The Regulation of Proton/Amino Acid Symport in Riccia fluitans L. by Cytosolic pH and Proton Pump Activity

In the aquatic liverwort Riccia fluitans the regulation of theplasma membrane H⁺/amino acid symport has been investigated.Cytosolic pH (pH_c), membrane potential (E_m) and membrane conductancehave been measured and related to transport data, (i) The releaseof [¹⁴C]amino acids is strongly stimulated by cytosolic acidification,induced by the external addition of acetic acid, a decreasein external K⁺, and in the change from light to dark. On average,a decrease in pHc of 0.5 to 0.6 units corresponded with a 4-foldstimulation in amino acid efflux. (ii) External pH changes havefar less effect on substrate transport than the cytosolic pHshifts of the same order. (iii) The inwardly directed positivecurrent, induced by amino acids, is severely inhibited by cytosolicacidification. (iv) Fusicoccin (FC) stimulates amino acid uptakewithout considerable change in proton motive force. (v) Whenthe proton motive force is kept constant, the uptake of aminoacids into Riccia thalli is much lower than when the pump isdeactivated. It is suggested that both the proton pump activityand cytosolic pH are the dominant factors in the regulationof the H⁺/amino acid symport across the plasma membrane of Ricciafluitans, and it is concluded that the proton motive force isnot a reliable quantity to predict and interpret transport kinetics. Key words: Amino acid, cytosolic pH, pH-sensitive electrode, proton motive force, regulation, Riccia fluitans     

New functions for the ancient globin family: bacterial responses to nitric oxide and nitrosative stress

Globin-like oxygen-binding proteins occur in bacteria, yeasts and other fungi, and protozoa. The simplest contain protohaem as sole prosthetic group, but show considerable variation in their similarity to the classical animal globins and plant globins. Flavohaemoglobins comprise a haem domain homologous to classical globins and a ferredoxin-NADP+ reductase (FNR)-like domain that converts the globin into an NAD(P)H-oxidizing protein with diverse reductase activities. In Escherichia coli, the prototype flavohaemoglobin (Hmp) is clearly involved in responses to nitric oxide (NO) and nitrosative stress: (i) the structural gene hmp is upregulated by NO and nitrosating agents; (ii) purified Hmp binds NO avidly, but also converts it to nitrate (aerobically) or nitrous oxide (anaerobically); (iii) hmp mutants are hypersensitive to NO and nitrosative stresses. Here, we review recent advances in E. coli and the growing number of microbes in which globins are known, draw particular attention to the essential chemistry of NO and related reactive species and their interactions with globins, and suggest that microbial globins have additional functions unrelated to 'NO' stresses.     

A member of the sugar transporter family, Stl1p is the glycerol/H+ symporter in Saccharomyces cerevisiae

Glycerol and other polyols are used as osmoprotectants by many organisms. Several yeasts and other fungi can take up glycerol by proton symport. To identify genes involved in active glycerol uptake in Saccharomyces cerevisiae we screened a deletion mutant collection comprising 321 genes encoding proteins with 6 or more predicted transmembrane domains for impaired growth on glycerol medium. Deletion of STL1, which encodes a member of the sugar transporter family, eliminates active glycerol transport. Stl1p is present in the plasma membrane in S. cerevisiae during conditions where glycerol symport is functional. Both the Stl1 protein and the active glycerol transport are subject to glucose-induced inactivation, following identical patterns. Furthermore, the Stl1 protein and the glycerol symporter activity are strongly but transiently induced when cells are subjected to osmotic shock. STL1 was heterologously expressed in Schizosaccharomyces pombe, a yeast that does not contain its own active glycerol transport system. In S. pombe, STL1 conferred the ability to take up glycerol against a concentration gradient in a proton motive force-dependent manner. We conclude that the glycerol proton symporter in S. cerevisiae is encoded by STL1.     

High capacity xylose transport in Candida intermedia PYCC 4715

Xylose-utilising yeasts were screened to identify strains with high xylose transport capacity. Among the fastest-growing strains in xylose medium, Candida intermedia PYCC 4715 showed the highest xylose transport capacity. Maximal specific growth rate was the same in glucose and xylose media (mu(max)=0.5 h-1, 30 degrees C). Xylose transport showed biphasic kinetics when cells were grown in either xylose- or glucose-limited culture. The high-affinity xylose/proton symport system (Km = 0.2 mM, Vmax = 7.5 mmol h-1 g-1) was more repressed by glucose than by xylose. The less specific low-affinity transport system (K = 50 mM, Vmax = 11 mmol h-1 g-1) appeared to operate through a facilitated-diffusion mechanism and was expressed constitutively. Inhibition experiments showed that glucose is a substrate of both xylose transport systems.     

