RESUMEN

In plant biology Fusicoccin (FC) is one of the most studied fungal metabolites to date. Since the structural identification in 1964, much has been learned about its effects on the physiology of plants, about the interference with the action of plant hormones, the molecular nature of the plant receptor(s) for FC and the biosynthetic pathway for FC in the fungus. The finding that the plasma membrane H+-ATPase in combination with 14-3-3 proteins acts as high-affinity receptor for FC was a breakthrough in the field. Ever since, the binding of FC to the ATPase|14-3-3 receptor has taken center stage in explaining all FC induced physiological effects. However, a more critical review shows that this is not at all evident for a number of FC induced effects. Examples of this are: the inhibition of outward rectifying K+-channels in guard cells, the phosphorylation/activation of PEP-carboxylase and malate accumulation, the antagonism with ABA induced production of H2O2 / NO and the effect on ethylene production. In addition, recently two other physiological processes were shown to be targeted by FC, viz. the activation of TORC1 and the interference of FC with the immune response to fungal elicitors. In this review, the notion will be challenged that all FC affected processes start with the binding to and activation of the PM-ATPase and the question is raised whether may be other proteins with a key role in the respective processes are directly targeted by FC. A second unresolved question is whether FC may be another example of a fungal molecule turning out to be a 'copy' of an as yet unknown plant molecule; in analogy to the fungal product and plant hormone gibberellic acid. A relevant question in this respect is whether it is a coincidence that proteins that act in a coordinated fashion during stomatal opening (the ATPases and K+-channels) are targeted by FC? Or are the sites where FC binds in the plant, conserved during evolution because they serve a physiological role, namely the accommodation of a plant produced molecule? In view of the evidence, albeit not conclusive, that plants indeed produce 'FC-like ligands', it is worthwhile to make a renewed attempt with current day improved technology to answer this question and may be upgrade FC or structural analogue(s) to a new level, the level of plant hormone.

RESUMEN

OBJECTIVES: The flora of Azerbaijan is represented by one species of the Macrosciadium genus: Macrosciadium alatum, belonging to the Apiaceae family. It is commonly found in the Greater and Lesser Caucasus regions of Azerbaijan, as part of subalpine meadow plant communities. M. alatum is characterized by its robust, thick, tuberous roots, long-petioled and several times pinnately divided leaves, numerous (30-50) white umbels, and oval-shaped fruits. The primary objective of this research is to determine the antimicrobial potential of the aqueous extract obtained from M. alatum against both Gram-negative and Gram-positive bacteria. The plant preparations utilized in in vitro experiments were in the form of maceration, infusion, and hydrodistillation as aqueous extracts. M. alatum extract exhibited maximum (measuring 22.3 ± 1.4 mm) inhibition zones against bacteria (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus, and Salmonella enteritidis) strains. Following exposure to the M. alatum plant extract, a significant reduction in bacterial cell cytoplasmic pH was observed (p≤0.04). METHODS: In order to investigate the antimicrobial effects of the plant extract, commonly accepted procedures were followed using well-known bacterial strains, including S. aureus, B. cereus, E. coli, S. enteritis and P. aeruginosa, which are principal causative agents of purulent-inflammatory processes. The 20 % aqueous extract was used. RESULTS: The conducted experiment to determine the impact of the plant extract on microorganisms revealed that the extract significantly affects the bacterial cell membrane. Specifically, there is a decrease in pH, and hyperpolarization of the cell membrane occurs. The efficacy of the preservative effect is highly dependent on the environmental pH. 1. The 20 % aqueous extract from exhibited antimicrobial activity and effectively preventing the development of foodborne pathogens and putrefactive microorganisms. 2. A 20 % aqueous extract of M. alatum exhibits antimicrobial activity, effectively inhibiting the growth of foodborne pathogens and spoilage microorganisms. 3. Extract led to an increase in H+ concentration within bacterial cell cytoplasm, surpassing the OH- concentration. 4. M. alatum species has a significant inhibitory effect on the growth of microorganisms such as S. aureus, E. coli, P. aeruginosa, and S. enteritidis. CONCLUSIONS: The results suggest that the extract from M. alatum possesses antimicrobial properties, making it a potential candidate for use as a natural food preservative. The observed hyperpolarization of the cell membrane and pH reduction further support its potential as an effective antibacterial agent.

