Single Strand Annealing Plays a Major Role in RecA-Independent Recombination between Repeated Sequences in the Radioresistant Deinococcus radiodurans Bacterium

© 2015 Ithurbide et al. The bacterium Deinococcus radiodurans is one of the most radioresistant organisms known. It is able to reconstruct a functional genome from hundreds of radiation-induced chromosomal fragments. Our work aims to highlight the genes involved in recombination between 438 bp direct repeats separated by intervening sequences of various lengths ranging from 1,479 bp to 10,500 bp to restore a functional tetA gene in the presence or absence of radiation-induced DNA double strand breaks. The frequency of spontaneous deletion events between the chromosomal direct repeats were the same in recA+ and in ΔrecA, ΔrecF, and ΔrecO bacteria, whereas recombination between chromosomal and plasmid DNA was shown to be strictly dependent on the RecA and RecF proteins. The presence of mutations in one of the repeated sequence reduced, in a MutS-dependent manner, the frequency of the deletion events. The distance between the repeats did not influence the frequencies of deletion events in recA+as well in ΔrecA bacteria. The absence of the UvrD protein stimulated the recombination between the direct repeats whereas the absence of the DdrB protein, previously shown to be involved in DNA double strand break repair through a single strand annealing (SSA) pathway, strongly reduces the frequency of RecA- (and RecO-) independent deletions events. The absence of the DdrB protein also increased the lethal sectoring of cells devoid of RecA or RecO protein. γ-irradiation of recA+cells increased about 10-fold the frequencies of the deletion events, but at a lesser extend in cells devoid of the DdrB protein. Altogether, our results suggest a major role of single strand annealing in DNA repeat deletion events in bacteria devoid of the RecA protein, and also in recA+bacteria exposed to ionizing radiation

Infection néonatale bactérienne précoce : Quand mettre sous antibiotique et quelle antibiothérapie ? Early bacterial neonatal infection: When to indicate antibiotic treatment and what antibiotic therapy ?

Objective. Propose a relevant management strategy that can identify newborns with a bacterial infectious risk and those under clinical monitoring alone or in combination with parenteral antibiotic therapy.Methods. Retrospective study carried out between SA < 42, suspected of early bacterial infection and monitored in Maternity and in the Neonatology Unit of the Hospital Group Carnelle Portes of Oise [Val France]. The clinical-biological and bacteriological data, the therapeutic strategy and the evolution are analyzed. Results. Two hundred and forty newborns were eligible and divided into three groups: 120 asymptomatic newborns with antenatal criteria for bacterial infectious risk [G1NAS], 70 symptomatic newborns with antenatal criteria for bacterial infectious risk [G2NSCARIB] and 50 symptomatic newborns without antenatal criteria of bacterial infectious risk [G3NSSCARIB]. Inflammatory biology is limited tocolonized G1NAS newborns and symptomatic groups. The identified bacteria [Peripheral samples, gastric fluid, blood and cerebrospinal fluid] were mainly the Streptococcus of the group and the E Coli. Antibiotic therapy has been shown to be useful in asymptomatic newborns with inflammatory syndrome and bacteria identified on peripheral samples and gastric fluid, but  also in all symptomatic newborns. Conclusion. In a early bacterial infection, an interventionist attitude is required, but early antibiotic therapy is only useful in the situation of symptomatic newborns. On the otherhand, in the asymptomatic newborns, antibiotic therapy will be reserved for those carrying both an identified bacteria and an inflammatory syndrome. Contexte et objectif. L‟infection néonatale bactérienne précoce est greffée d‟une forte mortalité et morbidité conduisant à une antibiothérapie probabiliste sans délai souvent à posteriori inutile. L‟objectif du présent travail était de proposer une stratégie de prise en charge pertinente susceptible de bien identifier les nouveau-nés à risque infectieux bactérien et ceux relevant d‟une surveillance clinique seule ou associée à une antibiothérapie parentérale.    Méthodes. Etude documentaire menée entre janvier 2014 et janvier 2016 sur des nouveau-nés de 36≥SA<42, suspects d‟infection bactérienne précoce et suivis en Maternité et dans l‟unité de Néonatologie du Groupe Hospitalier Carnelle Portes de l‟Oise [Val D‟Oise, France]. Les données clinico-biologiques et bactériologiques, la stratégie thérapeutique et l‟évolution sont analysées.  Résultats. Deux cent quarante nouveau-nés [NNES] ont été éligibles et repartis en trois groupes : 120 NNES asymptomatiques avec critères anténatals de risque infectieux bactérien [G1NAS], 70 NNES symptomatiques avec critères anténatals de risque infectieux bactérien [G2NSCARIB] et 50 NNES symptomatiques sans critères anténatals de risque infectieux bactérien [G3NSSCARIB]. La biologie inflammatoire est limitée aux NNES du groupe G1NAS colonisés et aux groupes symptomatiques. Les germes identifiés [Prélèvements périphériques, liquide gastrique, sang et liquide céphalorachidien] ont été principalement le Streptocoque du groupe β et l‟E Coli. L‟antibiothérapie s‟est avérée utile chez les NNES asymptomatiques avec syndrome inflam-matoire et germes identifiés sur les prélèvements périphériques et liquide gastrique, mais aussi chez tous les NNES symptomatiques.                                                                    Conclusion. Chez un NNE âgé de ≥ 36SA et suspect d‟infection bactérienne précoce, une attitude interventionniste est de rigueur, mais l‟antibiothérapie sans délai n‟est utile que dans les situations des NNES symptomatiques. En revanche, chez les NNES asymptomatiques, l‟antibiothérapie sera réservée à ceux porteurs à la fois d‟un germe et d‟un syndrome inflammatoire

