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
    Twitter feedGoogle ScholarORCID

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 Enuh
Blaise Manga Enuh
Picture of Enzien
Grace Enzien
Grad Student
Picture of Gnatzig
Curt Gnatzig
Picture of Hall
Benjamin Hall
Picture of Idowu
Daniel Idowu
Grad Student
Picture of Lahue
Caiti Kay Lahue
Grad Student
Picture of Lemke
Rachelle Lemke
Sr Research Specialist
Lab Manager
Picture of Metz
Fletcher Metz
Grad Student
Picture of Myers
Kevin Myers
Assistant Scientist
Picture of Rodriguez-Castro
Laura Rodriguez-Castro
Picture of Vilbert
Avery Vilbert
Assistant Scientist
Picture of Walters
Kevin Walters
Grad Student

Research Papers

  • Walters KA, Myers KS, Donohue TJ, Noguera DR (2024) Metagenome-assembled genomes from microbiomes fermenting dairy coproducts. Microbiology resource announcements :e0017324 · Pubmed · DOI

    To advance knowledge of microbial communities capable of fermenting agro-industrial residues into value-added products, we report metagenomes of microbial communities from six anaerobic bioreactors that were fed a mixture of ultra-filtered milk permeate and cottage cheese acid whey. These metagenomes produced 122 metagenome-assembled genomes that represent 34 distinct taxa.

  • Vilbert AC, Kontur WS, Gille D, Noguera DR, Donohue TJ (2023) Engineering Novosphingobium aromaticivorans to produce cis,cis -muconic acid from biomass aromatics. Applied and environmental microbiology 90((1)):e0166023 PMC5892560 · Pubmed · DOI

    The platform chemical cis,cis- muconic acid ( cc MA) provides facile access to a number of monomers used in the synthesis of commercial plastics. It is also a metabolic intermediate in the β-ketoadipic acid pathway of many bacteria and, therefore, a current target for microbial production from abundant renewable resources via metabolic engineering. This study investigates Novosphingobium aromaticivorans DSM12444 as a chassis for the production of cc MA from biomass aromatics. The N. aromaticivorans genome predicts that it encodes a previously uncharacterized protocatechuic acid (PCA) decarboxylase and a catechol 1,2-dioxygenase, which would be necessary for the conversion of aromatic metabolic intermediates to cc MA. This study confirmed the activity of these two enzymes in vitro and compared their activity to ones that have been previously characterized and used in cc MA production. From these results, we generated one strain that is completely derived from native genes and a second that contains genes previously used in microbial engineering synthesis of this compound. Both of these strains exhibited stoichiometric production of cc MA from PCA and produced greater than 100% yield of cc MA from the aromatic monomers that were identified in liquor derived from alkaline pretreated biomass. Our results show that a strain completely derived from native genes and one containing homologs from other hosts are both capable of stoichiometric production of cc MA from biomass aromatics. Overall, this work combines previously unknown aspects of aromatic metabolism in N. aromaticivorans and the genetic tractability of this organism to generate strains that produce cc MA from deconstructed biomass.IMPORTANCEThe production of commodity chemicals from renewable resources is an important goal toward increasing the environmental and economic sustainability of industrial processes. The aromatics in plant biomass are an underutilized and abundant renewable resource for the production of valuable chemicals. However, due to the chemical composition of plant biomass, many deconstruction methods generate a heterogeneous mixture of aromatics, thus making it difficult to extract valuable chemicals using current methods. Therefore, recent efforts have focused on harnessing the pathways of microorganisms to convert a diverse set of aromatics into a single product. Novosphingobium aromaticivorans DSM12444 has the native ability to metabolize a wide range of aromatics and, thus, is a potential chassis for conversion of these abundant compounds to commodity chemicals. This study reports on new features of N. aromaticivorans that can be used to produce the commodity chemical cis,cis -muconic acid from renewable and abundant biomass aromatics.

  • Ellis NA, Myers KS, Tung J, Davidson Ward A, Johnston K, Bonnington KE, Donohue TJ, Machner MP (2023) A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes. eLife 12: PMC6269432 · Pubmed · DOI

    Identifying virulence-critical genes from pathogens is often limited by functional redundancy. To rapidly interrogate the contributions of combinations of genes to a biological outcome, we have developed a mu ltiplex, r andomized C RISPR i nterference s equencing (MuRCiS) approach. At its center is a new method for the randomized self-assembly of CRISPR arrays from synthetic oligonucleotide pairs. When paired with PacBio long-read sequencing, MuRCiS allowed for near-comprehensive interrogation of all pairwise combinations of a group of 44 Legionella pneumophila virulence genes encoding highly conserved transmembrane proteins for their role in pathogenesis. Both amoeba and human macrophages were challenged with L. pneumophila bearing the pooled CRISPR array libraries, leading to the identification of several new virulence-critical combinations of genes. lpg2888 and lpg3000 were particularly fascinating for their apparent redundant functions during L. pneumophila human macrophage infection, while lpg3000 alone was essential for L. pneumophila virulence in the amoeban host Acanthamoeba castellanii . Thus, MuRCiS provides a method for rapid genetic examination of even large groups of redundant genes, setting the stage for application of this technology to a variety of biological contexts and organisms.

  • Hall BW, Kontur WS, Neri JC, Gille DM, Noguera DR, Donohue TJ (2023) Production of carotenoids from aromatics and pretreated lignocellulosic biomass by Novosphingobium aromaticivorans . Applied and environmental microbiology 89((12)):e0126823 PMC167454 · Pubmed · DOI

    There is economic and environmental interest in generating commodity chemicals from renewable resources, such as lignocellulosic biomass, that can substitute for chemicals derived from fossil fuels. The bacterium Novosphingobium aromaticivorans is a promising microbial platform for producing commodity chemicals from lignocellulosic biomass because it can produce these from compounds in pretreated lignocellulosic biomass, which many industrial microbial catalysts cannot metabolize. Here, we show that N. aromaticivorans can be engineered to produce several valuable carotenoids. We also show that engineered N. aromaticivorans strains can produce these lipophilic chemicals concurrently with the extracellular commodity chemical 2-pyrone-4,6-dicarboxylic acid when grown in a complex liquor obtained from alkaline pretreated lignocellulosic biomass. Concurrent microbial production of valuable intra- and extracellular products can increase the economic value generated from the conversion of lignocellulosic biomass-derived compounds into commodity chemicals and facilitate the separation of water- and membrane-soluble products.

  • Casadevall A, Cotter PA, Enquist L, Donohue TJ, Bertuzzi S, Nguyen NK (2023) An account of American Academy of Microbiology reforms and pandemic operations. mBio 14((6)):e0233423 PMC8262865 · Pubmed · DOI

    Change is an inevitable part of any organization if it wants to adapt and strive in a changing environment. That was what the American Academy of Microbiology (Academy) did from 2019-2023 when it transformed itself into a scientific think tank at ASM while maintaining the high standard of an honorific community of scholars. Here, we report on the recent history of the Academy and the changes that have taken place during this period. With the contribution of many thougtful leaders, the Academy refreshed its commitment to promote excellence and uphold its high values.

