Project description:Comparisson of expression profiling of a etrA deletion mutant strain (experimental sample) with that of the wild type Shewanella oneidensis MR-1 strain to assess global direct/indirect genetic regulation EtrA in Shewanella oneidensis MR-1 shares 73.6% and 50.8% amino acid sequence identity with the oxygen-sensing regulator Fnr in E. coli and Anr in Pseudomonas aeruginosa, respectively; however, its regulatory role of anaerobic metabolism in Shewanella spp. is complex and not well understood. Whole-genome expression profiling using a etrA gene deletion mutant as the experimental sample and the wild type strain as the reference, determine that EtrA fine-tunes the expression of genes involved in various anaerobic metabolic pathways, including nitrate, fumarate and dimethyl sulfoxide reduction. Moreover, genes involved in prophage activation and and genes implicated in aerobic metabolism were also differentially expressed. In contrast to previous studies that attributed a minor regulatory role to EtrA in Shewanella spp., this study demonstrates that EtrA acts as a global transcriptional regulator and cofers physiological advantages to the strain under certain growth conditions.
Project description:We combined high-resolution tiling microarrays and 5'-end RNA sequencing to obtain a genome-wide map of transcription start sites (TSSs) for Shewanella oneidensis MR-1. To test the reliability of these TSSs, we compared our result to those from differential RNA sequencing (dRNA-seq), which discriminates primary and processed ends of transcripts. We found that our identified TSSs tend to have significantly more mapped reads in the TEX(+) sample than the TEX(-) sample. Overall, the dRNA-seq results support the validity of our predictions for TSS.
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:We combined high-resolution tiling microarrays and 5'-end RNA sequencing to obtain a genome-wide map of transcription start sites (TSSs) for Shewanella oneidensis MR-1. To test the reliability of these TSSs, we compared our result to those from differential RNA sequencing (dRNA-seq), which discriminates primary and processed ends of transcripts. We found that our identified TSSs tend to have significantly more mapped reads in the TEX(+) sample than the TEX(-) sample. Overall, the dRNA-seq results support the validity of our predictions for TSS. S. oneidensis MR-1 was grown to mid-log phase in Luria-Bertani broth (LB) or defined lactate minimal medium, and total RNA was isolated and used for differential RNA-sequencing (dRNA-seq) by next-generation sequencing, which is used to verify genome-wide transcription start sites. For dRNA-seq, total RNA was partially treated with Terminator Exonuclease (TEX) to digest processed RNA and thereby enrich for primary transcript ends.
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:Anaerobic respiration in the metal reducer Shewanella oneidensis MR-1, unlike other bacteria, is regulated by the cAMP receptor protein, CRP. Three putative genes, cyaA, cyaB, and cyaC, predicted to encode class I, class IV, and class III adenylate cyclases respectively, have been identified in the genome sequence of this bacterium. Functional validation through complementation of an E. coli cya mutant confirmed that these genes encode proteins with adenylate cyclase activities. Chromosomal deletion of either cyaA or cyaB did not affect anaerobic respiration with fumarate, DMSO, or Fe(III), whereas the deletion of cyaC caused deficiencies in respiration with DMSO and Fe(III), and to a lesser extent with fumarate. A phenotype similar to that of a crp mutant, which lacks the ability to grow anaerobically with DMSO, fumarate, and Fe(III), was obtained when both cyaA and cyaC were deleted. Microarray analysis of gene expression in the crp and the cyaC mutants revealed the involvement of both genes in the regulation of key respiratory pathways that include DMSO, fumarate, and Fe(III) reduction. Additionally, several genes associated with plasmid replication, flagella biosynthesis, and electron transport, were differentially expressed in the cyaC mutant, but not in the crp mutant. Our results indicated that CyaC plays a major role in regulating anaerobic respiration, and may contribute to additional signaling pathways independent of CRP.
Project description:One of the most striking observations in the S. oneidensis genome regarding nitrate respiration is that napC is missing and nrfBCD is degenerated. To gain insights into candidate genes encoding the protein(s) in place of NapC and NrfBCD function, i.e., delivering electrons to NapA and NrfA, transcriptional profiling was carried out using the S. oneidensis whole-genome cDNA microarray. Cells of MR-1 grown on nitrate or fumarate under anaerobic conditions were sampled at the exponential phase for the analysis. The quality of the array data was statistically assessed using the method reported previously. Keywords: Compararive microarray study Compare transcriptional profiles of MR-1 grown on nitrate and Fumarate aiming at finding the electron transfer proteins to NapA and NrfA.