RESUMEN

Polycyclic aromatic hydrocarbons (PAHs) are a group of extremely carcinogenic organic pollutants. Our previous findings have demonstrated that plant roots actively take up PAHs through co-transport with H+ ions. Auxin serves as a pivotal regulator of plant growth and development. However, it remains unclear whether the hormone can enhance the uptake of PAHs by plant roots. Hence, the wheat root exposed to PAHs with/without auxins was set to investigate how the auxin promotes the PAHs uptake by roots. In our study, auxin could significantly enhance the uptake of PAHs after 4 h of exposure. After the addition of auxin, the root tissue cytoplasmic pH value was decreased and the H+ influx was observed, indicating that the extracellular space was alkalinized in a short time. The increased H+ influx rate enhanced the uptake of PAHs. In addition, the H+-ATPase activity was also increased, suggesting that auxin activated two distinct and antagonistic H+ flux pathways, and the H+ influx pathway was dominant. Our findings offer important information for exploring the mechanism underlying auxin regulation of PAHs uptake and the phytoremediation of PAH-contaminated soil and water.

Asunto(s)

Fenantrenos , Hidrocarburos Policíclicos Aromáticos , Contaminantes del Suelo , Hidrocarburos Policíclicos Aromáticos/análisis , Fenantrenos/metabolismo , Triticum/metabolismo , Concentración de Iones de Hidrógeno , Ácidos Indolacéticos , Contaminantes del Suelo/metabolismo , Raíces de Plantas/metabolismo

RESUMEN

In this report, we establish a straightforward method for estimating the equilibrium constant for the creatine kinase reaction (CK Keq″) over wide but physiologically and experimentally relevant ranges of pH, Mg2+ and temperature. Our empirical formula for CK Keq″ is based on experimental measurements. It can be used to estimate [ADP] when [ADP] is below the resolution of experimental measurements, a typical situation because [ADP] is on the order of micromolar concentrations in living cells and may be much lower in many in vitro experiments. Accurate prediction of [ADP] is essential for in vivo studies of cellular energetics and metabolism and for in vitro studies of ATP-dependent enzyme function under near-physiological conditions. With [ADP], we were able to obtain improved estimates of ΔGATP, necessitating the reinvestigation of previously reported ADP- and ΔGATP-dependent processes. Application to actomyosin force generation in muscle provides support for the hypothesis that, when [Pi] varies and pH is not altered, the maximum Ca2+-activated isometric force depends on ΔGATP in both living and permeabilized muscle preparations. Further analysis of the pH studies introduces a novel hypothesis around the role of submicromolar ADP in force generation.

Asunto(s)

Creatina Quinasa , Músculos , Transducción de Señal , Citoesqueleto de Actina , Adenosina Trifosfato

RESUMEN

Cetylpyridinium chloride (CPC) is an antimicrobial used in numerous personal care and janitorial products and food for human consumption at millimolar concentrations. Minimal information exists on the eukaryotic toxicology of CPC. We have investigated the effects of CPC on signal transduction of the immune cell type mast cells. Here, we show that CPC inhibits the mast cell function degranulation with antigen dose-dependence and at non-cytotoxic doses ∼1000-fold lower than concentrations in consumer products. Previously we showed that CPC disrupts phosphatidylinositol 4,5-bisphosphate, a signaling lipid critical for store-operated Ca2+ entry (SOCE), which mediates degranulation. Our results indicate that CPC inhibits antigen-stimulated SOCE: CPC restricts Ca2+ efflux from endoplasmic reticulum, reduces Ca2+ uptake into mitochondria, and dampens Ca2+ flow through plasma membrane channels. While inhibition of Ca2+ channel function can be caused by alteration of plasma membrane potential (PMP) and cytosolic pH, CPC does not affect PMP or pH. Inhibition of SOCE is known to depress microtubule polymerization, and here we show that CPC indeed dose-dependently shuts down formation of microtubule tracks. In vitro data reveal that CPC inhibition of microtubules is not due to direct CPC interference with tubulin. In summary, CPC is a signaling toxicant that targets Ca2+ mobilization.

