Jessica R. Sieber

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.

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.

The genome of Syntrophomonas wolfei: new insights into syntrophic metabolism and biohydrogen production

Syntrophomonas wolfei is a specialist, evolutionarily adapted for syntrophic growth with methanogens and other hydrogen- and/or formate-using microorganisms. This slow-growing anaerobe has three putative ribosome RNA operons, each of which has 16S rRNA and 23S rRNA genes of different length and multiple 5S rRNA genes. The genome also contains 10 RNA-directed, DNA polymerase genes. Genomic analysis shows that S. wolfei relies solely on the reduction of protons, bicarbonate or unsaturated fatty acids to re-oxidize reduced cofactors. Syntrophomonas wolfei lacks the genes needed for aerobic or anaerobic respiration and has an exceptionally limited ability to create ion gradients. An ATP synthase and a pyrophosphatase were the only systems detected capable of creating an ion gradient. Multiple homologues for β-oxidation genes were present even though S. wolfei uses a limited range of fatty acids from four to eight carbons in length.Syntrophomonas wolfei, other syntrophic metabolizers with completed genomic sequences, and thermophilic anaerobes known to produce high molar ratios of hydrogen from glucose have genes to produce H(2) from NADH by an electron bifurcation mechanism. Comparative genomic analysis also suggests that formate production from NADH may involve electron bifurcation. A membrane-bound, iron-sulfur oxidoreductase found in S. wolfei and Syntrophus aciditrophicus may be uniquely involved in reverse electron transport during syntrophic fatty acid metabolism. The genome sequence of S. wolfei reveals several core reactions that may be characteristic of syntrophic fatty acid metabolism and illustrates how biological systems produce hydrogen from thermodynamically difficult reactions.

The importance of hydrogen and formate transfer for syntrophic fatty, aromatic and alicyclic metabolism

We used a combination of genomic, transcriptional and enzymatic analyses to determine the mechanism of interspecies electron transfer by two model syntrophic microorganisms, Syntrophomonas wolfei and Syntrophus aciditrophicus. Both organisms contain multiple hydrogenase and formate dehydrogenase genes, but lack genes for outer membrane cytochromes and nanowire formation. Syntrophically grown cells and cell-free extracts of S. aciditrophicus and S. wolfei had both hydrogenase and formate dehydrogenase activities. Butyrate metabolism and CH4 production by washed cell suspensions of S. wolfei and Methanospirillum hungatei were inhibited by hydrogenase inhibitors (cyanide and carbon monoxide), but not by a formate dehydrogenase inhibitor (hypophosphite). Syntrophic benzoate oxidation and CH4 production by washed cell suspensions of S. aciditrophicus and M. hungatei were inhibited by hypophosphite, but not cyanide and carbon monoxide. All three inhibitors halted syntrophic cyclohexane-1-carboxylate metabolism. Two hydrogenase genes, hydA1 and hydA2, were more highly expressed when S. wolfei was grown syntrophically. S. aciditrophicus expressed multiple hydrogenase and formate dehydrogenase genes during syntrophic benzoate and cyclohexane-1-carboxylate growth, one of which (fdhA2) was highly differentially expressed during syntrophic benzoate growth. Thus, these syntrophic microorganisms have flexible metabolisms that allow them to use either H2 or formate transfer depending on the substrate involved.