A Major Role of the RecFOR Pathway in DNA Double-Strand-Break Repair through ESDSA in Deinococcus radiodurans

In Deinococcus radiodurans, the extreme resistance to DNA–shattering treatments such as ionizing radiation or desiccation is correlated with its ability to reconstruct a functional genome from hundreds of chromosomal fragments. The rapid reconstitution of an intact genome is thought to occur through an extended synthesis-dependent strand annealing process (ESDSA) followed by DNA recombination. Here, we investigated the role of key components of the RecF pathway in ESDSA in this organism naturally devoid of RecB and RecC proteins. We demonstrate that inactivation of RecJ exonuclease results in cell lethality, indicating that this protein plays a key role in genome maintenance. Cells devoid of RecF, RecO, or RecR proteins also display greatly impaired growth and an important lethal sectoring as bacteria devoid of RecA protein. Other aspects of the phenotype of recFOR knock-out mutants paralleled that of a ΔrecA mutant: ΔrecFOR mutants are extremely radiosensitive and show a slow assembly of radiation-induced chromosomal fragments, not accompanied by DNA synthesis, and reduced DNA degradation. Cells devoid of RecQ, the major helicase implicated in repair through the RecF pathway in E. coli, are resistant to γ-irradiation and have a wild-type DNA repair capacity as also shown for cells devoid of the RecD helicase; in contrast, ΔuvrD mutants show a markedly decreased radioresistance, an increased latent period in the kinetics of DNA double-strand-break repair, and a slow rate of fragment assembly correlated with a slow rate of DNA synthesis. Combining RecQ or RecD deficiency with UvrD deficiency did not significantly accentuate the phenotype of ΔuvrD mutants. In conclusion, RecFOR proteins are essential for DNA double-strand-break repair through ESDSA whereas RecJ protein is essential for cell viability and UvrD helicase might be involved in the processing of double stranded DNA ends and/or in the DNA synthesis step of ESDSA

A comparison between ultraviolet disinfection and copper alginate beads within a vortex bioreactor for the deactivation of bacteria in simulated waste streams with high levels of colour, humic acid and suspended solids.

