Physiology of Zymomonas mobilis: Some Unanswered Questions

The ethanol-producing bacterium Zymomonas mobilis can serve as a model organism for the study of rapid catabolism and inefficient energy conversion in bacteria. Some basic aspects of its physiology still remain poorly understood. Here, the energy-spilling pathways during uncoupled growth, the structure and function of electron transport chain, and the possible reasons for the inefficient oxidative phosphorylation are analysed. Also, the interaction between ethanol synthesis and respiration is considered. The search for mechanisms of futile transmembrane proton cycling, as well as identification of respiratory electron transport complexes, like the energy-coupling NAD(P)H:quinone oxidoreduct…

The inefficient aerobic energetics ofZymomonas mobilis: Identifying the bottleneck

To investigate the mechanisms of Zymomonas mobilis uncoupled aerobic metabolism, growth properties of the wild-type strain Zm6 were compared to those of its respiratory mutants cytB and cydB, and the effects of the ATPase inhibitor DCCD on growth and intracellular ATP concentration were studied. The effects of the ATPase inhibitor DCCD on growth and intracellular ATP concentration strongly indicated that the apparent lack of oxidative phosphorylation in aerobically growing Z. mobilis culture might be caused by the ATP hydrolyzing activity of the H(+) -dependent ATPase in all analyzed strains. Aerobic growth yields of the mutants, and their capacity of oxidative ATP synthesis with ethanol we…

Respiratory behaviour of a Zymomonas mobilis adhB::kan(r) mutant supports the hypothesis of two alcohol dehydrogenase isoenzymes catalysing opposite reactions.

AbstractPerturbation of the aerobic steady-state in a chemostat culture of the ethanol-producing bacterium Zymomonas mobilis with a small pulse of ethanol causes a burst of ethanol oxidation, although the reactant ratio of the alcohol dehydrogenase (ADH) reaction ([NADH][acetaldehyde][H+])/([ethanol][NAD+]) remains above the Keq value. Simultaneous catalysis of ethanol synthesis and oxidation by the two ADH isoenzymes, residing in different redox microenvironments, has been proposed previously. In the present study, this hypothesis is verified by construction of an ADH-deficient strain and by demonstration that it lacks the oxidative burst in response to perturbation of its aerobic steady-s…

NADH dehydrogenase deficiency results in low respiration rate and improved aerobic growth of Zymomonas mobilis.

The respiratory chain of the ethanol-producing bacterium Zymomonas mobilis is able to oxidize both species of nicotinamide cofactors, NADH and NADPH. A mutant strain with a chloramphenicol-resistance determinant inserted in ndh (encoding an NADH : CoQ oxidoreductase of type II) lacked the membrane NADH and NADPH oxidase activities, while its respiratory d-lactate oxidase activity was increased. Cells of the mutant strain showed a very low respiration rate with glucose and no respiration with ethanol. The aerobic growth rate of the mutant was elevated; exponential growth persisted longer, resulting in higher biomass densities. For the parent strain a similar effect of aerobic growth stimulat…

Miniature diamond-anvil cells for FTIR-microspectroscopy of small quantities of biosamples.

Fourier transform infrared (FTIR) spectroscopy techniques and data analyses have become widely available, are easy to use, and are convenient for studies of various biosamples, especially in biomedical science. Yet, cultivation of cells and purification of cell components are costly, often methodically challenging, and time and labor consuming. Therefore, reduction of the sample amount is of high value. Here we propose a novel method for the analysis of small quantities of biosamples by FTIR-microscopy of dry films using a diamond-anvil cell (DAC). This approach allows us to decrease the sample volume at least a hundred times compared to that for a high-throughput screening device (HTS-XT, …

Metabolic Profiling of Glucose-Fed Metabolically Active Resting Zymomonas mobilis Strains

Zymomonas mobilis is the most efficient bacterial ethanol producer and its physiology is potentially applicable to industrial-scale bioethanol production. However, compared to other industrially important microorganisms, the Z. mobilis metabolome and adaptation to various nutritional and genetic perturbations have been poorly characterized. For rational metabolic engineering, it is essential to understand how central metabolism and intracellular redox balance are maintained in Z. mobilis under various conditions. In this study, we applied quantitative mass spectrometry-based metabolomics to explore how glucose-fed non-growing Z. mobilis Zm6 cells metabolically adapt to change of oxygen avai…

Biotechnological potential of respiring Zymomonas mobilis: a stoichiometric analysis of its central metabolism.

