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

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

Construction of Lactose-Assimilating and High-Ethanol-Producing Yeasts by Protoplast Fusion

The availability of a yeast strain which is capable of fermenting lactose and at the same time is tolerant to high concentrations of ethanol would be useful for the production of ethanol from lactose. Kluyveromyces fragilis is capable of fermenting lactose, but it is not as tolerant as Saccharomyces cerevisiae to high concentrations of ethanol. In this study, we have used the protoplast fusion technique to construct hybrids between auxotrophic strains of S. cerevisiae having high ethanol tolerance and an auxotrophic strain of lactose-fermenting K. fragilis isolated by ethyl methanesulfonate mutagenesis. The fusants obtained were prototrophic and capable of assimilating lactose and producing ethanol in excess of 13% (vol/vol). The complementation frequency of fusion was about 0.7%. Formation of fusants was confirmed by the increased amount of chromosomal DNA per cell. Fusants contained 8 × 10⁻⁸ to 16 × 10⁻⁸ μg of DNA per cell as compared with about 4 × 10⁻⁸ μg of DNA per cell for the parental strains, suggesting that multiple fusions had taken place.     

Evidence of Bacteroides fragilis Protection from Bartonella henselae-Induced Damage

Bartonella henselae is able to internalize endothelial progenitor cells (EPCs), which are resistant to the infection of other common pathogens. Bacteroides fragilis is a gram-negative anaerobe belonging to the gut microflora. It protects from experimental colitis induced by Helicobacter hepaticus through the polysaccharide A (PSA). The aim of our study was to establish: 1) whether B. fragilis colonization could protect from B. henselae infection; if this event may have beneficial effects on EPCs, vascular system and tissues. Our in vitro results establish for the first time that B. fragilis can internalize EPCs and competes with B. henselae during coinfection. We observed a marked activation of the inflammatory response by Real-time PCR and ELISA in coinfected cells compared to B. henselae-infected cells (63 vs 23 up-regulated genes), and after EPCs infection with mutant B. fragilis ΔPSA (≅90% up-regulated genes) compared to B. fragilis. Interestingly, in a mouse model of coinfection, morphological and ultrastructural analyses by hematoxylin-eosin staining and electron microscopy on murine tissues revealed that damages induced by B. henselae can be prevented in the coinfection with B. fragilis but not with its mutant B. fragilis ΔPSA. Moreover, immunohistochemistry analysis with anti-Bartonella showed that the number of positive cells per field decreased of at least 50% in the liver (20±4 vs 50±8), aorta (5±1 vs 10±2) and spleen (25±3 vs 40±6) sections of mice coinfected compared to mice infected only with B. henselae. This decrease was less evident in the coinfection with ΔPSA strain (35±6 in the liver, 5±1 in the aorta and 30±5 in the spleen). Finally, B. fragilis colonization was also able to restore the EPC decrease observed in mice infected with B. henselae (0.65 vs 0.06 media). Thus, our data establish that B. fragilis colonization is able to prevent B. henselae damages through PSA.     

Chi hotspots trigger a conformational change in the helicase-like domain of AddAB to activate homologous recombination

In bacteria, the repair of double-stranded DNA breaks is modulated by Chi sequences. These are recognised by helicase-nuclease complexes that process DNA ends for homologous recombination. Chi activates recombination by changing the biochemical properties of the helicase-nuclease, transforming it from a destructive exonuclease into a recombination-promoting repair enzyme. This transition is thought to be controlled by the Chi-dependent opening of a molecular latch, which enables part of the DNA substrate to evade degradation beyond Chi. Here, we show that disruption of the latch improves Chi recognition efficiency and stabilizes the interaction of AddAB with Chi, even in mutants that are impaired for Chi binding. Chi recognition elicits a structural change in AddAB that maps to a region of AddB which resembles a helicase domain, and which harbours both the Chi recognition locus and the latch. Mutation of the latch potentiates the change and moderately reduces the duration of a translocation pause at Chi. However, this mutant displays properties of Chi-modified AddAB even in the complete absence of bona fide hotspot sequences. The results are used to develop a model for AddAB regulation in which allosteric communication between Chi binding and latch opening ensures quality control during recombination hotspot recognition.     

