Individual Interactions of the b Subunits within the Stator of the Escherichia coli ATP Synthase

F_OF₁ ATP synthases are rotary nanomotors that couple proton translocation across biological membranes to the synthesis/hydrolysis of ATP. During catalysis, the peripheral stalk, composed of two b subunits and subunit δ in Escherichia coli, counteracts the torque generated by the rotation of the central stalk. Here we characterize individual interactions of the b subunits within the stator by use of monoclonal antibodies and nearest neighbor analyses via intersubunit disulfide bond formation. Antibody binding studies revealed that the C-terminal region of one of the two b subunits is principally involved in the binding of subunit δ, whereas the other one is accessible to antibody binding without impact on the function of F_OF₁. Individually substituted cysteine pairs suitable for disulfide cross-linking between the b subunits and the other stator subunits (b-α, b-β, b-δ, and b-a) were screened and combined with each other to discriminate between the two b subunits (i.e. b_I and b_II). The results show the b dimer to be located at a non-catalytic α/β cleft, with b_I close to subunit α, whereas b_II is proximal to subunit β. Furthermore, b_I can be linked to subunit δ as well as to subunit a. Among the subcomplexes formed were a-b_I-α, b_II-β, α-b_I-b_II-β, and a-b_I-δ. Taken together, the data obtained define the different positions of the two b subunits at a non-catalytic interface and imply that each b subunit has a different role in generating stability within the stator. We suggest that b_I is functionally related to the single b subunit present in mitochondrial ATP synthase.     

S-Carboxymethylcysteine Synthase from Escherichia coli

An enzyme that catalyzes the synthesis of S-carboxymethyl- l-cysteine from 3-chloro- l-alanine (3-Cl-Ala) and thioglycolic acid was found in Escherichia coli W3110 and was designated as S- carboxymethyl-l-cysteine synthase. It was purified from the cell-free extract to electrophoretic homogeneity and was crystallized. The enzyme has a molecular weight of 84,000 and gave one band corresponding to a molecular weight of 37,000 on SDS-polyacrylamide gel electrophoresis. The purified enzyme catalyzed the β-replacement reactions between 3-CI-AIa and various thiol compounds. The apparent Km values for 3-Cl-Ala and thioglycolic acid were 40 mM and 15.4 mM. The enzyme showed very low activity as to the α,β-elimination reaction with 3-Cl-Ala and l-serine. It was not inactivated on the incubation with 3-Cl-Ala. The absorption spectrum of the enzyme shows a maximum at 412 nm, indicating that it contains pyridoxal phosphate as a cofactor. The N-terminal amino acid sequence was determined and the corresponding sequence was detected in the protein sequence data bank, but no homogeneous sequence was found.     

Sodium and Potassium Requirements for Active Transport of Glutamate by Escherichia coli K-12

Active transport of glutamate by Escherichia coli K-12 requires both Na(+) and K(+) ions. Increasing the concentration of Na(+) in the medium results in a decrease in the K(m) of the uptake system for glutamate; the capacity is not affected. Glutamate uptake by untreated cells is not stimulated by K(+). K(+)-depleted cells show a greatly reduced capacity for glutamate uptake. Preincubation of such cells in the presence of K(+) fully restores their capacity for glutamate uptake when Na(+) ions are also present in the uptake medium. Addition of either K(+) or Na(+) alone restores glutamate uptake to only about 20% of its maximum capacity in the presence of both cations. Changes in K(+) concentration affect the capacity for glutamate uptake but have no effect on the K(m) of the glutamate transport system. Ouabain does not inhibit the (Na(+)-K(+))-stimulated glutamate uptake by intact cells or spheroplasts of E. coli K-12.     

Efficient Production of Active Polyhydroxyalkanoate Synthase in Escherichia coli by Coexpression of Molecular Chaperones

The type I polyhydroxyalkanoate synthase from Cupriavidus necator was heterologously expressed in Escherichia coli with simultaneous overexpression of chaperone proteins. Compared to expression of synthase alone (14.55 mg liter⁻¹), coexpression with chaperones resulted in the production of larger total quantities of enzyme, including a larger proportion in the soluble fraction. The largest increase was seen when the GroEL/GroES system was coexpressed, resulting in approximately 6-fold-greater enzyme yields (82.37 mg liter⁻¹) than in the absence of coexpressed chaperones. The specific activity of the purified enzyme was unaffected by coexpression with chaperones. Therefore, the increase in yield was attributed to an enhanced soluble fraction of synthase. Chaperones were also coexpressed with a polyhydroxyalkanoate production operon, resulting in the production of polymers with generally reduced molecular weights. This suggests a potential use for chaperones to control the physical properties of the polymer.     

