An extensive survey of phytoviral RNA 3' uridylation identifies extreme variations and virus-specific patterns.

Viral RNAs can be uridylated in eukaryotic hosts. However, our knowledge of uridylation patterns and roles remains rudimentary for phytoviruses. Here, we report global 3' terminal RNA uridylation profiles for representatives of the main families of positive single-stranded RNA phytoviruses. We detected uridylation in all 47 viral RNAs investigated here, revealing its prevalence. Yet, uridylation levels of viral RNAs varied from 0.2% to 90%. Unexpectedly, most poly(A) tails of grapevine fanleaf virus (GFLV) RNAs, including encapsidated tails, were strictly monouridylated, which corresponds to an unidentified type of viral genomic RNA extremity. This monouridylation appears beneficial for GFLV because it became dominant when plants were infected with nonuridylated GFLV transcripts. We found that GFLV RNA monouridylation is independent of the known terminal uridylyltransferases (TUTases) HEN1 SUPPRESSOR 1 (HESO1) and UTP:RNA URIDYLYLTRANSFERASE 1 (URT1) in Arabidopsis (Arabidopsis thaliana). By contrast, both TUTases can uridylate other viral RNAs like turnip crinkle virus (TCV) and turnip mosaic virus (TuMV) RNAs. Interestingly, TCV and TuMV degradation intermediates were differentially uridylated by HESO1 and URT1. Although the lack of both TUTases did not prevent viral infection, we detected degradation intermediates of TCV RNA at higher levels in an Arabidopsis heso1 urt1 mutant, suggesting that uridylation participates in clearing viral RNA. Collectively, our work unveils an extreme diversity of uridylation patterns across phytoviruses and constitutes a valuable resource to further decipher pro- and antiviral roles of uridylation.

The Arabidopsis thaliana-Streptomyces Interaction Is Controlled by the Metabolic Status of the Holobiont.

How specific interactions between plant and pathogenic, commensal, or mutualistic microorganisms are mediated and how bacteria are selected by a plant are important questions to address. Here, an Arabidopsis thaliana mutant called chs5 partially deficient in the biogenesis of isoprenoid precursors was shown to extend its metabolic remodeling to phenylpropanoids and lipids in addition to carotenoids, chlorophylls, and terpenoids. Such a metabolic profile was concomitant to increased colonization of the phyllosphere by the pathogenic strain Pseudomonas syringae pv. tomato DC3000. A thorough microbiome analysis by 16S sequencing revealed that Streptomyces had a reduced colonization potential in chs5. This study revealed that the bacteria-Arabidopsis interaction implies molecular processes impaired in the chs5 mutant. Interestingly, our results revealed that the metabolic status of A. thaliana was crucial for the specific recruitment of Streptomyces into the microbiota. More generally, this study highlights specific as well as complex molecular interactions that shape the plant microbiota.

Sequence of the Mitochondrial Genome of Lactuca virosa Suggests an Unexpected Role in Lactuca sativa's Evolution.

The involvement of the different Lactuca species in the domestication and diversification of cultivated lettuce is not totally understood. Lactuca serriola is considered as the direct ancestor and the closest relative to Lactuca sativa, while the other wild species that can be crossed with L. sativa, Lactuca virosa, and Lactuca saligna, would have just contributed to the latter diversification of cultivated typologies. To contribute to the study of Lactuca evolution, we assembled the mtDNA genomes of nine Lactuca spp. accessions, among them three from L. virosa, whose mtDNA had not been studied so far. Our results unveiled little to no intraspecies variation among Lactuca species, with the exception of L. serriola where the accessions we sequenced diverge significantly from the mtDNA of a L. serriola accession already reported. Furthermore, we found a remarkable phylogenetic closeness between the mtDNA of L. sativa and the mtDNA of L. virosa, contrasting to the L. serriola origin of the nuclear and plastidial genomes. These results suggest that a cross between L. virosa and the ancestor of cultivated lettuce is at the origin of the actual mitochondrial genome of L. sativa.

