Papers by Author: Violaine Bonnefoy

Abstract: The elemental sulfur oxidising enzyme Sulfur Oxygenase Reductase (SOR) is very well investigated in acidothermophilic archaea, such as Acidianus brierleyi and Sulfolobus metallicus. In contrast, not much is known about the biochemistry of elemental sulfur oxidation in acidophilic bacteria. Recently, however, the SOR-encoding gene has been found also in a bacterial strain closely related to the moderate thermophile Acidithiobacillus caldus. Confusingly, for the latter species, also the involvement of the SOX system as well as thiosulfate:quinone oxidoreductase (TQO) and tetrathionate hydrolase (TTH) in sulfur compound oxidation has been proposed based on genome analysis. In this study, we have detected the sor-gene in other Acidithiobacillus caldus-like strains, isolated from various bioleaching habitats, indicating that SOR plays an important role in sulfur oxidation in this species. Based on sequence comparison, the new bacterial sor-genes are closely related and distant from the known archaeal sequences as well as from the SOR found in the neutrophilic bacterium Aquifex aeolicus. In addition, SOR activity has been detected in crude cell extracts from all Acidithiobacillus caldus-like strains tested. The enzyme is truly thermophilic as highest activities were achieved at 65 °C, which is far beyond the growth optimum of Acidithiobacillus caldus. This finding may give rise to the question whether the presence of SOR in Acidithiobacillus caldus is only relevant while growing at elevated temperatures. Currently, experiments are performed for testing this hypothesis (comparing growth and enzyme activities at 30 vs. 45 °C).

Abstract: Bioleaching is the extraction of metals, such as copper or gold, from ore by microorganisms. Bacterial attachment increases leaching activities due to the formation of a "reaction space" between the metal sulfide surface and the cell. This process depends on abiotic characteristics such as purity and degree of crystallization of the metal sulfide, as well as biotic ones such as the capacity of the bacteria for detecting favourable attachment sites and synthesizing a suitable cell envelope (EPS), for adhesion. Planktonic and sessile cells should differ significantly in their metabolic activities and therefore in their gene expression patterns. To help to understand At. ferrooxidans biofilm formation, microarray transcript profiling was carried out to compare planktonic and sessile cells. The high contents of EPS and ferric iron of the biofilms are interfering with RNA extraction, causing inhibition of DNAse, reverse transcriptase and/or polymerase activities required to get labelled target cDNA. In order to have sufficient high quality RNA suitable for transcriptomic analysis, we have optimized the biofilm formation of At. ferrooxidans on pyrite (FeS2) and the RNA extraction from the sessile cell population. DNA microarrays have been hybridized with labelled cDNAs from sessile and planktonic cells and preliminary data suggest that some genes are differently expressed between these two subpopulations. The understanding of these differences may help us to shift populations of leaching bacteria from the planktonic state towards the sessile state in order to influence bioleaching.

Abstract: It has long been recognized that isolates of iron- and sulfur-oxidizing acidophiles referred to as “Acidithiobacillus ferrooxidans” probably include more than one species, on the basis of differences in chromosomal GC contents and 16S rRNA gene sequences. Phylogenetic heterogeneity among these isolates was confirmed by phylogenetic analysis using the sequences of the 16S-23S intergenic spacers (ITS). Two main groups have been identified: the first includes the type strain of Acidithiobacillus ferrooxidans and a second comprises a cluster of newly isolated strains that have 98.5% 16S rRNA gene sequence identity with the type strain. Given that the new group of isolates have GC contents of 56 mol% as opposed to 58.8 mol% for At. ferrooxidansT, and that they share only 37% homologous DNA, these were given the new species name Acidithiobacillus ferrivorans. Further studies showed that, while strains of At. ferrivorans have many physiological traits in common with At. ferrooxidans, they also differ in some key characteristics. These include the ability to grow at temperatures as low as 4°C (as opposed to the lower limit of between 10 and 12°C for At. ferrooxidans) and the greater sensitivity of At. ferrivorans to low pH (minimum of 1.9 for growth as opposed to 1.3 for At. ferrooxidansT). Important genotypic differences include the fact that all strains of At. ferrivorans do not contain the archetypal rusticyanin gene (rusA), rather most contain a rusA homologue (rusB). Furthermore, the high potential iron-sulfur protein-encoding gene of all At. ferrivorans strains analyzed is more similar to the iro than to the hip gene characterized in At. ferrooxidansT. These results suggest that the iron oxidation pathways are different in At. ferrivorans and At. ferrooxidans.

Abstract: The acidophilic and strictly chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans oxidizes ferrous (Fe(II)) to ferric (Fe(III)) iron and reduced inorganic sulfur compounds (RISC) to sulfuric acid, in oxic conditions. The redox proteins involved in the electron transfer between Fe(II) and oxygen are encoded in the same transcriptional unit, the rus operon. The expression of this operon is induced in the presence of Fe(II), but not Fe(III), and is not repressed in the presence of sulfur (S0). A number of genes differentially expressed in iron or sulfur conditions have been identified by microarrays transcript profiling. We show here that the presence of Fe(II) induced the expression of the genes involved in iron oxidation and repressed the expression of the genes involved in RISC oxidation. Identification of the regulator(s) involved in this transcriptional regulation is underway. Two genes encoding putative regulators belonging to two transcriptional units located downstream from the rus operon have been cloned. One regulator with a putative ironsulfur cluster belongs to the IscR family and the other belongs to the two component sensor/regulator family. Expression of both genes is induced in the presence of Fe(II) and is not repressed by S0. The recombinant proteins have been purified and gel shift assays with the target regulatory regions are in progress.

Abstract: At the abandoned mining site of Carnoulès (Gard, France), weathering of the arsenopyrite rich tailings leads to the formation of acidic effluents heavily loaded with arsenite (As(III)). However, further downstream, the As(III) concentration decreases while the sediments are richer in arsenate (As(V)). A Thiomonas sp. able to oxidize arsenite to arsenate has been isolated and characterized. The aoxA and aoxB genes encoding the two subunits of this enzyme belong to an operon. Analysis of the genome sequence (Genoscope, Ivry, France) shows that this operon encodes also two cytochromes c, which could be the physiological partners of the arsenite oxidase, and a transcriptional regulator belonging to the metalloregulator ArsR/SmtB family, which could control the expression of the aox operon. The expression of this operon is higher in the presence than in the absence of As(III) and appears also to be repressed in the presence of thiosulfate, a more energetic substrate.