Iron availability enhances the cellular energetics of aerobic Escherichia coli cultures while upregulating anaerobic respiratory chains.

Aerobic Escherichia coli growth at restricted iron concentrations (≤ 1.75 ± 0.04 µM) is characterized by lower biomass yield, higher acetate accumulation and higher activation of the siderophore iron-acquisition systems. Although iron homeostasis in E. coli has been studied intensively, previous studies focused only on understanding the regulation of the iron import systems and the iron-requiring enzymes. Here, the effect of iron availability on the energy metabolism of E. coli has been investigated. It was established that aerobic cultures growing under limiting iron conditions showed lower ATP yield per glucose, lower growth rate and lower TCA cycle activity and respiration, at the same time as increased glucose consumption, acetate and pyruvate accumulation, practically mimicking microaerobic growth. However, at excess iron, independent of oxygen availability, the cultures showed high cellular energetics (5.8 ATP/mol of glucose) by using pathways requiring iron-rich complex proteins found in the TCA cycle and respiratory chain. In conditions of iron excess, some iron-requiring terminal reductases of the respiratory chain, that were thought to function only under anaerobiosis, were used by the E. coli, when in aerobic conditions, to maintain high respiratory activity. This allowed it to produce more biomass and more reactive oxygen species that were controlled by the higher activity of the antioxidant defenses (SOD, peroxidase and catalase) and the iron-sulfur cluster repair systems.

Bacillus cereus MH778713 elicits tomato plant protection against Fusarium oxysporum.

AIM: The genus Fusarium comprises plant pathogenic species with agricultural relevance. Fusarium oxysporum causes tomato wilt disease with significant production losses. The use of agrochemicals to control the Fusarium wilt of tomato is not environmentally friendly. Bacillus species, as biocontrol agents, provide a safe and sustainable means to control Fusarium-induced plant diseases. In this study, the ability of Bacillus cereus MH778713, a strain isolated from root nodules of Prosopis laevigata, to protect tomato plants against Fusarium wilt was evaluated. METHODS AND RESULTS: Bacillus cereus MH778713 and its volatiles inhibited the radial growth of F. oxysporum and stimulated tomato seedling growth in in vitro and in vivo tests. When tomato plants growing in the greenhouse were inoculated with B. cereus MH778713, the percentage of wilted plants decreased from 96% to 12%, indicating an effective crop protection against Fusarium wilt. Among the metabolites produced by B. cereus MH778713, hentriacontane and 2,4-di-tert-butylphenol promoted tomato seedling growth and showed antifungal activity against the target pathogen. CONCLUSION: The inoculation of B. cereus MH778713 on tomato seedlings helped plants to manage Fusarium wilt, suggesting the potential of B. cereus MH778713 as a biocontrol agent. SIGNIFICANCE AND IMPACT OF THE STUDY: These results complement our previous studies on chromium tolerance and bioremediation traits of B. cereus MH778713 by highlighting the potential of this metal-resistant micro-organism to boost crop growth and disease resistance.

Emerging Applications of Bacteriocins as Antimicrobials, Anticancer Drugs, and Modulators of The Gastrointestinal Microbiota.

The use of bacteriocins holds great promise in different areas such as health, food, nutrition, veterinary, nanotechnology, among others. Many research groups worldwide continue to advance the knowledge to unravel a novel range of therapeutic agents and food preservatives. This review addresses the advances of bacteriocins and their producer organisms as biocontrol agents for applications in the medical industry and agriculture. Furthermore, the bacteriocin mechanism of action and structural characteristics will be reviewed. Finally, the potential role of bacteriocins to modulate the signaling in host-associated microbial communities will be discussed.

Growth inhibition of pathogenic microorganisms by Pseudomonas protegens EMM-1 and partial characterization of inhibitory substances.