Sodium-Stimulated Transport of Glutamate in Escherichia coli

Wild-type Escherichia coli B grew poorly on glutamate as the sole carbon source, except at very high concentrations of the amino acid. The addition of sodium ion markedly stimulated the growth. It had the same effect in a mutant of E. coli B selected for the ability to grow at low glutamate concentrations. Sodium ion also potentiated growth inhibition by analogues of glutamate. The uptake of glutamate by nongrowing cells of the mutant was markedly stimulated by sodium ion in the presence of an energy source, chloramphenicol, and arsenite, which retarded glutamate degradation.     

Analysis of Glutamate Exit in Escherichia coli

Escherichia coli B exhibits carrier-mediated first-order exit of glutamate with a half-time of less than 4 min, similar to that observed in K-12 strains. Glutamate exit in both B and K-12 strains is inhibited by arsenite. Practically all of the radioactivity lost during exit by K-12 cells has been accounted for as glutamate in the cell filtrate.     

Genetic Analysis of Glutamate Transport and Glutamate Decarboxylase in Escherichia coli

The location of the Escherichia coli K-12 genes determining or regulating glutamate transport, and the location of the gene determining glutamate decarboxylase synthesis, were established by conjugation. The ability to grow on glutamate as the sole source of carbon and energy was used to select for glutamate transport recombinants. Two genes determining the ability to grow on glutamate as the sole source of carbon and energy were mapped. One (gltC) is located near mtl (mannitol), and the other (gltH) appears to be located between the gal (galactose) and trp (tryptophan) loci. The glutamate decarboxylase gene (gad) is strongly linked to gltC. The gltC(+) recombinants grow on glutamate much faster and accumulate this amino acid to a greater extent than do the gltH(+) recombinants. The gltH(+) gene functioned only in one female strain (P678), whereas the gltC gene functioned in all the female strains tested (P678, C600, W1).     

Human CCT4 and CCT5 Chaperonin Subunits Expressed in Escherichia coli Form Biologically Active Homo-oligomers

Chaperonins are a family of chaperones that encapsulate their substrates and assist their folding in an ATP-dependent manner. The ubiquitous eukaryotic chaperonin, TCP-1 ring complex (TRiC), is a hetero-oligomeric complex composed of two rings, each formed from eight different CCT (chaperonin containing TCP-1) subunits. Each CCT subunit may have distinct substrate recognition and ATP hydrolysis properties. We have expressed each human CCT subunit individually in Escherichia coli to investigate whether they form chaperonin-like double ring complexes. CCT4 and CCT5, but not the other six CCT subunits, formed high molecular weight complexes within the E. coli cells that sedimented about 20S in sucrose gradients. When CCT4 and CCT5 were purified, they were both organized as two back-to-back rings of eight subunits each, as seen by negative stain and cryo-electron microscopy. This morphology is consistent with that of the hetero-oligomeric double-ring TRiC purified from bovine testes and HeLa cells. Both CCT4 and CCT5 homo-oligomers hydrolyzed ATP at a rate similar to human TRiC and were active as assayed by luciferase refolding and human γD-crystallin aggregation suppression and refolding. Thus, both CCT4 and CCT5 homo-oligomers have the property of forming 8-fold double rings absent the other subunits, and these complexes carry out chaperonin reactions without other partner subunits.     

The b Subunit of Escherichia coli ATP Synthase

The b subunit of ATP synthase is a major component of the second stalk connecting the F₁and F₀ sectors of the enzyme and is essential for normal assembly and function. The156-residue b subunit of the Escherichia coli ATP synthase has been investigated extensivelythrough mutagenesis, deletion analysis, and biophysical characterization. The two copies ofb exist as a highly extended, helical dimer extending from the membrane to near the top ofF₁, where they interact with the subunit. The sequence has been divided into four domains:the N-terminal membrane-spanning domain, the tether domain, the dimerization domain, andthe C-terminal -binding domain. The dimerization domain, contained within residues 60–122,has many properties of a coiled-coil, while the -binding domain is more globular. Sites ofcrosslinking between b and the a, , , and subunits of ATP synthase have been identified,and the functional significance of these interactions is under investigation. The b dimer mayserve as an elastic element during rotational catalysis in the enzyme, but also directly influencesthe catalytic sites, suggesting a more active role in coupling.     

