Project description:S. oneidensis MR-1 was grown with different electron acceptors: an electrode at 0.4 V vs. SHE, 50 mM Fe(III)citrate, and oxygen. The gene expression pattern for each experiment was analyzed and the differences in gene expression for the different experimental conditions were compared.
Project description:S. oneidensis MR-1 was grown with different electron acceptors: an electrode at 0.4 V vs. SHE, 50 mM Fe(III)citrate, and oxygen. The gene expression pattern for each experiment was analyzed and the differences in gene expression for the different experimental conditions were compared. For two samples S. oneidensis was grown with a graphite anode electrode (at 0.4 V. vs. SHE) as the only electron acceptor - one sample was directly fed with 20 mM lactate, one sample was fed with lactate produced during fermentation of glucose by Lactococcus lactis. Four samples were grown anaerobically with 50 mM Fe(III)citrate as the only electron acceptor, and 20 mM lactate for 20 h. Three samples were grown aerobically with 20 mM lactate for 20 h. The same modified M4 medium was used for all samples. For RNA extraction, the biofilm was scraped off the frozen (-80M-BM-0C) carbon electrode with a sterile razor blade. Biofilm-carbon sludge was combined with 7 mL ice-cold phosphate buffer saline (PBS), vortexed, sonicated at 7 W for 30 s on ice (3 repetitions), and centrifuged. For the liquid cultures, 2 mL of each culture were combined with 2 mL RNA protect, vortexed, and centrifuged at 5,500g for 10 min. The pellets were resuspended in NAES buffer (50 mM sodium acetate buffer, 10 mM EDTA and 1 % SDS at pH 5). RNA was isolated with a phenol:chloroform extraction protocol.
Project description:High-resolution tiling analysis of the MR-1 transcriptome under diverse growth conditions The conditions include aerobic growth in Luria-Bertani broth (LB), aerobic growth in defined lactate minimal medium, anaerobic growth in defined lactate minimal medium with 20mM dimethyl sulfoxide as the electron acceptor, anaerobic growth in defined lactate minimal medium with 10mM iron (III) citrate as the electron acceptor, 10 minutes post heat shock at 42oC see GSE39468 for tiling data on lactate minimal media
Project description:High-resolution tiling analysis of the MR-1 transcriptome under diverse growth conditions The conditions include aerobic growth in Luria-Bertani broth (LB), aerobic growth in defined lactate minimal medium, anaerobic growth in defined lactate minimal medium with 20mM dimethyl sulfoxide as the electron acceptor, anaerobic growth in defined lactate minimal medium with 10mM iron (III) citrate as the electron acceptor, 10 minutes post heat shock at 42oC see GSE39468 for tiling data on lactate minimal media Four slides hybridized to mRNA and one “genomic control” array hybridized to genomic DNA
Project description:Shewanella oneidensis MR-1 is a facultative anaerobe that grows by respiration using a variety of electron acceptors. This organism serves as a model to study how bacteria thrive in redox-stratified environments. A glucose-utilizing engineered derivative of MR-1 has been reported to be unable to grow in glucose minimal medium (GMM) in the absence of electron acceptors, despite this strain having a complete set of genes for reconstructing glucose to lactate fermentative pathways. To gain insights into why MR-1 is incapable of fermentative growth, this study examined a hypothesis that this strain is programmed to repress the expression of some carbon metabolic genes in the absence of electron acceptors. Comparative transcriptomic analyses of the MR-1 derivative were conducted in the presence and absence of fumarate as an electron acceptor, and these found that the expression of many genes involved in carbon metabolism required for cell growth, including several tricarboxylic acid (TCA) cycle genes, was significantly downregulated in the absence of fumarate. This finding suggests a possibility that MR-1 is unable to grow fermentatively on glucose in minimal media owing to the shortage of nutrients essential for cell growth, such as amino acids. This idea was demonstrated in subsequent experiments that showed that the MR-1 derivative fermentatively grows in GMM containing tryptone or a defined mixture of amino acids. We suggest that gene regulatory circuits in MR-1 are tuned to minimize energy consumption under electron acceptor-depleted conditions, and that this results in defective fermentative growth in minimal media. IMPORTANCE It is an enigma why S. oneidensis MR-1 is incapable of fermentative growth despite having complete sets of genes for reconstructing fermentative pathways. Understanding the molecular mechanisms behind this defect will facilitate the development of novel fermentation technologies for the production of value-added chemicals from biomass feedstocks, such as electro-fermentation. The information provided in this study will also improve our understanding of the ecological strategies of bacteria living in redox-stratified environments.
Project description:We investigated the anode-specific responses of Shewanella oneidensis MR-1, an exoelectroactive ammaproteobacterium, using for the first time iTRAQ and 2D-LC MS/MS driven membrane proteomics to compare protein abundances in S. oneidensis when generating power in MFCs, and growing in a continuous culture.
Project description:In limiting oxygen as an electron acceptor, the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1 rapidly forms nanowires, extensions of its outer membrane containing the cytochromes MtrC and OmcA needed for extracellular electron transfer. RNA-Seq analysis was employed to determine differential gene expression over time from cultures maintained in a chemostat and limited for oxygen. We identified 465 genes with decreased expression and 677 genes with increased expression. The coordinated increased expression of heme biosynthesis, cytochrome maturation, and transport pathways indicates that S. oneidensis MR-1 increases cytochrome production, including transcription of genes encoding MtrA, MtrC and OmcA, and properly positions these decaheme cytochromes in or near the outer membrane during nanowire formation. In contrast, the expression of the mtrA and mtrC homologs mtrF and mtrD either remain unaffected or decrease during nanowire formation. The ompW gene, encoding a small outer membrane porin, has 50-fold higher expression during oxygen limitation, and it is proposed that OmpW plays a role in cation transport to maintain electrical neutrality during electron transfer. The genes encoding the anaerobic respiration regulator CRP and the extracytoplasmic function sigma factor RpoE are among transcription factor genes with increased expression. RpoE could function by signaling the initial response to oxygen limitation. Our results show that RpoE activates transcription from promoters upstream of mtrC and omcA. The transcriptome and mutant analysis of S. oneidensis MR-1 nanowire production are consistent with independent regulatory mechanisms for extending the outer membrane into tubular structures and for ensuring the electron transfer function of the nanowires.
Project description:. In this study we show successful use of SWATH-MS for quantitative proteomic analysis of a microbial electrochemically active biofilm. Shewanella oneidensis MR-1 was grown on carbon cloth electrodes under continuous anodic electrochemical polarizations in a bioelectrochemical system. Using lactate as the electron donor, anodes serving as terminal microbial electron acceptors were operated at three different electrode potentials (+0.71V, +0.21V & -0.19V vs. SHE) and the development of catalytic activity was monitored by measuring the current traces over time. Once maximum current was reached (usually within 21-29 hours) the electrochemical systems were shut off and biofilm proteins were extracted from the electrodes for proteomic assessment.