Scholarly Work - Center for Biofilm Engineering
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Item Robustness analysis of culturing perturbations on Escherichia coli colony biofilm beta-lactam and aminoglycoside antibiotic tolerance(2010-07) Zuroff, Trevor R.; Bernstein, Hans C.; Lloyd-Randolfi, J.; Jimenez-Taracido, L.; Stewart, Philip S.; Carlson, Ross P.BACKGROUND: Biofilms are ubiquitous. For instance, the majority of medical infections are thought to involve biofilms. However even after decades of investigation, the in vivo efficacy of many antimicrobial strategies is still debated, suggesting there is a need for better understanding of biofilm antimicrobial tolerances. The current study's goal is to characterize the robustness of biofilm antibiotic tolerance to medically and industrially relevant culturing perturbations. By definition, robust systems will return similar, predictable responses when perturbed, while non-robust systems will return very different, and potentially unpredictable, responses. The predictability of an antibiotic tolerance response is essential to developing, testing, and employing antimicrobial strategies. RESULTS: The antibiotic tolerance of Escherichia coli colony biofilms was tested against beta-lactam and aminoglycoside class antibiotics. Control scenario tolerances were compared to tolerances under culturing perturbations including 1) different nutritional environments 2) different temperatures 3) interruption of cellular quorum sensing and 4) different biofilm culture ages. Here, antibiotic tolerance was defined in terms of culturable biofilm cells recovered after a twenty four hour antibiotic treatment.Colony biofilm antibiotic tolerances were not robust to perturbations. Altering basic culturing parameters like nutritional environment or temperature resulted in very different, non-intuitive antibiotic tolerance responses. Some minor perturbations—like increasing the glucose concentration from 0.1 to 1 g/L—caused a ten-million fold difference in culturable cells over a twenty four hour antibiotic treatment. CONCLUSIONS: The current study presents a basis for robustness analysis of biofilm antibiotic tolerance. Biofilm antibiotic tolerance can vary in unpredictable manners based on modest changes in culturing conditions. Common antimicrobial testing methods, which only consider a single culturing condition, are not desirable since slight culturing variations can lead to very different outcomes. The presented data suggest it is essential to test antimicrobial strategies over a range of culturing perturbations relevant to the targeted application. In addition, the highly dynamic antibiotic tolerance responses observed here may explain why some current antimicrobial strategies occasionally fail.Item Molecular-level tradeoffs and metabolic adaptation to simultaneous stressors(2010-10) Carlson, Ross P.; Taffs, Reed L.Life is a dynamic process driven by the complex interplay between physical constraints and selection pressures, ranging from nutrient limitation to inhibitory substances to predators. These stressors are not mutually exclusive; microbes have faced concurrent challenges for eons. Genome-enabled systems biology approaches are adapting economic and ecological concepts like tradeoff curves and strategic resource allocation theory to analyze metabolic adaptations to simultaneous stressors. These methodologies can accurately describe and predict metabolic adaptations to concurrent stresses by considering the tradeoff between investment of limiting resources into enzymatic machinery and the resulting cellular function. The approaches represent promising links between computational biology and well-established economic and ecological methodologies for analyzing the interplay between physical constraints and microbial fitness.Item Synthetic Escherichia coli consortia engineered for syntrophy demonstrate enhanced biomass productivity(2012-01) Bernstein, Hans C.; Paulson, S. D.; Carlson, Ross P.Synthetic Escherichia coli consortia engineered for syntrophy demonstrated enhanced biomass productivity relative to monocultures. Binary consortia were designed to mimic a ubiquitous, naturally occurring ecological template of primary productivity supported by secondary consumption. The synthetic consortia replicated this evolution-proven strategy by combining a glucose positive E. coli strain, which served as the system's primary producer, with a glucose negative E. coli strain which consumed metabolic byproducts