Project description:Siderophore-binding proteins (SIPs) perform a key role in iron acquisition in multiple organisms. In the genome of the marine bacterium Shewanella frigidimarina NCIMB 400, the gene tagged as SFRI_RS12295 encodes a protein from this family. Here, the cloning, expression, purification and crystallization of this protein are reported, together with its preliminary X-ray crystallographic analysis to 1.35 Å resolution. The SIP crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 48.04, b = 78.31, c = 67.71 Å, α = 90, β = 99.94, γ = 90°, and are predicted to contain two molecules per asymmetric unit. Structure determination by molecular replacement and the use of previously determined ∼2 Å resolution SIP structures with ∼30% sequence identity as templates are ongoing.
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:Bacterial anaerobic respiration using extracellular electron acceptor plays a predominant role in global biogeochemical cycles. However, the bacterial adaptive mechanisms to the toxic organic pollutant as the extracellular electron acceptor during anaerobic respiration is not clear, which limits us to optimize the strategies for the bioremediation of contaminated environment. Here, we report the physiological characteristics and the global gene expression of an ecologically successful bacterium Shewanella decolorationis S12 when using a typical toxic organic pollutant, amaranth, as the extracellular electron acceptor. Our results revealed that filamentous shift (the cells stretched to fiber-like shapes as long as 18 μm) occurred under amaranth stress. Persistent stress led to higher filamentous cell rate and decolorization ability in subcultural cells compared with parental strains. Additionally, the expression of genes involved in cell division, chemotaxi system, energy conservation, damage repair, and material transport in filamentous cells were significantly stimulated. The detailed roles of some genes with significantly elevated expressions in filamentous cells were identified by site-directed mutagenesis, such as the outer membrane porin genes ompA and ompW, the cytochrome C genes arpC and arpD, the global regulatory factor gene rpoS and methyl-accepting chemotaxis proteins genes SHD_2793 and SHD_0015. Finally, a conceptual model was proposed to help deepen our insights into both the bacterial survival strategy when toxic organics were present, and the mechanisms in which these toxic organics were biodegraded as the extracellular electron acceptors.
Project description:Shewanella algae C6G3 can conduct dissimilative nitrate reduction into ammonium and MnIV reduction. This bacteria have the unusual ability to produce anaerobically nitrite from ammonium in the presence of MnIV. This property may explain NO2/3- accumulation observed in some anaerobic zones of marine sediments. To gain insight into their metabolic capabilities, global mRNA expression patterns were investigated by RNA-seq and qRT-PCR in cells growing with lactate and ammonium as carbon and nitrogen sources and with MnIV or nitrate as electron acceptors. Genes exhibiting higher expression levels in the presence of MnIV belonged to functional categories of carbohydrate, coenzyme, lipid metabolisms and inorganic ion transport. Furthermore, comparative transcriptomic pattern between MnIV and NO3 revealed that the strain presented an ammonium limitation status with MnIV, despite the presence of identical and non-limiting concentration of ammonium in both culture conditions. Regulators ntrB/nrtC, ammonium channel, nitrogen regulatory protein P-II, glutamine synthetase and asparagine synthetase glutamine dependent genes were over-expressed. In nitrate condition, genes involved in synthesis of several amino acids were over expressed. Among the genes associated with the stress response, Kat E was highly expressed particularly under manganese condition
Project description:We compared global transcriptional patterns in Pyrobaculum aerophilum cultures with oxygen, nitrate, arsenate and ferric iron as respiratory electron acceptors to identify genes and regulatory patterns that differentiate these pathways. Keywords: Time course study with different respiratory electron acceptors
Project description:Some microorganisms can respire with extracellular electron acceptors using an extended electron transport chain to the cell surface. These organisms apply flavin molecules as cofactors to facilitate one-electron transfer catalysed by the terminal reductases and as endogenous electron shuttles. In the model organism Shewanella oneidensis, riboflavin production and excretion triggers a specific biofilm formation response that is initiated at a specific threshold concentration, similar to canonical quorum sensing molecules. Riboflavin-mediated messaging is based on the overexpression of the gene encoding the putrescin decarboxylase speC which leads to posttranscriptional overproduction of proteins involved in biofilm formation. We performed a mass spectrometry-based analysis of cells with and without speC overexpression to identify the effect of SpeC overexpression on the cell proteome.
Project description:We compared global transcriptional patterns in Pyrobaculum aerophilum cultures with oxygen, nitrate, arsenate and ferric iron as respiratory electron acceptors to identify genes and regulatory patterns that differentiate these pathways. Keywords: Time course study with different respiratory electron acceptors To focus the microarrays on genes that are specifically affected by changes in terminal electron acceptors we analyzed gene expression in time courses with cultures shifted from aerobic growth to either NO3-, As(V), Fe(III)-citrate, or additional O2. Gene expression and substrate usage were measured at three time points (2.5, 4.5, and 7.5 hours), allowing sufficient time for changes in gene expression, while restricting cultures to a maximum of one or two cell divisions.