Major insights into the phylogenetic distribution, biochemistry, and evolutionary significance of organelles involved in ATP synthesis (energy metabolism) in eukaryotes that thrive in anaerobic environments for all or part of their life cycles have accrued in recent years. All known eukaryotic groups possess an organelle of mitochondrial origin, mapping the origin of mitochondria to the eukaryotic common ancestor, and genome sequence data are rapidly accumulating for eukaryotes that possess anaerobic mitochondria, hydrogenosomes, or mitosomes. Here we review the available biochemical data on the enzymes and pathways that eukaryotes use in anaerobic energy metabolism and summarize the metabolic end products that they generate in their anaerobic habitats, focusing on the biochemical roles that their mitochondria play in anaerobic ATP synthesis. We present metabolic maps of compartmentalized energy metabolism for 16 well-studied species. There are currently no enzymes of core anaerobic energy metabolism that are specific to any of the six eukaryotic supergroup lineages; genes present in one supergroup are also found in at least one other supergroup. The gene distribution across lineages thus reflects the presence of anaerobic energy metabolism in the eukaryote common ancestor and differential loss during the specialization of some lineages to oxic niches, just as oxphos capabilities have been differentially lost in specialization to anoxic niches and the parasitic life-style. Some facultative anaerobes have retained both aerobic and anaerobic pathways. Diversified eukaryotic lineages have retained the same enzymes of anaerobic ATP synthesis, in line with geochemical data indicating low environmental oxygen levels while eukaryotes arose and diversified.

Biochemistry and Evolution of Anaerobic Energy Metabolism in Eukaryotes

Occurrence, biosynthesis and function of isoprenoid quinones.

Select what you need: a comparative evaluation of the advantages and limitations of frequently used expression systems for foreign genes.

Polyhydroxyalkanoates, challenges and opportunities

Pseudomonas aeruginosa is a ubiquitously distributed opportunistic pathogen that inhabits soil and water as well as animal-, human-, and plant-host-associated environments. The ubiquity would be attributed to its very versatile energy metabolism. P. aeruginosa has a highly branched respiratory chain terminated by multiple terminal oxidases and denitrification enzymes. Five terminal oxidases for aerobic respiration have been identified in the P. aeruginosa cells. Three of them, the cbb3-1 oxidase, the cbb3-2 oxidase, and the aa3 oxidase, are cytochrome c oxidases and the other two, the bo3 oxidase and the cyanide-insensitive oxidase, are quinol oxidases. Each oxidase has a specific affinity for oxygen, efficiency of energy coupling, and tolerance to various stresses such as cyanide and reactive nitrogen species. These terminal oxidases are used differentially according to the environmental conditions. P. aeruginosa also has a complete set of the denitrification enzymes that reduce nitrate to molecular nitrogen via nitrite, nitric oxide (NO), and nitrous oxide. These nitrogen oxides function as alternative electron acceptors and enable P. aeruginosa to grow under anaerobic conditions. One of the denitrification enzymes, NO reductase, is also expected to function for detoxification of NO produced by the host immune defense system. The control of the expression of these aerobic and anaerobic respiratory enzymes would contribute to the adaptation of P. aeruginosa to a wide range of environmental conditions including in the infected hosts. Characteristics of these respiratory enzymes and the regulatory system that controls the expression of the respiratory genes in the P. aeruginosa cells are overviewed in this article.

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Regulation and Function of Versatile Aerobic and Anaerobic Respiratory Metabolism in Pseudomonas aeruginosa