Asunto(s)

Cetilpiridinio , Mastocitos , Humanos , Cetilpiridinio/metabolismo , Cetilpiridinio/farmacología , Calcio/metabolismo , Transducción de Señal , Preparaciones Farmacéuticas/metabolismo , Señalización del Calcio

RESUMEN

The intolerance of high CO2 in the exhaust gas is the "bottleneck" limiting the wide application of microalgae for CO2 biosequestration. Around this topic, we selected high-CO2-tolerant (LAMB 33 and 31) and nontolerant (LAMB 122) Chlorella strains to study their different energy metabolisms and cytoplasmic pH regulations in response to high CO2. Under 40 % CO2, LAMB 33 and 31 both showed elevated ATP synthesis, accelerated ATP consumption and fast cytoplasmic pH regulation while exhibiting different acclimating strategies therein: chloroplast acclimations were reflected by high chlorophyll contents in 33 but photosystem transitions in 31; faster mitochondrial acclimations occurred in 33 than in 31; cellular organic carbon mainly flowed to monosaccharide synthesis for 33 but to monosaccharide and protein synthesis for 31; and cytoplasmic pH regulation was attributed to V-ATPase in 31 but not in 33. All the above metabolic processes gradually collapsed in 122, leading to growth inhibition. Our study identified different metabolic acclimation strategies among Chlorella strains to high CO2 and provided new traits for breeding microalgae for CO2 biosequestration.

Asunto(s)

Chlorella , Microalgas , Dióxido de Carbono/metabolismo , Fitomejoramiento , Microalgas/metabolismo , Carbono/metabolismo , Clorofila/metabolismo , Metabolismo Energético , Aclimatación , Concentración de Iones de Hidrógeno , Monosacáridos , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Biomasa

RESUMEN

Indole is a signalling molecule produced by many bacterial species and involved in intraspecies, interspecies, and interkingdom signalling. Despite the increasing volume of research published in this area, many aspects of indole signalling remain enigmatic. There is disagreement over the mechanism of indole import and export and no clearly defined target through which its effects are exerted. Progress is hindered further by the confused and sometimes contradictory body of indole research literature. We explore the reasons behind this lack of consistency and speculate whether the discovery of a new, pulse mode of indole signalling, together with a move away from the idea of a conventional protein target, might help to overcome these problems and enable the field to move forward.

Asunto(s)

Biopelículas/crecimiento & desarrollo , Escherichia coli/metabolismo , Indoles/metabolismo , Percepción de Quorum/fisiología , Transducción de Señal/fisiología , Transporte Biológico/fisiología , Membrana Celular/metabolismo , Escherichia coli/crecimiento & desarrollo , Transducción de Señal/genética , Estrés Fisiológico/fisiología

RESUMEN

Glucose controls the phosphorylation of silent information regulator 2 (Sir2), a NAD+ -dependent protein deacetylase, which regulates the expression of the ATP-dependent proton pump Pma1 and replicative lifespan (RLS) in yeast. TORC1 signaling, which is a central regulator of cell growth and lifespan, is regulated by glucose as well as nitrogen sources. In this study, we demonstrate that TORC1 signaling controls Sir2 phosphorylation through casein kinase 2 (CK2) to regulate PMA1 expression and cytoplasmic pH (pHc) in yeast. Inhibition of TORC1 signaling by either TOR1 deletion or rapamycin treatment decreased PMA1 expression, pHc, and vacuolar pH, whereas activation of TORC1 signaling by expressing constitutively active GTR1 (GTR1Q65L) resulted in the opposite phenotypes. Deletion of SIR2 or expression of a phospho-mutant form of SIR2 increased PMA1 expression, pHc, and vacuolar pH in the tor1Δ mutant, suggesting a functional interaction between Sir2 and TORC1 signaling. Furthermore, deletion of TOR1 or KNS1 encoding a LAMMER kinase decreased the phosphorylation level of Sir2, suggesting that TORC1 signaling controls Sir2 phosphorylation. It was also found that Sit4, a protein phosphatase 2A (PP2A)-like phosphatase, and Kns1 are required for TORC1 signaling to regulate PMA1 expression and that TORC1 signaling and the cyclic AMP (cAMP)/protein kinase A (PKA) pathway converge on CK2 to regulate PMA1 expression through Sir2. Taken together, these findings suggest that TORC1 signaling regulates PMA1 expression and pHc through the CK2-Sir2 axis, which is also controlled by cAMP/PKA signaling in yeast.

