The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo

How living cells deal with head-on collisions of the replication and transcription complexes has been debated for a long time. Even in the widely studied model bacteria Escherichia coli, the enzymes that take care of such collisions are still unknown. We report here that in vivo, the DinG, Rep and UvrD helicases are essential for efficient replication across highly transcribed regions. We show that when rRNA operons (rrn) are inverted to face replication, the viability of the dinG mutant is affected and over-expression of RNase H rescues the growth defect, showing that DinG acts in vivo to remove R-loops. In addition, DinG, Rep and UvrD exert a common function, which requires the presence of two of these three helicases. After replication blockage by an inverted rrn, Rep in conjunction with DinG or UvrD removes RNA polymerase, a task that is fulfilled in its absence by the SOS-induced DinG and UvrD helicases. Finally, Rep and UvrD also act at inverted sequences other than rrn, and promote replication through highly transcribed regions in wild-type E. coli

Exploring the Phytobeneficial and Biocontrol Capacities of Endophytic Bacteria Isolated from Hybrid Vanilla Pods.

peer reviewedIn this study, 58 endophytic bacterial strains were isolated from pods of two hybrid vanilla plants from Madagascar, Manitra ampotony and Tsy taitra. They were genetically characterized and divided into four distinct phylotypes. Three were associated to genus Bacillus species, and the fourth to the genus Curtobacterium. A selection of twelve strains corresponding to the identified genetic diversity were tested in vitro for four phytobeneficial capacities: phosphate solubilisation, free nitrogen fixation, and phytohormone and siderophore production. They were also evaluated in vitro for their ability to biocontrol the growth of the vanilla pathogenic fungi, Fusarium oxysporum f. sp. radicis vanillae and Cholletotrichum orchidophilum. Three bacteria of phylotype 4, m62a, m64 and m65, showed a high nitrogen fixation capacity in vitro, similar to the Pseudomonas florescens F113 bacterium used as a control (phospate solubilizing efficiency respectively 0.50 ± 0.07, 0.43 ± 0.07 and 0.40 ± 0.06 against 0.48 ± 0.03). Strain t2 related to B. subtilis showed a higher siderophore production than F113 (respectively 1.40 ± 0.1 AU and 1.2 ± 0.1 AU). The strain m72, associated with phylotype 2, showed the highest rate of production of Indole-3-acetic acid (IAA) in vitro. Bacteria belonging to the pylotype 4 showed the best capacity to inhibit fungal growth, especially the strains m62b m64 and t24, which also induced a significant zone of inhibition, suggesting that they may be good candidates for controlling fungal diseases of vanilla. This competence was highlighted with spectral imaging showing the production of lipopeptides (Iturin A2 and A3, C16 and C15-Fengycin A and C14 and C15-Surfactin) by the bacterial strains m65 confronted with the pathogenic fungi of vanilla.FEDER BIOMED HUB Technology Suppor

Replication Fork Reversal after Replication–Transcription Collision

Replication fork arrest is a recognized source of genetic instability, and transcription is one of the most prominent causes of replication impediment. We analyze here the requirement for recombination proteins in Escherichia coli when replication–transcription head-on collisions are induced at a specific site by the inversion of a highly expressed ribosomal operon (rrn). RecBC is the only recombination protein required for cell viability under these conditions of increased replication-transcription collisions. In its absence, fork breakage occurs at the site of collision, and the resulting linear DNA is not repaired and is slowly degraded by the RecJ exonuclease. Lethal fork breakage is also observed in cells that lack RecA and RecD, i.e. when both homologous recombination and the potent exonuclease V activity of the RecBCD complex are inactivated, with a slow degradation of the resulting linear DNA by the combined action of the RecBC helicase and the RecJ exonuclease. The sizes of the major linear fragments indicate that DNA degradation is slowed down by the encounter with another rrn operon. The amount of linear DNA decreases nearly two-fold when the Holliday junction resolvase RuvABC is inactivated in recB, as well as in recA recD mutants, indicating that part of the linear DNA is formed by resolution of a Holliday junction. Our results suggest that replication fork reversal occurs after replication–transcription head-on collision, and we propose that it promotes the action of the accessory replicative helicases that dislodge the obstacle