  • Hall AN, Hall BW, Kinney KJ, Olsen GG, Banta AB, Noguera DR, Donohue TJ, Peters JM (2023) Tools for Genetic Engineering and Gene Expression Control in Novosphingobium aromaticivorans and Rhodobacter sphaeroides . bioRxiv : the preprint server for biology : PMC4837600 · Pubmed · DOI

    Alphaproteobacteria have a variety of cellular and metabolic features that provide important insights into biological systems and enable biotechnologies. For example, some species are capable of converting plant biomass into valuable biofuels and bioproducts have the potential to form the backbone of the sustainable bioeconomy. Among the Alphaproteobacteria, Novosphingobium aromaticivorans , Rhodobacter sphaeroides , and Zymomonas mobilis , show particular promise as organisms that can be engineered to convert extracted plant lignin or sugars into bioproducts and biofuels. Genetic manipulation of these bacteria is needed to introduce engineered pathways and modulate expression of native genes with the goal of enhancing bioproduct output. Although recent work has expanded the genetic toolkit for Z. mobilis , N. aromaticivorans and R. sphaeroides still need facile, reliable approaches to deliver genetic payloads to the genome and to control gene expression. Here, we expand the platform of genetic tools for N. aromaticivorans and R. sphaeroides to address these issues. We demonstrate that Tn 7 transposition is an effective approach for introducing engineered DNA into the chromosome of N. aromaticivorans and R. sphaeroides . We screen a synthetic promoter library to identify inducible promoters with strong, regulated activity in both organisms. Combining Tn 7 integration with promoters from our library, we establish CRISPR interference systems for N. aromaticivorans and R. sphaeroides that can target essential genes and modulate engineered pathways. We anticipate that these systems will greatly facilitate both genetic engineering and gene function discovery efforts in these industrially important species and other Alphaproteobacteria.

  • Hall, A., Donohue, T. J., and J. Peters. (2023) Complete sequences of conjugal helper plasmids pRK2013 and pEVS104. microPublication :0.17912/micropub.biology.000882. PMC10375283 · Pubmed · DOI

    We present the complete sequences of two commonly used conjugal helper plasmids: pRK2013 and pEVS104. These sequences will enable engineering of custom helper plasmids, for example, with different antibiotic markers or origins of replication. We provide both sequence information and plasmid maps to aid future engineering efforts.

  • Alberge F, Lakey BD, Schaub RE, Dohnalkova AC, Lemmer KC, Dillard JP, Noguera DR, Donohue TJ (2023) A previously uncharacterized divisome-associated lipoprotein, DalA, is needed for normal cell division in Rhodobacterales . mBio 14((4)):e0120323 PMC7265319 · Pubmed · DOI

    The bacterial cell envelope is a key subcellular compartment with important roles in antibiotic resistance, nutrient acquisition, and cell morphology. We seek to gain a better understanding of proteins that contribute to the function of the cell envelope in Alphaproteobacteria . Using Rhodobacter sphaeroides , we show that a previously uncharacterized protein, RSP_1200, is an outer membrane (OM) lipoprotein that non-covalently binds peptidoglycan (PG). Using a fluorescently tagged version of this protein, we find that RSP_1200 undergoes a dynamic repositioning during the cell cycle and is enriched at the septum during cell division. We show that the position of RSP_1200 mirrors the location of FtsZ rings, leading us to propose that RSP_1200 is a newly identified component of the R. sphaeroides ' divisome. Additional support for this hypothesis includes the co-precipitation of RSP_1200 with FtsZ, the Pal protein, and several predicted PG L,D-transpeptidases. We also find that a ∆ RSP_1200 mutation leads to defects in cell division, sensitivity to PG-active antibiotics, and results in the formation of OM protrusions at the septum during cell division. Based on these results, we propose to name RSP_1200 DalA (for division-associated lipoprotein A) and postulate that DalA serves as a scaffold to position or modulate the activity of PG transpeptidases that are needed to form envelope invaginations during cell division. We find that DalA homologs are present in members of the Rhodobacterales order within Alphaproteobacteria . Therefore, we propose that further analysis of this and related proteins will increase our understanding of the macromolecular machinery and proteins that participate in cell division in Gram-negative bacteria. IMPORTANCE Multi-protein complexes of the bacterial cell envelope orchestrate key processes like growth, division, biofilm formation, antimicrobial resistance, and production of valuable compounds. The subunits of these protein complexes are well studied in some bacteria, and differences in their composition and function are linked to variations in cell envelope composition, shape, and proliferation. However, some envelope protein complex subunits have no known homologs across the bacterial phylogeny. We find that Rhodobacter sphaeroides RSP_1200 is a newly identified lipoprotein (DalA) and that loss of this protein causes defects in cell division and changes the sensitivity to compounds, affecting cell envelope synthesis and function. We find that DalA forms a complex with proteins needed for cell division, binds the cell envelope polymer peptidoglycan, and colocalizes with enzymes involved in the assembly of this macromolecule. The analysis of DalA provides new information on the cell division machinery in this and possibly other Alphaproteobacteria.

  • Donohue, TJ (2023) Producing Transportation Fuels, Electrical Power, and Chemicals in a Circular Bioeconomy. The Bridge: Linking Engineering and Society 53(2):41-46 · DOI

    https://www.nae.edu/294933/Summer-Bridge-on-Engineering-the-Energy-Transition ISSN 0737-6278

  • Walters KA, Mohan G, Myers KS, Ingle AT, Donohue TJ, Noguera DR (2023) A metagenome-level analysis of a microbial community fermenting ultra-filtered milk permeate. Frontiers in bioengineering and biotechnology 11:1173656 PMC5399333 · Pubmed · DOI

    Fermentative microbial communities have the potential to serve as biocatalysts for the conversion of low-value dairy coproducts into renewable chemicals, contributing to a more sustainable global economy. To develop predictive tools for the design and operation of industrially relevant strategies that utilize fermentative microbial communities, there is a need to determine the genomic features of community members that are characteristic to the accumulation of different products. To address this knowledge gap, we performed a 282-day bioreactor experiment with a microbial community that was fed ultra-filtered milk permeate, a low-value coproduct from the dairy industry. The bioreactor was inoculated with a microbial community from an acid-phase digester. A metagenomic analysis was used to assess microbial community dynamics, construct metagenome-assembled genomes (MAGs), and evaluate the potential for lactose utilization and fermentation product synthesis of community members represented by the assembled MAGs. This analysis led us to propose that, in this reactor, members of the Actinobacteriota phylum are important in the degradation of lactose, via the Leloir pathway and the bifid shunt, and the production of acetic, lactic, and succinic acids. In addition, members of the Firmicutes phylum contribute to the chain-elongation-mediated production of butyric, hexanoic, and octanoic acids, with different microbes using either lactose, ethanol, or lactic acid as the growth substrate. We conclude that genes encoding carbohydrate utilization pathways, and genes encoding lactic acid transport into the cell, electron confurcating lactate dehydrogenase, and its associated electron transfer flavoproteins, are genomic features whose presence in Firmicutes needs to be established to infer the growth substrate used for chain elongation.

  • Lakey BD, Alberge F, Parrell D, Wright ER, Noguera DR, Donohue TJ (2023) The role of CenKR in the coordination of Rhodobacter sphaeroides cell elongation and division. mBio 14((4)):e0063123 PMC7969528 · Pubmed · DOI

    Cell elongation and division are essential aspects of the bacterial life cycle that must be coordinated for viability and replication. The impact of misregulation of these processes is not well understood as these systems are often not amenable to traditional genetic manipulation. Recently, we reported on the CenKR two-component system (TCS) in the Gram-negative bacterium Rhodobacter sphaeroides that is genetically tractable, widely conserved in α-proteobacteria, and directly regulates the expression of components crucial for cell elongation and division, including genes encoding subunit of the Tol-Pal complex. In this work, we show that overexpression of cenK results in cell filamentation and chaining. Using cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET), we generated high-resolution two-dimensional (2D) images and three-dimensional (3D) volumes of the cell envelope and division septum of wild-type cells and a cenK overexpression strain finding that these morphological changes stem from defects in outer membrane (OM) and peptidoglycan (PG) constriction. By monitoring the localization of Pal, PG biosynthesis, and the bacterial cytoskeletal proteins MreB and FtsZ, we developed a model for how increased CenKR activity leads to changes in cell elongation and division. This model predicts that increased CenKR activity decreases the mobility of Pal, delaying OM constriction, and ultimately disrupting the midcell positioning of MreB and FtsZ and interfering with the spatial regulation of PG synthesis and remodeling. IMPORTANCE By coordinating cell elongation and division, bacteria maintain their shape, support critical envelope functions, and orchestrate division. Regulatory and assembly systems have been implicated in these processes in some well-studied Gram-negative bacteria. However, we lack information on these processes and their conservation across the bacterial phylogeny. In R. sphaeroides and other α-proteobacteria, CenKR is an essential two-component system (TCS) that regulates the expression of genes known or predicted to function in cell envelope biosynthesis, elongation, and/or division. Here, we leverage unique features of CenKR to understand how increasing its activity impacts cell elongation/division and use antibiotics to identify how modulating the activity of this TCS leads to changes in cell morphology. Our results provide new insight into how CenKR activity controls the structure and function of the bacterial envelope, the localization of cell elongation and division machinery, and cellular processes in organisms with importance in health, host-microbe interactions, and biotechnology.

