in countering cellular toxicity of G4-stabilizing ligands. Together, our study reveals novel genome caretaking functions for Rep and RecA in resolving deleterious G4 structures., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

RAD51 plays a central role in homologous recombination during double-strand break repair and in replication fork dynamics. Misregulation of RAD51 is associated with genetic instability and cancer. RAD51 is regulated by many accessory proteins including the highly conserved Shu complex. Here, we report the function of the human Shu complex during replication to regulate RAD51 recruitment to DNA repair foci and, secondly, during replication fork restart following replication fork stalling. Deletion of the Shu complex members, SWS1 and SWSAP1, using CRISPR/Cas9, renders cells specifically sensitive to the replication fork stalling and collapse caused by methyl methanesulfonate and mitomycin C exposure, a delayed and reduced RAD51 response, and fewer sister chromatid exchanges. Our additional analysis identified SPIDR and PDS5B as novel Shu complex interacting partners and genetically function in the same pathway upon DNA damage. Collectively, our study uncovers a protein complex, which consists of SWS1, SWSAP1, SPIDR and PDS5B, involved in DNA repair and provides insight into Shu complex function and composition., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Homologous recombination is a fundamental process in all living organisms that allows the faithful repair of DNA double strand breaks, through the exchange of DNA strands between homologous regions of the genome. Results of three decades of investigation and recent fruitful observations have unveiled key elements of the reaction mechanism, which proceeds along nucleofilaments of recombinase proteins of the RecA family. Yet, one essential aspect of homologous recombination has largely been overlooked when deciphering the mechanism: while ATP is hydrolyzed in large quantity during the process, how exactly hydrolysis influences the DNA strand exchange reaction at the structural level remains to be elucidated. In this study, we build on a previous geometrical approach that studied the RecA filament variability without bound DNA to examine the putative implication of ATP hydrolysis on the structure, position, and interactions of up to three DNA strands within the RecA nucleofilament. Simulation results on modeled intermediates in the ATP cycle bring important clues about how local distortions in the DNA strand geometries resulting from ATP hydrolysis can aid sequence recognition by promoting local melting of already formed DNA heteroduplex and transient reverse strand exchange in a weaving type of mechanism., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Bacterial Y-family DNA polymerases are usually classified into DinB (Pol IV), UmuC (the catalytic subunit of Pol V) and ImuB, a catalytically dead essential component of the ImuA-ImuB-DnaE2 mutasome. However, the true diversity of Y-family polymerases is unknown. Furthermore, for most of them the structures are unavailable and interactions are poorly characterized. To gain a better understanding of bacterial Y-family DNA polymerases, we performed a detailed computational study. It revealed substantial diversity, far exceeding traditional classification. We found that a large number of subfamilies feature a C-terminal extension next to the common Y-family region. Unexpectedly, in most C-terminal extensions we identified a region homologous to the N-terminal oligomerization motif of RecA. This finding implies a universal mode of interaction between Y-family members and RecA (or ImuA), in the case of Pol V strongly supported by experimental data. In gram-positive bacteria, we identified a putative Pol V counterpart composed of a Y-family polymerase, a YolD homolog and RecA. We also found ImuA-ImuB-DnaE2 variants lacking ImuA, but retaining active or inactive Y-family polymerase, a standalone ImuB C-terminal domain and/or DnaE2. In summary, our analyses revealed that, despite considerable diversity, bacterial Y-family polymerases share previously unanticipated similarities in their structural domains/motifs and interactions., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Bacterial recombinational repair of double-strand breaks often begins with creation of initiating 3' single-stranded DNA (ssDNA) tails on each side of a double-strand break (DSB). Importantly, if the RecBCD pathway is followed, RecBCD creates a gap between the sequences at 3' ends of the initiating strands. The gap flanks the DSB and extends at least to the nearest Chi site on each strand. Once the initiating strands form ssDNA-RecA filaments, each ssDNA-RecA filament searches for homologous double-stranded DNA (dsDNA) to use as a template for the DNA synthesis needed to fill the gap created by RecBCD. Our experimental results show that the DNA synthesis requires formation of a heteroduplex dsDNA that pairs >20 contiguous bases in the initiating strand with sequence matched bases in a strand from the original dsDNA. To trigger synthesis, the heteroduplex must be near the 3' end of the initiating strand. Those experimentally determined requirements for synthesis combined with the Chi site dependence of the function of RecBCD and the distribution of Chi sites in bacterial genomes could allow the RecBCD pathway to avoid some genomic rearrangements arising from directly induced DSBs; however, the same three factors could promote other rearrangements., (© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Homologous recombination is essential to genome maintenance, and also to genome diversification. In virtually all organisms, homologous recombination depends on the RecA/Rad51-family recombinases, which catalyze ATP-dependent formation of homologous joints-critical intermediates in homologous recombination. RecA/Rad51 binds first to single-stranded (ss) DNA at a damaged site to form a spiral nucleoprotein filament, after which double-stranded (ds) DNA interacts with the filament to search for sequence homology and to form consecutive base pairs with ssDNA ('pairing'). How sequence homology is recognized and what exact role filament formation plays remain unknown. We addressed the question of whether filament formation is a prerequisite for homologous joint formation. To this end we constructed a nonpolymerizing (np) head-to-tail-fused RecA dimer (npRecA dimer) and an npRecA monomer. The npRecA dimer bound to ssDNA, but did not form continuous filaments upon binding to DNA; it formed beads-on-string structures exclusively. Although its efficiency was lower, the npRecA dimer catalyzed the formation of D-loops (a type of homologous joint), whereas the npRecA monomer was completely defective. Thus, filament formation contributes to efficiency, but is not essential to sequence-homology recognition and pairing, for which a head-to-tail dimer form of RecA protomer is required and sufficient.

