Structure of the exopolyphosphatase (PPX) from Zymomonas mobilis reveals a two-magnesium-ions PPX.

Rapid adaptation of metabolic capabilities is crucial for bacterial survival in habitats with fluctuating nutrient availability. In such conditions, the bacterial stringent response is a central regulatory mechanism activated by nutrient starvation or other stressors. This response is primarily controlled by exopolyphosphatase/guanosine pentaphosphate phosphohydrolase (PPX/GPPA) enzymes. To gain further insight into these enzymes, the high-resolution crystal structure of PPX from Zymomonas mobilis (ZmPPX) was determined at 1.8 Å. The phosphatase activity of PPX was strictly dependent on the presence of divalent metal cations. Notably, the structure of ZmPPX revealed the presence of two magnesium ions in the active site center, which is atypical compared to other PPX structures where only one divalent ion is observed. ZmPPX exists as a dimer in solution and belongs to the "long" PPX group consisting of four domains. Remarkably, the dimer configuration exhibits a substantial and deep aqueduct with positive potential along its interface. This aqueduct appears to extend towards the active site region, suggesting that this positively charged aqueduct could potentially serve as a binding site for polyP.

Investigating ethanol production using the Zymomonas mobilis crude extract.

Cell-free systems have become valuable investigating tools for metabolic engineering research due to their easy access to metabolism without the interference of the membrane. Therefore, we applied Zymomonas mobilis cell-free system to investigate whether ethanol production is controlled by the genes of the metabolic pathway or is limited by cofactors. Initially, different glucose concentrations were added to the extract to determine the crude extract's capability to produce ethanol. Then, we investigated the genes of the metabolic pathway to find the limiting step in the ethanol production pathway. Next, to identify the bottleneck gene, a systemic approach was applied based on the integration of gene expression data on a cell-free metabolic model. ZMO1696 was determined as the bottleneck gene and an activator for its enzyme was added to the extract to experimentally assess its effect on ethanol production. Then the effect of NAD+ addition at the high concentration of glucose (1 M) was evaluated, which indicates no improvement in efficiency. Finally, the imbalance ratio of ADP/ATP was found as the controlling factor by measuring ATP levels in the extract. Furthermore, sodium gluconate as a carbon source was utilized to investigate the expansion of substrate consumption by the extract. 100% of the maximum theoretical yield was obtained at 0.01 M of sodium gluconate while it cannot be consumed by Z. mobilis. This research demonstrated the challenges and advantages of using Z. mobilis crude extract for overproduction.

Cysteine supplementation enhanced inhibitor tolerance of Zymomonas mobilis for economic lignocellulosic bioethanol production.

Inhibitors in lignocellulosic hydrolysates are toxic to Zymomonas mobilis and reduce its bioethanol production. This study revealed cysteine supplementation enhanced furfural tolerance in Z. mobilis with a 2-fold biomass increase. Transcriptomic study illustrated that cysteine biosynthesis pathway was down-regulated while cysteine catabolism was up-regulated with cysteine supplementation. Mutants for genes involved in cysteine metabolism were constructed, and metabolites in cysteine metabolic pathway including methionine, glutathione, NaHS, glutamate, and pyruvate were supplemented into media. Cysteine supplementation boosted glutathione synthesis or H2S release effectively in Z. mobilis leading to the reduced accumulation of reactive oxygen species (ROS) induced by furfural, while pyruvate and glutamate produced in the H2S generation pathway promoted cell growth by serving as the carbon or nitrogen source. Finally, cysteine supplementation was confirmed to enhance Z. mobilis tolerance against ethanol, acetate, and corncob hydrolysate with an enhanced ethanol productivity from 0.38 to 0.55 g-1∙L-1∙h-1.

Biochemical and Mutational Analysis of Radical S-Adenosyl-L-Methionine Adenosylhopane Synthase HpnH from Zymomonas mobilis Reveals that the Conserved Residue Cysteine-106 Reduces a Radical Intermediate and Determines the Stereochemistry.

