Eliciting the impacts of cellular noise on metabolic trade-offs by quantitative mass imaging.

Optimal metabolic trade-offs between growth and productivity are key constraints in strain optimization by metabolic engineering; however, how cellular noise impacts these trade-offs and drives the emergence of subpopulations with distinct resource allocation strategies, remains largely unknown. Here, we introduce a single-cell strategy for quantifying the trade-offs between triacylglycerol production and growth in the oleaginous microorganism Yarrowia lipolytica. The strategy relies on high-throughput quantitative-phase imaging and, enabled by nanoscale secondary ion mass spectrometry analyses and dedicated image processing, allows us to image how resources are partitioned between growth and productivity. Enhanced precision over population-averaging biotechnologies and conventional microscopy demonstrates how cellular noise impacts growth and productivity differently. As such, subpopulations with distinct metabolic trade-offs emerge, with notable impacts on strain performance and robustness. By quantifying the self-degradation of cytosolic macromolecules under nutrient-limiting conditions, we discover the cell-to-cell heterogeneity in protein and fatty-acid recycling, unmasking a potential bet-hedging strategy under starvation.

Pyrobaculum yellowstonensis Strain WP30 Respires on Elemental Sulfur and/or Arsenate in Circumneutral Sulfidic Geothermal Sediments of Yellowstone National Park.

Thermoproteales (phylum Crenarchaeota) populations are abundant in high-temperature (>70°C) environments of Yellowstone National Park (YNP) and are important in mediating the biogeochemical cycles of sulfur, arsenic, and carbon. The objectives of this study were to determine the specific physiological attributes of the isolate Pyrobaculum yellowstonensis strain WP30, which was obtained from an elemental sulfur sediment (Joseph's Coat Hot Spring [JCHS], 80°C, pH 6.1, 135 µM As) and relate this organism to geochemical processes occurring in situ. Strain WP30 is a chemoorganoheterotroph and requires elemental sulfur and/or arsenate as an electron acceptor. Growth in the presence of elemental sulfur and arsenate resulted in the formation of thioarsenates and polysulfides. The complete genome of this organism was sequenced (1.99 Mb, 58% G+C content), revealing numerous metabolic pathways for the degradation of carbohydrates, amino acids, and lipids. Multiple dimethyl sulfoxide-molybdopterin (DMSO-MPT) oxidoreductase genes, which are implicated in the reduction of sulfur and arsenic, were identified. Pathways for the de novo synthesis of nearly all required cofactors and metabolites were identified. The comparative genomics of P. yellowstonensis and the assembled metagenome sequence from JCHS showed that this organism is highly related (∼95% average nucleotide sequence identity) to in situ populations. The physiological attributes and metabolic capabilities of P. yellowstonensis provide an important foundation for developing an understanding of the distribution and function of these populations in YNP.

METABOLIC SPATIAL VARIABILITY IN ELECTRODE-RESPIRING GEOBACTER SULFURREDUCENS BIOFILMS.

In this study, we quantified electron transfer rates, depth profiles of electron donor, and biofilm structure of Geobacter sulfurreducens biofilms using an electrochemical-nuclear magnetic resonance microimaging biofilm reactor. Our goal was to determine whether electron donor limitations existed in electron transfer processes of electrode-respiring G. sulfurreducens biofilms. Cells near the top of the biofilms consumed acetate and were metabolically active; however, acetate concentration decreased to below detection within the top 100 microns of the biofilms. Additionally, porosity in the biofilms fell below 10% near the electrode surface, exacerbating exclusion of acetate from the lower regions. The dense biofilm matrix in the acetate-depleted zone acted as an electrical conduit passing electrons generated at the top of the biofilm to the electrode. To verify the distribution of cell metabolic activity, we used uranium as a redox-active probe for localizing electron transfer activity and X-ray absorption spectroscopy to determine the uranium oxidation state. Cells near the top reduced UVI more actively than the cells near the base. High-resolution transmission electron microscopy images showed intact, healthy cells near the top and plasmolyzed cells near the base. Contrary to models proposed in the literature, which hypothesize that cells nearest the electrode surface are the most metabolically active because of a lower electron transfer resistance, our results suggest that electrical resistance through the biofilm does not restrict long-range electron transfer. Cells far from the electrode can respire across metabolically inactive cells, taking advantage of their extracellular infrastructure produced during the initial biofilm formation.

