Microbiology & Cell Biology
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Item Contribution of wild foods to diet, food security, and cultural values amidst climate change(2019-11) Smith, Erin; Ahmed, Selena; Running Crane, MaryAnn; Eggers, Margaret J.; Pierre, Mike; Flagg, Kenneth A.; Byker Shanks, CarmenWild foods are recognized to contribute to diet and food security through enhancing the availability of local, diverse, and nonmarket food sources. We investigated the contribution of wild foods to diet, food security, and cultural identity in a Native American[1] community in the context of climate change. Structured interviews were conducted with low-income residents of the Flathead Indian Reservation[2] in Northwestern Montana who participate in the federal Food Distribution Program on Indian Reservations, also known by participants as ‘Commodities.’ Responses to structured questions were analyzed for frequency, and open-ended responses were coded and analyzed to identify prevalent themes. Our analysis indicated that half of participants were food insecure. Approximately 28% of participants engaged in at least one wild food procurement activity, including hunting, fishing, and harvesting. On average, participants who engaged in one or more wild food procurement activities were more food secure than those who did not. Results highlight the multidimensional valuation of wild foods by participants including taste, freshness, nutritional quality, being a traditional community practice, and providing a sense of self-sufficiency. Climate change is perceived by participants to be adversely impacting wild food systems due to increased variability in seasonality and precipitation and increased incidences of wild fire. Findings point to the need for community-based strategies to strengthen wild food knowledge toward enhancing food sovereignty in Native American communities, in the context of climate change. [1] The term ‘Native American’ was determined to be the preferred term for referencing the Native American community in this study, based on consultation from our community advisory board. [2] The term ‘Flathead Indian Reservation’ was determined to be the preferred term for referencing the location in which this study was held, based on consultation from our community advisory board.Item DropSOAC: Stabilizing Microfluidic Drops for Time-Lapse Quantification of Single-Cell Bacterial Physiology(2019-09) Pratt, Shawna L.; Zath, Geoffrey K.; Williamson, Kelly S.; Franklin, Michael J.; Chang, Connie B.The physiological heterogeneity of cells within a microbial population imparts resilience to stresses such as antimicrobial treatments and nutrient limitation. This resilience is partially due to a subpopulation of cells that can survive such stresses and regenerate the community. Microfluidic approaches now provide a means to study microbial physiology and bacterial heterogeneity at the single cell level, improving our ability to isolate and examine these subpopulations. Drop-based microfluidics provides a high-throughput approach to study individual cell physiology within bacterial populations. Using this approach, single cells are isolated from the population and encapsulated in growth medium dispersed in oil using a 15 μm diameter drop making microfluidic device. The drops are arranged as a packed monolayer inside a polydimethylsiloxane (PDMS) microfluidic device. Growth of thousands of individual cells in identical microenvironments can then be imaged using confocal laser scanning microscopy (CLSM). A challenge for this approach has been the maintenance of drop stability during extended time-lapse imaging. In particular, the drops do not maintain their volume over time during incubation in PDMS devices, due to fluid transport into the porous PDMS surroundings. Here, we present a strategy for PDMS device preparation that stabilizes drop position and volume within a drop array on a microfluidic chip for over 20 h. The stability of water-in-oil drops is maintained by soaking the device in a reservoir containing both water and oil in thermodynamic equilibrium. This ensures that phase equilibrium of the drop emulsion fluids within the porous PDMS material is maintained during drop incubation and imaging. We demonstrate the utility of this approach, which we label DropSOAC (DropStabilization On AChip), for time-lapse studies of bacterial growth. We characterize growth of Pseudomonas aeruginosa and its Δhpf mutant derivative during resuscitation and growth following