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Faculty & Staff

  • Image of Timothy J. Donohue

    Timothy J. Donohue

    Ira L. Baldwin Professor of Bacteriology
    UW Foundation Chairman Fetzer-Bascom Professor
    Office: 608-262-4663
    Lab: 608-265-8465
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Job Listings

notifications_active Postdoctoral Research Associate – Bacterial Pathway Design

Start and Promotion Dates

  • Assistant Professor: 1986
  • Associate Professor: 1991
  • Full Professor: 1996


B.S., Life Sciences, Polytechnic Institute of Brooklyn 1975
M.S., Microbiology, Pennsylvania State University 1977
Ph.D., Microbiology,Pennsylvania State University 1980
Postdoctoral Research: Microbiology, University of Illinois at Urbana-Champaign

Research Overview

Our laboratory analyzes networks that microbes use to grow or produce bioproducts from renewable resources. To dissect this fundamentally important problem, we dissect genomic, metabolic and regulatory pathways of bacteria that convert renewable resources, such as non-edible lignocellulosic plant biomass, into products that are currently derived from fossil fuels. By mining genome sequence databases, coupling genomic (microarrays & RNAseq, proteomics, metabolomics), computational, molecular and synthetic biology techniques, we define how carbon and energy in nutrients is partitioned into cell growth or formation of bioproducts. The metabolic pathways, signal transduction networks, transcription factors, and signals that control these processes are identified, modelled or re-engineered using mutants, in vitro systems and in silico models. Our long range goals are to understand energy-conserving pathways of societal importance, and to combine computational and experimental systems to design microbial machines with increased capacity to utilize renewable resources, or enable a green production of fuels and chemicals.


Principal Investigator and Director, Great Lakes Bioenergy Research Center
Interim Director Wisconsin Energy Institute
Biotechnology Training Program
Molecular Biosciences Training Program
Genetics Training Program
Cell and Molecular Biology Graduate Program
Microbiology Doctoral Training Program


  • 2018 - Promega Biotechnology Research Award, given by the American Academy of Microbiology
  • 2016 - UW Foundation Chairman Fetzer-Bascom Professor
  • 2013 - The American Society for Microbiology (ASM) President
  • 2009 - American Association for the Advancement of Science (AAAS) Fellow
  • 2000 - American Academy of Microbiology Fellow
  • 1993 - CALS Pound Research Award

Lab Personnel

Picture of Alberge
Francois Alberge
Picture of Fortney
Nathaniel Fortney
Picture of Hall
Benjamin Hall
Grad Student
Picture of Kontour
Wayne Kontour
Picture of Lakey
Bryan Lakey
Grad Student
Picture of Lemke
Rachelle Lemke
Sr Research Specialist
Lab Manager
Picture of Linz
Alexandra Linz
Picture of Ma
Yanjun Ma
Picture of Myers
Kevin Myers
Assistant Scientist
Picture of Walters
Kevin Walters
Grad Student

Research Papers

  • Henry KK, Ross W, Myers KS, Lemmer KC, Vera JM, Landick R, Donohue TJ, Gourse RL (2020) A majority of promoters lack a crucial RNA polymerase recognition feature, enabling coordinated transcription activation. Proceedings of the National Academy of Sciences of the United States of America : · Pubmed · DOI

    Using an in vitro transcription system with purified RNA polymerase (RNAP) to investigate rRNA synthesis in the photoheterotrophic α-proteobacterium , we identified a surprising feature of promoters recognized by the major holoenzyme. Transcription from rRNA promoters was unexpectedly weak, correlating with absence of -7T, the very highly conserved thymine found at the last position in -10 elements of promoters in most bacterial species. Thymine substitutions for adenine at position -7 in the three rRNA promoters strongly increased intrinsic promoter activity, indicating that RNAP can utilize -7T when present. rRNA promoters were activated by purified CarD, a transcription factor found in many bacterial species but not in β- and γ-proteobacteria. Overall, CarD increased the activity of 15 of 16 native promoters tested in vitro that lacked -7T, whereas it had no effect on three of the four native promoters that contained -7T. Genome-wide bioinformatic analysis of promoters from and two other α-proteobacterial species indicated that 30 to 43% contained -7T, whereas 90 to 99% of promoters from non-α-proteobacteria contained -7T. Thus, promoters lacking -7T appear to be widespread in α-proteobacteria and may have evolved away from consensus to enable their coordinated regulation by transcription factors like CarD. We observed a strong reduction in CarD levels when cells enter stationary phase, suggesting that reduced activation by CarD may contribute to inhibition of rRNA transcription when cells enter stationary phase, the stage of growth when bacterial ribosome synthesis declines.

  • Scarborough MJ, Hamilton JJ, Erb EA, Donohue TJ, Noguera DR (2020) Diagnosing and Predicting Mixed-Culture Fermentations with Unicellular and Guild-Based Metabolic Models. mSystems 5((5)): PMC7527139 · Pubmed · DOI

    Multispecies microbial communities determine the fate of materials in the environment and can be harnessed to produce beneficial products from renewable resources. In a recent example, fermentations by microbial communities have produced medium-chain fatty acids (MCFAs). Tools to predict, assess, and improve the performance of these communities, however, are limited. To provide such tools, we constructed two metabolic models of MCFA-producing microbial communities based on available genomic, transcriptomic, and metabolomic data. The first model is a unicellular model (iFermCell215), while the second model (iFermGuilds789) separates fermentation activities into functional guilds. Ethanol and lactate are fermentation products known to serve as substrates for MCFA production, while acetate is another common cometabolite during MCFA production. Simulations with iFermCell215 predict that low molar ratios of acetate to ethanol favor MCFA production, whereas the products of lactate and acetate coutilization are less dependent on the acetate-to-lactate ratio. In simulations of an MCFA-producing community fed a complex organic mixture derived from lignocellulose, iFermGuilds789 predicted that lactate was an extracellular cometabolite that served as a substrate for butyrate (C4) production. Extracellular hexanoic (C6) and octanoic (C8) acids were predicted by iFermGuilds789 to be from community members that directly metabolize sugars. Modeling results provide several hypotheses that can improve our understanding of microbial roles in an MCFA-producing microbiome and inform strategies to increase MCFA production. Further, these models represent novel tools for exploring the role of mixed microbial communities in carbon recycling in the environment, as well as in beneficial reuse of organic residues. Microbiomes are vital to human health, agriculture, and protecting the environment. Predicting behavior of self-assembled or synthetic microbiomes, however, remains a challenge. In this work, we used unicellular and guild-based metabolic models to investigate production of medium-chain fatty acids by a mixed microbial community that is fed multiple organic substrates. Modeling results provided insights into metabolic pathways of three medium-chain fatty acid-producing guilds and identified potential strategies to increase production of medium-chain fatty acids. This work demonstrates the role of metabolic models in augmenting multi-omic studies to gain greater insights into microbiome behavior.

  • Myers KS, Vera JM, Lemmer KC, Linz AM, Landick R, Noguera DR, Donohue TJ (2020) Genome-Wide Identification of Transcription Start Sites in Two , Rhodobacter sphaeroides 2.4.1 and Novosphingobium aromaticivorans DSM 12444. Microbiology resource announcements 9((36)): PMC7471390 · Pubmed · DOI

    Here, we report the genome-wide identification of transcription start sites (TSSs) from two grown under conditions that result in significant changes in gene expression. TSSs that were identified as present in one condition or both will be an important resource for future studies of these, and possibly other, .

  • Myers KS, Place M, Noguera DR, Donohue TJ (2020) COnTORT: COmprehensive Transcriptomic ORganizational Tool for Simultaneously Retrieving and Organizing Numerous Gene Expression Data Sets from the NCBI Gene Expression Omnibus Database. Microbiology resource announcements 9((25)): PMC7303417 · Pubmed · DOI

    We introduce COnTORT (mprehensive ranscriptomic ganizational ool), a publicly available program that retrieves all available gene expression data and associated metadata for an organism from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database. The data are compiled into text files that can be used for downstream bioinformatic applications.

  • Lemke RAS, Olson SM, Morse K, Karlen SD, Higbee A, Beebe ET, Ralph J, Coon JJ, Fox BG, Donohue TJ (2020) A bacterial biosynthetic pathway for methylated furan fatty acids. The Journal of biological chemistry 295((29)):9786-9801 PMC7380195 · Pubmed · DOI

    Fatty acids play many important roles in cells and also in industrial processes. Furan fatty acids (FuFAs) are present in the lipids of some plant, fish, and microbial species and appear to function as second messengers in pathways that protect cells from membrane-damaging agents. We report here the results of chemical, genetic, and synthetic biology experiments to decipher the biosynthesis of the monomethylated FuFA, methyl 9-(3-methyl-5-pentylfuran-2-yl) nonanoate (9M5-FuFA), and its dimethyl counterpart, methyl 9-(3,4-dimethyl-5-pentylfuran-2-yl) nonanoate (9D5-FuFA), in two α-proteobacteria. Each of the steps in FuFA biosynthesis occurs on pre-existing phospholipid fatty acid chains, and we identified pathway intermediates and the gene products that catalyze 9M5-FuFA and 9D5-FuFA synthesis in 2.4.1 and CGA009. One previously unknown pathway intermediate was a methylated diunsaturated fatty acid, (1012)-11-methyloctadeca-10,12-dienoic acid (11Me-10,12-18:2), produced from (11)-methyloctadeca-11-enoic acid (11Me-12-18:1) by a newly identified fatty acid desaturase, UfaD. We also show that molecular oxygen (O) is the source of the oxygen atom in the furan ring of 9M5-FuFA, and our findings predict that an O-derived oxygen atom is incorporated into 9M5-FuFA via a protein, UfaO, that uses the 11Me-1012-18:2 fatty acid phospholipid chain as a substrate. We discovered that also contains a SAM-dependent methylase, FufM, that produces 9D5-FuFA from 9M5-FuFA. These results uncover the biochemical sequence of intermediates in a bacterial pathway for 9M5-FuFA and 9D5-FuFA biosynthesis and suggest the existence of homologs of the enzymes identified here that could function in FuFA biosynthesis in other organisms.

  • Lemmer KC, Alberge F, Myers KS, Dohnalkova AC, Schaub RE, Lenz JD, Imam S, Dillard JP, Noguera DR, Donohue TJ (2020) The NtrYX Two-Component System Regulates the Bacterial Cell Envelope. mBio 11((3)): PMC7240162 · Pubmed · DOI

    Activity of the NtrYX two-component system has been associated with important processes in diverse bacteria, ranging from symbiosis to nitrogen and energy metabolism. In the facultative alphaproteobacterium , loss of the two-component system NtrYX results in increased lipid production and sensitivity to some known cell envelope-active compounds. In this study, we show that NtrYX directly controls multiple properties of the cell envelope. We find that the response regulator NtrX binds upstream of cell envelope genes, including those involved in peptidoglycan biosynthesis and modification and in cell division. We show that loss of NtrYX impacts the cellular levels of peptidoglycan precursors and lipopolysaccharide and alters cell envelope structure, increasing cell length and the thickness of the periplasm. Cell envelope function is also disrupted in the absence of NtrYX, resulting in increased outer membrane permeability. Based on the properties of cells lacking NtrYX and the target genes under direct control of this two-component system, we propose that NtrYX plays a previously undescribed, and potentially conserved, role in the assembly, structure, and function of the cell envelope in a variety of bacteria. The bacterial cell envelope provides many important functions. It protects cells from harsh environments, serves as a selective permeability barrier, houses bioenergetic functions, defines sensitivity to antibacterial agents, and plays a crucial role in biofilm formation, symbiosis, and virulence. Despite the important roles of this cellular compartment, we lack a detailed understanding of the biosynthesis and remodeling of the cell envelope. Here, we report that the two-component signaling system NtrYX is a previously undescribed regulator of cell envelope processes, providing evidence that it is directly involved in controlling transcription of genes involved in cell envelope assembly, structure, and function in this and possibly other bacteria. Thus, our data report on a newly discovered process used by bacteria to assemble and remodel the cell envelope.