The TUTase URT1 connects decapping activators and prevents the accumulation of excessively deadenylated mRNAs to avoid siRNA biogenesis.

Uridylation is a widespread modification destabilizing eukaryotic mRNAs. Yet, molecular mechanisms underlying TUTase-mediated mRNA degradation remain mostly unresolved. Here, we report that the Arabidopsis TUTase URT1 participates in a molecular network connecting several translational repressors/decapping activators. URT1 directly interacts with DECAPPING 5 (DCP5), the Arabidopsis ortholog of human LSM14 and yeast Scd6, and this interaction connects URT1 to additional decay factors like DDX6/Dhh1-like RNA helicases. Nanopore direct RNA sequencing reveals a global role of URT1 in shaping poly(A) tail length, notably by preventing the accumulation of excessively deadenylated mRNAs. Based on in vitro and in planta data, we propose a model that explains how URT1 could reduce the accumulation of oligo(A)-tailed mRNAs both by favoring their degradation and because 3' terminal uridines intrinsically hinder deadenylation. Importantly, preventing the accumulation of excessively deadenylated mRNAs avoids the biogenesis of illegitimate siRNAs that silence endogenous mRNAs and perturb Arabidopsis growth and development.

Effect of arsenite and growth in biofilm conditions on the evolution of Thiomonas sp. CB2.

Thiomonas bacteria are ubiquitous at acid mine drainage sites and play key roles in the remediation of water at these locations by oxidizing arsenite to arsenate, favouring the sorption of arsenic by iron oxides and their coprecipitation. Understanding the adaptive capacities of these bacteria is crucial to revealing how they persist and remain active in such extreme conditions. Interestingly, it was previously observed that after exposure to arsenite, when grown in a biofilm, some strains of Thiomonas bacteria develop variants that are more resistant to arsenic. Here, we identified the mechanisms involved in the emergence of such variants in biofilms. We found that the percentage of variants generated increased in the presence of high concentrations of arsenite (5.33 mM), especially in the detached cells after growth under biofilm-forming conditions. Analysis of gene expression in the parent strain CB2 revealed that genes involved in DNA repair were upregulated in the conditions where variants were observed. Finally, we assessed the phenotypes and genomes of the subsequent variants generated to evaluate the number of mutations compared to the parent strain. We determined that multiple point mutations accumulated after exposure to arsenite when cells were grown under biofilm conditions. Some of these mutations were found in what is referred to as ICE19, a genomic island (GI) carrying arsenic-resistance genes, also harbouring characteristics of an integrative and conjugative element (ICE). The mutations likely favoured the excision and duplication of this GI. This research aids in understanding how Thiomonas bacteria adapt to highly toxic environments, and, more generally, provides a window to bacterial genome evolution in extreme environments.

Phylogenomic Classification and Biosynthetic Potential of the Fossil Fuel-Biodesulfurizing Rhodococcus Strain IGTS8.

Rhodococcus strain IGTS8 is the most extensively studied model bacterium for biodesulfurization of fossil fuels via the non-destructive sulfur-specific 4S pathway. This strain was initially assigned to Rhodococcus rhodochrous and later to Rhodococcus erythropolis thus making its taxonomic status debatable and reflecting the limited resolution of methods available at the time. In this study, phylogenomic analyses of the whole genome sequences of strain IGTS8 and closely related rhodococci showed that R. erythropolis and Rhodococcus qingshengii are very closely related species, that Rhodococcus strain IGTS8 is a R. qingshengii strain and that several strains identified as R. erythropolis should be re-classified as R. qingshengii. The genomes of strains assigned to these species contain potentially novel biosynthetic gene clusters showing that members of these taxa should be given greater importance in the search for new antimicrobials and other industrially important biomolecules. The plasmid-borne dsz operon encoding fossil fuel desulfurization enzymes was present in R. qingshengii IGTS8 and R. erythropolis XP suggesting that it might be transferable between members of these species.