Identification of a potent small-molecule inhibitor of bacterial DNA repair that potentiates quinolone antibiotic activity in methicillin-resistant Staphylococcus aureus

The global emergence of antibiotic resistance is one of the most serious challenges facing modern medicine. There is an urgent need for validation of new drug targets and the development of small molecules with novel mechanisms of action. We therefore sought to inhibit bacterial DNA repair mediated by the AddAB/RecBCD protein complexes as a means to sensitize bacteria to DNA damage caused by the host immune system or quinolone antibiotics. A rational, hypothesis-driven compound optimization identified IMP-1700 as a cell-active, nanomolar potency compound. IMP-1700 sensitized multidrug-resistant Staphylococcus aureus to the fluoroquinolone antibiotic ciprofloxacin, where resistance results from a point mutation in the fluoroquinolone target, DNA gyrase. Cellular reporter assays indicated IMP-1700 inhibited the bacterial SOS-response to DNA damage, and compound-functionalized Sepharose successfully pulled-down the AddAB repair complex. This work provides validation of bacterial DNA repair as a novel therapeutic target and delivers IMP-1700 as a tool molecule and starting point for therapeutic development to address the pressing challenge of antibiotic resistance.     

One-Step Reverse-Transcription FRET-PCR for Differential Detection of Five Ebolavirus Species

Ebola is an emerging infectious disease caused by a deadly virus belonging to the family Filoviridae, genus Ebolavirus. Based on their geographical distribution, Ebolavirus has been classified into total five species so far, mainly Zaire, Sudan, Taï Forest, Bundibugyo and Reston. It is important to be able to differentiate the Ebolavirus species as they significantly differ in pathogenicity and more than one species can be present in an area. We have developed a one-step step-down RT-PCR detecting all five Ebolavirus species with high sensitivity (1 copy of Ebolavirus DNA, 10 copies of RNA and 320 copies of RNA spiked in 1 ml whole blood). The primers and FRET-probes we designed enabled us to differentiate five Ebolavirus species by distinct T_m (Zaire: flat peaks between 53.0°C and 56.9°C; Sudan: 51.6°C; Reston: flat peaks between 47.5°C and 54.9°C; Tai Forest: 52.8°C; Bundibugyo: dual peaks at 48.9°C and 53.5°C), and by different amplicon sizes (Zaire 255bp, Sudan 211bp, Reston 192bp, Taï Forest 166bp, Bundibugyo 146bp). This one-size-fit-all assay enables the rapid detection and discrimination of the five Ebolavirus species in a single reaction.     

Different modes of spacer acquisition by the Staphylococcus epidermidis type III-A CRISPR-Cas system

CRISPR-Cas systems provide prokaryotic organisms with an adaptive defense mechanism that acquires immunological memories of infections. This is accomplished by integration of short fragments from the genome of invaders such as phages and plasmids, called ‘spacers’, into the CRISPR locus of the host. Depending on their genetic composition, CRISPR-Cas systems can be classified into six types, I-VI, however spacer acquisition has been extensively studied only in type I and II systems. Here, we used an inducible spacer acquisition assay to study this process in the type III-A CRISPR-Cas system of Staphylococcus epidermidis, in the absence of phage selection. Similarly to type I and II spacer acquisition, this type III system uses Cas1 and Cas2 to preferentially integrate spacers from the chromosomal terminus and free dsDNA ends produced after DNA breaks, in a manner that is enhanced by the AddAB DNA repair complex. Surprisingly, a different mode of spacer acquisition from rRNA and tRNA loci, which spans only the transcribed sequences of these genes and is not enhanced by AddAB, was also detected. Therefore, our findings reveal both common mechanistic principles that may be conserved in all CRISPR-Cas systems, as well as unique and intriguing features of type III spacer acquisition.     

Pulsed IR Heating Studies of Single-Molecule DNA Duplex Dissociation Kinetics and Thermodynamics

Single-molecule fluorescence spectroscopy is a powerful technique that makes it possible to observe the conformational dynamics associated with biomolecular processes. The addition of precise temperature control to these experiments can yield valuable thermodynamic information about equilibrium and kinetic rate constants. To accomplish this, we have developed a microscopy technique based on infrared laser overtone/combination band absorption to heat small (≈10⁻¹¹ liter) volumes of water. Detailed experimental characterization of this technique reveals three major advantages over conventional stage heating methods: 1), a larger range of steady-state temperatures (20–100°C); 2), substantially superior spatial (≤20 μm) control; and 3), substantially superior temporal (≈1 ms) control. The flexibility and breadth of this spatial and temporally resolved laser-heating approach is demonstrated in single-molecule fluorescence assays designed to probe the dissociation of a 21 bp DNA duplex. These studies are used to support a kinetic model based on nucleic acid end fraying that describes dissociation for both short (<10 bp) and long (>10 bp) DNA duplexes. These measurements have been extended to explore temperature-dependent kinetics for the 21 bp construct, which permit determination of single-molecule activation enthalpies and entropies for DNA duplex dissociation.     

