Publications by Colleges and Departments (MSU - Bozeman)
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Item Stoichiometric modelling of assimilatory and dissimilatory biomass utilisation in a microbial community(2016-08) Hunt, Kristopher A.; Jennings, Ryan deM.; Inskeep, William P.; Carlson, Ross P.Assimilatory and dissimilatory utilisation of autotroph biomass by heterotrophs is a fundamental mechanism for the transfer of nutrients and energy across trophic levels. Metagenome data from a tractable, thermoacidophilic microbial community in Yellowstone National Park was used to build an in silico model to study heterotrophic utilisation of autotroph biomass using elementary flux mode analysis and flux balance analysis. Assimilatory and dissimilatory biomass utilisation was investigated using 29 forms of biomass-derived dissolved organic carbon (DOC) including individual monomer pools, individual macromolecular pools and aggregate biomass. The simulations identified ecologically competitive strategies for utilizing DOC under conditions of varying electron donor, electron acceptor or enzyme limitation. The simulated growth environment affected which form of DOC was the most competitive use of nutrients; for instance, oxygen limitation favoured utilisation of less reduced and fermentable DOC while carbon-limited environments favoured more reduced DOC. Additionally, metabolism was studied considering two encompassing metabolic strategies: simultaneous versus sequential use of DOC. Results of this study bound the transfer of nutrients and energy through microbial food webs, providing a quantitative foundation relevant to most microbial ecosystems.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 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 Molecular imaging and depth profiling of biomaterials interfaces by femtosecond laser desorption postionization mass spectrometry(2013-10) Cui, Y.; Bhardwaj, C.; Milasinovic, S.; Carlson, Ross P.; Gordon, R. J.; Hanley, L.Mass spectrometry (MS) imaging is increasingly being applied to probe the interfaces of biomaterials with invasive microbial biofilms, human tissue, or other biological materials. Laser desorption vacuum ultraviolet postionization with 75 fs, 800 nm laser pulses (fs-LDPI-MS) was used to collect MS images of a yeast–Escherichia coli co-culture biofilm. The method was also used to depth profile a three-dimensionally structured, multispecies biofilm. Finally, fs-LDPI-MS analyses of yeast biofilms grown under different conditions were compared with LDPI-MS using ultraviolet, nanosecond pulse length laser desorption as well as with fs laser desorption ionization without postionization. Preliminary implications for the use of fs-LDPI-MS for the analysis of biomaterials interfaces are discussed and contrasted with established methods in MS imaging.Item Differentiation of microbial species and strains in coculture biofilms by multivariate analysis of laser desorption postionization mass spectra(2013-09) Bhardwaj, C.; Cui, Y.; Hofstetter, T.; Liu, S. Y.; Bernstein, Hans C.; Carlson, Ross P.; Ahmed, M.; Hanley, L.7.87 to 10.5 eV vacuum ultraviolet (VUV) photon energies were used in laser desorption postionization mass spectrometry (LDPI-MS) to analyze biofilms comprised of binary cultures of interacting microorganisms. The effect of photon energy was examined using both tunable synchrotron and laser sources of VUV radiation. Principal components analysis (PCA) was applied to the MS data to differentiate species in Escherichia coli–Saccharomyces cerevisiae coculture biofilms. PCA of LDPI-MS also differentiated individual E. coli strains in a biofilm comprised of two interacting gene deletion strains, even though these strains differed from the wild type K-12 strain by no more than four gene deletions each out of approximately 2000 genes. PCA treatment of 7.87 eV LDPI-MS data separated the E. coli strains into three distinct groups, two “pure†groups, and a mixed region. Furthermore, the “pure†regions of the E. coli cocultures showed greater variance by PCA at 7.87 eV photon energies compared to 10.5 eV radiation. This is consistent with the expectation that the 7.87 eV photoionization selects a subset of low ionization energy analytes while 10.5 eV is more inclusive, detecting a wider range of analytes. These two VUV photon energies therefore give different spreads via PCA and their respective use in LDPI-MS constitute an additional experimental parameter to differentiate strains and species.