scholarly journals Energy Conservation in Fermentations of Anaerobic Bacteria

2021 ◽  
Vol 12 ◽  
Author(s):  
Wolfgang Buckel
Keyword(s):  
Anaerobic Bacteria ◽  
Human Microbiome ◽  
Oxidative Decarboxylation ◽  
Free Energies ◽  
Glutamate Mutase ◽  
Available Energy ◽  
Glycyl Radical ◽  
Oxygen Sensitive ◽  
Coenzyme B ◽  
Radical Enzymes

Anaerobic bacteria ferment carbohydrates and amino acids to obtain energy for growth. Due to the absence of oxygen and other inorganic electron acceptors, the substrate of a fermentation has to serve as electron donor as well as acceptor, which results in low free energies as compared to that of aerobic oxidations. Until about 10 years ago, anaerobes were thought to exclusively use substrate level phosphorylation (SLP), by which only part of the available energy could be conserved. Therefore, anaerobes were regarded as unproductive and inefficient energy conservers. The discovery of electrochemical Na+ gradients generated by biotin-dependent decarboxylations or by reduction of NAD+ with ferredoxin changed this view. Reduced ferredoxin is provided by oxidative decarboxylation of 2-oxoacids and the recently discovered flavin based electron bifurcation (FBEB). In this review, the two different fermentation pathways of glutamate to ammonia, CO2, acetate, butyrate and H2 via 3-methylaspartate or via 2-hydroxyglutarate by members of the Firmicutes are discussed as prototypical examples in which all processes characteristic for fermentations occur. Though the fermentations proceed on two entirely different pathways, the maximum theoretical amount of ATP is conserved in each pathway. The occurrence of the 3-methylaspartate pathway in clostridia from soil and the 2-hydroxyglutarate pathway in the human microbiome of the large intestine is traced back to the oxygen-sensitivity of the radical enzymes. The coenzyme B12-dependent glutamate mutase in the 3-methylaspartate pathway tolerates oxygen, whereas 2-hydroxyglutaryl-CoA dehydratase is extremely oxygen-sensitive and can only survive in the gut, where the combustion of butyrate produced by the microbiome consumes the oxygen and provides a strict anaerobic environment. Examples of coenzyme B12-dependent eliminases are given, which in the gut are replaced by simpler extremely oxygen sensitive glycyl radical enzymes.

scholarly journals BIOME-Preserve: A Novel Storage and Transport Medium for Preserving Anaerobic Microbiota Samples for Culture Recovery

2020 ◽  
Author(s):  
Embriette R. Hyde ◽  
Hiram Lozano ◽  
Steven Cox
Keyword(s):  
Nucleic Acids ◽  
Human Health ◽  
Anaerobic Bacteria ◽  
Human Microbiome ◽  
Sample Collection ◽  
Culture Collections ◽  
Oxygen Sensitive ◽  
The One ◽  
The Relationship ◽  
Human Stool

AbstractCulture-based study design is critical to advance research into the relationship between human health and the microbiome. Traditional sample collection protocols are focused on preserving nucleic acids and metabolites and are largely inappropriate for preserving sensitive anaerobic bacteria alive for later culture recovery. Here we introduce a novel microbiome preservation kit (BIOME-Preserve) that facilitates recovery of anaerobic organisms from human stool held at room temperature. Using a combination of culture recovery and shallow whole-genome shotgun sequencing, we characterized the culturable anaerobes from fresh human stool and from human stool held in BIOME-Preserve for up to 120 hours. We recovered several species of interest to microbiome researchers, including Bifidobacterium spp., Bacteroides spp., Blautia spp., Eubacterium halii, Akkermansia muciniphila, and Faecalibacterium prausnitzii. Together, our results suggest BIOME-Preserve is practical for the collection, transport, and culture of anaerobic bacteria from human samples and can help provide the foundation for culture collections that can be used in further research and in the development of microbiome-based therapeutics.ImportanceSequencing-based protocols for studying the human microbiome have unearthed a wealth of information about the relationship between the microbiome and human health. But these microbes cannot be leveraged as therapeutic targets without culture-based studies to phenotype species of interest and to establish culture collections for use in animal models. Contrary to popular opinion, most gastrointestinal bacteria can be cultured, yet most sample collection strategies are optimized for the preservation of nucleic acids and/or metabolites only and do not take into account considerations for preserving oxygen-sensitive anaerobes and facultative anaerobes, which comprise the majority of the human gut microbiome. A human microbiome sample transport and preservation medium such as the one described here can play an important role in enabling researchers to better understand the link between the microbiome and human health and how to leverage that link through novel microbiome-based therapeutics.

