Microbiome analysis in Lascaux Cave in relation to black stain alterations of rock surfaces and collembola.

Anthropization of Palaeolithic caves open for tourism may favour collembola invasion and result in the formation of black stains attributed to pigmented fungi. However, ecological processes underpinning black stain formation are not fully understood. Here, we tested the hypotheses that black stains from the Apse room of Lascaux Cave display a specific microbiota enriched in pigmented fungi, and that collembola thriving on the stains have the potential to consume and disseminate these black fungi. Metabarcoding showed that the microbiota of black stains and neighbouring unstained parts strongly differed, with in black stains a higher prevalence of Ochroconis and other pigmented fungi and the strong regression of Pseudomonas bacteria (whose isolates inhibited in vitro the growth of pigmented fungi). Isotopic analyses indicated that Folsomia candida collembola thriving on stains could feed on black stain in situ and assimilate the pigmented fungi they were fed with in vitro. They could carry these fungi and disseminate them when tested with complex black stains from Lascaux. This shows that black stain formation is linked to the development of pigmented fungi, which coincides with the elimination of antagonistic pseudomonads, and points towards a key role of F. candida collembola in the dynamics of pigmented fungi.

Larval habitat determines the bacterial and fungal microbiota of the mosquito vector Aedes aegypti.

Mosquito larvae are naturally exposed to microbial communities present in a variety of larval development sites. Several earlier studies have highlighted that the larval habitat influences the composition of the larval bacterial microbiota. However, little information is available on their fungal microbiota, i.e. the mycobiota. In this study, we provide the first simultaneous characterization of the bacterial and fungal microbiota in field-collected Aedes aegypti larvae and their respective aquatic habitats. We evaluated whether the microbial communities associated with the breeding site may affect the composition of both the bacterial and fungal communities in Ae. aegypti larvae. Our results show a higher similarity in microbial community structure for both bacteria and fungi between larvae and the water in which larvae develop than between larvae from different breeding sites. This supports the hypothesis that larval habitat is a major factor driving microbial composition in mosquito larvae. Since the microbiota plays an important role in mosquito biology, unravelling the network of interactions that operate between bacteria and fungi is essential to better understand the functioning of the mosquito holobiont.

Small-Scale Variability in Bacterial Community Structure in Different Soil Types.

Microbial spatial distribution has mostly been studied at field to global scales (i.e., ecosystem scales). However, the spatial organization at small scales (i.e., centimeter to millimeter scales), which can help improve our understanding of the impacts of spatial communities structure on microbial functioning, has received comparatively little attention. Previous work has shown that small-scale spatial structure exists in soil microbial communities, but these studies have not compared soils from geographically distant locations, nor have they utilized community ecology approaches, such as the core and satellite hypothesis and/or abundance-occupancy relationships, often used in macro-ecology, to improve the description of the spatial organization of communities. In the present work, we focused on bacterial diversity (i.e., 16S rRNA gene sequencing) occurring in micro-samples from a variety of locations with different pedo-climatic histories (i.e., from semi-arid, alpine, and temperate climates) and physicochemical properties. The forms of ecological spatial relationships in bacterial communities (i.e., occupancy-frequency and abundance-occupancy) and taxa distributions (i.e., habitat generalists and specialists) were investigated. The results showed that bacterial composition differed in the four soils at the small scale. Moreover, one soil presented a satellite mode distribution, whereas the three others presented bimodal distributions. Interestingly, numerous core taxa were present in the four soils among which 8 OTUs were common to the four sites. These results confirm that analyses of the small-scale spatial distribution are necessary to understand consequent functional processes taking place in soils, affecting thus ecosystem functioning.

Ionizing-radiation-resistant Kocuria rhizophila PT10 isolated from the Tunisian Sahara xerophyte Panicum turgidum: Polyphasic characterization and proteogenomic arsenal.

A new strain belonging to the genus Kocuria, designed PT10, was isolated from irradiated roots of the xerophyte Panicum turgidum. Isolate PT10 is a Gram-positive, coccoid, aerobic and ionizing-radiation (IR)-resistant actinobacterium. PT10 has shown an ability to survive under extreme conditions, such as gamma irradiation, desiccation and high concentration of hydrogen peroxide. Phenotypic, chemotaxonomic and comparative genome analyses support the assignment of strain PT10 (LMG 31102 = DSM 108617) as Kocuria rhizophila. The complete genome sequence of PT10 consists of one chromosome (2,656,287 bps), with a 70.7% G + C content and comprises 2481 protein-coding sequences. A total of 1487 proteins were identified by LC-MS/MS profiling. In silico analyses revealed that the proteome of the oxidation-tolerant PT10 possesses several features explaining its IR-resistant phenotype and many adaptive pathways implicated in response to environmental pressures - desiccation, cold, reactive oxygen species and other stressors.