  • Myers KS, Ingle AT, Walters KA, Fortney NW, Scarborough MJ, Donohue TJ, Noguera DR (2023) Comparison of metagenomes from fermentation of various agroindustrial residues suggests a common model of community organization. Frontiers in bioengineering and biotechnology 11:1197175 PMC4585908 · Pubmed · DOI

    The liquid residue resulting from various agroindustrial processes is both rich in organic material and an attractive source to produce a variety of chemicals. Using microbial communities to produce chemicals from these liquid residues is an active area of research, but it is unclear how to deploy microbial communities to produce specific products from the different agroindustrial residues. To address this, we fed anaerobic bioreactors one of several agroindustrial residues (carbohydrate-rich lignocellulosic fermentation conversion residue, xylose, dairy manure hydrolysate, ultra-filtered milk permeate, and thin stillage from a starch bioethanol plant) and inoculated them with a microbial community from an acid-phase digester operated at the wastewater treatment plant in Madison, WI, United States. The bioreactors were monitored over a period of months and sampled to assess microbial community composition and extracellular fermentation products. We obtained metagenome assembled genomes (MAGs) from the microbial communities in each bioreactor and performed comparative genomic analyses to identify common microorganisms, as well as any community members that were unique to each reactor. Collectively, we obtained a dataset of 217 non-redundant MAGs from these bioreactors. This metagenome assembled genome dataset was used to evaluate whether a specific microbial ecology model in which medium chain fatty acids (MCFAs) are simultaneously produced from intermediate products (e.g., lactic acid) and carbohydrates could be applicable to all fermentation systems, regardless of the feedstock. MAGs were classified using a multiclass classification machine learning algorithm into three groups, organisms fermenting the carbohydrates to intermediate products, organisms utilizing the intermediate products to produce MCFAs, and organisms producing MCFAs directly from carbohydrates. This analysis revealed common biological functions among the microbial communities in different bioreactors, and although different microorganisms were enriched depending on the agroindustrial residue tested, the results supported the conclusion that the microbial ecology model tested was appropriate to explain the MCFA production potential from all agricultural residues.

  • Walters, KA, Mohan, G, Myers, KS, Ingle, AT, Donohue T., and DR Noguera (2023) A metagenome-level analysis of a microbial community fermenting ultra-filtered milk permeate Frontiers in Bioengineering and Biotechnology 11: · DOI

    Fermentative microbial communities have the potential to serve as biocatalysts for the conversion of low-value dairy coproducts into renewable chemicals, contributing to a more sustainable global economy. To develop predictive tools for the design and operation of industrially relevant strategies that utilize fermentative microbial communities, there is a need to determine the genomic features of community members that are characteristic to the accumulation of different products. To address this knowledge gap, we performed a 282-day bioreactor experiment with a microbial community that was fed ultra-filtered milk permeate, a low-value coproduct from the dairy industry. The bioreactor was inoculated with a microbial community from an acid-phase digester. A metagenomic analysis was used to assess microbial community dynamics, construct metagenome-assembled genomes (MAGs), and evaluate the potential for lactose utilization and fermentation product synthesis of community members represented by the assembled MAGs. This analysis led us to propose that, in this reactor, members of the Actinobacteriota phylum are important in the degradation of lactose, via the Leloir pathway and the bifid shunt, and the production of acetic, lactic, and succinic acids. In addition, members of the Firmicutes phylum contribute to the chain-elongation-mediated production of butyric, hexanoic, and octanoic acids, with different microbes using either lactose, ethanol, or lactic acid as the growth substrate. We conclude that genes encoding carbohydrate utilization pathways, and genes encoding lactic acid transport into the cell, electron confurcating lactate dehydrogenase, and its associated electron transfer flavoproteins, are genomic features whose presence in Firmicutes needs to be established to infer the growth substrate used for chain elongation.

  • Ellis NA, Myers KS, Tung J, Ward AD, Johnston K, Bonnington KE, Donohue TJ, Machner MP (2023) A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes. bioRxiv : the preprint server for biology : PMC7343393 · Pubmed · DOI

    Identifying virulence-critical genes from pathogens is often limited by functional redundancy. To rapidly interrogate the contributions of combinations of genes to a biological outcome, we have developed a multiplex, randomized CRISPR interference sequencing (MuRCiS) approach. At its center is a new method for the randomized self-assembly of CRISPR arrays from synthetic oligonucleotide pairs. When paired with PacBio long-read sequencing, MuRCiS allowed for near-comprehensive interrogation of all pairwise combinations of a group of 44 Legionella pneumophila virulence genes encoding highly conserved transmembrane proteins for their role in pathogenesis. Both amoeba and human macrophages were challenged with L. pneumophila bearing the pooled CRISPR array libraries, leading to the identification of several new virulence-critical combinations of genes. lpg2888 and lpg3000 were particularly fascinating for their apparent redundant functions during L. pneumophila human macrophage infection, while lpg3000 alone was essential for L. pneumophila virulence in the amoeban host Acanthamoeba castellanii . Thus, MuRCiS provides a method for rapid genetic examination of even large groups of redundant genes, setting the stage for application of this technology to a variety of biological contexts and organisms.

  • Hall BW, Bingman CA, Fox BG, Noguera DR, Donohue TJ (2022) A broad specificity β-propeller enzyme from Rhodopseudomonas palustris that hydrolyzes many lactones including γ-valerolactone. The Journal of biological chemistry 299((1)):102782 PMC8265157 · Pubmed · DOI

    Lactones are prevalent in biological and industrial settings, yet there is a lack of information regarding enzymes used to metabolize these compounds. One compound, γ-valerolactone (GVL), is used as a solvent to dissolve plant cell walls into sugars and aromatic molecules for subsequent microbial conversion to fuels and chemicals. Despite the promise of GVL as a renewable solvent for biomass deconstruction, residual GVL can be toxic to microbial fermentation. Here, we identified a Ca-dependent enzyme from Rhodopseudomonas palustris (Rpa3624) and showed that it can hydrolyze aliphatic and aromatic lactones and esters, including GVL. Maximum-likelihood phylogenetic analysis of other related lactonases with experimentally determined substrate preferences shows that Rpa3624 separates by sequence motifs into a subclade with preference for hydrophobic substrates. Additionally, we solved crystal structures of this β-propeller enzyme separately with either phosphate, an inhibitor, or a mixture of GVL and products to define an active site where calcium-bound water and calcium-bound aspartic and glutamic acid residues make close contact with substrate and product. Our kinetic characterization of WT and mutant enzymes combined with structural insights inform a reaction mechanism that centers around activation of a calcium-bound water molecule promoted by general base catalysis and close contacts with substrate and a potential intermediate. Similarity of Rpa3624 with other β-propeller lactonases suggests this mechanism may be relevant for other members of this emerging class of versatile catalysts.