  • Oshlag JZ, Ma Y, Morse K, Burger BT, Lemke RA, Karlen SD, Myers KS, Donohue TJ, Noguera DR (2019) Anaerobic Degradation of Syringic Acid by an Adapted Strain of Rhodopseudomonas palustris. Applied and environmental microbiology 86((3)): PMC6974649 · Pubmed · DOI

    While lignin represents a major fraction of the carbon in plant biomass, biological strategies to convert the components of this heterogeneous polymer into products of industrial and biotechnological value are lacking. Syringic acid (3,5-dimethoxy-4-hydroxybenzoic acid) is a by-product of lignin degradation, appearing in lignocellulosic hydrolysates, deconstructed lignin streams, and other agricultural products. CGA009 is a known degrader of phenolic compounds under photoheterotrophic conditions via the benzoyl coenzyme A (CoA) degradation (BAD) pathway. However, CGA009 is reported to be unable to metabolize -methoxylated phenolics, such as syringic acid. We isolated a strain of (strain SA008.1.07), adapted from CGA009, which can grow on syringic acid under photoheterotrophic conditions, utilizing it as a sole source of organic carbon and reducing power. An SA008.1.07 mutant with an inactive benzoyl-CoA reductase structural gene was able to grow on syringic acid, demonstrating that the metabolism of this aromatic compound is not through the BAD pathway. Comparative gene expression analyses of SA008.1.07 implicated the involvement of products of the operon (, , ), which has been described as catalyzing aerobic aromatic ring demethylation in other bacteria, in anaerobic syringic acid degradation. In addition, experiments with a deletion mutant demonstrated the involvement of the operon in anaerobic syringic acid degradation. These observations provide new insights into the anaerobic degradation of -methoxylated and other aromatics by Lignin is the most abundant aromatic polymer on Earth and a resource that could eventually substitute for fossil fuels as a source of aromatic compounds for industrial and biotechnological applications. Engineering microorganisms for the production of aromatic-based biochemicals requires detailed knowledge of the metabolic pathways for the degradation of aromatics that are present in lignin. Our isolation and analysis of a strain capable of syringic acid degradation reveal a previously unknown metabolic route for aromatic degradation in This study highlights several key features of this pathway and sets the stage for a more complete understanding of the microbial metabolic repertoire required to metabolize aromatic compounds from lignin and other renewable sources.

  • Scarborough MJ, Myers KS, Donohue TJ, Noguera DR (2019) Medium-Chain Fatty Acid Synthesis by " Weimeria bifida" gen. nov., sp. nov., and " Pseudoramibacter fermentans" sp. nov. Appl. Environ. Microbiol. 86(3): (PMC6974650) · Pubmed · DOI

    Chain elongation is emerging as a bioprocess to produce valuable medium-chain fatty acids (MCFA; 6 to 8 carbons in length) from organic waste streams by harnessing the metabolism of anaerobic microbiomes. Although our understanding of chain elongation physiology is still evolving, the reverse β-oxidation pathway has been identified as a key metabolic function to elongate the intermediate products of fermentation to MCFA. Here, we describe two uncultured chain-elongating microorganisms that were enriched in an anaerobic microbiome transforming the residues from a lignocellulosic biorefining process. Based on a multi-omic analysis, we describe " Weimeria bifida" gen. nov., sp. nov., and " Pseudoramibacter fermentans" sp. nov., both predicted to produce MCFA but using different substrates. The analysis of a time series metatranscriptomic data set suggests that " Weimeria bifida" is an effective xylose utilizer since both the pentose phosphate pathway and the bifid shunt are active. Furthermore, the metatranscriptomic data suggest that energy conservation during MCFA production in this organism is essential and occurs via the creation of an ion motive force using both the RNF complex and an energy-conserving hydrogenase. For " Pseudoramibacter fermentans," predicted to produce MCFA from lactate, the metatranscriptomic analysis reveals the activity of an electron-confurcating lactate dehydrogenase, energy conservation via the RNF complex, H production for redox balance, and glycerol utilization. A thermodynamic analysis also suggests the possibility of glycerol being a substrate for MCFA production by " Pseudoramibacter fermentans." In total, this work reveals unknown characteristics of MCFA production in two novel organisms. Chain elongation by medium-chain fatty acid (MCFA)-producing microbiomes offers an opportunity to produce valuable chemicals from organic streams that would otherwise be considered waste. However, the physiology and energetics of chain elongation are only beginning to be studied, and many of these organisms remain uncultured. We analyzed MCFA production by two uncultured organisms that were identified as the main MCFA producers in a microbial community enriched from an anaerobic digester; this characterization, which is based on meta-multi-omic analysis, complements the knowledge that has been acquired from pure-culture studies. The analysis revealed previously unreported features of the metabolism of MCFA-producing organisms.

  • Lonetto MA, Donohue TJ, Gross CA, Buttner MJ (2019) Discovery of the extracytoplasmic function σ factors. Molecular microbiology 112((2)):348-355 · Pubmed · DOI

    This special issue of Molecular Microbiology marks the 25 anniversary of the discovery of the extracytoplasmic function (ECF) σ factors, proteins that subsequently emerged as the largest group of alternative σ factors and one of the three major pillars of signal transduction in bacteria, alongside one- and two-component systems. A single bacterial genome can encode > 100 ECF σ factors, and combined with their cognate anti-σ factors, they represent a modular design that primarily functions in transmembrane signal transduction. Here, we first describe the immediate events that led to the 1994 publication in the Proceeding of the National Academy of Sciences USA, and then set them in the broader context of key events in the history of σ biology research.

  • Donohue TJ (2019) Shedding light on a Group IV (ECF11) alternative σ factor. Molecular microbiology 112((2)):374-384 PMC6852236 · Pubmed · DOI

    This year marks the 50 anniversary of the discovery of σ as a protein factor that was needed for bacterial RNA polymerase to accurately transcribe a promoter in vitro. It was 25 years later that the Group IV alternative σs were described as a distinct family of proteins related to σ . In the intervening time, there has been an ever-growing list of Group IV σs, numbers of genes they transcribe, insight into the diverse suite of processes they control, and appreciation for their impact on bacterial lifestyles. This work summarizes knowledge of the Rhodobacter sphaeroides σ -ChrR pair, a member of the ECF11 subfamily of Group IV alternative σs, in protecting cells from the reactive oxygen species, singlet oxygen. It describes lessons learned from analyzing ChrR, a zinc-dependent anti-σ factor, that are generally applicable to Group IV σs and relevant to the response to single oxygen. This MicroReview also illustrates insights into stress responses in this and other bacteria that have been acquired by analyzing or modeling the activity of the σ -ChrR across the bacterial phylogeny.

  • Perez JM, Kontur WS, Alherech M, Coplien J, Karlen SD, Stahl SS, Donohue TJ, Noguera DR (2019) Funneling aromatic products of chemically depolymerized lignin into 2-pyrone-4-6-dicarboxylic acid with Novosphingobium aromaticivorans. Green Chem 21:1340-1350 · DOI

    Lignin is an aromatic heteropolymer found in plant biomass. Depolymerization of lignin, either through biological or chemical means, invariably produces heterogenous mixtures of low molecular weight aromatic compounds. Microbes that can metabolize lignin-derived aromatics have evolved pathways that funnel these heterogeneous mixtures into a few common intermediates before opening the aromatic ring. In this work, we engineered Novosphingobium aromaticivorans DSM12444, via targeted gene deletions, to use its native funneling pathways to simultaneously convert plant-derived aromatic compounds containing syringyl (S), guaiacyl (G), and p-hydroxyphenyl (H) aromatic units into 2-pyrone-4,6-dicarboxylic acid (PDC), a potential polyester precursor. In batch cultures containing defined media, the engineered strain converted
    several of these depolymerization products, including S-diketone and G-diketone (non-natural compounds specifically produced by chemical depolymerization), into PDC with yields ranging from 22% to 100%. In batch cultures containing a heterogeneous mixture of aromatic monomers derived from chemical depolymerization of poplar lignin, 59% of the measured aromatic compounds were converted to PDC. Overall, our results show that N. aromaticivorans has ideal characteristics for its use as a microbial platform for funneling heterogeneous mixtures of lignin depolymerization products into PDC or other commodity chemicals.

  • Kontur WS, Olmsted CN, Yusko LM, Niles AV, Walters KA, Beebe ET, Vander Meulen KA, Karlen SD, Gall DL, Noguera DR, Donohue TJ (2018) A heterodimeric glutathione -transferase that stereospecifically breaks lignin's β()-aryl ether bond reveals the diversity of bacterial β-etherases. The Journal of biological chemistry 294((6)):1877-1890 PMC6369299 · Pubmed · DOI

    Lignin is a heterogeneous polymer of aromatic subunits that is a major component of lignocellulosic plant biomass. Understanding how microorganisms deconstruct lignin is important for understanding the global carbon cycle and could aid in developing systems for processing plant biomass into valuable commodities. Sphingomonad bacteria use stereospecific glutathione -transferases (GSTs) called β-etherases to cleave the β-aryl ether (β-O-4) bond, the most common bond between aromatic subunits in lignin. Previously characterized bacterial β-etherases are homodimers that fall into two distinct GST subclasses: LigE homologues, which cleave the β() stereoisomer of the bond, and LigF homologues, which cleave the β() stereoisomer. Here, we report on a heterodimeric β-etherase (BaeAB) from the sphingomonad that stereospecifically cleaves the β()-aryl ether bond of the di-aromatic compound β-(2-methoxyphenoxy)-γ-hydroxypropiovanillone (MPHPV). BaeAB's subunits are phylogenetically distinct from each other and from other β-etherases, although they are evolutionarily related to LigF, despite the fact that BaeAB and LigF cleave different β-aryl ether bond stereoisomers. We identify amino acid residues in BaeAB's BaeA subunit important for substrate binding and catalysis, including an asparagine that is proposed to activate the GSH cofactor. We also show that BaeAB homologues from other sphingomonads can cleave β()-MPHPV and that they may be as common in bacteria as LigE homologues. Our results suggest that the ability to cleave the β-aryl ether bond arose independently at least twice in GSTs and that BaeAB homologues may be important for cleaving the β()-aryl ether bonds of lignin-derived oligomers in nature.

  • Scarborough MJ, Lawson CE, Hamilton JJ, Donohue TJ, Noguera DR (2018) Metatranscriptomic and Thermodynamic Insights into Medium-Chain Fatty Acid Production Using an Anaerobic Microbiome. mSystems 3(6): (PMC6247018) · Pubmed · DOI

    Biomanufacturing from renewable feedstocks can offset fossil fuel-based chemical production. One potential biomanufacturing strategy is production of medium-chain fatty acids (MCFA) from organic feedstocks using either pure cultures or microbiomes. While the set of microbes in a microbiome can often metabolize organic materials of greater diversity than a single species can and while the role of specific species may be known, knowledge of the carbon and energy flow within and between organisms in MCFA-producing microbiomes is only now starting to emerge. Here, we integrated metagenomic, metatranscriptomic, and thermodynamic analyses to predict and characterize the metabolic network of an anaerobic microbiome producing MCFA from organic matter derived from lignocellulosic ethanol fermentation conversion residue. A total of 37 high-quality (>80% complete, <10% contamination) metagenome-assembled genomes (MAGs) were recovered from the microbiome, and metabolic reconstruction of the 10 most abundant MAGs was performed. Metabolic reconstruction combined with metatranscriptomic analysis predicted that organisms affiliated with and would degrade carbohydrates and ferment sugars to lactate and acetate. - and -affiliated organisms were predicted to transform these fermentation products to MCFA. Thermodynamic analyses identified conditions under which H is expected to be either produced or consumed, suggesting a potential role of H partial pressure in MCFA production. From an integrated systems analysis perspective, we propose that MCFA production could be improved if microbiomes were engineered to use homofermentative instead of heterofermentative and if MCFA-producing organisms were engineered to preferentially use a thioesterase instead of a coenzyme A (CoA) transferase as the terminal enzyme in reverse β-oxidation. Mixed communities of microbes play important roles in health, the environment, agriculture, and biotechnology. While tapping the combined activities of organisms within microbiomes may allow the utilization of a wider range of substrates in preference to the use of pure cultures for biomanufacturing, harnessing the metabolism of these mixed cultures remains a major challenge. Here, we predicted metabolic functions of bacteria in a microbiome that produces medium-chain fatty acids from a renewable feedstock. Our findings lay the foundation for efforts to begin addressing how to engineer and control microbiomes for improved biomanufacturing, how to build synthetic mixtures of microbes that produce valuable chemicals from renewable resources, and how to better understand the microbial communities that contribute to health, agriculture, and the environment.

