Stoichiometry of the H+-ATPase of Escherichia coli cells during anaerobic growth

The H+/ATP stoichiometry of the H+-ATPase was investigated in Escherichia coli cells growing under anaerobic conditions at pH 6 and 7. The protonmotive force was determined from the intracellular accumulation of benzoate and tetraphenylphosphonium ions, as well as the accumulation of lactose in this lac operon inducible, but beta-galactosidase negative strain. The phosphorylation potential was calculated from the cellular concentrations of ATP, ADP and inorganic phosphate. By comparing the phosphorylation potential and the proton motive force under these steady state conditions, the H+/ATP stoichiometry was determined to be 3, similar to the value previously found in the same cells growing under aerobic conditions.     

H2-dependent anaerobic growth of Escherichia coli on L-malate: succinate formation.

Escherichia coli grew anaerobically on L-malate only in the presence of H2; 91% of the L-malate utilized was converted to succinate. Anaerobically isolated membrane vesicles catalyzed the reduction of fumarate with H2 and contained a b-type cytochrome. Cytochrome c552 was present in the "periplasmic space."     

Cell yields of Escherichia coli during anaerobic growth on fumarate and molecular hydrogen

Escherichia coli was grown anaerobically on sodium fumarate and molecular hydrogen or sodium formate in continuous culture. The maximal growth yield and the maintenance coefficient were determined. In a mineral medium a Y _fum ^max value of 6.6 g dry weight per mol fumarate was found. This value increased to 7.5 when casamino acids were present in the medium. From these data and the corresponding Y _ATP ^max values it could be calculated that per mol of fumarate reduced, 0.4 mol of ATP became available for growth. In batch culture a Y_fum value of 4.8 g dry weight per mol fumarate was determined.     

Amplification of sex repressor function of one fi-+ R-factor during anaerobic growth of Escherichia coli.

It has been reported by Mitsuhashi (1965) that transfer of one R-factor was completely inhibited by anaerobic transfer conditions. In contrast, several workers have observed R-factor transfer, although at a reduced rate, in the animal intestines, a largely anaerobic environment. It is shown here that in vitro transfer of the R-factor R1 (F-type pilus, fi-+) in Escherichia coli K-12 is severely depressed, whereas transfer of R64 (I-type pilus, fi-minus) is slightly stimulated by anaerobiosis. Inhibition of R1 fertility is dependent on anaerobic conditions during pregrowth of the donor cells, whereas the oxygen tension during recipient pregrowth, transfer, and plating is of little importance. Anaerobic pregrowth has a less inhibitory effect on the fertility of R1drd19, a mutant of R1 having a defective sex repressor. The fi-+ property of R1 when introduced into F' or Hfr bacteria is amplified during anaerobic growth. These observations strongly indicate that the sex repressor is the mediator of the anaerobic fertility inhibition of the R-factor R1. This hypothesis was supported by studies of the formation of sex pili, the only gene product identified that is controlled by the sex repressor of R1. Using propagation of the F-type pilus-specific phage MS2 as a measure of the degree of sex piliation of a bacterial population, it is shown that in anaerobic cultures sex piliation due to R1 is strongly repressed, whereas piliation due to R1drd19 is repressed to a lesser extent. The possible survival value of the response of R1 towards oxygen tension is discussed.     

pH-dependent catabolic protein expression during anaerobic growth of Escherichia coli K-12

During aerobic growth of Escherichia coli, expression of catabolic enzymes and envelope and periplasmic proteins is regulated by pH. Additional modes of pH regulation were revealed under anaerobiosis. E. coli K-12 strain W3110 was cultured anaerobically in broth medium buffered at pH 5.5 or 8.5 for protein identification on proteomic two-dimensional gels. A total of 32 proteins from anaerobic cultures show pH-dependent expression, and only four of these proteins (DsbA, TnaA, GatY, and HdeA) showed pH regulation in aerated cultures. The levels of 19 proteins were elevated at the high pH; these proteins included metabolic enzymes (DhaKLM, GapA, TnaA, HisC, and HisD), periplasmic proteins (ProX, OppA, DegQ, MalB, and MglB), and stress proteins (DsbA, Tig, and UspA). High-pH induction of the glycolytic enzymes DhaKLM and GapA suggested that there was increased fermentation to acids, which helped neutralize alkalinity. Reporter lac fusion constructs showed base induction of sdaA encoding serine deaminase under anaerobiosis; in addition, the glutamate decarboxylase genes gadA and gadB were induced at the high pH anaerobically but not with aeration. This result is consistent with the hypothesis that there is a connection between the gad system and GabT metabolism of 4-aminobutanoate. On the other hand, 13 other proteins were induced by acid; these proteins included metabolic enzymes (GatY and AckA), periplasmic proteins (TolC, HdeA, and OmpA), and redox enzymes (GuaB, HmpA, and Lpd). The acid induction of NikA (nickel transporter) is of interest because E. coli requires nickel for anaerobic fermentation. The position of the NikA spot coincided with the position of a small unidentified spot whose induction in aerobic cultures was reported previously; thus, NikA appeared to be induced slightly by acid during aeration but showed stronger induction under anaerobic conditions. Overall, anaerobic growth revealed several more pH-regulated proteins; in particular, anaerobiosis enabled induction of several additional catabolic enzymes and sugar transporters at the high pH, at which production of fermentation acids may be advantageous for the cell.     

