Metabolism of model substrates by syntrophic microbial communities

Advanced surface proteomics of syntrophic and cellulolytic microbes

Cellulosome biogenesis in cellulolytic bacteria

Metabolism of model substrates by syntrophic microbial communities

In collaboration with the Atomic Structure and Enabling Capabilities team we will utilize next-gen omics methods to elucidate the molecular patterns, pathways and enzymes underlying community-driven syntrophic substrate bioconversions.

The essential anaerobic recycling of carbon in natural and manmade environments requires the actions of many fermentative and syntrophic groups of microbes. This cooperative process requires cell-cell interactions with back-and-forth adjustments of upstream fermentation reactions with downstream syntrophic community reactions that control optimal recycling rates. Many processes of syntrophic metabolism are not well understood, and this impacts the ability to predict and model their metabolism and roles in carbon recycling in nature. To address this, we use an integrated set of omics tools coupled with new informatics approaches to identify and characterize core metabolic pathways in model syntrophic communities when growing on unstudied or poorly studied classes of substrates (i.e., amino acids, carboxylates, and long chain fatty acids). Also targeted for study are their associated oxidation reduction reactions, electron transfer pathways, modes of energy conservation and other essential cell functions. Biochemical and structural approaches further complement these studies. Limited genomic sequencing is planned for other poorly characterized syntrophic strains to further dissect and predict their metabolic potential along with associated cell-cell interactions within the anaerobic communities. With the Atomic Structure and Enabling Capabilities team we further elucidate their shared versus unique features to fill gaps in our bioinformatic and structural understanding of these processes. Resulting information enables future prediction and modeling efforts of syntrophic and other anaerobic carbon recycling of plant materials by the larger BER DOE community.

The anaerobic decomposition of plant and animal polymers including polysaccharides, proteins, nucleic acids and lipids to CO2, H2O and methane requires syntrophic metabolism as an essential step. In this multi-stage process, fermentative bacteria hydrolyze the polymeric substrates present in all anaerobic habitats and then ferment the hydrolysis products to acetate and longer chain fatty acids, alcohols, aromatic compounds, CO2, formate, and H2.  Then, propionate and longer chain fatty acids, aromatic compounds, amino acids, carboxylates, and alcohols are syntrophically metabolized to acetate, H2, and formate. Lastly, two different groups, the hydrogen/formate-using and acetate-using methanogens, complete the process by converting the acetate, formate and hydrogen to CO2 and CH4.

In these environments, syntrophy is driven by the exchange of hydrogen and formate in tightly coupled mutualistic interaction among the cooperating microorganisms. The consumption of the acetate, hydrogen and formate by the methanogen partner keeps the intermediates at the very low concentrations necessary for the overall degradative process to be energetically favorable.

Disruption of syntrophic metabolism significantly impacts organic matter turnover in natural and anthropogenic environments: short-chain fatty acids accumulate, the pH drops, and methane production decreases. Such events may lead to sequestration of carbon in wetlands. On the negative side, disruption of syntrophy limits biofuel methane production from renewable resources. The ubiquity of syntrophic metabolism in many anoxic environments emphasizes that metabolic cooperation among microbial species is essential for the anaerobic conversion of organic matter in nature.

Although this tightly coupled mutualistic interaction among cooperating anaerobic syntrophic communities is required for the operation of the global carbon cycle, this essential cooperation is not well understood. At the cellular level, we have limited understanding of central metabolic pathways, electron transfer reactions and physiological interactions that occur among syntrophic partners. Our ability to analyze and predict impacts the ability to model methane flux in natural habitats, to control waste degradation, and to maximize the conversion of renewable resources to the energy-rich fuels.

We have established the pathways for carbon and electron flow in several model syntrophic metabolizing organisms that metabolize SCFA’s (Syntrophomonas wolfei) benzoate and cyclohexane carboxylate (Syntrophus aciditrophicus) and for crotonate and butyrate (S. wolfei subsp methylbutyrica) when each is grown with the methanogenic partner, Methanospirillum hungatei. For one of these, S. wolfei, the core pathways for carbon and electron flow is depicted in Figure 1A. Biochemical and structural analysis of key pathway proteins is in progress (e.g., Acs2, Fig. 1B) to determine the role(s) of post translational lysine modifications (PTMs) in regulating core pathways. Our team recently identified multiple types of PTMs in the three bacterial strains above, suggesting a common theme within this class of syntrophic microbes. To support the proposed studies, genomic sequencing of uncharacterized syntrophic strains metabolizing other model substrates is also in progress.

Figure 1. Electron flow during syntrophy by S. wolfei.

A. The postulated electron flow in S. wolfei is shown in red. Abbreviations: EtfAB, electron transfer flavoprotein subunits A and B; Fd, ferredoxin; Hyd, hydrogenase; FeS oxid., membrane-bound iron-sulfur oxidoreductase; Fix, Fix system for reverse electron transfer (Etf:Fd oxidoreductase); MQH2, reduced menaquinone.

B. Structure of the S. wolfei Acs2 paralog involved in CoA transfer (unpublished

Figure 2A. M. hungatei cell envelope

A cryo-EM image of the cell end with associated sheath, pili, and flagella structure, scale bar = 1 um.

Figure 2B. M. hungatei cell flagellum

A cross section of the M. hungatei flagellum with attached glycan moieties indicated in red.

Advanced surface proteomics of syntrophic and cellulolytic microbes

In collaboration with the Atomic Structure and Enabling Capabilities team we are developing and using next generation tools to explore microbial cell envelope components, structures, and functions.