The Escherichia coli RecA protein catalyzes the central step of homologous recombination using its homology search and strand exchange activity. RecA is a DNA-dependent ATPase, but its homology search and strand exchange activities are largely independent of its ATPase activity. ATP hydrolysis converts a high affinity DNA binding form, RecA-ATP, to a low affinity form RecA-ADP, thereby supporting an ATP hydrolysis-dependent dynamic cycle of DNA binding and dissociation. We provide evidence for a novel function of RecA's dynamic behavior; RecA's ATPase activity prevents accumulation of toxic complexes caused by direct binding of RecA to undamaged regions of dsDNA. We show that a mutant form of RecA, RecA-K250N, previously shown to be toxic to E. coli, is a loss-of-function ATPase-defective mutant. We use a new method for detecting RecA complexes involving nucleoid surface spreading and immunostaining. The method allows detection of damage-induced RecA foci; STED microscopy revealed these to typically be between 50 and 200 nm in length. RecA-K250N, and other toxic variants of RecA, form spontaneous DNA-bound complexes that are independent of replication and of accessory proteins required to load RecA onto tracts of ssDNA in vivo, supporting the hypothesis that RecA's expenditure of ATP serves an error correction function.

A limitation to the engineering of cellulolytic thermophiles is the availability of functional, thermostable (≥ 60 °C) replicating plasmid vectors for rapid expression and testing of genes that provide improved or novel fuel molecule production pathways. A series of plasmid vectors for genetic manipulation of the cellulolytic thermophile Caldicellulosiruptor bescii has recently been extended to Clostridium thermocellum, another cellulolytic thermophile that very efficiently solubilizes plant biomass and produces ethanol. While the C. bescii pBAS2 replicon on these plasmids is thermostable, the use of homologous promoters, signal sequences and genes led to undesired integration into the bacterial chromosome, a result also observed with less thermostable replicating vectors. In an attempt to overcome undesired plasmid integration in C. thermocellum, a deletion of recA was constructed. As expected, C. thermocellum ∆recA showed impaired growth in chemically defined medium and an increased susceptibility to UV damage. Interestingly, we also found that recA is required for replication of the C. bescii thermophilic plasmid pBAS2 in C. thermocellum, but it is not required for replication of plasmid pNW33N. In addition, the C. thermocellum recA mutant retained the ability to integrate homologous DNA into the C. thermocellum chromosome. These data indicate that recA can be required for replication of certain plasmids, and that a recA-independent mechanism exists for the integration of homologous DNA into the C. thermocellum chromosome. Understanding thermophilic plasmid replication is not only important for engineering of these cellulolytic thermophiles, but also for developing genetic systems in similar new potentially useful non-model organisms.