Adaptation bactérienne à un composé xénobiotique, le lindane

L'introduction des composés xénobiotiques confronte les bactéries à de nouveaux environnements. Leur dégradation implique le déroulement de stratégies adaptatives conduisant à la mise en place de nouvelles voies et de nouveaux gènes cataboliques. Cette stratégie peut résulter d'un remodelage interne des séquences d'ADN déjà présentes dans le génome des bactéries mais aussi de leur récupération par des transferts horizontaux auprès d'autres organismes bactériens. L'étude génétique d'une bactérie (Sphingobium) capable de dégrader le lindane a révèlé la remarquable plasticité de son génome (mouvements d'IS, réarrangements génomiques) dans la mise en place de la voie de dégradation. En parallèle, une approche bioinformatique et moléculaire nous aussi permis d'explorer les ressources génétiques présents dans le métagénome (ADN de l'environnement) afin de comprendre l'origine moléculaire d'un gène potentiellement nouveau (linA). Des fragments de gènes putatifs ont été identifiés et ont été intégrés dans une évolution in vitro (Shuffling) du gèneLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Development of metagenomic DNA shuffling for the construction of a xenobiotic gene

International audienceWe describe a metagenomic DNA shuffling process by combining protein engineering process mutation generator and the high potential diversity of metagenomic DNA derived from the environment. Numerous previous shuffling processes attempted to recombine more or less related parental sequences. At the same time, metagenomic approaches unveiled a huge diversity of DNA sequences and genomes, which have not yet been identified to date. In this study, we attempted to combine these two approaches in order to regenerate a novel gene. Here, we present the possibility that DNA fragments from an entire microbial community (metagenome) might be available for the creation of novel genes capable of degrading pollutants. Metagenomic DNA extracted from non-polluted soil was shuffled in vitro to recreate the linA gene responsible for the first steps of lindane degradation. In this work, 74% of the ORF came from separate subsets of the metagenomic pool from a lindane-free and linA-free soil. Our results demonstrate that microbial community genetic diversity can serve as a source for novel gene construction during in vitro manipulation. This in vitro gene construction might also simulate the mosaic nature of novel genes. This demonstration might lead to other attempts to mimic bacterial adaptation and to construct degradative genes for novel compounds not yet released into the environment

Plasmid-encoded gamma-hexachlorocyclohexane degradation genes and insertion sequences in Sphingobium francense (ex-Sphingomonas paucimobilis Sp+)

International audienceThe lin genes encode the gamma-hexachlorocyclohexane (gamma-HCH or lindane) catabolic pathway in lindane-degrading strains. The location and stability of these genes have been explored in the lindane-degrading Sphingobium francense strain Sp+, and in two nonlindane-degrading mutants (Sp1- and Sp2-). The lin genes, linA, linB, linE and linX were localized by hybridization on three of the six plasmids of the S. francense strain Sp+ showing dispersal within the genome. The linC gene was detected by PCR, but was not detected by hybridization on any of the plasmids. The hybridization of the linA and linX genes was negative with the two nonlindane-degrading mutants S. francense strains, Sp1- and Sp2-. The dynamic of this genome associated with gene loss and acquisition, and plasmid rearrangement was explored by a search for associated insertion sequences. A new insertion sequence, ISSppa4, belonging to the IS21 family was detected and compared with IS6100 and ISsp1. Insertion sequence localization was explored on different hybridization patterns (plasmid, total genome) with the lindane-degrading Sp+ strain and the two nondegrading derivatives (Sp1-, Sp2-). Insertion sequence movement and plasmid rearrangement could explain the emergence of the nonlindane-degrading mutants

Evaluation de la capacité des arbres à enregistrer une contamination métallique. Approche exploratoire sur les casiers Girardon du Rhône

Dans le cadre des projets de restauration écologique du Rhône, le devenir des casiers Girardon est questionné. Le démantèlement stratégique de certaines de ces structures a déjà eu lieu afin de répondre à des enjeux sécuritaires et écologiques. Néanmoins, un grand nombre de ces ouvrages sont encore en place et ont piégé des sédiments fins au cours du siècle dernier. Les travaux de recherche engagés récemment montrent que les ouvrages Girardon ont modifié le paysage rhodanien tout comme la construction des infrastructures hydroélectriques durant la seconde moitié du XXème siècle, conduisant à la terrestrialisation puis la végétalisation des marges alluviales. Les patrons géochimiques des sédiments fins piégés dans les casiers répondent à une chronologie spécifique résultant des phases d’aménagement et des périodes majeures de flux de contaminants (Clozel-Leloup et al., 2013). L’enjeu est donc d’évaluer le stockage des contaminants dans les sédiments en fonction de cette histoire mais aussi le transfert potentiel de ces contaminants à la végétation riparienne