Draft genome sequence of Promicromonospora panici sp. nov., a novel ionizing-radiation-resistant actinobacterium isolated from roots of the desert plant Panicum turgidum.

A novel strain of the genus Promicromonospora, designated PT9T, was recovered from irradiated roots of the xerophyte Panicum turgidum collected from the Ksar Ghilane oasis in southern Tunisia. Strain PT9T is aerobic, non-spore-forming, Gram- positive actinomycete that produces branched hyphae and forms white to yellowish-white colonies. Chemotaxonomic features, including fatty acids, whole cell sugars and polar lipid profiles, support the assignment of PT9T to the genus Promicromonospora. The genomic relatedness indexes based on DNA-DNA hybridization and average nucleotide identity values revealed a significant genomic divergence between strain PT9T and all sequenced type strains of the taxon. Phylogenomic analysis showed that isolate PT9T was most closely related to Promicromonospora soli CGMCC 4.7398T. Phenotypic and phylogenomic analyses suggest that isolate PT9T represents a novel species of the genus Promicromonospora, for which the name Promicromonospora panici sp. nov. is proposed. The type strain is PT9T (LMG 31103T = DSM 108613T).The isolate PT9T is an ionizing-radiation-resistant actinobacterium (D10 value = 2.6 kGy), with resistance to desiccation and hydrogen peroxide. The complete genome sequence of PT9T consists of 6,582,650 bps with 71.2% G+C content and 6291 protein-coding sequences. This genome will help to decipher the microbial genetic bases for ionizing-radiation resistance mechanisms including the response to oxidative stress.

Secondary metabolites from plant-associated Pseudomonas are overproduced in biofilm.

Plant rhizosphere soil houses complex microbial communities in which microorganisms are often involved in intraspecies as well as interspecies and inter-kingdom signalling networks. Some members of these networks can improve plant health thanks to an important diversity of bioactive secondary metabolites. In this competitive environment, the ability to form biofilms may provide major advantages to microorganisms. With the aim of highlighting the impact of bacterial lifestyle on secondary metabolites production, we performed a metabolomic analysis on four fluorescent Pseudomonas strains cultivated in planktonic and biofilm colony conditions. The untargeted metabolomic analysis led to the detection of hundreds of secondary metabolites in culture extracts. Comparison between biofilm and planktonic conditions showed that bacterial lifestyle is a key factor influencing Pseudomonas metabolome. More than 50% of the detected metabolites were differentially produced according to planktonic or biofilm lifestyles, with the four Pseudomonas strains overproducing several secondary metabolites in biofilm conditions. In parallel, metabolomic analysis associated with genomic prediction and a molecular networking approach enabled us to evaluate the impact of bacterial lifestyle on chemically identified secondary metabolites, more precisely involved in microbial interactions and plant-growth promotion. Notably, this work highlights the major effect of biofilm lifestyle on acyl-homoserine lactone and phenazine production in P. chlororaphis strains.

Candidatus Frankia nodulisporulans sp. nov., an Alnus glutinosa-infective Frankia species unable to grow in pure culture and able to sporulate in-planta.

We describe a new Frankia species, for three non-isolated strains obtained from Alnus glutinosa in France and Sweden, respectively. These strains can nodulate several Alnus species (A. glutinosa, A. incana, A. alnobetula), they form hyphae, vesicles and sporangia in the root nodule cortex but have resisted all attempts at isolation in pure culture. Their genomes have been sequenced, they are significantly smaller than those of other Alnus-infective species (5Mb instead of 7.5Mb) and are very closely related to one another (ANI of 100%). The name Candidatus Frankia nodulisporulans is proposed. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene and draft genome sequences reported in this study for AgTrS, AgUmASt1 and AgUmASH1 are MT023539/LR778176/LR778180 and NZ_CADCWS000000000.1/CADDZU010000001/CADDZW010000001, respectively.

Proposal of 'Candidatus Frankia alpina', the uncultured symbiont of Alnus alnobetula and A. incana that forms spore-containing nitrogen-fixing root nodules.

