Uwe Deppenmeier

Complete genome sequence of the acetic acid bacterium Gluconobacter oxydans.

Gluconobacter oxydans is unsurpassed by other organisms in its ability to incompletely oxidize a great variety of carbohydrates, alcohols and related compounds. Furthermore, the organism is used for several biotechnological processes, such as vitamin C production. To further our understanding of its overall metabolism, we sequenced the complete genome of G. oxydans 621H. The chromosome consists of 2,702,173 base pairs and contains 2,432 open reading frames. In addition, five plasmids were identified that comprised 232 open reading frames. The sequence data can be used for metabolic reconstruction of the pathways leading to industrially important products derived from sugars and alcohols. Although the respiratory chain of G. oxydans was found to be rather simple, the organism contains many membrane-bound dehydrogenases that are critical for the incomplete oxidation of biotechnologically important substrates. Moreover, the genome project revealed the unique biochemistry of G. oxydans with respect to the process of incomplete oxidation.

Biochemistry and biotechnological applications of Gluconobacter strains

Abstract. The genus Gluconobacter belongs to the group of acetic acid bacteria, which are characterized by their ability to incompletely oxidize a wide range of carbohydrates and alcohols. The corresponding products (aldehydes, ketones and organic acids) are excreted almost completely into the medium. In most cases, the reactions are catalyzed by dehydrogenases connected to the respiratory chain. Since the reactive centers of the enzymes are oriented towards the periplasmic space, transport of substrates and products into, and out of, the cell is not necessary. Thus, rapid accumulation of incompletely oxidized products in the medium is facilitated. These organisms are able to grow in highly concentrated sugar solutions and at low pH-values. High oxidation rates correlate with low biomass production, which makes Gluconobacter strains interesting organisms for industrial applications. Modern fermentation processes, such as the production of L-sorbose (vitamin C synthesis) and 6-amino-L-sorbose (synthesis of the antidiabetic drug miglitol) are carried out with members of this genus. Other important products are dihydroxyacetone, gluconate and ketogluconates. The bacteria belonging to the genus Gluconobacter exhibit extraordinary uniqueness not only in their biochemistry but also in their growth behavior and response to extreme culture conditions. This uniqueness makes them ideal organisms for microbial process development.

The unique biochemistry of methanogenesis.

Methanogenic archaea have an unusual type of metabolism because they use H2 + CO2, formate, methylated C1 compounds, or acetate as energy and carbon sources for growth. The methanogens produce methane as the major end product of their metabolism in a unique energy-generating process. The organisms received much attention because they catalyze the terminal step in the anaerobic breakdown of organic matter under sulfate-limiting conditions and are essential for both the recycling of carbon compounds and the maintenance of the global carbon flux on Earth. Furthermore, methane is an important greenhouse gas that directly contributes to climate changes and global warming. Hence, the understanding of the biochemical processes leading to methane formation are of major interest. This review focuses on the metabolic pathways of methanogenesis that are rather unique and involve a number of unusual enzymes and coenzymes. It will be shown how the previously mentioned substrates are converted to CH4 via the CO2-reducing, methylotrophic, or aceticlastic pathway. All catabolic processes finally lead to the formation of a mixed disulfide from coenzyme M and coenzyme B that functions as an electron acceptor of certain anaerobic respiratory chains. Molecular hydrogen, reduced coenzyme F420, or reduced ferredoxin are used as electron donors. The redox reactions as catalyzed by the membrane-bound electron transport chains are coupled to proton translocation across the cytoplasmic membrane. The resulting electrochemical proton gradient is the driving force for ATP synthesis as catalyzed by an A1A0-type ATP synthase. Other energy-transducing enzymes involved in methanogenesis are the membrane-integral methyltransferase and the formylmethanofuran dehydrogenase complex. The former enzyme is a unique, reversible sodium ion pump that couples methyl-group transfer with the transport of Na+ across the membrane. The formylmethanofuran dehydrogenase is a reversible ion pump that catalyzes formylation and deformylation of methanofuran. Furthermore, the review addresses questions related to the biochemical and genetic characteristics of the energy-transducing enzymes and to the mechanisms of ion translocation.