Identification and evolution of nsLTPs in the root nodule nitrogen fixation clade and molecular response of Frankia to AgLTP24

Abstract Non-specific lipid transfer proteins (nsLTPs) are antimicrobial peptides, involved in several plant biological processes including root nodule nitrogen fixation (RNF). Nodulating plants belonging to the RNF clade establish symbiosis with the nitrogen-fixing bacteria rhizobia (legumes symbiosis model) and Frankia (actinorhizal symbiosis model) leading to root nodule formation. nsLTPs are involved in processes active in early step of symbiosis and functional nodule in both models. In legumes, nsLTPs have been shown to regulate symbiont entry, promote root cortex infection, membrane biosynthesis, and improve symbiosis efficiency. More recently, a nsLTP, AgLTP24 has been described in the context of actinorhizal symbiosis between Alnus glutinosa and Frankia alni ACN14a. AgLTP24 is secreted at an early step of symbiosis on the deformed root hairs and targets the symbiont in the nitrogen-fixing vesicles in functional nodules. nsLTPs are involved in RNF, but their functions and evolutionary history are still largely unknown. Numerous putative nsLTPs were found up-regulated in functional nodules compared to non-infected roots in different lineages within the RNF clade. Here, results highlight that nodulating plants that are co-evolving with their nitrogen-fixing symbionts appear to have independently specialized nsLTPs for this interaction, suggesting a possible convergence of function, which opens perspectives to investigate nsLTPs functions in RNF

Genomic Insights of <i>Alnus</i>-Infective <i>Frankia</i> Strains Reveal Unique Genetic Features and New Evidence on Their Host-Restricted Lifestyle

The present study aimed to use comparative genomics to explore the relationships between Frankia and actinorhizal plants using a data set made of 33 Frankia genomes. The determinants of host specificity were first explored for “Alnus-infective strains” (i.e., Frankia strains belonging to Cluster Ia). Several genes were specifically found in these strains, including an agmatine deiminase which could possibly be involved in various functions as access to nitrogen sources, nodule organogenesis or plant defense. Within “Alnus-infective strains”, Sp+ Frankia genomes were compared to Sp− genomes in order to elucidate the narrower host specificity of Sp+ strains (i.e., Sp+ strains being capable of in planta sporulation, unlike Sp− strains). A total of 88 protein families were lost in the Sp+ genomes. The lost genes were related to saprophytic life (transcriptional factors, transmembrane and secreted proteins), reinforcing the proposed status of Sp+ as obligatory symbiont. The Sp+ genomes were also characterized by a loss of genetic and functional paralogs, highlighting a reduction in functional redundancy (e.g., hup genes) or a possible loss of function related to a saprophytic lifestyle (e.g., genes involved in gas vesicle formation or recycling of nutrients)

Chitinolytic actinobacteria isolated from an Algerian semi-arid soil: development of an antifungal chitinase-dependent assay and GH18 chitinase gene identification

The purpose of this study was to explore the microbial potential of a semi-arid sandy soil from south-central Algeria in order to isolate new chitinolytic actinobacteria. This soil is subjected to high temperatures (up to 43 degrees C) and has low nutrient content. Strains were isolated by plating soil suspensions on Bennett and Colloidal Chitin (CCM) medium. An initial clustering of isolates was made through BOX-PCR genetic profiling. Next, a 16S rRNA gene sequencing of representative isolates was realized. We also identified optimum physicochemical conditions for chitinolytic activity. A rapid in vitro assay based on glucose catabolic repression was developed to select isolates having a chitinase-dependent antifungal activity against two phytopathogenic fungi. Gene identification of glycosyl hydrolase family 18 (GH18) permitted us to assess the divergence of chitinase genes. Forty isolates were obtained from the semi-arid sandy soil. The molecular identification permitted us to assign them to Streptomyces or Micromonospora genera with seven possibly new bacterial species. For chitinolytic activity, 100% of isolates were able to grow and degrade colloidal chitin at pH 7 and at a temperature ranging from 30 to 40 degrees C. We also observed that Micromonospora strains had atypical activity patterns, with a strong chitinase activity maintained at high temperature. Finally, three strains presented an interesting chitinolytic potential to reduce fungal growth with new GH18 sequences. This study presents a new rapid method to detect antifungal chitinase-dependent activity that allowed to identify potentially new species of actinobacteria and new GH18 gene sequences