Adenosylhopane is a crucial precursor of C35 hopanoids, which are believed to modulate the fluidity and permeability of bacterial cell membranes. Adenosylhopane is formed by a crosslinking reaction between diploptene and a 5'-deoxyadenosyl radical that is generated by the radical S-adenosyl-L-methionine (SAM) enzyme HpnH. We previously showed that HpnH from Streptomyces coelicolor A3(2) (ScHpnH) converts diploptene to (22R)-adenosylhopane. However, the mechanism of the stereoselective C-C bond formation was unclear. Thus, here, we performed biochemical and mutational analysis of another HpnH, from the ethanol-producing bacterium Zymomonas mobilis (ZmHpnH). Similar to ScHpnH, wild-type ZmHpnH afforded (22R)-adenosylhopane. Conserved cysteine and tyrosine residues were suggested as possible hydrogen sources to quench the putative radical reaction intermediate. A Cys106Ala mutant of ZmHpnH had one-fortieth the activity of the wild-type enzyme and yielded both (22R)- and (22S)-adenosylhopane along with some related byproducts. Radical trapping experiments with a spin-trapping agent supported the generation of a radical intermediate in the ZmHpnH-catalyzed reaction. We propose that the thiol of Cys106 stereoselectively reduces the radical intermediate generated at the C22 position by the addition of the 5'-deoxadenosyl radical to diploptene, to complete the reaction.

Exometabolomics Assisted Design and Validation of Synthetic Obligate Mutualism.

Synthetic microbial ecology has the potential to enhance the productivity and resiliency of biotechnology processes compared to approaches using single isolates. Engineering microbial consortia is challenging; however, one approach that has attracted significant attention is the creation of synthetic obligate mutualism using auxotrophic mutants that depend on each other for exchange or cross-feeding of metabolites. Here, we describe the integration of mutant library fitness profiling with mass spectrometry based exometabolomics as a method for constructing synthetic mutualism based on cross-feeding. Two industrially important species lacking known ecological interactions, Zymomonas mobilis and Escherichia coli, were selected as the test species. Amino acid exometabolites identified in the spent medium of Z. mobilis were used to select three corresponding E. coli auxotrophs (proA, pheA and IlvA), as potential E. coli counterparts for the coculture. A pooled mutant fitness assay with a Z. mobilis transposon mutant library was used to identify mutants with improved growth in the presence of E. coli. An auxotroph mutant in a gene (ZMO0748) with sequence similarity to cysteine synthase A (cysK), was selected as the Z. mobilis counterpart for the coculture. Exometabolomic analysis of spent E. coli medium identified glutathione related metabolites as potentially available for rescue of the Z. mobilis cysteine synthase mutant. Three sets of cocultures between the Z. mobilis auxotroph and each of the three E. coli auxotrophs were monitored by optical density for growth and analyzed by flow cytometry to confirm high cell counts for each species. Taken together, our methods provide a technological framework for creating synthetic mutualisms combining existing screening based methods and exometabolomics for both the selection of obligate mutualism partners and elucidation of metabolites involved in auxotroph rescue.

Reconstruction of a charge balanced genome-scale metabolic model to study the energy-uncoupled growth of Zymomonas mobilis ZM1.

Zymomonas mobilis is an ethanologenic bacterium and is known to be an example microorganism with energy-uncoupled growth. A genome-scale metabolic model could be applicable for understanding the characteristics of Z. mobilis with rapid catabolism and inefficient energy conversion. In this study, a charge balanced genome-scale metabolic model (iEM439) of Z. mobilis ATCC 10988 (ZM1) including 439 genes, 692 metabolic reactions and 658 metabolites was reconstructed based on genome annotation and previously published information. The model presents a much better prediction for biomass and ethanol concentrations in a batch culture by using dynamic flux balance analysis compared with the two previous genome-scale metabolic models. Furthermore, intracellular flux distribution obtained from the model was consistent with the fluxes for glucose fermentation determined by (13)C NMR. The model predicts that there is no difference in growth rates of Z. mobilis under aerobic and anaerobic conditions whereas ethanol production is decreased and production of other metabolites including acetate and acetoin is increased under aerobic conditions. Experimental data confirm the predicted differences between the aerobic and anaerobic growth of Z. mobilis. Finally, the model was used to study the energy-uncoupled growth of Z. mobilis and to predict its effect on flux distribution in the central metabolism. Flux distribution obtained from the model indicates that coupling growth and energy reduces ethanol secretion and changes the flux distribution to produce more biomass. This coupling is also associated with a significant increase in the proton uptake rate based on the prediction of the charge balanced model. Hence, resistance to intracellular pH reduction could be the main reason for uncoupled growth and Z. mobilis uses ATPase to pump out the proton. Experimental observations are in accordance with the predicted relationship between growth, ATP dissipation and proton exchange.