The active, yet energetically inefficient electron transport chain of the ethanologenic bacterium Zymomonas mobilis could be used in metabolic engineering for redox-balancing purposes during synthesis of certain products. Although several reconstructions of Z. mobilis metabolism have been published, important aspects of redox balance and aerobic catabolism have not previously been considered. Here, annotated genome sequences and metabolic reconstructions have been combined with existing biochemical evidence to yield a medium-scale model of Z. mobilis central metabolism in the form of COBRA Toolbox model files for flux balance analysis (FBA). The stoichiometric analysis presented here sugges…

Metabolic response of bacteria to elevated concentrations of glyphosate-based herbicide.

Abstract Glyphosate-based herbicides (GBHs) are the most widespread commonly used broad-spectrum herbicides that contaminate soils and waters, are toxic to bacteria, plants and animals, and have been classified as ‘probably carcinogenic to humans’ by the International Agency for Research on Cancer in 2015. Particular soil bacteria and fungi can degrade GBHs, hence, search for new GBH-degrading strains or microbial consortia, effective under specific growth conditions and local environment, seems to be a promising solution for bio-remediation of glyphosate-contaminated environment. Consequently, there is a need for rapid and informative methods to evaluate the GBH-induced changes of the meta…

Respiratory type II NAD(P)H dehydrogenase of Zymomonas mobilis with altered cofactor specificity

Syntrophy of Crypthecodinium cohnii and immobilized Zymomonas mobilis for docosahexaenoic acid production from sucrose-containing substrates

Marine heterotrophic dinoflagellate Crypthecodinium cohnii is an aerobic oleaginous microorganism that accumulates intracellular lipid with high content of 4,7,10,13,16,19-docosahexaenoic acid (DHA), a polyunsaturated ω-3 (22:6) fatty acid with multiple health benefits. C. cohnii can grow on glucose and ethanol, but not on sucrose or fructose. For conversion of sucrose-containing renewables to C. cohnii DHA, we investigated a syntrophic process, involving immobilized cells of ethanologenic bacterium Zymomonas mobilis for fermenting sucrose to ethanol. The non-respiring, NADH dehydrogenase-deficient Z. mobilis strain Zm6-ndh, with high ethanol yield both under anaerobic and aerobic condition…

Structure of the Zymomonas mobilis respiratory chain: oxygen affinity of electron transport and the role of cytochrome c peroxidase

The genome of the ethanol-producing bacterium Zymomonas mobilis encodes a bd-type terminal oxidase, cytochrome bc 1 complex and several c-type cytochromes, yet lacks sequences homologous to any of the known bacterial cytochrome c oxidase genes. Recently, it was suggested that a putative respiratory cytochrome c peroxidase, receiving electrons from the cytochrome bc 1 complex via cytochrome c 552, might function as a peroxidase and/or an alternative oxidase. The present study was designed to test this hypothesis, by construction of a cytochrome c peroxidase mutant (Zm6-perC), and comparison of its properties with those of a mutant defective in the cytochrome b subunit of the bc 1 complex (Zm…

Ethanol cycle in an ethanologenic bacterium

AbstractA novel redox cycle is suggested, performing interconversion between acetaldehyde and ethanol in aerobically growing ethanologenic bacterium Zymomonas mobilis. It is formed by the two alcohol dehydrogenase (ADH) isoenzymes simultaneously catalyzing opposite reactions. ADH I is catalyzing acetaldehyde reduction. The local reactant ratio at its active site probably is shifted towards ethanol synthesis due to direct channeling of NADH from glycolysis. ADH II is oxidizing ethanol. The net result of the cycle operation is NADH shuttling from glycolysis to the membrane respiratory chain, and ensuring flexible distribution of reducing equivalents between the ADH reaction and respiration.

Erratum to “electron transport chain in aerobically cultivatedZymomonas mobilis” [FEMS microbiol. lett. 143 (1996) 185–189]

Membrane D-lactate oxidase in Zymomonas mobilis: evidence for a branched respiratory chain.

Respiratory chain composition of the ethanol-producing bacterium Zymomonas mobilis was studied. Its membrane D-lactate oxidase was characterised. With NADH, but not D-lactate as substrate, a cytochrome o-like component was seen in CO difference spectra. Chlorpromazine specifically inhibited reduction of cytochrome d, while myxothiazol eliminated the cytochrome o-like features in CO difference spectra. It is suggested that electrons from NADH are distributed between branches terminated by the cytochrome o-like component, cytochrome a, and cytochrome d. With D-lactate, electrons are transported to cytochrome a, or an unidentified CN(-)-sensitive oxidase, and cytochrome d.

