"Biofilmomics": functional protein expression in biofilm biotechnologies revealed by quantitative proteomics
Date
2020
Authors
Chignell, Jeremy, author
Reardon, Kenneth, advisor
De Long, Susan, advisor
Peebles, Christie, committee member
Sharvelle, Sybil, committee member
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Abstract
Microbial biotechnologies that utilize biofilms often exhibit superior performance compared with planktonic systems. Many details of biofilm metabolism that drive those improvements in performance remain unclear. Only recently have molecular tools emerged that can provide a holistic picture of life in a complex biosystem like a biofilm for the purposes of answering questions on a system level. The purpose of this work was to address four fundamental questions about protein expression in biofilms: what kind of protein expression is distinctive to biofilms? Which biofilm proteins are associated with a function of interest? How does co-culture with another species affect biofilm-related protein expression? When during multi-species biofilm development does a function of interest emerge and who in the community is responsible? Label-free quantitative proteomics was used in conjunction with physiological experimentation to address these four questions. In the first study we found that L. delbrueckii lactis protein expression in flow-cell biofilms was 31% more diverse than in planktonic cultures, and proteins related to catalytic activity were significantly increased in biofilms at the expense of proteins for cell motility and replication. Roles for riboflavin and fatty acid metabolism suggested modulations in redox functions and membrane turnover during life in a biofilm. The second study compared protein expression by S. onedensis MR-1 in electricity-generating biofilms with that in aerobic biofilms from the same microbial fuel cell reactor. Three novel proteins associated with electricity generation were identified, in addition to proteomic evidence of aerobic metabolism by anode biofilm cells. The latter result was shown to be consistent with kinetics of oxygen depletion and bulk cell growth in the MFC, suggesting operational conditions to reduce this bulk cell growth and thereby reduce fouling of the cathode and improve overall Coulombic efficiency of the single-chamber MFC system. In the third study, it was discovered through proteomic and physiological experiments that a virulent phenotype associated with biofilm formation was triggered in P. putida when co-cultured with B. atrophaeus. Dramatic shifts in protein expression at the initial trigger point of virulent biofilm formation by P. putida are described. Finally, a comparison of the meta-proteomes of microbial fuel cell biofilms at different stages of development indicated that proteins in metabolic pathways for carbon storage and competitive inhibition are differentially expressed when the biofilm becomes electrochemically active. Meta-proteomics and 16S rRNA gene sequencing agreed that it is possible for a microbial fuel cell community to maintain high diversity (and therefore potentially higher resilience) while generating electricity at levels comparable to a MFC community dominated by Geobacter. Each of these chapters was prepared as an independent manuscript, though the themes were integrated by the overall theme of quantifying differential protein expression in biofilms in order to reveal new details about their development and functionality. Since the performance of many engineered biosystems—including those that employ biofilms—often can be controlled adequately at an operational level, an attitude persists that any additional molecular investigation is superfluous. The work presented here provides evidence for the opposite viewpoint: a rich understanding of the molecular mechanisms behind biofilm functionality can inform strategies for continuous system improvement and suggest new capabilities and biotechnological applications of biofilms.
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Subject
biofilm
lactic
proteomics
engineering
bacteria
microbial fuel cell