Maddalena V Coppi’s research while affiliated with University of Massachusetts Amherst and other places

Clostridium phytofermentans was isolated from forest soil and is distinguished by its capacity to directly ferment plant cell wall polysaccharides into ethanol as the primary product, suggesting that it possesses unusual catabolic pathways. The objective of the present study was to understand the molecular mechanisms of biomass conversion to ethanol in a single organism, Clostridium phytofermentans, by analyzing its complete genome and transcriptome during growth on plant carbohydrates. The saccharolytic versatility of C. phytofermentans is reflected in a diversity of genes encoding ATP-binding cassette sugar transporters and glycoside hydrolases, many of which may have been acquired through horizontal gene transfer. These genes are frequently organized as operons that may be controlled individually by the many transcriptional regulators identified in the genome. Preferential ethanol production may be due to high levels of expression of multiple ethanol dehydrogenases and additional pathways maximizing ethanol yield. The genome also encodes three different proteinaceous bacterial microcompartments with the capacity to compartmentalize pathways that divert fermentation intermediates to various products. These characteristics make C. phytofermentans an attractive resource for improving the efficiency and speed of biomass conversion to biofuels.

Primer names, sequences and conditions used for qPCR analysis. (DOC)

Clostridium phytofermentans, an anaerobic soil bacterium, can directly convert plant biomass into biofuels. The genome of C. phytofermentans contains three loci with genes encoding shell proteins of bacterial microcompartments (BMC), organelles composed entirely of proteins. One of the BMC loci has homology to a BMC-encoding locus implicated in the conversion of fucose to propanol and propionate in a human gut commensal, Roseburia inulinivorans. We hypothesized that it had a similar role in C. phytofermentans. When C. phytofermentans was grown on fucose, the major products identified were ethanol, propanol and propionate. Transmission electron microscopy of fucose- and rhamnose-grown cultures revealed polyhedral structures, presumably BMCs. Microarray analysis indicated that during growth on fucose, operons coding for the BMC locus, fucose dissimilatory enzymes, and an ATP-binding cassette transporter became the dominant transcripts. These data are consistent with fucose fermentation producing a 1,2-propanediol intermediate that is further metabolized in the microcompartment encoded in the BMC locus. Growth on another deoxyhexose sugar, rhamnose, resulted in the expression of the same BMC locus and similar fermentation products. However, a different set of dissimilatory enzymes and transport system genes were induced. Quite surprisingly, growth on fucose or rhamnose also led to the expression of a diverse array of complex plant polysaccharide-degrading enzymes. Based on physiological, genomic, and microarray analyses, we propose a model for the fermentation of fucose and rhamnose in C. phytofermentans that includes enzymes encoded in the same BMC locus. Comparative genomic analysis suggests that this BMC may be present in other clostridial species.

Whole-genome microarray analysis of Geobacter sulfurreducens grown on insoluble Fe(III) oxide or Mn(IV) oxide versus soluble Fe(III) citrate revealed significantly different expression patterns. The most up-regulated genes, omcS and omcT, encode cell surface c-type cytochromes, OmcS being required for Fe(III) and Mn(IV) oxide reduction. Other electron transport genes up-regulated on both metal oxides included genes encoding putative menaquinol:ferricytochrome c oxidoreductase complexes Cbc4 and Cbc5, periplasmic c-type cytochromes Dhc2 and PccF, outer membrane c-type cytochromes OmcC, OmcG and OmcV, multicopper oxidase OmpB, the structural components of electrically conductive pili, PilA-N and PilA-C, and enzymes that detoxify reactive oxygen/nitrogen species. Genes up-regulated on Fe(III) oxide encode putative menaquinol:ferricytochrome c oxidoreductase complexes Cbc3 and Cbc6, periplasmic c-type cytochromes including PccG and PccJ, and outer membrane c-type cytochromes including OmcA, OmcE, OmcH, OmcL, OmcN, OmcO and OmcP. Electron transport genes up-regulated on Mn(IV) oxide encode periplasmic c-type cytochromes PccR, PgcA, PpcA and PpcD, outer membrane c-type cytochromes OmaB/OmaC, OmcB and OmcZ, multicopper oxidase OmpC, and menaquinone-reducing enzymes. Genetic studies indicated that MacA, OmcB, OmcF, OmcG, OmcH, OmcI, OmcJ, OmcM, OmcV and PccH, the putative Cbc5 complex subunit CbcC, and the putative Cbc3 complex subunit CbcV are important for reduction of Fe(III) oxide but not essential for Mn(IV) oxide. Gene expression patterns for Geobacter uraniireducens were similar. These results demonstrate that the physiology of Fe(III)-reducing bacteria differs significantly during growth on different insoluble and soluble electron acceptors and emphasize the importance of c-type cytochromes for extracellular electron transfer in G. sulfurreducens.