Ferritin-like family proteins in the anaerobe Bacteroides fragilis: when an oxygen storm is coming, take your iron to the shelter

Bacteroides are gram-negative anaerobes and one of the most abundant members the lower GI tract microflora where they play an important role in normal intestinal physiology. Disruption of this commensal relationship has a great impact on human health and disease. Bacteroides spp. are significant opportunistic pathogens causing infections when the mucosal barrier integrity is disrupted following predisposing conditions such as GI surgery, perforated or gangrenous appendicitis, perforated ulcer, diverticulitis, trauma and inflammatory bowel diseases. B. fragilis accounts for 60–90 % of all anaerobic infections despite being a minor component of the genus (<1 % of the flora). Clinical strains of B. fragilis are among the most aerotolerant anaerobes. When shifted from anaerobic to aerobic conditions B. fragilis responds to oxidative stress by inducing the expression of an extensive set of genes involved in protection against oxygen derived radicals and iron homeostasis. In Bacteroides, little is known about the metal/oxidative stress interactions and the mobilization of intra-cellular non-heme iron during the oxidative stress response has been largely overlooked. Here we present an overview of the work carried out to demonstrate that both oxygen-detoxifying enzymes and iron-storage proteins are essential for B. fragilis to survive an adverse oxygen-rich environment. Some species of Bacteroides have acquired multiple homologues of the iron storage and detoxifying ferritin-like proteins but some species contain none. The proteins found in Bacteroides are classical mammalian H-type non-heme ferritin (FtnA), non-specific DNA binding and starvation protein (Dps) and the newly characterized bacterial Dps-Like miniferritin protein. The full contribution of ferritin-like proteins to pathophysiology of commensal and opportunistic pathogen Bacteroides spp. still remains to be elucidated.     

Use of 16S rRNA Gene-Targeted Group-Specific Primers for Real-Time PCR Analysis of Predominant Bacteria in Human Feces

16S rRNA gene-targeted group-specific primers were designed and validated for specific detection and quantification of the Clostridium leptum subgroup and the Atopobium cluster. To monitor the predominant bacteria in human feces by real-time PCR, we used these specific primers together with four sets of group-specific primers for the Clostridium coccoides group, the Bacteroides fragilis group, Bifidobacterium, and Prevotella developed in a previous study (T. Matsuki, K. Watanabe, J. Fujimoto, Y. Miyamoto, T. Takada, K. Matsumoto, H. Oyaizu, and R. Tanaka, Appl. Environ. Microbiol. 68:5445-5451, 2002). Examination of DNA extracted from the feces of 46 healthy adults showed that the C. coccoides group was present in the greatest numbers (log₁₀ 10.3 ± 0.3 cells per g [wet weight] [average ± standard deviation]), followed by the C. leptum subgroup (log₁₀ 9.9 ± 0.7 cells per g [wet weight]), the B. fragilis group (log₁₀ 9.9 ± 0.3 cells per g [wet weight]), Bifidobacterium (log₁₀ 9.4 ± 0.7 cells per g [wet weight]), and the Atopobium cluster (log₁₀ 9.3 ± 0.7 cells per g [wet weight]). These five bacterial groups were detected in all 46 volunteers. Prevotella was found in only 46% of the subjects at a level of log₁₀ 9.7 ± 0.8 cells per g (wet weight). Examination of changes in the population and the composition of the intestinal flora for six healthy adults over an 8-month period revealed that the composition of the flora of each volunteer remained stable throughout the test period.     

