Center for Biofilm Engineering (CBE)
Permanent URI for this communityhttps://scholarworks.montana.edu/handle/1/9334
At the Center for Biofilm Engineering (CBE), multidisciplinary research teams develop beneficial uses for microbial biofilms and find solutions to industrially relevant biofilm problems. The CBE was established at Montana State University, Bozeman, in 1990 as a National Science Foundation Engineering Research Center. As part of the MSU College of Engineering, the CBE gives students a chance to get a head start on their careers by working on research teams led by world-recognized leaders in the biofilm field.
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Item Answer Set Programming for Computing Constraints-Based Elementary Flux Modes: Application to Escherichia coli Core Metabolism(MDPI AG, 2020-12) Mahout, Maxime; Carlson, Ross P.; Peres, SabineElementary Flux Modes (EFMs) provide a rigorous basis to systematically characterize the steady state, cellular phenotypes, as well as metabolic network robustness and fragility. However, the number of EFMs typically grows exponentially with the size of the metabolic network, leading to excessive computational demands, and unfortunately, a large fraction of these EFMs are not biologically feasible due to system constraints. This combinatorial explosion often prevents the complete analysis of genome-scale metabolic models. Traditionally, EFMs are computed by the double description method, an efficient algorithm based on matrix calculation; however, only a few constraints can be integrated into this computation. They must be monotonic with regard to the set inclusion of the supports; otherwise, they must be treated in post-processing and thus do not save computational time. We present aspefm, a hybrid computational tool based on Answer Set Programming (ASP) and Linear Programming (LP) that permits the computation of EFMs while implementing many different types of constraints. We apply our methodology to the Escherichia coli core model, which contains 226×106 EFMs. In considering transcriptional and environmental regulation, thermodynamic constraints, and resource usage considerations, the solution space is reduced to 1118 EFMs that can be computed directly with aspefm. The solution set, for E. coli growth on O2 gradients spanning fully aerobic to anaerobic, can be further reduced to four optimal EFMs using post-processing and Pareto front analysis.Item Role of hibernation promoting factor in ribosomal protein stability during Pseudomonas aeruginosa dormancy(MDPI, 2020-12) Theng, Sokuntheary; Williamson, Kerry S.; Franklin, Michael J.Pseudomonas aeruginosa is an opportunistic pathogen that causes biofilm-associated infections. P. aeruginosa can survive in a dormant state with reduced metabolic activity in nutrient-limited environments, including the interiors of biofilms. When entering dormancy, the bacteria undergo metabolic remodeling, which includes reduced translation and degradation of cellular proteins. However, a supply of essential macromolecules, such as ribosomes, are protected from degradation during dormancy. The small ribosome-binding proteins, hibernation promoting factor (HPF) and ribosome modulation factor (RMF), inhibit translation by inducing formation of inactive 70S and 100S ribosome monomers and dimers. The inactivated ribosomes are protected from the initial steps in ribosome degradation, including endonuclease cleavage of the ribosomal RNA (rRNA). Here, we characterized the role of HPF in ribosomal protein (rProtein) stability and degradation during P. aeruginosa nutrient limitation. We determined the effect of the physiological status of P. aeruginosa prior to starvation on its ability to recover from starvation, and on its rRNA and rProtein stability during cell starvation. The results show that the wild-type strain and a stringent response mutant (∆relA∆spoT strain) maintain high cellular abundances of the rProteins L5 and S13 over the course of eight days of starvation. In contrast, the abundances of L5 and S13 reduce in the ∆hpf mutant cells. The loss of rProteins in the ∆hpf strain is dependent on the physiology of the cells prior to starvation. The greatest rProtein loss occurs when cells are first cultured to stationary phase prior to starvation, with less rProtein loss in the ∆hpf cells that are first cultured to exponential phase or in balanced minimal medium. Regardless of the pre-growth conditions, P. aeruginosa recovery from starvation