Identification and properties of yeasts associated with the aerobic deterioration of wheat and alfalfa silages

Populations of fungi in aerobically deteriorating wheat and alfalfa silages were identified as: Endomycopsis burtonii, E. selenospora, Hansenula canadensis, Candida tenuis and C. silvicola. The yeasts recovered were similar for both silages, but H. canadensis was recovered only in wheat silages. All of these yeasts could utilize lactic acid aerobically, but not anaerobically. Only Endomycopsis spp. could utilize propionic acid aerobically and none of the yeasts utilized this acid anaerobically. However, all yeasts grew in complete media supplemented with propionate. Therefore, while lactic and propionic acids may contribute to stability under anaerobic conditions, they are much less less effective after the silage is exposed to air.     

Citrate Metabolism in Aerobacter cloacae

Growth of Aerobacter cloacae on citrate either anaerobically or aerobically did not require and was not stimulated by the presence of Na(+) in the medium. Citrate was metabolized anaerobically via the fermentation pathway as evidenced by the (i) presence of oxalacetate decarboxylase, (ii) induction of citrate lyase, and (iii) repression of alpha-ketoglutarate dehydrogenase under anaerobic conditions. Thus, although all the other enzymes of the citric acid cycle were present in anaerobic cells, this pathway was not available for the metabolism of citrate. Citrate was metabolized aerobically via the citric acid cycle, since (i) citrate lyase but not oxalacetate decarboxylase was repressed and (ii) alpha-ketoglutarate dehydrogenase was induced under these conditions. The presence of Na(+) in the medium did not lead to a repression of alpha-ketoglutarate dehydrogenase as in the case of Aerobacter aerogenes. The oxalacetate decarboxylase was a soluble, constitutive enzyme, not activated by Na(+) nor inhibited by avidin. It was slightly inhibited by ethylenediaminetetraacetate but was not stimulated by Mg(2+) or Mn(2+). Thus, this enzyme differed markedly in its properties from the same enzyme found in citrate-grown A. aerogenes.     

Identification of a telomeric fragment from the right arm of chromosome III of Aspergillus nidulans

The activities of pyruvate decarboxylase, alcohol dehydrogenase and certain glycosidases were measured for six species of yeast. Five of these yeasts could utilize one or more disaccharides aerobically, but not anaerobically, although all could use D-glucose anaerobically. That is, each of the five showed the Kluyver effect; but the sixth yeast, Saccharomyces cerevisiae, did not do so. When grown on a glycoside with which it gave the Kluyver effect, each yeast had much less pyruvate decarboxylase activity than when grown on D-glucose or another glycoside. There was no consistent corresponding lowering of activity of either alcohol dehydrogenase, or of the appropriate glycosidase. Hence, pyruvate decarboxylase may have a role in producing the Kluyver effect.     

Electrogenicity, pH-Dependence, and Stoichiometry of the Proton-Sucrose Symport

The electrogenicity, pH-dependence, and stoichiometry of the proton-sucrose symport were examined in plasma membrane vesicles isolated from sugar beet (Beta vulgaris L. cv Great Western) leaves. Symport mediated sucrose transport was electrogenic as demonstrated by the effect of membrane potential on ΔpH-dependent flux. In the absence of significant charge compensation, a low rate of sucrose transport was observed. When membrane potential was clamped at zero with symmetric potassium concentrations and valinomycin, the rate of sucrose flux was stimulated fourfold. In the presence of a negative membrane potential, transport increased six-fold. These results are consistent with electrogenic sucrose transport which results in a net flux of positive charge into the vesicles. The effect of membrane potential on the kinetics of sucrose transport was on V_max only with no apparent change in K_m. Sucrose transport rates driven by membrane potential only, i.e. in the absence of ΔpH, were comparable to ΔpH-driven flux. Both membrane potential and ΔpH-driven sucrose transport were used to examine proton binding to the symport and the apparent K_m for H⁺ was 0.7 micromolar. The kinetics of sucrose transport as a function of proton concentration exhibited a simple hyperbolic relationship. This observation is consistent with kinetic models of ion-cotransport systems when the stoichiometry of the system, ion:substrate, is 1:1. Quantitative measurements of proton and sucrose fluxes through the symport support a 1:1 stoichiometry. The biochemical details of protoncoupled sucrose transport reported here provide further evidence in support of the chemiosmotic hypothesis of nutrient transport across the plant cell plasma membrane.     