The bacterial strain, EMM-1, was isolated from the rhizosphere of red maize ("Rojo Criollo") and identified as Pseudomonas protegens EMM-1 based on phylogenetic analysis of 16S rDNA, rpoB, rpoD, and gyrB gene sequences. We uncovered genes involved in the production of antimicrobial compounds like 2,4-diacetylphloroglucinol (2,4-DAPG), pyoluteorin, and lectin-like bacteriocins. These antimicrobial compounds are also produced by other fluorescent pseudomonads alike P. protegens. Double-layer agar assay showed that P. protegens EMM-1 inhibited the growth of several multidrug-resistant (MDR) bacteria, especially clinical isolates of the genera Klebsiella and ß-hemolytic Streptococcus. This strain also displayed inhibitory effects against diverse fungi, such as Aspergillus, Botrytis, and Fusarium. Besides, a crude extract of inhibitory substances secreted into agar was obtained after the cold-leaching process, and physicochemical characterization was performed. The partially purified inhibitory substances produced by P. protegens EMM-1 inhibited the growth of Streptococcus sp. and Microbacterium sp., but no inhibitory effect was noted for other bacterial or fungal strains. The molecular weight determined after ultrafiltration was between 3 and 10 kDa. The inhibitory activity was thermally stable up to 60°C (but completely lost at 100°C), and the inhibitory activity remained active in a wide pH range (from 3 to 9). After treatment with a protease from Bacillus licheniformis, the inhibitory activity was decreased by 90%, suggesting the presence of proteic natural compounds. All these findings suggested that P. protegens EMM-1 is a potential source of antimicrobials to be used against pathogens for humans and plants.

Long-Chain Hydrocarbons (C21, C24, and C31) Released by Bacillus sp. MH778713 Break Dormancy of Mesquite Seeds Subjected to Chromium Stress.

Volatile organic compounds (VOCs) produced by rhizobacteria have been proven to stimulate plant growth during germination and seedling stages. However, the modulating effect of bacterial volatiles on the germination of seeds subjected to heavy metal stress is scarcely studied. In this work, the ability of volatiles released by Bacillus sp. MH778713 to induce seed dormancy breakage in Prosopis laevigata and Arabidopsis thaliana seeds were examined. The minimal inhibitory concentration of chromium (Cr) VI that prevents seed germination of P. laevigata and A. thaliana on water-Cr-agar plates was 2500 and 100 mg L-1, respectively. Remarkably, partitioned Petri-dish co-cultivation of Bacillus sp. MH778713 and plant seeds under Cr-stress showed the beneficial effect of volatiles emitted by Bacillus sp. MH778713, helping plant seeds to overcome Cr-stress. Among the metabolites emitted by Bacillus sp. MH778713, octadecane, heneicosane, 2,4-di-tert-butylphenol, hexadecane, eicosane, octacosane, and tetratriacontane were the most abundant. To confirm that these long-chain compounds produced by Bacillus sp. MH778713 could be responsible for the seed dormancy breakage, high pure organic compounds (2,4-di-tert-butylphenol, heneicosane, hentriacontane, and tetracosane) were used directly in germination assays of P. laevigata and A. thaliana seeds instead of volatiles emitted by Bacillus sp. MH778713. All organic compounds allowed Prosopis and Arabidopsis seeds to overcome Cr-toxicity and germinate. The results of this study provide new insight into the role of long-chain bacterial compounds produced by Bacillus sp. MH778713 as triggers of seed abiotic stress tolerance, surmounting chromium stress and stimulating seedling development.

Importancia de compuestos antimicrobianos producidos por bacterias benéficas en el biocontrol de fitopatógenos / The importance of antimicrobial compounds produced by beneficial bacteria on the biocontrol of phytopathogens

ABSTRACT Bacteria produce antimicrobial compounds to compete for nutrients and space in a particular habitat. Antagonistic interactions can be evaluated by several methodologies including the double-layer agar and simultaneous inhibition assays. Among the well-known inhibitory substances produced by bacteria are the broad-spectrum antibiotics, organic acids, siderophores, antifungal, and bacteriocins. The most studied bacterial genera able to produce these inhibitory substances are Enterococcus, Lactococcus, Streptomyces, Bacillus, Pseudomonas, Klebsiella, Escherichia, and Burkholderia. Some beneficial bacteria can promote plant growth and degrade toxic compounds in the environment representing an attractive solution to diverse issues in agriculture and soil pollution, particularly in fields with damaged soils where pesticides and fertilizers have been indiscriminately used. Beneficial bacteria may increase plant health by inhibiting pathogenic microorganisms; some examples include Gluconacetobacter diazotrophicus, Azospirullum brasilense, Pseudomonas fluorescens, Pseudomonas protegens, and Burkholderia tropica. However, most studies showing the antagonistic potential of these bacteria have been performed in vitro, and just a few of them have been evaluated in association with plants. Several inhibitory substances involved in pathogen antagonism have not been elucidated yet; in fact, we know only 1 % of the bacterial diversity in a natural environment leading us to assume that many other inhibitory substances remain unexplored. In this review, we will describe the characteristics of some antimicrobial compounds produced by beneficial bacteria, the principal methodologies performed to evaluate their production, modes of action, and their importance for biotechnological purposes.