Pathways of energy conservation in methanogenic archaea

Methanogenic archaea are strictly anaerobic organisms that derive their metabolic energy from the conversion of a restricted number of substrates to methane. H2+CO2 and formate are converted to CH4 via the CO2-reducing pathway, while methanol and methylamines are metabolized by the methylotrophic pathway. A limited number of methanogenic organisms utilize acetate by the aceticlastic pathway. Redox reactions involved in these processes are partly catalyzed by membrane-bound enzyme systems that generate or, in the case of endergonic reactions, use electrochemical ion gradients. The H2:heterodisulfide oxidoreductase, the F420H2:heterodisulfide oxidoreductase and the CO:heterodisulfide oxidoreductase, are novel systems that generate a proton motive force by redox-potential-driven H+ translocation. The methyltetrahydromethanopterin:coenzyme M methyltransferase is a unique, reversible sodium ion pump that couples methyl transfer with the transport of Na+ across the cytoplasmic membrane. Formylmethanofuran dehydrogenase is a reversible ion pump that catalyzes formylation and deformylation, of methanofuran. In summary, the pathways are coupled to the generation of an electrochemical sodium ion gradient and an electrochemical proton gradient. Both ion gradients are used directly for ATP synthesis via membrane integral ATP synthases. The function of the above-mentioned systems and their components in the metabolism of methanogens are described in detail.

Acquisition of 1,000 eubacterial genes physiologically transformed a methanogen at the origin of Haloarchaea

Archaebacterial halophiles (Haloarchaea) are oxygen-respiring heterotrophs that derive from methanogens—strictly anaerobic, hydrogen-dependent autotrophs. Haloarchaeal genomes are known to have acquired, via lateral gene transfer (LGT), several genes from eubacteria, but it is yet unknown how many genes the Haloarchaea acquired in total and, more importantly, whether independent haloarchaeal lineages acquired their genes in parallel, or as a single acquisition at the origin of the group. Here we have studied 10 haloarchaeal and 1,143 reference genomes and have identified 1,089 haloarchaeal gene families that were acquired by a methanogenic recipient from eubacteria. The data suggest that these genes were acquired in the haloarchaeal common ancestor, not in parallel in independent haloarchaeal lineages, nor in the common ancestor of haloarchaeans and methanosarcinales. The 1,089 acquisitions include genes for catabolic carbon metabolism, membrane transporters, menaquinone biosynthesis, and complexes I–IV of the eubacterial respiratory chain that functions in the haloarchaeal membrane consisting of diphytanyl isoprene ether lipids. LGT on a massive scale transformed a strictly anaerobic, chemolithoautotrophic methanogen into the heterotrophic, oxygen-respiring, and bacteriorhodopsin-photosynthetic haloarchaeal common ancestor.

Novel reactions involved in energy conservation by methanogenic archaea.

Methanogenic archaea of the order Methanosarcinales which utilize C1 compounds such as methanol, methylamines or H2+CO2, employ two novel membrane‐bound electron transport systems generating an electrochemical proton gradient: the H2:heterodisulfide oxidoreductase and the F420H2:heterodisulfide oxidoreductase. The systems are composed of the heterodisulfide reductase and either a membrane‐bound hydrogenase or a F420H2 dehydrogenase which is functionally homologous to the proton‐translocating NADH dehydrogenase. Cytochromes and the novel electron carrier methanophenazine are also involved. In addition, the methyl‐H4MPT:HS‐CoM methyltransferase is bioenergetically relevant. The enzyme couples methyl group transfer with the translocation of sodium ions and seems to be present in all methanogens. The proton‐translocating systems with the participation of cytochromes and methanophenazine have been found so far only in the Methanosarcinales.

Life close to the thermodynamic limit: how methanogenic archaea conserve energy.