The members of the genus Frankia are, with a few exceptions, a group of nitrogen-fixing symbiotic actinobacteria that nodulate mostly woody dicotyledonous plants belonging to three orders, eight families and 23 genera of pioneer dicots. These bacteria have been characterized phylogenetically and grouped into four molecular clusters. One of the clusters, cluster 1 contains strains that induce nodules on Alnus spp. (Betulaceae), Myrica spp., Morella spp. and Comptonia spp. (Myricaceae) that have global distributions. Some of these strains produce not only hyphae and vesicles, as other cluster 1 strains do, but also numerous sporangia in their host symbiotic tissues, hence their phenotype being described as spore-positive (Sp+). While Sp+ strains have resisted repeated attempts at cultivation, their genomes have recently been characterized and found to be different from those of all described species, being markedly smaller than their phylogenetic neighbours. We thus hereby propose to create a 'Candidatus Frankia alpina' species for some strains present in nodules of Alnus alnobetula and A. incana that grow in alpine environments at high altitudes or in subarctic environments at high latitudes.

Co-occurrence of rhizobacteria with nitrogen fixation and/or 1-aminocyclopropane-1-carboxylate deamination abilities in the maize rhizosphere.

The plant microbiota may differ depending on soil type, but these microbiota probably share the same functions necessary for holobiont fitness. Thus, we tested the hypothesis that phytostimulatory microbial functional groups are likely to co-occur in the rhizosphere, using groups corresponding to nitrogen fixation (nifH) and 1-aminocyclopropane-1-carboxylate deamination (acdS), i.e. two key modes of action in plant-beneficial rhizobacteria. The analysis of three maize fields in two consecutive years showed that quantitative PCR numbers of nifH and of acdS alleles differed according to field site, but a positive correlation was found overall when comparing nifH and acdS numbers. Metabarcoding analyses in the second year indicated that the diversity level of acdS but not nifH rhizobacteria in the rhizosphere differed across fields. Furthermore, between-class analysis showed that the three sites differed from one another based on nifH or acdS sequence data (or rrs data), and the bacterial genera contributing most to field differentiation were not the same for the three bacterial groups. However, co-inertia analysis indicated that the genetic structures of both functional groups and of the whole bacterial community were similar across the three fields. Therefore, results point to co-selection of rhizobacteria harboring nitrogen fixation and/or 1-aminocyclopropane-1-carboxylate deamination abilities.

Author Correction: Complex ecological interactions of Staphylococcus aureus in tampons during menstruation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genome Sequence of Pseudomonas sp. Strain ST1, Isolated from Olive (Olea europaea L.) Knot Galls in Croatia.

We report the genome sequence of a Pseudomonas sp. strain isolated from olive knot galls. The genome size is 6.101 Mbp with a G+C content of 58%. A total of 6,137 coding DNA sequences (CDS) were predicted, including 52 tRNA and 4 rRNA genes.

Whole-Genome Sequence of a Pantoea sp. Strain Isolated from an Olive (Olea europaea L.) Knot.

Here, we present the total genome sequence of Pantoea sp. strain paga, a plant-associated bacterium isolated from knots present on olive trees grown on the Adriatic Coast. The genome size of Pantoea sp. paga is 5.08 Mb, with a G+C content of 54%. The genome contains 4,776 predicted coding DNA sequences (CDSs), including 70 tRNA genes and 1 ribosomal operon. Obtained genome sequence data will provide insight on the physiology, ecology, and evolution of Pantoea spp.

Draft genome sequences for three unisolated Alnus-infective Frankia Sp+ strains, AgTrS, AiOr and AvVan, the first sequenced Frankia strains able to sporulate in-planta.

Actinobacteria from genus Frankia are able to form symbiotic associations with actinorhizal plants including alders. Among them, Sp+ strains are characterized by their ability to differentiate numerous sporangia inside host plant cells (unlike "Sp-" strains unable of in-planta sporulation). Here, we report the first genome sequences of three unisolated Sp+ strains: AgTrS, AiOr and AvVan obtained from Alnus glutinosa, A. incana and A. alnobetula (previously known as viridis), respectively (with genome completeness estimated at more than 98%). They represent new Frankia species based on Average Nucleotide Identity (ANI) calculations, and the smallest Alnus-infective Frankia genomes so far sequenced (~5 Mbp), with 5,178, 6,192 and 5,751 candidate protein-encoding genes for AgTrS, AiOr and AvVan, respectively.

Genome Sequences of Three Nocardia cyriacigeorgica Strains and One Nocardia asteroides Strain.