RESUMEN Las bacterias producen compuestos antimicrobianos para competir por nutrientes y espacio en un hábitat particular. Las interacciones antagónicas pueden evaluarse mediante varias metodologías, incluido el agar de doble capa y los ensayos de inhibición simultánea. Las sustancias inhibidoras mejor conocidas producidas por bacterias incluyen antibióticos, ácidos orgánicos, sideróforos, antifúngicos y bacteriocinas. Entre los géneros bacterianos más estudiados que producen sustancias inhibidoras se incluyen Enterococcus, Lactococcus, Streptomyces, Bacillus, Pseudomonas, Klebsiella, Escherichia y Burkholderia. Algunas bacterias beneficiosas tienen la capacidad de promover el crecimiento de las plantas y degradar compuestos tóxicos en el ambiente, por lo que podrían incrementar el rendimiento de los cultivos y disminuir problemas de contaminación del suelo, especialmente donde los pesticidas y fertilizantes han sido utilizados indiscriminadamente. Algunas bacterias beneficiosas pueden aumentar la salud de las plantas al inhibir microorganismos patógenos, por ejemplo, Gluconacetobacter diazotrophicus, Azospirullum brasilense, Pseudomonas fluorescens, Pseudomonas protegens y Burkholderia tropica. Sin embargo, la mayoría de los estudios que muestran el potencial antagónico de estas bacterias se han realizado in vitro, y pocos de ellos se han evaluado en asociación con plantas. Varias sustancias inhibitorias implicadas en el antagonismo de los patógenos aún son desconocidas; de hecho, sabemos que solo se ha aislado el 1 % de la diversidad bacteriana en un ambiente natural, lo que sugiere que hay muchas otras sustancias inhibitorias que no han sido exploradas. En esta revisión describimos las características de algunos compuestos antimicrobianos producidos por bacterias beneficiosas, las principales metodologías usadas para evaluar su producción, modos de acción y su importancia para fines biotecnológicos.

Structural characterization of scorpion peptides and their bactericidal activity against clinical isolates of multidrug-resistant bacteria.

Scorpion venom peptides represent a novel source of antimicrobial peptides (AMPs) with broad-spectrum activity. In this study, we determined the minimum bactericidal concentration (MBC) of three scorpion AMPs, Uy234, Uy17, and Uy192, which are found in the venomous glands of the Urodacus yaschenkoi scorpion, against the clinical isolates of multidrug-resistant (MDR) bacteria. In addition, we tested the activity of a consensus AMP designed in our laboratory based on some previously reported IsCT-type (cytotoxic linear peptide) AMPs with the aim of obtaining higher antimicrobial activity. All peptides tested showed high antimicrobial activity against MDR clinical isolates, with the highest activity against ß-hemolytic Streptococcus strains. The hemolytic activity was determined against human red blood cells and was significantly lower than that of previously reported AMPs. The α-helical structure of the four AMPs was confirmed by circular dichroism (CD). These results suggest that the four peptides can be valuable tools for the design and development of AMPs for use in the inhibition of MDR pathogenic bacteria. A clear index of synergism and additivity was found for the combination of QnCs-BUAP + Uy234, which makes these peptides the most promising candidates against pathogenic bacteria.

Chromium Hyper-Tolerant Bacillus sp. MH778713 Assists Phytoremediation of Heavy Metals by Mesquite Trees (Prosopis laevigata).

