The number of accessible SH-groups in Escherichia coli membrane vesicles is increased by ATP or by formate

The number of accessible SH-groups was determined in membrane vesicles prepared from Escherichia coli growing in fermentation conditions at slightly alkaline pH on glucose with or without added formate. Addition of ATP or formate to the vesicles caused a approximately 1.4-fold increase in the number of accessible SH-groups. The increase was inhibited by treatment with N-ethylmaleimide or the presence of the F(0)F(1)-ATPase inhibitors N,N(')-dicyclohexylcarbodiimide or sodium azide. The increase in accessible SH-groups was also absent in strains with the ATP synthase operon deleted or with the single F(0) domain cysteine Cysb21 changed to Ala. Using hyc and hyf mutants, it was shown that the increase was also largely dependent on hydrogenase 4 or hydrogenase 3, main components of formate hydrogen lyase, when bacteria were grown in the absence or presence of added formate. These results suggest a relationship between the F(0)F(1)-ATP synthase and hydrogenase 4 or hydrogenase 3 under fermentation conditions.     

Escherichia coli mutants tht require either pyridoxine or alanine

Some pyridoxineless mutants of genetic group V grow to normal cell yields in glucose-salts medium containing 0.11 mm d- or l-alanine as the sole supplement.     

Anaerobic iron uptake by Escherichia coli.

Assimilation and uptake of iron in anaerobic cultures of Escherichia coli were supported by iron supplied as ferrienterobactin, ferrichrome, and ferrous ascorbate; however, as in the aerobic cultures, ferrichrome A was a poor iron source. Albomycin inhibited both aerobically and anaerobically grown cells. The siderophore outer membrane receptor proteins FepA and FhuA were produced under anaerobic iron-deficient conditions. Anaerobic transport of ferrienterobactin and ferrichrome was inhibited by KCN and dinitrophenol. The Km for ferrienterobactin uptake in anaerobically grown cells was 0.8 microM, and the Vmax was 38 pmol/min per mg, compared with 0.1 microM and 80 pmol/min per mg, respectively, in aerobically grown cells.     

L-asparagine uptake in Escherichia coli.

The uptake of L-asparagine by Escherichia coli K-12 is characterized by two kinetic components with apparent Km values of 3.5 muM and 80 muM. The 3.5 muM Km system displays a maximum velocity of 1.1 nmol/min per mg of protein, which is a low value when compared with derepressed levels of other amino acid transport systems but is relatively specific for L-asparagine. Compounds providing effective competition for L-asparagine uptake were 4-carbon analogues of the L-isomer with alterations at the beta-amide position, i.e., 5-diazo-4-oxo-L-norvaline (Ki = 4.6 muM), beta-hydroxyamyl-L-aspartic acid (Ki = 10 muM), and L-aspartic acid (Ki = 50 muM). Asparagine uptake is energy dependent and is inhibited by a number of metabolic inhibitors. In a derived strain of E. coli deficient in cytoplasmic asparaginase activity asparagine can be accumulated several-fold above the apparent biosynthetic pool of the amino acid and 100-fold above the external medium. The high affinity system is repressed by culture of cells with L-asparagine supplements in excess of 1 mM and is suggested to be necessary for growth of E. coli asparagine auxotrophs with lower supplement concentrations.     

ATP synthesis driven by a potassium diffusion potential in Methanobacterium thermoautotrophicum is stimulated by sodium

Abstract ATP synthesis driven by a potassium diffusion potential was studied in cell suspensions of Methanobacterium thermoautotrophicum (Marburg). This transient increase in the intracellular ATP content was stimulated five-fold by the addition of sodium ions, from about 2 nmol ATP/min × mg cells (dry weight) at 0.07 mM Na⁺ to about 10 nmol ATP/min × mg cells at 25 mM Na⁺.     

A conformational change in the lactose permease of Escherichia coli is induced by ligand binding or membrane potential.

Lactose transport in membrane vesicles containing lactose permease with a single Cys residue in place of Val 315 is inactivated by N-ethylmaleimide in a manner that is stimulated by substrate or by a H+ electrochemical gradient (delta microH+; Sahin-Tóth M, Kaback HR, 1993, Protein Sci 2:1024-1033). The findings are confirmed and extended in this communication. Purified, reconstituted Val 315-->Cys permease reacts with N-ethylmaleimide or hydrophobic fluorescent maleimides but not with a membrane impermeant thiol reagent, and beta-galactosides specifically stimulate the rate of labeling. Furthermore, the reactivity of purified Val 315-->Cys permease is enhanced by imposition of a membrane potential (delta psi, interior negative). The results indicate that either ligand binding or delta psi induces a conformational change in the permease that brings the N-terminus of helix X into an environment that is more accessible from the lipid phase.     