from the primary producer. The engineered consortia utilized strategic division of labor to simultaneously optimize multiple tasks enhancing overall culture performance. Consortial interactions resulted in the emergent property of enhanced system biomass productivity which was demonstrated with three distinct culturing systems: batch, chemostat and biofilm growth. Glucose-based biomass productivity increased by ~15, 20 and 50% compared to appropriate monoculture controls for these three culturing systems, respectively. Interestingly, the consortial interactions also produced biofilms with predictable, self-assembling, laminated microstructures. This study establishes a metabolic engineering paradigm which can be easily adapted to existing E. coli based bioprocesses to improve productivity based on a robust ecological themeItem Resolution of volatile fuel compound profiles from Ascocoryne sarcoides: A comparison by proton transfer reaction-mass spectrometry and solid phase microextraction gas chromatography mass spectrometry(2012-04) Mallette, Natasha D.; Knighton, W. Berk; Strobel, Gary A.; Carlson, Ross P.; Peyton, Brent M.Volatile hydrocarbon production by Ascocoryne sacroides was studied over its growth cycle. Gas-phase compounds were measured continuously with a proton transfer reaction-mass spectrometry (PTR-MS) and at distinct time points with gas chromatography-mass spectrometry (GC-MS) using head space solid phase microextraction (SPME). The PTR-MS ion signal permitted temporal resolution of the volatile production while the SPME results revealed distinct compound identities. The quantitative PTR-MS results showed the volatile production was dominated by ethanol and acetaldehyde, while the concentration of the remainder of volatiles consistently reached 2,000 ppbv. The measurement of alcohols from the fungal culture by the two techniques correlated well. Notable compounds of fuel interest included nonanal, 1-octen-3-ol, 1-butanol, 3-methyl- and benzaldehyde. Abiotic comparison of the two techniques demonstrated SPME fiber bias toward higher molecular weight compounds, making quantitative efforts with SPME impractical. Together, PTR-MS and SPME GC-MS were shown as valuable tools for characterizing volatile fuel compound production from microbiological sources.Item Use of sodium bicarbonate to stimulate triacylglycerol accumulation in the chlorophyte Scenedesmus sp. and the diatom Phaeodactylum tricornutum(2012-10) Gardner, Robert D.; Cooksey, Keith E.; Mus, Florence; Macur, Richard E.; Moll, Karen M.; Eustance, E. O.; Carlson, Ross P.; Gerlach, Robin; Fields, Matthew W.; Peyton, Brent M.There is potential for algal-derived biofuel to help alleviate part of the world’s dependency on petroleum based fuels. However, research must still be done on strain selection, induction of triacylglycerol (TAG) accumulation, and fundamental algal metabolic studies, along with large-scale culturing techniques, harvesting, and biofuel/biomass processing. Here, we have advanced the knowledge on Scenedesmus sp. strain WC-1 by monitoring growth, pH, and TAG accumulation on a 14:10 light–dark cycle with atmospheric air or 5% CO2 in air (v/v) aeration. Under ambient aeration, there was a loss of pH-induced TAG accumulation, presumably due to TAG consumption during the lower culture pH observed during dark hours (pH 9.4). Under 5% CO2 aeration, the growth rate nearly doubled from 0.78 to 1.53 d−1, but the pH was circumneutral (pH 6.9) and TAG accumulation was minimal. Experiments were also performed with 5% CO2 during the exponential growth phase, which was then switched to aeration with atmospheric air when nitrate was close to depletion. These tests were run with and without the addition of 50 mM sodium bicarbonate. Cultures without added bicarbonate showed decreased growth rates with the aeration change, but there was no immediate TAG accumulation. The cultures with bicarbonate added immediately ceased cellular replication and rapid TAG accumulation was observed, as monitored by Nile Red fluorescence which has previously been correlated by gas chromatography to cellular TAG levels. Sodium bicarbonate addition (25 mM final concentration) was also tested with the marine diatom Phaeodactylum tricornutum strain Pt-1 and this organism also accumulated TAG.Item Microbial consortia engineering for cellular factories: In vitro to in silico systems(2012-10) Bernstein, Hans C.; Carlson, Ross P.This mini-review discusses the current state of experimental and computational