  • Umana, GE, Perez, JM, Unda, F, Lin, CY, Sener, C, Karlen, SD, Mansfield, SD, Eudes, A, Ralph, J, Donohue, TJ, and DR Noguera (2022) Biological funneling of phenolics from transgenic plants engineered to express the bacterial 3-dehydroshikimate dehydratase (qsuB) gene Frontiers in Chemical Engineering 4:1036084 · DOI

    The economic and environmental sustainability of lignocellulosic biomass biorefineries is predicated on generating biofuels and bioproducts from cellwall polysaccharide and lignin polymers. Historical efforts in plant genetic engineering have focused on the development of strategies that facilitate biomass deconstruction, with more recently efforts including the synthesis of high-value chemicals in planta. One such genetic modification is the expression of the bacterial quinate and shikimate utilization B (qsuB) gene that increases the accumulation of protocatechuic acid in lignocellulosic biomass. Herein, we evaluated the effectiveness of an alkaline pretreatment process to extract phenolics directly from wild-type and QsuB-transgenic lines of Arabidopsis, poplar, and sorghum, and then upgrade them to the polyester precursor 2-pyrone-4,6-dicarboxylic acid (PDC) with an engineered strain of Novosphingobium aromaticivorans. Protocatechuic acid extracted from all QsuB transgenic lines was found to be mostly in the glycosylated form. Glycosylated protocatechuic acid and other plant-derived phenolics were effectively metabolized by N. aromaticivorans, and PDC production was greatest using extracts from an Arabidopsis QsuB transgenic line (~5% w/w), followed by QsuB sorghum (~1.1% w/w), and QsuB poplar (~0.4% w/w) lines. The comparison of PDC production from wild-type and QsuB transgenic lines of Arabidopsis, poplar, and sorghum demonstrates the utility of a mild alkaline pretreatment to liberate phenolics from plant biomass that are either naturally present or that accumulate as a consequence of genetic engineering strategies. All QsuB transgenic lines outperformed their wild-type counterparts with respect to observed PDC yields. In addition, microbial funneling to PDC was effective even when most of the protocatechuic acid extracted was in glycosylated form, clearly demonstrating that this bacterium can metabolize these aromatic conjugates. These findings illustrate the benefits of combining plant and microbial engineering for bioproduct formation from phenolics in lignocellulosic biorefineries.

  • Ingle AT, Fortney NW, Myers KS, Walters KA, Scarborough MJ, Donohue TJ, Noguera DR (2022) Metagenome-Assembled Genomes from a Microbiome Grown in Dairy Manure Hydrolysate. Microbiology resource announcements 11((8)):e0029222 PMC8265157 · Pubmed · DOI

    Anaerobic microbiomes can be used to recover the chemical energy in agroindustrial and municipal wastes as useful products. Here, we report a total of 109 draft metagenome-assembled genomes from a bioreactor-fed carbohydrate-rich dairy manure hydrolysate. Studying these genomes will aid us in deciphering the metabolic networks in anaerobic microbiomes.

  • Fortney NW, Myers KS, Ingle AT, Walters KA, Scarborough MJ, Donohue TJ, Noguera DR (2022) Metagenomes and Metagenome-Assembled Genomes from Microbiomes Metabolizing Thin Stillage from an Ethanol Biorefinery. Microbiology resource announcements 11((8)):e0029022 PMC6210121 · Pubmed · DOI

    Here, we report the metagenomes from five anaerobic bioreactors, operated under different conditions, that were fed carbohydrate-rich thin stillage from a corn starch ethanol plant. The putative functions of the abundant taxa identified here will inform future studies of microbial communities involved in valorizing this and other low-value agroindustrial residues.

  • Walters KA, Myers KS, Wang H, Fortney NW, Ingle AT, Scarborough MJ, Donohue TJ, Noguera DR (2022) Metagenomes and Metagenome-Assembled Genomes from Microbial Communities Fermenting Ultrafiltered Milk Permeate. Microbiology resource announcements 11((7)):e0029322 PMC6210121 · Pubmed · DOI

    Fermentative microbial communities can be utilized for the conversion of various agroindustrial residues into valuable chemicals. Here, we report 34 metagenomes from anaerobic bioreactors fed lactose-rich ultrafiltered milk permeate and 278 metagenome-assembled genomes (MAGs). These MAGs can inform future studies aimed at generating renewable chemicals from dairy and other agroindustrial residues.

  • Lakey BD, Myers KS, Alberge F, Mettert EL, Kiley PJ, Noguera DR, Donohue TJ (2022) The essential Rhodobacter sphaeroides CenKR two-component system regulates cell division and envelope biosynthesis. PLoS genetics 18((6)):e1010270 PMC7265319 · Pubmed · DOI

    Bacterial two-component systems (TCSs) often function through the detection of an extracytoplasmic stimulus and the transduction of a signal by a transmembrane sensory histidine kinase. This kinase then initiates a series of reversible phosphorylation modifications to regulate the activity of a cognate, cytoplasmic response regulator as a transcription factor. Several TCSs have been implicated in the regulation of cell cycle dynamics, cell envelope integrity, or cell wall development in Escherichia coli and other well-studied Gram-negative model organisms. However, many α-proteobacteria lack homologs to these regulators, so an understanding of how α-proteobacteria orchestrate extracytoplasmic events is lacking. In this work we identify an essential TCS, CenKR (Cell envelope Kinase and Regulator), in the α-proteobacterium Rhodobacter sphaeroides and show that modulation of its activity results in major morphological changes. Using genetic and biochemical approaches, we dissect the requirements for the phosphotransfer event between CenK and CenR, use this information to manipulate the activity of this TCS in vivo, and identify genes that are directly and indirectly controlled by CenKR in Rb. sphaeroides. Combining ChIP-seq and RNA-seq, we show that the CenKR TCS plays a direct role in maintenance of the cell envelope, regulates the expression of subunits of the Tol-Pal outer membrane division complex, and indirectly modulates the expression of peptidoglycan biosynthetic genes. CenKR represents the first TCS reported to directly control the expression of Tol-Pal machinery genes in Gram-negative bacteria, and we predict that homologs of this TCS serve a similar function in other closely related organisms. We propose that Rb. sphaeroides genes of unknown function that are directly regulated by CenKR play unknown roles in cell envelope biosynthesis, assembly, and/or remodeling in this and other α-proteobacteria.

  • Wadler CS, Wolters JF, Fortney NW, Throckmorton KO, Zhang Y, Miller CR, Schneider RM, Wendt-Pienkowski E, Currie CR, Donohue TJ, Noguera DR, Hittinger CT, Thomas MG (2022) Utilization of lignocellulosic biofuel conversion residue by diverse microorganisms. Biotechnology for biofuels and bioproducts 15((1)):70 PMC8320890 · Pubmed · DOI

    Lignocellulosic conversion residue (LCR) is the material remaining after deconstructed lignocellulosic biomass is subjected to microbial fermentation and treated to remove the biofuel. Technoeconomic analyses of biofuel refineries have shown that further microbial processing of this LCR into other bioproducts may help offset the costs of biofuel generation. Identifying organisms able to metabolize LCR is an important first step for harnessing the full chemical and economic potential of this material. In this study, we investigated the aerobic LCR utilization capabilities of 71 Streptomyces and 163 yeast species that could be engineered to produce valuable bioproducts. The LCR utilization by these individual microbes was compared to that of an aerobic mixed microbial consortium derived from a wastewater treatment plant as representative of a consortium with the highest potential for degrading the LCR components and a source of genetic material for future engineering efforts.

  • Fremin, B. J. et al. (2022) Thousands of small, novel genes predicted in global phage genomes Cell Reports 39(12):110984 PMC9254267 · Pubmed · DOI

    Small genes (<150 nucleotides) have been systematically overlooked in phage genomes. We employ a large-scale comparative genomics approach to predict >40,000 small-gene families in ∼2.3 million phage genome contigs. We find that small genes in phage genomes are approximately 3-fold more prevalent than in host prokaryotic genomes. Our approach enriches for small genes that are translated in microbiomes, suggesting the small genes identified are coding. More than 9,000 families encode potentially secreted or transmembrane proteins, more than 5,000 families encode predicted anti-CRISPR proteins, and more than 500 families encode predicted antimicrobial proteins. By combining homology and genomic-neighborhood analyses, we reveal substantial novelty and diversity within phage biology, including small phage genes found in multiple host phyla, small genes encoding proteins that play essential roles in host infection, and small genes that share genomic neighborhoods and whose encoded proteins may share related functions.