Heavy metal accumulation in mesquite trees (Prosopis laevigata) growing in aluminum, titanium, chromium and zirconium-polluted soils of a semi-arid region in Mexico was investigated using wavelength dispersive X-ray fluorescence analysis. The results showed that P. laevigata trees can hyper accumulate up to 4100 mg/kg of Al, 14000 mg/kg of Fe, 1600 mg/kg of Ti, 2500 mg/kg of Zn, but not chromium, regarding high chromium concentrations found in soils (435 mg/kg). Since plant-associated microorganism can modulate phytoremediation efficiency, the biodiversity of P. laevigata associated bacteria was studied. Eighty-eight isolates from P. laevigata nodules were obtained; all isolates tolerated high concentrations of Al, Fe, Zn and Cr in vitro. The top-six chromium tolerant strains were identified by 16S rRNA sequence analysis as belonging to genus Bacillus. Bacillus sp. MH778713, close to Bacillus cereus group, showed to be the most resistant strain, tolerating up to 15000 mg/L Cr (VI) and 10000 mg/L of Al. Regarding the bioaccumulation traits, Bacillus sp. MH778713 accumulated up to 100 mg Cr(VI)/g of cells when it was exposed to 1474 mg/L of Cr VI. To assess Bacillus sp. MH778713 ability to assist Prosopis laevigata phytoremediation; twenty plants were inoculated or non-inoculated with Bacillus sp. MH778713 and grown in nitrogen-free Jensen's medium added with 0, 10 and 25 mg/L of Cr(VI). Only plants inoculated with Bacillus sp. grew in the presence of chromium showing the ability of this strain to assist chromium phytoremediation. P. laevigata and Bacillus spp. may be considered as good candidates for soil restoration of arid and semiarid sites contaminated with heavy metals.

Desiccation-induced viable but nonculturable state in Pseudomonas putida KT2440, a survival strategy.

The potential of Pseudomonas putida KT2440 to act as a plant-growth promoter or as a bioremediator of toxic compounds can be affected by desiccation. In the present work, the bacterial survival ratio (BSR) in response to air desiccation was evaluated for P. putida KT2440 in the presence of different protectors. The BSR in the presence of nonreducing disaccharides, such as trehalose, was high after 15 days of desiccation stress (occurring at 30°C and 50% relative humidity), whereas in the absence of a protector the bacterial counts diminished to nondetectable numbers (ca 2.8 log CFU/mL). The LIVE/DEAD staining method showed that bacteria protected with trehalose maintained increased numbers of green cells after desiccation while cells without protection were all observed to be red. This indicated that nonprotected bacteria had compromised membrane integrity. However, when nonprotected bacteria subjected to 18 days of desiccation stress were rehydrated for a short time with maize root exudates or for 48 h with water (prolonged rehydration), the bacterial counts were as high as that observed for those not subjected to desiccation stress, suggesting that the cells entered the viable but nonculturable (VBNC) state under desiccation and that they returned to a culturable state after those means of rehydration. Interestingly an increase in the green color intensity of cells that returned to a culturable state was observed using LIVE/DEAD staining method, indicating an improvement in their membrane integrity. Cellular activity in the VBNC state was determined. A GFP-tagged P. putida strain expressing GFP constitutively was subjected to desiccation. After 12 days of desiccation, the GFP-tagged strain lost culturability, but it exhibited active GFP expression, which in turn made the cells green. Furthermore, the expression of 16S rRNA, rpoN (housekeeping), mutL, mutS (encoding proteins from the mismatch repair complex), and oprH (encoding an outer membrane protein) were examined by RT-PCR. All evaluated genes were expressed by both types of cells, culturable and nonculturable, indicating active molecular processes during the VBNC state.

Effect of amino acids on transcription and translation of key genes in E. coli K and B grown at a steady state in minimal medium.

Growing E. coli to high densities is a common strategy for biologicals production. The process is implemented by using complex or minimal media with different feeding strategies. To understand the effect of amino acids, E. coli B and K were grown at a steady state of 0.35 h-1 in glucose minimal medium with and without amino acids, while their metabolism, protein abundance and gene expression were compared. The results showed that amino acids promoted higher acetate excretion, higher fatty acid biosynthesis (K strain), repressed glucose uptake rate, and decreased expression of proteins associated with the TCA cycle, glyoxylate shunt and amino acid biosynthesis. In presence of amino acids, E. coli K upregulated fatty acid biosynthesis and repressed more genes and proteins involved in amino acid biosynthesis than E. coli B. These findings are correlated with higher yield on glucose (Yx/s) and high specific biomass production rate (qx) in K strain in the presence of amino acids. In contrast, pre-formed precursor molecules such as amino acids did not affect fatty acid biosynthesis in E. coli B or Yx/s and qx, which were higher than those of E. coli K, suggesting that constitutive synthesis of energetically demanding precursors and higher fatty acid ß-oxidation activity is key for high biomass-performer E. coli B. Both strains turned off unnecessary pathways and directed their metabolism to proteome efficient overflow metabolism likely to generate energy and provide protein to functions supporting higher growth rate.

Compatible bacterial mixture, tolerant to desiccation, improves maize plant growth.

