Multi-substrate growth kinetics of Pseudomonas putida for phenol removal

The biodegradation of phenol by a pure culture of Pseudomonas putida was investigated in a continuously fed stirred-tank reactor, under aerobic conditions. The dilution rate was varied between 0.0174 h⁻¹ and 0.278 h⁻¹, covering a wide range of dissolved oxygen and the inhibition region of phenol. Through non-linear analysis of the data, a dual-substrate growth kinetics, Haldane kinetics for phenol and Monod kinetics for oxygen, was derived with high correlation coefficients. Respective biokinetic parameters were evaluated as μ_m = 0.569 h⁻¹, K _p = 18.539 mg/l, K _i = 99.374 mg/l, K _o = 0.048 mg/l, Y _x/p = 0.521 g microorganism/g phenol and Y _x/o = 0.338 g microorganism/g oxygen, being in good agreement with other studies in the literature. Maintenance factors for both phenol and oxygen were calculated for the first time for P. putida while the saturation coefficient for oxygen, K _o, was genuinely evaluated from the constructed model, not imported or adapted from other studies as reported in the literature. All pertinent biokinetic parameters for P. putida have been calculated from continuous system data, which are most appropriate for use in continuous bioprocess applications. Received: 29 July 1996 / Received revision: 18 November 1996 / Accepted: 23 November 1996     

Mechanism for phenol tolerance in phenol-degrading Comamonas testosteroni strain

Comamonas testosteroni P15 and its mutant strain E23 can tolerate and utilize phenol as the sole source of carbon and energy at up to 15 mM and 20 mM, respectively. Compared to the wild type P15, mutant E23 showed higher values of K _s and K _i but a lower μ_max value, and had lower phenol hydroxylase and catechol 2,3-dioxygenase activities. Without phenol exposure, mutant E23 demonstrated a two-fold greater amount of cardiolipin than the wild type P15. Upon exposure to phenol, an increase in cardiolipin at the expense of phosphatidylethanolamine was observed in the wild type P15. However, there was no significant difference in major phospholipid contents between mutant E23 cells grown in the presence or absence of phenol. It was noted that the ratio of trans/cis fatty acids of phosphatidylethanolamine and cardiolipin in mutant E23 was 65–70% higher than that in the wild type P15. In the absence of phenol, the degree of saturation of cardiolipin in mutant E23 was 33% higher than that in wild type P15. In contrast to earlier findings, an increase in C16:1 9trans with a simultaneous decrease in C18:1 11cis instead of C16:1 9cis was observed in specific classes of phospholipids. Received: 30 July 1998 / Received last revision: 16 November 1998 / Accepted: 12 December 1998     

Isochorismate hydroxymutase from a cell-suspension culture of Galium mollugo L.

Isochorismate hydroxymutase (i.e. isochorismate synthase, EC 5.4.99.6) was purified from an anthraquinone-producing cell-suspension culture of Galium mollugo L. Although attempts to stabilize the labile enzyme met with little success, a substantial increase in enzyme activity was observed in the presence of glycine betaine (500 mM). Column chromatography on solid supports other than diethylaminoethyl (DEAE)-Sephacel, Phenylsepharose Cl-4B or Cibacron Blue 3G-A did not give active enzyme preparations. In spite of these drawbacks the enzyme was purified 573-fold. Enzyme activity depended strictly on the presence of Mg²⁺. Kinetic data for chorismate in the forward reaction (K _m = 807 μM, V _max = 6.2 pkat · mg⁻¹) and for isochorismate in the reverse reaction (K _m = 675 μM, V _max = 5.9 pkat · mg⁻¹) were determined. Received: 18 November 1996 / Accepted: 28 December 1996     

Purification and characterization of a glucokinase from young tomato (Lycopersicon esculentum L. Mill.) fruit

In order to clearly establish the properties of the enzymes responsible for hexose phosphorylation we have undertaken the separation and characterization of these enzymes present in tomato fruit (Martinez-Barajas and Randall 1996). This report describes the partial purification and characterization of glucokinase (EC. 2.7.1.1) from young green tomato fruit. The procedure yielded a 360-fold enrichment of glucokinase. Tomato fruit glucokinase is a monomer with a molecular mass of 53 kDa. Glucokinase activity was optimal between pH 7.5 and 8.5, preferred ATP as the phosphate donor (K _m = 0.223 mM) and exhibited low activity with GTP or UTP. The tomato fruit glucokinase showed highest affinity for glucose (K _m = 65 μM). Activity observed with glucose was 4-fold greater than with mannose and 50-fold greater than with fructose. The tomato fruit glucokinase was sensitive to product inhibition by ADP (K _i = 36 μM). Little inhibition was observed with glucose 6-phosphate (up to 15 mM) at pH 8.0; however, at pH 7.0 glucokinase activity was inhibited 30–50% by physiological concentrations of glucose 6-phosphate. Received: 4 October 1997 / Accepted: 10 January 1998     

