Project description:BACKGROUND: Arsenic is present in numerous ecosystems and microorganisms have developed various mechanisms to live in such hostile environments. Herminiimonas arsenicoxydans, a bacterium isolated from arsenic contaminated sludge, has acquired remarkable capabilities to cope with arsenic. In particular our previous studies have suggested the existence of a temporal induction of arsenite oxidase, a key enzyme in arsenic metabolism, in the presence of As(III). RESULTS: Microarrays were designed to compare gene transcription profiles under a temporal As(III) exposure. Transcriptome kinetic analysis demonstrated the existence of two phases in arsenic response. The expression of approximatively 14% of the whole genome was significantly affected by an As(III) early stress and 4% by an As(III) late exposure. The early response was characterized by arsenic resistance, oxidative stress, chaperone synthesis and sulfur metabolism. The late response was characterized by arsenic metabolism and associated mechanisms such as phosphate transport and motility. The major metabolic changes were confirmed by chemical, transcriptional, physiological and biochemical experiments. These early and late responses were defined as general stress response and specific response to As(III), respectively. CONCLUSION: Gene expression patterns suggest that the exposure to As(III) induces an acute response to rapidly minimize the immediate effects of As(III). Upon a longer arsenic exposure, a broad metabolic response was induced. These data allowed to propose for the first time a kinetic model of the As(III) response in bacteria.
Project description:Both the speciation and toxicity of arsenic are affected by bacterial transformations, i.e. oxidation, reduction or methylation. These transformations have a major impact on environmental contamination and more particularly on arsenic contamination of drinking water. Herminiimonas arsenicoxydans has been isolated from an arsenic- contaminated environment and has developed various mechanisms for coping with arsenic, including the oxidation of As(III) to As(V) as a detoxification mechanism.In the present study, a differential transcriptome analysis was used to identify genes, including arsenite oxidase encoding genes, involved in the response of H. arsenicoxydans to As(III). To get insight into the molecular mechanisms of this enzyme activity, a Tn5 transposon mutagenesis was performed. Transposon insertions resulting in a lack of arsenite oxidase activity disrupted aoxR and aoxS genes, showing that the aox operon transcription is regulated by the AoxRS two-component system. Remarkably, transposon insertions were also identified in rpoN coding for the alternative N sigma factor (sigma54) of RNA polymerase and in dnaJ coding for the Hsp70 co-chaperone. Western blotting with anti-AoxB antibodies and quantitative RT-PCR experiments allowed us to demonstrate that the rpoN and dnaJ gene products are involved in the control of arsenite oxidase gene expression. Finally, the transcriptional start site of the aoxAB operon was determined using rapid amplification of cDNA ends (RACE) and a putative -12/-24 sigma54-dependent promoter motif was identified upstream of aoxAB coding sequences.These results reveal the existence of novel molecular regulatory processes governing arsenite oxidase expression in H. arsenicoxydans. These data are summarized in a model that functionally integrates arsenite oxidation in the adaptive response to As(III) in this microorganism.
Project description:Pseudomonas aeruginosa is a ubiquitous gram-negative bacterium capable of forming biofilms on living and non-living surfaces, frequently leading to undesirable consequences. We found that lauroyl arginate ethyl (LAE), a synthetic non-oxidizing biocide, inhibited biofilm formation by P. aeruginosa at sub-growth inhibitory concentrations in both static and flow conditions. To identify the genes targeted by LAE, a global transcriptome analysis was conducted using a gene chip microarray.
Project description:view of the global regulation of gene expression in Herminiimonas arsenicoxydans in response to As(III) stress, in particular those coding for arsenite oxidation.
Project description:Wild type G. sulfurreducens DL1 strain (see Caccavo, F., Jr., D. J. Lonergan, D. R. Lovley, M. Davis, J. F. Stolz, and M. J. McInerney. 1994. Geobacter sulfurreducens sp. nov., a hydrogen- and acetate-oxidizing dissimilatory metal-reducing microorganism. Appl Environ Microbiol 60:3752-9. see also Coppi, M. V., C. Leang, S. J. Sandler, and D. R. Lovley. 2001. Development of a genetic system for Geobacter sulfurreducens. Appl Environ Microbiol 67:3180-7.) and DLCN16 mutant (.rpoS::Km) (see Nuñez, C., L. Adams, S. Childers, and D. R. Lovley. 2004. The RpoS sigma factor in the dissimilatory Fe(III)-reducing bacterium Geobacter sulfurreducens. J Bacteriol 186:5543-6.) were grown under anaerobic conditions at 30 °C in continuous culture with a 200 ml working volume as previously described (see Esteve-Nunez, A., M. Rothermich, M. Sharma, and D. Lovley. 2005. Growth of Geobacter sulfurreducens under nutrient-limiting conditions in continuous culture. Environ Microbiol 7:641-8.). Cells were cultured at a growth rate of 0.05 h-1, steady-state cell growth was obtained after 5 volume refills and was confirmed by a constant cell density and concentrations of Fe(II). Acetate (5.5 mM) was the electron donor and the limiting substrate. The electron acceptor was Fe(III)-citrate (60mM). Two biological replicates of control and treatment cells were obtained to produce hybridizations for this experiment.
Project description:Wild type G. sulfurreducens DL1 strain (see Caccavo, F., Jr., D. J. Lonergan, D. R. Lovley, M. Davis, J. F. Stolz, and M. J. McInerney. 1994. Geobacter sulfurreducens sp. nov., a hydrogen- and acetate-oxidizing dissimilatory metal-reducing microorganism. Appl Environ Microbiol 60:3752-9. see also Coppi, M. V., C. Leang, S. J. Sandler, and D. R. Lovley. 2001. Development of a genetic system for Geobacter sulfurreducens. Appl Environ Microbiol 67:3180-7.) and DLCN16 mutant (.rpoS::Km) (see Nuñez, C., L. Adams, S. Childers, and D. R. Lovley. 2004. The RpoS sigma factor in the dissimilatory Fe(III)-reducing bacterium Geobacter sulfurreducens. J Bacteriol 186:5543-6.) were grown under anaerobic conditions at 30 °C in continuous culture with a 200 ml working volume as previously described (see Esteve-Nunez, A., M. Rothermich, M. Sharma, and D. Lovley. 2005. Growth of Geobacter sulfurreducens under nutrient-limiting conditions in continuous culture. Environ Microbiol 7:641-8.). Cells were cultured at a growth rate of 0.05 h-1, steady-state cell growth was obtained after 5 volume refills and was confirmed by a constant cell density and concentrations of fumarate and succinate. Acetate (5.5 mM) was the electron donor and the limiting substrate. The electron acceptor was fumarate (30mM). Three biological replicates of control and treatment cells were obtained to produce hybridizations for this experiment.