Analysis of the unwinding activity of the dimeric RECQ1 helicase in the presence of human replication protein A

RecQ helicases are required for the maintenance of genome stability. Characterization of the substrate specificity and identification of the binding partners of the five human RecQ helicases are essential for understanding their function. In the present study, we have developed an efficient baculovirus expression system that allows us to obtain milligram quantities of recombinant RECQ1. Our gel filtration and dynamic light scattering experiments show that RECQ1 has an apparent molecular mass of 158 kDa and a hydrodynamic radius of 5.4 ± 0.6 nm, suggesting that RECQ1 forms dimers in solution. The oligomeric state of RECQ1 remains unchanged upon binding to a single-stranded (ss)DNA fragment of 50 nt. We show that RECQ1 alone is able to unwind short DNA duplexes (<110 bp), whereas considerably longer substrates (501 bp) can be unwound only in the presence of human replication protein A (hRPA). The same experiments with Escherichia coli SSB show that RECQ1 is specifically stimulated by hRPA. However, hRPA does not affect the ssDNA-dependent ATPase activity of RECQ1. In addition, our far western, ELISA and co-immunoprecipitation experiments demonstrate that RECQ1 physically interacts with the 70 kDa subunit of hRPA and that this interaction is not mediated by DNA.     

Prod is a novel DNA-binding protein that binds to the 1.686 g/cm(3) 10 bp satellite repeat of Drosophila melanogaster

The proliferation disrupter (prod) gene of Drosophila melanogaster encodes a novel protein associated with centromeric chromosomal regions that is required for chromatin condensation and cell viability. We have examined the binding of the Prod protein to DNA in vitro. Co-immunoprecipitation experiments demonstrate that Prod is a DNA-binding protein that specifically recognizes the 10 bp AGAATAACAT satellite repeat of D.melanogaster. Footprinting experiments show that the protein interacts with a 5–8 bp target sequence in each 10 bp repeat and suggest that it can mediate condensation of this satellite into a superhelix. Gel retardation experiments indicate that Prod does not have a well defined DNA-binding domain and it binds the satellite in a co-operative manner, probably forming Prod multimers. Since Prod localizes to both heterochromatin and euchromatin in vivo, we discuss the possibility that the ability of pre-existing euchromatic proteins to bind DNA in a co-operative manner, might be a prerequisite of satellite compaction and satellite amplification, thereby providing a basic factor in heterochromatin evolution.     

Dual Nuclease and Helicase Activities of Helicobacter pylori AddAB Are Required for DNA Repair, Recombination, and Mouse Infectivity