We show in this study that the combination of a swirl flow reactor and an antimicrobial agent (in this case copper alginate beads) is a promising technique for the remediation of contaminated water in waste streams recalcitrant to UV-C treatment. This is demonstrated by comparing the viability of both common and UV-C resistant organisms in operating conditions where UV-C proves ineffective - notably high levels of solids and compounds which deflect UV-C. The swirl flow reactor is easy to construct from commonly available plumbing parts and may prove a versatile and powerful tool in waste water treatment in developing countries

Mre11-Rad50 Promotes Rapid Repair of DNA Damage in the Polyploid Archaeon Haloferax volcanii by Restraining Homologous Recombination

Polyploidy is frequent in nature and is a hallmark of cancer cells, but little is known about the strategy of DNA repair in polyploid organisms. We have studied DNA repair in the polyploid archaeon Haloferax volcanii, which contains up to 20 genome copies. We have focused on the role of Mre11 and Rad50 proteins, which are found in all domains of life and which form a complex that binds to and coordinates the repair of DNA double-strand breaks (DSBs). Surprisingly, mre11 rad50 mutants are more resistant to DNA damage than the wild-type. However, wild-type cells recover faster from DNA damage, and pulsed-field gel electrophoresis shows that DNA double-strand breaks are repaired more slowly in mre11 rad50 mutants. Using a plasmid repair assay, we show that wild-type and mre11 rad50 cells use different strategies of DSB repair. In the wild-type, Mre11-Rad50 appears to prevent the repair of DSBs by homologous recombination (HR), allowing microhomology-mediated end-joining to act as the primary repair pathway. However, genetic analysis of recombination-defective radA mutants suggests that DNA repair in wild-type cells ultimately requires HR, therefore Mre11-Rad50 merely delays this mode of repair. In polyploid organisms, DSB repair by HR is potentially hazardous, since each DNA end will have multiple partners. We show that in the polyploid archaeon H. volcanii the repair of DSBs by HR is restrained by Mre11-Rad50. The unrestrained use of HR in mre11 rad50 mutants enhances cell survival but leads to slow recovery from DNA damage, presumably due to difficulties in the resolution of DNA repair intermediates. Our results suggest that recombination might be similarly repressed in other polyploid organisms and at repetitive sequences in haploid and diploid species

The deinococcal DdrB protein is involved in an early step of DNA double strand break repair and in plasmid transformation through its single-strand annealing activity.

International audienceThe Deinococcus radiodurans bacterium exhibits an extreme resistance to ionizing radiation. Here, we investigated the in vivo role of DdrB, a radiation-induced Deinococcus specific protein that was previously shown to exhibit some in vitro properties akin to those of SSB protein from Escherichia coli but also to promote annealing of single stranded DNA. First we report that the deletion of the C-terminal motif of the DdrB protein, which is similar to the SSB C-terminal motif involved in recruitment to DNA of repair proteins, did neither affect cell radioresistance nor DNA binding properties of purified DdrB protein. We show that, in spite of their different quaternary structure, DdrB and SSB occlude the same amount of ssDNA in vitro. We also show that DdrB is recruited early and transiently after irradiation into the nucleoid to form discrete foci. Absence of DdrB increased the lag phase of the extended synthesis-dependent strand annealing (ESDSA) process, affecting neither the rate of DNA synthesis nor the efficiency of fragment reassembly, as indicated by monitoring DNA synthesis and genome reconstitution in cells exposed to a sub-lethal ionizing radiation dose. Moreover, cells devoid of DdrB were affected in the establishment of plasmid DNA during natural transformation, a process that requires pairing of internalized plasmid single stranded DNA fragments, whereas they were proficient in transformation by a chromosomal DNA marker that integrates into the host chromosome through homologous recombination. Our data are consistent with a model in which DdrB participates in an early step of DNA double strand break repair in cells exposed to very high radiation doses. DdrB might facilitate the accurate assembly of the myriad of small fragments generated by extreme radiation exposure through a single strand annealing (SSA) process to generate suitable substrates for subsequent ESDSA-promoted genome reconstitution