Publications by Colleges and Departments (MSU - Bozeman)
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Item Arsenic Exposure Causes Global Changes in the Metalloproteome of Escherichia coli(MDPI AG, 2023-02) Larson, James; Tokmina-Lukaszewska, Monika; Fausset, Hunter; Spurzem, Scott; Cox, Savannah; Cooper, Gwendolyn; Copié, Valérie; Bothner, BrianArsenic is a toxic metalloid with differential biological effects, depending on speciation and concentration. Trivalent arsenic (arsenite, AsIII) is more toxic at lower concentrations than the pentavalent form (arsenate, AsV). In E. coli, the proteins encoded by the arsRBC operon are the major arsenic detoxification mechanism. Our previous transcriptional analyses indicate broad changes in metal uptake and regulation upon arsenic exposure. Currently, it is not known how arsenic exposure impacts the cellular distribution of other metals. This study examines the metalloproteome of E. coli strains with and without the arsRBC operon in response to sublethal doses of AsIII and AsV. Size exclusion chromatography coupled with inductively coupled plasma mass spectrometry (SEC-ICPMS) was used to investigate the distribution of five metals (56Fe, 24Mg, 66Zn, 75As, and 63Cu) in proteins and protein complexes under native conditions. Parallel analysis by SEC-UV-Vis spectroscopy monitored the presence of protein cofactors. Together, these data reveal global changes in the metalloproteome, proteome, protein cofactors, and soluble intracellular metal pools in response to arsenic stress in E. coli. This work brings to light one outcome of metal exposure and suggests that metal toxicity on the cellular level arises from direct and indirect effects.Item Detection of cryptosporidium and E. coli using fluorescent in situ hybridization and solid phase laser cytometry(Montana State University - Bozeman, College of Letters & Science, 2013) Broadaway, Susan Cameron; Chairperson, Graduate Committee: Barry H. PyleCryptosporidium parvum is a protozoal pathogen transmitted through water by the fecal-oral route as oocysts. Because the oocysts are more resistant to environmental stresses than the bacteria conventionally used as indicators of fecal contamination, they can be present in water when indicator organisms, such as E. coli, are not found. In addition, because they are resistant to chlorine, they can pass from source water through water treatment into drinking water systems. The EPA method for detection of Cryptosporidium oocysts consists of identifying oocysts with fluorescently labeled antibodies, staining with 4',6-diamidino-2-phenylindole and examining slides with epifluorescent microscopy and differential interference contrast microscopy. This protocol is labor intensive and subject to technician error. A new method was developed for the rapid detection of Cryptosporidium parvum oocysts using fluorescent in situ hybridization (FISH) and the ScanRDI, a solid phase laser cytometer. Optimization of the FISH protocol for use with the ScanRDI was done with E. coli cells and known Cryptosporidium oocysts as a model. Source water and treated drinking water from the water treatment plant at Crow Agency on the Crow Indian Reservation in Montana was collected over the course of a year and concentrated using the EPA protocol for collection of oocysts. The samples were then examined for Cryptosporidium oocysts using both the ScanRDI method and the standard US EPA method. The combination of FISH for labeling Cryptosporidium and the ScanRDI for examination results in significantly higher numbers of Cryptosporidium detected as well as greater ease in identification. A statistical comparison was done that determined there was no correlation between the number of E. coli cells found in the water samples and the number of Cryptosporidium oocysts present. Additionally, although not tested on environmental samples, the FISH/ScanRDI method allowed for simultaneous detection of Cryptosporidium parvum oocysts and E. coli cells on the same membrane filter. Membranes were incubated before hybridization, hybridized concurrently with a Cryptosporidium specific probe and a probe specific for E. coli, followed by detection for both organisms with the ScanRDI.