The glycolytic flux in Escherichia coli is controlled by the demand for ATP

The nature of the control of glycolytic flux is one of the central, as-yet-uncharacterized issues in cellular metabolism. We developed a molecular genetic tool that specifically induces ATP hydrolysis in living cells without interfering with other aspects of metabolism. Genes encoding the F(1) part of the membrane-bound (F(1)F(0)) H(+)-ATP synthase were expressed in steadily growing Escherichia coli cells, which lowered the intracellular [ATP]/[ADP] ratio. This resulted in a strong stimulation of the specific glycolytic flux concomitant with a smaller decrease in the growth rate of the cells. By optimizing additional ATP hydrolysis, we increased the flux through glycolysis to 1.7 times that of the wild-type flux. The results demonstrate why attempts in the past to increase the glycolytic flux through overexpression of glycolytic enzymes have been unsuccessful: the majority of flux control (>75%) resides not inside but outside the pathway, i.e., with the enzymes that hydrolyze ATP. These data further allowed us to answer the question of whether catabolic or anabolic reactions control the growth of E. coli. We show that the majority of the control of growth rate resides in the anabolic reactions, i.e., the cells are mostly "carbon" limited. Ways to increase the efficiency and productivity of industrial fermentation processes are discussed.     

Natural DNA uptake by Escherichia coli

Escherichia coli has homologues of the competence genes other species use for DNA uptake and processing, but natural competence and transformation have never been detected. Although we previously showed that these genes are induced by the competence regulator Sxy as in other gamma-proteobacteria, no conditions are known that naturally induce sxy expression. We have now tested whether the competence gene homologues encode a functional DNA uptake machinery and whether DNA uptake leads to recombination, by investigating the effects of plasmid-borne sxy expression on natural competence in a wide variety of E. coli strains. High- and low-level sxy expression alone did not induce transformation in any of the strains tested, despite varying the transforming DNA, its concentration, and the incubation conditions used. Direct measurements of uptake of radiolabelled DNA were below the limit of detection, however transformants were readily detected when recombination functions were provided by the lambda Red recombinase. This is the first demonstration that E. coli sxy expression can induce natural DNA uptake and that E. coli's competence genes do encode a functional uptake machinery. However, the amount of transformation cells undergo is limited both by low levels of DNA uptake and by inefficient DNA processing/recombination.     

Sucrose uptake is driven by the Na+ electrochemical potential in the marine bacterium Vibrio alginolyticus.

Na+ was found to be essential for the accumulation of sucrose by Vibrio alginolyticus. Sucrose uptake was completely inhibited by the addition of proton conductor at neutral pH, but not at alkaline pH, where the primary electrogenic Na+ pump generates the Na+ electrochemical gradient. We therefore conclude that sucrose transport is driven by the electrochemical potential of Na+ in this organism.     

ATP hydrolysis during SOS induction in Escherichia coli.

Changes in cellular ATP concentration during SOS induction in strains of Escherichia coli with different levels of RecA and LexA proteins were studied. UV irradiation of RecA+ strains induced a twofold increase in the ATP concentration around the first 20 min, followed by a decrease to the values of nonirradiated cells. On the other hand, mutants defective in RecA protein or with either deficient RecA protease activity or cleavage-resistant LexA repressor did not show any decrease, suggesting that ATP consumption is related to LexA repressor hydrolysis. Furthermore, strains presenting a constitutive synthesis of RecA protein showed the same changes in ATP concentration as the wild-type strain. Likewise, the presence in a RecA+ strain of a LexA(Def) protein, which is defective in its capacity for binding specifically to SOS operators, did not disturb the changes in ATP when compared with the LexA+ RecA+ strain. Moreover, after UV irradiation, a LexA(Def) RecA- double mutant showed an important increase in ATP concentration, which remained elevated for at least 120 min after UV treatment.     

Cadmium uptake in Escherichia coli K-12.

109Cd2+ uptake by Escherichia coli occurred by means of an active transport system which has a Km of 2.1 microM Cd2+ and a Vmax of 0.83 mumol/min X g (dry weight) in uptake buffer. 109Cd2+ accumulation was both energy dependent and temperature sensitive. The addition of 20 microM Cd2+ or Zn2+ (but not Mn2+) to the cell suspensions preloaded with 109Cd2+ caused the exchange of Cd2+. 109Cd2+ (0.1 microM) uptake by cells was inhibited by the addition of 20 microM Zn2+ but not Mn2+. Zn2+ was a competitive inhibitor of 109Cd2+ uptake with an apparent Ki of 4.6 microM Zn2+. Although Mn2+ did not inhibit 109Cd2+ uptake, the addition of either 20 microM Cd2+ or Zn2+ prevented the uptake of 0.1 microM 54Mn2+, which apparently occurs by a separate transport system. The inhibition of 54Mn2+ accumulation by Cd2+ or Zn2+ did not follow Michaelis-Menten kinetics and had no defined Ki values. Co2+ was a competitive inhibitor of Mn2+ uptake with an apparent Ki of 34 microM Co2+. We were unable to demonstrate an active transport system for 65Zn2+ in E. coli.     

