Project description:Shewanella oneidensis MR-1 exhibits diverse metal ion-reducing capabilities and thus is of potential utility as a bioremediation agent. Knowledge of the molecular components and regulatory mechanisms dictating cellular responses to heavy metal stress, however, remains incomplete. In a previous work, the S. oneidensis so2426 gene, annotated as a DNA-binding response regulator, was demonstrated to be specifically responsive at both the transcript and protein levels to acute chromate [Cr(VI)] challenge. To delineate the cellular function of SO2426 and its contribution to metal stress response, we integrated genetic and physiological approaches with a genome-wide screen for target gene candidates comprising the SO2426 regulon. Keywords: Time course
Project description:BACKGROUND: Although solid surface-associated biofilm development of S. oneidensis has been extensively studied in recent years, pellicles formed at the air-liquid interface are largely overlooked. The goal of this work was to understand basic requirements and mechanism of pellicle formation in S. oneidensis. RESULTS: We demonstrated that pellicle formation can be completed when oxygen and certain cations were present. Ca(II), Mn(II), Cu(II), and Zn(II) were essential for the process evidenced by fully rescuing pellicle formation of S. oneidensis from the EDTA treatment while Mg (II), Fe(II), and Fe(III) were much less effective. Proteins rather than DNA were crucial in pellicle formation and the major exopolysaccharides may be rich in mannose. Mutational analysis revealed that flagella were not required for pellicle formation but flagellum-less mutants delayed pellicle development substantially, likely due to reduced growth in static media. The analysis also demonstrated that AggA type I secretion system was essential in formation of pellicles but not of solid surface-associated biofilms in S. oneidensis. CONCLUSION: This systematic characterization of pellicle formation shed lights on our understanding of biofilm formation in S. oneidensis and indicated that the pellicle may serve as a good research model for studying bacterial communities.
Project description:Biofilms, or surface-attached microbial communities, are both ubiquitous and resilient in the environment. Although much is known about how biofilms form, develop, and detach, very little is understood about how these events are related to metabolism and its dynamics. It is commonly thought that large subpopulations of cells within biofilms are not actively producing proteins or generating energy and are therefore dead. An alternative hypothesis is that within the growth-inactive domains of biofilms, significant populations of living cells persist and retain the capacity to dynamically regulate their metabolism. To test this, we employed unstable fluorescent reporters to measure growth activity and protein synthesis in vivo over the course of biofilm development and created a quantitative routine to compare domains of activity in independently grown biofilms. Here we report that Shewanella oneidensis biofilm structures reproducibly stratify with respect to growth activity and metabolism as a function of size. Within domains of growth-inactive cells, genes typically upregulated under anaerobic conditions are expressed well after growth has ceased. These findings reveal that, far from being dead, the majority of cells in mature S. oneidensis biofilms have actively turned-on metabolic programs appropriate to their local microenvironment and developmental stage.
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:The physiology and transcriptome dynamics of the metal ion-reducing bacterium Shewanella oneidensis strain MR-1 in response to nonradioactive strontium (Sr) exposure were investigated. Studies indicated that MR-1 was able to grow aerobically in complex medium in the presence of 180 mM SrCl2 but showed severe growth inhibition at levels above that concentration. Temporal gene expression profiles were generated from aerobically grown, mid-exponential-phase MR-1 cells shocked with 180 mM SrCl2 and analyzed for significant differences in mRNA abundance with reference to data for nonstressed MR-1 cells. Genes with annotated functions in siderophore biosynthesis and iron transport were among the most highly induced (>100-fold [P < 0.05]) open reading frames in response to acute Sr stress, and a mutant (SO3032::pKNOCK) defective in siderophore production was found to be hypersensitive to SrCl2 exposure, compared to parental and wild-type strains. Transcripts encoding multidrug and heavy metal efflux pumps, proteins involved in osmotic adaptation, sulfate ABC transporters, and assimilative sulfur metabolism enzymes also were differentially expressed following Sr exposure but at levels that were several orders of magnitude lower than those for iron transport genes. Precipitate formation was observed during aerobic growth of MR-1 in broth cultures amended with 50, 100, or 150 mM SrCl2 but not in cultures of the SO3032::pKNOCK mutant or in the abiotic control. Chemical analysis of this precipitate using laser-induced breakdown spectroscopy and static secondary ion mass spectrometry indicated extracellular solid-phase sequestration of Sr, with at least a portion of the heavy metal associated with carbonate phases.
Project description:Shewanella oneidensis couples oxidation of lactate to respiration of many substrates. Here we report that llpR (L-lactate-positive regulator, SO_3460) encodes a positive regulator of L-lactate utilization distinct from previously studied regulators. We also demonstrate D-lactate inhibition of L-lactate utilization in S. oneidensis, resulting in preferential utilization of the D isomer.
Project description:Shewanella oneidensis is a highly motile organism by virtue of a polar, glycosylated flagellum composed of flagellins FlaA and FlaB. In this study, the functional flagellin FlaB was isolated and analyzed with nano-liquid chromatography-mass spectrometry (MS) and tandem MS. In combination with the mutational analysis, we propose that the FlaB flagellin protein from S. oneidensis is modified at five serine residues with a series of novel O-linked posttranslational modifications (PTMs) that differ from each other by 14 Da. These PTMs are composed in part of a 274-Da sugar residue that bears a resemblance to the nonulosonic acids. The remainder appears to be composed of a second residue whose mass varies by 14 Da depending on the PTM. Further investigation revealed that synthesis of the glycans initiates with PseB and PseC, the first two enzymes of the Pse pathway. In addition, a number of lysine residues are found to be methylated by SO4160, an analogue of the lysine methyltransferase of Salmonella enterica serovar Typhimurium.