Succinate production in dual-phase Escherichia coli fermentations depends on the time of transition from aerobic to anaerobic conditions

We examined succinic acid production in Escherichia coli AFP111 using dual-phase fermentations, which comprise an initial aerobic growth phase followed by an anaerobic production phase. AFP111 has mutations in the pfl, ldhA, and ptsG genes, and we additionally transformed this strain with the pyc gene (AFP111/pTrc99A-pyc) to provide metabolic flexibility at the pyruvate node. Aerobic fermentations with these two strains were completed to catalog physiological states during aerobic growth that might influence succinate generation in the anaerobic phase. Activities of six key enzymes were also determined for these aerobic fermentations. From these results, six transition times based on physiological states were selected for studying dual-phase fermentations. The final succinate yield and productivity depend greatly on the physiological state of the cells at the time of transition. Using the best transition time, fermentations achieved a final succinic acid concentration of 99.2 g/l with an overall yield of 110% and productivity of 1.3 g/l h. Journal of Industrial Microbiology & Biotechnology (2002) 28, 325–332 DOI: 10.1038/sj/jim/7000250 Received 01 October 2001/ Accepted in revised form 12 March 2002     

Divergent roles of RpoS in Escherichia coli under aerobic and anaerobic conditions

Escherichia coli exhibited different levels of rpoS expression and general stress resistance under aerobiosis and anaerobiosis. Expression measured using reporter gene fusions and protein levels was lower under anaerobic conditions. Consistent with earlier findings, rpoS mutants were selected in aerobic nutrient-limited cultures but rpoS mutants were not enriched under anaerobiosis. This result suggested that, despite its decreased level, RpoS had a function under anaerobic conditions not essential under aerobiosis. Competition experiments between rpoS(+) and rpoS bacteria confirmed the advantage conferred by RpoS under anaerobiosis. In contrast, stress resistance assays suggested RpoS made a greater contribution to general stress resistance under aerobiosis than anaerobiosis. These results indicate a significant, but different role of RpoS in aerobic and anaerobic environments.     

Changes in the pool of polyamines [correction of polyvitamins] during transition from anaerobic to aerobic conditions and localization of enzymes for their synthesis in Escherichia coli cells

The content of intra- and extracellular polyamines and the activity of enzymes mediating their synthesis change depending on the regime of cell aeration. The pool of putrescine rises abruptly upon the transition from anaerobic to aerobic conditions owing to its liberation from the bound state as well as due to an increase in the activity of ornithine decarboxylase; as a result, the structural-functional organisation of membranes is restored. The free pool of cadaverine appears because, presumably, its binding to membranes is upset and the activity of lysine decarboxylase rises. The localisation of the enzymes for polyamine synthesis in the cell seems to be determined by the specific action of their products on particular cellular structures and metabolic processes in Escherichia coli cells.     

Reductive dechlorination of Tri- and tetrachloroethylenes depends on transition from aerobic to anaerobic conditions

Aerobic enrichment cultures from contaminated groundwaters dechlorinated trichloroethylene (TCE) (14.6 mg/liter; 111 mumol/liter) and tetrachloroethylene (PCE) (16.2 mg/liter; 98 mumol/liter) reductively within 4 days after the transition from aerobic to anaerobic conditions. The transformation products were equimolar amounts of cis-1,2-dichloroethylene and traces of 1,1-dichloroethylene. No other chlorinated product and no methane were detected. The change was accompanied by the release of sulfide, which caused a decrease in the redox potential from 0 to -150 mV. In sterile control experiments, sulfide led to the abiotic formation of traces of 1,1-dichloroethylene without cis-1,2-dichloroethylene production. The reductive dechlorination of PCE via TCE depended on these specific transition conditions after consumption of the electron acceptor oxygen or nitrate. Repeated feeding of TCE or PCE to cultures after the change to anaerobic conditions yielded no further dechlorination. Only aerobic subcultures with an air/liquid ratio of 1:4 maintained dechlorination activities; anaerobic subcultures showed no transformation. Bacteria from noncontaminated sites showed no reduction under the same conditions.     

Reductive dechlorination of Tri- and tetrachloroethylenes depends on transition from aerobic to anaerobic conditions.