microbial consortia engineering with a focus on cellular factories. A discussion of promising ecological theories central to community resource usage is presented to facilitate interpretation of consortial designs. Recent case studies exemplifying different resource usage motifs and consortial assembly templates are presented. The review also highlights in silico approaches to design and to analyze consortia with an emphasis on stoichiometric modeling methods. The discipline of microbial consortia engineering possesses a widely accepted potential to generate highly novel and effective bio-catalysts for applications from biofuels to specialty chemicals to enhanced mineral recovery.Item Potential role of multiple carbon fixation pathways during lipid accumulation in Phaeodactylum tricornutum(2012-06) Valenzuela, Jacob J.; Mazurie, Aurélien J.; Carlson, Ross P.; Gerlach, Robin; Cooksey, Keith E.; Peyton, Brent M.; Fields, Matthew W.BACKGROUND: Phaeodactylum tricornutum is a unicellular diatom in the class Bacillariophyceae. The full genome hasbeen sequenced (<30 Mb), and approximately 20 to 30% triacylglyceride (TAG) accumulation on a dry cell basis hasbeen reported under different growth conditions. To elucidate P. tricornutum gene expression profiles duringnutrient-deprivation and lipid-accumulation, cell cultures were grown with a nitrate to phosphate ratio of 20:1 (N:P)and whole-genome transcripts were monitored over time via RNA-sequence determination.RESULTS: The specific Nile Red (NR) fluorescence (NR fluorescence per cell) increased over time; however, theincrease in NR fluorescence was initiated before external nitrate was completely exhausted. Exogenous phosphatewas depleted before nitrate, and these results indicated that the depletion of exogenous phosphate might be anearly trigger for lipid accumulation that is magnified upon nitrate depletion. As expected, many of the genesassociated with nitrate and phosphate utilization were up-expressed. The diatom-specific cyclins cyc7 and cyc10were down-expressed during the nutrient-deplete state, and cyclin B1 was up-expressed during lipid-accumulationafter growth cessation. While many of the genes associated with the C3 pathway for photosynthetic carbonreduction were not significantly altered, genes involved in a putative C4 pathway for photosynthetic carbonassimilation were up-expressed as the cells depleted nitrate, phosphate, and exogenous dissolved inorganic carbon(DIC) levels. P. tricornutum has multiple, putative carbonic anhydrases, but only two were significantly up-expressed(2-fold and 4-fold) at the last time point when exogenous DIC levels had increased after the cessation of growth.Alternative pathways that could utilize HCO-3 were also suggested by the gene expression profiles (e.g., putativepropionyl-CoA and methylmalonyl-CoA decarboxylases).CONCLUSION: The results indicate that P. tricornutum continued carbon dioxide reduction when population growthwas arrested and different carbon-concentrating mechanisms were used dependent upon exogenous DIC levels.Based upon overall low gene expression levels for fatty acid synthesis, the results also suggest that the build-up ofprecursors to the acetyl-CoA carboxylases may play a more significant role in TAG synthesis rather than the actualenzyme levels of acetyl-CoA carboxylases per se. The presented insights into the types and timing of cellularresponses to inorganic carbon will help maximize photoautotrophic carbon flow to lipid accumulation.Item Identification and imaging of peptides and proteins on Enterococcus faecalis biofilms by matrix assisted laser desportion ionization mass spectrometry(2012-09) Blaze, M. T.; Aydin, B.; Carlson, Ross P.; Hanley, L.The heptapeptide ARHPHPH was identified from biofilms and planktonic cultures of two different strains of Enterococcus faecalis, V583 and ATCC 29212, using matrix assisted laser desorption ionization mass spectrometry (MALDI-MS). ARHPHPH was also imaged at the boundary of cocultured, adjacent E. faecalis and Escherichia coli (ATCC 25922) biofilms, appearing only on the E. faecalis side. ARHPHPH was proteolyzed from κ-casein, a component in the growth media, by E. faecalis microbes. Additionally, top down and bottom up proteomic approaches were combined to identify and spatially locate multiple proteins within intact E. faecalis V583 biofilms by MALDI-MS. The resultant tandem MS data were searched against the NCBInr E. faecalis V583 database to identify thirteen cytosolic and membrane proteins