Purification and properties of a novel raw starch degrading cyclomaltodextrin glucanotransferase from Bacillus firmus

A novel raw starch degrading cyclomaltodextrin glucanotransferase (CGTase; E.C. 2.4.1.19), produced by Bacillus firmus, was purified to homogeneity by ultrafiltration, affinity and gel filtration chromatography. The molecular weight of the pure protein was estimated to be 78 000 and 82 000 Da, by SDS-PAGE and gel filtration, respectively. The pure enzyme had a pH optimum in the range 5.5–8.5. It was stable over the pH range 7–11 at 10 °C, and at pH 7.0 at 60 °C. The optimum temperature for enzyme activity was 65 °C. In the absence of substrate, the enzyme rapidly lost its activity above 30 °C. K _m and k _cat for the pure enzyme were 1.21 mg/ml and 145.17 μM/mg per minute respectively, with soluble starch as the substrate. For cyclodextrin production, tapioca starch was the best substrate used when gelatinized, while wheat starch was the best substrate used when raw. This CGTase could degrade raw wheat starch very efficiently; up to 50% conversion to cyclodextrins was obtained from 150 g/l starch without using any additives. The enzyme produced α-, β- and γ-cyclodextrins in the ratio of 0.2:9.2:0.6 and 0.2:8.6:1.2 from gelatinized tapioca starch and raw wheat starch with 150 g/l concentration respectively, after 18 h incubation. Received: 25 September 1998 / Received revision: 15 December 1998 / Accepted: 21 December 1998     

Mode of depolymerisation of hemicellulose by various mannanases and xylanases in relation to their ability to bleach softwood pulp

Endo-mannanases and endo-xylanases cleave different heteromannans and xylans yielding mainly dimers and trimers of the corresponding sugars as end-products. However, in the early stages of hydrolysis, four purified mannanases and four xylanases from fungal and bacterial origin, examined in this study, showed a different pattern of released oligomers (determined up to the pentamers). Furthermore, some of these enzymes showed a preference for cleaving the polysaccharides in the middle of the chain while others acted more at the end. When the increase in the specific fluidity of mannan and xylan solutions per reducing sugar released (K _v) was measured against the bleaching effect of the enzymes on softwood kraft pulp, a correlation was found. A xylanase from Penicillium simplicissimum (K _v = 0.15 l mPa⁻¹s⁻¹g⁻¹) and a mannanase from Sclerotium rolfsii (K _v = 0.12 l mPa⁻¹s⁻¹g⁻¹) applied in a O(QX)P bleaching sequence (O = oxygen delignification, X = treatment with hemicellulolytic enzymes, Q = chelation of metals, P = treatment with hydrogen peroxide in alkaline solution) gave a high brightness increase of 3.0% and 1.9% ISO respectively. A less significant brightness increase was obtained with enzymes showing lower K _v values, such as a xylanase from Schizophyllum commune (K_v = 0.051  l mPa⁻¹s⁻¹g⁻¹, 0.2% ISO) and a bacterial mannanase (K _v = 0.061 l mPa⁻¹s⁻¹g⁻¹,0.5% ISO). Received: 19 December 1996 / Received revision: 20 February 1997 / Accepted: 22 February 1997     

Equilibrium dialysis study and mechanistic implications of coenzyme A binding to acetyl-CoA synthase/carbon monoxide dehydrogenase from Clostridium thermoaceticum

Clostridium thermoaceticum were determined by equilibrium dialysis. CoA bound to as-isolated native α ₂ β ₂ enzyme with K _D = 10 ± 8 μM and n = 0.2 ± 0.1 moles per αβ dimer, where K _D is the thermodynamic dissociation constant and n is the number of CoAs bound per αβ dimer of the enzyme. The enzyme is heterogeneous; for example, only  ∼ 30% of α subunits contain A-clusters with labile Ni ions (the remainder have nonlabile Ni ions and are nonfunctional). The observed n value suggests that CoA binds only to αβ units with Ni-labile A-clusters. The CoA binding properties of enzyme lacking labile Ni was essentially the same, indicating that CoA does not bind directly to the Ni of the A-cluster. This was further evidenced by the observation that bound CoA did not inhibit removal of the labile Ni by 1,10-phenanthroline. CoA did not bind CO-reduced enzyme, and the EPR signal exhibited by the one-electron reduced and CO-bound form of the A-cluster was unaffected by the presence of up to 200 μM CoA. In contrast, CoA did bind Ti(III)-citrate-reduced enzyme (K _D = 36 ± 16 μM, n = 0.16 ± 0.08). Implications of these results for the mechanism of catalysis are discussed. Received: 29 March 1999 / Accepted: 8 September 1999     

Ion channels in guard cells of Arabidopsis thaliana (L.) Heynh.