Hydrogen-producingEscherichia colistrains overexpressing lactose permease: FT-IR analysis of the lactose-induced stress

The lactose permease gene (lacY) was overexpressed in the septuple knockout mutant of Escherichia coli, previously engineered for hydrogen production from glucose. It was expected that raising the lactose transporter activity would elevate the intracellular lactose concentration, inactivate the lactose repressor, induce the lactose operon, and as a result stimulate overall lactose consumption and conversion. However, overexpression of the lactose transporter caused a considerable growth delay in the recombinant strain on lactose, resembling to some extent the "lactose killing" phenomenon. Therefore, the recombinant strain was subjected to selection on lactose-containing media. Selection on …

Metabolic Engineering of Bacterial Respiration: High vs. Low P/O and the Case of Zymomonas mobilis

Respiratory chain plays a pivotal role in the energy and redox balance of aerobic bacteria. By engineering respiration, it is possible to alter the efficiency of energy generation and intracellular redox state, and thus affect the key bioprocess parameters: cell yield, productivity and stress resistance. Here we summarize the current metabolic engineering and synthetic biology approaches to bacterial respiratory metabolism, with a special focus on the respiratory chain of the ethanologenic bacterium Zymomonas mobilis. Electron transport in Z. mobilis can serve as a model system of bacterial respiration with low oxidative phosphorylation efficiency. Its application for redox balancing and re…

Zymomonas mobilis metabolism: Novel tools and targets for its rational engineering

Abstract Zymomonas mobilis is an α-proteobacterium that interests the biofuel industry due to its perfect ethanol fermentation yields. From its first description as a bacterial isolate in fermented alcoholic beverages to date, Z. mobilis has been rigorously studied in directions basic and applied. The Z. mobilis powerful Entner-Doudoroff glycolytic pathway has been the center of rigorous biochemical studies and, aside from ethanol, it has attracted interest in terms of high-added-value chemical manufacturing. Energetic balances and the effects of respiration have been explored in fundamental directions as also in applications pursuing strain enhancement and the utilization of alternative ca…

Kinetic modelling of the Zymomonas mobilis Entner-Doudoroff pathway: insights into control and functionality.

Zymomonas mobilis, an ethanol-producing bacterium, possesses the Entner-Doudoroff (E-D) pathway, pyruvate decarboxylase and two alcohol dehydrogenase isoenzymes for the fermentative production of ethanol and carbon dioxide from glucose. Using available kinetic parameters, we have developed a kinetic model that incorporates the enzymic reactions of the E-D pathway, both alcohol dehydrogenases, transport reactions and reactions related to ATP metabolism. After optimizing the reaction parameters within likely physiological limits, the resulting kinetic model was capable of simulating glycolysis in vivo and in cell-free extracts with good agreement with the fluxes and steady-state intermediate …

Translocation of Zymomonas mobilis pyruvate decarboxylase to periplasmic compartment for production of acetaldehyde outside the cytosol

Abstract Acetaldehyde, a valuable commodity chemical, is a volatile inhibitory byproduct of aerobic fermentation in Zymomonas mobilis and in several other microorganisms. Attempting to improve acetaldehyde production by minimizing its contact with the cell interior and facilitating its removal from the culture, we engineered a Z. mobilis strain with acetaldehyde synthesis reaction localized in periplasm. For that, the pyruvate decarboxylase (PDC) was transferred from the cell interior to the periplasmic compartment. This was achieved by the construction of a Z. mobilis Zm6 PDC‐deficient mutant, fusion of PDC with the periplasmic signal sequence of Z. mobilis gluconolactonase, and the follow…

Electron transport chain in aerobically cultivated Zymomonas mobilis

Respiratory chain composition and energy coupling in cytoplasmic membrane of Zymomonas mobilis was shown to depend on culture aeration. Aerobically grown cells contained mainly the non-energy-generating NADH dehydrogenase with kM for NADH 58 mM. In anaerobically cultivated bacteria, the energy-coupling NADH dehydrogenase complex with km for NADH 7 mM predominated. In aerobically cultivated Z. mobilis, CoQ content and absorption peaks of cytochromes at 554–556 nm and 525–528 nm were significantly increased. Energy-coupling site I, linked to the NADH:CoQ oxidoreductase complex, could be eliminated under sulfate-deficient cultivation conditions. For anaerobically grown cells this resulted in l…

An increased sensitivity of hydrocarbons-degrading bacteria to heavy metals in the presence of diesel oil