High-Resolution Mapping of 3' Extremities of RNA Exosome Substrates by 3' RACE-Seq.

The main 3'-5' exoribonucleolytic activity of eukaryotic cells is provided by the RNA exosome. The exosome is constituted by a core complex of nine subunits (Exo9), which coordinates the recruitment and the activities of distinct types of cofactors. The RNA exosome cofactors confer distributive and processive 3'-5' exoribonucleolytic, endoribonucleolytic, and RNA helicase activities. In addition, several RNA binding proteins and terminal nucleotidyltransferases also participate in the recognition of exosome RNA substrates.To fully understand the biological roles of the exosome, the respective functions of its cofactors must be deciphered. This entails the high-resolution analysis of 3' extremities of degradation or processing intermediates in different mutant backgrounds or growth conditions. Here, we describe a detailed 3' RACE-seq procedure for targeted mapping of exosome substrate 3' ends. This procedure combines a 3' RACE protocol with Illumina sequencing to enable the high-resolution mapping of 3' extremities and the identification of untemplated nucleotides for selected RNA targets.

In situ metabolic activities of uncultivated Ferrovum sp. CARN8 evidenced by metatranscriptomic analysis.

Amongst iron-oxidizing bacteria playing a key role in the natural attenuation of arsenic in acid mine drainages (AMDs), members of the Ferrovum genus were identified in mine effluent or water treatment plants, and were shown to dominate biogenic precipitates in field pilot experiments. In order to address the question of the in situ activity of the uncultivated Ferrovum sp. CARN8 strain in the Carnoulès AMD, we assembled its genome using metagenomic and metatranscriptomic sequences and we determined standardized expression values for protein-encoding genes. Our results showed that this microorganism was indeed metabolically active and allowed us to sketch out its metabolic activity in its natural environment. Expression of genes related to the respiratory chain and carbon fixation suggests aerobic energy production coupled to ferrous iron oxidation and chemolithoautotrophic growth. Notwithstanding the presence of nitrogenase genes in its genome, expression data also indicated that Ferrovum sp. CARN8 relied on ammonium import rather than nitrogen fixation. The expression of flagellum and chemotaxis genes hints that at least a proportion of this strain population was motile. Finally, apart from some genes related to metal resistance showing surprisingly low expression values, genes involved in stress response were well expressed as expected in AMDs.

Efficient Replication of the Plastid Genome Requires an Organellar Thymidine Kinase.

Thymidine kinase (TK) is a key enzyme of the salvage pathway that recycles thymidine nucleosides to produce deoxythymidine triphosphate. Here, we identified the single TK of maize (Zea mays), denoted CPTK1, as necessary in the replication of the plastidial genome (cpDNA), demonstrating the essential function of the salvage pathway during chloroplast biogenesis. CPTK1 localized to both plastids and mitochondria, and its absence resulted in an albino phenotype, reduced cpDNA copy number and a severe deficiency in plastidial ribosomes. Mitochondria were not affected, indicating they are less reliant on the salvage pathway. Arabidopsis (Arabidopsis thaliana) TKs, TK1A and TK1B, apparently resulted from a gene duplication after the divergence of monocots and dicots. Similar but less-severe effects were observed for Arabidopsis tk1a tk1b double mutants in comparison to those in maize cptk1 TK1B was important for cpDNA replication and repair in conditions of replicative stress but had little impact on the mitochondrial phenotype. In the maize cptk1 mutant, the DNA from the small single-copy region of the plastidial genome was reduced to a greater extent than other regions, suggesting preferential abortion of replication in this region. This was accompanied by the accumulation of truncated genomes that resulted, at least in part, from unfaithful microhomology-mediated repair. These and other results suggest that the loss of normal cpDNA replication elicits the mobilization of new replication origins around the rpoB (beta subunit of plastid-encoded RNA polymerase) transcription unit and imply that increased transcription at rpoB is associated with the initiation of cpDNA replication.