Asunto(s)

Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sirtuinas/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Citoplasma/metabolismo , Fosforilación , Proteína Fosfatasa 2/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , ATPasas de Translocación de Protón/biosíntesis , ATPasas de Translocación de Protón/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal

RESUMEN

Phosphoinositides, diacylglycerolpyrophosphate, ceramide-1-phosphate, and phosphatidic acid belong to a unique class of membrane signaling lipids that contain phosphomonoesters in their headgroups having pKa values in the physiological range. The phosphomonoester headgroup of phosphatidic acid enables this lipid to act as a pH biosensor as changes in its protonation state with intracellular pH regulate binding to effector proteins. Here, we demonstrate that binding of pleckstrin homology (PH) domains to phosphatidylinositol 4-phosphate (PI4P) in the yeast trans-Golgi network (TGN) is dependent on intracellular pH, indicating PI4P is a pH biosensor. pH biosensing by TGN PI4P in response to nutrient availability governs protein sorting at the TGN, likely by regulating sterol transfer to the TGN by Osh1, a member of the conserved oxysterol-binding protein (OSBP) family of lipid transfer proteins. Thus, pH biosensing by TGN PI4P allows for direct metabolic regulation of protein trafficking and cell growth.

Asunto(s)

Proteínas Portadoras/metabolismo , Glucosa/farmacología , Fosfatos de Fosfatidilinositol/metabolismo , Receptores de Esteroides/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Red trans-Golgi/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Transporte de Proteínas , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/crecimiento & desarrollo , Transducción de Señal , Edulcorantes/farmacología , Red trans-Golgi/efectos de los fármacos

RESUMEN

Local illumination of the characean internode with a 30-s pulse of white light was found to induce the delayed transient increase of modulated chlorophyll fluorescence in shaded cell parts, provided the analyzed region is located downstream in the cytoplasmic flow at millimeter distances from the light spot. The fluorescence response to photostimulation of a remote cell region indicates that the metabolites produced by source chloroplasts in an illuminated region are carried downstream with the cytoplasmic flow, thus ensuring long-distance communications between anchored plastids in giant internodal cells. The properties of individual stages of metabolite signaling are not yet well known. We show here that the export of assimilates and/or reducing equivalents from the source chloroplasts into the flowing cytoplasm is largely insensitive to the direction of plasma-membrane H+ flows, whereas the events in sink regions where these metabolites are delivered to the acceptor chloroplasts under dim light are controlled by H+ fluxes across the plasma membrane. The fluorescence response to local illumination of remote cell regions was best pronounced under weak background light and was also observed in a modified form within 1-2 min after the transfer of cell to darkness. The fluorescence transients in darkened cells were suppressed by antimycin A, an inhibitor of electron transfer from ferredoxin to plastoquinone, whereas the fluorescence response under background light was insensitive to this inhibitor. We conclude that the accumulation of reduced metabolites in the stroma leads to the reduction of photosystem II primary quinone acceptor (QA) via two separate (photochemical and non-photochemical) pathways.

Asunto(s)

Membrana Celular/metabolismo , Chara/metabolismo , Cloroplastos/metabolismo , Oscuridad , Protones , Antimicina A/análogos & derivados , Antimicina A/farmacología , Membrana Celular/efectos de los fármacos , Chara/efectos de los fármacos , Clorofila/metabolismo , Cloroplastos/efectos de los fármacos , Fluorescencia , Concentración de Iones de Hidrógeno , Complejo de Proteína del Fotosistema II/metabolismo , Transducción de Señal/efectos de los fármacos