Secretion of Alkaline Phosphatase Subunits by Spheroplasts of Escherichia coli

Under conditions that permitted continued protein synthesis, spheroplasts of Escherichia coli were unable to form active alkaline phosphatase, although they synthesized protein that was antigenically related to alkaline phosphatase subunits. This cross-reacting protein was primarily detected in the medium of the spheroplast culture, and it had properties that closely resembled those of the alkaline phosphatase subunit. These results suggest that formation of the active alkaline phosphatase dimer by intact E. coli cells proceeds by a pathway in which inactive subunits released from polyribosomes diffuse through the bacterial cell membrane to a periplasmic space where subsequent dimerization to active enzyme occurs. This pathway provides a possible mechanism for the specific localization of this enzyme to the E. coli periplasmic space.     

Pathway Choice in Glutamate Synthesis in Escherichia coli

Escherichia coli has two primary pathways for glutamate synthesis. The glutamine synthetase-glutamate synthase (GOGAT) pathway is essential for synthesis at low ammonium concentration and for regulation of the glutamine pool. The glutamate dehydrogenase (GDH) pathway is important during glucose-limited growth. It has been hypothesized that GDH is favored when the organism is stressed for energy, because the enzyme does not use ATP as does the GOGAT pathway. The results of competition experiments between the wild-type and a GDH-deficient mutant during glucose-limited growth in the presence of the nonmetabolizable glucose analog α-methylglucoside were consistent with the hypothesis. Enzyme measurements showed that levels of the enzymes of the glutamate pathways dropped as the organism passed from unrestricted to glucose-restricted growth. However, other conditions influencing pathway choice had no substantial effect on enzyme levels. Therefore, substrate availability and/or modulation of enzyme activity are likely to be major determinants of pathway choice in glutamate synthesis.     

Intergeneric Complementation of Anthranilate Synthase Subunits

Partially purified subunits of anthranilate synthase were prepared from Bacillus subtilis and Pseudomonas aeruginosa. The large component from B. subtilis (I(B)) complements well with the small component from P. aeruginosa (II(P)) to reconstitute a glutamine-reactive anthranilate synthase. This interaction can be demonstrated with crude extracts from a B. subtilis trpX mutant and a P. aeruginosa trpA mutant. Complementation was also observed with the large component from P. aeruginosa (I(P)) and the small subunit from B. subtilis (II(B)). At saturation the heterologous complex I(B)II(P) has 93% of the activity of the homologous complex I(B)II(B), whereas the hybrid I(P)II(B) is only 22% as active as the homologous complex I(P)II(P).     

Rapid Glutamate Decarboxylase Assay for Detection of Escherichia coli

Volume 59, no. 12, p. 4347, column 1, line 3 from bottom: "500 x g" should read "2,500 x g." [This corrects the article on p. 4347 in vol. 59.].     

Ribonucleic Acid Synthesis and Glutamate Excretion in Escherichia coli

Cultures of Escherichia coli excreted glutamate into the medium when protein synthesis was blocked in RC(rel) strains or when it was blocked with chloramphenicol in either RC(str) or RC(rel) strains. Both of these conditions resulted in continued ribonucleic acid (RNA) synthesis in the absence of protein synthesis. Glutamate was also excreted by both RC(str) and RC(rel) strains when RNA synthesis was inhibited by uracil starvation or by treatment with actinomycin D. It is proposed that, in each of these cases, glutamate excretion resulted from an increase in the permeability of the cell membrane.     

Manganese Active Transport in Escherichia coli

Manganese was accumulated by cells of Escherichia coli by means of an active transport system quite independent of the magnesium transport system. When the radioisotope (54)Mn was used, manganese transport showed saturation kinetics with a K(m) of 2 x 10(-7)m and a V(max) of 1 to 4 nmoles/min per 10(12) cells at 25 C. The manganese transport system is highly specific; magnesium and calcium did not stimulate, inhibit, or compete with manganese for cellular uptake. Cobalt and iron specifically interfered with (54)Mn uptake, but only when added at concentrations 100 times higher than the K(m) for manganese. Active transport of manganese is temperature- and energy-dependent: uptake of (54)Mn was inhibited by cyanide, dinitrophenol, and m-chlorophenyl carbonylcyanide hydrazone (CCCP). Furthermore, the turnover or exit of manganese from intact cells was inhibited by energy poisons such as dinitrophenol and CCCP.     

