Predominant heterotrophic diazotrophic bacteria are involved in Sargassum proliferation in the Great Atlantic Sargassum Belt.

Since 2011, the Caribbean coasts have been subject to episodic influxes of floating Sargassum seaweed of unprecedented magnitude originating from a new area "the Great Atlantic Sargassum Belt" (GASB), leading in episodic influxes and mass strandings of floating Sargassum. For the biofilm of both holopelagic and benthic Sargassum as well as in the surrounding waters, we characterized the main functional groups involved in the microbial nitrogen cycle. The abundance of genes representing nitrogen fixation (nifH), nitrification (amoA), and denitrification (nosZ) showed the predominance of diazotrophs, particularly within the GASB and the Sargasso Sea. In both location, the biofilm associated with holopelagic Sargassum harboured a more abundant proportion of diazotrophs than the surrounding water. The mean δ15N value of the GASB seaweed was very negative (-2.04‰), and lower than previously reported, reinforcing the hypothesis that the source of nitrogen comes from the nitrogen-fixing activity of diazotrophs within this new area of proliferation. Analysis of the diversity of diazotrophic communities revealed for the first time the predominance of heterotrophic diazotrophic bacteria belonging to the phylum Proteobacteria in holopelagic Sargassum biofilms. The nifH sequences belonging to Vibrio genus (Gammaproteobacteria) and Filomicrobium sp. (Alphaproteobacteria) were the most abundant and reached, respectively, up to 46.0% and 33.2% of the community. We highlighted the atmospheric origin of the nitrogen used during the growth of holopelagic Sargassum within the GASB and a contribution of heterotrophic nitrogen-fixing bacteria to a part of the Sargassum proliferation.

Genome-wide transcriptional responses of two metal-tolerant symbiotic Mesorhizobium isolates to zinc and cadmium exposure.

BACKGROUND: Mesorhizobium metallidurans STM 2683T and Mesorhizobium sp. strain STM 4661 were isolated from nodules of the metallicolous legume Anthyllis vulneraria from distant mining spoils. They tolerate unusually high Zinc and Cadmium concentrations as compared to other mesorhizobia. This work aims to study the gene expression profiles associated with Zinc or Cadmium exposure and to identify genes involved in metal tolerance in these two metallicolous Mesorhizobium strains of interest for mine phytostabilization purposes. RESULTS: The draft genomes of the two Mezorhizobium strains were sequenced and used to map RNAseq data obtained after Zinc or Cadmium stresses. Comparative genomics and transcriptomics allowed the rapid discovery of metal-specific or/and strain-specific genes. Respectively 1.05% (72/6,844) and 0.97% (68/6,994) predicted Coding DNA Sequences (CDS) for STM 2683 and STM 4661 were significantly differentially expressed upon metal exposure. Among these, a significant number of CDS involved in transport (13/72 and 13/68 for STM 2683 and STM 4661, respectively) and sequestration (15/72 and 16/68 for STM 2683 and STM 4661, respectively) were identified. Thirteen CDS presented homologs in both strains and were differentially regulated by Zinc and/or Cadmium. For instance, several PIB-type ATPases and genes likely to participate in metal sequestration were identified. Among the conserved CDS that showed differential regulation in the two isolates, we also found znuABC homologs encoding for a high affinity ABC-type Zinc import system probably involved in Zinc homeostasis. Additionally, global analyses suggested that both metals also repressed significantly the translational machinery. CONCLUSIONS: The comparative RNAseq-based approach revealed a relatively low number of genes significantly regulated in the two Mesorhizobium strains. Very few of them were involved in the non-specific metal response, indicating that the approach was well suited for identifying genes that specifically respond to Zinc and Cadmium. Among significantly up-regulated genes, several encode metal efflux and sequestration systems which can be considered as the most widely represented mechanisms of rhizobial metal tolerance. Downstream functional studies will increase successful phytostabilization strategies by selecting appropriate metallicolous rhizobial partners.

Antibiotic-resistant soil bacteria in transgenic plant fields.