Glycyl Radical Enzymes and Sulfonate Metabolism in the Microbiome

2021 ◽  
Vol 90 (1) ◽  
Author(s):  
Yifeng Wei ◽  
Yan Zhang
Keyword(s):  
Bond Cleavage ◽  
Anaerobic Bacteria ◽  
Sulfur Cycle ◽  
Annual Review ◽  
Publication Date ◽  
Strictly Anaerobic ◽  
Glycyl Radical ◽  
Free Radical Chemistry ◽  
Reducing Bacteria ◽  
Radical Enzymes

Sulfonates include diverse natural products and anthropogenic chemicals and are widespread in the environment. Many bacteria can degrade sulfonates and obtain sulfur, carbon, and energy for growth, playing important roles in the biogeochemical sulfur cycle. Cleavage of the inert sulfonate C–S bond involves a variety of enzymes, cofactors, and oxygen-dependent and oxygen-independent catalytic mechanisms. Sulfonate degradation by strictly anaerobic bacteria was recently found to involve C–S bond cleavage through O2-sensitive free radical chemistry, catalyzed by glycyl radical enzymes (GREs). The associated discoveries of new enzymes and metabolic pathways for sulfonate metabolism in diverse anaerobic bacteria have enriched our understanding of sulfonate chemistry in the anaerobic biosphere. An anaerobic environment of particular interest is the human gut microbiome, where sulfonate degradation by sulfate- and sulfite-reducing bacteria (SSRB) produces H2S, a process linked to certain chronic diseases and conditions. Expected final online publication date for the Annual Review of Biochemistry, Volume 90 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

New glycyl radical enzymes catalysing key metabolic steps in anaerobic bacteria

2005 ◽  
Vol 386 (10) ◽  
Author(s):  
Thorsten Selmer ◽  
Antonio J. Pierik ◽  
Johann Heider
Keyword(s):  
Anaerobic Bacteria ◽  
Glycyl Radical ◽  
Radical Enzymes

scholarly journals Decarboxylating and Nondecarboxylating Glutaryl-Coenzyme A Dehydrogenases in the Aromatic Metabolism of Obligately Anaerobic Bacteria

2009 ◽  
Vol 191 (13) ◽  
pp. 4401-4409 ◽  
Author(s):  
Simon Wischgoll ◽  
Martin Taubert ◽  
Franziska Peters ◽  
Nico Jehmlich ◽  
Martin von Bergen ◽  
...  
Keyword(s):  
Coenzyme A ◽  
Anaerobic Bacteria ◽  
Oxidative Decarboxylation ◽  
Kinetic Properties ◽  
Strictly Anaerobic ◽  
Amino Acid Residues ◽  
Geobacter Metallireducens ◽  
Sulfate Reducing ◽  
Reverse Transcription Pcr ◽  
Gene Coding