The mechanism by which pre-synaptic RecA nucleoprotein filaments efficiently locate sequence homology across genomic DNA remains unclear. Here, using atomic force microscopy, we directly investigate the intermediates of the RecA-mediated homologous recombination process and find it to be highly cooperative, involving multiple phases. Initially, the process is dominated by a rapid 'association' phase, where multiple filaments interact on the same dsDNA simultaneously. This cooperative nature is reconciled by the observation of localized dense clusters of pre-synaptic filaments interacting with the observed dsDNA molecules. This confinement of reactive species within the vicinity of the dsDNA, is likely to play an important role in ensuring that a high interaction rate between the nucleoprotein filaments and the dsDNA can be achieved. This is followed by a slower 'resolution' phase, where the synaptic joints either locate sequence homology and progress to a post-synaptic joint, or dissociate from the dsDNA. Surprisingly, the number of simultaneous synaptic joints decreases rapidly after saturation of the dsDNA population, suggesting a reduction in interaction activity of the RecA filaments. We find that the time-scale of this decay is in line with the time-scale of the dispersion of the RecA filament clusters, further emphasising the important role this cooperative phenomena may play in the RecA-facilitated homology search., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The correct identification of bacteria from the Burkholderia cepacia complex (Bcc) is crucial for epidemiological studies and treatment of cystic fibrosis infections. However, genome-based identification tools are revealing many controversial Bcc species assignments. The aim of this work is to re-examine the taxonomic position of the soil bacterium B. cepacia 89 through polyphasic and genomic approaches. recA and 16S rRNA gene sequence analysis positioned strain 89 inside the Bcc group. However, based on the divergence score of seven concatenated allele sequences, and values of average nucleotide identity, and digital DNA:DNA hybridization, our results suggest that strain 89 is different from other Bcc species formerly described. Thus, we propose to classify Burkholderia sp. 89 as the novel species Burkholderia catarinensis sp. nov. with strain 89T (=DSM 103188T = BR 10601T) as the type strain. Moreover, our results call the attention to some probable misidentifications of Bcc genomes at the National Center for Biotechnology Information database., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The genetic diversity of bacterial populations nodulating Lupinus luteus (yellow lupine) in Northern Tunisia was examined. Phylogenetic analyses of 43 isolates based on recA and gyrB partial sequences grouped them in three clusters, two of which belong to genus Bradyrhizobium (41 isolates) and one, remarkably, to Microvirga (2 isolates), a genus never previously described as microsymbiont of this lupine species. Representatives of the three clusters were analysed in-depth by multilocus sequence analysis of five housekeeping genes (rrs, recA, glnII, gyrB and dnaK). Surprisingly, the Bradyrhizobium cluster with the two isolates LluI4 and LluTb2 may constitute a new species defined by a separate position between Bradyrhizobium manausense and B. denitrificans. A nodC-based phylogeny identified only two groups: one formed by Bradyrhizobium strains included in the symbiovar genistearum and the other by the Microvirga strains. Symbiotic behaviour of representative isolates was tested, and among the seven legumes inoculated only a difference was observed i.e. the Bradyrhizobium strains nodulated Ornithopus compressus unlike the two strains of Microvirga. On the basis of these data, we conclude that L. luteus root nodule symbionts in Northern Tunisia are mostly strains within the B. canariense/B. lupini lineages, and the remaining strains belong to two groups not previously identified as L. luteus endosymbionts: one corresponding to a new clade of Bradyrhizobium and the other to the genus Microvirga., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

RecA-family recombinase-catalyzed ATP-dependent homologous joint formation is critical for homologous recombination, in which RecA or Rad51 binds first to single-stranded (ss)DNA and then interacts with double-stranded (ds)DNA. However, when RecA or Rad51 interacts with dsDNA before binding to ssDNA, the homologous joint-forming activity of RecA or Rad51 is quickly suppressed. We found that under these and adenosine diphosphate (ADP)-generating suppressive conditions for the recombinase activity, RecA or Rad51 at similar optimal concentrations enhances the DNA ligase-catalyzed dsDNA end-joining (DNA ligation) about 30- to 40-fold. The DNA ligation enhancement by RecA or Rad51 transforms most of the substrate DNA into multimers within 2-5 min, and for this enhancement, ADP is the common and best cofactor. Adenosine triphosphate (ATP) is effective for RecA, but not for Rad51. Rad51/RecA-enhanced DNA ligation depends on dsDNA-binding, as shown by a mutant, and is independent of physical interactions with the DNA ligase. These observations demonstrate the common and unique activities of RecA and Rad51 to juxtapose dsDNA-ends in preparation for covalent joining by a DNA ligase. This new in vitro function of Rad51 provides a simple explanation for our genetic observation that Rad51 plays a role in the fidelity of the end-joining of a reporter plasmid DNA, by yeast canonical non-homologous end-joining (NHEJ) in vivo., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Unlabelled: Yersinia enterocolitica is currently divided into two subspecies: subsp. enterocolitica including highly pathogenic strains of biotype 1B and subsp. palearctica including nonpathogenic strains of biotype 1A and moderately pathogenic strains of biotypes 2-5. In this work, we characterized 162 Y. enterocolitica strains of biotype 1A and 50 strains of biotypes 2-4 isolated from human, animal and food samples by restriction fragment length polymorphism using the HindIII restriction enzyme. Phylogenetic relatedness of 20 representative Y. enterocolitica strains including 15 biotype 1A strains was further studied by the multilocus sequence analysis of four housekeeping genes (glnA, gyrB, recA and HSP60). In all the analyses, biotype 1A strains formed a separate genomic group, which differed from Y. enterocolitica subsp. enterocolitica and from the strains of biotypes 2-4 of Y. enterocolitica subsp. palearctica. Based on these results, biotype 1A strains considered nonpathogenic should not be included in subspecies palearctica containing pathogenic strains of biotypes 2-5., Significance and Impact of the Study: Yersinia enterocolitica strains are currently divided into six biotypes and two subspecies. Strains of biotype 1A, which are phenotypically and genotypically very heterogeneous, are classified as subspecies palearctica. In this study, European Y. enterocolitica 1A strains isolated from both human and nonhuman sources were characterized using restriction fragment length polymorphism and multilocus sequence analysis. The European biotype 1A strains formed a separate group, which differed from strains belonging to subspecies enterocolitica and palearctica. This may indicate that the current division between the two subspecies is not sufficient considering the strain diversity within Y. enterocolitica., (© 2016 The Society for Applied Microbiology.)