Aerobic enrichment cultures from contaminated groundwaters dechlorinated trichloroethylene (TCE) (14.6 mg/liter; 111 mumol/liter) and tetrachloroethylene (PCE) (16.2 mg/liter; 98 mumol/liter) reductively within 4 days after the transition from aerobic to anaerobic conditions. The transformation products were equimolar amounts of cis-1,2-dichloroethylene and traces of 1,1-dichloroethylene. No other chlorinated product and no methane were detected. The change was accompanied by the release of sulfide, which caused a decrease in the redox potential from 0 to -150 mV. In sterile control experiments, sulfide led to the abiotic formation of traces of 1,1-dichloroethylene without cis-1,2-dichloroethylene production. The reductive dechlorination of PCE via TCE depended on these specific transition conditions after consumption of the electron acceptor oxygen or nitrate. Repeated feeding of TCE or PCE to cultures after the change to anaerobic conditions yielded no further dechlorination. Only aerobic subcultures with an air/liquid ratio of 1:4 maintained dechlorination activities; anaerobic subcultures showed no transformation. Bacteria from noncontaminated sites showed no reduction under the same conditions.     

Inactivation of Mg chelatase during transition from anaerobic to aerobic growth in Rhodobacter capsulatus

The facultative photosynthetic bacterium Rhodobacter capsulatus can adapt from an anaerobic photosynthetic mode of growth to aerobic heterotrophic metabolism. As this adaptation occurs, the cells must rapidly halt bacteriochlorophyll synthesis to prevent phototoxic tetrapyrroles from accumulating, while still allowing heme synthesis to continue. A likely control point is Mg chelatase, the enzyme that diverts protoporphyrin IX from heme biosynthesis toward the bacteriochlorophyll biosynthetic pathway by inserting Mg(2+) to form Mg-protoporphyrin IX. Mg chelatase is composed of three subunits that are encoded by the bchI, bchD, and bchH genes in R. capsulatus. We report that BchH is the rate-limiting component of Mg chelatase activity in cell extracts. BchH binds protoporphyrin IX, and BchH that has been expressed and purified from Escherichia coli is red in color due to the bound protoporphyrin IX. Recombinant BchH is rapidly inactivated by light in the presence of O(2), and the inactivation results in the formation of a covalent adduct between the protein and the bound protoporphyrin IX. When photosynthetically growing R. capsulatus cells are transferred to aerobic conditions, Mg chelatase is rapidly inactivated, and BchH is the component that is most rapidly inactivated in vivo when cells are exposed to aerobic conditions. The light- and O(2)-stimulated inactivation of BchH could account for the rapid inactivation of Mg chelatase in vivo and provide a mechanism for inhibiting the synthesis of bacteriochlorophyll during adaptation of photosynthetically grown cells to aerobic conditions while still allowing heme synthesis to occur for aerobic respiration.     

Are the aerobic and anaerobic phosphofructokinases of Escherichia coli different?

Phosphofructokinase has been purified from Escherichia coli strain K-12 grown in a glucose-limited chemostat, both aerobically and anaerobically. The enzymes migrated together in polyacrylamide gel electrophoresis, had the same subunit size in denaturing (dodecylsulfate) gels (Mr approx. 34000) and the same kinetic characteristics as described earlier for E. coli phosphofructokinase [e.g. Blangy et al. (1968) J. Mol. Biol. 31, 13-35]: a sigmoid curve of velocity vs. fructose 6-phosphate concentration, activation by ADP, and inhibition by phosphoenolpyruvate. Findings [e.g. Doelle (1975) Eur. J. Biochem, 50, 335-342] of quite different enzymes in aerobic and anaerobic cells were not confirmed.     

On culturing Escherichia coli on a mineral salts medium during anaerobic conditions

The substrate and products of the hydrogenlyase complex, formic acid, carbon dioxide, and molecular hydrogen, are co-operatively implicated in maintaining growth of E. coli under anaerobic conditions. Growth is observed in the presence of a combination of carbon dioxide + molecular hydrogen, or carbon dioxide + formic acid in the medium. The study shows that it is possible to culture E. coli under anaerobic conditions while sparging with nitrogen, without supplementing exogenous carbon dioxide, formic acid or molecular hydrogen. This condition occurs when the strain is allowed an appropriate induction period and is present at a sufficiently high cell density, since the cell density affects the rate of e.g. CO₂ production. In a system sparged with nitrogen gas, the removal of CO₂ due to this sparging must be balanced with a cell density dependent production rate of CO₂. It is concluded that the formic hydrogenlyase complex should be considered as an integral part of the general maintenance of the anabolism of E. coli during anaerobic conditions on a mineral salts medium, as well as being a net producer of end products in E. coli metabolism.This work was supported by the Swedish National Board for Industrial and Technical Development. A. Askendahl is acknowledged for valuable assistance in the preparation of figures and P. Warkentin for language editing.     