which have functional association with the cell surface. Two of these proteins, enolase and GAPDH, are glycolytic enzymes known to display multiple functions in bacterial virulence in related bacterial strains. This work illustrates a powerful approach for discovering and localizing multiple peptides and proteins within intact biofilms.Item Physiological and molecular analysis of carbon source supplementation and pH stress-induced lipid accumulation in the marine diatom Phaeodactylum tricornutum(2013-03) Mus, Florence; Toussaint, Jean-Paul; Cooksey, Keith E.; Fields, Matthew W.; Gerlach, Robin; Peyton, Brent M.; Carlson, Ross P.A detailed physiological and molecular analysis of lipid accumulation under a suite of conditions including nitrogen limitation, alkaline pH stress, bicarbonate supplementation, and organic acid supplementation was performed on the marine diatom Phaeodactylum tricornutum. For all tested conditions, nitrogen limitation was a prerequisite for lipid accumulation and the other culturing strategies only enhanced accumulation highlighting the importance of compounded stresses on lipid metabolism. Volumetric lipid levels varied depending on condition; the observed rankings from highest to lowest were for inorganic carbon addition (15 mM bicarbonate), organic acid addition (15 carbon mM acetate), and alkaline pH stress (pH9.0). For all lipidaccumulating cultures except acetate supplementation, a common series of physiological steps were observed. Upon extracellular nitrogen exhaustion, culture growth continued for approximately 1.5 cell doublings with decreases in specific protein and photosynthetic pigment content. As nitrogen limitation arrested cell growth, carbohydrate content decreased with a corresponding increase in lipid content. Addition of the organic carbon source acetate appeared to activate alternative metabolic pathways for lipid accumulation. Molecular level data on more than 50 central metabolism transcripts were measured using real-time PCR. Analysis of transcripts suggested the central metabolism pathways associated with bicarbonate transport, carbonic anhydrases, and C4 carbon fixations were important for lipid accumulation. Transcriptomic data also suggested that repurposing of phospholipids may play a role in lipid accumulation. This study provides a detailed physiological and molecular-level foundation for improved understanding of diatom nutrient cycling and contributes to a metabolic blueprint for controlling lipid accumulation in diatoms.Item Nutrient resupplementation arrests bio-oil accumulation in Phaeodactylum tricornutum(2013-08) Valenzuela, Jacob J.; Carlson, Ross P.; Gerlach, Robin; Cooksey, Keith E.; Peyton, Brent M.; Bothner, Brian; Fields, Matthew W.Phaeodactylum tricornutum is a marine diatom in the class Bacillariophyceae and is important ecologically and industrially with regards to ocean primary production and lipid accumulation for biofuel production, respectively. Triacylglyceride (TAG) accumulation has been reported in P. tricornutum under different nutrient stresses, and our results show that lipid accumulation can occur with nitrate or phosphate depletion. However, greater lipid accumulation was observed when both nutrients were depleted as observed using a Nile Red assay and fatty acid methyl ester (FAME) profiles. Nitrate depletion had a greater effect on lipid accumulation than phosphate depletion. Lipid accumulation in P. tricornutum was arrested upon resupplementation with the depleted nutrient. Cells depleted of nitrogen showed a distinct shift from a lipid accumulation mode to cellular growth post resupplementation with nitrate, as observed through increased cell numbers and consumption of accumulated lipid. Phosphate depletion caused lipid accumulation that was arrested upon phosphate resupplementation. The cessation of lipid accumulation was followed by lipid consumption without an increase in cell numbers. Cells depleted in both nitrate and phosphate displayed cell growth upon the addition of both nitrate and phosphate and had the largest observed lipid consumption upon resupplementation. These results indicate that phosphate resupplementation can shut down lipid accumulation but does not cause cells to shift into cellular growth, unlike nitrate resupplementation. These data suggest that nutrient resupplementation will arrest lipid accumulation and that switching between cellular growth and lipid accumulation can be regulated upon the availability of nitrogen and phosphorus.