  • Scarborough MJ, Lynch G, Dickson M, McGee M, Donohue TJ, Noguera DR (2018) Increasing the economic value of lignocellulosic stillage through medium-chain fatty acid production. Biotechnol Biofuels 11:200 (PMC6052542) · Pubmed · DOI

    Lignocellulosic biomass is seen as an abundant renewable source of liquid fuels and chemicals that are currently derived from petroleum. When lignocellulosic biomass is used for ethanol production, the resulting liquid residue (stillage) contains large amounts of organic material that could be further transformed into recoverable bioproducts, thus enhancing the economics of the biorefinery. Here we test the hypothesis that a bacterial community could transform the organics in stillage into valuable bioproducts. We demonstrate the ability of this microbiome to convert stillage organics into medium-chain fatty acids (MCFAs), identify the predominant community members, and perform a technoeconomic analysis of recovering MCFAs as co-products of ethanol production. Steady-state operation of a stillage-fed bioreactor showed that 18% of the organic matter in stillage was converted to MCFAs. Xylose and complex carbohydrates were the primary substrates transformed. During the MCFA production period, the five major genera represented more than 95% of the community, including , , , , and . To assess the potential benefits of producing MCFAs from stillage, we modeled the economics of ethanol and MCFA co-production, at MCFA productivities observed during reactor operation. The analysis predicts that production of MCFAs, ethanol, and electricity could reduce the minimum ethanol selling price from $2.15 to $1.76 gal ($2.68 gal gasoline equivalents) when compared to a lignocellulosic biorefinery that produces only ethanol and electricity.

  • Kontur WS, Bingman CA, Olmsted CN, Wassarman DR, Ulbrich A, Gall DL, Smith RW, Yusko LM, Fox BG, Noguera DR, Coon JJ, Donohue TJ (2018) uses a Nu-class glutathione -transferase as a glutathione lyase in breaking the β-aryl ether bond of lignin. J. Biol. Chem. 293(14):4955-4968 (PMC5892560) · Pubmed · DOI

    As a major component of plant cell walls, lignin is a potential renewable source of valuable chemicals. Several sphingomonad bacteria have been identified that can break the β-aryl ether bond connecting most phenylpropanoid units of the lignin heteropolymer. Here, we tested three sphingomonads predicted to be capable of breaking the β-aryl ether bond of the dimeric aromatic compound guaiacylglycerol-β-guaiacyl ether (GGE) and found that metabolizes GGE at one of the fastest rates thus far reported. After the ether bond of racemic GGE is broken by replacement with a thioether bond involving glutathione, the glutathione moiety must be removed from the resulting two stereoisomers of the phenylpropanoid conjugate β-glutathionyl-γ-hydroxypropiovanillone (GS-HPV). We found that the Nu-class glutathione -transferase NaGST is the only enzyme needed to remove glutathione from both ()- and ()-GS-HPV in We solved the crystal structure of NaGST and used molecular modeling to propose a mechanism for the glutathione lyase (deglutathionylation) reaction in which an enzyme-stabilized glutathione thiolate attacks the thioether bond of GS-HPV, and the reaction proceeds through an enzyme-stabilized enolate intermediate. Three residues implicated in the proposed mechanism (Thr, Tyr, and Tyr) were found to be critical for the lyase reaction. We also found that Nu-class GSTs from sp. SYK-6 (which can also break the β-aryl ether bond) and (which cannot break the β-aryl ether bond) can also cleave ()- and ()-GS-HPV, suggesting that glutathione lyase activity may be common throughout this widespread but largely uncharacterized class of glutathione -transferases.

  • Gall DL, Kontur WS, Lan W, Kim H, Li Y, Ralph J, Donohue TJ, Noguera DR (2017) Enzymatic Depolymerization of Lignin with Release of Syringyl, Guaiacyl, and Tricin Units. Appl. Environ. Microbiol. 84(3): (PMC5772236) · Pubmed · DOI

    New environmentally sound technologies are needed to derive valuable compounds from renewable resources. Lignin, an abundant polymer in terrestrial plants comprised predominantly of guaiacyl and syringyl monoaromatic phenylpropanoid units, is a potential natural source of aromatic compounds. In addition, the plant secondary metabolite tricin is a recently discovered and moderately abundant flavonoid in grasses. The most prevalent interunit linkage between guaiacyl, syringyl, and tricin units is the β-ether linkage. Previous studies have shown that bacterial β-etherase pathway enzymes catalyze glutathione-dependent cleavage of β-ether bonds in dimeric β-ether lignin model compounds. To date, however, it remains unclear whether the known β-etherase enzymes are active on lignin polymers. Here we report on enzymes that catalyze β-ether cleavage from bona fide lignin, under conditions that recycle the cosubstrates NAD and glutathione. Guaiacyl, syringyl, and tricin derivatives were identified as reaction products when different model compounds or lignin fractions were used as substrates. These results demonstrate an enzymatic system that can recycle cosubstrates while releasing aromatic monomers from model compounds as well as natural and engineered lignin oligomers. These findings can improve the ability to produce valuable aromatic compounds from a renewable resource like lignin. Many bacteria are predicted to contain enzymes that could convert renewable carbon sources into substitutes for compounds that are derived from petroleum. The β-etherase pathway present in sphingomonad bacteria could cleave the abundant β-O-4-aryl ether bonds in plant lignin, releasing a biobased source of aromatic compounds for the chemical industry. However, the activity of these enzymes on the complex aromatic oligomers found in plant lignin is unknown. Here we demonstrate biodegradation of lignin polymers using a minimal set of β-etherase pathway enzymes, the ability to recycle needed cofactors (glutathione and NAD) , and the release of guaiacyl, syringyl, and tricin as depolymerized products from lignin. These observations provide critical evidence for the use and future optimization of these bacterial β-etherase pathway enzymes for industrial-level biotechnological applications designed to derive high-value monomeric aromatic compounds from lignin.

  • Burger BT, Imam S, Scarborough MJ, Noguera DR, Donohue TJ (2017) Combining Genome-Scale Experimental and Computational Methods To Identify Essential Genes in . mSystems 2(3): (PMC5513736) · Pubmed · DOI

    is one of the best-studied alphaproteobacteria from biochemical, genetic, and genomic perspectives. To gain a better systems-level understanding of this organism, we generated a large transposon mutant library and used transposon sequencing (Tn-seq) to identify genes that are essential under several growth conditions. Using newly developed Tn-seq analysis software (TSAS), we identified 493 genes as essential for aerobic growth on a rich medium. We then used the mutant library to identify conditionally essential genes under two laboratory growth conditions, identifying 85 additional genes required for aerobic growth in a minimal medium and 31 additional genes required for photosynthetic growth. In all instances, our analyses confirmed essentiality for many known genes and identified genes not previously considered to be essential. We used the resulting Tn-seq data to refine and improve a genome-scale metabolic network model (GEM) for . Together, we demonstrate how genetic, genomic, and computational approaches can be combined to obtain a systems-level understanding of the genetic framework underlying metabolic diversity in bacterial species. Knowledge about the role of genes under a particular growth condition is required for a holistic understanding of a bacterial cell and has implications for health, agriculture, and biotechnology. We developed the Tn-seq analysis software (TSAS) package to provide a flexible and statistically rigorous workflow for the high-throughput analysis of insertion mutant libraries, advanced the knowledge of gene essentiality in , and illustrated how Tn-seq data can be used to more accurately identify genes that play important roles in metabolism and other processes that are essential for cellular survival.

  • Lemmer KC, Zhang W, Langer SJ, Dohnalkova AC, Hu D, Lemke RA, Piotrowski JS, Orr G, Noguera DR, Donohue TJ (2017) Mutations That Alter the Bacterial Cell Envelope Increase Lipid Production. MBio 8(3): (PMC5442454) · Pubmed · DOI

    Lipids from microbes offer a promising source of renewable alternatives to petroleum-derived compounds. In particular, oleaginous microbes are of interest because they accumulate a large fraction of their biomass as lipids. In this study, we analyzed genetic changes that alter lipid accumulation in By screening an Tn mutant library for insertions that increased fatty acid content, we identified 10 high-lipid (HL) mutants for further characterization. These HL mutants exhibited increased sensitivity to drugs that target the bacterial cell envelope and changes in shape, and some had the ability to secrete lipids, with two HL mutants accumulating ~60% of their total lipids extracellularly. When one of the highest-lipid-secreting strains was grown in a fed-batch bioreactor, its lipid content was comparable to that of oleaginous microbes, with the majority of the lipids secreted into the medium. Based on the properties of these HL mutants, we conclude that alterations of the cell envelope are a previously unreported approach to increase microbial lipid production. We also propose that this approach may be combined with knowledge about biosynthetic pathways, in this or other microbes, to increase production of lipids and other chemicals. This paper reports on experiments to understand how to increase microbial lipid production. Microbial lipids are often cited as one renewable replacement for petroleum-based fuels and chemicals, but strategies to increase the yield of these compounds are needed to achieve this goal. While lipid biosynthesis is often well understood, increasing yields of these compounds to industrially relevant levels is a challenge, especially since genetic, synthetic biology, or engineering approaches are not feasible in many microbes. We show that altering the bacterial cell envelope can be used to increase microbial lipid production. We also find that the utility of some of these alterations can be enhanced by growing cells in bioreactor configurations that can be used industrially. We propose that our findings can inform current and future efforts to increase production of microbial lipids, other fuels, or chemicals that are currently derived from petroleum.

  • Gall DL, Ralph J, Donohue TJ, Noguera DR (2017) Biochemical transformation of lignin for deriving valued commodities from lignocellulose. Curr. Opin. Biotechnol. 45:120-126 · Pubmed · DOI

    The biochemical properties of lignin present major obstacles to deriving societally beneficial entities from lignocellulosic biomass, an abundant and renewable feedstock. Similar to other biopolymers such as polysaccharides, polypeptides, and ribonucleic acids, lignin polymers are derived from multiple types of monomeric units. However, lignin's renowned recalcitrance is largely attributable to its racemic nature and the variety of covalent inter-unit linkages through which its aromatic monomers are linked. Indeed, unlike other biopolymers whose monomers are consistently inter-linked by a single type of covalent bond, the monomeric units in lignin are linked via non-enzymatic, combinatorial radical coupling reactions that give rise to a variety of inter-unit covalent bonds in mildly branched racemic polymers. Yet, despite the chemical complexity and stability of lignin, significant strides have been made in recent years to identify routes through which valued commodities can be derived from it. This paper discusses emerging biological and biochemical means through which degradation of lignin to aromatic monomers can lead to the derivation of commercially valuable products.

  • Warwick Anderson, Rolf Apweiler, Alex Bateman, Guntram A Bauer, Helen Berman, Judith A Blake, Niklas Blomberg, Stephen K Burley, Guy Cochrane, Valentina Di Francesco, Tim Donohue, Christine Durinx, Alfred Game, Eric Green, Takashi Gojobori, Peter Goodhand, Ada Hamosh, Henning Hermjakob, Minoru Kanehisa, Robert Kiley, Johanna McEntyre, Rowan McKibbin, Satoru Miyano, Barbara Pauly, Norbert Perrimon, Mark A Ragan, Geoffrey Richards, Yik-Ying Teo, Monte Westerfield, Eric Westhof, Paul F Lasko (2017) Towards Coordinated International Support Of Core Data Resources For The Life Sciences BioRxiv : · DOI

    On November 18-19, 2016, the Human Frontier Science Program Organization (HFSPO) hosted a meeting of senior managers of key data resources and leaders of several major funding organizations to discuss the challenges associated with sustaining biological and biomedical (i.e., life sciences) data resources and associated infrastructure. A strong consensus emerged from the group that core data resources for the life sciences should be supported through a coordinated international effort(s) that better ensure long-term sustainability and that appropriately align funding with scientific impact. Ideally, funding for such data resources should allow for access at no charge, as is presently the usual (and preferred) mechanism. Below, the rationale for this vision is described, and some important considerations for developing a new international funding model to support core data resources for the life sciences are presented.

  • Blaser MJ, Cardon ZG, Cho MK, Dangl JL, Donohue TJ, Green JL, Knight R, Maxon ME, Northen TR, Pollard KS, Brodie EL (2016) Toward a Predictive Understanding of Earth's Microbiomes to Address 21st Century Challenges. MBio 7(3): (PMC4895116) · Pubmed

    Microorganisms have shaped our planet and its inhabitants for over 3.5 billion years. Humankind has had a profound influence on the biosphere, manifested as global climate and land use changes, and extensive urbanization in response to a growing population. The challenges we face to supply food, energy, and clean water while maintaining and improving the health of our population and ecosystems are significant. Given the extensive influence of microorganisms across our biosphere, we propose that a coordinated, cross-disciplinary effort is required to understand, predict, and harness microbiome function. From the parallelization of gene function testing to precision manipulation of genes, communities, and model ecosystems and development of novel analytical and simulation approaches, we outline strategies to move microbiome research into an era of causality. These efforts will improve prediction of ecosystem response and enable the development of new, responsible, microbiome-based solutions to significant challenges of our time.

