Robert L Hettich

• Doctor of Philosophy
• Oak Ridge National Laboratory

Metaproteomics is an emerging approach for studying microbiomes, offering the ability to characterize proteins that underpin microbial functionality within diverse ecosystems. As the primary catalytic and structural components of microbiomes, proteins provide unique insights into the active processes and ecological roles of microbial communities. B...

Metaproteomics is an emerging approach for studying microbiomes, offering the ability to characterize proteins that underpin microbial functionality within diverse ecosystems. As the primary catalytic and structural components of microbiomes, proteins provide unique insights into the active processes and ecological roles of microbial communities. B...

Background Fecal samples are widely used as a proxy for studying gut microbiome composition in both human and animal research. Fecal metaproteomics provides valuable insights by tracking changes in the relative abundance of microbial taxa and their protein functions. To ensure reliable results, it is crucial to minimize alterations in the metaprote...

Metaproteomics is an emerging approach for studying microbiomes, offering the ability to characterize proteins that underpin microbial functionality within diverse ecosystems. As the primary catalytic and structural components of microbiomes, proteins provide unique insights into the active processes and ecological roles of microbial communities. B...

The mode of transport of lignin monomers to the sites of polymerization in the apoplast remains controversial. C‐Lignin is a recently discovered form of lignin found in some seed coats that is composed exclusively of units derived from caffeyl alcohol. RNA‐seq and proteome analyses identified a number of transporters co‐expressed with C‐lignin depo...

Metaproteomics is an increasingly popular methodology that provides information regarding the metabolic functions of specific microbial taxa and has potential for contributing to ocean ecology and biogeochemical studies. A blinded multi-laboratory intercomparison was conducted to assess comparability and reproducibility of taxonomic and functional...

Numerous genetic codes developed during the evolution of Eukaryotes and three are known in Bacteria, yet no alternative genetic code has been established for Archaea. Some bacterial and archaeal proteins include selenocysteine or pyrrolysine, the 21 st and 22 nd amino acids, but no evidence establishes the adoption of a genetic code in which a stop...

The rhizosphere is a dynamic environment where rhizodeposits that include primary and secondary metabolites and mucilage serve as nutrient sources for soil microorganisms, attracting them toward plant roots. However, understanding how these microbes specifically respond to plant root chemical signals has been hindered by the challenges of disentang...

Background Clostridium autoethanogenum is an acetogenic bacterium that autotrophically converts carbon monoxide (CO) and carbon dioxide (CO2) gases into bioproducts and fuels via the Wood–Ljungdahl pathway (WLP). To facilitate overall carbon capture efficiency, the reaction stoichiometry requires supplementation of hydrogen at an increased ratio of...

Ectomycorrhizal fungi establish mutually beneficial relationships with trees, trading nutrients for carbon. Suillus are ectomycorrhizal fungi that are critical to the health of boreal and temperate forest ecosystems. Comparative genomics has identified a high number of non-ribosomal peptide synthetase and terpene biosynthetic gene clusters (BGC) po...

Understanding the organizational principles of microbial communities is essential for interpreting ecosystem stability. Previous studies have investigated the formation of bacterial communities under nutrient-poor conditions or obligate relationships to observe cooperative interactions among different species. How microorganisms form stabilized com...

In every omics experiment, genes or their products are identified for which even state of the art tools are unable to assign a function. In the biotechnology chassis organism Pseudomonas putida, these proteins of unknown function make up 14% of the proteome. This missing information can bias analyses since these proteins can carry out functions whi...

Metaproteomics is an increasingly popular methodology that provides information regarding the metabolic functions of specific microbial taxa and has potential for contributing to ocean ecology and biogeochemical studies. A blinded multi-laboratory intercomparison was conducted to assess comparability and reproducibility of taxonomic and functional...

Ectomycorrhizal fungi establish mutually beneficial relationships with trees, trading nutrients for carbon. Suillus are ectomycorrhizal fungi that are critical to the health of boreal and temperate forest ecosystems. Comparative genomics has identified a high number of non-ribosomal peptide synthetase and terpene biosynthetic gene clusters (BGC) po...

The Salicaceae family is of growing interest in the study of dioecy in plants because the sex determination region (SDR) has been shown to be highly dynamic, with differing locations and heterogametic systems between species. Without the ability to transform and regenerate Salix in tissue culture, previous studies investigating the mechanisms regul...

The Salicaceae family is of growing interest in the study of dioecy in plants because the sex determination region (SDR) has been shown to be highly dynamic, with differing locations and heterogametic systems between species. Without the ability to transform and regenerate Salix in tissue culture, previous studies investigating the mechanisms regul...

