Deciphering the role of Paenibacillus strain Q8 in the organic matter recycling in the acid mine drainage of Carnoulès

<p>Abstract</p> <p>Background</p> <p>The recycling of the organic matter is a crucial function in any environment, especially in oligotrophic environments such as Acid Mine Drainages (AMDs). Polymer-degrading bacteria might play an important role in such ecosystem, at least by releasing by-products useful for the rest of the community. In this study, physiological, molecular and biochemical experiments were performed to decipher the role of a <it>Paenibacillus </it>strain isolated from the sediment of Carnoulès AMD.</p> <p>Results</p> <p>Even though <it>Paenibacillus </it>sp. strain Q8 was isolated from an oligotrophic AMD showing an acidic pH, it developed under both acidic and alkaline conditions and showed a heterotrophic metabolism based on the utilization of a broad range of organic compounds. It resisted to numerous metallic stresses, particularly high arsenite (As(III)) concentrations (> 1,800 mg/L). Q8 was also able to efficiently degrade polymers such as cellulose, xylan and starch. Function-based screening of a Q8 DNA-library allowed the detection of 15 clones with starch-degrading activity and 3 clones with xylan-degrading activity. One clone positive for starch degradation carried a single gene encoding a "protein of unknown function". Amylolytic and xylanolytic activities were measured both in growing cells and with acellular extracts of Q8. The results showed the ability of Q8 to degrade both polymers under a broad pH range and high As(III) and As(V) concentrations. Activity measurements allowed to point out the constitutive expression of the amylase genes and the mainly inducible expression of the xylanase genes. PACE demonstrated the endo-acting activity of the amylases and the exo-acting activity of the xylanases.</p> <p>Conclusions</p> <p>AMDs have been studied for years especially with regard to interactions between bacteria and the inorganic compartment hosting them. To date, no study reported the role of microorganisms in the recycling of the organic matter. The present work suggests that the strain Q8 might play an important role in the community by recycling the scarce organic matter (cellulose, hemicellulose, starch...), especially when the conditions change. Furthermore, function-based screening of a Q8 DNA library allowed to assign an amylolytic function to a gene previously unknown. AMDs could be considered as a reservoir of genes with potential biotechnological properties.</p

Subinhibitory Arsenite Concentrations Lead to Population Dispersal in Thiomonas sp.

Biofilms represent the most common microbial lifestyle, allowing the survival of microbial populations exposed to harsh environmental conditions. Here, we show that the biofilm development of a bacterial species belonging to the Thiomonas genus, frequently found in arsenic polluted sites and playing a key role in arsenic natural remediation, is markedly modified when exposed to subinhibitory doses of this toxic element. Indeed, arsenite [As(III)] exposure led to a considerable impact on biofilm maturation by strongly increasing the extracellular matrix synthesis and by promoting significant cell death and lysis within microcolonies. These events were followed by the development of complex 3D-biofilm structures and subsequently by the dispersal of remobilized cells observed inside the previously formed hollow voids. Our results demonstrate that this biofilm community responds to arsenite stress in a multimodal way, enhancing both survival and dispersal. Addressing this complex bacterial response to As(III) stress, which might be used by other microorganisms under various adverse conditions, may be essential to understand how Thiomonas strains persist in extreme environments

A Tale of Two Oxidation States: Bacterial Colonization of Arsenic-Rich Environments

Microbial biotransformations have a major impact on contamination by toxic elements, which threatens public health in developing and industrial countries. Finding a means of preserving natural environments—including ground and surface waters—from arsenic constitutes a major challenge facing modern society. Although this metalloid is ubiquitous on Earth, thus far no bacterium thriving in arsenic-contaminated environments has been fully characterized. In-depth exploration of the genome of the β-proteobacterium Herminiimonas arsenicoxydans with regard to physiology, genetics, and proteomics, revealed that it possesses heretofore unsuspected mechanisms for coping with arsenic. Aside from multiple biochemical processes such as arsenic oxidation, reduction, and efflux, H. arsenicoxydans also exhibits positive chemotaxis and motility towards arsenic and metalloid scavenging by exopolysaccharides. These observations demonstrate the existence of a novel strategy to efficiently colonize arsenic-rich environments, which extends beyond oxidoreduction reactions. Such a microbial mechanism of detoxification, which is possibly exploitable for bioremediation applications of contaminated sites, may have played a crucial role in the occupation of ancient ecological niches on earth

Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children

Para-infectious brain injury in COVID-19 persists at follow-up despite attenuated cytokine and autoantibody responses