Purple nonsulfur bacteria (PNSB) are facultative photosynthetic bacteria and exhibit an extremely versatile metabolism. A central focus of research on PNSB dealt with the elucidation of mechanisms by which they manage to balance cellular redox under diverse conditions, in particular under photoheterotrophic growth. Given the complexity of the central metabolism of PNSB, metabolic modeling becomes crucial for an integrated analysis of the accumulated biological knowledge. We reconstructed a stoichiometric model capturing the central metabolism of three important representatives of PNSB (Rhodospirillum rubrum, Rhodobacter sphaeroides and Rhodopseudomonas palustris). Using flux variability analysis, the model reveals key metabolic constraints related to redox homeostasis in these bacteria. With the help of the model we can (i) give quantitative explanations for non-intuitive, partially species-specific phenomena of photoheterotrophic growth of PNSB, (ii) reproduce various quantitative experimental data, and (iii) formulate several new hypotheses. For example, model analysis of photoheterotrophic growth reveals that - despite a large number of utilizable catabolic pathways - substrate-specific biomass and CO2 yields are fixed constraints, irrespective of the assumption of optimal growth. Furthermore, our model explains quantitatively why a CO2 fixing pathway such as the Calvin cycle is required by PNSB for many substrates (even if CO2 is released). We also analyze the role of other pathways potentially involved in redox metabolism and how they affect quantitatively the required capacity of the Calvin cycle. Our model also enables us to discriminate between different acetate assimilation pathways that were proposed recently for R. sphaeroides and R. rubrum, both lacking the isocitrate lyase. Finally, we demonstrate the value of the metabolic model also for potential biotechnological applications: we examine the theoretical capabilities of PNSB for photoheterotrophic hydrogen production and identify suitable genetic interventions to increase the hydrogen yield. Taken together, the metabolic model (i) explains various redox-related phenomena of the versatile metabolism of PNSB, (ii) delivers new hypotheses on the operation and relevance of several metabolic pathways, and (iii) holds significant potential as a tool for rational metabolic engineering of PNSB in biotechnological applications.

https://link.springer.com/content/pdf/10.1186%2F1752-0509-5-150.pdf

Metabolic network modeling of redox balancing and biohydrogen production in purple nonsulfur bacteria

Purple non-sulfur bacteria (Rhodospirillaceae) have been extensively employed for studying principles of photosynthetic and respiratory electron transport phosphorylation and for investigating the regulation of gene expression in response to redox signals. Here, we use mathematical modeling to evaluate the steady-state behavior of the electron transport chain (ETC) in these bacteria under different environmental conditions. Elementary-modes analysis of a stoichiometric ETC model reveals nine operational modes. Most of them represent well-known functional states, however, two modes constitute reverse electron flow under respiratory conditions, which has been barely considered so far. We further present and analyze a kinetic model of the ETC in which rate laws of electron transfer steps are based on redox potential differences. Our model reproduces well-known phenomena of respiratory and photosynthetic operation of the ETC and also provides non-intuitive predictions. As one key result, model simulations demonstrate a stronger reduction of ubiquinone when switching from high-light to low-light conditions. This result is parameter insensitive and supports the hypothesis that the redox state of ubiquinone is a suitable signal for controlling photosynthetic gene expression.

Modeling the electron transport chain of purple non-sulfur bacteria

The biosynthesis of the major carotenoid spirilloxanthin by the purple nonsulfur bacterium Rhodospirillum rubrum is thought to occur via a linear pathway proceeding through phytoene and, later, lycopene as intermediates. This assumption is based solely on early chemical evidence (B. H. Davies, Biochem. J. 116:93-99, 1970). In most purple bacteria, the desaturation of phytoene, catalyzed by the enzyme phytoene desaturase (CrtI), leads to neurosporene, involving only three dehydrogenation steps and not four as in the case of lycopene. We show here that the chromosomal insertion of a kanamycin resistance cassette into the crtC-crtD region of the partial carotenoid gene cluster, whose gene products are responsible for the downstream processing of lycopene, leads to the accumulation of the latter as the major carotenoid. We provide spectroscopic and biochemical evidence that in vivo, lycopene is incorporated into the light-harvesting complex 1 as efficiently as the methoxylated carotenoids spirilloxanthin (in the wild type) and 3,4,3',4'-tetrahydrospirilloxanthin (in a crtD mutant), both under semiaerobic, chemoheterotrophic, and photosynthetic, anaerobic conditions. Quantitative growth experiments conducted in dark, semiaerobic conditions, using a growth medium for high cell density and high intracellular membrane levels, which are suitable for the conventional industrial production in the absence of light, yielded lycopene at up to 2 mg/g (dry weight) of cells or up to 15 mg/liter of culture. These values are comparable to those of many previously described Escherichia coli strains engineered for lycopene production. This study provides the first genetic proof that the R. rubrum CrtI produces lycopene exclusively as an end product.