Microbial envelopes serve as the gateway to the cell as they control the entry and exit of small molecules, while also providing a protective barrier against harsh environmental chemicals. They also play key roles in cell biosynthesis, energy harvesting and cell rigidity, plus serve as the platform for assembling and operating specialized cell structures such as flagella, pili, cell surface attached receptors, other proteins, and extracellular machines such as the cellulosome. Our knowledge of these processes in many environmentally relevant anaerobic microbes is quite limited. This contrasts with our understanding of major envelope components in the well-studied bacteria E. coli and B. subtilis. We propose to develop and apply enabling tools to improve the study of microbial surfaces at the molecular level. MS methods will be devised to identify low abundance cell envelope proteins involved in membrane associated nutrient uptake and secretion, protein export, cell surface display, and the biogenesis of extracellular structures. We will apply MS and NMR methods to identify and structurally characterize the secondary cell wall polymer in C. thermocellum that serves to attach and anchor the extracellular cellulosome machine. While many of these tasks by nature are exploratory and descriptive, they hold significant potential to improve our understanding of basic cellular processes in anaerobic microbes of interest. They also have applications in improving predictions and modeling of processes in other poorly studied microbes, and for microbial design tasks to meet the needs and objectives of DOE BER laboratories and investigators.

With experience applying advanced MS methods to elucidate surface structures of bacteria and archaea, much of our effort has focused on syntrophic systems that recycle carbon. In several microbes we have identified surface exposed proteins, for example in Methanosarcina acetivorans, previously undescribed surface layer proteins in Methanospirillum hungatgei (Figure 2A and manuscripts in preparation), and flagella (aka, archaella).

We also identified, purified, and characterized the major flagella protein from archaeon Methanospirillum hungatei JF1. We determined its structure by cryo-EM with our UCLA collaborator, Hong Zhou. At 3.4 Å resolution, the structure provided the first high resolution view of any archaeal appendage (Figure 2B). It reveals properties distinct from bacterial counterparts. This cryo-EM structure of FlaB3 (Mhun_3140) also revealed several post-translational modifications (PTMs) that includes six glycans. Enzymatic digestion and mass spectrometry (MS) of the appendages localized 5 of those 6 modification sites, each bearing a nominally identical disaccharide of composition consistent with threonine-acetamido-deoxyhexuronic acid linked to C8H14O5 (potentially dimethylated hexose). T106, the sixth modification site, was localized from a peptide mass map and cryoEM information and found to be identically glycosylated. All 6 glycosylation sites were found to be O-linked, making M. hungatei FlaB3 the first archaellin identified with O-linked glycosylation. Experiments are in progress to characterize the glycans and their linkages further.Proteomics has been a powerful tool for study of biological systems and has been advanced tremendously over the past few years. We and others have developed label free MS techniques that allow for sensitive and rapid quantification of proteins from whole cells and cell communitie. We have also devised new techniques based on MS and “native” MS to facilitate the characterization of protein assemblies and ligand binding. Such methods can be integrated with other analyses to model and refine 3D structures for biological complexes and assemblies.

Surface proteomics of syntrophic and cellulolytic microbes are addressed in two broad research areas:

  • Microbial cell envelope protein identification and characterization
  • Molecular basis of cellulosome tethering to the cell wall

Cellulosome biogenesis in cellulolytic bacteria

Dwindling petroleum supplies and climate change have increased the need for renewable fuels, chemicals and materials that can be produced from lignocellulosic biomass. However, the high costs associated with degrading recalcitrant lignocellulose into its component sugars has greatly reduced its utility as a feedstock. It is generally believed that this limitation can be overcome by using consolidated bioprocessing (CBP) microbes, microorganisms that can both degrade lignocellulose and utilize the resulting sugars to produce high-levels of a bio-commodity.

The thermophile, C. thermocellum is a promising microbial platform to produce renewable biofuels from plant biomass because it efficiently degrades lignocellulose and possesses ethanologenic capabilities. Its potent cellulolytic activity originates from surface displayed cellulosome structures that deconstruct biomass by properly positioning an array of cellulase enzymes with complementary activities. Current efforts have discovered these supramolecular structures in a variety of anaerobic bacterial species and determined the enzymes and scaffolding proteins that are required for their construction. However, relatively little is known about how C. thermocellum and other bacteria assemble cellulosomes or how they tailor its enzyme composition to degrade different types of biomass.

Our goal is to gain insight into the gene expression networks that control the composition of the cellulosome in C. thermocellum and to define the mechanism through which they are modulated by extracellular signals. We seek to define the regulons of the nine extracytoplasmic function (ECF) σ-factors that encode cellulosome components and the molecular mechanism through which membrane-embedded RsgI anti-σ factors modulate σ-factor function in response to binding to extracellular carbohydrates. Relatively few species of cellulolytic anaerobic bacteria have developed the capacity to display cellulosomes, suggesting that they have evolved unique export systems to produce these massive structures. Therefore, we also aim to identify components of this export machinery through comparative genomic analyses and functional testing in C. thermocellum. The results of this work further the objectives of the DOE BER by identifying fundamental genome-encoded properties that confer potent, tunable cellulolytic activity to bacteria, which is an important step toward developing cost-effective microbial solutions to convert plant biomass into renewable biofuels and chemicals.

Figure 3. Proposed RsgI-SigI regulation system. 

Nine RsgI anti-σ factors are embedded in the membrane (RsgI [#1-9]. Signaling is triggered when extracellular carbohydrates and possibly other types of signals bind to the ectodomain. The released cognate σ factors (either SigI [#1-8]) then directs gene transcription of cellulosome components.