ABSTRACT In anaerobic bacteria using aromatic growth substrates, glutaryl-coenzyme A (CoA) dehydrogenases (GDHs) are involved in the catabolism of the central intermediate benzoyl-CoA to three acetyl-CoAs and CO2. In this work, we studied GDHs from the strictly anaerobic, aromatic compound-degrading organisms Geobacter metallireducens (GDHGeo) (Fe[III] reducing) and Desulfococcus multivorans (GDHDes) (sulfate reducing). GDHGeo was purified from cells grown on benzoate and after the heterologous expression of the benzoate-induced bamM gene. The gene coding for GDHDes was identified after screening of a cosmid gene library. Reverse transcription-PCR revealed that its expression was induced by benzoate; the product was heterologously expressed and isolated. Both wild-type and recombinant GDHGeo catalyzed the oxidative decarboxylation of glutaryl-CoA to crotonyl-CoA at similar rates. In contrast, recombinant GDHDes catalyzed only the dehydrogenation to glutaconyl-CoA. The latter compound was decarboxylated subsequently to crotonyl-CoA by the addition of membrane extracts from cells grown on benzoate in the presence of 20 mM NaCl. All GDH enzymes were purified as homotetramers of a 43- to 44-kDa subunit and contained 0.6 to 0.7 flavin adenine dinucleotides (FADs)/monomer. The kinetic properties for glutaryl-CoA conversion were as follows: for GDHGeo, the Km was 30 ± 2 μM and the V max was 3.2 ± 0.2 μmol min−1 mg−1, and for GDHDes, the Km was 52 ± 5 μM and the V max was 11 ± 1 μmol min−1 mg−1. GDHDes but not GDHGeo was inhibited by glutaconyl-CoA. Highly conserved amino acid residues that were proposed to be specifically involved in the decarboxylation of the intermediate glutaconyl-CoA were identified in GDHGeo but are missing in GDHDes. The differential use of energy-yielding/energy-demanding enzymatic processes in anaerobic bacteria that degrade aromatic compounds is discussed in view of phylogenetic relationships and constraints of overall energy metabolism.

scholarly journals An Aerobic Hybrid Phthalate Degradation Pathway via Phthaloyl-Coenzyme A in Denitrifying Bacteria

2020 ◽  
Vol 86 (11) ◽  
Author(s):  
Christa Ebenau-Jehle ◽  
Christina I. S. L. Soon ◽  
Jonathan Fuchs ◽  
Robin Geiger ◽  
Matthias Boll
Keyword(s):  
Phthalic Acid ◽  
Coenzyme A ◽  
Anaerobic Bacteria ◽  
Degradation Pathway ◽  
Phthalic Acid Esters ◽  
Content Type ◽  
Facultatively Anaerobic ◽  
Oxygen Sensitive ◽  
Acid Esters ◽  
Facultatively Anaerobic Bacteria

ABSTRACT The degradation of the xenobiotic phthalic acid esters by microorganisms is initiated by the hydrolysis to the respective alcohols and ortho-phthalate (hereafter, phthalate). In aerobic bacteria and fungi, oxygenases are involved in the conversion of phthalate to protocatechuate, the substrate for ring-cleaving dioxygenases. In contrast, anaerobic bacteria activate phthalate to the extremely unstable phthaloyl-coenzyme A (CoA), which is decarboxylated by oxygen-sensitive UbiD-like phthaloyl-CoA decarboxylase (PCD) to the central benzoyl-CoA intermediate. Here, we demonstrate that the facultatively anaerobic, denitrifying Thauera chlorobenzoica 3CB-1 and Aromatoleum evansii KB740 strains use phthalate as a growth substrate under aerobic and denitrifying conditions. In vitro assays with extracts from cells grown aerobically with phthalate demonstrated the succinyl-CoA-dependent activation of phthalate followed by decarboxylation to benzoyl-CoA. In T. chlorobenzoica 3CB-1, we identified PCD as a highly abundant enzyme in both aerobically and anaerobically grown cells, whereas genes for phthalate dioxygenases are missing in the genome. PCD was highly enriched from aerobically grown T. chlorobenzoica cells and was identified as an identical enzyme produced under denitrifying conditions. These results indicate that the initial steps of aerobic phthalate degradation in denitrifying bacteria are accomplished by the anaerobic enzyme inventory, whereas the benzoyl-CoA oxygenase-dependent pathway is used for further conversion to central intermediates. Such a hybrid pathway requires intracellular oxygen homeostasis at concentrations low enough to prevent PCD inactivation but sufficiently high to supply benzoyl-CoA oxygenase with its cosubstrate. IMPORTANCE Phthalic acid esters (PAEs) are industrially produced on a million-ton scale per year and are predominantly used as plasticizers. They are classified as environmentally relevant xenobiotics with a number of adverse health effects, including endocrine-disrupting activity. Biodegradation by microorganisms is considered the most effective process to eliminate PAEs from the environment. It is usually initiated by the hydrolysis of PAEs to alcohols and o-phthalic acid. Degradation of o-phthalic acid fundamentally differs in aerobic and anaerobic microorganisms; aerobic phthalate degradation heavily depends on dioxygenase-dependent reactions, whereas anaerobic degradation employs the oxygen-sensitive key enzyme phthaloyl-CoA decarboxylase. We demonstrate that aerobic phthalate degradation in facultatively anaerobic bacteria proceeds via a previously unknown hybrid degradation pathway involving oxygen-sensitive and oxygen-dependent key enzymes. Such a strategy is essential for facultatively anaerobic bacteria that frequently switch between oxic and anoxic environments.