Clostridium thermocellum, a promising candidate for consolidated bioprocessing, has been subjected to numerous engineering strategies for enhanced bioethanol production. Measurements of intracellular metabolites at substrate concentrations high enough (>50 g/L) to allow the production of industrially relevant titers of ethanol would inform efforts...

Mutualistic association can improve a plant’s health and productivity. G-type lectin receptor-like kinase (PtLecRLK1) is a susceptibility factor in Populus trichocarpa that permits root colonization by a beneficial fungus, Laccaria bicolor. Engineering PtLecRLK1 also permits L. bicolor root colonization in non-host plants similar to Populus trichoc...

Cell-free systems derived from crude cell extracts have developed into tools for gene expression, with applications in prototyping, biosensing, and protein production. Key to the development of these systems is optimization of cell extract preparation methods. However, the applied nature of these optimizations often limits investigation into the co...

A major threat to forest ecosystems and plantation forestry is the introduction of a non-native pathogen. Among non-domesticated populations with relatively high levels of genetic diversity, a measurable range of susceptibility to resistance can be expected. Identifying genetic determinants of resistant and susceptible individuals can inform the de...

The human gut microbiome, which develops and stabilizes during the early stages of infant life, plays an essential role in host health through the production of metabolic resources and the stimulation and training of the immune system. To study colonization and community functional dynamics of the microbiota based on responses to host immune proces...

The ability to control the distribution of thermodynamic driving force throughout a metabolic pathway is likely to be an important tool for metabolic engineering. The phosphofructokinase reaction is a key enzyme in Embden-Mayerhof-Parnas glycolysis and therefore improving the thermodynamic driving force of this reaction in C. thermocellum is believ...

Microorganisms are critical drivers of biological processes that contribute significantly to plant sustainability and productivity. In recent years, emerging research on plant holobiont theory and microbial invasion ecology has radically transformed how we study plant–microbe interactions. Over the last few years, we have witnessed an accelerating...

Previous bioinformatic analyses of metagenomic data have indicated that bacteriophages can use genetic codes different from those of their host bacteria. In particular, reassignment of stop codon TAG to glutamine (a variation known as ‘genetic code 15’) has been predicted. Here, we use LC-MS/MS-based metaproteomics of human fecal samples to provide...

A bifunctional peroxidase enzyme, 4-coumarate 3-hydroxylase (C3H/APX), provides a parallel route to the shikimate shunt pathway for the conversion of 4-coumarate to caffeate in the early steps of lignin biosynthesis. Knockdown of C3H/APX (C3H/APX-KD) expression has been shown to reduce the lignin content in Brachypodium distachyon. However, like ma...

Clostridium autoethanogenum is a model gas-fermenting acetogen for commercial ethanol production. It is also a platform organism being developed for the carbon-negative production of acetone and isopropanol by gas fermentation. We have assembled a 5.5 kb pCA plasmid for type strain DSM10061 (JA1-1) using three genome sequence datasets. pCA is predi...

There are known associations between opioids, obesity, and the gut microbiome, but the molecular connection/mediation of these relationships is not understood. To better clarify the interplay of physiological, genetic, and microbial factors, this study investigated the microbiome and host inflammatory responses to chronic opioid administration in g...

Economically viable production of cellulosic biofuels requires operation at high solids loadings—on the order of 15 wt%. To this end we characterize Nature’s ability to deconstruct and utilize mid-season switchgrass at increasing solid loadings using an anaerobic methanogenic microbiome. This community exhibits undiminished fractional carbohydrate...

Coenzyme F 420 -dependent sulfite reductase (Fsr) protects methanogenic archaea inhabiting deep-sea hydrothermal vents from the inactivation of methyl coenzyme M reductase (Mcr), one of their essential energy production enzymes. Anaerobic methanotrophic archaea (ANME) that oxidize methane and rely on Mcr, carry Fsr homologs that form a distinct cla...

Lignin biosynthesis begins with the deamination of phenylalanine and tyrosine as a key branch point between primary and secondary metabolism in land plants. Here, we used a systems biology approach to investigate the global metabolic responses to lignin pathway perturbations in the model grass Brachypodium distachyon. We identified the lignin biosy...

The only known required component of the newly described Type XI secretion system (TXISS) is an outer membrane protein (OMP) of the DUF560 family. TXISSOMPs are broadly distributed across proteobacteria, but properties of the cargo proteins they secrete are largely unexplored. We report biophysical, histochemical, and phenotypic evidence that Xenor...