Cyanide inhibits respiration yet stimulates aerobic growth of Zymomonas mobilis

Potassium cyanide at submillimolar concentrations (20-500 microM) inhibited the high respiration rates of aerobic cultures of Zymomonas mobilis but, remarkably, stimulated culture growth. In batch culture, after an extended lag phase, exponential growth persisted longer, resulting in higher biomass densities. In aerobic chemostat cultures, elevated biomass concentration was observed in the presence of cyanide. This growth stimulation effect is attributed to decreased production of the inhibitory metabolite acetaldehyde at lowered respiration rates, when more reducing equivalents are channelled to alcohol dehydrogenase. Growth in the presence of cyanide did not alter the membrane cytochrome …

The paradoxical cyanide-stimulated respiration of Zymomonas mobilis: cyanide sensitivity of alcohol dehydrogenase (ADH II)

The respiratory inhibitor cyanide stimulates growth of the ethanologenic bacteriumZymomonas mobilis, perhaps by diverting reducing equivalents from respiration to ethanol synthesis, thereby minimizing accumulation of toxic acetaldehyde. This study sought to identify cyanide-sensitive components of respiration. In aerobically grown, permeabilizedZ. mobiliscells, addition of 200 μM cyanide caused gradual inhibition of ADH II, the iron-containing alcohol dehydrogenase isoenzyme, which, in aerobic cultures, might be oxidizing ethanol and supplying NADH to the respiratory chain. In membrane preparations, NADH oxidase was inhibited more rapidly, but to a lesser extent, than ADH II. The time-cours…

Effect of ADH II Deficiency on the Intracellular Redox Homeostasis in Zymomonas mobilis

Mutant strain of the facultatively anaerobic, ethanol-producing bacteriumZymomonas mobilis, deficient in the Fe-containing alcohol dehydrogenase isoenzyme (ADH II), showed impaired homeostasis of the intracellular NAD(P)H during transition from anaerobic to aerobic conditions, and also in steady-state continuous cultures at various oxygen supplies. At the same time, ADH II deficiency in aerobically grown cells was accompanied by a threefold increase of catalase activity and by about 50% increase of hydrogen peroxide excretion. It is concluded that ADH II under aerobic conditions functions to maintain intracellular redox homeostasis and to protect the cells from endogenous hydrogen peroxide.

The Low Energy-Coupling Respiration in Zymomonas mobilis Accelerates Flux in the Entner-Doudoroff Pathway.

Performing oxidative phosphorylation is the primary role of respiratory chain both in bacteria and eukaryotes. Yet, the branched respiratory chains of prokaryotes contain alternative, low energy-coupling electron pathways, which serve for functions other than oxidative ATP generation (like those of respiratory protection, adaptation to low-oxygen media, redox balancing, etc.), some of which are still poorly understood. We here demonstrate that withdrawal of reducing equivalents by the energetically uncoupled respiratory chain of the bacterium Zymomonas mobilis accelerates its fermentative catabolism, increasing the glucose consumption rate. This is in contrast to what has been observed in o…

Fourier transform infrared spectroscopy as a tool for investigation of microbial response to the multi-component organic contamination

Ecotoxicological study on multi-component contamination with hydrocarbons and heavy metals

High aerobic growth with low respiratory rate: The ndh-deficient Zymomonas mobilis

Improvement of acetaldehyde production in Zymomonas mobilis by engineering of Its aerobic metabolism

Acetaldehyde is a valuable product of microbial biosynthesis, which can be used by the chemical industry as the entry point for production of various commodity chemicals. In ethanologenic microorganisms, like yeast or the bacterium Zymomonas mobilis, this compound is the immediate metabolic precursor of ethanol. In aerobic cultures of Z. mobilis, it accumulates as a volatile, inhibitory byproduct, due to the withdrawal of reducing equivalents from the alcohol dehydrogenase reaction by respiration. The active respiratory chain of Z. mobilis with its low energy-coupling efficiency is well-suited for regeneration of NAD+ under conditions when acetaldehyde, but not ethanol, is the desired catab…

Aerobic catabolism and respiratory lactate bypass in Ndh-negative Zymomonas mobilis

Ability to ferment in the presence of oxygen increases the robustness of bioprocesses and opens opportunity for novel industrial setups. The ethanologenic bacterium Zymomonas mobilis performs rapid and efficient anaerobic ethanol fermentation, yet its respiratory NADH dehydrogenase (Ndh)-deficient strain (ndh-) is known to produce ethanol with high yield also under oxic conditions. Compared to the wild type, it has a lower rate of oxygen consumption, and an increased expression of the respiratory lactate dehydrogenase (Ldh). Here we present a quantitative study of the product spectrum and carbon balance for aerobically growing ndh-. Ldh-deficient and Ldh-overexpressing ndh- strains were con…