The Mre11 protein interacts with both Rad50 and the HerA bipolar helicase and is recruited to DNA following gamma irradiation in the archaeon Sulfolobus acidocaldarius

Background: The ubiquitous Rad50 and Mre11 proteins play a key role in many processes involved in the maintenance of genome integrity in Bacteria and Eucarya, but their function in the Archaea is presently unknown. We showed previously that in most hyperthermophilic archaea, rad50-mre11 genes are linked to nurA encoding both a single-strand endonuclease and a 5' to 3' exonuclease, and herA, encoding a bipolar DNA helicase which suggests the involvement of the four proteins in common molecular pathway(s). Since genetic tools for hyperthermophilic archaea are just emerging, we utilized immuno-detection approaches to get the first in vivo data on the role(s) of these proteins in the hyperthermophilic crenarchaeon Sulfolobus acidocaldarius. Results: We first showed that S. acidocaldarius can repair DNA damage induced by high doses of gamma rays, and we performed a time course analysis of the total levels and sub-cellular partitioning of Rad50, Mre11, HerA and NurA along with the RadA recombinase in both control and irradiated cells. We found that during the exponential phase, all proteins are synthesized and display constant levels, but that all of them exhibit a different sub-cellular partitioning. Following gamma irradiation, both Mre11 and RadA are immediately recruited to DNA and remain DNA-bound in the course of DNA repair. Furthermore, we show by immuno-precipitation assays that Rad50, Mre11 and the HerA helicase interact altogether. Conclusion: Our analyses strongly support that in Sulfolobus acidocaldarius, the Mre11 protein and the RadA recombinase might play an active role in the repair of DNA damage introduced by gamma rays and/or may act as DNA damage sensors. Moreover, our results demonstrate the functional interaction between Mre11, Rad50 and the HerA helicase and suggest that each protein play different roles when acting on its own or in association with its partners. This report provides the first in vivo evidence supporting the implication of the Mre11 protein in DNA repair processes in the Archaea and showing its interaction with both Rad50 and the HerA bipolar helicase. Further studies on the functional interactions between these proteins, the NurA nuclease and the RadA recombinase, will allow us to define their roles and mechanism of action.Publisher PDFPeer reviewe

Unraveling the Stratification of an Iron-Oxidizing Microbial Mat by Metatranscriptomics

International audienceA metatranscriptomic approach was used to study community gene expression in a naturally occurring iron-rich microbial mat. Total microbial community RNA was reversely transcribed and sequenced by pyrosequencing. Characterization of expressed gene sequences provided accurate and detailed information of the composition of the transcriptionally active community and revealed phylogenetic and functional stratifications within the mat. Comparison of 16S rRNA reads and delineation of OTUs showed significantly lower values of metatranscriptomic-based richness and diversity in the upper parts of the mat than in the deeper regions. Taxonomic affiliation of rRNA sequences and mRNA genome recruitments indicated that iron-oxidizing bacteria affiliated to the genus Leptothrix, dominated the community in the upper layers of the mat. Surprisingly, type I methanotrophs contributed to the majority of the sequences in the deep layers of the mat. Analysis of mRNA expression patterns showed that genes encoding the three subunits of the particulate methane monooxygenase (pmoCAB) were the most highly expressed in our dataset. These results provide strong hints that iron-oxidation and methane-oxidation occur simultaneously in microbial mats and that both groups of microorganisms are major players in the functioning of this ecosystem

Groundwater Isolation Governs Chemistry and Microbial Community Structure along Hydrologic Flowpaths

International audienceThis study deals with the effects of hydrodynamic functioning of hard-rock aquifers on microbial communities. In hard-rock aquifers, the heterogeneous hydrologic circulation strongly constrains groundwater residence time, hydrochemistry, and nutrient supply. Here, residence time and a wide range of environmental factors were used to test the influence of groundwater circulation on active microbial community composition, assessed by high throughput sequencing of 16S rRNA. Groundwater of different ages was sampled along hydrogeologic paths or loops, in three contrasting hard-rock aquifers in Brittany (France). Microbial community composition was driven by groundwater residence time and hydrogeologic loop position. In recent groundwater, in the upper section of the aquifers or in their recharge zone, surface water inputs caused high nitrate concentration and the predominance of putative denitrifiers. Although denitrification does not seem to fully decrease nitrate concentrations due to low dissolved organic carbon concentrations, nitrate input has a major effect on microbial communities. The occurrence of taxa possibly associated with the application of organic fertilizers was also noticed. In ancient isolated groundwater, an ecosystem based on Fe(II)/Fe(III) and S/SO4 redox cycling was observed down to several 100 of meters below the surface. In this depth section, microbial communities were dominated by iron oxidizing bacteria belonging to Gallionellaceae. The latter were associated to old groundwater with high Fe concentrations mixed to a small but not null percentage of recent groundwater inducing oxygen concentrations below 2.5 mg/L. These two types of microbial community were observed in the three sites, independently of site geology and aquifer geometry, indicating hydrogeologic circulation exercises a major control on microbial communities

Comparative metagenomics and bioinformatics tools

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Molekularbiologische Charakterisierung von nicht-kultivierten Crenarchaeota und Acidobacteria in Metagenombanken aus Bodenproben