Identification and characterization of the first cholesterol-dependent cytolysins from gram-negative bacteria

ABSTRACT The cholesterol-dependent cytolysins (CDCs) are pore-forming toxins that have been exclusively associated with a wide variety of bacterial pathogens and opportunistic pathogens from the Firmicutes and Actinobacteria, which exhibit a Gram-positive type of cell structure. We have characterized the first CDCs from Gram-negative bacterial species, which include Desulfobulbus propionicus type species Widdel 1981 (DSM 2032) (desulfolysin [DLY]) and Enterobacter lignolyticus (formerly Enterobacter cloacae) SCF1 (enterolysin [ELY]). The DLY and ELY primary structures show that they maintain the signature motifs of the CDCs but lack an obvious secretion signal. Recombinant, purified DLY (rDLY) and ELY (rELY) exhibited cholesterol-dependent binding and cytolytic activity and formed the typical large CDC membrane oligomeric pore complex. Unlike the CDCs from Gram-positive species, which are human- and animal-opportunistic pathogens, neither D. propionicus nor E. lignolyticus is known to be a pathogen or commensal of humans or animals: the habitats of both organisms appear to be restricted to anaerobic soils and/or sediments. These studies reveal for the first time that the genes for functional CDCs are present in bacterial species that exhibit a Gram-negative cell structure. These are also the first bacterial species containing a CDC gene that are not known to inhabit or cause disease in humans or animals, which suggests a role of these CDCs in the defense against eukaryote bacterial predators.

Proteomic analysis reveals metabolic and regulatory systems involved in the syntrophic and axenic lifestyle of Syntrophomonas wolfei

Microbial syntrophy is a vital metabolic interaction necessary for the complete oxidation of organic biomass to methane in all-anaerobic ecosystems. However, this process is thermodynamically constrained and represents an ecosystem-level metabolic bottleneck. To gain insight into the physiology of this process, a shotgun proteomics approach was used to quantify the protein landscape of the model syntrophic metabolizer, Syntrophomonas wolfei, grown axenically and syntrophically with Methanospirillum hungatei. Remarkably, the abundance of most proteins as represented by normalized spectral abundance factor (NSAF) value changed very little between the pure and coculture growth conditions. Among the most abundant proteins detected were GroEL and GroES chaperonins, a small heat shock protein, and proteins involved in electron transfer, beta-oxidation, and ATP synthesis. Several putative energy conservation enzyme systems that utilize NADH and ferredoxin were present. The abundance of an EtfAB2 and the membrane-bound iron-sulfur oxidoreductase (Swol_0698 gene product) delineated a potential conduit for electron transfer between acyl-CoA dehydrogenases and membrane redox carriers. Proteins detected only when S. wolfei was grown with M. hungatei included a zinc-dependent dehydrogenase with a GroES domain, whose gene is present in genomes in many organisms capable of syntrophy, and transcriptional regulators responsive to environmental stimuli or the physiological status of the cell. The proteomic analysis revealed an emphasis on macromolecular stability and energy metabolism by S. wolfei and presence of regulatory mechanisms responsive to external stimuli and cellular physiological status.

Pyrophosphate-Dependent ATP Formation from Acetyl Coenzyme A in Syntrophus aciditrophicus, a New Twist on ATP Formation