Plant growth-promoting rhizobacteria (PGPR) increase plant growth and crop productivity. The inoculation of plants with a bacterial mixture (consortium) apparently provides greater benefits to plant growth than inoculation with a single bacterial strain. In the present work, a bacterial consortium was formulated containing four compatible and desiccation-tolerant strains with potential as PGPR. The formulation had one moderately (Pseudomonas putida KT2440) and three highly desiccation-tolerant (Sphingomonas sp. OF178, Azospirillum brasilense Sp7 and Acinetobacter sp. EMM02) strains. The four bacterial strains were able to adhere to seeds and colonize the rhizosphere of plants when applied in both mono-inoculation and multi-inoculation treatments, showing that they can also coexist without antagonistic effects in association with plants. The effects of the bacterial consortium on the growth of blue maize were evaluated. Seeds inoculated with either individual bacterial strains or the bacterial consortium were subjected to two experimental conditions before sowing: normal hydration or desiccation. In general, inoculation with the bacterial consortium increased the shoot and root dry weight, plant height and plant diameter compared to the non-inoculated control or mono-inoculation treatments. The bacterial consortium formulated in this work had greater benefits for blue maize plants even when the inoculated seeds underwent desiccation stress before germination, making this formulation attractive for future field applications.

Increasing dissolved-oxygen disrupts iron homeostasis in production cultures of Escherichia coli.

The damaging effect of high oxygen concentration on growth of Escherichia coli is well established. Over-oxygenation increases the intracellular concentration of reactive oxygen species (ROS), causing the destruction of the [4Fe-4S] cluster of dehydratases and limiting the biosynthesis of both branched-chain amino acids and nicotinamide adenine dinucleotide. A key enzyme that reduces the damaging effect of superoxide is superoxide dismutase (SOD). Its transcriptional regulation is controlled by global transcription regulators that respond to changes in oxygen and iron concentrations and pH. Production of biological compounds from E. coli is currently achieved using cultures grown to high cell densities which require oxygen-enriched air supply. It is, therefore, important to study the effect of over-oxygenation on E. coli metabolism and the bacterial protecting mechanism. The effect of over-oxygenation on the superoxide dismutase regulation system was evaluated in cultures grown in a bioreactor by increasing the oxygen concentration from 30 to 300 % air saturation. Following the change in the dissolved oxygen (DO), the expression of sodC, the periplasmic CuZn-containing SOD, and sodA, the cytosolic Mn-containing SOD, was higher in all the tested strains, while the expression of the sodB, the cytosolic Fe-containing SOD, was lower. The down-regulation of the sodB was found to be related to the activation of the small RNA RyhB. It was revealed that iron homeostasis, in particular ferric iron, was involved in the RyhB activation and in sodB regulation but not in sodA. Supplementation of amino acids to the culture medium reduced the intracellular ROS accumulation and reduced the activation of both SodA and SodC following the increase in the oxygen concentration. The study provides evidence that at conditions of over-oxygenation, sodA and sodC are strongly regulated by the amount of ROS, in particular superoxide; and sodB is regulated by iron availability through the small RNA RyhB. In addition, information on the impact of NADH, presence of amino acids and type of iron on SOD regulation, and consequently, on the ROS concentration is provided.

Effect of elevated oxygen concentration on bacteria, yeasts, and cells propagated for production of biological compounds.

The response of bacteria, yeast, and mammalian and insects cells to oxidative stress is a topic that has been studied for many years. However, in most the reported studies, the oxidative stress was caused by challenging the organisms with H2O2 and redox-cycling drugs, but not by subjecting the cells to high concentrations of molecular oxygen. In this review we summarize available information about the effect of elevated oxygen concentrations on the physiology of microorganisms and cells at various culture conditions. In general, increased oxygen concentrations promote higher leakage of reactive oxygen species (superoxide and H2O2) from the respiratory chain affecting metalloenzymes and DNA that in turn cause impaired growth and elevated mutagenesis. To prevent the potential damage, the microorganisms and cells respond by activating antioxidant defenses and repair systems. This review described the factors that affect growth properties and metabolism at elevated oxygen concentrations that cells may be exposed to, in bioreactor sparged with oxygen enriched air which could affect the yield and quality of the recombinant proteins produced by high cell density schemes.

Production of recombinant protein by a novel oxygen-induced system in Escherichia coli.