Chemostat cultivation as a tool for studies on sugar transport in yeasts.

Chemostat cultivation enables investigations into the effects of individual environmental parameters on sugar transport in yeasts. Various means are available to manipulate the specific rate of sugar uptake (qs) in sugar-limited chemostat cultures. A straightforward way to manipulate qs is variation of the dilution rate, which, in substrate-limited chemostat cultures, is equal to the specific growth rate. Alternatively, qs can be varied independently of the growth rate by mixed-substrate cultivation or by variation of the biomass yield on sugar. The latter can be achieved, for example, by addition of nonmetabolizable weak acids to the growth medium or by variation of the oxygen supply. Such controlled manipulation of metabolic fluxes cannot be achieved in batch cultures, in which various parameters that are essential for the kinetics of sugar transport cannot be controlled. In sugar-limited chemostat cultures, yeasts adapt their sugar transport systems to cope with the low residual sugar concentrations, which are often in the micromolar range. Under the conditions, yeasts with high-affinity proton symport carriers have a competitive advantage over yeasts that transport sugars via facilitated-diffusion carriers. Chemostat cultivation offers unique possibilities to study the energetic consequences of sugar transport in growing cells. For example, anaerobic, sugar-limited chemostat cultivation has been used to quantify the energy requirement for maltose-proton symport in Saccharomyces cerevisiae. Controlled variation of growth conditions in chemostat cultures can be used to study the differential expression of genes involved in sugar transport and as such can make an important contribution to the ongoing studies on the molecular biology of sugar transport in yeasts.     

Photoresponses in Rhodobacter sphaeroides: role of photosynthetic electron transport.

Rhodobacter sphaeroides responds to a decrease in light intensity by a transient stop followed by adaptation. There is no measurable response to increases in light intensity. We confirmed that photosynthetic electron transport is essential for a photoresponse, as (i) inhibitors of photosynthetic electron transport inhibit photoresponses, (ii) electron transport to oxidases in the presence of oxygen reduces the photoresponse, and (iii) the magnitude of the response is dependent on the photopigment content of the cells. The photoresponses of cells grown in high light, which have lower concentrations of light-harvesting photopigment and reaction centers, saturated at much higher light intensities than the photoresponses of cells grown in low light, which have high concentrations of light-harvesting pigments and reaction centers. We examined whether the primary sensory signal from the photosynthetic electron transport chain was a change in the electrochemical proton gradient or a change in the rate of electron transport itself (probably reflecting redox sensing). R. sphaeroides showed no response to the addition of the proton ionophore carbonyl cyanide 4-trifluoromethoxyphenylhydrazone, which decreased the electrochemical proton gradient, although a behavioral response was seen to a reduction in light intensity that caused an equivalent reduction in proton gradient. These results strongly suggest that (i) the photosynthetic apparatus is the primary photoreceptor, (ii) the primary signal is generated by a change in the rate of electron transport, (iii) the change in the electrochemical proton gradient is not the primary photosensory signal, and (iv) stimuli affecting electron transport rates integrate via the electron transport chain.     

Anaerobic transport of serine and 2-aminoisobutyric acid by Staphylococcus epidermidis.

A membrane-bound ATPase detected in extracts of anaerobically grown Staphylococcus epidermidis was inhibited by a variety of compounds which inhibit ATPases in other organisms. Serine and 2-aminoisobutyric acid (AIB) were shown to enter the organism via the same transport system. The transport of AIB, the membrane potential and the transmembrane pH gradient were partially or completely abolished by the same inhibitors and also by uncoupling agents and lipid-soluble ions. It is proposed therefore that this ATPase generates and maintains an electrochemical gradient of protons across the cytoplasmic membrane of S. epidermidis capable of driving AIB uptake. Studies of AIB-induced proton movements suggested that AIB enters via a proton symport mechanism.     