ABSTRACT Syntrophus aciditrophicus is a model syntrophic bacterium that degrades key intermediates in anaerobic decomposition, such as benzoate, cyclohexane-1-carboxylate, and certain fatty acids, to acetate when grown with hydrogen-/formate-consuming microorganisms. ATP formation coupled to acetate production is the main source for energy conservation by S. aciditrophicus. However, the absence of homologs for phosphate acetyltransferase and acetate kinase in the genome of S. aciditrophicus leaves it unclear as to how ATP is formed, as most fermentative bacteria rely on these two enzymes to synthesize ATP from acetyl coenzyme A (CoA) and phosphate. Here, we combine transcriptomic, proteomic, metabolite, and enzymatic approaches to show that S. aciditrophicus uses AMP-forming, acetyl-CoA synthetase (Acs1) for ATP synthesis from acetyl-CoA. acs1 mRNA and Acs1 were abundant in transcriptomes and proteomes, respectively, of S. aciditrophicus grown in pure culture and coculture. Cell extracts of S. aciditrophicus had low or undetectable acetate kinase and phosphate acetyltransferase activities but had high acetyl-CoA synthetase activity under all growth conditions tested. Both Acs1 purified from S. aciditrophicus and recombinantly produced Acs1 catalyzed ATP and acetate formation from acetyl-CoA, AMP, and pyrophosphate. High pyrophosphate levels and a high AMP-to-ATP ratio (5.9 ± 1.4) in S. aciditrophicus cells support the operation of Acs1 in the acetate-forming direction. Thus, S. aciditrophicus has a unique approach to conserve energy involving pyrophosphate, AMP, acetyl-CoA, and an AMP-forming, acetyl-CoA synthetase. IMPORTANCE Bacteria use two enzymes, phosphate acetyltransferase and acetate kinase, to make ATP from acetyl-CoA, while acetate-forming archaea use a single enzyme, an ADP-forming, acetyl-CoA synthetase, to synthesize ATP and acetate from acetyl-CoA. Syntrophus aciditrophicus apparently relies on a different approach to conserve energy during acetyl-CoA metabolism, as its genome does not have homologs to the genes for phosphate acetyltransferase and acetate kinase. Here, we show that S. aciditrophicus uses an alternative approach, an AMP-forming, acetyl-CoA synthetase, to make ATP from acetyl-CoA. AMP-forming, acetyl-CoA synthetases were previously thought to function only in the activation of acetate to acetyl-CoA. Bacteria use two enzymes, phosphate acetyltransferase and acetate kinase, to make ATP from acetyl-CoA, while acetate-forming archaea use a single enzyme, an ADP-forming, acetyl-CoA synthetase, to synthesize ATP and acetate from acetyl-CoA. Syntrophus aciditrophicus apparently relies on a different approach to conserve energy during acetyl-CoA metabolism, as its genome does not have homologs to the genes for phosphate acetyltransferase and acetate kinase. Here, we show that S. aciditrophicus uses an alternative approach, an AMP-forming, acetyl-CoA synthetase, to make ATP from acetyl-CoA. AMP-forming, acetyl-CoA synthetases were previously thought to function only in the activation of acetate to acetyl-CoA.

Membrane Complexes of Syntrophomonas wolfei Involved in Syntrophic Butyrate Degradation and Hydrogen Formation

Syntrophic butyrate metabolism involves the thermodynamically unfavorable production of hydrogen and/or formate from the high potential electron donor, butyryl-CoA. Such redox reactions can occur only with energy input by a process called reverse electron transfer. Previous studies have demonstrated that hydrogen production from butyrate requires the presence of a proton gradient, but the biochemical machinery involved has not been clearly elucidated. In this study, the gene and enzyme systems involved in reverse electron transfer by Syntrophomonas wolfei were investigated using proteomic and gene expression approaches. S. wolfei was grown in co-culture with Methanospirillum hungatei or Dehalococcoides mccartyi under conditions requiring reverse electron transfer and compared to both axenic S. wolfei cultures and co-cultures grown in conditions that do not require reverse electron transfer. Blue native gel analysis of membranes solubilized from syntrophically grown cells revealed the presence of a membrane-bound hydrogenase, Hyd2, which exhibited hydrogenase activity during in gel assays. Bands containing a putative iron-sulfur (FeS) oxidoreductase were detected in membranes of crotonate-grown and butyrate grown S. wolfei cells. The genes for the corresponding hydrogenase subunits, hyd2ABC, were differentially expressed at higher levels during syntrophic butyrate growth when compared to growth on crotonate. The expression of the FeS oxidoreductase gene increased when S. wolfei was grown with M. hungatei. Additional membrane-associated proteins detected included FoF1 ATP synthase subunits and several membrane transporters that may aid syntrophic growth. Furthermore, syntrophic butyrate metabolism can proceed exclusively by interspecies hydrogen transfer, as demonstrated by growth with D. mccartyi, which is unable to use formate. These results argue for the importance of Hyd2 and FeS oxidoreductase in reverse electron transfer during syntrophic butyrate degradation.