We report four draft genome sequences of Nocardia spp. The strains are the Nocardia cyriacigeorgica DSM 44484 pathogenic type strain; two environmental isolates, Nocardia cyriacigeorgica EML446 and EML1456; and the Nocardia asteroides ATCC 19247 nonpathogenic type strain, with estimated genome sizes of 6.3 to 6.8 Mb. The study of these isolates will provide insight into physiology, evolution, and pathogenicity of Nocardia spp.

Anthropization level of Lascaux Cave microbiome shown by regional-scale comparisons of pristine and anthropized caves.

Limestone areas across the world develop karstic caves, which are populated by a wide range of macro- and microorganisms. Many of these caves display Paleolithic art or outstanding speleothems, and in the last century they have been subjected to anthropization due to touristic management and intense human frequentation. Despite their cultural importance and associated conservation issues, the impact of anthropization on cave biodiversity is not known. Here, we show that anthropization is associated with specific cave biota modifications. We compared diversity in four pristine caves, four anthropized show caves, and the iconic Lascaux Cave with even stronger anthropization. The predominant microbial higher taxa were the same in all caves, but the most anthropized cave (Lascaux) was unique as it differed from the eight others by a higher proportion of Bacteroidetes bacteria and the absence of Euryarchaeota and Woesearchaeota archaea. Anthropization resulted in lower diversity and altered community structure for bacteria and archaea on cave walls, especially in Lascaux, but with a more limited effect on microeukaryotes and arthropods. Our findings fill a key gap in our understanding of the response of karstic communities to anthropization, by revealing that tourism-related anthropization impacts on the prokaryotic microbiome rather than on eukaryotic residents, and that it shapes cave biota irrespective of cave natural features.

Genomic, phylogenetic and catabolic re-assessment of the Pseudomonas putida clade supports the delineation of Pseudomonas alloputida sp. nov., Pseudomonas inefficax sp. nov., Pseudomonas persica sp. nov., and Pseudomonas shirazica sp. nov.

Bacteria of the Pseudomonas putida group are studied for a large panel of properties ranging from plant growth promotion and bioremediation to pathogenicity. To date, most of the classification of individual pseudomonads from this group relies on 16S RNA gene analysis, which is insufficient for accurate taxonomic characterization within bacterial species complexes of the Pseudomonas putida group. Here, a collection of 20 of these bacteria, isolated from various soils, was assessed via multi-locus sequence analysis of rpoD, gyrB and rrs genes. The 20 strains clustered in 7 different clades of the P. putida group. One strain per cluster was sequenced and results were compared to complete genome sequences of type strains of the P. putida group. Phylogenetic analyses, average nucleotide identity data and digital DNA hybridizations, combined to phenotypic characteristics, resulted in the proposition and description of four new species i.e. Pseudomonas alloputida Kh7 T (= LMG 29756 T = CFBP 8484 T) sp. nov., Pseudomonas inefficax JV551A3 T (= DSM108619 T = CFBP 8493 T) sp. nov., Pseudomonas persica RUB6 T (= LMG 29757 T = CFBP 8486 T) sp. nov. and Pseudomonas shirazica VM14 T (= LMG 29953 T = CFBP 8487 T) sp. nov.

Rock substrate rather than black stain alterations drives microbial community structure in the passage of Lascaux Cave.

BACKGROUND: The World-famous UNESCO heritage from the Paleolithic human society, Lascaux Cave (France), has endeavored intense microclimatic perturbations, in part due to high touristic pressure. These perturbations have resulted in numerous disturbances of the cave ecosystem, including on its microbial compartment, which resulted in the formation of black stains especially on the rock faces of the passage. We investigated the cave microbiome in this part of Lascaux by sampling three mineral substrates (soil, banks, and inclined planes) on and outside stains to assess current cave microbial assemblage and explore the possibility that pigmented microorganisms involved in stain development occur as microbial consortia. METHODS: Microbial abundance and diversity were assessed by means of quantitative PCR and high-throughput sequencing (Illumina MiSeq) of several DNA and cDNA taxonomic markers. Five sampling campaigns were carried out during winter and summer to embrace potential seasonal effect in this somewhat stable environment (based on measurements of temperature and CO2 concentration). RESULTS: While the season or type of mineral substrate did not affect the abundances of bacteria and micro-eukaryotes on or outside stains, mineral substrate rather than stain presence appears to be the most significant factor determining microbial diversity and structuring microbial community, regardless of whether DNA or cDNA markers were considered. A phylogenetic signal was also detected in relation to substrate types, presence of stains but not with season among the OTUs common to the three substrates. Co-occurrence network analyses showed that most bacterial and fungal interactions were positive regardless of the factor tested (season, substrate, or stain), but these networks varied according to ecological conditions and time. Microorganisms known to harbor pigmentation ability were well established inside but also outside black stains, which may be prerequisite for subsequent stain formation. CONCLUSIONS: This first high throughput sequencing performed in Lascaux Cave showed that black stains were secondary to mineral substrate in determining microbiome community structure, regardless of whether total or transcriptionally active bacterial and micro-eukaryotic communities were considered. These results revealed the potential for new stain formation and highlight the need for careful microbiome management to avoid further cave wall degradation.