  • Beach NK, Myers KS, Donohue TJ, Noguera DR (2022) Metagenomes from 25 Low-Abundance Microbes in a Partial Nitritation Anammox Microbiome. Microbiology resource announcements 11((6)):e0021222 PMC3998144 · Pubmed · DOI

    Microbial communities using anammox bacteria to remove nitrogen are increasingly important in wastewater treatment. We report on 25 metagenome-assembled genomes of low-abundance microbes from a partial nitritation anammox bioreactor system that have not been described previously. These data add to the body of information about this important wastewater treatment system.

  • Linz AM, Ma Y, Scholz S, Noguera DR, Donohue TJ (2022) iNovo479: Metabolic Modeling Provides a Roadmap to Optimize Bioproduct Yield from Deconstructed Lignin Aromatics by Novosphingobium aromaticivorans . Metabolites 12((4)): PMC9028409 · Pubmed · DOI

    Lignin is an abundant renewable source of aromatics and precursors for the production of other organic chemicals. However, lignin is a heterogeneous polymer, so the mixture of aromatics released during its depolymerization can make its conversion to chemicals challenging. Microbes are a potential solution to this challenge, as some can catabolize multiple aromatic substrates into one product. Novosphingobium aromaticivorans has this ability, and its use as a bacterial chassis for lignin valorization could be improved by the ability to predict product yields based on thermodynamic and metabolic inputs. In this work, we built a genome-scale metabolic model of N. aromaticivorans , iNovo479, to guide the engineering of strains for aromatic conversion into products. iNovo479 predicted product yields from single or multiple aromatics, and the impact of combinations of aromatic and non-aromatic substrates on product yields. We show that enzyme reactions from other organisms can be added to iNovo479 to predict the feasibility and profitability of producing additional products by engineered strains. Thus, we conclude that iNovo479 can help guide the design of bacteria to convert lignin aromatics into valuable chemicals.

  • Beach NK, Myers KS, Owen BR, Seib M, Donohue TJ, Noguera DR (2022) Correction for Beach et al., "Exploring the Meta-regulon of the CRP/FNR Family of Global Transcriptional Regulators in a Partial-Nitritation Anammox Microbiome". mSystems 7((3)):e0021322 PMC9239163 · Pubmed · DOI

    No abstract available.

  • Scarborough MJ, Myers KS, Fortney NW, Ingle AT, Donohue TJ, Noguera DR (2022) Metagenome-Assembled Genomes from a Microbiome Converting Xylose to Medium-Chain Carboxylic Acids. Microbiology resource announcements 11((4)):e0115121 PMC9022542 · Pubmed · DOI

    There is growing interest in producing beneficial products from wastes using microbiomes. We previously performed multiomic analyses of a bioreactor microbiome that converted carbohydrate-rich lignocellulosic residues to medium-chain carboxylic acids. Here, we present draft metagenome-assembled genomes from this microbiome, obtained from reactors in which xylose was the primary carbon source.

  • Donohue TJ (2022) Editorial overview: Microbial activities powering society. Current opinion in microbiology 67:102144 · Pubmed · DOI

    No abstract available.

  • Perez, J. M., Umana, G., Sener, C., Coplien, J., Haak, D., Li, Y., Karlen, S. D., Ralph, J., Donohue, T. J., and D. R. Noguera (2021) Integrating lignin depolymerization with microbial funneling processes using agronomically relevant feedstocks Green Chemistry 24:2795-2811 · DOI

    The economic feasibility of the lignocellulosic biomass refinery requires the valorization of lignin in addition to its polysaccharide fraction. One promising approach is the combination of chemical methods for lignin fractionation and depolymerization with microbial funneling of the resulting phenolic monomers into valuable chemicals. In this work, we explored the integration of γ-valerolactone (GVL) for biomass pretreatment, catalytic hydrogenolysis for lignin depolymerization, and microbial funneling to 2-pyrone-4,6-dicarboxylic acid (PDC) by the engineered bacterium Novosphingobium aromaticivorans strain PDC. We first investigated the microbial PDC production feasibility from common phenolic compounds previously identified in lignin hydrogenolysis products. Next, we studied the PDC production potential from maple, poplar, sorghum, and switchgrass using the proposed integrated pipeline and, finally, we performed a technoeconomic analysis (TEA) of the system to identify parameters that affect its economic feasibility. We found that N. aromaticivorans strain PDC is able to produce PDC from phenolic compounds with propanol, methyl, or methyl ester sidechains. Using Pd/C as a catalyst for hydrogenolysis to favor the production of these phenolics from lignin extracted with the GVL process, we obtained microbial PDC production yields of 88, 139, 103, and 79 g PDC per kg lignin from maple, poplar, sorghum, and switchgrass, respectively. Using these yields, we estimated a baseline minimum selling price of $12.10 per kg of purified PDC, and identified options to further improve the integrated pipeline.

  • Myers KS, Behari Lal P, Noguera DR, Donohue TJ (2021) Using Genome Scale Mutant Libraries to Identify Essential Genes. Methods in molecular biology (Clifton, N.J.) 2377:215-236 · Pubmed · DOI

    Identification of essential genes is key to understanding the required processes and gene products of organisms under one or more conditions. Transposon sequencing (Tn-seq) has been used to predict essential genes or ones that conditionally impact fitness in a wide variety of organisms. Here, we describe the generation of genome-scale mutant libraries and the analysis of Tn-seq data to identify essential genes from cultures grown in a single condition as well as those that are conditionally important by analyzing the behavior of these mutant libraries in different growth environments. While we illustrate the approach using data derived from Tn-seq analysis of the α-proteobacteria Rhodobacter sphaeroides and Zymomonas mobilis, the protocols and systems we describe should be generally applicable to a variety of organisms.

  • Beach NK, Myers KS, Owen BR, Seib M, Donohue TJ, Noguera DR (2021) Exploring the Meta-regulon of the CRP/FNR Family of Global Transcriptional Regulators in a Partial-Nitritation Anammox Microbiome. mSystems 6((5)):e0090621 PMC8510549 · Pubmed · DOI

    Microorganisms must respond to environmental changes to survive, often by controlling transcription initiation. Intermittent aeration during wastewater treatment presents a cyclically changing environment to which microorganisms must react. We used an intermittently aerated bioreactor performing partial nitritation and anammox (PNA) to investigate how the microbiome responds to recurring change. Meta-transcriptomic analysis revealed a dramatic disconnect between the relative DNA abundance and gene expression within the metagenome-assembled genomes (MAGs) of community members, suggesting the importance of transcriptional regulation in this microbiome. To explore how community members responded to cyclic aeration via transcriptional regulation, we searched for homologs of the catabolite repressor protein/fumarate and nitrate reductase regulatory protein (CRP/FNR) family of transcription factors (TFs) within the MAGs. Using phylogenetic analyses, evaluation of sequence conservation in important amino acid residues, and prediction of genes regulated by TFs in the MAGs, we identified homologs of the oxygen-sensing FNR in Nitrosomonas and Rhodocyclaceae , nitrogen-sensing dissimilative nitrate respiration regulator that responds to nitrogen species (DNR) in Rhodocyclaceae , and nitrogen-sensing nitrite and nitric oxide reductase regulator that responds to nitrogen species (NnrR) in Nitrospira MAGs. Our data also predict that CRP/FNR homologs in Ignavibacteria , Flavobacteriales , and Saprospiraceae MAGs sense carbon availability. In addition, a CRP/FNR homolog in a Brocadia MAG was most closely related to CRP TFs known to sense carbon sources in well-studied organisms. However, we predict that in autotrophic Brocadia, this TF most likely regulates a diverse set of functions, including a response to stress during the cyclic aerobic/anoxic conditions. Overall, this analysis allowed us to define a meta-regulon of the PNA microbiome that explains functions and interactions of the most active community members. IMPORTANCE Microbiomes are important contributors to many ecosystems, including ones where nutrient cycling is stimulated by aeration control. Optimizing cyclic aeration helps reduce energy needs and maximize microbiome performance during wastewater treatment; however, little is known about how most microbial community members respond to these alternating conditions. We defined the meta-regulon of a PNA microbiome by combining existing knowledge of how the CRP/FNR family of bacterial TFs respond to stimuli, with metatranscriptomic analyses to characterize gene expression changes during aeration cycles. Our results indicated that, for some members of the community, prior knowledge is sufficient for high-confidence assignments of TF function, whereas other community members have CRP/FNR TFs for which inferences of function are limited by lack of prior knowledge. This study provides a framework to begin elucidating meta-regulons in microbiomes, where pure cultures are not available for traditional transcriptional regulation studies. Defining the meta-regulon can help in optimizing microbiome performance.