BACKGROUND: The SoxRS regulon of E. coli is activated in response to elevated dissolved oxygen concentration likely to protect the bacteria from possible oxygen damage. The soxS expression can be increased up to 16 fold, making it a possible candidate for recombinant protein expression. Compared with the existing induction approaches, oxygen induction is advantageous because it does not involve addition or depletion of growth factors or nutrients, addition of chemical inducers or temperature changes that can affect growth and metabolism of the producing bacteria. It also does not affect the composition of the growth medium simplifying the recovery and purification processes. RESULTS: The soxS promoter was cloned into the commercial pGFPmut3.1 plasmid creating pAB49, an expression vector that can be induced by increasing oxygen concentration. The efficiency and the regulatory properties of the soxS promoter were characterized by measuring the GFP expression when the culture dissolved oxygen concentration was increased from 30% to 300% air saturation. The expression level of recombinant GFP was proportional to the oxygen concentration, demonstrating that pAB49 is a controllable expression vector. A possible harmful effect of elevated oxygen concentration on the recombinant product was found to be negligible by determining the protein-carbonyl content and its specific fluorescence. By performing high density growth in modified LB medium, the cells were induced by increasing the oxygen concentration. After 3 hours at 300% air saturation, GFP fluorescence reached 109000 FU (494 mg of GFP/L), representing 3.4% of total protein, and the cell concentration reached 29.1 g/L (DW). CONCLUSIONS: Induction of recombinant protein expression by increasing the dissolved oxygen concentration was found to be a simple and efficient alternative expression strategy that excludes the use of chemical, nutrient or thermal inducers that have a potential negative effect on cell growth or the product recovery.

Escherichia coli avoids high dissolved oxygen stress by activation of SoxRS and manganese-superoxide dismutase.

BACKGROUND: High concentrations of reactive oxygen species (ROS) were reported to cause oxidative stress to E. coli cells associated with reduced or inhibited growth. The high ROS concentrations described in these reports were generated by exposing the bacteria to H2O2 and superoxide-generating chemicals which are non-physiological growth conditions. However, the effect of molecular oxygen on oxidative stress response has not been evaluated. Since the use of oxygen-enriched air is a common strategy to support high density growth of E. coli, it was important to investigate the effect of high dissolved oxygen concentrations on the physiology and growth of E. coli and the way it responds to oxidative stress. RESULTS: To determine the effect of elevated oxygen concentrations on the growth characteristics, specific gene expression and enzyme activity in E. coli, the parental and SOD-deficient strain were evaluated when the dissolved oxygen (dO2) level was increased from 30% to 300%. No significant differences in the growth parameters were observed in the parental strain except for a temporary decrease of the respiration and acetate accumulation profile. By performing transcriptional analysis, it was determined that the parental strain responded to the oxidative stress by activating the SoxRS regulon. However, following the dO2 switch, the SOD-deficient strain activated both the SoxRS and OxyR regulons but it was unable to resume its initial growth rate. CONCLUSION: The transcriptional analysis and enzyme activity results indicated that when E. coli is exposed to dO2 shift, the superoxide stress regulator SoxRS is activated and causes the stimulation of the superoxide dismutase system. This enables the E. coli to protect itself from the poisoning effects of oxygen. The OxyR protecting system was not activated, indicating that H2O2 did not increase to stressing levels.

Simulation of dissolved CO(2) gradients in a scale-down system: a metabolic and transcriptional study of recombinant Escherichia coli.

Deficient mixing in industrial-scale bioreactors is an important concern as it results in a heterogeneous environment that may affect microbial cell physiology. Dissolved carbon dioxide (dCO(2) ) fluctuations, which can occur in large-scale bioreactors, were simulated for the first time and their effects were evaluated on Escherichia coli expressing recombinant green fluorescent protein (GFP). The dCO(2) gradients were simulated by continuously circulating the medium between two vessels of a scale-down system to mimic mean circulation times (t(c) ) of 50, 170, and 375 s. Specific growth rate (µ) decreased by 11% and acetate concentration increased by 23% at the highest t(c) compared to reference cultures. An increase in the time needed for attaining maximum GFP concentration was also observed. The effect of dCO(2) fluctuations on the transcriptional levels of genes involved in the glutamate decarboxylase (gadA and gadC) and α-ketoglutarate dehydrogenase (sucA and sucB) were analyzed by quantitative real time PCR. Such genes are known to be highly over- or underexpressed at elevated constant dCO(2) . Expression levels of gadA and gadC increased up to 60% and 72%, and sucA decreased 1.8-fold in the culture performed at the highest t(c) . Only a minor decrease of sucB expression was observed at t(c) of 170 and 375 s. Although exposure to continuous high dCO(2) can affect culture performance, in this work it was shown that E. coli is able to rescue its metabolism in very short times when cells are intermittently returned to low dCO(2) conditions after being exposed to high dCO(2) .