To understand neurological complications of COVID-19 better both acutely and for recovery, we measured markers of brain injury, inflammatory mediators, and autoantibodies in 203 hospitalised participants; 111 with acute sera (1–11 days post-admission) and 92 convalescent sera (56 with COVID-19-associated neurological diagnoses). Here we show that compared to 60 uninfected controls, tTau, GFAP, NfL, and UCH-L1 are increased with COVID-19 infection at acute timepoints and NfL and GFAP are significantly higher in participants with neurological complications. Inflammatory mediators (IL-6, IL-12p40, HGF, M-CSF, CCL2, and IL-1RA) are associated with both altered consciousness and markers of brain injury. Autoantibodies are more common in COVID-19 than controls and some (including against MYL7, UCH-L1, and GRIN3B) are more frequent with altered consciousness. Additionally, convalescent participants with neurological complications show elevated GFAP and NfL, unrelated to attenuated systemic inflammatory mediators and to autoantibody responses. Overall, neurological complications of COVID-19 are associated with evidence of neuroglial injury in both acute and late disease and these correlate with dysregulated innate and adaptive immune responses acutely

Analyse génétique et moléculaire de stress arsenic de souches bactériennes isolées d'environnements contaminés par l'arsenic

L'arsenic est un métalloïde présent dans différents compartiments de l'environnement, provenant de sources naturelles ou de sources anthropiques. Les formes inorganiques, l'arsénite (As[III]) et l'arséniate (As[V]), sont les plus abondantes et aussi les plus toxiques. La proportion relative de ces états d'oxydation dans un environnement donné sera fonction de transformations chimiques spontanées, et surtout de biotransformations liées aux métabolismes bactériens, tels que la réduction et l'oxydation.Ce travail de thèse a eu comme objectif principal l'étude moléculaire et génétique de l'arsénite oxydase d'une ß-protéobactérie, Cenibacterium arsenoxidans. L'analyse de mutants obtenus par transposition aléatoire d'un mini-Tn5 a conduit à l'identification de deux gènes aoxA et aoxB, organisés en opéron et codant les deux sous-unités de l'arsénite oxydase. L'analyse phylogénétique des deux sous-unités de cette enzyme a révélé que celle-ci possède des homologues dans d'autres familles enzymatiques assurant des fonctions diverses. La comparaison entre ces différentes familles suggère une apparition très ancienne des enzymes de type arsénite oxydase (pré-LUCA). Par ailleurs nous avons étudié la réponse globale des bactéries se trouvant en contact avec l'arsenic. Pour ce faire, nous avons combiné deux approches, d'une part l'analyse des mutants dont le gène rapporteur est induit par l'arsenic, d'autre part l'étude des protéines dont la synthèse est régulée par l'arsenic. Nous avons montré que les gènes régulés par l'arsenic interviennent dans une grande variété de fonctions incluant l'information, le métabolisme intermédiaire, la structure des enveloppes, la transformation de différentes formes d'arsenic. Cette étude suggère, que la bactérie C. arsenoxidans, est capable de réaliser également la réduction de l'arséniate en arsénite en plus de l'oxydation de l'arsénite en arséniate. Au moins trois opérons de type ars (réduction de l'As[V]) seraient présents dans cette souche.Arsenic is ubiquitous in the biosphere and frequently reported as an environmental pollutant. Many studies have shown that arsenic is cytotoxic at micromolar concentrations. Bacteria overcome the toxic effects of arsenic by either reducing arsenate (As[V]) to As[III], which is actively exported (ars operon), or by oxidizing As[III] to the lesser toxic form As[V]. In a first part, the effects of arsenic stress on the arsenic oxidizing ß-proteobacterial strain C. arsenoxidans were studied. More than 4000 mutants were generated by random insertion of the lacZ-based reporter gene transposon Tn5lacZ2. Increased gene expression in the presence of As[III] was observed in twenty-two mutants. Two were deficient in As[III] oxidation. Sequence analysis of the DNA flanking the inserted transposon insertions allowed us to characterise for the first time two adjacent genes coding the two subunit of the arsenite oxidase, named aoxA and aoxB, organised in an operonic structure. Phylogenetic analysis for the two constituent subunits of the arsenite oxidase indicate an early origin of this enzyme, before the divergence of Archaea and Bacteria. In a second part, the global response to arsenic stress was studied by combining a proteomic approach with the mutants analysis. Twenty-two proteins and sixteen genes were identified as differentially expressed in cells grown in the presence of As[III]. We identified genes and proteins belonging to various functional classes including information and regulation pathways, intermediary metabolism, cell envelope and cellular processes. Moreover, the presence of As[V] reduction mechanisms in a As[III] oxidizing bacteria was shown for the first time.Our results suggest that the adaptation of C. arsenoxidans to an arsenic-contaminated environment is not limited to transformation of arsenic species, but is rather pleiotropic, since a great variety of biological effects were involved.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Composition et fonctionnement d'une communauté microbienne au sein d'un drainage minier acide (approches culturales et fonctionnelles)