GUP1 and its close homologue GUP2, encoding multimembrane-spanning proteins involved in active glycerol uptake in Saccharomyces cerevisiae

Many yeast species can utilize glycerol, both as a sole carbon source and as an osmolyte. In Saccharomyces cerevisiae, physiological studies have previously shown the presence of an active uptake system driven by electrogenic proton symport. We have used transposon mutagenesis to isolate mutants affected in the transport of glycerol into the cell. Here we present the identification of YGL084c, encoding a multimembrane-spanning protein, as being essential for proton symport of glycerol into S. cerevisiae. The gene is named GUP1 (glycerol uptake) and, for growth on glycerol, is important as a carbon and energy source. In addition, in strains deficient in glycerol production it also provides osmotic protection by the addition of glycerol. Another open reading frame (ORF), YPL189w, presenting a high degree of homology to YGL084c, similarly appears to be involved in active glycerol uptake in salt-containing glucose-based media in strains deficient in glycerol production. Analogously, this gene is named GUP2. To our knowledge, this is the first report on a gene product involved in active transport of glycerol in yeasts. Mutations with the same phenotypes occurred in two other ORFs of previously unknown function, YDL074c and YPL180w.     

Pentose utilization and transport by the ruminal bacterium Prevotella ruminicola

Plant cell wall polysaccharides are primarily composed of hexose or hexose derivatives, but a significant fraction is hemicellulose which contains pentose sugars. Prevotella ruminicola B₁4, a predominant ruminal bacterium, simultaneously metabolized pentoses and glucose or maltose, but the organism preferentially fermented pentoses over cellobiose and preferred xylose to sucrose. Xylose and arabinose transport at either low (2 M) or high (1 mM) substrate concentrations were observed only in the presence of sodium and if oxygen was excluded during the harvest and assay procedures. An artificial electrical potential () or chemical gradient of sodium (pNa) drove transport in anaerobically prepared membrane vesicles. Because (i) transport was electrogenic, (ii) a pNa drove uptake, and (iii) the number of sodium binding sites was approximately 1, it appeared that P. ruminicola possessed pentose/sodium symport mechanisms for the transport of arabinose and xylose at low substrate concentrations. Pentose uptake exhibited a low affinity for xylose or arabinose (>300M), and transport of xylose exhibited bi-phasic kinetics which suggested that a second sodium-dependent xylose transport system was present. Little study has been made on solute transport by Prevotella (Bacteroides) species and this work represents the first use of isolated membrane vesicles from these organisms.     

Transport of xylose and glucose in the xylose-fermenting yeast Pichia stipitis

Summary A low-affinity and a high-affinity sylose proton symport operated simultaneously in both starved and non-starved cells of Pichia stipitis. Glucose competed with xylose for transport by the low-affinity system and inhibited xylose transport by the high-affinity system non-competitively. The low affinity system was subject to substrate inhibition when glucose but not when xylose was the substrate. The differences between the characteristics of monosaccharide transport by Pichia stipitis and its imperfect state, Candida shehatae, are discussed.     

The nature of H+-K+-exchange in anaerobically and aerobically grown S. typhimurium

H+-K+-exchange via the Trk-like system of K+ accumulation takes place in anaerobically grown S. typhimurium LT-2 with stable ratio of DCC-sensitive ionic fluxes, equal to 2H+ of a cell for one K+ of the medium. This exchange is now observed in the mutant S. typhimurium TH-31 with unfunctional H+-ATPase. H+-K+-exchange in aerobically grown S. typhimurium LT-2 has unstable ratio of ionic fluxes. The rate of K+ uptake in anaerobically grown bacteria is higher than that in the aerobically grown ones. Q10 is about 1.8 both for H+ transfer and K+ uptake in anaerobically grown bacteria, but it is 1.7 and 0.9 respectively in the aerobically grown ones. Delta psi is not changed by different temperatures both in anaerobically and aerobically grown bacteria. The distribution of K+ in anaerobically grown bacteria is higher than 10(3) and the potassium equilibrium potential is much higher than the measured delta psi. In aerobically grown bacteria the distribution of K+ is in good conformity with the measured delta psi. H+ and K+ transport in anaerobically grown cells is likely to proceed by the same mechanism, which includes H+-ATPase and the Trk-like system. In aerobically grown bacteria these transport systems work separately, and the Trk-like system as K+-ionophore serving for K+ uptake across the electrical field on the membrane.     

Nitrous oxide reduction in nodules: denitrification or n(2) fixation?