RESUMEN

The combination of salt and acid is commonly used in the production of many foods, including pickles and fermented foods. However, in our previous studies, the addition of salt significantly reduced the inhibitory effect of acetic acid on Escherichia coli O157:H7 in laboratory media and pickled cucumbers. Therefore, this study was conducted to determine the mechanism by which salt confers resistance against acetic acid in E. coli O157:H7. The addition of high concentrations (up to 9% or 15% [w/v]) of salt increased the resistance of E. coli O157:H7 to acetic acid treatment. Combined treatment with acetic acid and salt showed varying results among different bacterial strains (an antagonistic effect for E. coli O157:H7 and Shigella and a synergistic effect for Listeria monocytogenes and Staphylococcus aureus). The addition of salt increased the cytoplasmic pH of E. coli O157:H7, but decreased the cytoplasmic pH of L. monocytogenes and S. aureus on treatment with acetic acid. Therefore, the addition of salt increases the acid resistance of E. coli O157:H7 possibly by increasing its acid resistance response and consequently preventing the acidification of its cytoplasm by organic acids.

Asunto(s)

Ácido Acético/farmacología , Escherichia coli O157/efectos de los fármacos , Escherichia coli O157/crecimiento & desarrollo , Cloruro de Sodio/farmacología , Concentración de Iones de Hidrógeno , Viabilidad Microbiana/efectos de los fármacos

RESUMEN

PURPOSE: The nonpigmented ciliary epithelium (NPE) is rich in soluble adenylyl cyclase (sAC), a proposed cytoplasmic bicarbonate sensor. Here, we examine the contribution of sAC to an increase in cyclic AMP (cAMP) and changes in a key ion transporter, H(+)-ATPase, in NPE exposed to acetazolamide, a carbonic anhydrase inhibitor (CAI). METHODS: Cyclic AMP was measured by radioimmunoassay in primary cultured porcine NPE. The pH-sensitive dye BCECF was used to examine cytoplasmic pH regulation. Subcellular protein translocation was examined by Western blot. RESULTS: A transient cAMP increase, detectable within minutes of acetazolamide treatment, was prevented by KH7, a specific sAC inhibitor. Following 10-minute exposure to acetazolamide, the abundance of H(+)-ATPase B1 subunit and sAC was doubled in a plasma membrane-rich fraction, suggesting subcellular translocation. Similar evidence of H(+)-ATPase translocation was observed in NPE exposed to 8-Bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP). Consistent with increased capacity for proton export, acetazolamide increased the rate of pH recovery from acidification. KH7 and bafilomycin A1, an inhibitor of H(+)-ATPase, both prevented the stimulatory effect of acetazolamide on pH recovery. In a parallel study, H(+)-ATPase abundance was found to be higher in the plasma membrane of HEK293 cells that overexpress sAC compared to the normal HEK293 cells. HEK cells that overexpress sAC and had higher H(+)-ATPase abundance displayed a faster rate of pH recovery and greater sensitivity to KH7. CONCLUSIONS: Acetazolamide increases cAMP in a response that involves activation of sAC. Subcellular translocation of H(+)-ATPase and an increase in the capacity for proton export by acetazolamide-treated NPE cells is a cAMP-dependent response.

Asunto(s)

Acetazolamida/farmacología , Adenilil Ciclasas/metabolismo , Cuerpo Ciliar/enzimología , Células Epiteliales/efectos de los fármacos , Animales , Inhibidores de Anhidrasa Carbónica/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Cuerpo Ciliar/citología , Cuerpo Ciliar/efectos de los fármacos , Células Epiteliales/citología , Células Epiteliales/enzimología , Humanos , Transporte Iónico , Porcinos

RESUMEN

The anion transport inhibitor DIDS is known to reduce aqueous humor secretion but questions remain about anion dependence of the effect. In some tissues, DIDS is reported to cause Na-K-ATPase inhibition. Here, we report on the ability of DIDS to inhibit Na-K-ATPase activity in nonpigmented ciliary epithelium (NPE) and investigate the underlying mechanism. Porcine NPE cells were cultured to confluence on permeable supports, treated with drugs added to both sides of the membrane, and then used for (86)Rb uptake measurements or homogenized to measure Na-K-ATPase activity or to detect protein phosphorylation. DIDS inhibited ouabain-sensitive (86)Rb uptake, activated Src family kinase (SFK), and caused a reduction of Na-K-ATPase activity. PP2, an SFK inhibitor, prevented the DIDS responses. In BCECF-loaded NPE, DIDS was found to reduce cytoplasmic pH (pHi). PP2-sensitive Na-K-ATPase activity inhibition, (86)Rb uptake suppression, and SFK activation were observed when a similar reduction of pHi was imposed by low-pH medium or an ammonium chloride withdrawal maneuver. PP2 and the ERK inhibitor U0126 prevented robust ERK1/2 activation in cells exposed to DIDS or subjected to pHi reduction, but U0126 did not prevent SFK activation or the Na-K-ATPase activity response. The evidence points to an inhibitory influence of DIDS on NPE Na-K-ATPase activity by a mechanism that hinges on SFK activation associated with a reduction of cytoplasmic pH.