Despite the availability of many mutants for signal transduction, Arabidopsis thaliana guard cells have so far not been used in electrophysiological research. Problems with the isolation of epidermal strips and the small size of A. thaliana guard cells were often prohibiting. In the present study these difficulties were overcome and guard cells were impaled with double-barreled microelectrodes. Membrane-potential recordings were often stable for over half an hour and voltage-clamp measurements could be conducted. The guard cells were found to exhibit two states. The majority of the guard cells had depolarized membrane potentials, which were largely dependent on external K⁺ concentrations. Other cells displayed spontaneous transitions to a more hyperpolarized state, at which the free-running membrane potential (E_m) was not sensitive to the external K⁺ concentration. Two outward-rectifying conductances were identified in cells in the depolarized state. A slow outward-rectifying channel (s-ORC) had properties resembling the K⁺-selective ORC of Vicia faba guard cells (Blatt, 1988, J Membr Biol 102: 235–246). The activation and inactivation times and the activation potential, all depended on the reversal potential (E_rev) of the s-ORC conductance. The s-ORC was blocked by Ba²⁺ (K_1/2 = 0.3–1.3mM) and verapamil (K_1/2 = 15–20 μM). A second rapid outward-rectifying conductance (r-ORC) activated instantaneously upon stepping the voltage to positive values and was stimulated by Ba²⁺. Inward-rectifying channels (IRC) were only observed in cells in the hyperpolarized state. The activation time and activation potential of this channel were not sensitive to the external K⁺ concentration. The slow activation of the IRC (t_1/2 ≈ 0.5 s) and its negative activation potential (V_threshold = −155 mV) resemble the values found for the KAT1 channel expressed in Saccharomyces cerevisiae (Bertl et al., 1995, Proc Natl Acad Sci USA 92: 2701–2705). The results indicate that A. thaliana guard cells provide an excellent system for the study of signal transduction processes. Received: 28 March 1996 / Accepted: 11 November 1996     

pH-mediated release of betalains from transformed root cultures of Beta vulgaris L.

Brief exposure of Beta vulgaris root cultures to acidic medium resulted in release of betalain pigments while the capability for regrowth and continued pigment accumulation was retained. A 10-min exposure to pH 2 followed by return to standard growth medium (pH 5.5, 1.1 mM PO₄) resulted in release of 0.59 mg pigment/g dry weight over the subsequent 24-h period. The released pigment corresponds to 36.8% of the total pigments. Further improvement in culture productivity was achieved through phosphate limitation. Specific pigment productivity increased fivefold for cultures grown in phosphate-free medium as compared to cultures grown in control medium (1.1 mM PO₄). A maximum total pigment production of 25.2 mg/l was observed at an initial medium phosphate level 0.3 mM. When combined with phosphate limitation, low pH facilitated the release of 3.03 mg pigment/g dry weight, which corresponds to 50% of the total pigment. The permeabilized roots were capable of regrowth and continued pigment accumulation. A cytochemical assay for respiratory activity revealed that the basis of regrowth was lateral root initials that were unaffected during the acidic pH treatment. Received: 16 December 1997 / Received revision: 7 May 1998 / Accepted: 16 May 1998     

Biodegradation of methyl violet by Pseudomonas mendocina MCM B-402

Pseudomonas mendocina MCM B-402 was found to utilize a triphenylmethane dye, methyl violet as the sole source of carbon when incorporated in synthetic medium. Almost complete decolorization of methyl violet by P. mendocina was observed within 48 h of incubation at ambient temperature (28 ± 2 °C) under aerated culture conditions, when the bacteria were inoculated into Davis Mingioli's synthetic medium at a concentration of 100 mg/l medium. Methyl violet was mineralized to CO₂ through three unknown intermediate metabolites and phenol. The decolorization of the dye involved demethylation. Received: 27 November 1998 / Received revision: 2 March 1999 / Accepted: 5 March 1999     

Production of sophorolipids from whey: development of a two-stage process with Cryptococcus curvatus ATCC 20509 and Candida bombicola ATCC 22214 using deproteinized whey concentrates as substrates

