Archaeal diversity: temporal variation in the arsenic-rich creek sediments of Carnoulès Mine, France.

The Carnoulès mine is an extreme environment located in the South of France. It is an unusual ecosystem due to its acidic pH (2-3), high concentration of heavy metals, iron, and sulfate, but mainly due to its very high concentration of arsenic (up to 10 g L⁻¹ in the tailing stock pore water, and 100-350 mg L⁻¹ in Reigous Creek, which collects the acid mine drainage). Here, we present a survey of the archaeal community in the sediment and its temporal variation using a culture-independent approach by cloning of 16S rRNA encoding genes. The taxonomic affiliation of Archaea showed a low degree of biodiversity with two different phyla: Euryarchaeota and Thaumarchaeota. The archaeal community varied in composition and richness throughout the sampling campaigns. Many sequences were phylogenetically related to the order Thermoplasmatales represented by aerobic or facultatively anaerobic, thermoacidophilic autotrophic or heterotrophic organisms like the organotrophic genus Thermogymnomonas. Some members of Thermoplasmatales can also derive energy from sulfur/iron oxidation or reduction. We also found microorganisms affiliated with methanogenic Archaea (Methanomassiliicoccus luminyensis), which are involved in the carbon cycle. Some sequences affiliated with ammonia oxidizers, involved in the first and rate-limiting step in nitrification, a key process in the nitrogen cycle were also observed, including Candidatus Nitrososphaera viennensis and Candidatus nitrosopumilus sp. These results suggest that Archaea may be important players in the Reigous sediments through their participation in the biochemical cycles of elements, including those of carbon and nitrogen.

Comparative responses of river biofilms at the community level to common organic solvent and herbicide exposure.

Residual pesticides applied to crops migrate from agricultural lands to surface and ground waters. River biofilms are the first aquatic non-target organisms which interact with pesticides. Therefore, ecotoxicological experiments were performed at laboratory scale under controlled conditions to investigate the community-level responses of river biofilms to a chloroacetanilide herbicide (alachlor) and organic solvent (methanol) exposure through the development referenced to control. Triplicate rotating annular bioreactors, inoculated with river water, were used to cultivate river biofilms under the influence of 1 and 10 µg L(-1) of alachlor and 25 mg L(-1) of methanol. For this purpose, functional (thymidine incorporation and carbon utilization spectra) and structural responses of microbial communities were assessed after 5 weeks of development. Structural aspects included biomass (chlorophyll a, confocal laser scanning microscopy) and composition (fluor-conjugated lectin binding, molecular fingerprinting, and diatom species composition). The addition of alachlor resulted in a significant reduction of bacterial biomass at 1 µg L(-1), whereas at 10 µg L(-1), it induced a significant reduction of exopolymer lectin binding, algal, bacterial, and cyanobacterial biomass. However, there were no changes in biofilm thickness or thymidine incorporation. No significant difference between the bacterial community structures of control and alachlor-treated biofilms was revealed by terminal restriction fragment length polymorphism (T-RFLP) analyses. However, the methanol-treated bacterial communities appeared different from control and alachlor-treated communities. Moreover, methanol treatment resulted in an increase of bacterial biomass and thymidine incorporation as well. Changes in dominant lectin binding suggested changes in the exopolymeric substances and community composition. Chlorophyll a and cyanobacterial biomass were also altered by methanol. This study suggested that the concentration-dependent effect of alachlor mainly remains limited to biomass and growth inhibition without apparent changes of structural and functional characteristics measured. Our work also establishes the potential toxic effects of organic solvents on river biofilm in ecotoxicological experiments. For the ecotoxicological experiments, the alternative of dissolution in organic solvent followed by its evaporation, depositing the chemical on a glass surface prior to dissolution in river water used here, appears to allow exposure while minimizing the effect of organic solvent.

Isolation of a type 2 metallothionein-like gene preferentially expressed in the tapetum in Zea mays.

A Zea mays cDNA, MZm3-4, was isolated by differential screening of a cDNA library obtained from meiotic stage anthers against a cDNA of 3-week-old seedlings. Northern blot analysis of RNA from different maize tissues and from male reproductive organs at various developmental stages demonstrated expression of a single transcript in anthers, from the pollen mother cell stage through the uninucleated microspore stage. In situ hybridization to anther sections resulted in a distinct signal only in the tapetum. The MZm3-4 cDNA is 743 nucleotides in length and has an open reading frame encoding a protein of 75 amino acids. Sequence comparisons with various databases revealed that MZm3-4 exhibits high similarities with type 2 plant metallothioneins at both the nucleotide and the amino-acid level. Primer extension analysis indicated that MZm3-4 cDNA is deleted of 13bp at the 5' end. Southern blot analysis showed that the MZm3-4 gene may be present in one or two copies in a Z. mays inbred line genome. This is the first report of the isolation of a type 2 metallothionein-like protein in maize. Moreover, the expression of this type 2 metallothionein-like gene is high in the male reproductive organs engaged in microsporogenesis.