n-Chloroaniline transformation by Escherichia coli under anaerobic conditions

In a medium without oxygen in the presence of nitrates, E. coli transforms p-chloranilin (p-CA) to yield a more hydrophilic compound which cannot be extracted with an organic solvent from water. The conditions for consecutive transformation of p-nitro-chlorobenzene (p-NCB) and p-CA have been determined: the reaction p-NCB leads to p-CA is inhibited by nitrates, p-CA transformation occurs in the presence of nitrates in the medium and depends on their concentration.     

Differential effect of YidC depletion on the membrane proteome of Escherichia coli under aerobic and anaerobic growth conditions

YidC of Escherichia coli belongs to the evolutionarily conserved Oxa1/Alb3/YidC family. Members of the family have all been implicated in membrane protein biogenesis of respiratory and energy transducing proteins. The number of proteins identified thus far to require YidC for their membrane biogenesis remains limited and the identification of new substrates may allow the elucidation of properties that define the YidC specificity. To this end we investigated changes in the membrane proteome of E. coli upon YidC depletion using metabolic labeling of proteins with ¹⁵N/¹⁴N combined with a MS‐centered proteomics approach and compared the effects of YidC depletion under aerobic and anaerobic growth conditions. We found that YidC depletion resulted in protein aggregation/misfolding in the cytoplasm as well as in the inner membrane of E. coli. A dramatic increase was observed in the chaperone‐mediated stress response upon YidC depletion and this response was limited to aerobically grown cells. A number of transporter proteins were identified as possible candidates for the YidC‐dependent insertion and/or folding pathway. These included the small metal ion transporter CorA, numerous ABC transporters, as well as the MFS transporters KgtP and ProP, providing a new subset of proteins potentially requiring YidC for membrane biogenesis.     

Differential sensitivities of the growth of Escherichia coli to acrylate under aerobic and anaerobic conditions and its effect on product formation

The effect of acrylate on the growth of Escherichia coli was determined under aerobic and anaerobic conditions in glucose-defined medium. Growth occurred with up to 35 mM acrylate under aerobic conditions but ceased at 5 mM acrylate under anaerobic conditions. This differential sensitivity can be attributed to inhibition of pyruvate formate lyase and/or pflB gene repression, as this enzyme is necessary for anaerobic growth of E. coli. The effect of acrylate on end-product distribution was also determined by growing E. coli first aerobically, then switching to anaerobic conditions. In the absence of acrylate, E. coli generated the typical distribution of mixed-acid products, with about 12 % of pyruvate being metabolically converted to lactate. In contrast, in the presence of 5 mM acrylate, E. coli converted 83 % of pyruvate to lactate, consistent with a reduction in pyruvate formate lyase activity.     

Role of fatty acids in the transition from anaerobic to aerobic metabolism in skeletal muscle during exercise

In moderate physical exercise, the transition from predominantly anaerobic towards predominantly aerobic metabolism is a key step to improve performance. Increase in the supply of oxygen and nutrients, such as free fatty acids (FFA) and glucose, which accompanies high blood flow, is required for this transition. The mechanisms involved in the vasodilation in skeletal muscle during physical activity are not completely known yet. In this article, we postulate a role of FFA and heat production in this process. The presence of uncoupling protein-2 and -3 (UCP-2 and -3) in skeletal muscle, whose activity is dependent on FFA, suggests that these metabolites can act as mitochondrial uncouplers in this tissue. Evidence indicates however that UCPs act as uncouplers only when coenzyme Q is predominantly in the reduced state (i.e. under nonphosphorylation conditions or state 4 respiration) as is observed in resting muscles and in the beginning of physical activity (predominantly anaerobic metabolism). The increase in the lipolytic activity in adipose tissue in the beginning of physical activity results in elevated plasma FFA levels. The FFA can then act on the UCPs, increasing the local heat production. We propose that this calorigenic effect of FFA is important to activate nitric oxide synthase, resulting in nitric oxide production and consequent vasodilation. Therefore, FFA would be important mediators for the changes that occur in muscle metabolism during prolonged physical activity, ensuring the appropriate supply of oxygen and nutrients by increasing blood flow at the beginning of exercise in the contracting skeletal muscles.