  • Pereira JH, Heins RA, Gall DL, McAndrew RP, Deng K, Holland KC, Donohue TJ, Noguera DR, Simmons BA, Sale KL, Ralph J, Adams PD (2016) Structural and Biochemical Characterization of the Early and Late Enzymes in the Lignin β-Aryl Ether Cleavage Pathway from Sphingobium sp. SYK-6. J. Biol. Chem. 291(19):10228-38 (PMC4858972) · Pubmed

    There has been great progress in the development of technology for the conversion of lignocellulosic biomass to sugars and subsequent fermentation to fuels. However, plant lignin remains an untapped source of materials for production of fuels or high value chemicals. Biological cleavage of lignin has been well characterized in fungi, in which enzymes that create free radical intermediates are used to degrade this material. In contrast, a catabolic pathway for the stereospecific cleavage of β-aryl ether units that are found in lignin has been identified in Sphingobium sp. SYK-6 bacteria. β-Aryl ether units are typically abundant in lignin, corresponding to 50-70% of all of the intermonomer linkages. Consequently, a comprehensive understanding of enzymatic β-aryl ether (β-ether) cleavage is important for future efforts to biologically process lignin and its breakdown products. The crystal structures and biochemical characterization of the NAD-dependent dehydrogenases (LigD, LigO, and LigL) and the glutathione-dependent lyase LigG provide new insights into the early and late enzymes in the β-ether degradation pathway. We present detailed information on the cofactor and substrate binding sites and on the catalytic mechanisms of these enzymes, comparing them with other known members of their respective families. Information on the Lig enzymes provides new insight into their catalysis mechanisms and can inform future strategies for using aromatic oligomers derived from plant lignin as a source of valuable aromatic compounds for biofuels and other bioproducts.

  • Spero MA, Brickner JR, Mollet JT, Pisithkul T, Amador-Noguez D, Donohue TJ (2016) Different Functions of Phylogenetically Distinct Bacterial Complex I Isozymes. J. Bacteriol. 198(8):1268-80 (PMC4859585) · Pubmed

    NADH:quinone oxidoreductase (complex I) is a bioenergetic enzyme that transfers electrons from NADH to quinone, conserving the energy of this reaction by contributing to the proton motive force. While the importance of NADH oxidation to mitochondrial aerobic respiration is well documented, the contribution of complex I to bacterial electron transport chains has been tested in only a few species. Here, we analyze the function of two phylogenetically distinct complex I isozymes in Rhodobacter sphaeroides, an alphaproteobacterium that contains well-characterized electron transport chains. We found that R. sphaeroides complex I activity is important for aerobic respiration and required for anaerobic dimethyl sulfoxide (DMSO) respiration (in the absence of light), photoautotrophic growth, and photoheterotrophic growth (in the absence of an external electron acceptor). Our data also provide insight into the functions of the phylogenetically distinct R. sphaeroidescomplex I enzymes (complex IA and complex IE) in maintaining a cellular redox state during photoheterotrophic growth. We propose that the function of each isozyme during photoheterotrophic growth is either NADH synthesis (complex IA) or NADH oxidation (complex IE). The canonical alphaproteobacterial complex I isozyme (complex IA) was also shown to be important for routing electrons to nitrogenase-mediated H2 production, while the horizontally acquired enzyme (complex IE) was dispensable in this process. Unlike the singular role of complex I in mitochondria, we predict that the phylogenetically distinct complex I enzymes found across bacterial species have evolved to enhance the functions of their respective electron transport chains. Cells use a proton motive force (PMF), NADH, and ATP to support numerous processes. In mitochondria, complex I uses NADH oxidation to generate a PMF, which can drive ATP synthesis. This study analyzed the function of complex I in bacteria, which contain more-diverse and more-flexible electron transport chains than mitochondria. We tested complex I function in Rhodobacter sphaeroides, a bacterium predicted to encode two phylogenetically distinct complex I isozymes. R. sphaeroides cells lacking both isozymes had growth defects during all tested modes of growth, illustrating the important function of this enzyme under diverse conditions. We conclude that the two isozymes are not functionally redundant and predict that phylogenetically distinct complex I enzymes have evolved to support the diverse lifestyles of bacteria.

  • Helmich KE, Pereira JH, Gall DL, Heins RA, McAndrew RP, Bingman C, Deng K, Holland KC, Noguera DR, Simmons BA, Sale KL, Ralph J, Donohue TJ, Adams PD, Phillips GN (2015) Structural Basis of Stereospecificity in the Bacterial Enzymatic Cleavage of β-Aryl Ether Bonds in Lignin. J. Biol. Chem. 291(10):5234-46 (PMC4777856) · Pubmed

    Lignin is a combinatorial polymer comprising monoaromatic units that are linked via covalent bonds. Although lignin is a potential source of valuable aromatic chemicals, its recalcitrance to chemical or biological digestion presents major obstacles to both the production of second-generation biofuels and the generation of valuable coproducts from lignin's monoaromatic units. Degradation of lignin has been relatively well characterized in fungi, but it is less well understood in bacteria. A catabolic pathway for the enzymatic breakdown of aromatic oligomers linked via β-aryl ether bonds typically found in lignin has been reported in the bacterium Sphingobium sp. SYK-6. Here, we present x-ray crystal structures and biochemical characterization of the glutathione-dependent β-etherases, LigE and LigF, from this pathway. The crystal structures show that both enzymes belong to the canonical two-domain fold and glutathione binding site architecture of the glutathione S-transferase family. Mutagenesis of the conserved active site serine in both LigE and LigF shows that, whereas the enzymatic activity is reduced, this amino acid side chain is not absolutely essential for catalysis. The results include descriptions of cofactor binding sites, substrate binding sites, and catalytic mechanisms. Because β-aryl ether bonds account for 50-70% of all interunit linkages in lignin, understanding the mechanism of enzymatic β-aryl ether cleavage has significant potential for informing ongoing studies on the valorization of lignin.

  • Lin TY, Santos TM, Kontur WS, Donohue TJ, Weibel DB (2015) A Cardiolipin-Deficient Mutant of Rhodobacter sphaeroides Has an Altered Cell Shape and Is Impaired in Biofilm Formation. J. Bacteriol. 197(21):3446-55 (PMC4621061) · Pubmed

    Cell shape has been suggested to play an important role in the regulation of bacterial attachment to surfaces and the formation of communities associated with surfaces. We found that a cardiolipin synthase (Δcls) mutant of the rod-shaped bacterium Rhodobacter sphaeroides--in which synthesis of the anionic, highly curved phospholipid cardiolipin (CL) is reduced by 90%--produces ellipsoid-shaped cells that are impaired in biofilm formation. Reducing the concentration of CL did not cause significant defects in R. sphaeroides cell growth, swimming motility, lipopolysaccharide and exopolysaccharide production, surface adhesion protein expression, and membrane permeability. Complementation of the CL-deficient mutant by ectopically expressing CL synthase restored cells to their rod shape and increased biofilm formation. Treating R. sphaeroides cells with a low concentration (10 μg/ml) of the small-molecule MreB inhibitor S-(3,4-dichlorobenzyl)isothiourea produced ellipsoid-shaped cells that had no obvious growth defect yet reduced R. sphaeroides biofilm formation. This study demonstrates that CL plays a role in R. sphaeroides cell shape determination, biofilm formation, and the ability of the bacterium to adapt to its environment. Membrane composition plays a fundamental role in the adaptation of many bacteria to environmental stress. In this study, we build a new connection between the anionic phospholipid cardiolipin (CL) and cellular adaptation in Rhodobacter sphaeroides. We demonstrate that CL plays a role in the regulation of R. sphaeroides morphology and is important for the ability of this bacterium to form biofilms. This study correlates CL concentration, cell shape, and biofilm formation and provides the first example of how membrane composition in bacteria alters cell morphology and influences adaptation. This study also provides insight into the potential of phospholipid biosynthesis as a target for new chemical strategies designed to alter or prevent biofilm formation.

  • Alivisatos AP, Blaser MJ, Brodie EL, Chun M, Dangl JL, Donohue TJ, Dorrestein PC, Gilbert JA, Green JL, Jansson JK, Knight R, Maxon ME, McFall-Ngai MJ, Miller JF, Pollard KS, Ruby EG, Taha SA, Alivisatos AP, Balskus EP, Biteen JS, Blaser MJ, Brodie EL, Browning ND, Cardon ZG, Cavanaugh CM, Chun M, Cliffel DE, Colwell RR, Dangl JL, Donohue TJ, Dorrestein PC, Fraser SE, Friesen ML, Gilbert JA, Gilbert SF, Green JL, Harwood CS, Henriksen JR, Highlander SK, Huang Y, Jansson JK, Johnson AT, Kasper DL, Knight R, Kujawinski EB, Martin CL, Maxon ME, McFall-Ngai MJ, Miller JF, Moran MA, Nelson KE, Orphan VJ, Ozcan A, Paša-Tolić L, Pollard KS, Regev A, Rubin EM, Ruby EG, Segre JA, Silver PA, Taha SA, Vivanco JM, Weinstock GM, Weiss PS, Yang P (2015) MICROBIOME. A unified initiative to harness Earth's microbiomes. Science 350(6260):507-8 · Pubmed

    No abstract available.

  • Austin S, Kontur WS, Ulbrich A, Oshlag JZ, Zhang W, Higbee A, Zhang Y, Coon JJ, Hodge DB, Donohue TJ, Noguera DR (2015) Metabolism of Multiple Aromatic Compounds in Corn Stover Hydrolysate by Rhodopseudomonas palustris. Environ. Sci. Technol. 49(14):8914-22 (PMC5031247) · Pubmed

    Lignocellulosic biomass hydrolysates hold great potential as a feedstock for microbial biofuel production, due to their high concentration of fermentable sugars. Present at lower concentrations are a suite of aromatic compounds that can inhibit fermentation by biofuel-producing microbes. We have developed a microbial-mediated strategy for removing these aromatic compounds, using the purple nonsulfur bacterium Rhodopseudomonas palustris. When grown photoheterotrophically in an anaerobic environment, R. palustris removes most of the aromatics from ammonia fiber expansion (AFEX) treated corn stover hydrolysate (ACSH), while leaving the sugars mostly intact. We show that R. palustris can metabolize a host of aromatic substrates in ACSH that have either been previously described as unable to support growth, such as methoxylated aromatics, and those that have not yet been tested, such as aromatic amides. Removing the aromatics from ACSH with R. palustris, allowed growth of a second microbe that could not grow in the untreated ACSH. By using defined mutants, we show that most of these aromatic compounds are metabolized by the benzoyl-CoA pathway. We also show that loss of enzymes in the benzoyl-CoA pathway prevents total degradation of the aromatics in the hydrolysate, and instead allows for biological transformation of this suite of aromatics into selected aromatic compounds potentially recoverable as an additional bioproduct.

  • Lemmer KC, Dohnalkova AC, Noguera DR, Donohue TJ (2015) Oxygen-dependent regulation of bacterial lipid production. J. Bacteriol. 197(9):1649-58 (PMC4403652) · Pubmed

    Understanding the mechanisms of lipid accumulation in microorganisms is important for several reasons. In addition to providing insight into assembly of biological membranes, lipid accumulation has important applications in the production of renewable fuels and chemicals. The photosynthetic bacterium Rhodobacter sphaeroides is an attractive organism to study lipid accumulation, as it has the ability to increase membrane production at low O2 tensions. Under these conditions, R. sphaeroides develops invaginations of the cytoplasmic membrane to increase its membrane surface area for housing of the membrane-bound components of its photosynthetic apparatus. Here we use fatty acid levels as a reporter of membrane lipid content. We show that, under low-O2 and anaerobic conditions, the total fatty acid content per cell increases 3-fold. We also find that the increases in the amount of fatty acid and photosynthetic pigment per cell are correlated as O2 tensions or light intensity are changed. To ask if lipid and pigment accumulation were genetically separable, we analyzed strains with mutations in known photosynthetic regulatory pathways. While a strain lacking AppA failed to induce photosynthetic pigment-protein complex accumulation, it increased fatty acid content under low-O2 conditions. We also found that an intact PrrBA pathway is required for low-O2-induced fatty acid accumulation. Our findings suggest a previously unknown role of R. sphaeroides transcriptional regulators in increasing fatty acid and phospholipid accumulation in response to decreased O2 tension. Lipids serve important functions in living systems, either as structural components of membranes or as a form of carbon storage. Understanding the mechanisms of lipid accumulation in microorganisms is important for providing insight into the assembly of biological membranes and additionally has important applications in the production of renewable fuels and chemicals. In this study, we investigate the ability of Rhodobacter sphaeroides to increase membrane production at low O2 tensions in order to house its photosynthetic apparatus. We demonstrate that this bacterium has a mechanism to increase lipid content in response to decreased O2 tension and identify a transcription factor necessary for this response. This is significant because it identifies a transcriptional regulatory pathway that can increase microbial lipid content.