starvation. We demonstrate that growth rate and lag time heterogeneity of hundreds of individual bacterial cells can be determined starting from single isolated cells. The results show that the DropSOAC capsule provides a high-throughput approach toward studies of microbial physiology at the single cell level, and can be used to characterize physiological differences of cells from within a larger population.Item Janthinobacterium CG23_2: comparative genome analysis reveals enhanced environmental sensing and transcriptional regulation for adaptation to life in an Antarctic supraglacial stream(2019-10) Dieser, Markus; Smith, Heidi J.; Ramaraj, Thiruvarangan; Foreman, Christine M.As many bacteria detected in Antarctic environments are neither true psychrophiles nor endemic species, their proliferation in spite of environmental extremes gives rise to genome adaptations. Janthinobacterium sp. CG23_2 is a bacterial isolate from the Cotton Glacier stream, Antarctica. To understand how Janthinobacterium sp. CG23_2 has adapted to its environment, we investigated its genomic traits in comparison to genomes of 35 published Janthinobacterium species. While we hypothesized that genome shrinkage and specialization to narrow ecological niches would be energetically favorable for dwelling in an ephemeral Antarctic stream, the genome of Janthinobacterium sp. CG23_2 was on average 1.7 ± 0.6 Mb larger and predicted 1411 ± 499 more coding sequences compared to the other Janthinobacterium spp. Putatively identified horizontal gene transfer events contributed 0.92 Mb to the genome size expansion of Janthinobacterium sp. CG23_2. Genes with high copy numbers in the species-specific accessory genome of Janthinobacterium sp. CG23_2 were associated with environmental sensing, locomotion, response and transcriptional regulation, stress response, and mobile elements—functional categories which also showed molecular adaptation to cold. Our data suggest that genome plasticity and the abundant complementary genes for sensing and responding to the extracellular environment supported the adaptation of Janthinobacterium sp. CG23_2 to this extreme environment.Item Long-Term Flow through Human Intestinal Organoids with the Gut Organoid Flow Chip (GOFlowChip)(2019-09) Sidar, Barkan; Jenkins, Brittany R.; Huang, Sha; Spence, Jason R.; Walk, Seth T.Human intestinal organoids (HIOs) are millimeter-scale models of the human intestinal epithelium and hold tremendous potential for advancing fundamental and applied biomedical research. HIOs resemble the native gut in that they consist of a fluid-filled lumen surrounded by a polarized epithelium and associated mesenchyme; however, their topologically-closed, spherical shape prevents flow through the interior luminal space, making the system less physiological and leading to the buildup of cellular and metabolic waste. These factors ultimately limit experimentation inside the HIOs. Here, we present a millifluidic device called the gut organoid flow chip (GOFlowChip), which we use to “port” HIOs and establish steady-state liquid flow through the lumen for multiple days. This long-term flow is enabled by the use of laser-cut silicone gaskets, which allow liquid in the device to be slightly pressurized, suppressing bubble formation. To demonstrate the utility of the device, we establish separate luminal and extraluminal flow and use luminal flow to remove accumulated waste. This represents the first demonstration of established liquid flow through the luminal space of a gastrointestinal organoid over physiologically relevant time scales. Flow cytometry results reveal that HIO cell viability is unaffected by long-term porting and luminal flow. We expect the real-time, long-term control over luminal and extraluminal contents provided by the GOFlowChip will enable a wide variety of studies including intestinal secretion, absorption, transport, and co-culture with intestinal microorganisms.Item A Novel Gastric Spheroid Co-culture Model Reveals Chemokine-Dependent Recruitment of Human Dendritic Cells to the Gastric Epithelium(2019-03) Sebrell, Thomas A.; Hashimi, Marziah; Sidar, Barkan; Wilkinson, Royce A.; Kirpotina, Liliya; Quinn, Mark T.; Malkoc, Zeynep; Taylor, Paul J.; Wilking, James N.; Bimczok, DianeBackground & Aims Gastric dendritic cells (DCs) control the adaptive response to infection with Helicobacter pylori, a major risk factor for peptic ulcer disease and gastric cancer. We hypothesize that DC interactions with the gastric epithelium position gastric