Robert P. Gunsalus

EcoCyc: fusing model organism databases with systems biology

EcoCyc (http://EcoCyc.org) is a model organism database built on the genome sequence of Escherichia coli K-12 MG1655. Expert manual curation of the functions of individual E. coli gene products in EcoCyc has been based on information found in the experimental literature for E. coli K-12-derived strains. Updates to EcoCyc content continue to improve the comprehensive picture of E. coli biology. The utility of EcoCyc is enhanced by new tools available on the EcoCyc web site, and the development of EcoCyc as a teaching tool is increasing the impact of the knowledge collected in EcoCyc.

EcoCyc: a comprehensive database of Escherichia coli biology

EcoCyc (http://EcoCyc.org) is a comprehensive model organism database for Escherichia coli K-12 MG1655. From the scientific literature, EcoCyc captures the functions of individual E. coli gene products; their regulation at the transcriptional, post-transcriptional and protein level; and their organization into operons, complexes and pathways. EcoCyc users can search and browse the information in multiple ways. Recent improvements to the EcoCyc Web interface include combined gene/protein pages and a Regulation Summary Diagram displaying a graphical overview of all known regulatory inputs to gene expression and protein activity. The graphical representation of signal transduction pathways has been updated, and the cellular and regulatory overviews were enhanced with new functionality. A specialized undergraduate teaching resource using EcoCyc is being developed.

EcoCyc: A comprehensive view of Escherichia coli biology

EcoCyc (http://EcoCyc.org) provides a comprehensive encyclopedia of Escherichia coli biology. EcoCyc integrates information about the genome, genes and gene products; the metabolic network; and the regulatory network of E. coli. Recent EcoCyc developments include a new initiative to represent and curate all types of E. coli regulatory processes such as attenuation and regulation by small RNAs. EcoCyc has started to curate Gene Ontology (GO) terms for E. coli and has made a dataset of E. coli GO terms available through the GO Web site. The curation and visualization of electron transfer processes has been significantly improved. Other software and Web site enhancements include the addition of tracks to the EcoCyc genome browser, in particular a type of track designed for the display of ChIP-chip datasets, and the development of a comparative genome browser. A new Genome Omics Viewer enables users to paint omics datasets onto the full E. coli genome for analysis. A new advanced query page guides users in interactively constructing complex database queries against EcoCyc. A Macintosh version of EcoCyc is now available. A series of Webinars is available to instruct users in the use of EcoCyc.

Syntrophy in Anaerobic Global Carbon Cycles

Syntrophy is an essential intermediary step in the anaerobic conversion of organic matter to methane where metabolically distinct microorganisms are tightly linked by the need to maintain the exchanged metabolites at very low concentrations. Anaerobic syntrophy is thermodynamically constrained, and is probably a prime reason why it is difficult to culture microbes as these approaches disrupt consortia. Reconstruction of artificial syntrophic consortia has allowed uncultured syntrophic metabolizers and methanogens to be optimally grown and studied biochemically. The pathways for syntrophic acetate, propionate and longer chain fatty acid metabolism are mostly understood, but key steps involved in benzoate breakdown and cyclohexane carboxylate formation are unclear. Syntrophic metabolism requires reverse electron transfer, close physical contact, and metabolic synchronization of the syntrophic partners. Genomic analyses reveal that multiple mechanisms exist for reverse electron transfer. Surprisingly, the flagellum functions were implicated in ensuring close physical proximity and synchronization of the syntrophic partners.

Physiology, Ecology, Phylogeny, and Genomics of Microorganisms Capable of Syntrophic Metabolism

Syntrophic metabolism is diverse in two respects: phylogenetically with microorganisms capable of syntrophic metabolism found in the Deltaproteobacteria and in the low G+C gram‐positive bacteria, and metabolically given the wide variety of compounds that can be syntrophically metabolized. The latter includes saturated fatty acids, unsaturated fatty acids, alcohols, and hydrocarbons. Besides residing in freshwater and marine anoxic sediments and soils, microbes capable of syntrophic metabolism also have been observed in more extreme habitats, including acidic soils, alkaline soils, thermal springs, and permanently cold soils, demonstrating that syntrophy is a widely distributed metabolic process in nature. Recent ecological and physiological studies show that syntrophy plays a far larger role in carbon cycling than was previously thought. The availability of the first complete genome sequences for four model microorganisms capable of syntrophic metabolism provides the genetic framework to begin dissecting the biochemistry of the marginal energy economies and interspecies interactions that are characteristic of the syntrophic lifestyle.