Understanding the prevalence and polymorphism of antibiotic resistance genes in soil bacteria and their potential to be transferred horizontally is required to evaluate the likelihood and ecological (and possibly clinical) consequences of the transfer of these genes from transgenic plants to soil bacteria. In this study, we combined culture-dependent and -independent approaches to study the prevalence and diversity of bla genes in soil bacteria and the potential impact that a 10-successive-year culture of the transgenic Bt176 corn, which has a blaTEM marker gene, could have had on the soil bacterial community. The bla gene encoding resistance to ampicillin belongs to the beta-lactam antibiotic family, which is widely used in medicine but is readily compromised by bacterial antibiotic resistance. Our results indicate that soil bacteria are naturally resistant to a broad spectrum of beta-lactam antibiotics, including the third cephalosporin generation, which has a slightly stronger discriminating effect on soil isolates than other cephalosporins. These high resistance levels for a wide range of antibiotics are partly due to the polymorphism of bla genes, which occur frequently among soil bacteria. The blaTEM116 gene of the transgenic corn Bt176 investigated here is among those frequently found, thus reducing any risk of introducing a new bacterial resistance trait from the transgenic material. In addition, no significant differences were observed in bacterial antibiotic-resistance levels between transgenic and nontransgenic corn fields, although the bacterial populations were different.

Microbial sequences retrieved from environmental samples from seasonal arctic snow and meltwater from Svalbard, Norway.

16S rRNA gene (rrs) clone libraries were constructed from two snow samples (May 11, 2007 and June 7, 2007) and two meltwater samples collected during the spring of 2007 in Svalbard, Norway (79 degrees N). The libraries covered 19 different microbial classes, including Betaproteobacteria (21.3%), Sphingobacteria (16.4%), Flavobacteria (9.0%), Acidobacteria (7.7%) and Alphaproteobacteria (6.5%). Significant differences were detected between the two sets of sample libraries. First, the meltwater libraries had the highest community richness (Chao1: 103.2 and 152.2) and Shannon biodiversity indices (between 3.38 and 3.59), when compared with the snow libraries (Chao1: 14.8 and 59.7; Shannon index: 1.93 and 3.01). Second, integral-LIBSHUFF analyses determined that the bacterial communities in the snow libraries were significantly different from those of the meltwater libraries. Despite these differences, our data also support the theory that a common core group of microbial populations exist within a variety of cryohabitats. Electronic supplementary material The online version of this article (doi:10.1007/s00792-009-0299-2) contains supplementary material, which is available to authorized users.

Characterization of denitrification gene clusters of soil bacteria via a metagenomic approach.

We characterized operons encoding enzymes involved in denitrification, a nitrogen-cycling process involved in nitrogen losses and greenhouse gas emission, using a metagenomic approach which combines molecular screening and pyrosequencing. Screening of 77,000 clones from a soil metagenomic library led to the identification and the subsequent characterization of nine denitrification gene clusters.

Evaluation of functional gene enrichment in a soil metagenomic clone library.

We evaluated the use of mixed oligonucleotide probes hybridized to metagenomic clones spotted on high density membranes. The pooled probes included oligonucleotides designed for genes associated with denitrification, antibiotic resistance, and dehalogenation among others. Pyrosequence comparison between the clones and the original DNA demonstrated the utility of clone screening with pooled probes.

Complete Genome Sequence of Bradyrhizobium sp. Strain ORS3257, an Efficient Nitrogen-Fixing Bacterium Isolated from Cowpea in Senegal.

Here, we report the complete genome sequence of Bradyrhizobium sp. strain ORS3257, which forms efficient symbioses with cowpea, peanut, or groundnut. These genomic data will be useful to identify genes associated with symbiotic performance and host compatibility on several legumes, including Aeschynomene species, with which a Nod-independent type III secretion system (T3SS)-dependent symbiosis can be established.

Impact of heavy metal contamination on oxidative stress of Eisenia andrei and bacterial community structure in Tunisian mine soil.