Plant-microbe interactions in the rhizosphere play a vital role in plant health and productivity. The composition and function of root-associated microbiomes is strongly influenced by their surrounding environment, which is often customized by their host. How microbiomes change with respect to space and time across plant roots remains poorly unders...

Background Microbial-driven solubilization of lignocellulosic material is a natural mechanism that is exploited in anaerobic digesters (ADs) to produce biogas and other valuable bioproducts. Glycoside hydrolases (GHs) are the main enzymes that bacterial and archaeal populations use to break down complex polysaccharides in these reactors. Methodolog...

Membrane potential homeostasis is essential for cell survival. Defects in membrane potential lead to pleiotropic phenotypes, consistent with the central role of membrane energetics in cell physiology. Homologs of the progestin and AdipoQ receptors (PAQRs) are conserved in multiple phyla of Bacteria and Eukarya. In eukaryotes, PAQRs are proposed to...

The transformation of 4-hydroxybenzoate (4-HBA) to protocatechuate (PCA) is catalyzed by flavoprotein oxygenases known as para-hydroxybenzoate-3-hydroxylases (PHBHs). In Pseudomonas putida KT2440 (P. putida) strains engineered to convert lignin-related aromatic compounds to muconic acid (MA), PHBH activity is rate-limiting, as indicated by the accu...

Soil microbiomes harbour unparalleled functional and phylogenetic diversity. However, extracting isolates with a targeted function from complex microbiomes is not straightforward, particularly if the associated phenotype does not lend itself to high-throughput screening. Here, we tackle the methylation of arsenic (As) in anoxic soils. As methylatio...

Many industrial chemicals that are produced from fossil resources could be manufactured more sustainably through fermentation. Here we describe the development of a carbon-negative fermentation route to producing the industrially important chemicals acetone and isopropanol from abundant, low-cost waste gas feedstocks, such as industrial emissions a...

The role of lipo-chitooligosaccharides (LCOs) as signaling molecules that mediate the establishment of symbiotic relationships between fungi and plants is being redefined. New evidence suggests that the production of these molecular signals may be more of a common trait in fungi than what was previously thought. LCOs affect different aspects of gro...

Microbial life is widespread in the terrestrial subsurface and present down to several kilometers depth, but the energy sources that fuel metabolism in deep oligotrophic and anoxic environments remain unclear. In the deep crystalline bedrock of the Fennoscandian Shield at Olkiluoto, Finland, opposing gradients of abiotic methane and ancient seawate...

Soil microbiomes harbour unparalleled functional and phylogenetic diversity. However, extracting isolates with a targeted function from complex microbiomes is not straightforward, particularly if the associated phenotype does not lend itself to high-throughput screening. Here, we tackle the methylation of arsenic (As) in anoxic soils. As methylatio...

Metagenomic findings suggesting that bacteriophages (phages) can use genetic codes different from those of their host bacteria reveal a new dimension of phage-host interaction dynamics. Whereas reassignment of stop codons to code for amino acids has been predicted, there has been no proteomic validation of alternative coding in phages. In fact, one...

Anthropogenic activities and natural processes release dichloromethane (DCM, methylene chloride), a toxic chemical with substantial ozone-depleting capacity. Specialized anaerobic bacteria metabolize DCM; however, the genetic basis for this process has remained elusive. Comparative genomics of the three known anaerobic DCM-degrading bacterial speci...

The human gut microbiome produces a complex mixture of biomolecules that interact with human physiology and play essential roles in health and disease. Crosstalk between micro-organisms and host cells is enabled by different direct contacts, but also by the export of molecules through secretion systems and extracellular vesicles. The resulting mole...

Metaproteomics has matured into a powerful tool to assess functional interactions in microbial communities. While many metaproteomic workflows are available, the impact of method choice on results remains unclear. Here, we carry out a community-driven, multi-laboratory comparison in metaproteomics: the critical assessment of metaproteome investigat...

Through connecting genomic and metabolic information, metaproteomics is an essential approach for understanding how microbiomes function in space and time. The international metaproteomics community is delighted to announce the launch of the Metaproteomics Initiative (www.metaproteomics.org), the goal of which is to promote dissemination of metapro...

Background Microbe-microbe interactions between members of the plant rhizosphere are important but remain poorly understood. A more comprehensive understanding of the molecular mechanisms used by microbes to cooperate, compete, and persist has been challenging because of the complexity of natural ecosystems and the limited control over environmenta...