Three new shuttle vectors for heterologous expression in Zymomonas mobilis

Background: Zymomonas mobilis, as a novel platform for bio-ethanol production, has been attracted more attention and it is very important to construct vectors for the efficient expression of foreign genes in this bacterium. Results: Three shuttle vectors ( pSUZM 1, pSUZM2 and pSUZM3 ) were first constructed with the origins of replication from the chromosome and two native plasmids (pZZM401 and pZZM402) of Z. mobilis ZM4, respectively. The three shuttle vectors were stable in Z. mobilis ZM4 and have 3,32 and 27 copies, respectively. The promoter Ppdc (a), from the pyruvate decarboxylase gene, was clonedinto the shuttle vectors, generatingthe expressionvectors pSUZM1(2, 3)a. The codon-optimized glucoamylase gene from Aspergillus awamori combined with the signal peptide sequence from the alkaline phosphatase gene of Z. mobilis was cloned into pSUZM1(2, 3)a, resulting in the plasmids pSUZM1a-GA, pSUZM2a-GA and pSUZM3a-GA, respectively. After transforming these plasmids into Z. mobilis ZM4, the host was endowed with glucoamylase activity for starch hydrolysis. Both pSUZM2a-GA and pSUZM3a-GA were more efficientatproducingglucoamylase thanpSUZM1a-GA. Conclusions: These results indicated that these expression vectors are useful tools for gene expression in Z. mobilis and this could provide a solid foundation for further studies of heterologous gene expression in Z. mobilis.

Genetic variability analysis of Zymomonas mobilis strains from the UFPEDA microorganisms collection.

Zymomonas mobilis is a Gram-negative bacterium that has drawn attention in the bioethanol industry. Besides bioethanol, this bacterium also produces other biotechnological products such as levans, which show antitumor activity. Molecular studies involving Z. mobilis have advanced to the point that allows us to characterize interspecies genetic diversity and understand their metabolism, and these data are essential for better utilization of this species. In this study, the genetic diversity of 24 strains from the Microorganisms Collection of Departamento de Antibióticos (UFPEDA) from Universidade Federal de Pernambuco were characterized. The methods used were amplified ribosomal DNA restriction analysis and diversity analysis of the internally transcribed 16S-23S rDNA spacer region (ISR). These analyses revealed low genetic variability of the 16S rDNA gene. These data confirm that these isolates are, or are closely related to, Z. mobilis. Moreover, the analysis of the ISR confirmed the genetic variability of strains deposited in the UFPEDA collection of microorganisms and grouped these strains into ten ribotypes, which can be used in the future for breeding programs and for the preservation of biodiversity. Furthermore, this study characterized the genetic variability between the UFPEDA 205/ ZAP, UFPEDA 98/AG11, and ZAG strains, which were obtained by spheroplast fusion among them. The data also indicate that there is genetic variability among the UFPEDA 202/CP4 and UFPEDA 633/ ZM4 strains, demonstrating that these important Z. mobilis strains are distinct, as suggested in previous studies.

N2 gas is an effective fertilizer for bioethanol production by Zymomonas mobilis.

A nascent cellulosic ethanol industry is struggling to become cost-competitive against corn ethanol and gasoline. Millions of dollars are spent on nitrogen supplements to make up for the low nitrogen content of the cellulosic feedstock. Here we show for the first time to our knowledge that the ethanol-producing bacterium, Zymomonas mobilis, can use N2 gas in lieu of traditional nitrogen supplements. Despite being an electron-intensive process, N2 fixation by Z. mobilis did not divert electrons away from ethanol production, as the ethanol yield was greater than 97% of the theoretical maximum. In a defined medium, Z. mobilis produced ethanol 50% faster per cell and generated half the unwanted biomass when supplied N2 instead of ammonium. In a cellulosic feedstock-derived medium, Z. mobilis achieved a similar cell density and a slightly higher ethanol yield when supplied N2 instead of the industrial nitrogen supplement, corn steep liquor. We estimate that N2-utilizing Z. mobilis could save a cellulosic ethanol production facility more than $1 million/y.