DCs for uptake of luminal H pylori and promote DC responses to epithelial-derived mediators. The aim of this study was to determine whether the gastric epithelium actively recruits DCs using a novel co-culture model of human gastric epithelial spheroids and monocyte-derived DCs. Methods Spheroid cultures of primary gastric epithelial cells were infected with H pylori by microinjection. Co-cultures were established by adding human monocyte-derived DCs to the spheroid cultures and were analyzed for DC recruitment and antigen uptake by confocal microscopy. Protein array, gene expression polymerase chain reaction array, and chemotaxis assays were used to identify epithelial-derived chemotactic factors that attract DCs. Data from the co-culture model were confirmed using human gastric tissue samples. Results Human monocyte-derived DCs co-cultured with gastric spheroids spontaneously migrated to the gastric epithelium, established tight interactions with the epithelial cells, and phagocytosed luminally applied H pylori. DC recruitment was increased upon H pylori infection of the spheroids and involved the activity of multiple chemokines including CXCL1, CXCL16, CXCL17, and CCL20. Enhanced chemokine expression and DC recruitment to the gastric epithelium also was observed in H pylori–infected human gastric tissue samples. Conclusions Our results indicate that the gastric epithelium actively recruits DCs for immunosurveillance and pathogen sampling through chemokine-dependent mechanisms, with increased recruitment upon active H pylori infection.Item Competitive resource allocation to metabolic pathways contributes to overflow metabolisms and emergent properties in cross-feeding microbial consortia(2018-04) Carlson, Ross P.; Beck, Ashley E.; Phalak, Poonam; Fields, Matthew W.; Gedeon, Tomas; Hanley, Luke; Harcombe, W. R.; Henson, Michael A.; Heys, Jeffrey J.Resource scarcity is a common stress in nature and has a major impact on microbial physiology. This review highlights microbial acclimations to resource scarcity, focusing on resource investment strategies for chemoheterotrophs from the molecular level to the pathway level. Competitive resource allocation strategies often lead to a phenotype known as overflow metabolism; the resulting overflow byproducts can stabilize cooperative interactions in microbial communities and can lead to cross-feeding consortia. These consortia can exhibit emergent properties such as enhanced resource usage and biomass productivity. The literature distilled here draws parallels between in silico and laboratory studies and ties them together with ecological theories to better understand microbial stress responses and mutualistic consortia functioning.Item Draft genome sequence and description of Janthinobacterium sp. strain CG3, a psychrotolerant antarctic Supraglacial stream bacterium(2013-11) Smith, Heidi J.; Akiyama, Tatsuya; Foreman, Christine M.; Franklin, Michael J.; Woyke, Tanja; Teshima, H; Davenport, K.; Daligault, H.; Erkkila, T.; Goodwin, L. A.; Gu, W.; Xu, Yan; Chain, P. S.Here we present the draft genome sequence of Janthinobacterium sp. strain CG3, a psychrotolerant non-violacein-producing bacterium that was isolated from the Cotton Glacier supraglacial stream. The genome sequence of this organism will provide insight into the mechanisms necessary for bacteria to survive in UV-stressed icy environments.Item Microbes in mercury-enriched geothermal springs in western North America(2016-11) Geesey, Gill G.; Barkay, Tamar; King, SueBecause geothermal environments contain mercury (Hg) from natural sources, microorganisms that evolved in these systems have likely adapted to this element. Knowledge of the interactions between microorganisms and Hg in geothermal systems may assist in understanding the long-term evolution of microbial adaptation to Hg with relevance to other environments where Hg is introduced from anthropogenic sources. A number of microbiological studies with supporting geochemistry have been conducted in geothermal systems across western North America. Approximately 1 in 5 study sites include measurements of Hg. Of all prokaryotic taxa reported across sites with microbiological and accompanying physicochemical data, 42% have been detected at sites in which Hg was measured. Genes specifying Hg reduction and detoxification by microorganisms were detected in a number of hot springs across the region. Archaeal-like sequences, representing two crenarchaeal orders and one order each of the Euryarchaeota and Thaumarchaeota, dominated