RecBCD enzyme is a complex, three-subunit protein machine essential for the major pathway of DNA double-strand break repair and homologous recombination in Escherichia coli Upon encountering a Chi recombination-hotspot during DNA unwinding, RecBCD nicks DNA to produce a single-stranded DNA end onto which it loads RecA protein. Conformational changes that regulate RecBCD's helicase and nuclease activities are induced upon its interaction with Chi, defined historically as 5' GCTGGTGG 3'. Chi is thought to be recognized as single-stranded DNA passing through a tunnel in RecC. To define the Chi recognition-domain in RecC and thus the mechanism of the RecBCD-Chi interaction, we altered by random mutagenesis eight RecC amino acids lining the tunnel. We screened for loss of Chi activity with Chi at one site in bacteriophage λ. The 25 recC mutants analyzed thoroughly had undetectable or strongly reduced Chi-hotspot activity with previously reported Chi sites. Remarkably, most of these mutants had readily detectable, and some nearly wild-type, activity with Chi at newly generated Chi sites. Like wild-type RecBCD, these mutants had Chi activity that responded dramatically (up to fivefold, equivalent to Chi's hotspot activity) to nucleotide changes flanking 5' GCTGGTGG 3'. Thus, these and previously published RecC mutants thought to be Chi-recognition mutants are actually Chi context-dependence mutants. Our results fundamentally alter the view that Chi is a simple 8-bp sequence recognized by the RecC tunnel. We propose that Chi hotspots have dual nucleotide sequence interactions, with both the RecC tunnel and the RecB nuclease domain., (Copyright © 2016 by the Genetics Society of America.)

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., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Unlabelled: The Burkholderia cepacia complex (BCC) is a group of closely related species which includes opportunistic pathogens causing chronic respiratory infections in immunocompromised patients, or individuals affected by cystic fibrosis (CF). Other Burkholderia species causing infection in the CF population are Burkholderia gladioli and Burkholderia pseudomallei. Traditional phenotypic analyses have been demonstrated to be inadequate for reliable identifications of isolates of BCC and B. gladioli. A pan-genomic analysis approach was used to design species-specific probes for Burkholderia cenocepacia, B. cepacia, Burkholderia multivorans, Burkholderia vietnamiensis, Burkholderia ambifaria, Burkholderia dolosa, Burkholderia pyrrocinia and B. gladioli. Multiplex real-time PCR assay was developed and tested using sputum specimens collected from CF patients spiked with Burkholderia species. The assay exhibited 100% sensitivity for all eight target species and detected 10(2) to 10(3)  CFU ml(-1) when applied to spiked sputum. Our PCR assay resulted highly specific for each of the Burkholderia species tested, allowing discrimination among Burkholderia and non-Burkholderia pathogens. Analysis carried out on 200 sputa positive for the presence of Burkholderia revealed that PCR assay and recA sequencing were fully comparable for identification of Burkholderia at the level of species., Significance and Impact of the Study: Burkholderia cepacia complex (BCC) has a complex taxonomic organization and its identification is a challenge for microbiology laboratories. Nonidentification or misidentification of BCC isolates represent a problem in epidemiology and treatment of cystic fibrosis patients. The high specificity and sensitivity of the multiplex Real-time PCR assay developed in this study indicates its potential to be a rapid and reliable method for the detection of Burkholderia at the level of species from sputum samples of cystic fibrosis patients., (© 2015 The Society for Applied Microbiology.)