Defining cell culture conditions to improve human norovirus infectivity assays.

Significant difficulties remain for determining whether human noroviruses (hNoV) recovered from water, food, and environmental samples are infectious. Three-dimensional (3-D) tissue culture of human intestinal cells has shown promise in developing an infectivity assay, but reproducibility, even within a single laboratory, remains problematic. From the literature and our observations, we hypothesized that the common factors that lead to more reproducible hNoV infectivity in vitro requires that the cell line be (1) of human gastrointestinal origin, (2) expresses apical microvilli, and (3) be a positive secretor cell line. The C2BBe1 cell line, which is a brush-border producing clone of Caco-2, meets these three criteria. When challenged with Genogroup II viruses, we observed a 2 Log(10) increase in viral RNA titer. A passage experiment with GII viruses showed evidence of the ability to propagate hNoV by both quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microscopy. In our hands, using 3-D C2BBe1 cells improves reproducibility of the infectivity assay for hNoV, but the assay can still be variable. Two sources of variability include the cells themselves (mixed phenotypes of small and large intestine) and initial titer measurements using qRT-PCR that measures all RNA vs. plaque assays that measure infectious virus.

Role of outer-membrane cytochromes MtrC and OmcA in the biomineralization of ferrihydrite by Shewanella oneidensis MR-1.

In an effort to improve the understanding of electron transfer mechanisms at the microbe-mineral interface, Shewanella oneidensis MR-1 mutants with in-frame deletions of outer-membrane cytochromes (OMCs), MtrC and OmcA, were characterized for the ability to reduce ferrihydrite (FH) using a suite of microscopic, spectroscopic, and biochemical techniques. Analysis of purified recombinant proteins demonstrated that both cytochromes undergo rapid electron exchange with FH in vitro with MtrC displaying faster transfer rates than OmcA. Immunomicroscopy with cytochrome-specific antibodies revealed that MtrC co-localizes with iron solids on the cell surface while OmcA exhibits a more diffuse distribution over the cell surface. After 3-day incubation of MR-1 with FH, pronounced reductive transformation mineral products were visible by electron microscopy. Upon further incubation, the predominant phases identified were ferrous phosphates including vivianite [Fe(3)(PO(4))(2)x8H(2)O] and a switzerite-like phase [Mn(3),Fe(3)(PO(4))(2)x7H(2)O] that were heavily colonized by MR-1 cells with surface-exposed outer-membrane cytochromes. In the absence of both MtrC and OmcA, the cells ability to reduce FH was significantly hindered and no mineral transformation products were detected. Collectively, these results highlight the importance of the outer-membrane cytochromes in the reductive transformation of FH and support a role for direct electron transfer from the OMCs at the cell surface to the mineral.

Alkaliphilus transvaalensis gen. nov., sp. nov., an extremely alkaliphilic bacterium isolated from a deep South African gold mine.

A novel extreme alkaliphile was isolated from a mine water containment dam at 3.2 km below land surface in an ultra-deep gold mine near Carletonville, South Africa. The cells of this bacterium were straight to slightly curved rods, motile by flagella and formed endospores. Growth was observed over the temperature range 20-50 degrees C (optimum 40 degrees C; 45 min doubling time) and pH range 8.5-12.5 (optimum pH 10.0). The novel isolate, one of the most alkaliphilic micro-organisms yet described, was a strictly anaerobic chemo-organotroph capable of utilizing proteinaceous substrates such as yeast extract, peptone, tryptone and casein. Elemental sulfur, thiosulfate or fumarate, when included as accessory electron acceptors, improved growth. The G+C content of genomic DNA was 36.4 mol %. Phylogenetic analysis based on the 16S rDNA sequence indicated that the isolate is a member of cluster XI within the low G+C gram-positive bacteria, but only distantly related to previously described members. On the basis of physiological and molecular properties, the isolate represents a novel species, for which the name Alkaliphilus transvaalensis gen. nov., sp. nov. is proposed (type strain SAGM1T = JCM 10712T = ATCC 700919T). The mechanism of generation of the highly alkaline microbial habitat and the possible source of the alkaliphile are discussed.