Helicobacter pylori infection of the human stomach is associated with disease-causing inflammation that elicits DNA damage in both bacterial and host cells. Bacteria must repair their DNA to persist. The H. pylori AddAB helicase-exonuclease is required for DNA repair and efficient stomach colonization. To dissect the role of each activity in DNA repair and infectivity, we altered the AddA and AddB nuclease (NUC) domains and the AddA helicase (HEL) domain by site-directed mutagenesis. Extracts of Escherichia coli expressing H. pylori addA^NUCB or addAB^NUC mutants unwound DNA but had approximately half of the exonuclease activity of wild-type AddAB; the addA^NUCB^NUC double mutant lacked detectable nuclease activity but retained helicase activity. Extracts with AddA^HELB lacked detectable helicase and nuclease activity. H. pylori with the single nuclease domain mutations were somewhat less sensitive to the DNA-damaging agent ciprofloxacin than the corresponding deletion mutant, suggesting that residual nuclease activity promotes limited DNA repair. The addA^NUC and addA^HEL mutants colonized the stomach less efficiently than the wild type; addB^NUC showed partial attenuation. E. coli ΔrecBCD expressing H. pylori addAB was recombination-deficient unless H. pylori recA was also expressed, suggesting a species-specific interaction between AddAB and RecA and also that H. pylori AddAB participates in both DNA repair and recombination. These results support a role for both the AddAB nuclease and helicase in DNA repair and promoting infectivity.Infection of the stomach with Helicobacter pylori causes a variety of diseases including gastritis, peptic ulcers, and gastric cancer (1). A central feature of the pathology of these conditions is the establishment of a chronic inflammatory response that acts both on the host and the infecting bacteria (2). Both epithelial (3, 4) and lymphoid (5, 6) cells in the gastric mucosa of infected individuals release DNA-damaging agents that can introduce double-stranded (ds)² breaks into the bacterial chromosome (7). The ds breaks must be repaired for the bacteria to survive and establish chronic colonization of the stomach. Homologous recombination is required for the faithful repair of DNA damage and bacterial survival. Alteration of the expression of one of a series of cell surface proteins on H. pylori occurs by an apparent gene conversion of babA, the frequency of which is reduced in repair-deficient strains (8, 9). This change in the cell surface, which may allow H. pylori to evade the host immune response, is a second means by which recombination can promote efficient colonization of the stomach by H. pylori.The initiation or presynaptic steps of recombination at dsDNA breaks in most bacteria involves the coordinated action of nuclease and helicase activities provided by one of two multisubunit enzymes, the AddAB and RecBCD enzymes (10). Escherichia coli recBCD null mutants have reduced cell viability, are hypersensitive to DNA-damaging agents, and are homologous recombination-deficient (11–14). Similarly, H. pylori addA and addB null mutants are hypersensitive to DNA-damaging agents, have reduced frequencies of babA gene conversion, and colonize the stomach of mice less efficiently than wild-type strains (8).The activities of RecBCD enzyme from E. coli (15–19) and AddAB from H. pylori (8) or Bacillus subtilis (20–23) indicate some common general features of the presynaptic steps of DNA repair. In the case of E. coli, repair begins when the RecBCD enzyme binds to a dsDNA end and unwinds the DNA using its ATP-dependent helicase activities (17, 24). Single-stranded (ss) DNA produced during unwinding, with or without accompanying nuclease, is coated with RecA protein (16, 25). This recombinogenic substrate engages in strand exchange with a homologous intact duplex to form a joint molecule. Joint molecules are thought to be converted into intact, recombinant DNA either by replication or by cutting and ligation of exchanged strands (26).Although the AddAB and RecBCD enzymes appear to play similar roles in promoting recombination and DNA repair, they differ in several ways. RecBCD is a heterotrimer, composed of one copy of the RecB, RecC, and RecD gene products (27), whereas AddAB has two subunits, encoded by the addA and addB genes (21, 28). The enzyme subunit(s) responsible for helicase activity can be inferred from the presence of conserved protein domains or the activity of purified proteins. AddA, RecB, and RecD are superfamily I helicases with six highly conserved helicase motifs, including the conserved Walker A box found in many enzymes that bind ATP (29–32). A Walker A box is defined by the consensus sequence (G/A)XXGXGKT (X is any amino acid (29). RecBCD enzymes in which the conserved Lys in this motif is changed to Gln have a reduced affinity for ATP binding (33, 34) and altered helicase activity (17, 35–37).A nuclease domain with the conserved amino acid sequence LDYK is found in RecB, AddA, AddB, and many other nucleases (38). The conserved Asp plays a role in Mg²⁺ binding at the active site; Mg²⁺ is required for nuclease activity (39). The recB1080 mutation, which changes codon 1080 from the conserved Asp in this motif to Ala, eliminates nuclease activity (39).We have recently shown that addA and addB deletion mutants are hypersensitive to DNA-damaging agents and impaired in colonization of the mouse stomach compared with wild-type strains (8). To determine the roles of the individual helicase and nuclease activities of H. pylori AddAB in DNA repair and infectivity, we used site-directed mutagenesis to inactivate the conserved nuclease domains of addA and addB and the conserved ATPase (helicase) domain of AddA. Here, we report that loss of the AddAB helicase is sufficient to impair H. pylori DNA repair and infectivity and, when the genes are expressed in E. coli, homologous recombination. AddAB retains partial activity in biochemical and genetic assays when either of the two nuclease domains is inactivated but loses all detectable nuclease activity when both domains are inactivated. Remarkably, H. pylori AddAB can produce recombinants in E. coli only in the presence of H. pylori RecA, suggesting a species-specific interaction in which AddAB facilitates the production of ssDNA-coated with RecA protein. Our results show that both the helicase and nuclease activities are required for the biological roles of H. pylori AddAB.     