and the integrity of its rRNA are impaired in the absence of HPF. The results indicate that protein remodeling during P. aeruginosa starvation includes the degradation of rProteins, and that HPF is essential to prevent rProtein loss in starved P. aeruginosa. The results also indicate that HPF is produced throughout cell growth, and that regardless of the cellular physiological status, HPF is required to protect against ribosome loss when the cells subsequently enter starvation phase.Item Subsurface hydrocarbon degradation strategies in low- and high-sulfate coal seam communities identified with activity-based metagenomics(Springer Science and Business Media LLC, 2022-02) Schweitzer, Hannah D.; Smith, Heidi J.; Barnhart, Elliott P.; McKay, Luke J.; Gerlach, Robin; Cunningham, Alfred B.; Malmstrom, Rex R.; Goudeau, Danielle; Fields, Matthew W.Environmentally relevant metagenomes and BONCAT-FACS derived translationally active metagenomes from Powder River Basin coal seams were investigated to elucidate potential genes and functional groups involved in hydrocarbon degradation to methane in coal seams with high- and low-sulfate levels. An advanced subsurface environmental sampler allowed the establishment of coal-associated microbial communities under in situ conditions for metagenomic analyses from environmental and translationally active populations. Metagenomic sequencing demonstrated that biosurfactants, aerobic dioxygenases, and anaerobic phenol degradation pathways were present in active populations across the sampled coal seams. In particular, results suggested the importance of anaerobic degradation pathways under high-sulfate conditions with an emphasis on fumarate addition. Under low-sulfate conditions, a mixture of both aerobic and anaerobic pathways was observed but with a predominance of aerobic dioxygenases. The putative low-molecular-weight biosurfactant, lichysein, appeared to play a more important role compared to rhamnolipids. The methods used in this study—subsurface environmental samplers in combination with metagenomic sequencing of both total and translationally active metagenomes—offer a deeper and environmentally relevant perspective on community genetic potential from coal seams poised at different redox conditions broadening the understanding of degradation strategies for subsurface carbon.Item In Situ Enhancement and Isotopic Labeling of Biogenic Coalbed Methane(American Chemical Society, 2022-02) Barnhart, Elliott P.; Ruppert, Leslie; Hiebert, Randy; Smith, Heidi J.; Schweitzer, Hannah D.; Clark, Arthur C.; Weeks, Edwin P.; Orem, William H.; Varonka, Matthew S.; Platt, George; Shelton, Jenna L.; Davis, Katherine J.; Hyatt, Robert J.; McIntosh, Jennifer C.; Ashley, Kilian; Ono, Shuhei; Martini, Anna M.; Hackley, Keith C.; Gerlach, Robin; Spangler, Lee; Phillips, Adrienne J.; Barry, Mark; Cunningham, Alfred B.; Fields, Matthew W.Subsurface microbial (biogenic) methane production is an important part of the global carbon cycle that has resulted in natural gas accumulations in many coal beds worldwide. Laboratory studies suggest that complex carbon-containing nutrients (e.g., yeast or algae extract) can stimulate methane production, yet the effectiveness of these nutrients within coal beds is unknown. Here, we use downhole monitoring methods in combination with deuterated water (D2O) and a 200-liter injection of 0.1% yeast extract (YE) to stimulate and isotopically label newly generated methane. A total dissolved gas pressure sensor enabled real time gas measurements (641 days preinjection and for 478 days postinjection). Downhole samples, collected with subsurface environmental samplers, indicate that methane increased 132% above preinjection levels based on isotopic labeling from D2O, 108% based on pressure readings, and 183% based on methane measurements 266 days postinjection. Demonstrating that YE enhances biogenic coalbed methane production in situ using multiple novel measurement methods has immediate implications for other field-scale biogenic methane investigations, including in situ methods to detect and track microbial activities related to the methanogenic turnover of recalcitrant carbon in the subsurface.Item Experimental Designs to Study the Aggregation and Colonization of Biofilms by Video Microscopy With Statistical Confidenc(Frontiers Media SA, 2022-01) Pettygrove, Brian A.; Smith, Heidi J.; Pallister, Kyler B.; Voyich, Jovanka M.; Stewart, Philip S.; Parker, Albert E.The goal of this study was to