High-level production of the industrial product lycopene by the photosynthetic bacterium Rhodospirillum rubrum.

The purple nonsulfur bacterium Rhodospirillum rubrum has been employed to study physiological adaptation to limiting oxygen tensions (microaerophilic conditions). R. rubrum produces maximal levels of photosynthetic membranes when grown with both succinate and fructose as carbon sources under microaerophilic conditions in comparison to the level (only about 20% of the maximum) seen in the absence of fructose. Employing a unique partial O2 pressure (pO2) control strategy to reliably adjust the oxygen tension to values below 0.5%, we have used bioreactor cultures to investigate the metabolic rationale for this effect. A metabolic profile of the central carbon metabolism of these cultures was obtained by determination of key enzyme activities under microaerophilic as well as aerobic and anaerobic phototrophic conditions. Under aerobic conditions succinate and fructose were consumed simultaneously, whereas oxygen-limiting conditions provoked the preferential breakdown of fructose. Fructose was utilized via the Embden-Meyerhof-Parnas pathway. High levels of pyrophosphate-dependent phosphofructokinase activity were found to be specific for oxygen-limited cultures. No glucose-6-phosphate dehydrogenase activity was detected under any conditions. We demonstrate that NADPH is supplied mainly by the pyridine-nucleotide transhydrogenase under oxygen-limiting conditions. The tricarboxylic acid cycle enzymes are present at significant levels during microaerophilic growth, albeit at lower levels than those seen under fully aerobic growth conditions. Levels of the reductive tricarboxylic acid cycle marker enzyme fumarate reductase were also high under microaerophilic conditions. We propose a model by which the primary “switching” of oxidative and reductive metabolism is performed at the level of the tricarboxylic acid cycle and suggest how this might affect redox signaling and gene expression in R. rubrum.

Microaerophilic Cooperation of Reductive and Oxidative Pathways Allows Maximal Photosynthetic Membrane Biosynthesis in Rhodospirillum rubrum

It is now well established that, for photosynthetic bacteria, the aerobic-to-microaerophilic transition activates the membrane-bound sensor kinase RegB, which subsequently phosphorylates the transcriptional activator RegA, thereby inducing elevated levels of intracellular photosynthetic membranes. The mechanism of RegB activation—in particular, the role of ubiquinone-10—is controversial at present. One problem here is that very limited quantitative in vivo data for the response of the ubiquinone redox state to different cultivation conditions exist. Here, we utilize Rhodospirillum rubrum to study the correlation of the quinone redox state to the expression level of photosynthetic membranes and determine an effective response function directly. Our results show that changes in the photosynthetic membrane levels between 50 and 95% of that maximally attainable are associated with only a twofold change in the ubiquinol/ubiquinone ratio and are not necessarily proportional to the total levels of either quinone or [NAD+ + NADH]. There is no correlation between the redox potentials of the quinone and pyridine nucleotide pools. Hill function analysis of the photosynthetic membrane induction in response to the quinone redox state suggests that the induction process is highly cooperative. Our results are probably generally applicable to quinone redox regulation in bacteria.

Hartmut Grammel

Papers

Metabolic network modeling of redox balancing and biohydrogen production in purple nonsulfur bacteria

Modeling the electron transport chain of purple non-sulfur bacteria

High-level production of the industrial product lycopene by the photosynthetic bacterium Rhodospirillum rubrum.

Microaerophilic Cooperation of Reductive and Oxidative Pathways Allows Maximal Photosynthetic Membrane Biosynthesis in Rhodospirillum rubrum

Redox-State Dynamics of Ubiquinone-10 Imply Cooperative Regulation of Photosynthetic Membrane Expression in Rhodospirillum rubrum