Anthropogenic activities and natural processes release dichloromethane (DCM), a toxic chemical with substantial ozone depleting capacity. Specialized anaerobic bacteria metabolize DCM; however, the genetic basis for this process has remained elusive. Comparative genomics of the three known anaerobic DCM-degrading bacterial species revealed a homolo...

Microbial life is widespread in the terrestrial subsurface and present down to several kilometers depth, but the energy sources that fuel metabolism in deep oligotrophic and anoxic environments remain unclear. In the deep crystalline bedrock of the Fennoscandian Shield at Olkiluoto, Finland, opposing gradients of abiotic methane and ancient seawate...

Soil‐borne microbes can establish compatible relationships with host plants, providing a large variety of nutritive and protective compounds in exchange for photosynthesized sugars. However, the molecular mechanisms mediating the establishment of these beneficial relationships remain unclear. Our previous genetic mapping and whole‐genome resequenci...

Background Candida parapsilosis is a common cause of invasive candidiasis, especially in newborn infants, and infections have been increasing over the past two decades. C. parapsilosis has been primarily studied in pure culture, leaving gaps in understanding of its function in a microbiome context. Results Here, we compare five unique C. parapsilo...

Owing to rising levels of greenhouse gases in our atmosphere and oceans, climate change poses significant environmental, economic, and social challenges globally. Technologies that enable carbon capture and conversion of greenhouse gases into useful products will help mitigate climate change by enabling a new circular carbon economy. Gas fermentati...

Background Total DNA (intracellular, iDNA and extracellular, eDNA) from ancient permafrost records the mixed genetic repository of the past and present microbial populations through geological time. Given the exceptional preservation of eDNA under perennial frozen conditions, typical metagenomic sequencing of total DNA precludes the discrimination...

Background Mass spectrometry-based proteomics can identify and quantify thousands of proteins from individual microbial species, but a significant percentage of these proteins are unannotated and hence classified as proteins of unknown function (PUFs). Due to the difficulty in extracting meaningful metabolic information, PUFs are often overlooked o...

Dichloroacetate (DCA) commonly occurs in the environment due to natural production and anthropogenic releases, but its fate under anoxic conditions is uncertain. Mixed culture RM comprising "Candidatus Dichloromethanomonas elyunquensis" strain RM utilizes DCA as an energy source, and the transient formation of formate, H2, and carbon monoxide (CO)...

Microbial communities colonize plant tissues and contribute to host function. How these communities form and how individual members contribute to shaping the microbial community are not well understood. Synthetic microbial communities, where defined individual isolates are combined, can serve as valuable model systems for uncovering the organizatio...

Bioremediation of chlorinated ethenes in anoxic aquifers hinges on organohalide-respiring Dehalococcoidia expressing vinyl chloride (VC) reductive dehalogenase (RDase). The tceA gene encoding the trichloroethene-dechlorinating RDase TceA is frequently detected in contaminated groundwater but not recognized as a biomarker for VC detoxification. We d...

Metaproteomics has matured into a powerful tool to assess functional interactions in microbial communities. While many metaproteomic workflows are available, the impact of method choice on results remains unclear. Here, we carried out the first community-driven, multi-lab comparison in metaproteomics: the critical assessment of metaproteome investi...

Methods for measuring gut microbiota biochemical activities in vivo are needed to characterize its functional states in health and disease. To illustrate one approach, an arabinan-containing polysaccharide was isolated from pea fiber, its structure defined, and forward genetic and proteomic analyses used to compare its effects, versus unfractionate...

Valorization of lignin, an abundant component of plant cell walls, is critical to enabling the lignocellulosic bioeconomy. Biological funneling using microbial biocatalysts has emerged as an attractive approach to convert complex mixtures of lignin depolymerization products to value-added compounds. Ideally, biocatalysts would convert aromatic comp...

Bacterial chemotaxis affords motile bacteria the ability to navigate the environment to locate niches for growth and survival. At the molecular level, chemotaxis depends on chemoreceptor signaling arrays that interact with cytoplasmic proteins to control the direction of movement. In Azospirillum brasilense , chemotaxis is mediated by two distinct...

Rising temperatures in the Arctic have led to the thawing of tundra soils, which is rapidly changing terrain, hydrology, and plant and microbial communities, causing hotspots of biogeochemical activity across the landscape. Despite this, little is known about how nutrient-rich low molecular weight dissolved organic matter (LMW DOM) varies within an...

Cell-free systems present a significant opportunity to harness the metabolic potential of diverse organisms. Removing the cellular context provides the ability to produce biological products without the need to maintain cell viability and enables metabolic engineers to explore novel chemical transformation systems. Crude extracts maintain much of a...