Changes in tolerance to herbicide toxicity throughout development stages of phototrophic biofilms.

Ecotoxicological experiments have been performed in laboratory-scale microcosms to investigate the sensitivity of phototrophic biofilm communities to the alachlor herbicide, in relation to the stages of phototrophic biofilm maturation (age of the phototrophic biofilms) and physical structure (intact biofilm versus recolonization). The phototrophic biofilms were initially cultivated on artificial supports in a prototype rotating annular bioreactor (RAB) with Taylor-Couette type flow under constant operating conditions. Biofilms were collected after 1.6 and 4.4 weeks of culture providing biofilms with different maturation levels, and then exposed to nominal initial alachlor concentration of 10 µg L(-1) in either intact or recolonized biofilms for 15 days in microcosms (mean time-weighted average concentration - TWAC of 5.52 ± 0.74 µg L(-1)). At the end of the exposure period, alachlor effects were monitored by a combination of biomass descriptors (ash-free dry mass - AFDM, chlorophyll a), structural molecular fingerprinting (T-RFLP), carbon utilization spectra (Biolog) and diatom species composition. We found significant effects that in terms of AFDM, alachlor inhibited growth of the intact phototrophic biofilms. No effect of alachlor was observed on diatom composition or functional and structural properties of the bacterial community regardless of whether they were intact or recolonized. The intact three-dimensional structure of the biofilm did not appear to confer protection from the effects of alachlor. Bacterial community structure and biomass level of 4.4 weeks - intact phototrophic biofilms were significantly influenced by the biofilm maturation processes rather than alachlor exposure. The diatom communities which were largely composed of mobile and colonizer life-form populations were not affected by alachlor. This study showed that the effect of alachlor (at initial concentration of 10 µg L(-1) or mean TWAC of 5.52 ± 0.74 µg L(-1)) is mainly limited to biomass reduction without apparent changes in the ecological succession trajectories of bacterial and diatom communities and suggested that carbon utilization spectra of the biofilm are not damaged resulting. These results confirmed the importance of considering the influence of maturation processes or community age when investigating herbicide effects. This is particularly important with regard to the use of phototrophic biofilms as bio-indicators.

A photosynthetic rotating annular bioreactor (Taylor-Couette type flow) for phototrophic biofilm cultures.

In their natural environment, the structure and functioning of microbial communities from river phototrophic biofilms are driven by biotic and abiotic factors. An understanding of the mechanisms that mediate the community structure, its dynamics and the biological succession processes during phototrophic biofilm development can be gained using laboratory-scale systems operating with controlled parameters. For this purpose, we present the design and description of a new prototype of a rotating annular bioreactor (RAB) (Taylor-Couette type flow, liquid working volume of 5.04 L) specifically adapted for the cultivation and investigation of phototrophic biofilms. The innovation lies in the presence of a modular source of light inside of the system, with the biofilm colonization and development taking place on the stationary outer cylinder (onto 32 removable polyethylene plates). The biofilm cultures were investigated under controlled turbulent flowing conditions and nutrients were provided using a synthetic medium (tap water supplemented with nitrate, phosphate and silica) to favour the biofilm growth. The hydrodynamic features of the water flow were characterized using a tracer method, showing behaviour corresponding to a completely mixed reactor. Shear stress forces on the surface of plates were also quantified by computer simulations and correlated with the rotational speed of the inner cylinder. Two phototrophic biofilm development experiments were performed for periods of 6.7 and 7 weeks with different inoculation procedures and illumination intensities. For both experiments, biofilm biomasses exhibited linear growth kinetics and produced 4.2 and 2.4 mg cm(-)² of ash-free dry matter. Algal and bacterial community structures were assessed by microscopy and T-RFLP, respectively, and the two experiments were different but revealed similar temporal dynamics. Our study confirmed the performance and multipurpose nature of such an innovative photosynthetic bioreactor for phototrophic biofilm investigations.

Characterization of MZm3-3, a Zea mays tapetum-specific transcript.

A cDNA, MZm3-3, was isolated by differential screening of a cDNA library from Zea mays meiotic stage anthers against cDNA of 3-week-old seedlings. Characterization of this cDNA indicated that the MZm3-3 gene is expressed specifically during male gametogenesis. Its expression is highly and preferentially detected in the tapetum, from the pollen mother cell to uninucleated microspore stages. It encodes a short alkaline protein of 10.6 kDa, with a conserved pattern of eight cysteine residues. Sequence analysis showed that these features are shared with lipid transfer proteins and some male-flower-specific proteins. The presence of a putative signal peptide indicates that MZm3-3 enters into the secretory pathway to then be released into the anther loculus. Based on these features, the secretory activity of the tapetum and the temporal expression pattern of MZm3-3, a contribution to pollen coat formation is suggested. Southern blot analyses demonstrated the presence of closely related genes, indicating that MZm3-3 belongs to a multigene family.