The inefficient aerobic energetics of Zymomonas 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 were closely similar, not supporting presence of one major, yet energetically inefficient electron transport branch causing the observed poor aerobic growth and lack of oxidative phosphorylation in Z. mobilis. We suggest that rapidly operating Entner-Doudoroff pathway generates too high phosphorylation potential for the weakly coupled respiratory system to shift the H(+) -dependent ATPase toward ATP synthesis.

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 concentrations reported in previous experimental studies. In addition, the model is shown to be consistent with experimental results for the coupled response of ATP concentration and glycolytic flux to ATPase inhibition. Metabolic control analysis of the model revealed that the majority of flux control resides not inside, but outside the E-D pathway itself, predominantly in ATP consumption, demonstrating why past attempts to increase the glycolytic flux through overexpression of glycolytic enzymes have been unsuccessful. Co-response analysis indicates how homeostasis of ATP concentrations starts to deteriorate markedly at the highest glycolytic rates. This kinetic model has potential for application in Z. mobilis metabolic engineering and, since there are currently no E-D pathway models available in public databases, it can serve as a basis for the development of models for other micro-organisms possessing this type of glycolytic pathway.

Hydrogen sulfide formation as well as ethanol production in different media by cysND- and/or cysIJ-inactivated mutant strains of Zymomonas mobilis ZM4.

Many bacteria reduce inorganic sulfate to sulfide to satisfy their need for sulfur, one of the most important elements for biological life. But little is known about the metabolic pathways involving hydrogen sulfide (H2S) in mesophilic bacteria. By genomic sequence analysis, a complete set of genes for the assimilatory sulfate reduction pathway has been identified in the ethanologen Zymomonas mobilis. In this study, the first ATP sulfurylase- and final sulfite reductase-encoding genes cysND and cysIJ, respectively, in the putative pathway from sulfate to sulfite in Z. mobilis ZM4 was singly or doubly inactivated by homologous recombination and a site-specific FLP-FRT recombination. The resultant mutants, ∆cysND, ∆cysIJ and ∆cysND-cat∆cysIJ, were unable to produce detectable H2S in glucose or sucrose-containing rich medium and sweet sorghum juice, in which the wild-type ZM4 produced detectable H2S. While adding sulfite (SO3²â») into media impaired the growth of the mutants and ZM4 to varying degrees, the sulfite restored the H2S formation in the ∆cysND in the above media, but not in the ∆cysIJ and ∆cysND-cat∆cysIJ mutants. Although it seemed that the inactivation of cysND and cysIJ did not exert a significant negative effect on the cell growth at least in glucose or sucrose medium, the ethanol production of all mutants was inferior to that of ZM4 in sucrose medium and sweet sorghum juice. In addition, adding L-cysteine to glucose-containing rich media restored H2S formation of all mutants, indicating the existence of another pathway for producing H2S in Z. mobilis. All these results would help to further elucidate the metabolic pathways involving H2S in Z. mobilis and exploit the biotechnological applications of this industrially important bacterium.

Structures of iron-dependent alcohol dehydrogenase 2 from Zymomonas mobilis ZM4 with and without NAD+ cofactor.