The phylogenetic diversity of 29 rhizobial strains nodulating Phaseolus vulgaris in Iran was analysed on the basis of their core and symbiotic genes. These strains displayed five 16S rRNA-RFLP patterns and belong to eight ERIC-PCR clusters. The phylogenetic analyses of 16S rRNA, recA and atpD core genes allowed the identification of several strains as Rhizobium sophoriradicis, R. leguminosarum, R. tropici and Pararhizobium giardinii, whereas other strains represented a new phylogenetic lineage related to R. vallis. These strains and those identified as R. sophoriradicis and R. leguminosarum belong to the symbiovar phaseoli carrying the γ nodC allele distributed in P. vulgaris endosymbionts in America, Europe, Africa and Asia. The strain identified as R. tropici belongs to the symbiovar tropici carried by strains of R. tropici, R. leucaenae, R. lusitanum and R. freirei nodulating P. vulgaris in America, Africa and Asia. The strain identified as P. giardinii belongs to the symbiovar giardinii together with the type strain of this species nodulating P. vulgaris in France. It is remarkable that the recently described species R. sophoriradicis is worldwide distributed in P. vulgaris nodules carrying the γ nodC allele of symbiovar phaseoli harboured by rhizobia isolated in the American distribution centers of this legume., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Rhizogenic Agrobacterium biovar 1 strains have been found to cause extensive root proliferation on hydroponically grown Cucurbitaceae and Solanaceae crops, resulting in substantial economic losses. As these agrobacteria live under similar ecological conditions, infecting a limited number of crops, it may be hypothesized that genetic and phenotypic variation among such strains is relatively low. In this study we assessed the phenotypic diversity as well as the phylogenetic and evolutionary relationships of several rhizogenic Agrobacterium biovar 1 strains from cucurbit and solanaceous crops. A collection of 41 isolates was subjected to a number of phenotypic assays and characterized by MLSA targeting four housekeeping genes (16S rRNA gene, recA, rpoB and trpE) and two loci from the root-inducing Ri-plasmid (part of rolB and virD2). Besides phenotypic variation, remarkable genotypic diversity was observed, especially for some chromosomal loci such as trpE. In contrast, genetic diversity was lower for the plasmid-borne loci, indicating that the studied chromosomal housekeeping genes and Ri-plasmid-borne loci might not exhibit the same evolutionary history. Furthermore, phylogenetic and network analyses and several recombination tests suggested that recombination could be contributing in some extent to the evolutionary dynamics of rhizogenic Agrobacterium populations. Finally, a genomospecies-level identification analysis revealed that at least four genomospecies may occur on cucurbit and tomato crops (G1, G3, G8 and G9). Together, this study gives a first glimpse at the genetic and phenotypic diversity within this economically important plant pathogenic bacterium., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The Escherichia coli SOS system is a well-established model for the cellular response to DNA damage. Control of SOS depends largely on the RecA protein. When RecA is activated by single-stranded DNA in the presence of a nucleotide triphosphate cofactor, it mediates cleavage of the LexA repressor, leading to expression of the 30(+)-member SOS regulon. RecA activation generally requires the introduction of DNA damage. However, certain recA mutants, like recA730, bypass this requirement and display constitutive SOS expression as well as a spontaneous (SOS) mutator effect. Presently, we investigated the possible interaction between SOS and the cellular deoxynucleoside triphosphate (dNTP) pools. We found that dNTP pool changes caused by deficiencies in the ndk or dcd genes, encoding nucleoside diphosphate kinase and dCTP deaminase, respectively, had a strongly suppressive effect on constitutive SOS expression in recA730 strains. The suppression of the recA730 mutator effect was alleviated in a lexA-deficient background. Overall, the findings suggest a model in which the dNTP alterations in the ndk and dcd strains interfere with the activation of RecA, thereby preventing LexA cleavage and SOS induction., (Published by Oxford University Press on behalf of Nucleic Acids Research 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Rhizobial diversity and host preferences were assessed in 65 native Fynbos legumes of the papilionoid legume tribes Astragaleae, Crotalarieae, Genisteae, Indigofereae, Millettieae, Phaseoleae, Podalyrieae, Psoraleeae and Sesbanieae. Sequence analyses of chromosomal 16S rRNA, recA, atpD and symbiosis-related nodA, nifH genes in parallel with immunogold labelling assays identified the symbionts as alpha- (Azorhizobium, Bradyrhizobium, Ensifer, Mesorhizobium and Rhizobium) and beta-rhizobial (Burkholderia) lineages with the majority placed in the genera Mesorhizobium and Burkholderia showing a wide range of host interactions. Despite a degree of symbiotic promiscuity in the tribes Crotalarieae and Indigofereae nodulating with both alpha- and beta-rhizobia, Mesorhizobium symbionts appeared to exhibit a general host preference for the tribe Psoraleeae, whereas Burkholderia prevailed in the Podalyrieae. Although host genotype was the main factor determining rhizobial diversity, ecological factors such as soil acidity and site elevation were positively correlated with genetic variation within Mesorhizobium and Burkholderia, respectively, indicating an interplay of host and environmental factors on the distribution of Fynbos rhizobia., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In all organisms, RecA-family recombinases catalyze homologous joint formation in homologous genetic recombination, which is essential for genome stability and diversification. In homologous joint formation, ATP-bound RecA/Rad51-recombinases first bind single-stranded DNA at its primary site and then interact with double-stranded DNA at another site. The underlying reason and the regulatory mechanism for this conserved binding order remain unknown. A comparison of the loop L1 structures in a DNA-free RecA crystal that we originally determined and in the reported DNA-bound active RecA crystals suggested that the aspartate at position 161 in loop L1 in DNA-free RecA prevented double-stranded, but not single-stranded, DNA-binding to the primary site. This was confirmed by the effects of the Ala-replacement of Asp-161 (D161A), analyzed directly by gel-mobility shift assays and indirectly by DNA-dependent ATPase activity and SOS repressor cleavage. When RecA/Rad51-recombinases interact with double-stranded DNA before single-stranded DNA, homologous joint-formation is suppressed, likely by forming a dead-end product. We found that the D161A-replacement reduced this suppression, probably by allowing double-stranded DNA to bind preferentially and reversibly to the primary site. Thus, Asp-161 in the flexible loop L1 of wild-type RecA determines the preference for single-stranded DNA-binding to the primary site and regulates the DNA-binding order in RecA-catalyzed recombinase reactions., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

