College of Agriculture
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As the foundation of the land grant mission at Montana State University, the College of Agriculture and the Montana Agricultural Experiment Station provide instruction in traditional and innovative degree programs and conduct research on old and new challenges for Montana’s agricultural community. This integration creates opportunities for students and faculty to excel through hands-on learning, to serve through campus and community engagement, to explore unique solutions to distinct and interesting questions and to connect Montanans with the global community through research discoveries and outreach.
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Item Real-Time Digitization of Metabolomics Patterns from a Living System Using Mass Spectrometry(2014-10) Heinemann, Joshua; Noon, Brigit; Mohigmi, Mohammad J.; Mazurie, Aurélien J.; Dickensheets, David L.; Bothner, BrianThe real-time quantification of changes in intracellular metabolic activities has the potential to vastly improve upon traditional transcriptomics and metabolomics assays for the prediction of current and future cellular phenotypes. This is in part because intracellular processes reveal themselves as specific temporal patterns of variation in metabolite abundance that can be detected with existing signal processing algorithms. Although metabolite abundance levels can be quantified by mass spectrometry (MS), large-scale real-time monitoring of metabolite abundance has yet to be realized because of technological limitations for fast extraction of metabolites from cells and biological fluids. To address this issue, we have designed a microfluidic-based inline small molecule extraction system, which allows for continuous metabolomic analysis of living systems using MS. The system requires minimal supervision, and has been successful at real-time monitoring of bacteria and blood. Feature-based pattern analysis of Escherichia coli growth and stress revealed cyclic patterns and forecastable metabolic trajectories. Using these trajectories, future phenotypes could be inferred as they exhibit predictable transitions in both growth and stress related changes. Herein, we describe an interface for tracking metabolic changes directly from blood or cell suspension in real-time.Item IgG Endopeptidase SeMac does not Inhibit Opsonophagocytosis of Streptococcus equi Subspecies equi by Horse PolymorphonuclearLeukocytes(2010-08) Liu, Mengyao; Lei, BenfangThe secreted Mac protein made by group A Streptococcus (GAS) inhibits opsonophagocytosis of GAS by human polymorphonuclear leukocytes (PMNs). This protein also has the endopeptidase activity against human immunoglobulin G (IgG), and the Cys94, His262 and Asp284 are critical for the enzymatic activity. The horse pathogen Streptococcus equi subspecies equi produces a homologue of Mac (SeMac). SeMac was characterized to determine whether SeMac has IgG endopeptidase activity and inhibits opsonophagocytosis of S. equi by horse PMNs. The gene was cloned and recombinant SeMac was overexpressed in Escherichia coli and purified to homogeneity. Mice with experimental S. equi infection and horses with strangles caused by S. equi seroconverted to SeMac, indicating that SeMac is produced in vivo during infection. SeMac has endopeptidase activity against human IgG. However, the protein just cleaves a small fraction, which may be IgG1 only, of horse IgG. Replacement of Cys102 with Ser or His272 with Ala abolishes the enzymatic activity of SeMac, and the Asp294Ala mutation greatly decreases the enzymatic activity. SeMac does not inhibit opsonophagocytosis of S. equi by horse PMNs but opsonophagocytosis of GAS by human PMNs. Thus, SeMac is a cysteine endopeptidase with a limited activity against horse IgG and must have other function.Item Direct Heme Transfer Reactions in the Group A Streptococcus Heme Acquisition Pathway(2012-05) Lu, C.; Xie, G.; Liu, Mengyao; Zhu, Hui; Lei, BenfangThe heme acquisition machinery in Group A Streptococcus (GAS) consists of the surface proteins Shr and Shp and ATP-binding cassette transporter HtsABC. Shp cannot directly acquire heme from methemoglobin (metHb) but directly transfers its heme to HtsA. It has not been previously determined whether Shr directly relays heme from metHb to Shp. Thus, the complete pathway for heme acquisition from metHb by the GAS heme acquisition machinery has remained unclear. In this study, the metHb-to-Shr and Shr-to-Shp heme transfer reactions were characterized by spectroscopy, kinetics and protein-protein interaction analyses. Heme is efficiently transferred from the β and α subunits of metHb to Shr with rates that are 7 and 60 times greater than those of the passive heme release from metHb, indicating that Shr directly acquires heme from metHb. The rapid heme transfer from Shr to Shp involves an initial heme donor/acceptor complex and a spectrally and kinetically detectable transfer intermediate, implying that heme is directly channeled from Shr to Shp. The present results show that Shr speeds up heme transfer from metHb to Shp, whereas Shp speeds up heme transfer from Shr to HtsA. Furthermore, the findings demonstrate that Shr can interact with metHb and Shp but not HtsA. Taken together with our published results on the Shp/HtsA reaction, these findings establish a model of the heme acquisition pathway in GAS in which Shr directly extracts heme from metHb and Shp relays it from Shr to HtsA.Item Non-Heme-Binding Domains and Segments of the Staphylococcus aureus IsdB Protein Critically Contribute to the Kinetics and Equilibriumof Heme Acquisition from Methemoglobin(2014-06) Zhu, Hui; Li, Dengfeng; Liu, Mengyao; Copie, Valerie; Lei, BenfangThe hemoglobin receptor IsdB rapidly acquires heme from methemoglobin (metHb) in the heme acquisition pathway of Staphylococcus aureus. IsdB consists of N-terminal segment (NS), NEAT1 (N1), middle (MD), and heme binding NEAT2 (N2) domains, and C-terminal segment (CS). This study aims to elucidate the roles of these domains or segments in the metHb/IsdB reaction. Deletion of CS does not alter the kinetics and equilibrium of the reaction. Sequential deletions of NS and N1 in NS-N1-MD-N2 progressively reduce heme transfer rates and change the kinetic pattern from one to two phases, but have no effect on the equilibrium of the heme transfer reaction, whereas further deletion of MD reduces the percentage of transferred metHb heme. MD-N2 has higher affinity for heme than N2. MD in trans reduces rates of heme dissociation from holo-N2 and increases the percentage of metHb heme captured by N2 by 4.5 fold. NS-N1-MD and N2, but not NS-N1, MD, and N2, reconstitute the rapid metHb/IsdB reaction. NS-N1-MD-NIsdC, a fusion protein of NS-N1-MD and the NEAT domain of IsdC, slowly acquires heme from metHb by itself but together with N2 results in rapid heme loss from metHb. Thus, NS-N1 and MD domains specifically and critically contribute to the kinetics and equilibrium of the metHb/IsdB reaction, respectively. These findings support a mechanism of direct heme acquisition by IsdB in which MD enhances the affinity of N2 for heme to thermodynamically drive heme transfer from metHb to IsdB and in which NS is required for the rapid and single phase kinetics of the metHb/IsdB reaction.