Mutations permitting the anaerobic growth of Escherichia coli on trehalose

Wild-type strains of Escherichia coli K-12 do not grow anaerobically on trehalose or galactose. We isolated two operon fusion mutants of E. coli which gained the ability to grow on trehalose anaerobically (tan). The tanA-lac mutation was located at 41 min on the E. coli genetic map and also abolished growth on glucuronic acid both aerobically and anaerobically. The tanB-lac mutation was mapped to 68 min and permitted anaerobic growth on galactose as well as trehalose. The tanB-lac fusion was induced anaerobically whereas tanA-lac showed more or less constitutive beta-galactosidase expression.     

Effect of diethylsulphoxide on growth,survival and ion exchange of Escherichia coli

AIMS: To study the effect of diethylsulphoxide (DESO) on Escherichia coli growth, survival and ionic exchange in comparison with dimethylsulphoxide (DMSO). METHODS AND RESULTS: Bacterial survival was estimated by counting colony-forming units and by the most probable number (five-tube) technique; the K+ and H+ transport and H(2) formation were determined electrochemically. Diethylsulphoxide at concentrations between 0.01 and 0.5% (w/v) stimulated and above 5% decreased the anaerobic growth rate and survival. 2H+ : K+ exchange and H(2) formation were lost at 5% DESO. At 0.05% DESO the kinetic characteristics of H+ : K+ exchange and H(2) formation were typical for Delta micro (H(+)) -dependent TrkA uncoupled with F(0)F(1) under respiration. CONCLUSIONS: Diethylsulphoxide at low concentrations serves as an electron acceptor for an anaerobic respiratory chain stimulating bacterial growth and survival through the modulation of H+ : K+ exchange and H(2) formation activity. The effects of DESO were more pronounced than those of DMSO. SIGNIFICANCE AND IMPACT OF THE STUDY: Diethylsulphoxide determines essential biological and therapeutic properties that make its application preferable.     

Electroneutral H+-K+ exchange in liver mitochondria. Regulation by membrane potential

The paper analyzes the factors affecting the H+-K+ exchange catalyzed by rat liver mitochondria depleted of endogenous Mg2+ by treatment with the ionophore A23187. The exchange has been monitored as the rate of K+ efflux following addition of A23187 in low-K+ media. (1) The H+-K+ exchange is abolished by uncouplers and respiratory inhibitors. The inhibition is not related to the depression of delta pH, whereas a dependence is found on the magnitude of the transmembrane electrical potential, delta psi. Maximal rate of K+ efflux is observed at 180-190 mV, whereas K+ efflux is inhibited below 140-150 mV. (2) Activation of H+-K+ exchange leads to depression of delta pH but not of delta psi. Respiration is only slightly stimulated by the onset of H+-K+ exchange in the absence of valinomycin. These findings indicate that the exchange is electroneutral, and that the delta psi control presumably involves conformational changes of the carrier. (3) Incubation in hypotonic media at pH 7.4 or in isotonic media at alkaline pH results in a marked activation of the rate of H+-K+ exchange, while leaving unaffected the level of Mg2+ depletion. This type of activation results in partial 'uncoupling' from the delta psi control, suggesting that membrane stretching and alkaline pH induce conformational changes on the exchange carrier equivalent to those induced by high delta psi. (4) The available evidence suggests that the activity of the H+-K+ exchanger is modulated by the electrical field across the inner mitochondrial membrane.     

The effect of cyclic AMP on anaerobic growth of Escherichia coli

Adenosine 3′,5′-cyclic phosphate (cyclic AMP) stimulated a cyclic AMP-deficient mutant strain of $\text{Escherichia coli}$ to grow anaerobically on glucose in a minimal medium and in media supplemented with nitrate or casein hydrolysate. Cyclic AMP was found to stimulate the production of the formic hydrogenlyase system in this mutant strain, but had no effect on its ability to carry out anaerobic reductions of nitrate or nitrite. It was also observed that CO₂ stimulated the anaerobic growth of the mutant in the absence of cyclic AMP.     

Expression of R-plasmid functions during anaerobic growth of an Escherichia coli K-12 host.

The normal habitat of enteric bacteria is largely anaerobic. Expression of the three characteristic properties of R-plasmids, drug resistance, vegetative replication, and fertility, was therefore studied in Escherichia coli K-12 during anaerobiosis. Replication and drug resistance functions were not altered in the 45 R-plasmids studies, whereas the expression of fertility varied considerably among different R-plasmids during anaerobiosis. The R-plasmids could be divided into three groups, one showing a strong, the second a moderate, and the third little or no reduction of fertility by anaerobiosis. Plasmid-determined sensitivity to F-, N-, and I-specific phage, respectively, was well, although not absolutely, correlated with each of three groups mentioned. Anaerobiosis-aerobiosis appears to change the fertility of type F R-plasmids by influencing the degree of repression of their fertility functions such as the formation of sex pili. Although the minimum inhibitory concentrations of ampicillin, chloramphenicol, streptomycin, and tetracycline were unaltered by anaerobiosis, sulfonamide was found to be four- to eightfold less active under this condition in both resistant and sensitive strains. A surprisingly high frequency and uniformity of minimal inhibitory concentrations was observed for R-plasmid-mediated resistance to streptomycin and chloramphenicol.     

Effect of acetate on the growth of Escherichia coli during aerobiosis and anaerobiosis

The dynamics of acetate accumulation was studied in Escherichia coli K-12 batch cultures with a substrate addition. At pH 7.0, the growth stopped at 150 mmoles of acetate per litre of the medium under the aerobic conditions or at 35 mmoles of acetate per litre of the medium under the anaerobic conditions. Experiments with extraneous addition of acetate suggest that acetic acid plays a key role in inhibiting the growth of E. coli by acid metabolites. The authors propose a hypothetical mechanism to account for the inhibiting action of acetate.     

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.