Methane-forming archaea are strictly anaerobic, ancient microbes that are widespread in nature. These organisms are commonly found in anaerobic environments such as rumen, anaerobic sediments of rivers and lakes, hyperthermal deep sea vents and even hypersaline environments. From an evolutionary standpoint they are close to the origin of life. Common to all methanogens is the biological production of methane by a unique pathway currently only found in archaea. Methanogens can grow on only a limited number of substrates such as H(2) + CO(2), formate, methanol and other methyl group-containing substrates and some on acetate. The free energy change associated with methanogenesis from these compounds allows for the synthesis of 1 (acetate) to a maximum of only 2 mol of ATP under standard conditions while under environmental conditions less than one ATP can be synthesized. Therefore, methanogens live close to the thermodynamic limit. To cope with this problem, they have evolved elaborate mechanisms of energy conservation using both protons and sodium ions as the coupling ion in one pathway. These energy conserving mechanisms are comprised of unique enzymes, cofactors and electron carriers present only in methanogens. This review will summarize the current knowledge of energy conservation of methanogens and focus on recent insights into structure and function of ion translocating enzymes found in these organisms.

The membrane-bound electron transport system of Methanosarcina species.

Members of the genus Methanosarcina are strictly anaerobic archaea that derive their metabolic energy from the conversion of a restricted number of substrates to methane. H2 + CO2 are converted to CH4 via the CO2-reducing pathway, while methanol and methylamines are metabolized by the methylotrophic pathway. Two novel electron transport systems are involved in the process of methanogenesis. Both systems are able to use a heterodisulfide as electron acceptor and either H2 or F420H2 as electron acceptors and generate a proton-motive force by redox potential-driven H+-translocation. The H2:heterodisulfide oxidoreductase is composed of an F420-nonreducing hydrogenase and the heterodisulfide reductase. The latter protein is also part of the F420H2:heterodisulfide oxidoreductase system. The second component of this system is referred to as F420H2 dehydrogenase. The archaeal protein is a homologue of complex I of the respiratory chain from bacteria and mitochondria. This review focuses on the biochemical and genetic characteristics of the three energy-transducing enzymes and on the mechanisms of ion translocation.

DNA microarray analysis of Methanosarcina mazei Gö1 reveals adaptation to different methanogenic substrates

Methansarcina mazei Gö1 DNA arrays were constructed and used to evaluate the genomic expression patterns of cells grown on either of two alternative methanogenic substrates, acetate or methanol, as sole carbon and energy source. Analysis of differential transcription across the genome revealed two functionally grouped sets of genes that parallel the central biochemical pathways in, and reflect many known features of, acetate and methanol metabolism. These include the acetate-induced genes encoding acetate activating enzymes, acetyl-CoA synthase/CO dehydrogenase, and carbonic anhydrase. Interestingly, additional genes expressed at significantly higher levels during growth on acetate included two energy-conserving complexes (the Ech hydrogenase, and the A1A0-type ATP synthase). Many previously unknown features included the induction by acetate of genes coding for ferredoxins and flavoproteins, an aldehyde:ferredoxin oxidoreductase, enzymes for the synthesis of aromatic amino acids, and components of iron, cobalt and oligopeptide uptake systems. In contrast, methanol-grown cells exhibited elevated expression of genes assigned to the methylotrophic pathway of methanogenesis. Expression of genes for components of the translation apparatus was also elevated in cells grown in the methanol medium relative to acetate, and was correlated with the faster growth rate observed on the former substrate. These experiments provide the first comprehensive insight into substrate-dependent gene expression in a methanogenic archaeon. This genome-wide approach, coupled with the complementary molecular and biochemical tools, should greatly accelerate the exploration of Methanosarcina cell physiology, given the present modest level of our knowledge of these large archaeal genomes.

Redox-driven proton translocation in methanogenic Archaea

Abstract. Methanogenic archaea of the genus Methanosarcina are able to utilize H2 + CO2, methylated C1 compounds or acetate as energy and carbon source, thereby producing methane as the major end product. The methanogenic pathways lead to the formation of a mixed disulfide derived from coenzyme M and coenzyme B. This disulfide is of major importance for methanogens because it is the terminal electron acceptor of a branched respiratory chain. Molecular hydrogen, reduced coenzyme F420 or reduced ferredoxin are used as electron donors. Four enzymes are involved in the membrane-bound electron transport system of Methanosarcina species, all of which are involved in the generation of an electrochemical proton gradient that is used for ATP synthesis. This review focuses on the membrane-bound electron transport chains of Methanosarcina species with respect to the biochemical and genetic characteristics of the unusual energy transducing enzymes. Furthermore, the review addresses questions concerning the relationship between methanogenic proteins and components of respiratory chains found in bacteria and eukarya.