quantify the variability of confocal laser scanning microscopy (CLSM) time-lapse images of early colonizing biofilms to aid in the design of future imaging experiments. To accomplish this a large imaging dataset consisting of 16 independent CLSM microscopy experiments was leveraged. These experiments were designed to study interactions between human neutrophils and single cells or aggregates of Staphylococcus aureus (S. aureus) during the initial stages of biofilm formation. Results suggest that in untreated control experiments, variability differed substantially between growth phases (i.e., lag or exponential). When studying the effect of an antimicrobial treatment (in this case, neutrophil challenge), regardless of the inoculation level or of growth phase, variability changed as a frown-shaped function of treatment efficacy (i.e., the reduction in biofilm surface coverage). These findings were used to predict the best experimental designs for future imaging studies of early biofilms by considering differing (i) numbers of independent experiments; (ii) numbers of fields of view (FOV) per experiment; and (iii) frame capture rates per hour. A spreadsheet capable of assessing any user-specified design is included that requires the expected mean log reduction and variance components from user-generated experimental results. The methodology outlined in this study can assist researchers in designing their CLSM studies of antimicrobial treatments with a high level of statistical confidence.Item Change Rippling through Our Waters and Culture(Wiley, 2020-04) Martin, Christine; Doyle, John; LaFrance, JoRee; Lefthand, Myra J.; Young, Sara L.; Three Irons, Emery; Eggers, Margaret J.It is well established that climate change is already causing a wide variety of human health impacts in the United States and globally, and that for many reasons Native Americans are particularly vulnerable. Tribal water security is particularly threatened; the ways in which climate changes are damaging community health and well-being through impacts on water resources have been addressed more thoroughly for Tribes in coastal, arid, and sub-arctic/arctic regions of the United States. In this article, Crow Tribal members from the Northern Plains describe the impacts of climate and environmental change on local water resources and ecosystems, and thereby on Tribal community health and well-being. Formal, qualitative research methodology was employed drawing on interviews with 26 Crow Tribal Elders. Multiple determinants of health are addressed, including cultural, social, economic, and environmental factors. The sense of environmental-cultural-health loss and despair at the inability to address the root causes of climate change are widespread. Yet the co-authors and many other Tribal members are actively prioritizing, addressing, and coping with some of the local impacts of these changes, and are carrying on Apsáalooke [Crow] lifeways and values.Item The widespread IS200/IS605 transposon family encodes diverse programmable RNA-guided endonucleases(American Association for the Advancement of Science, 2021-10) Altae-Tran, Han; Kannan, Soumya; Demircioglu, F. Esra; Oshiro, Rachel; Nety, Suchita P.; McKay, Luke J.; Dlakić, Mensur; Inskeep, William P.; Makarova, Kira S.; Macrae, Rhiannon K.; Koonin, Eugene V.; Zhang, FengIscB proteins are putative nucleases encoded in a distinct family of IS200/IS605 transposons and are likely ancestors of the RNA-guided endonuclease Cas9, but the functions of IscB and its interactions with any RNA remain uncharacterized. Using evolutionary analysis, RNA sequencing, and biochemical experiments, we reconstructed the evolution of CRISPR-Cas9 systems from IS200/IS605 transposons. We found that IscB uses a single noncoding RNA for RNA-guided cleavage of double-stranded DNA and can be harnessed for genome editing in human cells. We also demonstrate the RNA-guided nuclease activity of TnpB, another IS200/IS605 transposon-encoded protein and the likely ancestor of Cas12 endonucleases. This work reveals a widespread class of transposon-encoded RNA-guided nucleases, which we name OMEGA (obligate mobile element–guided activity), with strong potential for developing as biotechnologies.Item The establishment of the CBE launched biofilms as a field of specialized research(The establishment of the CBE launched biofilms as a field of specialized research, 2020-12) Fields, Matthew W.; Sturman, Paul; Anderson, SkipThe Center for Biofilm Engineering was the