  • Spero MA, Aylward FO, Currie CR, Donohue TJ (2015) Phylogenomic analysis and predicted physiological role of the proton-translocating NADH:quinone oxidoreductase (complex I) across bacteria. MBio 6(2): (PMC4453560) · Pubmed

    The proton-translocating NADH:quinone oxidoreductase (complex I) is a multisubunit integral membrane enzyme found in the respiratory chains of both bacteria and eukaryotic organelles. Although much research has focused on the enzyme's central role in the mitochondrial respiratory chain, comparatively little is known about its role in the diverse energetic lifestyles of different bacteria. Here, we used a phylogenomic approach to better understand the distribution of complex I across bacteria, the evolution of this enzyme, and its potential roles in shaping the physiology of different bacterial groups. By surveying 970 representative bacterial genomes, we predict complex I to be present in ~50% of bacteria. While this includes bacteria with a wide range of energetic schemes, the presence of complex I is associated with specific lifestyles, including aerobic respiration and specific types of phototrophy (bacteria with only a type II reaction center). A phylogeny of bacterial complex I revealed five main clades of enzymes whose evolution is largely congruent with the evolution of the bacterial groups that encode complex I. A notable exception includes the gammaproteobacteria, whose members encode one of two distantly related complex I enzymes predicted to participate in different types of respiratory chains (aerobic versus anaerobic). Comparative genomic analyses suggest a broad role for complex I in reoxidizing NADH produced from various catabolic reactions, including the tricarboxylic acid (TCA) cycle and fatty acid beta-oxidation. Together, these findings suggest diverse roles for complex I across bacteria and highlight the importance of this enzyme in shaping diverse physiologies across the bacterial domain. Living systems use conserved energy currencies, including a proton motive force (PMF), NADH, and ATP. The respiratory chain enzyme, complex I, connects these energy currencies by using NADH produced during nutrient breakdown to generate a PMF, which is subsequently used for ATP synthesis. Our goal is to better understand the role of complex I in bacteria, whose energetic diversity allows us to view its function in a range of biological contexts. We analyzed sequenced bacterial genomes to predict the presence, evolution, and function of complex I in bacteria. We identified five main classes of bacterial complex I and predict that different classes participate in different types of respiratory chains (aerobic and anaerobic). We also predict that complex I helps maintain a cellular redox state by reoxidizing NADH produced from central metabolism. Our findings suggest diverse roles for complex I in bacterial physiology, highlighting the need for future laboratory-based studies.

  • Imam S, Noguera DR, Donohue TJ (2015) An integrated approach to reconstructing genome-scale transcriptional regulatory networks. PLoS Comput. Biol. 11(2):e1004103 (PMC4344238) · Pubmed

    Transcriptional regulatory networks (TRNs) program cells to dynamically alter their gene expression in response to changing internal or environmental conditions. In this study, we develop a novel workflow for generating large-scale TRN models that integrates comparative genomics data, global gene expression analyses, and intrinsic properties of transcription factors (TFs). An assessment of this workflow using benchmark datasets for the well-studied γ-proteobacterium Escherichia coli showed that it outperforms expression-based inference approaches, having a significantly larger area under the precision-recall curve. Further analysis indicated that this integrated workflow captures different aspects of the E. coli TRN than expression-based approaches, potentially making them highly complementary. We leveraged this new workflow and observations to build a large-scale TRN model for the α-Proteobacterium Rhodobacter sphaeroides that comprises 120 gene clusters, 1211 genes (including 93 TFs), 1858 predicted protein-DNA interactions and 76 DNA binding motifs. We found that ~67% of the predicted gene clusters in this TRN are enriched for functions ranging from photosynthesis or central carbon metabolism to environmental stress responses. We also found that members of many of the predicted gene clusters were consistent with prior knowledge in R. sphaeroides and/or other bacteria. Experimental validation of predictions from this R. sphaeroides TRN model showed that high precision and recall was also obtained for TFs involved in photosynthesis (PpsR), carbon metabolism (RSP_0489) and iron homeostasis (RSP_3341). In addition, this integrative approach enabled generation of TRNs with increased information content relative to R. sphaeroides TRN models built via other approaches. We also show how this approach can be used to simultaneously produce TRN models for each related organism used in the comparative genomics analysis. Our results highlight the advantages of integrating comparative genomics of closely related organisms with gene expression data to assemble large-scale TRN models with high-quality predictions.

  • Imam S, Fitzgerald CM, Cook EM, Donohue TJ, Noguera DR (2015) Quantifying the effects of light intensity on bioproduction and maintenance energy during photosynthetic growth of Rhodobacter sphaeroides. Photosyn. Res. 123(2):167-82 · Pubmed

    Obtaining a better understanding of the physiology and bioenergetics of photosynthetic microbes is an important step toward optimizing these systems for light energy capture or production of valuable commodities. In this work, we analyzed the effect of light intensity on bioproduction, biomass formation, and maintenance energy during photoheterotrophic growth of Rhodobacter sphaeroides. Using data obtained from steady-state bioreactors operated at varying dilution rates and light intensities, we found that irradiance had a significant impact on biomass yield and composition, with significant changes in photopigment, phospholipid, and biopolymer storage contents. We also observed a linear relationship between incident light intensity and H2 production rate between 3 and 10 W m(-2), with saturation observed at 100 W m(-2). The light conversion efficiency to H2 was also higher at lower light intensities. Photosynthetic maintenance energy requirements were also significantly affected by light intensity, with links to differences in biomass composition and the need to maintain redox homeostasis. Inclusion of the measured condition-dependent biomass and maintenance energy parameters and the measured photon uptake rate into a genome-scale metabolic model for R. sphaeroides (iRsp1140) significantly improved its predictive performance. We discuss how our analyses provide new insights into the light-dependent changes in bioenergetic requirements and physiology during photosynthetic growth of R. sphaeroides and potentially other photosynthetic organisms.

  • Imam S, Noguera DR, Donohue TJ (2015) CceR and AkgR regulate central carbon and energy metabolism in alphaproteobacteria. MBio 6(1): (PMC4323418) · Pubmed

    Many pathways of carbon and energy metabolism are conserved across the phylogeny, but the networks that regulate their expression or activity often vary considerably among organisms. In this work, we show that two previously uncharacterized transcription factors (TFs) are direct regulators of genes encoding enzymes of central carbon and energy metabolism in the alphaproteobacterium Rhodobacter sphaeroides. The LacI family member CceR (RSP_1663) directly represses genes encoding enzymes in the Entner-Doudoroff pathway, while activating those encoding the F1F0 ATPase and enzymes of the tricarboxylic acid (TCA) cycle and gluconeogenesis, providing a direct transcriptional network connection between carbon and energy metabolism. We identified bases that are important for CceR DNA binding and showed that DNA binding by this TF is inhibited by 6-phosphogluconate. We also showed that the GntR family TF AkgR (RSP_0981) directly activates genes encoding several TCA cycle enzymes, and we identified conditions where its activity is increased. The properties of single and double ΔCceR and ΔAkgR mutants illustrate that these 2 TFs cooperatively regulate carbon and energy metabolism. Comparative genomic analysis indicates that CceR and AkgR orthologs are found in other alphaproteobacteria, where they are predicted to have a conserved function in regulating central carbon metabolism. Our characterization of CceR and AkgR has provided important new insight into the networks that control central carbon and energy metabolism in alphaproteobacteria that can be exploited to modify or engineer new traits in these widespread and versatile bacteria. To extract and conserve energy from nutrients, cells coordinate a set of metabolic pathways into integrated networks. Many pathways that conserve energy or interconvert metabolites are conserved across cells, but the networks regulating these processes are often highly variable. In this study, we characterize two previously unknown transcriptional regulators of carbon and energy metabolism that are conserved in alphaproteobacteria, a group of abundant, environmentally and biotechnologically important organisms. We identify the genes they regulate, the DNA sequences they recognize, the metabolite that controls the activity of one of the regulators, and conditions where they are required for growth. We provide important new insight into conserved cellular networks that can also be used to improve a variety of hosts for converting feedstock into valuable products.

  • Imam S, Noguera DR, Donohue TJ (2014) Global analysis of photosynthesis transcriptional regulatory networks. PLoS Genet. 10(12):e1004837 (PMC4263372) · Pubmed

    Photosynthesis is a crucial biological process that depends on the interplay of many components. This work analyzed the gene targets for 4 transcription factors: FnrL, PrrA, CrpK and MppG (RSP_2888), which are known or predicted to control photosynthesis in Rhodobacter sphaeroides. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) identified 52 operons under direct control of FnrL, illustrating its regulatory role in photosynthesis, iron homeostasis, nitrogen metabolism and regulation of sRNA synthesis. Using global gene expression analysis combined with ChIP-seq, we mapped the regulons of PrrA, CrpK and MppG. PrrA regulates ∼34 operons encoding mainly photosynthesis and electron transport functions, while CrpK, a previously uncharacterized Crp-family protein, regulates genes involved in photosynthesis and maintenance of iron homeostasis. Furthermore, CrpK and FnrL share similar DNA binding determinants, possibly explaining our observation of the ability of CrpK to partially compensate for the growth defects of a ΔFnrL mutant. We show that the Rrf2 family protein, MppG, plays an important role in photopigment biosynthesis, as part of an incoherent feed-forward loop with PrrA. Our results reveal a previously unrealized, high degree of combinatorial regulation of photosynthetic genes and significant cross-talk between their transcriptional regulators, while illustrating previously unidentified links between photosynthesis and the maintenance of iron homeostasis.

  • Peterson AC, Hauschild JP, Quarmby ST, Krumwiede D, Lange O, Lemke RA, Grosse-Coosmann F, Horning S, Donohue TJ, Westphall MS, Coon JJ, Griep-Raming J (2014) Development of a GC/Quadrupole-Orbitrap mass spectrometer, part I: design and characterization. Anal. Chem. 86(20):10036-43 (PMC4204906) · Pubmed

    Identification of unknown compounds is of critical importance in GC/MS applications (metabolomics, environmental toxin identification, sports doping, petroleomics, and biofuel analysis, among many others) and remains a technological challenge. Derivation of elemental composition is the first step to determining the identity of an unknown compound by MS, for which high accuracy mass and isotopomer distribution measurements are critical. Here, we report on the development of a dedicated, applications-grade GC/MS employing an Orbitrap mass analyzer, the GC/Quadrupole-Orbitrap. Built from the basis of the benchtop Orbitrap LC/MS, the GC/Quadrupole-Orbitrap maintains the performance characteristics of the Orbitrap, enables quadrupole-based isolation for sensitive analyte detection, and includes numerous analysis modalities to facilitate structural elucidation. We detail the design and construction of the instrument, discuss its key figures-of-merit, and demonstrate its performance for the characterization of unknown compounds and environmental toxins.

  • Gall DL, Ralph J, Donohue TJ, Noguera DR (2014) A group of sequence-related sphingomonad enzymes catalyzes cleavage of β-aryl ether linkages in lignin β-guaiacyl and β-syringyl ether dimers. Environ. Sci. Technol. 48(20):12454-63 (PMC4207535) · Pubmed

    Lignin biosynthesis occurs via radical coupling of guaiacyl and syringyl hydroxycinnamyl alcohol monomers (i.e., "monolignols") through chemical condensation with the growing lignin polymer. With each chain-extension step, monolignols invariably couple at their β-positions, generating chiral centers. Here, we report on activities of bacterial glutathione-S-transferase (GST) enzymes that cleave β-aryl ether bonds in lignin dimers that are composed of different monomeric units. Our data reveal that these sequence-related enzymes from Novosphingobium sp. strain PP1Y, Novosphingobium aromaticivorans strain DSM12444, and Sphingobium sp. strain SYK-6 have conserved functions as β-etherases, catalyzing cleavage of each of the four dimeric α-keto-β-aryl ether-linked substrates (i.e., guaiacyl-β-guaiacyl, guaiacyl-β-syringyl, syringyl-β-guaiacyl, and syringyl-β-syringyl). Although each β-etherase cleaves β-guaiacyl and β-syringyl substrates, we have found that each is stereospecific for a given β-enantiomer in a racemic substrate; LigE and LigP β-etherase homologues exhibited stereospecificity toward β(R)-enantiomers whereas LigF and its homologues exhibited β(S)-stereospecificity. Given the diversity of lignin's monomeric units and the racemic nature of lignin polymers, we propose that bacterial catabolic pathways have overcome the existence of diverse lignin-derived substrates in nature by evolving multiple enzymes with broad substrate specificities. Thus, each bacterial β-etherase is able to cleave β-guaiacyl and β-syringyl ether-linked compounds while retaining either β(R)- or β(S)-stereospecificity.