During meiosis programmed DNA double-strand breaks (DSBs) are repaired by homologous recombination using the sister chromatid or the homologous chromosome (homolog) as a template. This repair results in crossover (CO) and non-crossover (NCO) recombinants. Only CO formation between homologs provides the physical linkages guiding correct chromosome segregation, which are essential to produce healthy gametes. The factors that determine the CO/NCO decision are still poorly understood. Using Schizosaccharomyces pombe as a model we show that the Rad51/Dmc1-paralog complexes Rad55-Rad57 and Rdl1-Rlp1-Sws1 together with Swi5-Sfr1 play a major role in antagonizing both the FANCM-family DNA helicase/translocase Fml1 and the RecQ-type DNA helicase Rqh1 to limit hybrid DNA formation and promote Mus81-Eme1-dependent COs. A common attribute of these protein complexes is an ability to stabilize the Rad51/Dmc1 nucleoprotein filament, and we propose that it is this property that imposes constraints on which enzymes gain access to the recombination intermediate, thereby controlling the manner in which it is processed and resolved., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Here, we report the surprising and, to our knowledge, unique example of horizontal interkingdom transfer of a human opportunistic pathogen (Propionibacterium acnes) to a crop plant (the domesticated grapevine Vitis vinifera L.). Humans, like most organisms, have established a long-lasting cohabitation with a variety of microbes, including pathogens and gut-associated bacteria. Studies which have investigated the dynamics of such associations revealed numerous cases of bacterial host switches from domestic animals to humans. Much less is, however, known about the exchange of microbial symbionts between humans and plants. Fluorescent in situ hybridization localized P. acnes in the bark, in xylem fibers, and, more interestingly, inside pith tissues. Phylogenetic and population genetic analyses suggest that the establishment of the grapevine-associated P. acnes as obligate endophyte is compatible with a recent transfer event, likely during the Neolithic, when grapevine was domesticated.

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.

Tandem genetic duplications arise frequently between the seven directly repeated 5.5-kb rrn loci that encode ribosomal RNAs in Salmonella enterica. The closest rrn genes, rrnB and rrnE, flank a 40-kb region that includes the purHD operon. Duplications of purHD arise by exchanges between rrn loci and form at a high rate (10(-3)/cell/division) that remains high in strains blocked for early steps in recombination (recA, recB, and/or recF), but drops 30-fold in mutants blocked for later Holliday junction resolution (ruvC recG). The duplication defect of a ruvC recG mutant was fully corrected by an added mutation in any one of the recA, recB, or recF genes. To explain these results, we propose that early recombination defects activate an alternative single-strand annealing pathway for duplication formation. In wild-type cells, rrn duplications form primarily by the action of RecFORA on single-strand gaps. Double-strand breaks cannot initiate rrn duplications because rrn loci lack Chi sites, which are essential for recombination between two separated rrn sequences. A recA or recF mutation allows unrepaired gaps to accumulate such that different rrn loci can provide single-strand rrn sequences that lack the RecA coating that normally inhibits annealing. A recB mutation activates annealing by allowing double-strand ends within rrn to avoid digestion by RecBCD and provide a new source of rrn ends for use in annealing. The equivalent high rates of rrn duplication by recombination and annealing pathways may reflect a limiting economy of gaps and breaks arising in heavily transcribed, palindrome-rich rrn sequences.

During meiosis, homologous recombination (HR) is essential to repair programmed DNA double-strand breaks (DSBs), and a dedicated protein machinery ensures that the homologous chromosome is favored over the nearby sister chromatid as a repair template. The homologous-pairing protein2/meiotic nuclear division protein1 (HOP2/MND1) protein complex has been identified as a crucial factor of meiotic HR in Arabidopsis thaliana, since loss of either MND1 or HOP2 results in failure of DNA repair. We isolated two mutant alleles of HOP2 (hop2-2 and hop2-3) that retained the capacity to repair meiotic DSBs via the sister chromatid but failed to use the homologous chromosome. We show that in these alleles, the recombinases radiation sensitive51 (RAD51) and disrupted meiotic cDNA1 (DMC1) are loaded, but only the intersister DNA repair pathway is activated. The hop2-2 phenotype is correlated with a decrease in HOP2/MND1 complex abundance. In hop2-3, a truncated HOP2 protein is produced that retains its ability to bind to DMC1 and DNA but forms less stable complexes with MND1 and fails to efficiently stimulate DMC1-driven D-loop formation. Genetic analyses demonstrated that in the absence of DMC1, HOP2/MND1 is dispensable for RAD51-mediated intersister DNA repair, while in the presence of DMC1, a minimal amount of functional HOP2/MND1 is essential to drive intersister DNA repair.