The ethanologenic bacterium Zymomonas mobilis ZM4 is of special interest because it has a high ethanol yield. This is made possible by the two alcohol dehydrogenases (ADHs) present in Z. mobilis ZM4 (zmADHs), which shift the equilibrium of the reaction toward the synthesis of ethanol. They are metal-dependent enzymes: zinc for zmADH1 and iron for zmADH2. However, zmADH2 is inactivated by oxygen, thus implicating zmADH2 as the component of the cytosolic respiratory system in Z. mobilis. Here, we show crystal structures of zmADH2 in the form of an apo-enzyme and an NAD+­cofactor complex. The overall folding of the monomeric structure is very similar to those of other functionally related ADHs with structural variations around the probable substrate and NAD+ cofactor binding region. A dimeric structure is formed by the limited interactions between the two subunits with the bound NAD+ at the cleft formed along the domain interface. The catalytic iron ion binds near to the nicotinamide ring of NAD+, which is likely to restrict and locate the ethanol to the active site together with the oxidized Cys residue and several nonpolar bulky residues. The structures of the zmADH2 from the proficient ethanologenic bacterium Z. mobilis, with and without NAD+ cofactor, and modeling ethanol in the active site imply that there is a typical metal-dependent catalytic mechanism.

Production of ethanol from mesquite (Prosopis juliflora (SW) D. C) pods mash by Zymomonas mobilis and Saccharomyces cerevisiae

This study aimed to assess the use of mesquite pods hydrated mash as biomass for the growth of Saccharomyces cerevisiae UFEPEDA-1012 and Zymomonas mobilis UFEPEDA-205 and for ethanol production using a submerged fermentation. A 2³ factorial design was used to analyze the effects of the type of microorganism, time of fermentation and condition of cultivation on the ethanol production in mesquite pods mash (30 g 100 mL-1). From the obtained results the hydrated mesquite pods mash presented as a good substrate for the growth of S. cerevisiae and Z. mobilis in comparison to the standard media. The effect that most affected the ethanol production was the type of microorganism. The highest ethanol concentration (141.1 gL-1) was found when Z. mobilis was cultivated in mesquite pods mash under static condition for 36 hrs. Ethanol production by S. cerevisiae was higher (44.32 gL-1) after 18 hrs of fermentation under static condition. According to these results, the mesquite pods could be known as an alternative substrate to be used for biotechnological purposes, mainly for ethanol production.

Measurement and analysis of intracellular ATP levels in metabolically engineered Zymomonas mobilis fermenting glucose and xylose mixtures.

Intracellular adenosine-5'-triphosphate (ATP) levels were measured in a metabolically engineered Zymomonas mobilis over the course of batch fermentations of glucose and xylose mixtures. Fermentations were conducted over a range of pH (5-6) in the presence of varying initial amounts of acetic acid (0-8 g/L) using a 10% (w/v) total sugar concentration (glucose only, xylose only, or 5% glucose/5% xylose mixture). Over the design space investigated, ethanol process yields varied between 56.6% and 92.3% +/- 1.3% of theoretical, depending upon the test conditions. The large variation in process yields reflects the strong effect pH plays in modulating the inhibitory effect of acetic acid on fermentation performance. A corresponding effect was observed on maximum cellular specific growth rates, with the rates varying between a low of 0.15 h(-1) observed at pH 5 in the presence of 8 g/L acetic acid to a high of 0.32 +/- 0.02 h(-1) obtained at pH 5 or 6 when no acetic acid was initially present. While substantial differences were observed in intracellular specific ATP concentration profiles depending upon fermentation conditions, maximum intracellular ATP accumulation levels varied within a relatively narrow range (1.5-3.8 mg ATP/g dry cell mass). Xylose fermentations produced and accumulated ATP at much slower rates than mixed sugar fermentations (5% glucose, 5% xylose), and the ATP production and accumulation rates in the mixed sugar fermentations were slightly slower than in glucose fermentations. Results demonstrate that higher levels of acetic acid delay the onset and influence the extent of intracellular ATP accumulation. ATP production and accumulation rates were most sensitive to acetic acid at lower values of pH.

Performance of immobilized Zymomonas mobilis 31821 (pZB5) on actual hydrolysates produced by Arkenol technology.

By applying the Arkenol process using highly concentrated sulfuric acid, various biomass feedstocks, including cedar tree, rice straw, newspaper, and bagasse, were successfully processed and converted into glucose and xylose for fermentation usage in a flash fermentation reactor in which the performance of National Renewable Energy Laboratory's patented rec-Zymomonas mobilis 31821 (pZB5) after immobilization was investigated. The immobilization medium is a photocrosslinked resin made from polyethylene glycols or polypropylene glycols. Recombinant or rec-Z. mobilis used in the study has been shown to efficiently ferment glucose and xylose at a relatively high concentration (12-15%), that is a typical hydrolysate produced from cellulosic feedstocks. The application of immobilized rec-Z. mobilis and flash fermentation technology, together with concentrated acid technology producing a high concentration sugar solution, promises to speed the development of the cellulose-to-ethanol industry.