Identification and Characterization of Conjugative Transposons CTn86 and CTn9343 in Bacteroides fragilis Strains

The related genetic elements flanking the Bacteroides fragilis pathogenicity island (PAI) in enterotoxigenic B. fragilis (ETBF) 86-5443-2-2 and also present in pattern III nontoxigenic B. fragilis (NTBF) NCTC 9343 were defined as putative conjugative transposons (CTns), designated CTn86 and CTn9343, respectively (A. A. Franco, J. Bacteriol. 181:6623-6633, 2004). CTn86 and CTn9343 have the same basic structures except that their encoded transposases have low similarity and CTn9343 lacks the B. fragilis PAI and contains an extra 7-kb region not present in CTn86. In this study, using DNA hybridization and PCR analysis, we characterized the genetic element flanking the PAI in a collection of ETBF strains and the related genetic elements in a collection of NTBF pattern III strains. We found that in all 123 ETBF strains, the PAI is contained in a genetic element similar to CTn86. Of 73 pattern III strains, 26 (36%) present a genetic element similar to CTn9343, 38 (52%) present a genetic element similar to CTn9343 but lack the 7-kb region that is also absent in CTn86 (CTn9343-like element), and 9 (12%) present a genetic element similar to CTn86 but lacking the PAI (CTn86-like element). In addition to containing CTn86, ETBF strains can also contain CTn9343, CTn9343-like, or CTn86-like elements. CTn86, CTn9343, CTn86-like, and CTn9343-like elements were found exclusively in B. fragilis strains and predominantly in division I, cepA-positive strains.     

Phylogenetic Ubiquity and Shuffling of the Bacterial RecBCD and AddAB Recombination Complexes