  • Kyrpides NC, Hugenholtz P, Eisen JA, Woyke T, Göker M, Parker CT, Amann R, Beck BJ, Chain PS, Chun J, Colwell RR, Danchin A, Dawyndt P, Dedeurwaerdere T, DeLong EF, Detter JC, De Vos P, Donohue TJ, Dong XZ, Ehrlich DS, Fraser C, Gibbs R, Gilbert J, Gilna P, Glöckner FO, Jansson JK, Keasling JD, Knight R, Labeda D, Lapidus A, Lee JS, Li WJ, Ma J, Markowitz V, Moore ER, Morrison M, Meyer F, Nelson KE, Ohkuma M, Ouzounis CA, Pace N, Parkhill J, Qin N, Rossello-Mora R, Sikorski J, Smith D, Sogin M, Stevens R, Stingl U, Suzuki K, Taylor D, Tiedje JM, Tindall B, Wagner M, Weinstock G, Weissenbach J, White O, Wang J, Zhang L, Zhou YG, Field D, Whitman WB, Garrity GM, Klenk HP (2014) Genomic encyclopedia of bacteria and archaea: sequencing a myriad of type strains. PLoS Biol. 12(8):e1001920 (PMC4122341) · Pubmed

    Microbes hold the key to life. They hold the secrets to our past (as the descendants of the earliest forms of life) and the prospects for our future (as we mine their genes for solutions to some of the planet's most pressing problems, from global warming to antibiotic resistance). However, the piecemeal approach that has defined efforts to study microbial genetic diversity for over 20 years and in over 30,000 genome projects risks squandering that promise. These efforts have covered less than 20% of the diversity of the cultured archaeal and bacterial species, which represent just 15% of the overall known prokaryotic diversity. Here we call for the funding of a systematic effort to produce a comprehensive genomic catalog of all cultured Bacteria and Archaea by sequencing, where available, the type strain of each species with a validly published name (currently∼11,000). This effort will provide an unprecedented level of coverage of our planet's genetic diversity, allow for the large-scale discovery of novel genes and functions, and lead to an improved understanding of microbial evolution and function in the environment.

  • Lemke RA, Peterson AC, Ziegelhoffer EC, Westphall MS, Tjellström H, Coon JJ, Donohue TJ (2014) Synthesis and scavenging role of furan fatty acids. Proc. Natl. Acad. Sci. U.S.A. 111(33):E3450-7 (PMC4143029) · Pubmed

    Fatty acids play important functional and protective roles in living systems. This paper reports on the synthesis of a previously unidentified 19 carbon furan-containing fatty acid, 10,13-epoxy-11-methyl-octadecadienoate (9-(3-methyl-5-pentylfuran-2-yl)nonanoic acid) (19Fu-FA), in phospholipids from Rhodobacter sphaeroides. We show that 19Fu-FA accumulation is increased in cells containing mutations that increase the transcriptional response of this bacterium to singlet oxygen ((1)O2), a reactive oxygen species generated by energy transfer from one or more light-excited donors to molecular oxygen. We identify a previously undescribed class of S-adenosylmethionine-dependent methylases that convert a phospholipid 18 carbon cis unsaturated fatty acyl chain to a 19 carbon methylated trans unsaturated fatty acyl chain (19M-UFA). We also identify genes required for the O2-dependent conversion of this 19M-UFA to 19Fu-FA. Finally, we show that the presence of (1)O2 leads to turnover of 19Fu-Fa in vivo. We propose that furan-containing fatty acids like 19Fu-FA can act as a membrane-bound scavenger of (1)O2, which is naturally produced by integral membrane enzymes of the R. sphaeroides photosynthetic apparatus.

  • Lennon CW, Lemmer KC, Irons JL, Sellman MI, Donohue TJ, Gourse RL, Ross W (2014) A Rhodobacter sphaeroides protein mechanistically similar to Escherichia coli DksA regulates photosynthetic growth. MBio 5(3):e01105-14 (PMC4010833) · Pubmed

    ABSTRACT DksA is a global regulatory protein that, together with the alarmone ppGpp, is required for the "stringent response" to nutrient starvation in the gammaproteobacterium Escherichia coli and for more moderate shifts between growth conditions. DksA modulates the expression of hundreds of genes, directly or indirectly. Mutants lacking a DksA homolog exhibit pleiotropic phenotypes in other gammaproteobacteria as well. Here we analyzed the DksA homolog RSP2654 in the more distantly related Rhodobacter sphaeroides, an alphaproteobacterium. RSP2654 is 42% identical and similar in length to E. coli DksA but lacks the Zn finger motif of the E. coli DksA globular domain. Deletion of the RSP2654 gene results in defects in photosynthetic growth, impaired utilization of amino acids, and an increase in fatty acid content. RSP2654 complements the growth and regulatory defects of an E. coli strain lacking the dksA gene and modulates transcription in vitro with E. coli RNA polymerase (RNAP) similarly to E. coli DksA. RSP2654 reduces RNAP-promoter complex stability in vitro with RNAPs from E. coli or R. sphaeroides, alone and synergistically with ppGpp, suggesting that even though it has limited sequence identity to E. coli DksA (DksAEc), it functions in a mechanistically similar manner. We therefore designate the RSP2654 protein DksARsp. Our work suggests that DksARsp has distinct and important physiological roles in alphaproteobacteria and will be useful for understanding structure-function relationships in DksA and the mechanism of synergy between DksA and ppGpp. IMPORTANCE The role of DksA has been analyzed primarily in the gammaproteobacteria, in which it is best understood for its role in control of the synthesis of the translation apparatus and amino acid biosynthesis. Our work suggests that DksA plays distinct and important physiological roles in alphaproteobacteria, including the control of photosynthesis in Rhodobacter sphaeroides. The study of DksARsp, should be useful for understanding structure-function relationships in the protein, including those that play a role in the little-understood synergy between DksA and ppGpp.

  • Gall DL, Kim H, Lu F, Donohue TJ, Noguera DR, Ralph J (2014) Stereochemical features of glutathione-dependent enzymes in the Sphingobium sp. strain SYK-6 β-aryl etherase pathway. J. Biol. Chem. 289(12):8656-67 (PMC3961688) · Pubmed

    Glutathione-dependent enzymes play important protective, repair, or metabolic roles in cells. In particular, enzymes in the glutathione S-transferase (GST) superfamily function in stress responses, defense systems, or xenobiotic detoxification. Here, we identify novel features of bacterial GSTs that cleave β-aryl ether bonds typically found in plant lignin. Our data reveal several original features of the reaction cycle of these GSTs, including stereospecific substrate recognition and stereoselective formation of β-S-thioether linkages. Products of recombinant GSTs (LigE, LigP, and LigF) are β-S-glutathionyl-α-keto-thioethers that are degraded by a β-S-thioetherase (LigG). All three Lig GSTs produced the ketone product (β-S-glutathionyl-α-veratrylethanone) from an achiral side chain-truncated model substrate (β-guaiacyl-α-veratrylethanone). However, when β-etherase assays were conducted with a racemic model substrate, β-guaiacyl-α-veratrylglycerone, LigE- or LigP-catalyzed reactions yielded only one of two potential product (β-S-glutathionyl-α-veratrylglycerone) epimers, whereas the other diastereomer (differing in configuration at the β-position (i.e. its β-epimer)) was produced only in the LigF-catalyzed reaction. Thus, β-etherase catalysis causes stereochemical inversion of the chiral center, converting a β(R)-substrate to a β(S)-product (LigE and LigP), and a β(S)-substrate to a β(R)-product (LigF). Further, LigG catalyzed glutathione-dependent β-S-thioether cleavage with β-S-glutathionyl-α-veratrylethanone and with β(R)-configured β-S-glutathionyl-α-veratrylglycerone but exhibited no or significantly reduced β-S-thioether-cleaving activity with the β(S)-epimer, demonstrating that LigG is a stereospecific β-thioetherase. We therefore propose that multiple Lig enzymes are needed in this β-aryl etherase pathway in order to cleave the racemic β-ether linkages that are present in the backbone of the lignin polymer.

  • Imam S, Noguera DR, Donohue TJ (2013) Global insights into energetic and metabolic networks in Rhodobacter sphaeroides. BMC Syst Biol 7:89 (PMC3849096) · Pubmed

    Improving our understanding of processes at the core of cellular lifestyles can be aided by combining information from genetic analyses, high-throughput experiments and computational predictions. We combined data and predictions derived from phenotypic, physiological, genetic and computational analyses to dissect the metabolic and energetic networks of the facultative photosynthetic bacterium Rhodobacter sphaeroides. We focused our analysis on pathways crucial to the production and recycling of pyridine nucleotides during aerobic respiratory and anaerobic photosynthetic growth in the presence of an organic electron donor. In particular, we assessed the requirement for NADH/NADPH transhydrogenase enzyme, PntAB during respiratory and photosynthetic growth. Using high-throughput phenotype microarrays (PMs), we found that PntAB is essential for photosynthetic growth in the presence of many organic electron donors, particularly those predicted to require its activity to produce NADPH. Utilizing the genome-scale metabolic model iRsp1095, we predicted alternative routes of NADPH synthesis and used gene expression analyses to show that transcripts from a subset of the corresponding genes were conditionally increased in a ΔpntAB mutant. We then used a combination of metabolic flux predictions and mutational analysis to identify flux redistribution patterns utilized in the ΔpntAB mutant to compensate for the loss of this enzyme. Data generated from metabolic and phenotypic analyses of wild type and mutant cells were used to develop iRsp1140, an expanded genome-scale metabolic reconstruction for R. sphaeroides with improved ability to analyze and predict pathways associated with photosynthesis and other metabolic processes. These analyses increased our understanding of key aspects of the photosynthetic lifestyle, highlighting the added importance of NADPH production under these conditions. It also led to a significant improvement in the predictive capabilities of a metabolic model for the different energetic lifestyles of a facultative organism.

  • Gall DL, Ralph J, Donohue TJ, Noguera DR (2013) Benzoyl coenzyme a pathway-mediated metabolism of meta-hydroxy-aromatic acids in Rhodopseudomonas palustris. J. Bacteriol. 195(18):4112-20 (PMC3754758) · Pubmed

    Photoheterotrophic metabolism of two meta-hydroxy-aromatic acids, meta-, para-dihydroxybenzoate (protocatechuate) and meta-hydroxybenzoate, was investigated in Rhodopseudomonas palustris. When protocatechuate was the sole organic carbon source, photoheterotrophic growth in R. palustris was slow relative to cells using compounds known to be metabolized by the benzoyl coenzyme A (benzoyl-CoA) pathway. R. palustris was unable to grow when meta-hydroxybenzoate was provided as a sole source of organic carbon under photoheterotrophic growth conditions. However, in cultures supplemented with known benzoyl-CoA pathway inducers (para-hydroxybenzoate, benzoate, or cyclohexanoate), protocatechuate and meta-hydroxybenzoate were taken up from the culture medium. Further, protocatechuate and meta-hydroxybenzoate were each removed from cultures containing both meta-hydroxy-aromatic acids at equimolar concentrations in the absence of other organic compounds. Analysis of changes in culture optical density and in the concentration of soluble organic compounds indicated that the loss of these meta-hydroxy-aromatic acids was accompanied by biomass production. Additional experiments with defined mutants demonstrated that enzymes known to participate in the dehydroxylation of para-hydroxybenzoyl-CoA (HbaBCD) and reductive dearomatization of benzoyl-CoA (BadDEFG) were required for metabolism of protocatechuate and meta-hydroxybenzoate. These findings indicate that, under photoheterotrophic growth conditions, R. palustris can degrade meta-hydroxy-aromatic acids via the benzoyl-CoA pathway, apparently due to the promiscuity of the enzymes involved.