Plant Nutrient Resource Use Strategies Shape Active Rhizosphere Microbiota Through Root Exudation.

Plant strategies for soil nutrient uptake have the potential to strongly influence plant-microbiota interactions, due to the competition between plants and microorganisms for soil nutrient acquisition and/or conservation. In the present study, we investigate whether these plant strategies could influence rhizosphere microbial activities via root exudation, and contribute to the microbiota diversification of active bacterial communities colonizing the root-adhering soil (RAS) and inhabiting the root tissues. We applied a DNA-based stable isotope probing (DNA-SIP) approach to six grass species distributed along a gradient of plant nutrient resource strategies, from conservative species, characterized by low nitrogen (N) uptake, a long lifespans and low root exudation level, to exploitative species, characterized by high rates of photosynthesis, rapid rates of N uptake and high root exudation level. We analyzed their (i) associated microbiota composition involved in root exudate assimilation and soil organic matter (SOM) degradation by 16S-rRNA-based metabarcoding. (ii) We determine the impact of root exudation level on microbial activities (denitrification and respiration) by gas chromatography. Measurement of microbial activities revealed an increase in denitrification and respiration activities for microbial communities colonizing the RAS of exploitative species. This increase of microbial activities results probably from a higher exudation rate and more diverse metabolites by exploitative plant species. Furthermore, our results demonstrate that plant nutrient resource strategies have a role in shaping active microbiota. We present evidence demonstrating that plant nutrient use strategies shape active microbiota involved in root exudate assimilation and SOM degradation via root exudation.

Short-term impacts of anthropogenic stressors on Aedes albopictus mosquito vector microbiota.

Recent studies have highlighted the potential role of microbiota in the biology of the Aedes albopictus mosquito vector. This species is highly anthropogenic and exhibits marked ecological plasticity, with a resulting high potential to colonize a wide range of habitats-including anthropized areas-under various climatic conditions. We put forward the hypothesis that climate and anthropogenic activities, such as the use of antibiotics in agriculture and human medicine, might affect the mosquito-associated bacterial community. We thus studied the additive impact of a temperature decrease and antibiotic ingestion on the temporal dynamics of Ae. albopictus survival and its associated bacterial communities. The results showed no effects of disturbances on mosquito survival. However, short-term temperature impacts on bacterial diversity were observed, while both the community structure and bacterial diversity were affected by early antibiotic ingestion. The genera Elizabethkingia, Chryseobacterium and Wolbachia, as well as an unclassified member of the Bacteroidales order were particularly affected. Antibiotics negatively impacted Elizabethkingia abundance, while Chryseobacterium was completely eliminated following both disturbances, to the benefit of Wolbachia and the unclassified Bacteroidales species. These results generated fresh insight into the effects of climate and anthropogenic activities such as the use of antibiotics on mosquito microbiota.

Complex ecological interactions of Staphylococcus aureus in tampons during menstruation.

Menstrual toxic shock syndrome (mTSS) is a severe disease that occurs in healthy women vaginally colonized by Staphylococcus aureus producing toxic shock toxin 1 and who use tampons. The aim of the present study was to determine the impact of the composition of vaginal microbial communities on tampon colonisation by S. aureus during menses. We analysed the microbiota in menstrual fluids extracted from tampons from 108 healthy women and 7 mTSS cases. Using culture, S. aureus was detected in menstrual fluids of 40% of healthy volunteers and 100% of mTSS patients. Between class analysis of culturomic and 16S rRNA gene metabarcoding data indicated that the composition of the tampons' microbiota differs according to the presence or absence of S. aureus and identify discriminating genera. However, the bacterial communities of tampon fluid positive for S. aureus did not cluster together. No difference in tampon microbiome richness, diversity, and ecological distance was observed between tampon vaginal fluids with or without S. aureus, and between healthy donors carrying S. aureus and mTSS patients. Our results show that the vagina is a major niche of. S. aureus in tampon users and the composition of the tampon microbiota control its virulence though more complex interactions than simple inhibition by lactic acid-producing bacterial species.