The aims of this work were firstly to study the effect of heavy metal-polluted soils from Tunisian mine on earthworm biochemical biomarkers and on bacterial communities and therefore to analyze the interaction between earth worms and bacterial communities in these contaminated soils. For this purpose, we had introduced earthworm Eisenia andrei in six soils: one from mine spoils and five from agricultural soils, establishing a gradient of contamination. The response of worms to the presence of heavy metal was analyzed at the biochemical and transcriptional levels. In a second time, the impact of worm on bacterial community structure was investigated using automated ribosomal intergenic spacer analysis (ARISA) fingerprinting. An impact of heavy metal-contaminated soils on the oxidative status of E. andrei was observed, but this effect was dependent of the level of heavy metal contamination. Moreover, our results demonstrate that the introduction of earthworms E. andrei has an impact on bacterial community; however, the major change was observed in the less contaminated site. Furthermore, a significant correlation between earthworm oxidative status biomarkers and bacterial community structure was observed, mainly in the mine spoils. Therefore, we contribute to a better understanding of the relationships between epigenic earthworms and bacterial communities in heavy metal-contaminated soils.

Effect of carbon and nitrogen input on the bacterial community structure of Neocaledonian nickel mine spoils.

Neocaledonian mine spoils are considered as an extreme environment because of their edaphic conditions, which are unfavourable for life. The principal characteristics of this soil are the high nickel content (20,000 ppm) and the very low carbon (0.2%) and nitrogen (0.01%) levels, which are certainly among the major limiting factors for heterotrophic bacterial growth. The aim of this work was to determine what changes could occur in the bacterial community structure of the mine spoils when a carbon and a nitrogen source were added. Soil bacterial response to nutrient addition was examined in both the mine spoils and an agricultural soil, which is characterized by normal levels of nutrients. 16S rRNA gene clone libraries constructed to characterize changes occurring in the different soil bacterial communities showed an important selection of Actinobacteria in the mine spoils as a consequence of nutrient amendment: Actinobacteria represented 75% and 96% of the bacterial community structure after succinate and glucose addition, respectively. This was observed only in the mine spoils and is probably a consequence of the extreme environmental conditions. Carbon amendment in the agricultural soil led to an increase in Firmicutes, mainly Bacillus sp.

RisaAligner software for aligning fluorescence data between Agilent 2100 Bioanalyzer chips: Application to soil microbial community analysis.

Ribosomal Intergenic Spacer Analysis (RISA) is a high-resolution and highly reproducible fingerprinting technique for discriminating between microbial communities. The community profiles can be visualized using the Agilent 2100 Bioanalyzer. Comparison between fingerprints relies upon precise estimation of all amplified DNA fragment lengths; however, size standard computation can vary between gel runs. For complex samples such as soil microbial communities, discrimination by fragment size is not always sufficient. In such cases, the comparison of whole fluorescence data as a function of time (electrophoregrams) is more appropriate. When electrophoregrams [fluorescence = f (time)] are used, and more than one chip is involved, electrophoregram comparisons are challenging due to experimental variations between chips and the lack of correction by the Agilent software in such situations. Here we present RisaAligner software for analyzing and comparing electrophoregrams from Agilent chips using a nonlinear ladder-alignment algorithm. We demonstrate the robustness and substantial improvement of data analysis by analyzing soil microbial profiles obtained with Agilent DNA 1000 and High Sensitivity chips.

Extracellular plant DNA in Geneva groundwater and traditional artesian drinking water fountains.

DNA, as the signature of life, has been extensively studied in a wide range of environments. While DNA analysis has become central to work on natural gene exchange, forensic analyses, soil bioremediation, genetically modified organisms, exobiology, and palaeontology, fundamental questions about DNA resistance to degradation remain. This paper investigated on the presence of plant DNA in groundwater and artesian fountain (groundwater-fed) samples, which relates to the movement and persistence of DNA in the environment. The study was performed in the groundwater and in the fountains, which are considered as a traditional artesian drinking water in Geneva Champagne Basin. DNA from water samples was extracted, analysed and quantified. Plant gene sequences were detected using PCR amplification based on 18S rRNA gene primers specific for eukaryotes. Physicochemical parameters of water samples including temperature, pH, conductivity, organic matter, dissolved organic carbon (DOC) and total organic carbon (TOC) were measured throughout the study. The results revealed that important quantities of plant DNA can be found in the groundwater. PCR amplification based on 18S rDNA, cloning, RFLP analysis and sequencing demonstrated the presence of plant DNA including Vitis rupestris, Vitis berlandieri, Polygonum sp. Soltis, Boopis graminea, and Sinapis alba in the water samples. Our observations support the notion of plant DNA release, long-term persistence and movement in the unsaturated medium as well as in groundwater aquifers.

