Publications by Colleges and Departments (MSU - Bozeman)
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Item Activity-based, genome-resolved metagenomics uncovers key populations and pathways involved in subsurface conversions of coal to methane(Springer Science and Business Media LLC, 2021-10) McKay, Luke J.; Smith, Heidi J.; Barnhart, Elliott P.; Schweitzer, Hannah D.; Malmstrom, Rex R.; Goudeau, Danielle; Fields, Matthew W.Microbial metabolisms and interactions that facilitate subsurface conversions of recalcitrant carbon to methane are poorly understood. We deployed an in situ enrichment device in a subsurface coal seam in the Powder River Basin (PRB), USA, and used BONCAT-FACS-Metagenomics to identify translationally active populations involved in methane generation from a variety of coal-derived aromatic hydrocarbons. From the active fraction, high-quality metagenome-assembled genomes (MAGs) were recovered for the acetoclastic methanogen, Methanothrix paradoxum, and a novel member of the Chlorobi with the potential to generate acetate via the Pta-Ack pathway. Members of the Bacteroides and Geobacter also encoded Pta-Ack and together, all four populations had the putative ability to degrade ethylbenzene, phenylphosphate, phenylethanol, toluene, xylene, and phenol. Metabolic reconstructions, gene analyses, and environmental parameters also indicated that redox fluctuations likely promote facultative energy metabolisms in the coal seam. The active "Chlorobi PRB" MAG encoded enzymes for fermentation, nitrate reduction, and multiple oxygenases with varying binding affinities for oxygen. "M. paradoxum PRB" encoded an extradiol dioxygenase for aerobic phenylacetate degradation, which was also present in previously published Methanothrix genomes. These observations outline underlying processes for bio-methane from subbituminous coal by translationally active populations and demonstrate activity-based metagenomics as a powerful strategy in next generation physiology to understand ecologically relevant microbial populations.Item Global transcriptional, physiological, and metabolite analyses of the responses of Desulfovibrio vulgaris Hildenborough to salt adaptation(2009-12) He, Zhili; Zhou, Aifen; Baidoo, Edward E. K.; He, Q.; Joachimiak, M. P.; Benke, P.; Phan, R.; Mukhopadhyay, A.; Hemme, C. L.; Huang, K.; Alm, E. J.; Fields, Matthew W.; Wall, Judy D.; Stahl, David A.; Hazen, Terry C.; Keasling, J. D.; Arkin, Adam P.; Zhou, JizhongThe response of Desulfovibrio vulgaris Hildenborough to salt adaptation (long-term NaCl exposure) was examined by performing physiological, global transcriptional, and metabolite analyses. Salt adaptation was reflected by increased expression of genes involved in amino acid biosynthesis and transport, electron transfer, hydrogen oxidation, and general stress responses (e.g., heat shock proteins, phage shock proteins, and oxidative stress response proteins). The expression of genes involved in carbon metabolism, cell growth, and phage structures was decreased. Transcriptome profiles of D. vulgaris responses to salt adaptation were compared with transcriptome profiles of D. vulgaris responses to salt shock (short-term NaCl exposure). Metabolite assays showed that glutamate and alanine accumulated under salt adaptation conditions, suggesting that these amino acids may be used as osmoprotectants in D. vulgaris. Addition of amino acids (glutamate, alanine, and tryptophan) or yeast extract to the growth medium relieved salt-related growth inhibition. A conceptual model that links the observed results to currently available knowledge is proposed to increase our understanding of the mechanisms of D. vulgaris adaptation to elevated NaCl levels.Item Shewanella oneidensis MR-1 sensory box protein involved in aerobic and anoxic growth(2011-03) Sundararajan, Anitha; Kurowski, J.; Yan, T.; Klingeman, D. M.; Joachimiak, M. P.; Zhou, Jizhong; Naranjo, B.; Gralnick, J. A.; Fields, Matthew W.Although little is known of potential function for conserved signaling proteins, it is hypothesized that such proteins play important roles to coordinate cellular responses to environmental stimuli. In order to elucidate the function of a putative sensory box protein (PAS domains) in Shewanella oneidensis MR-1, the physiological role of SO3389 was characterized. The predicted open reading frame (ORF) encodes a putative sensory box protein that has PAS, GGDEF, and