Asunto(s)

Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Células Epiteliales/efectos de los fármacos , Proteínas del Ojo/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Familia-src Quinasas/metabolismo , Animales , Butadienos/farmacología , Activación Enzimática , Células Epiteliales/citología , Células Epiteliales/metabolismo , Proteínas del Ojo/genética , Regulación de la Expresión Génica/efectos de los fármacos , Concentración de Iones de Hidrógeno/efectos de los fármacos , Transporte Iónico/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Nitrilos/farmacología , Potasio/metabolismo , Cultivo Primario de Células , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Transducción de Señal , Sodio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Porcinos

RESUMEN

The influence of anoxia and hypoxia on dynamic of intracellurar pH and ATP content in rice and wheat root tips was investigated with (31)P-NMR spectroscopy. Both cereals responded to hypoxia similarly, by rapid cytoplasmic acidification (from pH 7.6-7.7 to 7.1), which was followed by slow partial recovery (0.3 units). Anoxia led to a dramatic pH(cyt) drop in tissues of both species (from pH 7.6-7.7 to less than 7.0) and partial recovery took place in rice only. In wheat, the acidification continued to pH 6.8 after 6 h of exposure. Anoxic wheat root tips were deficient in ADH induction, whereas increased activity of alcoholic fermentation enzymes took place in anoxic rice root tips, as well as in both species after hypoxic treatment. In both plants, NTP content followed the dynamics of pH(cyt). There was a strong correlation between NTP content and cytoplasmic H(+) activity ([H(+)](cyt) = 10(-pHcyt)) for both hypoxic and anoxic conditions. In this addendum we want to focus the reader's attention on the importance of adequate experimental design when hypoxia is under investigation and on some further perspectives of intracellular pH regulation in plants under anaerobic conditions.

RESUMEN

The role of cytosolic pH (pHc ) in growing germ tubes of the filamentous fungus Magnaporthe grisea was analysed by confocal ratio imaging of the pH-sensitive fluorescent dye 5(6)-carboxyseminaphthorhodafluor-1 (SNARF-1). The cytosol of these cells was successfully loaded with the acetoxymethyl ester of the dye and the pHc was visualized and quantified during conidium germination, germ tube growth and appressorium induction by simultaneous dual-emission confocal ratio imaging. Calibrations of the free acid in vitro and calibrations in vivo produced results indicating a similar dynamic response in the pH range 6.0-8.0 for both methods. The pHc in growing germ tubes was consistently pH 7.2±0.1 during all developmental stages analysed. Only slight changes in pHc (<0.1 pH unit) were found in response to alkaline external pH (pH 8.0). No changes in pHc occurred in response to an acidic extracellular pH (pH 6.0) or to the presence of nutrients. There was no observation of either pronounced gradients or changes in pHc in growing germ tubes accompanying conidium germination, germ tube growth or early appressorium formation.

RESUMEN

A method was established to measure the cytoplasmic pH of the facultative alkaliphilic strain, Bacillus lentus C-125. The bacterium was loaded with a pH-sensitive fluorescent probe, 2',7'-bis-(2-carboxyethyl)-5 (and -6)-carboxyfluorescein (BCECF), and cytoplasmic pH was determined from the intensity of fluorescence of the intracellular BCECF. The activity of the organism to maintain neutral cytoplasmic pH was assessed by measuring the cytoplasmic pH of the cells exposed to various pH conditions. The cytoplasmic pH maintenance activity of C-125 increased with increasing culture pH, indicating that the activity was regulated in response to the culture pH.