Molekular-phylogenetische Studien, basierend auf der Charakterisierung von 16S rRNA-Genen, haben gezeigt, dass Bodenhabitate besonders reich an mikrobieller Diversität sind. So wurde z.B. eine bestimmte Gruppe der Crenarchaeota (Archaea), die bislang als Extremophile galten, durch 16S rDNA-Studien häufig in Bodenhabitaten entdeckt. Weiterhin konnte das Phylum der Acidobacteria, das derzeit von ca. 200 16S rDNA Umwelt-Sequenzen und von nur 3 kultivierten Vertretern repräsentiert wird, definiert werden. Es ist aufgrund seiner phylogenetischen Breite mit den Proteobacteria vergleichbar. Acidobacteria stellen häufig eine dominierende Fraktion der Mikroorganismen in Bodenhabitaten dar, sind jedoch bis heute ebenso wie die mesophilen Crenarchaeota weitgehend uncharakterisiert. Aus Mangel an geeigneten Techniken bleiben bislang insgesamt mehr als 99% der Mikroorganismen in den meisten Habitaten uncharakterisiert, weil sie nicht im Labor kultiviert werden können. Inspiriert von den schnellen Fortschritten der mikrobiellen Genomik wurde in dieser Arbeit die Strategie der Umweltgenomik auf Bodenhabitate angewendet, um erstmals mesophile, unkultivierte Crenarchaeota und Acidobacteria aus Böden zu charakterisieren. Voraussetzung dafür war die direkte Isolierung von hochmolekularer DNA aus Bodenproben, die aufgrund des Vorliegens von grossen Mengen an polyphenolischen Komponenten (Humin- und Fulvinsäuren) eine besondere Herausforderung darstellte. Es wurde eine Methodik entwickelt, die auf einer 2-phasigen Gelelektrophoresetechnik basierte, und die eine schnelle und reproduzierbare Isolierung von klonierbarer, hochmolekularer DNA aus unterschiedlichen Habitaten erlaubte. Von diesen Nukleinsäuren wurden komplexe Genbibliotheken mit großen Inserts konstruiert. Die Charakterisierung der Genbibliotheken zeigte, dass die in den Habitaten durch PCR-Studien dargestellte große mikrobielle Diversität tatsächlich auf genomischer Ebene ab-gebildet werden konnte. Mit Archaea-spezifischen und Acidobacteria-spezifischen Sonden wurden je ein Genomfragment der mesophilen nicht-kultivierten Crenarchaeota und der Acidobacteria aus einer 960 Mbp großen Fosmid-Genbank einer Bodenprobe isoliert und charakterisiert. Der archaeale Klon enthielt ein 34 kb Fragment, auf dem ein komplettes rRNA-Operon und 17 Protein-kodierende Gene lokalisiert waren. Die phylogenetischen Analysen der rRNA-Gene und eines Proteingens bestätigten die Zugehörigkeit des Genomfragments zur Gruppe der nicht-thermophilen Crenarchaeota. Die Genomstruktur und die auf dem Genomfragment lokalisierten Gene zeigten deutliche Unterschiede zu den bereits untersuchten, nahe-verwandten marinen Vertretern. Die detaillierte Analyse eines 35 kb Fragments der Acidobacteria bestätigte die Zugehörigkeit des Genomfragmentes zu dieser noch unbeschriebenen, ökologisch aber relevanten Gruppe. Durch vergleichende Genomanalysen und phylogenetische Rekonstruktionen wurden Hinweise auf einen horizontalen Gentransfer zwischen Rhizobiaceae (a-Proteobacteria) und Acidobacteria erhalten. Ein Bereich, der 10 kb mit 8 potentiellen Protein-kodierenden Genen überspannte, war colinear und zeigte große Sequenzähnlichkeiten mit einem Genombereich von Rhodopseudomonas palustris. Die Charakterisierung der Protein-kodierenden Gene ermöglichte erste Einblicke in das physiologische Potential der mesophilen Crenarchaeota und der Acidobacteria und stellt eine Basis für weiterführende genomische Untersuchungen dar

Functional community analysis of a microbial mat involved in the oxydation of iron by metatranscriptomics

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Community analysis of a microbial mat involved in the oxydation of iron by metatranscriptomics

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Stable Isotope Probing-RNA Strategy to Study Plant/Fungus Interactions

International audienceThe use of stable-isotope probing (SIP) allows tracing specific labeled substrates into fungi leading to a better understanding of their role in biogeochemical cycles and their relationship with their environment. Stable isotope probing combined with ribosomal RNA molecule, conserved in the three kingdoms of life, and messenger RNA analysis permits the linkage of diversity and function. Here, we describe two methods designed to investigate the interactions between plants and their associated mycorrhizal compartment by tracing carbon flux from the host plant to its symbionts

Analysis of the first genome fragment from the marine sponge-associated, novel candidate phylum Poribacteria by environmental genomics

The novel candidate phylum Poribacteria is specifically associated with several marine demosponge genera. Because no representatives of Poribacteria have been cultivated, an environmental genomic approach was used to gain insights into genomic properties and possibly physiological/functional features of this elusive candidate division. In a large-insert library harbouring an estimated 1.1 Gb of microbial community DNA from Aplysina aerophoba, a Poribacteria-positive 16S rRNA gene locus was identified. Sequencing and sequence annotation of the 39 kb size insert revealed 27 open reading frames (ORFs) and two genes for stable RNAs. The fragment exhibited an overall G+C content of 50.5% and a coding density of 86.1%. The 16S rRNA gene was unlinked from a conventional rrn operon. Its flanking regions did not show any synteny to other 16S rRNA encoding loci from microorganisms with unlinked rrn operons. Two of the predicted hypothetical proteins were highly similar to homologues from Rhodopirellula baltica. Furthermore, a novel kind of molybdenum containing oxidoreductase was predicted as well as a series of eight ORFs encoding for unusual transporters, channel or pore forming proteins. This environmental genomics approach provides, for the first time, genomic and, by inference, functional information on the so far uncultivated, sponge-associated candidate division Poribacteria