  • Linz AM, Ma Y, Perez JM, Myers KS, Kontur WS, Noguera DR, Donohue TJ (2021) Aromatic Dimer Dehydrogenases from Novosphingobium aromaticivorans Reduce Monoaromatic Diketones. Applied and environmental microbiology 87((24)):e0174221 PMC8612281 · Pubmed · DOI

    Lignin is a potential source of valuable chemicals, but its chemical depolymerization results in a heterogeneous mixture of aromatics and other products. Microbes could valorize depolymerized lignin by converting multiple substrates into one or a small number of products. In this study, we describe the ability of Novosphingobium aromaticivorans to metabolize 1-(4-hydroxy-3-methoxyphenyl)propane-1,2-dione (G-diketone), an aromatic Hibbert diketone that is produced during formic acid-catalyzed lignin depolymerization. By assaying genome-wide transcript levels from N. aromaticivorans during growth on G-diketone and other chemically-related aromatics, we hypothesized that the Lig dehydrogenases, previously characterized as oxidizing β-O-4 linkages in aromatic dimers, were involved in G-diketone metabolism by N. aromaticivorans. Using purified N. aromaticivorans Lig dehydrogenases, we found that LigL, LigN, and LigD each reduced the Cα ketone of G-diketone in vitro but with different substrate specificities and rates. Furthermore, LigL, but not LigN or LigD, also reduced the Cα ketone of 2-hydroxy-1-(4-hydroxy-3-methoxyphenyl)propan-1-one (GP-1) in vitro , a derivative of G-diketone with the Cβ ketone reduced, when GP-1 was provided as a substrate. The newly identified activity of these Lig dehydrogenases expands the potential range of substrates utilized by N. aromaticivorans beyond what has been previously recognized. This is beneficial both for metabolizing a wide range of natural and non-native depolymerized lignin substrates and for engineering microbes and enzymes that are active with a broader range of aromatic compounds. IMPORTANCE Lignin is a major plant polymer composed of aromatic units that have value as chemicals. However, the structure and composition of lignin have made it difficult to use this polymer as a renewable source of industrial chemicals. Bacteria like Novosphingobium aromaticivorans have the potential to make chemicals from lignin not only because of their natural ability to metabolize a variety of aromatics but also because there are established protocols to engineer N. aromaticivorans strains to funnel lignin-derived aromatics into valuable products. In this work, we report a newly discovered activity of previously characterized dehydrogenase enzymes with a chemically modified by-product of lignin depolymerization. We propose that the activity of N. aromaticivorans enzymes with both native lignin aromatics and those produced by chemical depolymerization will expand opportunities for producing industrial chemicals from the heterogenous components of this abundant plant polymer.

  • Ingle AT, Fortney NW, Walters KA, Donohue TJ, Noguera DR (2021) Mixed Acid Fermentation of Carbohydrate-Rich Dairy Manure Hydrolysate. Frontiers in bioengineering and biotechnology 9:724304 PMC8370043 · Pubmed · DOI

    Dairy manure (DM) is an abundant agricultural residue that is largely composed of lignocellulosic biomass. The aim of this study was to investigate if carbon derived from DM fibers can be recovered as medium-chain fatty acids (MCFAs), which are mixed culture fermentation products of economic interest. DM fibers were subjected to combinations of physical, enzymatic, chemical, and thermochemical pretreatments to evaluate the possibility of producing carbohydrate-rich hydrolysates suitable for microbial fermentation by mixed cultures. Among the pretreatments tested, decrystalization dilute acid pretreatment (DCDA) produced the highest concentrations of glucose and xylose, and was selected for further experiments. Bioreactors fed DCDA hydrolysate were operated. Acetic acid and butyric acid comprised the majority of end products during operation of the bioreactors. MCFAs were transiently produced at a maximum concentration of 0.17 mg COD/mg COD. Analyses of the microbial communities in the bioreactors suggest that lactic acid bacteria, Megasphaera , and Caproiciproducens were involved in MCFA and C4 production during DCDA hydrolysate metabolism.

  • Fortney NW, Hanson NJ, Rosa PRF, Donohue TJ, Noguera DR (2021) Diverse Profile of Fermentation Byproducts From Thin Stillage. Frontiers in bioengineering and biotechnology 9:695306 PMC8320890 · Pubmed · DOI

    The economy of biorefineries is influenced not only by biofuel production from carbohydrates but also by the production of valuable compounds from largely underutilized industrial residues. Currently, the demand for many chemicals that could be made in a biorefinery, such as succinic acid (SA), medium-chain fatty acids (MCFAs), and lactic acid (LA), is fulfilled using petroleum, palm oil, or pure carbohydrates as raw materials, respectively. Thin stillage (TS), the residual liquid material following distillation of ethanol, is an underutilized coproduct from the starch biofuel industry. This carbon-rich material has the potential for chemical upgrading by microorganisms. Here, we explored the formation of different fermentation products by microbial communities grown on TS using different bioreactor conditions. At the baseline operational condition (6-day retention time, pH 5.5, 35°C), we observed a mixture of MCFAs as the principal fermentation products. Operation of a bioreactor with a 1-day retention time induced an increase in SA production, and a temperature increase to 55°C resulted in the accumulation of lactic and propionic acids. In addition, a reactor operated with a 1-day retention time at 55°C conditions resulted in LA accumulation as the main fermentation product. The prominent members of the microbial community in each reactor were assessed by 16S rRNA gene amplicon sequencing and phylogenetic analysis. Under all operating conditions, members of the Lactobacillaceae family within Firmicutes and the Acetobacteraceae family within Proteobacteria were ubiquitous. Members of the Prevotellaceae family within Bacteroidetes and Lachnospiraceae family within the Clostridiales order of Firmicutes were mostly abundant at 35°C and not abundant in the microbial communities of the TS reactors incubated at 55°C. The ability to adjust bioreactor operating conditions to select for microbial communities with different fermentation product profiles offers new strategies to explore and compare potentially valuable fermentation products from TS and allows industries the flexibility to adapt and switch chemical production based on market prices and demands.

  • Myers KS, Noguera DR, Donohue TJ (2021) Promoter Architecture Differences among Alphaproteobacteria and Other Bacterial Taxa. mSystems 6((4)):e0052621 PMC8407463 · Pubmed · DOI

    Much of our knowledge of bacterial transcription initiation has been derived from studying the promoters of Escherichia coli and Bacillus subtilis. Given the expansive diversity across the bacterial phylogeny, it is unclear how much of this knowledge can be applied to other organisms. Here, we report on bioinformatic analyses of promoter sequences of the primary σ factor (σ) by leveraging publicly available transcription start site (TSS) sequencing data sets for nine bacterial species spanning five phyla. This analysis identifies previously unreported differences in the -35 and -10 elements of σ-dependent promoters in several groups of bacteria. We found that Actinobacteria and Betaproteobacteria σ-dependent promoters lack the TTG triad in their -35 element, which is predicted to be conserved across the bacterial phyla. In addition, the majority of the Alphaproteobacteria σ-dependent promoters analyzed lacked the thymine at position -7 that is highly conserved in other phyla. Bioinformatic examination of the Alphaproteobacteria σ-dependent promoters identifies a significant overrepresentation of essential genes and ones encoding proteins with common cellular functions downstream of promoters containing an A, C, or G at position -7. We propose that transcription of many σ-dependent promoters in Alphaproteobacteria depends on the transcription factor CarD, which is an essential protein in several members of this phylum. Our analysis expands the knowledge of promoter architecture across the bacterial phylogeny and provides new information that can be used to engineer bacteria for use in medical, environmental, agricultural, and biotechnological processes. IMPORTANCE Transcription of DNA to RNA by RNA polymerase is essential for cells to grow, develop, and respond to stress. Understanding the process and control of transcription is important for health, disease, the environment, and biotechnology. Decades of research on a few bacteria have identified promoter DNA sequences that are recognized by the σ subunit of RNA polymerase. We used bioinformatic analyses to reveal previously unreported differences in promoter DNA sequences across the bacterial phylogeny. We found that many Actinobacteria and Betaproteobacteria promoters lack a sequence in their -35 DNA recognition element that was previously assumed to be conserved and that Alphaproteobacteria lack a thymine residue at position -7, also previously assumed to be conserved. Our work reports important new information about bacterial transcription, illustrates the benefits of studying bacteria across the phylogenetic tree, and proposes new lines of future investigation.

