Project description:Microtoming Coupled with Microarray Analysis to Evaluate Potential Differences in the Metabolic Status of Geobacter sulfurreducens at Different Depths in Anode Biofilms Differences in the Metabolic Status of Geobacter sulfurreducens at Different Depths in A Current Producing Biofilm Further insight into the metabolic status of cells within anode biofilms is essential for understanding the functioning of microbial fuel cells and developing strategies to optimize their power output. In order to further compare the metabolic status of cells growing close to the anode versus cells in the outer portion of the anode biofilm, mature anode biofilms were treated to stop turnover over of mRNA and then encased in resin which was sectioned into 100 nm shavings with a diamond knife and pooled into inner (0-20 µm from anode surface) and outer (30-60 µm) fractions. Whole genome DNA microarray analysis of RNA extracted from the shavings revealed that, at a 2-fold lower threshold, there were 146 genes that had significant (p<0.05), differences in transcript abundance between the inner and outer portions of the biofilm. Only 1 gene, GSU0093, a hypothetical ABC transporter, had significantly higher transcript abundances in the outer biofilm. Genes with lower transcript abundance in the outer biofilm included genes for ribosomal proteins and NADH dehydrogenase, suggesting that cells in the outer biofilm had lower metabolic rates. However, the differences in transcript abundance were relatively low (<3-fold) and the outer biofilm did not have significantly lower expression of the genes for TCA cycle enzymes which previous studies have demonstrated are sensitive indicators of changes in rates of metabolism in G. sulfurreducens. There also was no significant difference in the transcript levels for outer-surface cell components thought to be important in electron transfer in anode biofilms. Lower expression of genes involved in stress responses in the outer biofilm may reflect the development of low pH near the surface of the anode. The results of the metabolic staining and gene expression studies suggest that cells throughout the biofilm are metabolically active and can potentially contribute to current production. The microtoming/microarray strategy described here may be useful for evaluating gene expression with depth in a diversity of microbial biofilms.

Project description:Microtoming Coupled with Microarray Analysis to Evaluate Potential Differences in the Metabolic Status of Geobacter sulfurreducens at Different Depths in Anode Biofilms Differences in the Metabolic Status of Geobacter sulfurreducens at Different Depths in A Current Producing Biofilm Further insight into the metabolic status of cells within anode biofilms is essential for understanding the functioning of microbial fuel cells and developing strategies to optimize their power output. In order to further compare the metabolic status of cells growing close to the anode versus cells in the outer portion of the anode biofilm, mature anode biofilms were treated to stop turnover over of mRNA and then encased in resin which was sectioned into 100 nm shavings with a diamond knife and pooled into inner (0-20 µm from anode surface) and outer (30-60 µm) fractions. Whole genome DNA microarray analysis of RNA extracted from the shavings revealed that, at a 2-fold lower threshold, there were 146 genes that had significant (p<0.05), differences in transcript abundance between the inner and outer portions of the biofilm. Only 1 gene, GSU0093, a hypothetical ABC transporter, had significantly higher transcript abundances in the outer biofilm. Genes with lower transcript abundance in the outer biofilm included genes for ribosomal proteins and NADH dehydrogenase, suggesting that cells in the outer biofilm had lower metabolic rates. However, the differences in transcript abundance were relatively low (<3-fold) and the outer biofilm did not have significantly lower expression of the genes for TCA cycle enzymes which previous studies have demonstrated are sensitive indicators of changes in rates of metabolism in G. sulfurreducens. There also was no significant difference in the transcript levels for outer-surface cell components thought to be important in electron transfer in anode biofilms. Lower expression of genes involved in stress responses in the outer biofilm may reflect the development of low pH near the surface of the anode. The results of the metabolic staining and gene expression studies suggest that cells throughout the biofilm are metabolically active and can potentially contribute to current production. The microtoming/microarray strategy described here may be useful for evaluating gene expression with depth in a diversity of microbial biofilms. Three biological replicates were hybridized in triplicate on a coustom affimetrix tilling array using prokaryotic protocol (p69Affy, p75 Adobe) for labeling, hybridization and scanning.

Project description:Multispecies biofilms are the predominant form of bacterial growth in natural and human-associated environments. Although the pathways involved in monospecies biofilm have been well characterized, less is known about the metabolic pathways and emergent traits of a multispecies biofilm community. Here, we performed a transcriptome survey of the developmental stages of a 3-species biofilm community and combined it with quantitative imaging and growth experiments. We report the remodelling of central metabolism of two of the three species in this community. Specifically, we observed an increase in the expression of genes associated with glycolysis and pentose phosphate pathways in K. pneumoniae. Similarly, a decrease in the expression of the same pathways in P. protegens was observed along with an increase in expression of glyoxalate cycle genes when grown as a mixed species biofilm, suggesting reorganisation of metabolic pathways and metabolite sharing for the community biofilms. To test the possibility of cross-feeding for the community, planktonic growth experiments revealed that both the Pseudomonads grew well in TCA cycle intermediates, while K. pneumoniae grew poorly when given those carbon sources. Despite this poor growth in mono-culture, K. pneumoniae was still the dominant species in mixed species biofilms cultivated in TCA intermediates as the sole source of carbon. The biofilm growth data, combined with the transcriptomics data, suggests there is reorganisation of metabolism for the community members and may allow for cross-feeding that allows K. pneumoniae to dominate the community. We also demonstrated that sdsA1 of P. aeruginosa was induced upon exposure to the surfactant SDS and that this gene was essential in protecting mono and mixed species biofilms from surfactant stress. This also suggests that the community members can share defence mechanisms. Overall, this study describes a comprehensive transcriptomics level investigation of shared resources, metabolites and stress defence that may underpin the emergent properties of mixed species biofilm communities.

Project description:G. sulfurreducens can generate electricity from the oxidation of organic compounds. This is because it can take electrons from organic compounds and ship them out to the outer surface of the cell where they can then be deposited on various insoluble electron acceptors including electrodes. Cells attatched to the surface of an electrode oxidize acetate and and deposit the electrons derived from acetate onto the surface of the electrode after which they can travel through an electrical circuit, producing a current. Microbial fuel cells powered by acetate oxidation by Geobacter species are called Geobatteries. In this experiment we compared gene expression in a biofilm of the wild type strain growing on the surface of an electrode within a current-producing Geobattery to gene expression in a wild type biofilm that is not producing current, but is growing on the surface of an electrode. In both cases, the cells were growing in a flow-through two chambered H-cell Geobattery setup. This consists of two glass chambers, an anoxic anode chamber containing G. sulfurreducens, a graphite electrode, a reference electrode and growth medium and an oxic chamber containing the counter electrode. The two chambers are connected by a cation selective membrane and a wire connected to a potentionstat. A potentiostat is an instrument which maintains the redox potential of the anode at a fixed value relative to a reference electrode. Media continuously flowed through the anoxic anode chamber at a dilution rate of 0.15. In the experimental condition, the Geobattery was operational. The circuit was closed and G. sulfurreducens attached to the electrode generated current as it oxidized acetate. The redox potential at the anode was poised at 300 mV by the potentiostat. In the control condition, everything was the same, except that the medium in the anode chamber contained fumarate as electron acceptor, and the anode was not hooked up to the potentiostat i.e. the circuit was open. This prevented the anode from serving as an electron acceptor. Nevertheless a thick G. sulfurreducens biofilm grew on the surface of the electrode. The control and experimental geobatteries were harvested when current in the operational/experimental Geobatteries reached 10 mA. Keywords: two condition comparison

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