High-flux isobutanol production using engineered Escherichia coli: a bioreactor study with in situ product removal.

Promising approaches to produce higher alcohols, e.g., isobutanol, using Escherichia coli have been developed with successful results. Here, we translated the isobutanol process from shake flasks to a 1-L bioreactor in order to characterize three E. coli strains. With in situ isobutanol removal from the bioreactor using gas stripping, the engineered E. coli strain (JCL260) produced more than 50 g/L in 72 h. In addition, the isobutanol production by the parental strain (JCL16) and the high isobutanol-tolerant mutant (SA481) were compared with JCL260. Interestingly, we found that the isobutanol-tolerant strain in fact produced worse than either JCL16 or JCL260. This result suggests that in situ product removal can properly overcome isobutanol toxicity in E. coli cultures. The isobutanol productivity was approximately twofold and the titer was 9% higher than n-butanol produced by Clostridium in a similar integrated system.

Driving forces enable high-titer anaerobic 1-butanol synthesis in Escherichia coli.

1-Butanol, an important chemical feedstock and advanced biofuel, is produced by Clostridium species. Various efforts have been made to transfer the clostridial 1-butanol pathway into other microorganisms. However, in contrast to similar compounds, only limited titers of 1-butanol were attained. In this work, we constructed a modified clostridial 1-butanol pathway in Escherichia coli to provide an irreversible reaction catalyzed by trans-enoyl-coenzyme A (CoA) reductase (Ter) and created NADH and acetyl-CoA driving forces to direct the flux. We achieved high-titer (30 g/liter) and high-yield (70 to 88% of the theoretical) production of 1-butanol anaerobically, comparable to or exceeding the levels demonstrated by native producers. Without the NADH and acetyl-CoA driving forces, the Ter reaction alone only achieved about 1/10 the level of production. The engineered host platform also enables the selection of essential enzymes with better catalytic efficiency or expression by anaerobic growth rescue. These results demonstrate the importance of driving forces in the efficient production of nonnative products.

Metabolic and transcriptional response of recombinant Escherichia coli to elevated dissolved carbon dioxide concentrations.

The effect of dissolved carbon dioxide (dCO(2)) concentration on the stoichiometric and kinetic constants and by-product accumulation was determined for Escherichia coli cells producing recombinant green fluorescent protein (GFP). Constant dCO(2), in the range of 20-300 mbar, was maintained during batch cultures by manipulating the inlet gas composition. As dCO(2) increased, specific growth rate (micro) decreased, and acetate accumulation and the time for onset of GFP production increased. Maximum biomass yield on glucose and GFP concentration were affected for dCO(2) above 70 and 150 mbar, respectively. Expression analysis of 16 representative genes showed that E. coli can respond at the transcriptional level upon exposure to increasing dCO(2), and revealed possible mechanisms responsible for the detrimental effects of high dCO(2). Genes studied included those involved in decarboxylation reactions (aceF, icdA, lpdA, sucA, sucB), genes from pathways of production and consumption of acetate (ackA, poxB, acs, aceA, fadR), genes from gluconeogenic and anaplerotic metabolism (pckA, ppc), genes from the acid resistance (AR) systems (adiA, gadA, gadC), and the heterologous gene (gfp). The transcription levels of tricarboxylic acid (TCA) cycle genes (icdA, sucA, sucB) and glyoxylate shunt (aceA) decreased as dCO(2) increased, whereas fadR (that codes for a negative regulator of the glyoxylate operon) and poxB (that codes for PoxB which is involved in acetate production from pyruvate) were up-regulated as dCO(2) increased up to 150 mbar. Furthermore, transcription levels of genes from the AR systems increased as dCO(2) increased up to 150 mbar, indicating that elevated dCO(2) triggers an acid stress response in E. coli cells. Altogether, such results suggest that the increased acetate accumulation and reduction in mu, biomass yield and maximum GFP concentration under high dCO(2) resulted from a lower carbon flux to TCA cycle, the concomitant accumulation of acetyl-CoA or pyruvate, and the acidification of the cytoplasm.