Structural diversity of the triterpenic hydrocarbons from the bacterium Zymomonas mobilis: the signature of defective squalene cyclization by the squalene/hopene cyclase.

Twelve polycyclic triterpenic hydrocarbons (alpha- and gamma-polypodatetraenes, dammara-20(21),24-diene, 17-isodammara-12,24-diene, eupha-7,24-diene, hop-17(21)-ene, neohop-13(18)-ene, 17-isodammara-20(21),24-diene, neohop-12-ene, fern-8-ene, diploptene and hop-21-ene) were detected in the hydrocarbon fraction from the bacterium Zymomonas mobilis. Some of them have never been reported from bacteria. These triterpenes were present in Z. mobilis in significant amounts, comparable to those of diploptene, which is usually the major triterpenic hydrocarbon in hopanoid-producing bacteria. The occurrence of such compounds confirms the lack of specificity of bacterial squalene cyclases and the possibility of alternative cyclization routes induced by the existence in the cyclization process of intermediate carbocations of sufficient lifetime.

Comparative energetics of glucose and xylose metabolism in recombinant Zymomonas mobilis.

Recombinant Zymomonas mobilis CP4:pZB5 was grown with pH control in batch and continuous modes with either glucose or xylose as the sole carbon and energy source. In batch cultures in which the ratio of the final cell mass concentration to the amount of sugar in the medium was constant (i.e., under conditions that promote "coupled growth"), maximum specific rates of glucose and xylose consumption were 8.5 and 2.1 g/(g of cell.h), respectively; maximum specific rates of ethanol production for glucose and xylose were 4.1 and 1.0 g/(g of cell.h), respectively; and average growth yields from glucose and xylose were 0.055 and 0.034 g of dry cell mass (DCM)/g of sugar, respectively. The corresponding value of YATP for glucose and xylose was 9.9 and 5.1 g of DCM/mol of ATP, respectively. YATP for the wild-type culture CP4 with glucose was 10.4 g of DCM/mol of ATP. For single substrate chemostat cultures in which the growth rate was varied as the dilution rate (D), the maximum or "true" growth yield (max Yx/s) was calculated from Pirt plots as the inverse of the slope of the best-fit linear regression for the specific sugar utilization rate as a function of D, and the "maintenance coefficient" (m) was determined as the y-axis intercept. For xylose, values of max Yx/s and m were 0.0417 g of DCM/g of xylose (YATP = 6.25) and 0.04 g of xylose/(g of cell.h), respectively. However, with glucose there was an observed deviation from linearity, and the data in the Pirt plot was best fit with a second-order polynomial in D. At D > 0.1/h, YATP = 8.71 and m = 2.05 g of glu/(g of cell.h) whereas at D < 0.1/h, YATP = 4.9 g of DCM/mol of ATP and m = 0.04 g of glu/(g of cell.h). This observation provides evidence to question the validity of the unstructured growth model and the assumption that Pirt's maintenance coefficient is a constant that is independent of the growth rate. Collectively, these observations with individual sugars and the values assigned to various growth and fermentation parameters will be useful in the development of models to predict the behavior of rec Zm in mixed substrate fermentations of the type associated with biomass-to-ethanol processes.

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 content. In non-growing cyanide-preincubated cells, with ethanol as the respiratory substrate, cyanide increased ATP levels; in such cells, a large part of the cyanide-sensitive respiration was inhibited within a few seconds after ethanol addition, while inhibition of the rest of respiration took several minutes. The more cyanide-sensitive respiration was apparently energy-nongenerating, and was absent in membrane preparations. Pelleting of membranes from cell-free extracts produced 'soluble' fractions in which a b-type haem was detectable by reduced minus oxidized difference spectroscopy. The function of the Z. mobilis respiratory chain in cell growth and respiratory protection, and the possible physiological role of aerobic generation of inhibitory metabolites, are discussed.