Organization of the TC and TE cellular T-DNA regions in Nicotiana otophora and functional analysis of three diverged TE-6b genes.

Nicotiana otophora contains Agrobacterium-derived T-DNA sequences introduced by horizontal gene transfer (Chen et al., 2014). Sixty-nine contigs were assembled into four different cellular T-DNAs (cT-DNAs) totalling 83 kb. TC and TE result from two successive transformation events, each followed by duplication, yielding two TC and two TE inserts. TC is also found in other Nicotiana species, whereas TE is unique to N. otophora. Both cT-DNA regions are partially duplicated inverted repeats. Analysis of the cT-DNA divergence patterns allowed reconstruction of the evolution of the TC and TE regions. TC and TE carry 10 intact open reading frames. Three of these are TE-6b genes, derived from a single 6b gene carried by the Agrobacterium strain which inserted TE in the N. otophora ancestor. 6b genes have so far only been found in Agrobacterium tumefaciens or Agrobacterium vitis T-DNAs and strongly modify plant growth (Chen and Otten, 2016). The TE-6b genes were expressed in Nicotiana tabacum under the constitutive 2 × 35S promoter. TE-1-6b-R and TE-2-6b led to shorter plants, dark-green leaves, a strong increase in leaf vein development and modified petiole wings. TE-1-6b-L expression led to a similar phenotype, but in addition leaves show outgrowths at the margins, flowers were modified and plants became viviparous, i.e. embryos germinated in the capsules at an early stage of their development. Embryos could be rescued by culture in vitro. The TE-6b phenotypes are very different from the earlier described 6b phenotypes and could provide new insight into the mode of action of the 6b genes.

Comparison of biofilm formation and motility processes in arsenic-resistant Thiomonas spp. strains revealed divergent response to arsenite.

Bacteria of the genus Thiomonas are found ubiquitously in arsenic contaminated waters such as acid mine drainage (AMD), where they contribute to the precipitation and the natural bioremediation of arsenic. In these environments, these bacteria have developed a large range of resistance strategies among which the capacity to form particular biofilm structures. The biofilm formation is one of the most ubiquitous adaptive response observed in prokaryotes to various stresses, such as those induced in the presence of toxic compounds. This study focused on the process of biofilm formation in three Thiomonas strains (CB1, CB2 and CB3) isolated from the same AMD. The results obtained here show that these bacteria are all capable of forming biofilms, but the architecture and the kinetics of formation of these biofilms differ depending on whether arsenite is present in the environment and from one strain to another. Indeed, two strains favoured biofilm formation, whereas one favoured motility in the presence of arsenite. To identify the underlying mechanisms, the patterns of expression of some genes possibly involved in the process of biofilm formation were investigated in Thiomonas sp. CB2 in the presence and absence of arsenite, using a transcriptomic approach (RNA-seq). The findings obtained here shed interesting light on how the formation of biofilms, and the motility processes contribute to the adaptation of Thiomonas strains to extreme environments.

Arsenite response in Coccomyxa sp. Carn explored by transcriptomic and non-targeted metabolomic approaches.

Arsenic is a toxic metalloid known to generate an important oxidative stress in cells. In the present study, we focused our attention on an alga related to the genus Coccomyxa, exhibiting an extraordinary capacity to resist high concentrations of arsenite and arsenate. The integrated analysis of high-throughput transcriptomic data and non-targeted metabolomic approaches highlighted multiple levels of protection against arsenite. Indeed, Coccomyxa sp. Carn induced a set of transporters potentially preventing the accumulation of this metalloid in the cells and presented a distinct arsenic metabolism in comparison to another species more sensitive to that compound, i.e. Euglena gracilis, especially in regard to arsenic methylation. Interestingly, Coccomyxa sp. Carn was characterized by a remarkable accumulation of the strong antioxidant glutathione (GSH). Such observation could explain the apparent low oxidative stress in the intracellular compartment, as suggested by the transcriptomic analysis. In particular, the high amount of GSH in the cell could play an important role for the tolerance to arsenate, as suggested by its partial oxidation into oxidized glutathione in presence of this metalloid. Our results therefore reveal that this alga has acquired multiple and original defence mechanisms allowing the colonization of extreme ecosystems such as acid mine drainages.