Regulation of Escherichia coli polynucleotide phosphorylase by ATP

Polynucleotide phosphorylase (PNPase), an enzyme conserved in bacteria and eukaryotic organelles, processively catalyzes the phosphorolysis of RNA, releasing nucleotide diphosphates, and the reverse polymerization reaction. In Escherichia coli, both reactions are implicated in RNA decay, as addition of either poly(A) or heteropolymeric tails targets RNA to degradation. PNPase may also be associated with the RNA degradosome, a heteromultimeric protein machine that can degrade highly structured RNA. Here, we report that ATP binds to PNPase and allosterically inhibits both its phosphorolytic and polymerization activities. Our data suggest that PNPase-dependent RNA tailing and degradation occur mainly at low ATP concentrations, whereas other enzymes may play a more significant role at high energy charge. These findings connect RNA turnover with the energy charge of the cell and highlight unforeseen metabolic roles of PNPase.     

Mutants of Escherichia coli with a growth requirement for either lysine or pyridoxine

Two distinct phenotypic classes of lysine requiring auxotrophs of Escherichia coli are described. Mutants of the LysA class produce little or no active diaminopimelic acid (DAP) decarboxylase and specifically require lysine for growth. Mutants of the LysB class produce a cryptic DAP decarboxylase which can be activated both in vivo and in vitro by higher than normal levels of its cofactor, pyridoxal 5'-phosphate. The LysB mutants have an alternate requirement for lysine or pyridoxine. Both LysA and LysB mutations map at 55 min, close to the thyA locus of E. coli. The association between pyridoxal phosphate and DAP decarboxylase appears to be much weaker in LysB mutants than in wild-type bacteria, and the mutant enzyme also sediments more slowly than wild-type enzyme in sucrose density gradients. The results suggest that the LysB mutations alter a specific region (or subunit) of the enzyme molecule which is needed to stabilize the binding of pyridoxal phosphate. These studies help to resolve certain contradictory observations on DAP decarboxylase reported earlier and may have relevance to pyridoxal phosphate enzymes in general. Prototrophic revertants of LysB mutants arise by second site mutations that result in increased availability of intracellular pyridoxal phosphate. These revertants appear to be derepressed for pyridoxine biosynthesis.     

Iron uptake is essential for Escherichia coli survival in drinking water

AIMS: The aim of this study was to elucidate if the need for iron for Escherichia coli to remain cultivable in a poorly nutritive medium such as the drinking water uses the iron transport system via the siderophores. METHODS AND RESULTS: Environmental strains of E. coli (isolated from a drinking water network), referenced strains of E. coli and mutants deficient in TonB, an essential protein for iron(III) acquisition, were incubated for 3 weeks at 25 degrees C, in sterile drinking water with and without lepidocrocite (gamma-FeOOH), an insoluble iron corrosion product. Only cells with a functional iron transport system were able to survive throughout the weeks. CONCLUSIONS: The iron transport system via protein TonB plays an essential role on the survival of E. coli in a weakly nutritive medium like drinking water. SIGNIFICANCE AND IMPACTS OF THE STUDY: Iron is a key parameter involved in coliform persistence in drinking water distribution systems.     

Energy coupling in the uptake of hexose phosphates by Escherichia coli.

Several methods were used to study the source of energy in the uptake of hexose phosphates by Escherichia coli K12. The uptake was sensitive to inhibition by agents that affect electron transport, such as lack of oxygen, cyanide, and heptylhydroxyquinoline-N-oxide, and by agents that affect ATP utilization, such as dicyclohexylcarbodiimide and arsenate. It was also sensitive to uncouplers in the presence of absence of oxygen. The strain of E. coli used extruded protons during respiration. Uncer anaerobic conditions, the uptake of approximately 1 eg to H+ per glucose 6-phosphate. These observations are consistent with a chemiosmotic mechanism of genergized glucose 6-phosphate uptake. The rate of glucose 6-phosphate uptake was maximal in KC1, but was also stimulated by MgC12 or CaC12. Inhibition by A217, a nigericin-like antibiotic, was prevented by K+ whereas valinomycin and gramicidin inhibited in the presence or absence of K+.     

ATP hydrolysis and DNA binding by the Escherichia coli RecF protein.

The Escherichia coli RecF protein possesses a weak ATP hydrolytic activity. ATP hydrolysis leads to RecF dissociation from double-stranded (ds)DNA. The RecF protein is subject to precipitation and an accompanying inactivation in vitro when not bound to DNA. A mutant RecF protein that can bind but cannot hydrolyze ATP (RecF K36R) does not readily dissociate from dsDNA in the presence of ATP. This is in contrast to the limited dsDNA binding observed for wild-type RecF protein in the presence of ATP but is similar to dsDNA binding by wild-type RecF binding in the presence of the nonhydrolyzable ATP analog, adenosine 5'-O-(3-thio)triphosphate (ATPgammaS). In addition, wild-type RecF protein binds tightly to dsDNA in the presence of ATP at low pH where its ATPase activity is blocked. A transfer of RecF protein from labeled to unlabeled dsDNA is observed in the presence of ATP but not ATPgammaS. The transfer is slowed considerably when the RecR protein is also present. In competition experiments, RecF protein appears to bind at random locations on dsDNA and exhibits no special affinity for single strand/double strand junctions when bound to gapped DNA. Possible roles for the ATPase activity of RecF in the regulation of recombinational DNA repair are discussed.