Thymoquinone Inhibits Escherichia coli ATP Synthase and Cell Growth

We examined the thymoquinone induced inhibition of purified F₁ or membrane bound F₁F_O E. coli ATP synthase. Both purified F₁ and membrane bound F₁F_O were completely inhibited by thymoquinone with no residual ATPase activity. The process of inhibition was fully reversible and identical in both membrane bound F₁F_o and purified F₁ preparations. Moreover, thymoquinone induced inhibition of ATP synthase expressing wild-type E. coli cell growth and non-inhibition of ATPase gene deleted null control cells demonstrates that ATP synthase is a molecular target for thymoquinone. This also links the beneficial dietary based antimicrobial and anticancer effects of thymoquinone to its inhibitory action on ATP synthase.     

Glutamate transport in wild-type and mutant strains of Escherichia coli

Halpern, Yeheskel S. (Hebrew University-Hadassah Medical School, Jerusalem, Israel), and Meir Lupo. Glutamate transport in wild-type and mutant strains of Escherichia coli. J. Bacteriol. 90:1288-1295. 1965.-Mutants of Escherichia coli able to grow on glutamate as their source of carbon showed glutamate dehydrogenase and glutamate-oxaloacetate transaminase activities similar to those possessed by the parent strain. The mutants took up glutamate at a much faster rate and showed a several-fold greater capacity for concentrating the amino acid than did the corresponding parent strains. Curvilinear double reciprocal plots of velocity of uptake versus glutamate concentration were obtained with the E. coli H strains. A break in the curve of glutamate uptake was observed with the E. coli K-12 strains when incubated in a glucose medium. It is suggested that these findings may be due to allosteric activation of glutamate permease by its substrate.     

Active accumulation of tetracycline by Escherichia coli

1. At low concentrations of tetracycline (10mug/ml) net accumulation of the drug by Escherichia coli cells ceased after 7-10min. 2. At higher concentrations of tetracycline (>30mug/ml) the period of net accumulation of the drug was significantly extended. 3. The efflux of tetracycline from E. coli cells transferred from medium containing 10mug of tetracycline/ml to drug-free medium was a rapid temperature-dependent process and was accelerated by 2,4-dinitrophenol. 4. As the concentration of tetracycline in the preloading phase was increased, the rate of subsequent efflux of the drug progressively declined. The efflux of drug from cells preloaded in medium containing 200mug of tetracycline/ml was negligible, although efflux was readily provoked by 2,4-dinitrophenol, by N-ethylmaleimide or by omission of glucose from the medium. 5. The initial rate of uptake of tetracycline by E. coli cells was linearly proportional to the concentration of tetracycline in the medium up to the maximum concentration of drug obtainable under the experimental conditions used (400mug/ml, 0.83mm). 6. Although N-ethylmaleimide strongly inhibited the accumulation of tetracycline by E. coli, no evidence was obtained for the direct involvement of thiol groups in the transport process. It was concluded that N-ethylmaleimide inhibited accumulation by interruption of the energy supply of the cells. 7. Osmotic shock of E. coli cells did not significantly affect the influx of tetracycline, but promoted both efflux of tetracycline and cell lysis in cells treated with a high concentration of tetracycline. 8. A study of the distribution of tetracycline among the subcellular fractions of penicillin-induced spheroplasts preincubated with various concentrations of tetracycline indicated that 60-70% of the accumulated tetracycline was in the high-speed supernatant fraction. Sephadex chromatography showed that the tetracycline of this fraction was present as the free drug. Sephadex chromatography of a detergent extract of the membrane fraction, however, indicated that a significant proportion of the tetracycline radioactivity of this fraction was apparently bound to some macromolecular component. 9. Cellulose phosphate paper chromatography of cold-acid extracts of spheroplasts preloaded with tetracycline indicated that the accumulated drug was chemically unchanged. 10. Membrane preparations isolated from osmotically lysed penicillin-induced spheroplasts showed a temperature-dependent binding of tetracycline that was not energy-dependent and was not inhibited by N-ethylmaleimide. The binding process was stimulated by omitting Mg(2+) from the medium, but conversely was profoundly inhibited by EDTA. 11. The relevance of these findings to the probable mechanism of active tetracycline accumulation by E. coli is discussed.