Thiomonas sp. CB2 is able to degrade urea and promote toxic metal precipitation in acid mine drainage waters supplemented with urea.

The acid mine drainage (AMD) in Carnoulès (France) is characterized by the presence of toxic metals such as arsenic. Several bacterial strains belonging to the Thiomonas genus, which were isolated from this AMD, are able to withstand these conditions. Their genomes carry several genomic islands (GEIs), which are known to be potentially advantageous in some particular ecological niches. This study focused on the role of the "urea island" present in the Thiomonas CB2 strain, which carry the genes involved in urea degradation processes. First, genomic comparisons showed that the genome of Thiomonas sp. CB2, which is able to degrade urea, contains a urea genomic island which is incomplete in the genome of other strains showing no urease activity. The urease activity of Thiomonas sp. CB2 enabled this bacterium to maintain a neutral pH in cell cultures in vitro and prevented the occurrence of cell death during the growth of the bacterium in a chemically defined medium. In AMD water supplemented with urea, the degradation of urea promotes iron, aluminum and arsenic precipitation. Our data show that ureC was expressed in situ, which suggests that the ability to degrade urea may be expressed in some Thiomonas strains in AMD, and that this urease activity may contribute to their survival in contaminated environments.

Adaptation in Toxic Environments: Arsenic Genomic Islands in the Bacterial Genus Thiomonas.

Acid mine drainage (AMD) is a highly toxic environment for most living organisms due to the presence of many lethal elements including arsenic (As). Thiomonas (Tm.) bacteria are found ubiquitously in AMD and can withstand these extreme conditions, in part because they are able to oxidize arsenite. In order to further improve our knowledge concerning the adaptive capacities of these bacteria, we sequenced and assembled the genome of six isolates derived from the Carnoulès AMD, and compared them to the genomes of Tm. arsenitoxydans 3As (isolated from the same site) and Tm. intermedia K12 (isolated from a sewage pipe). A detailed analysis of the Tm. sp. CB2 genome revealed various rearrangements had occurred in comparison to what was observed in 3As and K12 and over 20 genomic islands (GEIs) were found in each of these three genomes. We performed a detailed comparison of the two arsenic-related islands found in CB2, carrying the genes required for arsenite oxidation and As resistance, with those found in K12, 3As, and five other Thiomonas strains also isolated from Carnoulès (CB1, CB3, CB6, ACO3 and ACO7). Our results suggest that these arsenic-related islands have evolved differentially in these closely related Thiomonas strains, leading to divergent capacities to survive in As rich environments.

Toxic metal resistance in biofilms: diversity of microbial responses and their evolution.

Since biofilms are an important issue in the fields of medicine and health, several recent microbiological studies have focused on their formation and their contribution to toxic compound resistance mechanisms. In this review, we describe how metals impact biofilm formation and resistance, and how biofilms can help cells resist toxic metals. First, the organic matrix acts as a barrier isolating the cells from many environmental stresses. Secondly, the metabolism of the cells changes, and a slowly-growing or non-growing sub-population of cells known as persisters emerges. Thirdly, in the case of multispecies biofilms, metabolic interactions are developed, allowing cells to be more persistent or to have greater capacity to survive than a single species biofilm. Finally, we discuss how the high density of the cells may promote horizontal gene transfer processes, resulting in the acquisition of new features. All these crucial mechanisms enable microorganisms to survive and colonize toxic environments, and probably accelerate ongoing evolutionary processes.

Constitutive arsenite oxidase expression detected in arsenic-hypertolerant Pseudomonas xanthomarina S11.