  • Rothamer DA, Donohue TJ (2013) Chemistry and combustion of fit-for-purpose biofuels. Curr Opin Chem Biol 17(3):522-8 · Pubmed · DOI

    From the inception of internal combustion engines, biologically derived fuels (biofuels) have played a role. Nicolaus Otto ran a predecessor to today's spark-ignition engine with an ethanol fuel blend in 1860. At the 1900 Paris world's fair, Rudolf Diesel ran his engine on peanut oil. Over 100 years of petroleum production has led to consistency and reliability of engines that demand standardized fuels. New biofuels can displace petroleum-based fuels and produce positive impacts on the environment, the economy, and the use of local energy sources. This review discusses the combustion, performance and other requirements of biofuels that will impact their near-term and long-term ability to replace petroleum fuels in transportation applications.

  • Nam TW, Ziegelhoffer EC, Lemke RA, Donohue TJ (2013) Proteins needed to activate a transcriptional response to the reactive oxygen species singlet oxygen. MBio 4(1):e00541-12 (PMC3546557) · Pubmed

    Singlet oxygen ((1)O(2)) is a reactive oxygen species generated by energy transfer from one or more excited donors to molecular oxygen. Many biomolecules are prone to oxidation by (1)O(2), and cells have evolved systems to protect themselves from damage caused by this compound. One way that the photosynthetic bacterium Rhodobacter sphaeroides protects itself from (1)O(2) is by inducing a transcriptional response controlled by ChrR, an anti-σ factor which releases an alternative sigma factor, σ(E), in the presence of (1)O(2). Here we report that induction of σ(E)-dependent gene transcription is decreased in the presence of (1)O(2) when two conserved genes in the σ(E) regulon are deleted, including one encoding a cyclopropane fatty acid synthase homologue (RSP2144) or one encoding a protein of unknown function (RSP1091). Thus, we conclude that RSP2144 and RSP1091 are each necessary to increase σ(E) activity in the presence of (1)O(2). In addition, we found that unlike in wild-type cells, where ChrR is rapidly degraded when (1)O(2) is generated, turnover of this anti-σ factor is slowed when cells lacking RSP2144, RSP1091, or both of these proteins are exposed to (1)O(2). Further, we demonstrate that the organic hydroperoxide tert-butyl hydroperoxide promotes ChrR turnover in both wild-type cells and mutants lacking RSP2144 or RSP1091, suggesting differences in the ways different types of oxidants increase σ(E) activity. Oxygen serves many crucial functions on Earth; it is produced during photosynthesis and needed for other pathways. While oxygen is relatively inert, it can be converted to reactive oxygen species (ROS) that destroy biomolecules, cause disease, or kill cells. When energy is transferred to oxygen, the ROS singlet oxygen is generated. To understand how singlet oxygen impacts cells, we study the stress response to this ROS in Rhodobacter sphaeroides, a bacterium that, like plants, generates this compound as a consequence of photosynthesis. This paper identifies proteins that activate a stress response to singlet oxygen and shows that they act in a specific response to this ROS. The identified proteins are found in many free-living, symbiotic, or pathogenic bacteria that can encounter singlet oxygen in nature. Thus, our findings provide new information about a stress response to a ROS of broad biological, agricultural, and biomedical importance.

  • Kontur WS, Schackwitz WS, Ivanova N, Martin J, Labutti K, Deshpande S, Tice HN, Pennacchio C, Sodergren E, Weinstock GM, Noguera DR, Donohue TJ (2012) Revised sequence and annotation of the Rhodobacter sphaeroides 2.4.1 genome. J. Bacteriol. 194(24):7016-7 (PMC3510577) · Pubmed

    No abstract available.

  • Dufour YS, Donohue TJ (2012) Signal correlations in ecological niches can shape the organization and evolution of bacterial gene regulatory networks Adv Microb Physiol. 61:1-36 · Pubmed · DOI

    No abstract available.

  • Dufour YS, Imam S, Koo BM, Green HA, Donohue TJ (2012) Convergence of the transcriptional responses to heat shock and singlet oxygen stresses. PLoS Genet. 8(9):e1002929 (PMC3441632) · Pubmed

    No abstract available.

  • Kim MS, Dufour YS, Yoo JS, Cho YB, Park JH, Nam GB, Kim HM, Lee KL, Donohue TJ, Roe JH (2012) Conservation of thiol-oxidative stress responses regulated by SigR orthologues in actinomycetes. Mol. Microbiol. 85(2):326-44 · Pubmed

    No abstract available.

  • Kontur WS, Noguera DR, Donohue TJ (2012) Maximizing reductant flow into microbial H2 production. Curr. Opin. Biotechnol. 23(3):382-9 · Pubmed

    No abstract available.

  • Wecke T, Halang P, Staroń A, Dufour YS, Donohue TJ, Mascher T. (2012) Extracytoplasmic function σ factors of the widely distributed group ECF41 contain a fused regulatory domain. Microbiologyopen 1(2):194-213 PMC3426412 · Pubmed · DOI

    Bacteria need signal transducing systems to respond to environmental changes. Next to one- and two-component systems, alternative σ factors of the extra-cytoplasmic function (ECF) protein family represent the third fundamental mechanism of bacterial signal transduction. A comprehensive classification of these proteins identified more than 40 phylogenetically distinct groups, most of which are not experimentally investigated. Here, we present the characterization of such a group with unique features, termed ECF41. Among analyzed bacterial genomes, ECF41 σ factors are widely distributed with about 400 proteins from 10 different phyla. They lack obvious anti-σ factors that typically control activity of other ECF σ factors, but their structural genes are often predicted to be cotranscribed with carboxymuconolactone decarboxylases, oxidoreductases, or epimerases based on genomic context conservation. We demonstrate for Bacillus licheniformis and Rhodobacter sphaeroides that the corresponding genes are preceded by a highly conserved promoter motif and are the only detectable targets of ECF41-dependent gene regulation. In contrast to other ECF σ factors, proteins of group ECF41 contain a large C-terminal extension, which is crucial for σ factor activity. Our data demonstrate that ECF41 σ factors are regulated by a novel mechanism based on the presence of a fused regulatory domain.

  • Kontur WS, Ziegelhoffer EC, Spero MA, Imam S, Noguera DR, Donohue TJ (2011) Pathways involved in reductant distribution during photobiological H(2) production by Rhodobacter sphaeroides. Appl. Environ. Microbiol. 77(20):7425-9 (PMC3194864) · Pubmed

    No abstract available.

  • Imam S, Yilmaz S, Sohmen U, Gorzalski AS, Reed JL, Noguera DR, Donohue TJ (2011) iRsp1095: a genome-scale reconstruction of the Rhodobacter sphaeroides metabolic network. BMC Syst Biol 5:116 (PMC3152904) · Pubmed

    No abstract available.

  • Greenwell R, Nam TW, Donohue TJ (2011) Features of Rhodobacter sphaeroides ChrR required for stimuli to promote the dissociation of ?(E)/ChrR complexes. J. Mol. Biol. 407(4):477-91 (PMC3061837) · Pubmed

    No abstract available.

  • Suen G, Scott JJ, Aylward FO, Adams SM, Tringe SG, Pinto-Tomás AA, Foster CE, Pauly M, Weimer PJ, Barry KW, Goodwin LA, Bouffard P, Li L, Osterberger J, Harkins TT, Slater SC, Donohue TJ, Currie CR (2010) An insect herbivore microbiome with high plant biomass-degrading capacity. PLoS Genet. 6(9): (PMC2944797) · Pubmed

    No abstract available.

  • Dufour YS, Wesenberg GE, Tritt AJ, Glasner JD, Perna NT, Mitchell JC, Donohue TJ (2010) chipD: a web tool to design oligonucleotide probes for high-density tiling arrays. Nucleic Acids Res. 38(Web Server):W321-5 (PMC2896189) · Pubmed

    No abstract available.

  • Dufour YS, Kiley PJ, Donohue TJ (2010) Reconstruction of the core and extended regulons of global transcription factors. PLoS Genet. 6(7):e1001027 (PMC2908626) · Pubmed

    No abstract available.

  • Wang Y, Dufour YS, Carlson HK, Donohue TJ, Marletta MA, Ruby EG (2010) H-NOX-mediated nitric oxide sensing modulates symbiotic colonization by Vibrio fischeri. Proc. Natl. Acad. Sci. U.S.A. 107(18):8375-80 (PMC2889544) · Pubmed

    No abstract available.

  • Steven Slater, Kenneth Keegstra and Timothy J. Donohue (2010) The US Department of Energy Great Lakes Bioenergy Research Center: Midwestern Biomass as a Resource for Renewable Fuels BioEnergy Research 3(1):3-5 · DOI

    No abstract available.

  • Raman Lall, Timothy J. Donohue, Simeone Marino, Julie C. Mitchell (2010) Optimizing ethanol production selectivity Mathematical and Computer Modelling : · DOI

    No abstract available.

  • Luftu Safak Yilmaz, Wayne S. Kontur, Alison P. Sanders, Ugur Sohmen, Timothy J. Donohue and Daniel R. Noguera (2010) Electron Partitioning During Light- and Nutrient-Powered Hydrogen Production by Rhodobacter sphaeroides BioEnergy Research 3(1):55-66 · DOI

    No abstract available.

  • Ziegelhoffer EC, Donohue TJ (2009) Bacterial responses to photo-oxidative stress. Nat. Rev. Microbiol. 7(12):856-63 (PMC2793278) · Pubmed

    No abstract available.

  • Donohue TJ (2009) Targeted sigma factor turnover inserts negative control into a positive feedback loop. Mol. Microbiol. 73(5):747-50 (PMC2770264) · Pubmed

    No abstract available.

  • Gomelsky L, Moskvin OV, Stenzel RA, Jones DF, Donohue TJ, Gomelsky M (2008) Hierarchical regulation of photosynthesis gene expression by the oxygen-responsive PrrBA and AppA-PpsR systems of Rhodobacter sphaeroides. J. Bacteriol. 190(24):8106-14 (PMC2593241) · Pubmed

    No abstract available.

  • Dufour YS, Landick R, Donohue TJ (2008) Organization and evolution of the biological response to singlet oxygen stress. J. Mol. Biol. 383(3):713-30 (PMC2579311) · Pubmed

    No abstract available.

  • Du X, Callister SJ, Manes NP, Adkins JN, Alexandridis RA, Zeng X, Roh JH, Smith WE, Donohue TJ, Kaplan S, Smith RD, Lipton MS (2008) A computational strategy to analyze label-free temporal bottom-up proteomics data. J. Proteome Res. 7(7):2595-604 (PMC2574618) · Pubmed

    No abstract available.

  • Cho YK, Donohue TJ, Tejedor I, Anderson MA, McMahon KD, Noguera DR (2008) Development of a solar-powered microbial fuel cell. J. Appl. Microbiol. 104(3):640-50 · Pubmed

    No abstract available.

  • Wilson SM, Gleisten MP, Donohue TJ (2008) Identification of proteins involved in formaldehyde metabolism by Rhodobacter sphaeroides. Microbiology (Reading, Engl.) 154(Pt 1):296-305 (PMC2440690) · Pubmed

    No abstract available.

  • Zeng X, Roh JH, Callister SJ, Tavano CL, Donohue TJ, Lipton MS, Kaplan S (2007) Proteomic characterization of the Rhodobacter sphaeroides 2.4.1 photosynthetic membrane: identification of new proteins. J. Bacteriol. 189(20):7464-74 (PMC2168454) · Pubmed

    No abstract available.

  • Campbell EA, Greenwell R, Anthony JR, Wang S, Lim L, Das K, Sofia HJ, Donohue TJ, Darst SA (2007) A conserved structural module regulates transcriptional responses to diverse stress signals in bacteria. Mol. Cell 27(5):793-805 (PMC2390684) · Pubmed

    No abstract available.

  • Tavano CL, Donohue TJ (2006) Development of the bacterial photosynthetic apparatus. Curr. Opin. Microbiol. 9(6):625-31 (PMC2765710) · Pubmed

    No abstract available.

  • Callister SJ, Nicora CD, Zeng X, Roh JH, Dominguez MA, Tavano CL, Monroe ME, Kaplan S, Donohue TJ, Smith RD, Lipton MS (2006) Comparison of aerobic and photosynthetic Rhodobacter sphaeroides 2.4.1 proteomes. J. Microbiol. Methods 67(3):424-36 (PMC2794424) · Pubmed

    No abstract available.