Le drainage minier acide de Carnoulès est caractérisé par un pH très acide et une forte concentration en métaux et en arsenic. Par différentes approches moléculaires, des études précédentes ont montré une faible biodiversité et ont permis d'établir un modèle de fonctionnement de la communauté bactérienne. Le but de ce travail de thèse a été de préciser la composition et le fonctionnement de cette communauté bactérienne, en utilisant pour cela des approches culturales et fonctionnelles, en se focalisant particulièrement sur le recyclage de la matière organique. L'élaboration de différents milieux a permis l'isolement de 49 souches bactériennes appartenant à 19 genres, augmentant ainsi de 10 % la diversité bactérienne détectée à Carnoulès par rapport aux approches métagénomiques précédentes. Parmi les 19 genres, 3 sont nouveaux dont un, inféodé aux écosystèmes acides, a été caractérisé taxonomiquement et dénommé Acidiminas carnoulesii. La capacité de l'isolat Q8 appartenant au genre Paenibacillus à dégrader I'amidon et la xylane, dans de larges gammes de pH et de concentrations en arsenic, a permis d'attribuer à Paenibacillus un rôle dans la résilience de la communauté pour ces fonctions. Un criblage fonctionnel de I'ADN de Q8 dans Escherichia coli apermis d'isoler les gènes codant les protéines de dégradation de ces polymères. Par ailleurs, un criblage de 80000 clones de la banque d'ADN métagénomique de Carnoulès a permis la détection de 28 clones positifs pour l'activité amylolytique. Deux protéines ne présentant aucune similarité de séquence avec des amylases connues ont été caractérisées in vitro, confirmant leurs activités amylolyiques et démontrant que la bioprospection dans des sites a priori incongrus, autorise des découvertes insoupçonnées. Ces travaux ont ainsi permis de montrer que les approches culturales et fonctionnelles apportent des informations nouvelles par rapport à celles obtenues par les approches moléculaires. La complémentarité de ces approches est vérifiée, et elle apparaît indispensable dans l'analyse de la complexité des écosystèmes. Cependant, la compréhension de leur fonctionnement exigera des efforts redoublés.The Carnoulès acid mine drainage is characterized by an acidic pH and high metal and arsenicconcentrations. Based on the low bacterial biodiversity, molecular approaches allowed the determination of abacterial community functioning model. The aim of the PhD work was to clariff both the composition andthe functioning of this community, using cultural and functional approaches, focusing on the organic matterrecycling.Different media were designed that allowed the isolation of 49 bacterial strains belonging to 19 genera,leading to a 70 o increase in the bacterial diversity compared with previous metagenomic approaches.Among the 19 genera,3 are new, one of which, a previously uncultured genus frequently detected in acidicenvironments, has been taxonomically characterized and named Acidiminos cornoulesii.The ability of strain Q8, belonging to the genus Paenibacillus, to degrade starch and xylan over a wide rangeof pH values as well as 4rsenic concentrations allows to assign to Paenibacillus a role in the resilience of thecommunity for these functions. A function-based screening of the Q8-DNA in Escherichia coli led to isolatethe genes encoding the polymer-degrading proteins.Moreover, a function-based screening of 80,000 clones from a Carnoulès metagenomic DNA library led tothe detection of 28 positive clones for the amylolyic activity. Two proteins sharing no sequence similaritywith known amylases were characterized in vitro, demonstrating that bioprospecting in a priori incongruous sites can lead to unsuspected discoveries.These works show that new informations using cultural and functional approaches can be obtained comparedto those gained with molecular approaches. Results confirmed the complementarity between both approachesis crucial to analyze complex ecosystems. Nevertheless, more research efforts still have to be undertaken tounderstand their functioning.STRASBOURG-Bib.electronique 063 (674829902) / SudocSudocFranceF