RecBCD and AddAB are bacterial enzymes that share similar helicase and nuclease activities and initiate repair of DNA double-strand breaks by homologous recombination. Examination of the phylogenetic distribution of AddAB and RecBCD revealed that one or the other complex is present in most sequenced bacteria. In addition, horizontal gene transfer (HGT) events involving addAB and recBCD appear to be common, with the genes encoding one complex frequently replacing those encoding the other. HGT may also explain the unexpected identification of archaeal addAB genes. More than 85% of addAB and recBCD genes are clustered on the genome, suggesting operon structures. A few organisms, including the Mycobacteria, encode multiple copies of these complexes of either the same or mixed classes. The possibility that the enzymatic activities of the AddAB and RecBCD enzymes promote their horizontal transfer is discussed, and the distribution of AddAB/RecBCD is compared to that of the RecU/RuvC resolvases. Finally, it appears that two sequence motifs, the Walker A box involved in ATP binding and an iron-sulfur-cysteine cluster, are present only in subsets of AddB proteins, suggesting the existence of mechanistically distinct classes of AddB.Homologous recombination is central to the repair of DNA damaged by single-strand (SS) and double-strand (DS) breaks and gaps. Such discontinuities can occur after exposure to exogenous agents such as ionizing radiation but also when DNA replication forks break and as intermediates in DNA repair processes. Homologous recombination can also rearrange genetic information, making it important in processes such as phase variation of bacterial outer membrane proteins (for an example, see reference 2).Homologous recombination is highly conserved among viruses, bacteria, archaea, and eukaryotes. There are three recognizable stages (for reviews, see references 25 and 35). In the first stage, presynapsis, the DNA substrate is processed to give a SS region coated with strand exchange protein(s). In bacteria, this protein is RecA. In the second stage, synapsis, the protein-DNA complex pairs with its complementary homologous DNA target, displacing the other strand at the target site and forming a joint molecule. In the final stage, postsynapsis, DNA replication fills in any gaps in the joint molecule, which is then resolved to give recombinant DNA products.In bacteria, there appear to be two different presynaptic pathways that use either the related AddAB or RecBCD holoenzymes (Fig. ​(Fig.1)1) or, alternatively, the RecFOR proteins. RecFOR proteins appear to operate on SS gaps to ensure RecA is loaded there, whereas RecBCD and AddAB act on DNA DS breaks (DSBs), processing them to yield SS 3′ DNA ends coated with RecA (3, 43).Open in a separate windowFIG. 1.Structure of the RecBCD and AddAB proteins. The RecB, RecD, and AddA proteins include a helicase domain (solid green) with the canonical six-helicase motifs of helicase superfamily I. The most N-terminal of these motifs is the Walker A box (orange). The RecC and AddB proteins include an inactivated helicase domain (striped green). The RecB, AddA, and AddB proteins additionally possess similar short nuclease domains toward their C termini (red). Some, but not all, AddB proteins possess an N-terminal Walker A and/or a mostly C-terminal iron-sulfur motif made up of four cysteines (blue).The RecBCD complex has most thoroughly been studied in Escherichia coli, a member of the Gamma class of the phylum Proteobacteria. RecB and RecD are superfamily I helicases, and RecB is also a nuclease. After the RecBCD complex loads onto a DS DNA end, the RecB and RecD helicases separate the strands, with the slower RecB helicase traveling on one strand in the 3′-to-5′ direction and the faster RecD helicase traveling on the other strand from 5′ to 3′ (38). During translocation, E. coli RecBCD recognizes a specific DNA sequence, Chi (5′-GCTGGTGG-3′), perhaps via the RecC protein, which appears to be an inactivated helicase (Fig. ​(Fig.1)1) (32, 34). Chi is a recombination hot spot that switches the complex to a recombinogenic mode, producing a 3′ SS end at the Chi site via the nuclease activity of RecB (for reviews, see references 25 and 35). In addition to producing a 3′ SS “tail” needed for recombination, the RecBCD complex actively loads the RecA strand exchange protein onto this DNA (5). In E. coli, the RecBCD complex is responsible for essentially all recombinational repair of DSBs.Bacillus subtilis and some other bacteria lack RecBCD but possess a different complex with many of the same activities. This complex is composed of the AddA and AddB proteins (22). Like RecB, AddA is a superfamily I helicase and a nuclease (18, 21), organized with the same domain structure as RecB (Fig. ​(Fig.1).1). Similar to RecC, AddB appears to be an inactivated helicase, but it also possesses a nuclease domain similar to those of RecB and AddA (Fig. ​(Fig.1)1) (2, 11, 43). Like RecBCD, AddAB is an ATP-dependent helicase that acts as a nuclease in conjunction with its helicase activity, with the AddA nuclease acting on the 3′-end strand and the AddB nuclease on the 5′-end strand (43). Also like RecBCD, AddAB recognizes Chi-like control sequences, for instance, 5′-GCGCGTG-3′ in Lactococcus lactis and 5′-AGCGG-3′ in B. subtilis (7, 12). The AddAB complex is required for recombinational repair of DSBs (1, 2, 44), but it is not known if, like RecBCD, it actively loads RecA.Both RecBCD and AddAB are important for bacterial pathogenicity. addAB mutations reduce the ability of Helicobacter pylori to colonize mouse stomachs, and recBC mutants of Salmonella enterica serovar Typhimurium are severely compromised for infection and killing (2, 8).Using a strict set of criteria, Rocha et al. (30) examined the distribution across bacteria of the RecBCD and AddAB enzymes, along with other recombination and DNA repair proteins. They determined that the AddAB proteins are ubiquitous in some taxa and the RecBCD proteins in others, while yet other taxa have examples of both complexes in different species. In addition, they classified several species as lacking both the AddAB and RecBCD complexes. Recently, addAB genes from Epsilonproteobacteria were identified (2, 27, 40). Previously, all examined members of this proteobacterial class had been classified by Rocha et al. as lacking every component of the RecBCD and AddAB systems. This finding suggests that the stringent criteria used by Rocha et al. (30) might have led them to miss other examples of AddAB and RecBCD from sequenced bacteria. On this basis, and to gain a fuller understanding of the importance of the AddAB and RecBCD enzymes, their phylogenetic distribution was reexamined.     

The half-life of DNA in bone: measuring decay kinetics in 158 dated fossils

Claims of extreme survival of DNA have emphasized the need for reliable models of DNA degradation through time. By analysing mitochondrial DNA (mtDNA) from 158 radiocarbon-dated bones of the extinct New Zealand moa, we confirm empirically a long-hypothesized exponential decay relationship. The average DNA half-life within this geographically constrained fossil assemblage was estimated to be 521 years for a 242 bp mtDNA sequence, corresponding to a per nucleotide fragmentation rate (k) of 5.50 × 10^–6 per year. With an effective burial temperature of 13.1°C, the rate is almost 400 times slower than predicted from published kinetic data of in vitro DNA depurination at pH 5. Although best described by an exponential model (R² = 0.39), considerable sample-to-sample variance in DNA preservation could not be accounted for by geologic age. This variation likely derives from differences in taphonomy and bone diagenesis, which have confounded previous, less spatially constrained attempts to study DNA decay kinetics. Lastly, by calculating DNA fragmentation rates on Illumina HiSeq data, we show that nuclear DNA has degraded at least twice as fast as mtDNA. These results provide a baseline for predicting long-term DNA survival in bone.     