  • Myers KS, Place M, Kominek J, Noguera DR, Donohue TJ (2021) Delila-PY, a Pipeline for Utilizing the Delila Suite of Software to Identify Potential DNA Binding Motifs. Microbiology resource announcements 10((15)): PMC8050967 · Pubmed · DOI

    Predicting potential DNA binding motifs is a critical part of understanding gene expression across all domains of life. Here, we report the development of Delila-PY, an easy-to-use pipeline for utilizing the Delila suite of software to identify DNA binding motifs.

  • Ma Y, Donohue TJ, Noguera DR (2021) Kinetic modeling of anaerobic degradation of plant-derived aromatic mixtures by Rhodopseudomonas palustris. Biodegradation 32((2)):179-192 PMC7997838 · Pubmed · DOI

    Rhodopseudomonas palustris is a model microorganism for studying the anaerobic metabolism of aromatic compounds. While it is well documented which aromatics can serve as sole organic carbon sources, co-metabolism of other aromatics is poorly understood. This study used kinetic modeling to analyze the simultaneous degradation of aromatic compounds present in corn stover hydrolysates and model the co-metabolism of aromatics not known to support growth of R. palustris as sole organic substrates. The simulation predicted that p-coumaroyl amide and feruloyl amide were hydrolyzed to p-coumaric acid and ferulic acid, respectively, and further transformed via p-coumaroyl-CoA and feruloyl-CoA. The modeling also suggested that metabolism of p-hydroxyphenyl aromatics was slowed by substrate inhibition, whereas the transformation of guaiacyl aromatics was inhibited by their p-hydroxyphenyl counterparts. It also predicted that substrate channeling may occur during degradation of p-coumaroyl-CoA and feruloyl-CoA, resulting in no detectable accumulation of p-hydroxybenzaldehyde and vanillin, during the transformation of these CoA ligated compounds to p-hydroxybenzoic acid and vanillic acid, respectively. While the simulation correctly represented the known transformation of p-hydroxybenzoic acid via the benzoyl-CoA pathway, it also suggested co-metabolism of vanillic acid and syringic acid, which are known not to serve as photoheterotrophic growth substrate for R. palustris.

  • Perez JM, Kontur WS, Gehl C, Gille DM, Ma Y, Niles AV, Umana G, Donohue TJ, Noguera DR (2021) Redundancy in aromatic O-demethylation and ring opening reactions in Novosphingobium aromaticivorans and their impact in the metabolism of plant derived phenolics. Applied and environmental microbiology : PMC8091115 · Pubmed · DOI

    Lignin is a plant heteropolymer composed of phenolic subunits. Because of its heterogeneity and recalcitrance, the development of efficient methods for its valorization still remains an open challenge. One approach to utilize lignin is its chemical deconstruction into mixtures of monomeric phenolic compounds followed by biological funneling into a single product. Novosphingobium aromaticivorans DSM12444 has been previously engineered to produce 2-pyrone-4,6-dicarboxylic acid (PDC) from depolymerized lignin by simultaneously metabolizing multiple aromatics through convergent routes involving the intermediates 3-methoxygallic acid (3-MGA) and protocatechuic acid (PCA). We investigated enzymes predicted to be responsible for O -demethylation and oxidative aromatic ring opening, two critical reactions involved in the metabolism of phenolics compounds by N. aromaticivorans The results showed the involvement of DesA in O -demethylation of syringic and vanillic acids, LigM in O- demethylation of vanillic acid and 3-MGA, and a new O- demethylase, DmtS, in the conversion of 3-MGA into gallic acid (GA). In addition, we found that LigAB was the main aromatic ring opening dioxygenase involved in 3-MGA, PCA, and GA metabolism, and that a previously uncharacterized dioxygenase, LigAB2, had high activity with GA. Our results indicate a metabolic route not previously identified in N. aromaticivorans that involves O -demethylation of 3-MGA to GA. We predict this pathway channels ∼15% of the carbon flow from syringic acid, with the rest following ring opening of 3-MGA. The new knowledge obtained in this study allowed for the creation of an improved engineered strain for the funneling of aromatic compounds that exhibits stoichiometric conversion of syringic acid into PDC. IMPORTANCE For lignocellulosic biorefineries to effectively contribute to reduction of fossil fuel use, they need to become efficient at producing chemicals from all major components of plant biomass. Making products from lignin will require engineering microorganisms to funnel multiple phenolic compounds to the chemicals of interest, and N. aromaticivorans is a promising chassis for this technology. The ability of N. aromaticivorans to efficiently and simultaneously degrade many phenolic compounds may be linked to having functionally redundant aromatic degradation pathways and enzymes with broad substrate specificity. A detailed knowledge of aromatic degradation pathways is thus essential to identify genetic engineering targets to maximize product yields. Furthermore, knowledge of enzyme substrate specificity is critical to redirect flow of carbon to desired pathways. This study described an uncharacterized pathway in N. aromaticivorans and the enzymes that participate in this pathway, allowing the engineering of an improved strain for production of PDC from lignin.

  • Glanville DG, Mullineaux-Sanders C, Corcoran CJ, Burger BT, Imam S, Donohue TJ, Ulijasz AT (2021) A High-Throughput Method for Identifying Novel Genes That Influence Metabolic Pathways Reveals New Iron and Heme Regulation in Pseudomonas aeruginosa. mSystems 6((1)): PMC7857532 · Pubmed · DOI

    Heme is an essential metabolite for most life on earth. Bacterial pathogens almost universally require iron to infect a host, often acquiring this nutrient in the form of heme. The Gram-negative pathogen Pseudomonas aeruginosa is no exception, where heme acquisition and metabolism are known to be crucial for both chronic and acute infections. To unveil unknown genes and pathways that could play a role with heme metabolic flux in this pathogen, we devised an omic-based approach we dubbed "Met-Seq," for met abolite-coupled transposon seq uencing. Met-Seq couples a biosensor with fluorescence-activated cell sorting (FACS) and massively parallel sequencing, allowing for direct identification of genes associated with metabolic changes. In this work, we first construct and validate a heme biosensor for use with P. aeruginosa and exploit Met-Seq to identify 188 genes that potentially influence intracellular heme levels. Identified genes largely consisted of metabolic pathways not previously associated with heme, including many secreted virulence effectors, as well as 11 predicted small RNAs (sRNAs) and riboswitches whose functions are not currently understood. We verify that five Met-Seq hits affect intracellular heme levels; a predicted extracytoplasmic function (ECF) factor, a phospholipid acquisition system, heme biosynthesis regulator Dnr, and two predicted antibiotic monooxygenase (ABM) domains of unknown function (PA0709 and PA3390). Finally, we demonstrate that PA0709 and PA3390 are novel heme-binding proteins. Our data suggest that Met-Seq could be extrapolated to other biological systems and metabolites for which there is an available biosensor, and provides a new template for further exploration of iron/heme regulation and metabolism in P. aeruginosa and other pathogens. IMPORTANCE The ability to simultaneously and more directly correlate genes with metabolite levels on a global level would provide novel information for many biological platforms yet has thus far been challenging. Here, we describe a method to help address this problem, which we dub "Met-Seq" ( met abolite-coupled Tn seq uencing). Met-Seq uses the powerful combination of fluorescent biosensors, fluorescence-activated cell sorting (FACS), and next-generation sequencing (NGS) to rapidly identify genes that influence the levels of specific intracellular metabolites. For proof of concept, we create and test a heme biosensor and then exploit Met-Seq to identify novel genes involved in the regulation of heme in the pathogen Pseudomonas aeruginosa Met-Seq-generated data were largely comprised of genes which have not previously been reported to influence heme levels in this pathogen, two of which we verify as novel heme-binding proteins. As heme is a required metabolite for host infection in P. aeruginosa and most other pathogens, our studies provide a new list of targets for potential antimicrobial therapies and shed additional light on the balance between infection, heme uptake, and heme biosynthesis.