Double-strand breaks (DSB) occur in chromatin following replication fork collapse and chemical or physical damage [Symington and Gautier (Double-strand break end resection and repair pathway choice. Annu. Rev. Genet. 2011;45:247-271.)] and may be repaired by homologous recombination (HR) and non-homologous end-joining. Nucleosomes are the fundamental units of chromatin and must be remodeled during DSB repair by HR [Andrews and Luger (Nucleosome structure(s) and stability: variations on a theme. Annu. Rev. Biophys. 2011;40:99-117.)]. Physical initiation of HR requires RAD51, which forms a nucleoprotein filament (NPF) that catalyzes homologous pairing and strand exchange (recombinase) between DNAs that ultimately bridges the DSB gap [San Filippo, Sung and Klein. (Mechanism of eukaryotic HR. Annu. Rev. Biochem. 2008;77:229-257.)]. RAD51 forms an NPF on single-stranded DNA and double-stranded DNA (dsDNA). Although the single-stranded DNA NPF is essential for recombinase initiation, the role of the dsDNA NPF is less clear. Here, we demonstrate that the human RAD51 (HsRAD51) dsDNA NPF disassembles nucleosomes by unwrapping the DNA from the core histones. HsRAD51 that has been constitutively or biochemically activated for recombinase functions displays significantly reduced nucleosome disassembly activity. These results suggest that HsRAD51 can perform ATP hydrolysis-dependent nucleosome disassembly in addition to its recombinase functions.

Objectives: This study screened hospital sewage for the presence of blaNDM., Methods: Colonies grown from plates containing meropenem streaked with hospital sewage were screened for blaNDM by PCR. Species identification was performed by sequencing 16S rRNA genes. Clonal relatedness of isolates was determined by Enterobacterial repetitive intergenic consensus sequence-PCR, sequencing recA and PFGE. Mating was attempted to obtain self-transmissible plasmids carrying blaNDM. The genetic context of blaNDM was investigated by PCR mapping using pNDM-BJ01, a blaNDM-1-carrying plasmid from Acinetobacter lwoffii as the reference., Results: Two Acinetobacter johnsonii isolates, designated XBB1 and XBC1, from sewage were found carrying blaNDM-1, but were of different clonal origins. In both cases, blaNDM-1 was carried by a self-transmissible plasmid. PCR mapping and sequencing revealed that the blaNDM-1-carrying plasmid of XBB1, pXBB1, was the same as pNDM-BJ01, whereas that of XBC1, pXBC1, was a variant of pNDM-BJ01. A large region downstream of blaNDM-1, including groES/groEL, ISCR27 and ISAba125, was absent from pXBC1. On pXBB1, blaNDM-1 was carried by Tn125, an ISAba125-formed composite transposon, which was inserted into a sequence downstream of aphA6., Conclusions: Sewage of a hospital in China was found to contain blaNDM-1, suggesting that hospital sewage is an important but often overlooked reservoir of antimicrobial resistance determinants. A variety of Acinetobacter species in different locations have been found to harbour blaNDM-1, so the introduction of blaNDM-1 into Acinetobacter is unlikely to be a single event. The identification of blaNDM-1-carrying plasmid pNDM-BJ01/pXBB1 and its variants suggests that a common plasmid has been transferred among different host species at different locations.

The IncF plasmid p1658/97 (c. 125 kb) from Escherichia coli isolates recovered during a clonal outbreak in a hospital in Warsaw, Poland, in 1997 contains the extended-spectrum β-lactamase (ESBL) gene bla(SHV-5), originated from the Klebsiella pneumoniae chromosome. A region containing the bla(SHV-5) gene is flanked by two IS26 copies and its copy number multiplies spontaneously within p1658/97 and RecA-deficient E. coli strains. Here, we demonstrate that the amplified IS26-bla(SHV-5) units were arranged in tandems, containing up to more than 10 units, which could raise ceftazidime MICs for host strains from 4 μg mL(-1) to more than 128 μg mL(-1). Successive deletions within p1658/97, located outside the amplifiable module and encompassing even as little as c. 15% of the plasmid, blocked the amplification. Moreover, the complementing re-introduction of the deleted fragments in trans did not restore the process. Similarly, insertions of a 1-kb DNA fragment into the amplicon inhibited its self-multiplication ability. The module was able to transmit into another IS26-containing plasmid by recombination. The results prompted us to speculate that local DNA structure, especially favorable in p1658/97, might have been responsible for the IS26-bla(SHV-5) multiplication ability., (© 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Escherichia coli lyses by lambda phage propagation. Circular plasmid DNA was present during E. coli lysis as an extracellular plasmid DNA (excpDNA) that was stable enough to transform coexisting competent Bacillus subtilis cells. Detailed investigations unveiled that excpDNA is transient in both quality and quantity, with stability lasting no more than several hours. A survey using E. coli lambda lysogens with various genetic backgrounds demonstrated that the loss of Endonuclease I (ΔendA::kan) conferred extraordinary stability upon excpDNA for as long as 48 h. Studies on endA mutants suggested that excpDNA remained localized in cell debris, in contrast to E. coli genome DNA, which diffused into medium at an early point in lysis. Lambda lysogens constructed on endA recA mutants are presented for potential pipelines in delivery to other competent proficient microbes.

