Agnès Richaume

Heterogeneous Cell Density and Genetic Structure of Bacterial Pools Associated with Various Soil Microenvironments as Determined by Enumeration and DNA Fingerprinting Approach (RISA)

A bstractThe cell density and the genetic structure of bacterial subcommunities (further named pools) present in the various microenvironments of a silt loam soil were investigated. The microenvironments were isolated first using a procedure of soil washes that separated bacteria located outside aggregates (outer part) from those located inside aggregates (inner part). A nondestructive physical fractionation was then applied to the inner part in order to separate bacteria located inside stable aggregates of different size (size fractions, i.e., two macroaggregate fractions, two microaggregate fractions, and the dispersible day fraction). Bacterial densities measured by acridine orange direct counts (AODC) and viable heterotrophic (VH) cell enumerations showed the heterogeneous quantitative distribution of cells in soil. Bacteria were preferentially located in the inner part with 87.6% and 95.4% of the whole AODC and VH bacteria, respectively, and in the microaggregate and dispersible clay fractions of this part with more than 70% and 80% of the whole AODC and VH bacteria, respectively. The rRNA intergenic spacer analysis (RISA) was used to study the genetic structure of the bacterial pools. Different fingerprints and consequently different genetic structures were observed between the unfractionated soil and the microenvironments, and also among the various microenvironments, giving evidence that some populations were specific to a given location in addition to the common populations of all the microenvironments. Cluster and multivariate analysis of RISA profiles showed the weak contribution of the pools located in the macroaggregate fractions to the whole soil community structure, as well as the clear distinction between the pool associated to the macroaggregate fractions and the pools associated to the microaggregate ones. Furthermore, these statistical analyses allowed us to ascertain the influence of the clay and organic matter content of microenvironments on the genetic structure relatedness between pools.

A single procedure to recover DNA from the surface or inside aggregates and in various size fractions of soil suitable for PCR-based assays of bacterial communities

Abstract A single DNA procedure to recover bacterial DNA from various soil microenvironments which differ in their physical, chemical and structural properties was developed. These microenvironments, obtained by a combination of soil washes and physical fractionation, were the outer part or macroporosity (outside and surface of aggregates), the inner part or microporosity (inside of aggregates) and various size and stability classes of soil aggregates and particles. The DNA extraction method involved sample homogenization and cell disruption by grinding in liquid nitrogen, followed by enzymatic lysis with lysozyme and proteinase K. High yields of high molecular weight DNA (≥ 23 kb) were obtained for all microenvironments. Crude DNA yields for the various soil microenvironments were between 0.7 and 51.4 μg DNA·g−1 soil sample and were positively correlated with bacterial cell abundance (r = 0.91). Further purification steps allowed to recover at least 60 % of the DNA extracted from the various microenvironments. The suitability of the extracted DNA to undergo enzymatic amplification reactions and the effectiveness of the extraction procedure in recovering DNA from various native bacterial groups was tested using primers for archaebacterial 16S rDNAs, universal and group-specific eubacterial 16S rDNAs primers (β- and γ-proteobacteria, High G+C Gram-positive bacteria, and Bacillus species and relatives). Successful amplification of less ubiquitous genes was also obtained with primers targeting nitrogen fixation (nifH) and mercury resistance (merRTΔP) genes.

Use of polyclonal antibodies to detect and quantify the NOR protein of nitrite oxidizers in complex environments

In the approaches or models which aim to understand and/or predict how the functioning of ecosystems may be affected by perturbations or disturbances, little attention is generally given to microorganisms. Even when they are taken into account as indicators, variables which are poorly informative about the changes in the microbial functioning (microbial biomass or diversity or total number of microorganisms) are often used. To be able to estimate, in complex environments, the quantity of enzymes involved in key ecosystem processes may constitute a useful complementary tool. Here, we describe an immunological method for detecting and quantifying, in complex environments, the nitrite oxidoreductase (NOR), responsible for the oxidation of nitrite to nitrate. The alpha-catalytic subunit of the enzyme was purified from Nitrobacter hamburgensis and used for the production of polyclonal antibodies. These antibodies were used to detect and quantify the NOR by a chemifluorescence technique on Western blots after separation of total proteins from pure cultures and soil samples. They recognized the alpha-NOR of all the Nitrobacter species described to date, but no reaction was observed with members of other nitrite-oxidizing genera. The detection threshold and reproducibility of the proposed method were evaluated. The feasibility of its use to quantify NOR in a soil was tested.

Titanium dioxide nanoparticles strongly impact soil microbial function by affecting archaeal nitrifiers.

Soils are facing new environmental stressors, such as titanium dioxide nanoparticles (TiO2-NPs). While these emerging pollutants are increasingly released into most ecosystems, including agricultural fields, their potential impacts on soil and its function remain to be investigated. Here we report the response of the microbial community of an agricultural soil exposed over 90 days to TiO2-NPs (1 and 500 mg kg−1 dry soil). To assess their impact on soil function, we focused on the nitrogen cycle and measured nitrification and denitrification enzymatic activities and by quantifying specific representative genes (amoA for ammonia-oxidizers, nirK and nirS for denitrifiers). Additionally, diversity shifts were examined in bacteria, archaea, and the ammonia-oxidizing clades of each domain. With strong negative impacts on nitrification enzyme activities and the abundances of ammonia-oxidizing microorganism, TiO2-NPs triggered cascading negative effects on denitrification enzyme activity and a deep modification of the bacterial community structure after just 90 days of exposure to even the lowest, realistic concentration of NPs. These results appeal further research to assess how these emerging pollutants modify the soil health and broader ecosystem function.

L'impact du mercure sur les bactéries telluriques

Les polluants toxiques, tels que le mercure, affectent largement les ecosystemes aquatiques, mais aussi les sols. Comment les bacteries telluriques reagissent-elles ?