Geobacter sulfurreducens is one of the few microorganisms available in pure culture known to directly accept electrons from a negatively poised electrode. Microarray analysis was used to compare gene transcript abundance in biofilms of G. sulfurreducens using a graphite electrode as the sole electron donor for fumarate reduction compared with transcript abundance in biofilms growing on the same material, but not consuming current. Surprisingly, genes for putative cell-electrode connections, such as outer-surface cytochromes and pili, which are highly expressed in current-producing biofilms, were not highly expressed in current-consuming biofilms. Microarray analysis of G. sulfurreducens gene transcript abundance in current-consuming biofilms versus current-producing biofilms gave similar results. In both comparative studies current-consuming biofilms had greater transcript abundance for a gene (GSU3274) encoding a putative monoheme, c-type cytochrome. Deletion of genes for outer-surface proteins previously shown to be essential for optimal electron transfer to electrodes had no impact on electron transfer from electrodes. Deletion of GSU3274 completely inhibited electron transfer from electrodes, but had no impact on electron transfer to electrodes. These differences in gene expression patterns and the impact of gene deletions suggest that the mechanisms for electron transfer from electrodes to G. sulfurreducens differ significantly from the mechanisms for electron transfer to electrodes.

Geobacter sulfurreducens is capable of coupling the complete oxidation of organic compounds to iron reduction. The metabolic response of G. sulfurreducens towards variations in electron donors (acetate, hydrogen) and acceptors (Fe(III), fumarate) was investigated via (13)C-based metabolic flux analysis. We examined the (13)C-labeling patterns of proteinogenic amino acids obtained from G. sulfurreducens cultured with (13)C-acetate. Using (13)C-based metabolic flux analysis, we observed that donor and acceptor variations gave rise to differences in gluconeogenetic initiation, tricarboxylic acid cycle activity, and amino acid biosynthesis pathways. Culturing G. sulfurreducens cells with Fe(III) as the electron acceptor and acetate as the electron donor resulted in pyruvate as the primary carbon source for gluconeogenesis. When fumarate was provided as the electron acceptor and acetate as the electron donor, the flux analysis suggested that fumarate served as both an electron acceptor and, in conjunction with acetate, a carbon source. Growth on fumarate and acetate resulted in the initiation of gluconeogenesis by phosphoenolpyruvate carboxykinase and a slightly elevated flux through the oxidative tricarboxylic acid cycle as compared to growth with Fe(III) as the electron acceptor. In addition, the direction of net flux between acetyl-CoA and pyruvate was reversed during growth on fumarate relative to Fe(III), while growth in the presence of Fe(III) and acetate which provided hydrogen as an electron donor, resulted in decreased flux through the tricarboxylic acid cycle. We gained detailed insight into the metabolism of G. sulfurreducens cells under various electron donor/acceptor conditions using (13)C-based metabolic flux analysis. Our results can be used for the development of G. sulfurreducens as a chassis for a variety of applications including bioremediation and renewable biofuel production.

GC/MS mass isotopomer distributions. Carbon mass isotopomer distributions of different amino acid fragments computed from GC/MS measurements of TBDMS-amino acid derivatives are listed.

Anabolic precursor demand. Precursor stoichiometry given for the anabolic demand of G. sulfurreducens are listed.

Mass spectrometric (MS) isotopomer analysis has become a standard tool for investigating biological systems using stable isotopes. In particular, metabolic flux analysis uses mass isotopomers of metabolic products typically formed from (13)C-labeled substrates to quantitate intracellular pathway fluxes. In the current work, we describe a model-driven method of numerical bias estimation regarding MS isotopomer analysis. Correct bias estimation is crucial for measuring statistical qualities of measurements and obtaining reliable fluxes. The model we developed for bias estimation corrects a priori unknown systematic errors unique for each individual mass isotopomer peak. For validation, we carried out both computational simulations and experimental measurements. From stochastic simulations, it was observed that carbon mass isotopomer distributions and measurement noise can be determined much more precisely only if signals are corrected for possible systematic errors. By removing the estimated background signals, the residuals resulting from experimental measurement and model expectation became consistent with normality, experimental variability was reduced, and data consistency was improved. The method is useful for obtaining systematic error-free data from (13)C tracer experiments and can also be extended to other stable isotopes. As a result, the reliability of metabolic fluxes that are typically computed from mass isotopomer measurements is increased.

Limitations on the availability of Fe(III) as an electron acceptor are thought to play an important role in restricting the growth and activity of Geobacter species during bioremediation of contaminated subsurface environments, but the possibility that these organisms might also be limited in the subsurface by the availability of iron for assimilatory purposes was not previously considered because copious quantities of Fe(II) are produced as the result of Fe(III) reduction. Analysis of multiple Geobacteraceae genomes revealed the presence of a three-gene cluster consisting of homologues of two iron-dependent regulators, fur and dtxR (ideR), separated by a homologue of feoB, which encodes an Fe(II) uptake protein. This cluster appears to be conserved among members of the Geobacteraceae and was detected in several environments. Expression of the fur-feoB-ideR cluster decreased as Fe(II) concentrations increased in chemostat cultures. The number of Geobacteraceae feoB transcripts in groundwater samples from a site undergoing in situ uranium bioremediation was relatively high until the concentration of dissolved Fe(II) increased near the end of the field experiment. These results suggest that, because much of the Fe(II) is sequestered in solid phases, Geobacter species, which have a high requirement for iron for iron-sulfur proteins, may be limited by the amount of iron available for assimilatory purposes. These results demonstrate the ability of transcript analysis to reveal previously unsuspected aspects of the in situ physiology of microorganisms in subsurface environments.