  • Donohue TJ, Cogdell RJ (2006) Microorganisms and clean energy. Nat. Rev. Microbiol. 4(11):800 (PMC2605648) · Pubmed

    No abstract available.

  • Callister SJ, Dominguez MA, Nicora CD, Zeng X, Tavano CL, Kaplan S, Donohue TJ, Smith RD, Lipton MS (2006) Application of the accurate mass and time tag approach to the proteome analysis of sub-cellular fractions obtained from Rhodobacter sphaeroides 2.4.1. Aerobic and photosynthetic cell cultures. J. Proteome Res. 5(8):1940-7 (PMC2794423) · Pubmed

    No abstract available.

  • Green HA, Donohue TJ (2006) Activity of Rhodobacter sphaeroides RpoHII, a second member of the heat shock sigma factor family. J. Bacteriol. 188(16):5712-21 (PMC1540091) · Pubmed

    No abstract available.

  • Laguri C, Stenzel RA, Donohue TJ, Phillips-Jones MK, Williamson MP (2006) Activation of the global gene regulator PrrA (RegA) from Rhodobacter sphaeroides. Biochemistry 45(25):7872-81 (PMC2517121) · Pubmed

    No abstract available.

  • Ranson-Olson B, Jones DF, Donohue TJ, Zeilstra-Ryalls JH (2006) In vitro and in vivo analysis of the role of PrrA in Rhodobacter sphaeroides 2.4.1 hemA gene expression. J. Bacteriol. 188(9):3208-18 (PMC1447469) · Pubmed

    No abstract available.

  • Jones DF, Stenzel RA, Donohue TJ (2005) Mutational analysis of the C-terminal domain of the Rhodobacter sphaeroides response regulator PrrA. Microbiology (Reading, Engl.) 151(Pt 12):4103-10 (PMC2800098) · Pubmed

    No abstract available.

  • Mackenzie C, Choudhary M, Larimer FW, Predki PF, Stilwagen S, Armitage JP, Barber RD, Donohue TJ, Hosler JP, Newman JE, Shapleigh JP, Sockett RE, Zeilstra-Ryalls J, Kaplan S (2005) The home stretch, a first analysis of the nearly completed genome of Rhodobacter sphaeroides 2.4.1. Photosyn. Res. 70(1):19-41 · Pubmed

    No abstract available.

  • Tavano CL, Podevels AM, Donohue TJ (2005) Identification of genes required for recycling reducing power during photosynthetic growth. J. Bacteriol. 187(15):5249-58 (PMC1196016) · Pubmed

    No abstract available.

  • Anthony JR, Warczak KL, Donohue TJ (2005) A transcriptional response to singlet oxygen, a toxic byproduct of photosynthesis. Proc. Natl. Acad. Sci. U.S.A. 102(18):6502-7 (PMC1088386) · Pubmed

    No abstract available.

  • Hickman JW, Witthuhn VC, Dominguez M, Donohue TJ (2004) Positive and negative transcriptional regulators of glutathione-dependent formaldehyde metabolism. J. Bacteriol. 186(23):7914-25 (PMC529062) · Pubmed

    No abstract available.

  • Donohue TJ, Thomas CM (2004) Policy proposal for publication of papers with data sets from genome-wide studies. Int. J. Syst. Evol. Microbiol. 54(Pt 6):1915 · Pubmed

    No abstract available.

  • Donohue TJ, Thomas CM (2004) Policy proposal for publication of papers with data sets from genome-wide studies. Microbiology (Reading, Engl.) 150(Pt 11):3521-2 (PMC2798886) · Pubmed

    No abstract available.

  • Anthony JR, Newman JD, Donohue TJ (2004) Interactions between the Rhodobacter sphaeroides ECF sigma factor, sigma(E), and its anti-sigma factor, ChrR. J. Mol. Biol. 341(2):345-60 (PMC2796631) · Pubmed

    No abstract available.

  • Tavano CL, Comolli JC, Donohue TJ (2004) The role of dor gene products in controlling the P2 promoter of the cytochrome c2 gene, cycA, in Rhodobacter sphaeroides. Microbiology (Reading, Engl.) 150(Pt 6):1893-9 (PMC2802839) · Pubmed

    No abstract available.

  • Comolli JC, Donohue TJ (2004) Differences in two Pseudomonas aeruginosa cbb3 cytochrome oxidases. Mol. Microbiol. 51(4):1193-203 · Pubmed

    No abstract available.

  • Anthony JR, Green HA, Donohue TJ (2004) Purification of Rhodobacter sphaeroides RNA polymerase and its sigma factors. Meth. Enzymol. 370:54-65 · Pubmed

    No abstract available.

  • Rios-Velazquez C, Coller R, Donohue TJ (2003) Features of Rhodobacter sphaeroides CcmFH. J. Bacteriol. 185(2):422-31 (PMC145331) · Pubmed

    No abstract available.

  • Comolli JC, Donohue TJ (2002) Pseudomonas aeruginosa RoxR, a response regulator related to Rhodobacter sphaeroides PrrA, activates expression of the cyanide-insensitive terminal oxidase. Mol. Microbiol. 45(3):755-68 · Pubmed

    No abstract available.

  • Hickman JW, Barber RD, Skaar EP, Donohue TJ (2002) Link between the membrane-bound pyridine nucleotide transhydrogenase and glutathione-dependent processes in Rhodobacter sphaeroides. J. Bacteriol. 184(2):400-9 (PMC139586) · Pubmed

    No abstract available.

  • Newman JD, Anthony JR, Donohue TJ (2001) The importance of zinc-binding to the function of Rhodobacter sphaeroides ChrR as an anti-sigma factor. J. Mol. Biol. 313(3):485-99 · Pubmed

    No abstract available.

  • Cox RL, Patterson C, Donohue TJ (2001) Roles for the Rhodobacter sphaeroides CcmA and CcmG proteins. J. Bacteriol. 183(15):4643-7 (PMC95360) · Pubmed

    No abstract available.

  • Newman JD, Falkowski MJ, Schilke BA, Anthony LC, Donohue TJ (1999) The Rhodobacter sphaeroides ECF sigma factor, sigma(E), and the target promoters cycA P3 and rpoE P1. J. Mol. Biol. 294(2):307-20 · Pubmed

    No abstract available.

  • Karls RK, Wolf JR, Donohue TJ (1999) Activation of the cycA P2 promoter for the Rhodobacter sphaeroides cytochrome c2 gene by the photosynthesis response regulator. Mol. Microbiol. 34(4):822-35 · Pubmed

    No abstract available.

  • He Y, Gaal T, Karls R, Donohue TJ, Gourse RL, Roberts GP (1999) Transcription activation by CooA, the CO-sensing factor from Rhodospirillum rubrum. The interaction between CooA and the C-terminal domain of the alpha subunit of RNA polymerase. J. Biol. Chem. 274(16):10840-5 · Pubmed

    No abstract available.

  • Barber RD, Donohue TJ (1998) Pathways for transcriptional activation of a glutathione-dependent formaldehyde dehydrogenase gene. J. Mol. Biol. 280(5):775-84 · Pubmed

    No abstract available.

  • Barber RD, Donohue TJ (1998) Function of a glutathione-dependent formaldehyde dehydrogenase in Rhodobacter sphaeroides formaldehyde oxidation and assimilation. Biochemistry 37(2):530-7 · Pubmed

    No abstract available.

  • Karls RK, Brooks J, Rossmeissl P, Luedke J, Donohue TJ (1998) Metabolic roles of a Rhodobacter sphaeroides member of the sigma32 family. J. Bacteriol. 180(1):10-9 (PMC106842) · Pubmed

    No abstract available.

  • MacGregor BJ, Karls RK, Donohue TJ (1998) Transcription of the Rhodobacter sphaeroides cycA P1 promoter by alternate RNA polymerase holoenzymes. J. Bacteriol. 180(1):1-9 (PMC106841) · Pubmed

    No abstract available.

  • Flory JE, Donohue TJ (1997) Transcriptional control of several aerobically induced cytochrome structural genes in Rhodobacter sphaeroides. Microbiology (Reading, Engl.) 143 ( Pt 1:3101-10 · Pubmed

    No abstract available.

  • Donohue TJ (1997) Eubacterial signal transduction by ligands of the mammalian peripheral benzodiazepine receptor complex. Proc. Natl. Acad. Sci. U.S.A. 94(10):4821-2 (PMC33664) · Pubmed

    No abstract available.

  • Witthuhn VC, Gao J, Hong S, Halls S, Rott MA, Wraight CA, Crofts AR, Donohue TJ (1997) Reactions of isocytochrome c2 in the photosynthetic electron transfer chain of Rhodobacter sphaeroides. Biochemistry 36(4):903-11 · Pubmed

    No abstract available.

  • Bintrim SB, Donohue TJ, Handelsman J, Roberts GP, Goodman RM (1997) Molecular phylogeny of Archaea from soil. Proc. Natl. Acad. Sci. U.S.A. 94(1):277-82 (PMC19314) · Pubmed

    No abstract available.

  • Barber RD, Rott MA, Donohue TJ (1996) Characterization of a glutathione-dependent formaldehyde dehydrogenase from Rhodobacter sphaeroides. J. Bacteriol. 178(5):1386-93 (PMC177813) · Pubmed

    No abstract available.

  • Flory JE, Donohue TJ (1995) Organization and expression of the Rhodobacter sphaeroides cycFG operon. J. Bacteriol. 177(15):4311-20 (PMC177178) · Pubmed

    No abstract available.

  • Schilke BA, Donohue TJ (1995) ChrR positively regulates transcription of the Rhodobacter sphaeroides cytochrome c2 gene. J. Bacteriol. 177(8):1929-37 (PMC176832) · Pubmed

    No abstract available.

  • Brandner JP, Donohue TJ (1994) The Rhodobacter sphaeroides cytochrome c2 signal peptide is not necessary for export and heme attachment. J. Bacteriol. 176(3):602-9 (PMC205096) · Pubmed

    No abstract available.

  • Mecsas J, Rouviere PE, Erickson JW, Donohue TJ, Gross CA (1993) The activity of sigma E, an Escherichia coli heat-inducible sigma-factor, is modulated by expression of outer membrane proteins. Genes Dev. 7(12B):2618-28 · Pubmed

    No abstract available.

  • Karls RK, Jin DJ, Donohue TJ (1993) Transcription properties of RNA polymerase holoenzymes isolated from the purple nonsulfur bacterium Rhodobacter sphaeroides. J. Bacteriol. 175(23):7629-38 (PMC206919) · Pubmed

    No abstract available.

  • Rott MA, Witthuhn VC, Schilke BA, Soranno M, Ali A, Donohue TJ (1993) Genetic evidence for the role of isocytochrome c2 in photosynthetic growth of Rhodobacter sphaeroides Spd mutants. J. Bacteriol. 175(2):358-66 (PMC196149) · Pubmed

    No abstract available.

  • Schilke BA, Donohue TJ (1992) delta-Aminolevulinate couples cycA transcription to changes in heme availability in Rhodobacter sphaeroides. J. Mol. Biol. 226(1):101-15 · Pubmed

    No abstract available.

  • Rott MA, Fitch J, Meyer TE, Donohue TJ (1992) Regulation of a cytochrome c2 isoform in wild-type and cytochrome c2 mutant strains of Rhodobacter sphaeroides. Arch. Biochem. Biophys. 292(2):576-82 · Pubmed

    No abstract available.


  • Donohue, T. J. and P. J. Kiley. 2010. Bacterial responses to O2 limitation. pp 175-189. In Bacterial Stress Responses, 2nd edition. G. Storz and R. Hengge (ed). American Society for Microbiology Press, American Society for Microbiology, Washington, D.C.
  • Fortenbery, T. R., and T. J. Donohue. 2010. Opportunities and challenges for next generation biofuels. In Controversies in Science and Technology. Vol. 3. pp. 203-213. D. Kleinman, J. Delborne, K. Cloud-Hansen, and J. Handelsman (ed). Mary Ann Libert Inc, New Rochelle, NY.
  • Choudhary, M., Mackenzie, C., Donohue, T. J., and S. Kaplan. 2008. pp. 691-706. Purple Bacterial Genomics. In Advances in Photosynthesis and Respiration. Vol. 28. C. N. Hunter, F. Daldal, T. J. Beatty, and M. Thurnauer (ed). Springer Verlag Inc.