Detached cowpea nodules that contained a nitrous oxide reductase-positive (Nor) rhizobium strain (8A55) and a nitrous oxide reductase-negative (Nor) rhizobium strain (32H1) were incubated with 1% N(2)O (95 atom% N) in the following three atmospheres: (i) aerobic with C(2)H(2) (10%), (ii) aerobic without C(2)H(2), and (iii) anaerobic (argon atmosphere) without C(2)H(2). The greatest production of N(2) occurred anaerobically with 8A55, yet very little was formed with 32H1. Although acetylene reduction activity was slightly higher with 32H1, about 10 times more N(2) was produced aerobically by 8A55 than by 32H1 in the absence of acetylene. The major reductive pathway of N(2)O reduction by denitrifying rhizobium strain 8A55 is by nitrous oxide reductase rather than nitrogenase.     

Changes in the activities of the protoheme-synthesizing system during the growth of yeast under different conditions.

The levels of some enzymatic activities involved in protoheme synthesis have been measured in subcellular fractions obtained at different stages of the growth of the yeast Saccharomyces cerevisiae grown anaerobically and aerobically with glucose (50 or 6 g/ liter), and ethanol (20 g/liter) as the carbon source. The degree of repression of the respiratory system is estimated by the respiratory capacity of whole cells, by the activities of succinate-cytochrome c reductase and cytochrome c oxidase of the mitochondrial particles, and by the cytochrome spectra. The results show that (i) the more porphyrins (cytochromes) that are synthesized by the cells, the lower is the specific activity of δ-aminolevulinic acid (ALA) synthetase and the higher is the specific activity of ALA dehydratase, the activity ratio ALA synthetase/ALA dehydratase decreasing at least 10-fold compared to the repressed cells; (ii) the amount of intracellular ALA found under all conditions tested (from 0.05 to 1.5 mm in the cell sap) correlates well with the measured ALA synthetase activity; its presence argues against a rate-limiting function for ALA synthetase and rather favors such a role for the ALA dehydratase in the formation of heme in yeast; (iii) the rate of porphyrin synthesis measured in vitro is higher in the case of cells with high cytochrome contents; and (iv) the specific activities of succinyl CoA synthetase and protoheme ferrolyase are always present in nonlimiting amounts. Some experiments are described showing that the values of the activities which are calculated from these in situ and in vivo experiments compare well with the values measured in vitro in the acellular extracts. The results concerning the enzymatic activities, together with (i) the excretion of coproporphyrin(ogen) and the accumulation of protoporphyrin + Zn-protoporphyrin in anaerobiosis, (ii) the presence of protoporpho(di)methene (P503) in anaerobic and repressed cells, and (iii) the presence of intracellular ALA under all growth conditions, are discussed in terms of possible control(s) of heme synthesis in yeast.     

Continuous-culture study of the regulation of glucose and fructose transport in Kluyveromyces marxianus CBS 6556.

Regulation of transport of D-glucose and D-fructose was studied in Kluyveromyces marxianus grown in continuous culture. Both substrates could be transported by at least two different transport systems, low-affinity transport and high-affinity proton-sugar symport. The low-affinity transporter, specific for both glucose and fructose, was constitutively present and was apparently not regulated by carbon catabolite repression. Regulation of the activity of the glucose- and fructose-specific proton symport systems appeared to proceed mainly through catabolite repression. Activation of symport did not need the presence of specific inductor molecules in the medium. Nevertheless, the capacities of the proton-sugar symporters varied in cells grown on a wide variety of carbon sources. The possibility that the control of proton symport activity is related to the presence of specific intracellular metabolites is discussed.     

Effects on transport of rapidly penetrating,competing substrates: Activation and inhibition of the choline carrier in erythrocytes by imidazole

Summary The properties of the choline transport system are fundamentally altered in saline solution containing 5mm imidazole buffer instead of 5mm phosphate: (i) The system no longer exhibits accelerated exchange. (ii) Choline in the external compartment fails to increase the rate of inactivation of the carrier by N-ethylmaleimide. (iii) Depending on the relative concentrations of choline and imidazole, transport may be activated or inhibited. The maximum rates are increased more than fivefold by imidazole, but at moderate substrate concentrations activation is observed with low concentrations of imidazole and inhibition with high concentrations. (iv) The imidazole effect is asymmetric, there being a greater tendency to activate exit than entry. All this behavior is predicted by the carrier model if imidazole is a substrate of the choline carrier having a high maximum transport rate but a relatively low affinity, and if imidazole rapidly enters the cell by simple diffusion, so that it can add to carrier sites on both sides of the membrane. Addition at thecis side inhibits, and at thetrans side activates. According to the carrier model, asymmetry is a necessary consequence of the potassium ion gradient in red cells, potassium ion being another substrate of the choline system.