Hunting the Extinct Steppe Bison (Bison priscus) Mitochondrial Genome in the Trois-Frères Paleolithic Painted Cave

Despite the abundance of fossil remains for the extinct steppe bison (Bison priscus), an animal that was painted and engraved in numerous European Paleolithic caves, a complete mitochondrial genome sequence has never been obtained for this species. In the present study we collected bone samples from a sector of the Trois-Frères Paleolithic cave (Ariège, France) that formerly functioned as a pitfall and was sealed before the end of the Pleistocene. Screening the DNA content of the samples collected from the ground surface revealed their contamination by Bos DNA. However, a 19,000-year-old rib collected on a rock apart the pathway delineated for modern visitors was devoid of such contaminants and reproducibly yielded Bison priscus DNA. High-throughput shotgun sequencing combined with conventional PCR analysis of the rib DNA extract enabled to reconstruct a complete mitochondrial genome sequence of 16,318 bp for the extinct steppe bison with a 10.4-fold coverage. Phylogenetic analyses robustly established the position of the Bison priscus mitochondrial genome as basal to the clade delineated by the genomes of the modern American Bison bison. The extinct steppe bison sequence, which exhibits 93 specific polymorphisms as compared to the published Bison bison mitochondrial genomes, provides an additional resource for the study of Bovinae specimens. Moreover this study of ancient DNA delineates a new research pathway for the analysis of the Magdalenian Trois-Frères cave.     

Functional analysis of four Class III peroxidases from Chinese pear fruit: a critical role in lignin polymerization

Pear fruit could be used as good medicine to relieve coughs, promote salivation, nourish lungs, and reduce the risk of many diseases for its phytochemical action. Lignin is a major secondary metabolite in Chinese pear fruit. Class III peroxidase (Class III PRX) is an important enzyme in the biosynthesis of lignin in plants. However, we poorly understand the role of PRXs in lignin biosynthesis in Chinese pear fruit. In our study, we cloned five PRXs from Chinese pear (Pyrus bretschneideri), namely PbPRX2, PbPRX22, PbPRX34, PbPRX64, and PbPRX75, which contained 978 bp encoded 326 amino acids (AA), 2607 bp encoded 869 AA, 972 bp encoded 324 AA, 687 bp encoded 229 AA, and 1020 bp encoded 340 AA, respectively. Enzyme activity analysis showed that four recombinant PbPRX proteins had catalytic activities for pyrogallol, guaiacol, ferulic acid, coniferyl alcohol, and sinapyl alcohol. Subcellular localization experiments showed that these genes were located in the cell wall or cell membrane. Enzyme activity and kinetics of PbPRX2 revealed its role in polymerization of lignin in Chinese pear fruit. The present study suggested that PbPRXs played critical roles in lignin biosynthesis in Chinese pear fruit.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-00949-9.     

In Vivo Relationship between Intestinal Bifidobacteria Overgrowth and Bacteroides fragilis Repression Induced by Consumption of Bifidobacterial Cell-free Whey

Cell-free whey from a selected strain, Bifidobacterium breve C50, induced an increase in bifidobacteria associated with a Bacteroides fragilis reduction in the gut of conventional mice and humans. The purpose of our study was to investigate the mechanism of B. fragilis repression. C50 cell-free whey was given for 15 days to conventional or ex-germ-free mice mono-associated to the strain B. fragilis CFPL 358. Conventional and ex-germ-free control mice received whey which was incapable of promoting intestinal bifidobacteria and of reducing B. fragilis. Bacterial counting was carried out in the ileum, caecum and colon of both mouse models. The C50 cell-free whey induced a significant increase in endogenous bifidobacteria in the ileum of conventional mice, whereas B. fragilis was below detectable levels throughout the intestine. In ex-germ-free mice mono-associated with B. fragilis, the strain was seen to be at a high level through the whole intestine and no significant difference in counts was observed according to the whey administered to animals. The data indicated that a prerequisite for C50 cell-free whey repressive activity against B. fragilis is colonization of the mouse gut with complex bacterial microflora. With the exception of the distal ileum, the bifidobacterial overgrowth did not, however, support B. fragilis reduction. It is likely that in the caecum and colon some other bacteria participated in the process.