Pseudomonas xanthomarina S11 is an arsenite-oxidizing bacterium isolated from an arsenic-contaminated former gold mine in Salsigne, France. This bacterium showed high resistance to arsenite and was able to oxidize arsenite to arsenate at concentrations up to 42.72 mM As[III]. The genome of this strain was sequenced and revealed the presence of three ars clusters. One of them is located on a plasmid and is organized as an "arsenic island" harbouring an aio operon and genes involved in phosphorous metabolism, in addition to the ars genes. Neither the aioXRS genes nor a specific sigma-54-dependent promoter located upstream of aioBA genes, both involved in regulation of arsenite oxidase expression in other arsenite-oxidizing bacteria, could be identified in the genome. This observation is in accordance with the fact that no difference was observed in expression of arsenite oxidase in P. xanthomarina S11, whether or not the strain was grown in the presence of As[III].

Genome Sequence of Halomonas sp. Strain A3H3, Isolated from Arsenic-Rich Marine Sediments.

We report the genome sequence of Halomonas sp. strain A3H3, a bacterium with a high tolerance to arsenite, isolated from multicontaminated sediments of the l'Estaque harbor in Marseille, France. The genome is composed of a 5,489,893-bp chromosome and a 157,085-bp plasmid.

Metagenomic insights into microbial metabolism affecting arsenic dispersion in Mediterranean marine sediments.

Microorganisms dwelling in sediments have a crucial role in biogeochemical cycles and are expected to have a strong influence on the cycle of arsenic, a metalloid responsible for severe water pollution and presenting major health risks for human populations. We present here a metagenomic study of the sediment from two harbours on the Mediterranean French coast, l'Estaque and St Mandrier. The first site is highly polluted with arsenic and heavy metals, while the arsenic concentration in the second site is below toxicity levels. The goal of this study was to elucidate the potential impact of the microbial community on the chemical parameters observed in complementary geochemical studies performed on the same sites. The metagenomic sequences, along with those from four publicly available metagenomes used as control data sets, were analysed with the RAMMCAP workflow. The resulting functional profiles were compared to determine the over-represented Gene Ontology categories in the metagenomes of interest. Categories related to arsenic resistance and dissimilatory sulphate reduction were over-represented in l'Estaque. More importantly, despite very similar profiles, the identification of specific sequence markers for sulphate-reducing bacteria and sulphur-oxidizing bacteria showed that sulphate reduction was significantly more associated with l'Estaque than with St Mandrier. We propose that biotic sulphate reduction, arsenate reduction and fermentation may together explain the higher mobility of arsenic observed in l'Estaque in previous physico-chemical studies of this site. This study also demonstrates that it is possible to draw sound conclusions from comparing complex and similar unassembled metagenomes at the functional level, even with very low sequence coverage.

Life in an arsenic-containing gold mine: genome and physiology of the autotrophic arsenite-oxidizing bacterium rhizobium sp. NT-26.

Arsenic is widespread in the environment and its presence is a result of natural or anthropogenic activities. Microbes have developed different mechanisms to deal with toxic compounds such as arsenic and this is to resist or metabolize the compound. Here, we present the first reference set of genomic, transcriptomic and proteomic data of an Alphaproteobacterium isolated from an arsenic-containing goldmine: Rhizobium sp. NT-26. Although phylogenetically related to the plant-associated bacteria, this organism has lost the major colonizing capabilities needed for symbiosis with legumes. In contrast, the genome of Rhizobium sp. NT-26 comprises a megaplasmid containing the various genes, which enable it to metabolize arsenite. Remarkably, although the genes required for arsenite oxidation and flagellar motility/biofilm formation are carried by the megaplasmid and the chromosome, respectively, a coordinate regulation of these two mechanisms was observed. Taken together, these processes illustrate the impact environmental pressure can have on the evolution of bacterial genomes, improving the fitness of bacterial strains by the acquisition of novel functions.