Etude des biofilms bactériens arsénite-oxydants

Les biofilms sont des communautés hautement organisées permettant aux cellules de se maintenir dans une niche écologique donnée. Ces structures sont capables de séquestrer des composés toxiques tels que l'arsenic. Des biofilms bactériens présentant en plus une activité d oxydation de l arsénite [As(III)] en arséniate [As(V)], qui est une forme moins mobile de l arsenic, pourraient être avantageusement mis à profit dans un procédé de bioremédiation. Le but de ce travail de thèse était de caractériser les souches du genre Thiomonas, considérées comme particulièrement adaptées pour le traitement d eaux arséniées, et d étudier l impact de l As(III) sur la formation et le développement de biofilms arsénite-oxydants. La physiologie et la génomique de ces souches ont été étudiées par des analyses de protéomique différentielle et à l aide de puces CGH (Comparative Genomic Hybridization). Ces approches ont souligné de fortes différences physiologiques entre ces souches phylogénétiquement proches, qui peuvent être expliquées en partie par la grande plasticité de leur génome qui évolue par l acquisition d ilots génomiques. L impact de l As(III) sur la cinétique de développement des biofilms a ensuite été analysé par microscopie confocale. Cette étude a mis en évidence divers mécanismes induits en présence d As(III) et contrôlant l'initiation, la maturation et la dispersion des biofilms. Ainsi, l As(III) retarde la formation du biofilm d Herminiimonas arsenicoxydans en induisant la mobilité des cellules, alors qu il favorise le développement d un biofilm chez Thiomonas sp. CB2 en induisant la synthèse d exopolysaccharides. Ces travaux soulignent la diversité des réponses adaptatives des souches bactériennes au stress arsénié. A terme, ils devraient faciliter la mise en œuvre d une stratégie de bioremédiation des eaux arséniées en permettant d anticiper le comportement de la population bactérienne d intérêt.A biofilm is a highly organized microbial community, allowing the resident cells to persist in a given ecological niche. These structures are able to trap toxic compounds such as arsenic. In addition, bacterial biofilms catalyzing arsenite [As(III)] oxidation into the more easily immobilized form arsenate [As(V)] are also of particular interest for their use in an arsenic bioremediation system. The aim of this work was to characterize strains of the Thiomonas genus, which seem to be well-suited for the treatment of arsenic contaminated waters, and to assess As(III) effects on the formation and development of arsenite oxidizing biofilms. The physiology and genomics of the Thiomonas strains were investigated using differential proteomics analyses and a comparative genomic hybridization (CGH) approach. These studies highlighted strong physiological differences between these closely related strains. These divergences may be explained, at least in part, by a high genome plasticity and the horizontal transfer of genomic islands. As(III) effects on arsenite oxidizing biofilms development were then assessed using confocal microscopy. This approach revealed various As(III) induced mechanisms affecting multiple biofilm developmental steps. Indeed, As(III) induces Herminiimonas arsenicoxydans flagellar motility what delays biofilm formation, whereas in Thiomonas sp. CB2 it promotes biofilm development through the induction of exopolysaccharides synthesis. These results highlight the high diversity existing in the bacterial adaptive responses to arsenic. Moreover, they might be helpful to develop a bioremediation process, allowing the anticipation of the bacterial population behavior.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Novel and unexpected bacterial diversity in an arsenic-rich ecosystem revealed by culture-dependent approaches

<p>Abstract</p> <p>Background</p> <p>Acid Mine Drainages (AMDs) are extreme environments characterized by very acid conditions and heavy metal contaminations. In these ecosystems, the bacterial diversity is considered to be low. Previous culture-independent approaches performed in the AMD of Carnoulès (France) confirmed this low species richness. However, very little is known about the cultured bacteria in this ecosystem. The aims of the study were firstly to apply novel culture methods in order to access to the largest cultured bacterial diversity, and secondly to better define the robustness of the community for 3 important functions: As(III) oxidation, cellulose degradation and cobalamine biosynthesis.</p> <p>Results</p> <p>Despite the oligotrophic and acidic conditions found in AMDs, the newly designed media covered a large range of nutrient concentrations and a pH range from 3.5 to 9.8, in order to target also non-acidophilic bacteria. These approaches generated 49 isolates representing 19 genera belonging to 4 different phyla. Importantly, overall diversity gained 16 extra genera never detected in Carnoulès. Among the 19 genera, 3 were previously uncultured, one of them being novel in databases. This strategy increased the overall diversity in the Carnoulès sediment by 70% when compared with previous culture-independent approaches, as specific phylogenetic groups (<it>e.g.</it> the subclass <it>Actinobacteridae</it> or the order <it>Rhizobiales</it>) were only detected by culture. Cobalamin auxotrophy, cellulose degradation and As(III)-oxidation are 3 crucial functions in this ecosystem, and a previous meta- and proteo-genomic work attributed each function to only one taxon. Here, we demonstrate that other members of this community can also assume these functions, thus increasing the overall community robustness.</p> <p>Conclusions</p> <p>This work highlights that bacterial diversity in AMDs is much higher than previously envisaged, thus pointing out that the AMD system is functionally more robust than expected. The isolated bacteria may be part of the rare biosphere which remained previously undetected due to molecular biases. No matter their current ecological relevance, the exploration of the full diversity remains crucial to decipher the function and dynamic of any community. This work also underlines the importance to associate culture-dependent and -independent approaches to gain an integrative view of the community function.</p> <p>Reviewers</p> <p>This paper was reviewed by Sándor Pongor, Eugene V. Koonin and Brett Baker (nominated by Purificacion Lopez-Garcia).</p