in metagenomes' MerA (the mercuric reductase protein) inventories, while bacterial homologs were mostly found in one deeply sequenced metagenome. MerA homologs were more frequently found in metagenomes of microbial communities in acidic springs than in circumneutral or high pH geothermal systems, possibly reflecting higher bioavailability of Hg under acidic conditions. MerA homologs were found in hot springs prokaryotic isolates affiliated with Bacteria and Archaea taxa. Acidic sites with high Hg concentrations contain more of Archaea than Bacteria taxa, while the reverse appears to be the case in circumneutral and high pH sites with high Hg concentrations. However, MerA was detected in only a small fraction of the Archaea and Bacteria taxa inhabiting sites containing Hg. Nevertheless, the presence of MerA homologs and their distribution patterns in systems, in which Hg has yet to be measured, demonstrates the potential for detoxification by Hg reduction in these geothermal systems, particularly the low pH springs that are dominated by Archaea.Item Mineral formation during bacterial sulfate reduction in the presence of different electron donors and carbon sources(2016-04) Han, Xiqiu; Schultz, Logan N.; Zhang, Weiyan; Zhu, Jihao; Meng, Fanxu; Geesey, Gill G.Sulfate-reducing bacteria have long been known to promote mineral precipitation. However, the influence of electron donors (energy sources) and carbon sources on the minerals formed during sulfate reduction is less well understood. An investigation was therefore undertaken to determine how these nutrients affect sulfate reduction by the bacterium Desulfovibrio alaskensis G20 in a marine sediment pore water medium. Monohydrocalcite and a small amount of calcite formed during sulfate reduction with formate as the electron donor; Mg-phosphates and calcite precipitated when hydrogen served as the electron donor and when acetate and dissolved inorganic carbon served as carbon sources; and greigite and elemental sulfur were deposited when lactate was used as the electron donor and carbon source. The experimental results were generally consistent with geochemical modeling, suggesting that it may be possible to predict the processes and conditions during formation of these minerals in natural environments.Item A Lipid-Accumulating Alga Maintains Growth in Outdoor, Alkaliphilic Raceway Pond with Mixed Microbial Communities(2016-01) Bell, Tisza A. S.; Prithiviraj, Bharath; Wahlen, Brad D.; Fields, Matthew W.; Peyton, Brent M.Algal biofuels and valuable co-products are being produced in both open and closed cultivation systems. Growing algae in open pond systems may be a more economical alternative, but this approach allows environmental microorganisms to colonize the pond and potentially infect or outcompete the algal “crop.” In this study, we monitored the microbial community of an outdoor, open raceway pond inoculated with a high lipid-producing alkaliphilic alga, Chlorella vulgaris BA050. The strain C. vulgaris BA050 was previously isolated from Soap Lake, Washington, a system characterized by a high pH (∼9.8). An outdoor raceway pond (200 L) was inoculated with C. vulgaris and monitored for 10 days and then the culture was transferred to a 2,000 L raceway pond and cultivated for an additional 6 days. Community DNA samples were collected over the 16-day period in conjunction with water chemistry analyses and cell counts. Universal primers for the SSU rRNA gene sequences for Eukarya, Bacteria, and Archaea were used for barcoded pyrosequence determination. The environmental parameters that most closely correlated with C. vulgaris abundance were pH and phosphate. Community analyses indicated that the pond system remained dominated by the Chlorella population (93% of eukaryotic sequences), but was also colonized by other microorganisms. Bacterial sequence diversity increased over time while archaeal sequence diversity declined over the same time period. Using SparCC co-occurrence network analysis, a positive correlation was observed between C. vulgaris and Pseudomonas sp. throughout the experiment, which may suggest a symbiotic relationship between the two organisms. The putative relationship coupled with high pH may have contributed to the success of C. vulgaris. The characterization of the microbial community dynamics of an alkaliphilic open pond system provides significant insight into open pond systems that could be used to control photoautotrophic biomass productivity in an open, non-sterile environment.