Duplications are often attributed to "unequal recombination" between separated, directly repeated sequence elements (>100 bp), events that leave a recombinant element at the duplication junction. However, in the bacterial chromosome, duplications form at high rates (10(-3)-10(-5)/cell/division) even without recombination (RecA). Here we describe 1800 spontaneous lac duplications trapped nonselectively on the low-copy F'(128) plasmid, where lac is flanked by direct repeats of the transposable element IS3 (1258 bp) and by numerous quasipalindromic REP elements (30 bp). Duplications form at a high rate (10(-4)/cell/division) that is reduced only about 11-fold in the absence of RecA. With and without RecA, most duplications arise by recombination between IS3 elements (97%). Formation of these duplications is stimulated by IS3 transposase (Tnp) and plasmid transfer functions (TraI). Three duplication pathways are proposed. First, plasmid dimers form at a high rate stimulated by RecA and are then modified by deletions between IS3 elements (resolution) that leave a monomeric plasmid with an IS3-flanked lac duplication. Second, without RecA, duplications occur by single-strand annealing of DNA ends generated in different sister chromosomes after transposase nicks DNA near participating IS3 elements. The absence of RecA may stimulate annealing by allowing chromosome breaks to persist. Third, a minority of lac duplications (3%) have short (0-36 bp) junction sequences (SJ), some of which are located within REP elements. These duplication types form without RecA, Tnp, or Tra by a pathway in which the palindromic junctions of a tandem inversion duplication (TID) may stimulate deletions that leave the final duplication.

In the DNA damage response of most bacteria, UmuD forms part of the error-prone (UmuD'(2) )C polymerase V and is activated for this function by self-cleavage after DNA damage. However, the umuD homolog (umuDAb) present throughout the Acinetobacter genus encodes an extra N-terminal region, and in Acinetobacter baylyi, regulates transcription of DNA damage-induced genes. UmuDAb expressed in cells was correspondingly larger (24 kDa) than the Escherichia coli UmuD (15 kDa). DNA damage from mitomycin C or UV exposure caused UmuDAb cleavage in both E. coli wild-type and ΔumuD cells on a timescale resembling UmuD, but did not require UmuD. Like the self-cleaving serine proteases LexA and UmuD, UmuDAb required RecA for cleavage. This cleavage produced a UmuDAb' fragment of a size consistent with the predicted cleavage site of Ala83-Gly84. Site-directed mutations at Ala83 abolished cleavage, as did mutations at either the Ser119 or Lys156 predicted enzymatic residues. Co-expression of the cleavage site mutant and an enzymatic mutant did not allow cleavage, demonstrating a strictly intramolecular mechanism of cleavage that more closely resembles the LexA-type repressors than UmuD. These data show that UmuDAb undergoes a post-translational, LexA-like cleavage event after DNA damage, possibly to achieve its regulatory action., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Rhizobia are soil bacteria able to develop a nitrogen-fixing symbiosis with legumes. They are taxonomically spread among the alpha and beta subclasses of the Proteobacteria. Mimosa pudica, a tropical invasive weed, has been found to have an affinity for beta-rhizobia, including species within the Burkholderia and Cupriavidus genera. In this study, we describe the diversity of M. pudica symbionts in the island of New Caledonia, which is characterized by soils with high heavy metal content, especially of Ni. By using a plant-trapping approach on four soils, we isolated 96 strains, the great majority of which belonged to the species Cupriavidus taiwanensis (16S rRNA and recA gene phylogenies). A few Rhizobium strains in the newly described species Rhizobium mesoamericanum were also isolated. The housekeeping and nod gene phylogenies supported the hypothesis of the arrival of the C. taiwanensis and R. mesoamericanum strains together with their host at the time of the introduction of M. pudica in New Caledonia (NC) for its use as a fodder. The C. taiwanensis strains exhibited various tolerances to Ni, Zn and Cr, suggesting their adaptation to the specific environments in NC. Specific metal tolerance marker genes were found in the genomes of these symbionts, and their origin was investigated by phylogenetic analyses., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

The xnp1 remnant P2-type prophage of Xenorhabdus nematophila produces xenorhabdicin that is active against closely related species. Xenorhabdicin had not been characterized previously in other Xenorhabdus species. Here, we show xenorhabdicin production in six different strains of Xenorhabdus bovienii. The sequenced genome of X. bovienii SS-2004 was found to possess a highly conserved remnant P2-type cluster (xbp1). Inactivation of the xbpS1 sheath gene resulted in loss of bacteriocin activity, indicating that the xbp1 locus was required for xenorhabdicin production. xbp1 and xnp1 contain a CI-type repressor, a dinI gene involved in stabilization of ssDNA-RecA complexes and are inducible with mitomycin C, suggesting that both loci are regulated by cleavage of the CI repressor. Both xnp1 and xbp1 lack typical P2-type lysis genes but contain a predicted endolysin gene (enp) that may be involved in cell lysis. The main tail fibers of xnp1 and xbp1 are mosaic structures with divergent C-terminal regions suggesting they differ in host specificity. Several genes encoding C-terminal tail fiber fragments are present in the same position in xnp1 and xbp1. Recombination between the main fiber genes and the C-terminal fragments could potentially expand the host range specificity of xenorhabdicin in the respective strains., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)