EAL domains, and an in-frame deletion mutant was constructed (ΔSO3389) with approximately 95% of the ORF deleted. Under aerated conditions, wild-type and mutant cultures had similar growth rates, but the mutant culture had a lower growth rate under static, aerobic conditions. Oxygen consumption rates were lower for mutant cultures (1.5-fold), and wild-type cultures also maintained lower dissolved oxygen concentrations under aerated growth conditions. When transferred to anoxic conditions, the mutant did not grow with fumarate, iron(III), or dimethyl sulfoxide (DMSO) as electron acceptors. Biochemical assays demonstrated the expression of different c-type cytochromes as well as decreased fumarate reductase activity in the mutant transferred to anoxic growth conditions. Transcriptomic studies showed the inability of the mutant to up-express and down-express genes, including c-type cytochromes (e.g., SO4047/SO4048, SO3285/SO3286), reductases (e.g., SO0768, SO1427), and potential regulators (e.g., SO1329). The complemented strain was able to grow when transferred from aerobic to anoxic growth conditions with the tested electron acceptors. The modeled structure for the SO3389 PAS domains was highly similar to the crystal structures of FAD-binding PAS domains that are known O2/redox sensors. Based on physiological, genomic, and bioinformatic results, we suggest that the sensory box protein, SO3389, is an O2/redox sensor that is involved in optimization of aerobic growth and transitions to anoxia in S. oneidensis MR-1.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 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 Identification of a cyclic-di-GMP-modulating response regulator that impacts biofilm formation in a model sulfate reducing bacterium(2014-07) Rajeev, L.; Luning, E. G.; Altenburg, Sara; Zane, Grant M.; Baidoo, Edward E. K.; Catena, M.; Keasling, J. D.; Wall, Judy D.; Fields, Matthew W.; Mukhopadhyay, A.We surveyed the eight putative cyclic-di-GMP-modulating response regulators (RRs) in Desulfovibrio vulgaris Hildenborough that are predicted to function via two-component signaling. Using purified proteins, we examined cyclic-di-GMP (c-di-GMP) production or turnover in vitro of all eight proteins. The two RRs containing only GGDEF domains (DVU2067, DVU0636) demonstrated c-di-GMP production activity in vitro. Of the remaining proteins, three RRs with HD-GYP domains (DVU0722, DVUA0086, and DVU2933) were confirmed to be Mn(2+)-dependent phosphodiesterases (PDEs) in vitro and converted c-di-GMP to its linear form, pGpG. DVU0408, containing both c-di-GMP production (GGDEF) and degradation domains (EAL), showed c-di-GMP turnover activity in vitro also with production of pGpG. No c-di-GMP related activity could be assigned to the RR DVU0330, containing a metal-dependent phosphohydrolase HD-OD domain, or to the HD-GYP domain RR, DVU1181. Studies included examining the impact of overexpressed cyclic-di-GMP-modulating RRs in the heterologous host E. coli and led to the identification of one RR, DVU0636, with increased cellulose production. Evaluation of a transposon mutant in DVU0636 indicated that the strain was impaired in biofilm formation and demonstrated an altered carbohydrate:protein ratio relative to the D. vulgaris wild type biofilms. However, grown in liquid lactate/sulfate medium, the DVU0636 transposon mutant showed no growth impairment relative to the wild-type strain. Among the eight candidates, only the transposon disruption mutant in the DVU2067 RR presented a growth defect in liquid culture. Our results indicate that, of the two diguanylate cyclases (DGCs) that function as part of two-component signaling, DVU0636 plays an important role in biofilm formation while the function of DVU2067 has pertinence in planktonic growth.Item Complete genome sequence of Pelosinus fermentans JBW45, a member of a remarkably competitive group of Negativicutes in the Firmicutes Phylum(2015-09) Bowen De León, Kara; Utturkar, S. M.; Camilleri, Laura B.; Elias, Dwayne A.; Arkin, Adam P.; Fields, Matthew W.; Brown, S. D.; Wall, Judy D.The genome of Pelosinus fermentans JBW45, isolated from a chromium-contaminated site in Hanford, Washington, USA, has been completed with PacBio sequencing. Nine copies of the rRNA gene operon and multiple transposase genes with identical sequences resulted in breaks in the original draft genome and may suggest genomic instability of JBW45.