first center of excellence focused on biofilms and was originally funded through the Engineering Research Center Program from the U.S. National Science Foundation. After almost 30 years, biofilm continues to be a stand-alone scientific topic of inquiry that has broad implications for fundamental and applied science and engineering of bio-systems. However, much remains to be done, not only for research discovery but also education and outreach, to increase and grow the biofilm paradigm as well as our understanding of the microbial world.Item Sulfenate Esters of Simple Phenols Exhibit Enhanced Activity against Biofilms(American Chemical Society, 2020-03) Walsh, Danica J.; Livinghouse, Tom; Durling, Greg M.; Chase-Bayless, Yenny; Arnold, Adrienne D.; Stewart, Philip S.The recalcitrance exhibited by microbial biofilms to conventional disinfectants has motivated the development of new chemical strategies to control and eradicate biofilms. The activities of several small phenolic compounds and their trichloromethylsulfenyl ester derivatives were evaluated against planktonic cells and mature biofilms of Staphylococcus epidermidis and Pseudomonas aeruginosa. Some of the phenolic parent compounds are well-studied constituents of plant essential oils, for example, eugenol, menthol, carvacrol, and thymol. The potency of sulfenate ester derivatives was markedly and consistently increased toward both planktonic cells and biofilms. The mean fold difference between the parent and derivative minimum inhibitory concentration against planktonic cells was 44 for S. epidermidis and 16 for P. aeruginosa. The mean fold difference between the parent and derivative biofilm eradication concentration for 22 tested compounds against both S. epidermidis and P. aeruginosa was 3. This work demonstrates the possibilities of a new class of biofilm-targeting disinfectants deploying a sulfenate ester functional group to increase the antimicrobial potency toward microorganisms in biofilms.Item Characterization of subsurface media from locations up- and down-gradient of a uranium-contaminated aquifer(Elsevier BV, 2020-05) Moon, Ji-Won; Paradis, Charles J.; Joyner, Dominique C.; von Netzer, Frederick; Majumder, Erica L.; Dixon, Emma R.; Podar, Mircea; Ge, Xiaoxuan; Walian, Peter J.; Smith, Heidi J.; Wu, Xiaoqin; Zane, Grant M.; Walker, Kathleen F.; Thorgersen, Michael P.; Poole, Farris L. II; Lui, Lauren M.; Adams, Benjamin G.; De León, Kara B.; Brewer, Sheridan S.; Williams, Daniel E.; Lowe, Kenneth A.; Rodriguez, Miguel; Mehlhorn, Tonia L.; Pfiffner, Susan M.; Chakraborty, Romy; Arkin, Adam P.; Wall, Judy D.; Fields, Matthew W.; Adams, Michael W.W.; Stahl, David A.; Elias, Dwayne A.; Hazen, Terry C.The processing of sediment to accurately characterize the spatially-resolved depth profiles of geophysical and geochemical properties along with signatures of microbial density and activity remains a challenge especially in complex contaminated areas. This study processed cores from two sediment boreholes from background and contaminated core sediments and surrounding groundwater. Fresh core sediments were compared by depth to capture the changes in sediment structure, sediment minerals, biomass, and pore water geochemistry in terms of major and trace elements including pollutants, cations, anions, and organic acids. Soil porewater samples were matched to groundwater level, flow rate, and preferential flows and compared to homogenized groundwater-only samples from neighboring monitoring wells. Groundwater analysis of nearby wells only revealed high sulfate and nitrate concentrations while the same analysis using sediment pore water samples with depth was able to suggest areas high in sulfate-and nitrate-reducing bacteria based on their decreased concentration and production of reduced by-products that could not be seen in the groundwater samples. Positive correlations among porewater content, total organic carbon, trace metals and clay minerals revealed a more complicated relationship among contaminant, sediment texture, groundwater table, and biomass. The fluctuating capillary interface had high concentrations of Fe and Mn-oxides combined with trace elements including U, Th, Sr, Ba, Cu, and Co. This suggests the mobility of potentially hazardous elements, sediment structure, and biogeochemical factors are all linked together to impact microbial communities, emphasizing that solid interfaces play an important role in determining the abundance of bacteria in the sediments.