The genome of Syntrophus aciditrophicus: Life at the thermodynamic limit of microbial growth

Biochemically, the syntrophic bacteria constitute the missing link in our understanding of anaerobic flow of carbon in the biosphere. The completed genome sequence of Syntrophus aciditrophicus SB, a model fatty acid- and aromatic acid-degrading syntrophic bacterium, provides a glimpse of the composition and architecture of the electron transfer and energy-transducing systems needed to exist on marginal energy economies of a syntrophic lifestyle. The genome contains 3,179,300 base pairs and 3,169 genes where 1,618 genes were assigned putative functions. Metabolic reconstruction of the gene inventory revealed that most biosynthetic pathways of a typical Gram-negative microbe were present. A distinctive feature of syntrophic metabolism is the need for reverse electron transport; the presence of a unique Rnf-type ion-translocating electron transfer complex, menaquinone, and membrane-bound Fe-S proteins with associated heterodisulfide reductase domains suggests mechanisms to accomplish this task. Previously undescribed approaches to degrade fatty and aromatic acids, including multiple AMP-forming CoA ligases and acyl-CoA synthetases seem to be present as ways to form and dissipate ion gradients by using a sodium-based energy strategy. Thus, S. aciditrophicus, although nutritionally self-sufficient, seems to be a syntrophic specialist with limited fermentative and respiratory metabolism. Genomic analysis confirms the S. aciditrophicus metabolic and regulatory commitment to a nonconventional mode of life compared with our prevailing understanding of microbiology.

Succinate dehydrogenase and fumarate reductase from Escherichia coli

Succinate-ubiquinone oxidoreductase (SQR) as part of the trichloroacetic acid cycle and menaquinol-fumarate oxidoreductase (QFR) used for anaerobic respiration by Escherichia coli are structurally and functionally related membrane-bound enzyme complexes. Each enzyme complex is composed of four distinct subunits. The recent solution of the X-ray structure of QFR has provided new insights into the function of these enzymes. Both enzyme complexes contain a catalytic domain composed of a subunit with a covalently bound flavin cofactor, the dicarboxylate binding site, and an iron-sulfur subunit which contains three distinct iron-sulfur clusters. The catalytic domain is bound to the cytoplasmic membrane by two hydrophobic membrane anchor subunits that also form the site(s) for interaction with quinones. The membrane domain of E. coli SQR is also the site where the heme b556 is located. The structure and function of SQR and QFR are briefly summarized in this communication and the similarities and differences in the membrane domain of the two enzymes are discussed.

Genomic insights into syntrophy: the paradigm for anaerobic metabolic cooperation.

Syntrophy is a tightly coupled mutualistic interaction between hydrogen-/formate-producing and hydrogen-/formate-using microorganisms that occurs throughout the microbial world. Syntrophy is essential for global carbon cycling, waste decomposition, and biofuel production. Reverse electron transfer, e.g., the input of energy to drive critical redox reactions, is a defining feature of syntrophy. Genomic analyses indicate multiple systems for reverse electron transfer, including ion-translocating ferredoxin:NAD(+) oxidoreductase and hydrogenases, two types of electron transfer flavoprotein:quinone oxidoreductases, and other quinone reactive complexes. Confurcating hydrogenases that couple the favorable production of hydrogen from reduced ferredoxin with the unfavorable production of hydrogen from NADH are present in almost all syntrophic metabolizers, implicating their critical role in syntrophy. Transcriptomic analysis shows upregulation of many genes without assigned functions in the syntrophic lifestyle. High-throughput technologies provide insight into the mechanisms used to establish and maintain syntrophic consortia and conserve energy from reactions that operate close to thermodynamic equilibrium.

Dimerization allows DNA target site recognition by the NarL response regulator.

Two-component signal transduction systems are modular phosphorelay regulatory pathways common in prokaryotes. In the co-crystal structure of the Escherichia coli NarL signal output domain bound to DNA, we observe how the NarL family of two-component response regulators can bind DNA. DNA recognition is accompanied by the formation of a new dimerization interface, which could occur only in the full-length protein via a large intramolecular domain rearrangement. The DNA is recognized by the concerted effects of solvation, van der Waals forces and inherent DNA deformability, rather than determined primarily by major groove hydrogen bonding. These subtle forces permit a small DNA-binding domain to perturb the DNA helix, leading to major DNA curvature and a transition from B- to A-form DNA at the binding site, where valine on the recognition helix interacts unexpectedly with the polar major groove floor.

Regulation of succinate dehydrogenase (sdhCDAB) operon expression in Escherichia coli in response to carbon supply and anaerobiosis: role of ArcA and Fnr

Succinate dehydrogenase (SDH) of Escherichia coli, the sole membrane‐bound enzyme of the tricarboxylic acid cycle, participates in the aerobic electron‐transport pathway to generate energy via oxidative phosphorylation reactions. Previous studies have established that succinate dehydrogenase (SDiH) synthesis is elevated by aerobiosis and supressed during growth with glucose. To examine how the sdhCDAB genes that encode SDH are regulated by changes in the environment, sdh–lacZ fusions were constructed and analysed in vivo following cell growth under a variety of alternative culture conditions. Expression of sdh–lacZ was highest under aerobic conditions and was decreased 10‐foid in the absence of oxygen. The fnr and arcA gene products are required for this oxygen control and each acts to repress sdhC–lacZ expression. Expression of sdh–lacZ also varied 10‐ to 14‐foid depending on the type of carbon substrate used or the medium richness. This control was shown to be independent of the crp and fruR gene products, and indicates that some other regulatory element exists in the ceil to adjust SDH enzyme levels accordingly. Iron and haem availability affected sdhC–lacZ expression by two‐ to threefold. Lastly, fold. Lastly, sdhC–lacZ expression was shown to vary with the cell growth rate during aerobic and anaerobic conditions.