Development and validation of a prototype 16S rRNA-based taxonomic microarray for Alphaproteobacteria.

The microarray approach has been proposed for high throughput analysis of the microbial community by providing snapshots of the microbial diversity under different environmental conditions. For this purpose, a prototype of a 16S rRNA-based taxonomic microarray was developed and evaluated for assessing bacterial community diversity. The prototype microarray is composed of 122 probes that target bacteria at various taxonomic levels from phyla to species (mostly Alphaproteobacteria). The prototype microarray was first validated using bacteria in pure culture. Differences in the sequences of probes and potential target DNAs were quantified as weighted mismatches (WMM) in order to evaluate hybridization reliability. As a general feature, probes having a WMM > 2 with target DNA displayed only 2.8% false positives. The prototype microarray was subsequently tested with an environmental sample, which consisted of an Agrobacterium-related polymerase chain reaction amplicon from a maize rhizosphere bacterial community. Microarray results were compared to results obtained by cloning-sequencing with the same DNA. Microarray analysis enabled the detection of all 16S rRNA gene sequences found by cloning-sequencing. Sequences representing only 1.7% of the clone library were detected. In conclusion, this prototype 16S rRNA-based taxonomic microarray appears to be a promising tool for the analysis of Alphaproteobacteria in complex ecosystems.

Nickel mine spoils revegetation attempts: effect of pioneer plants on two functional bacterial communities involved in the N-cycle.

Nickel mine spoils in New Caledonia represent an extreme environment, rich in nickel and strongly deficient in elementary elements such as carbon and nitrogen. To rehabilitate these sites, revegetation attempts are performed with endemic plant species establishing dinitrogen-fixation symbiosis (Gymnostoma webbianum and Serianthes calycina). As this biological fixation process provides the major source of available nitrogen in this extreme environment, it could be expected that nitrogen cycling would be stimulated. To study the revegetation effect on mine spoils, the effect of the two pioneer plants on the structure and activity of two functional bacterial communities involved in the N-cycle was investigated. nifH and narG genes were used as molecular markers for dinitrogen-fixers and dissimilatory nitrate reducers respectively. In order to assess the influence of the plants on both communities, nine clone libraries were constructed for each targeted gene. Libraries containing 602 and 513 nifH and narG clones, respectively, were screened by restriction fragment length polymorphism (RFLP) analysis. One hundred and forty-one and 78 representative clones from at least all RFLP families containing more than one clone were sequenced from nifH and narG clone libraries respectively. Both pioneer plants modified the diversity and activity of the two functional communities. However, distinct effects were observed depending on the plant species and the community considered. Serianthes calycina strongly selected a diazotroph phylotype and restored the potential activity of both communities. In contrast, G. webbianum selected no particular phylotype and only restored a fixing activity.

Adaptation to nickel spiking of bacterial communities in neocaledonian soils.

Adaptation to nickel of bacterial communities of two extreme neocaledonian soils (an ultramafic soil and an acidic soil) was investigated by nickel spiking and compared with adaptation in a non-neocaledonian soil used as reference. Soil microcosms were amended with nickel chloride (NiCl2), and bacterial community structure was analysed with the ribosomal intergenic spacer analysis (RISA) technique. Then, bacterial populations that respond to nickel stress were identified by cloning and sequencing. In the ultramafic soil, a shift occurred on day zero on the assay profiles and consisted of the emergence of a bacterial group closely related to the Ralstonia/Oxalobacter/Burkholderia group. It is hypothesized that NiCl2 had a physico-chemical impact on soil structure. Fourteen days after nickel spiking, another shift occurred in the two soils that concerned a bacterial group belonging to the Actinomycete group. Only a few changes occurred in the bacterial community structure of the neocaledonian soils compared with those of the reference soil, which is more affected by nickel spiking. These results suggest that neocaledonian soil bacteria are particularly well adapted to nickel.