Background The cellulolytic thermophile Clostridium thermocellum is an important biocatalyst due to its ability to solubilize lignocellulosic feedstocks without the need for pretreatment or exogenous enzyme addition. At low concentrations of substrate, C. thermocellum can solubilize corn fiber > 95% in 5 days, but solubilization declines markedly a...

Correction for ‘Thermochemical wastewater valorization via enhanced microbial toxicity tolerance’ by Lahiru N. Jayakody et al. , Energy Environ. Sci. , 2018, 11 , 1625–1638, DOI: 10.1039/C8EE00460A.

Progress in cell-free protein synthesis (CFPS) has spurred resurgent interest in engineering complex biological metabolism outside of the cell. Unlike purified enzyme systems, crude cell-free systems can be prepared for a fraction of the cost and contain endogenous cellular pathways that can be activated for biosynthesis. Endogenous activity perfor...

Methods for measuring gut microbiota biochemical activities in vivo are needed to characterize its functional states in health and disease. To illustrate one approach, an arabinan-containing polysaccharide was purified from pea fiber, its structure defined, and forward genetic and proteomic analyses used to compare its effects, versus unfractionate...

Membrane organization plays an important role in signaling, transport, and defense. In eukaryotes, the stability, organization, and function of membrane proteins are influenced by certain lipids and sterols, such as cholesterol. Bacteria lack cholesterol, but carotenoids and hopanoids are predicted to play a similar role in modulating membrane prop...

Viruses modulate the function(s) of environmentally relevant microbial populations, yet considerations of the metabolic capabilities of individual virus particles themselves are rare. We used shotgun proteomics to quantitatively identify 43 virus-encoded proteins packaged within purified Aureococcus anophagefferens Virus (AaV) particles, normalizin...

Background The cellulolytic thermophile Clostridium thermocellum is an important biocatalyst due to its ability to solubilize lignocellulosic feedstocks without the need for pretreatment or exogenous enzyme addition. At low concentrations of substrate, C. thermocellum can solubilize corn fiber >95% in 5 days, but solubilization declines markedly at...

Background The cellulolytic thermophile Clostridium thermocellum is an important biocatalyst due to its ability to solubilize lignocellulosic feedstocks without the need for pretreatment or exogenous enzyme addition. At low concentrations of substrate, C. thermocellum can solubilize corn fiber >95% in 5 days, but solubilization declines markedly at...

Soil sulfur metabolism surprise Soil bacteria have a range of metabolic pathways that contribute to acquiring and recycling nutrients and carbon. Curiously, some of these organisms give off ethylene gas when starved for sulfur under anaerobic conditions. North et al. traced the source of ethylene to a small, sulfur-containing organic molecule produ...

Plant endo‐β‐1,4‐glucanases belonging to the Glycoside Hydrolase Family 9 have functional roles in cell wall biosynthesis and remodeling via endohydrolysis of (1→4)‐β‐D‐glucosidic linkages. Modification of cell wall chemistry via RNAi‐mediated downregulation of Populus deltoides KOR1 (PdKOR), a endo‐β‐1,4‐glucanase gene, in Populus deltoides has be...

Genomic and transcriptomic studies generate a rich source of molecular information; however, neither a static genome nor the presence of a messenger RNA provides the most direct insight into the functional state of a microbial cell at any given time. Rather, it is the ensemble of proteins (i.e., proteome) that is the primary workhorse for most biol...

Bile acids, synthesized from cholesterol by the liver, are chemically transformed along the intestinal tract by the gut microbiota and the products of these transformations signal through host receptors, impacting overall host health. These transformations include bile acid deconjugation, oxidation, and 7α-dehydroxylation. An understanding of the b...

Small peptides that are proteolytic cleavage products (PCPs) less than 100 amino acids are emerging as key signaling molecules that mediate cell-to-cell communication and biological processes that occur between and within plants, fungi, and bacteria. Yet, the discovery and characterization of these molecules is largely overlooked. Today, selective...

Developing cultivation methods that yield chemically and isotopically defined fatty acid (FA) compositions within bacterial cytoplasmic membranes establishes an in vivo experimental platform to study membrane biophysics and cell membrane regulation using novel approaches. Yet before fully realizing the potential of this method, it is prudent to und...

Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis which offers the potential to engineer improved water use efficiency (WUE) and drought resilience in C3 plants while sustaining productivity in hotter and drier climates that are predicted for much of the world. CAM species show an inverted pattern of stomatal opening and cl...