scholarly journals Enzymology and Evolution of the Pyruvate Pathway to 2-Oxobutyrate in Methanocaldococcus jannaschii

2007 ◽  
Vol 189 (12) ◽  
pp. 4391-4400 ◽  
Author(s):  
Randy M. Drevland ◽  
Abdul Waheed ◽  
David E. Graham
Keyword(s):  
Functional Divergence ◽  
Small Subunit ◽  
Isomerization Reaction ◽  
Oxidative Decarboxylation ◽  
Methanocaldococcus Jannaschii ◽  
Iron Sulfur Center ◽  
Gene Recruitment ◽  
Or Gene ◽  
Isopropylmalate Dehydrogenase ◽  
Coenzyme B

ABSTRACT The archaeon Methanocaldococcus jannaschii uses three different 2-oxoacid elongation pathways, which extend the chain length of precursors in leucine, isoleucine, and coenzyme B biosyntheses. In each of these pathways an aconitase-type hydrolyase catalyzes an hydroxyacid isomerization reaction. The genome sequence of M. jannaschii encodes two homologs of each large and small subunit that forms the hydrolyase, but the genes are not cotranscribed. The genes are more similar to each other than to previously characterized isopropylmalate isomerase or homoaconitase enzyme genes. To identify the functions of these homologs, the four combinations of subunits were heterologously expressed in Escherichia coli, purified, and reconstituted to generate the iron-sulfur center of the holoenzyme. Only the combination of MJ0499 and MJ1277 proteins catalyzed isopropylmalate and citramalate isomerization reactions. This pair also catalyzed hydration half-reactions using citraconate and maleate. Another broad-specificity enzyme, isopropylmalate dehydrogenase (MJ0720), catalyzed the oxidative decarboxylation of β-isopropylmalate, β-methylmalate, and d-malate. Combined with these results, phylogenetic analysis suggests that the pyruvate pathway to 2-oxobutyrate (an alternative to threonine dehydratase in isoleucine biosynthesis) evolved several times in bacteria and archaea. The enzymes in the isopropylmalate pathway of leucine biosynthesis facilitated the evolution of 2-oxobutyrate biosynthesis through the introduction of a citramalate synthase, either by gene recruitment or gene duplication and functional divergence.

scholarly journals The recovery of anaerobic bacteria from swabs

1974 ◽  
Vol 72 (3) ◽  
pp. 339-347 ◽  
Author(s):  
J. G. Collee ◽  
B. Watt ◽  
R. Brown ◽  
Sandra Johnstone
Keyword(s):  
Solid Medium ◽  
Standard Sample ◽  
Agar Medium ◽  
Anaerobic Bacteria ◽  
Original Sample ◽  
Progressive Loss ◽  
Apparent Loss ◽  
Oxygen Sensitive ◽  
Wild Strains ◽  
Sampling Procedures

SUMMARYWhen a standard sample of a simulated exudate containing known numbers of anaerobic bacteria was taken up on a swab and plated on solid medium, the number of colonies subsequently cultured represented a very small proportion of the original sample. Evidence is produced that the apparent loss is not primarily attributable to inactivation on the swab but rather to retention of organisms on the swab. This was demonstrable withClostridium welchiiand withBacteroidesspecies that have hitherto been regarded as relatively oxygen-sensitive.When stock strains ofBacteroidesspecies were held for some hours on swabs, some progressive loss of viability was demonstrable. A measure of protection was afforded when these organisms were held aerobically on blood agar medium, but a very exacting anaerobe and some wild strains of faecal anaerobes showed gradual inactivation under these conditions.These findings may have important implications in relation to currently employed bacteriological sampling procedures with swabs in clinical practice.

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