  • Todor H, Osadnik H, Campbell EA, Myers KS, Li H, Donohue TJ, Gross CA (2020) Rewiring the specificity of extracytoplasmic function sigma factors. Proceedings of the National Academy of Sciences of the United States of America 117((52)):33496-33506 PMC7776599 · Pubmed · DOI

    Bacterial genomes are being sequenced at an exponentially increasing rate, but our inability to decipher their transcriptional wiring limits our ability to derive new biology from these sequences. De novo determination of regulatory interactions requires accurate prediction of regulators' DNA binding and precise determination of biologically significant binding sites. Here we address these challenges by solving the DNA-specificity code of extracytoplasmic function sigma factors (ECF σs), a major family of bacterial regulators, and determining their putative regulons. We generated an aligned collection of ECF σs and their promoters by leveraging the autoregulatory nature of ECF σs as a means of promoter discovery and analyzed it to identify and characterize the conserved amino acid-nucleotide interactions that determine promoter specificity. This enabled de novo prediction of ECF σ specificity, which we combined with a statistically rigorous phylogenetic footprinting pipeline based on precomputed orthologs to predict the direct targets of ∼67% of ECF σs. This global survey indicated that some ECF σs are conserved global regulators controlling many genes throughout the genome, which are important under many conditions, while others are local regulators, controlling a few closely linked genes in response to specific stimuli in select species. This analysis reveals important organizing principles of bacterial gene regulation and presents a conceptual and computational framework for deciphering gene regulatory networks.

  • Henry KK, Ross W, Myers KS, Lemmer KC, Vera JM, Landick R, Donohue TJ, Gourse RL (2020) A majority of Rhodobacter sphaeroides 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 117((47)):29658-29668 PMC7703639 · Pubmed · DOI

    Using an in vitro transcription system with purified RNA polymerase (RNAP) to investigate rRNA synthesis in the photoheterotrophic α-proteobacterium Rhodobacter sphaeroides , we identified a surprising feature of promoters recognized by the major holoenzyme. Transcription from R. sphaeroides 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 R. sphaeroides RNAP can utilize -7T when present. rRNA promoters were activated by purified R. sphaeroides 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 R. sphaeroides 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 R. sphaeroides 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 R. sphaeroides 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.

  • Nayfach, S. et al., (2021) A genomic catalog of Earth’s microbiomes Nature biotechnology 39(4):499-509 PMC8041624 · Pubmed · DOI

    The reconstruction of bacterial and archaeal genomes from shotgun metagenomes has enabled insights into the ecology and evolution of environmental and host-associated microbiomes. Here we applied this approach to >10,000 metagenomes collected from diverse habitats covering all of Earth's continents and oceans, including metagenomes from human and animal hosts, engineered environments, and natural and agricultural soils, to capture extant microbial, metabolic and functional potential. This comprehensive catalog includes 52,515 metagenome-assembled genomes representing 12,556 novel candidate species-level operational taxonomic units spanning 135 phyla. The catalog expands the known phylogenetic diversity of bacteria and archaea by 44% and is broadly available for streamlined comparative analyses, interactive exploration, metabolic modeling and bulk download. We demonstrate the utility of this collection for understanding secondary-metabolite biosynthetic potential and for resolving thousands of new host linkages to uncultivated viruses. This resource underscores the value of genome-centric approaches for revealing genomic properties of uncultivated microorganisms that affect ecosystem processes.

  • 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. IMPORTANCE 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 Alphaproteobacteria , 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 Alphaproteobacteria 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, Alphaproteobacteria .

  • 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 ( CO mprehensive T ranscriptomic OR ganizational T 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 Rhodobacter sphaeroides 2.4.1 and Rhodopseudomonas palustris CGA009. One previously unknown pathway intermediate was a methylated diunsaturated fatty acid, (10 E, 12 E )-11-methyloctadeca-10,12-dienoic acid (11Me-10 t ,12 t -18:2), produced from (11 E )-methyloctadeca-11-enoic acid (11Me-12 t -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-10 t, 12 t -18:2 fatty acid phospholipid chain as a substrate. We discovered that R. palustris 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 Rhodobacter sphaeroides , 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 R. sphaeroides 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. IMPORTANCE 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 R. sphaeroides 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. Rhodopseudomonas palustris CGA009 is a known degrader of phenolic compounds under photoheterotrophic conditions via the benzoyl coenzyme A (CoA) degradation (BAD) pathway. However, R. palustris CGA009 is reported to be unable to metabolize meta -methoxylated phenolics, such as syringic acid. We isolated a strain of R. palustris (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 vanARB operon ( rpa3619 , rpa3620 , rpa3621 ), which has been described as catalyzing aerobic aromatic ring demethylation in other bacteria, in anaerobic syringic acid degradation. In addition, experiments with a vanARB deletion mutant demonstrated the involvement of the vanARB operon in anaerobic syringic acid degradation. These observations provide new insights into the anaerobic degradation of meta -methoxylated and other aromatics by R. palustris IMPORTANCE 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 Rhodopseudomonas palustris strain capable of syringic acid degradation reveal a previously unknown metabolic route for aromatic degradation in R. palustris 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 " Candidatus Weimeria bifida" gen. nov., sp. nov., and " Candidatus Pseudoramibacter fermentans" sp. nov. Applied and environmental microbiology 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 " Candidatus Weimeria bifida" gen. nov., sp. nov., and " Candidatus Pseudoramibacter fermentans" sp. nov., both predicted to produce MCFA but using different substrates. The analysis of a time series metatranscriptomic data set suggests that " Ca 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 " Ca 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 " Ca Pseudoramibacter fermentans." In total, this work reveals unknown characteristics of MCFA production in two novel organisms. IMPORTANCE 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 PMC8384415 · 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, J., M., Kontur, W. S., Alherech, M., Karlen, S., Ralph, J., Stahl, S., Donohue, T. J., and D. R. Noguera (2019) Funneling chemically depolymerized lignin products into 2,4-pyridinedicarboxylic acid with Novosphingobium aromaticivorans Green Chemistry 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 S -transferase that stereospecifically breaks lignin's β( R )-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 S -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( R ) stereoisomer of the bond, and LigF homologues, which cleave the β( S ) stereoisomer. Here, we report on a heterodimeric β-etherase (BaeAB) from the sphingomonad Novosphingobium aromaticivorans that stereospecifically cleaves the β( R )-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( R )-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( R )-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

    No abstract available.

  • 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. Biotechnology for biofuels 11:200 PMC6052542 · Pubmed · DOI

    No abstract available.

  • 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) Novosphingobium aromaticivorans uses a Nu-class glutathione S -transferase as a glutathione lyase in breaking the β-aryl ether bond of lignin. The Journal of biological chemistry 293((14)):4955-4968 PMC5892560 · Pubmed · DOI

    No abstract available.

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

    No abstract available.

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

    No abstract available.

  • 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

    No abstract available.

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

    No abstract available.

  • 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.

  • 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.

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  • 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.
  • 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.
  • 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.