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Title:Metabolic and molecular responses to interspecies hydrogen transfer between Ruminococcus albus and Wolinella succinogenes
Author(s):Geier, Renae Rose
Advisor(s):Mackie, Roderick
Contributor(s):Donovan, Sharon; Swanson, Kelly
Department / Program:Nutritional Sciences
Discipline:Nutritional Sciences
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:M.S.
Genre:Thesis
Subject(s):interspecies hydrogen transfer
Ruminococcus albus
Wolinella succinogenes
electron bifurcation
hydrogenases
co-culture
Abstract:The mutually beneficial interdependence of hydrogen-producing and hydrogen-utilizing bacteria was discovered by M. P. Bryant, M. J. Wolin and R. S. Wolfe at the University of Illinois in 1967. Based on thermodynamic principles, interspecies hydrogen transfer is a central process in anaerobic environments linking transfer of reducing power from fermentation of organic molecules to inorganic electron acceptors via hydrogen. Interspecies hydrogen transfer is the most significant example of unidirectional substrate supply enabling the syntrophic metabolic association between interacting microbial species and plays a significant role in the global methane cycle. Ruminococcus albus 7 is a hydrogen-producing, fermentative bacterium with two known hydrogen-producing hydrogenase complexes, HydABC and HydA2, as well as a putative hydrogen-sensing protein, HydS. HydABC is the only chromosomal hydrogenase, while HydA2 and HydS form a transcriptional unit on its plasmid pRumal01. The electron-bifurcating ferredoxin- and NAD-dependent [FeFe]-hydrogenase, HydABC, couples proton reduction using nicotinamide adenine dinucleotide (NADH) to proton reduction using reduced ferredoxin (Fdred), producing molecular hydrogen: 3 H+ + NADH + Fdred → 2 H2 + NAD+ + Fdox. HydA2, a ferredoxin-dependent [FeFe]-hydrogenase, reduces protons to molecular hydrogen using only reduced ferredoxin: 2 H+ + Fdred → H2 + Fdox. HydS contains a PAS domain, which often are present on sensory proteins. In addition, HydS contains a putative redox-sensing [4Fe:4S] cluster. We hypothesized HydS transcriptionally regulates HydA2 in a manner dependent on the presence of a hydrogen-utilizing syntroph. To test this hypothesis, R. albus 7 and a hydrogen-utilizing bacterium, Wolinella succinogenes DSM 1740, were grown in pure culture and in co-culture. W. succinogenes uses hydrogen as an electron acceptor for fumarate respiration. Cell growth was monitored by optical density (OD600) and quantitative polymerase chain reaction (qPCR). Metabolites were measured to observe changes caused by the interaction of the two bacteria. Lastly, RNA was extracted at mid-log phase for sequencing to compare whole genome transcriptomic profiles. Hydrogen accumulated in the R. albus pure culture, but not in the co-culture. Production of acetate increased and ethanol decreased when R. albus was grown in co-culture with W. succinogenes. Transcript abundance of HydA2 was 90-fold lower in co-culture, relative to pure culture. The electron-bifurcating hydrogenase, HydABC, had a small change in transcript abundance in co-culture relative to pure culture (1.2- to 1.3-fold increase). This suggests HydS might be sensing hydrogen levels and regulating the transcription of HydA2. These results also suggest the electron-bifurcating hydrogenase (HydABC) functions in central metabolism regardless of external hydrogen concentration. In addition, many genes in central carbon metabolism, de novo thiamin biosynthesis, and methionine transport were significantly increased. W. succinogenes reduced all the fumarate to succinate in both the pure culture and the co-culture with R. albus. Two of the three subunits of the [NiFe]-hydrogenase in W. succinogenes had an increase in transcript abundance of 2.7-fold to 2.9-fold. The transcripts for fumarate reductase had a small increase in abundance in co-culture (1.2-fold). W. succinogenes had an increased growth rate in co-culture. Other respiratory genes in W. succinogenes had increased transcriptional abundance, including formate dehydrogenase and genes involved in nitrate reduction. Transcripts for fumarate respiration were much higher than for nitrate respiration. This is the first study to show at the genome and metabolite levels that R. albus and W. succinogenes benefit from symbiotic IHT, although formate transfer may have been occurring in co-culture as well.
Issue Date:2016-07-19
Type:Thesis
URI:http://hdl.handle.net/2142/92962
Rights Information:Copyright 2016 Renae R. Geier
Date Available in IDEALS:2016-11-10
Date Deposited:2016-08


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