In order to produce sophorolipids from whey, thereby lowering the lactose content and biological oxygen demand, a two-step batch cultivation process was developed including medium sterilization by filtration. In the first step, whey was sterilized by a combination of crossflow and sterile filtration. Because the sophorolipid-producing yeast Candida bombicola ATCC 22214 was not able to use lactose as a carbon source directly, the oleaginous yeast Cryptococcus curvatus ATCC 20509 was grown on deproteinized whey concentrates (DWC). With 1: 1 diluted DWC-20, lactose was consumed as the carbon source and biomass (24 g/l dry weight content) as well as single-cell oil (SCO, 10 g/l) were produced. The cultivation broth was disrupted with a glass bead mill and it served as medium for growth (29 g cell dry mass/l) and sophorolipid production (12 g/l) of the yeast C. bombicola. Received: 29 July 1998 / Received revision: 5 October 1998 / Accepted: 11 October 1998     

Formation of benzaldehyde by Pseudomonas putida ATCC 12633

Aromatic and heterocyclic aldehydes may be produced by the mandelate pathway of Pseudomonas putida ATCC 12633 via the biotransformation of benzoyl formate and substrate analogues. Under optimised biotransformation conditions (37 °C, pH 5.4) and with benzoyl formate as a substrate, benzaldehyde may be accumulated with yields above 85%. Benzaldehyde is toxic to P. putida ATCC 12633; levels above 0.5 g/l (5 mM) reduce the biotransformation activity. Total activity loss occurs at an aldehyde concentration of 2.1 g/l (20 mM). To overcome this limitation, the rapid removal of the aldehyde is desirable via in situ product removal. The biotransformation of benzoyl formate (working volume 1 l) without in situ product removal accumulates 2.1 g/l benzaldehyde. Benzaldehyde removal by gas stripping produces a total of 3.5 g/l before inhibition. However, the most efficient method is solid-phase adsorption using activated charcoal as the sorbant, this allows the production of over 4.1 g/l benzaldehyde. Addition of bisulphite as a complexing agent causes inhibition of the biotransformation and bisulphite is therefore is not suitable for in situ product removal. Received: 16 March 1998 / Received revision: 20 May 1998 / Accepted: 21 May 1998     

Effects of culture conditions on β-poly(l-malate) production by Physarum polycephalum

Culture conditions for the fermentative production of β-poly(l-malate) (PMLA) by microplasmodia of Physarum polycephalum were investigated and optimized. Optimal production was achieved in the presence of CaCO₃. For 1.5% (w/v) d-glucose, 1% bactotryptone and 1% CaCO₃, a maximum of 1.7 g PMLA/l was secreted in 3 days. For 4.5% glucose and otherwise the same conditions, 2.7 g PMLA/l was produced in 6 days. The contribution of carbonate was inhibited by avidin. PMLA and biomass production were not strictly coupled: PMLA was also synthesized at the beginning of the stationary phase. At pH 5.5 PMLA production was twice that at pH 4.0, while biomass was not changed. Optimal temperatures were 24–28 °C. Received: 12 November 1998 / Received revision: 10 February 1999 / Accepted: 12 February 1999     

Production of cellulase by a wild strain of Chaetomium globosumusing delignified oil palm empty-fruit-bunch fibre as substrate

Studies on the feasibility of using delignified oil palm empty-fruit-bunch (OPEFB) fibres as a substrate for cellulase production by Chaetomium globosum strain 414 were carried out in shake-flask cultures containing different types and concentrations of nitrogen source. Peptone, as nitrogen source, gave maximum production of all the three main components of the cellulase complex (endoglucanase or carboxymethylcellulase, cellobiohydrolase or filter-paper-hydrolysing enzyme and β-glucosidase), followed by yeast extract, urea, KNO₃ and (NH₄)₂SO₄. The maximum specific growth rate (μ_max) of C. globosum strain 414 grown in medium containing OPEFB and peptone was 0.038 h⁻¹. In all the fermentations, the fungus was able to produce all the three cellulases with significant amounts of β-glucosidase, except when using (NH₄)₂SO₄ as nitrogen source, where β-glucosidase was not produced. With 6 g/l peptone and 10 g/l delignified OPEFB fibres, the fungus produced maximum concentrations of FPase, carboxymethylcellulase and β-glucosidase: 1.4, 30.8 and 9.8 U/ml, giving productivities of 10, 214 and 24 U l⁻¹h⁻¹, respectively. The cellulase mixture, partially purified by ammonium sulphate precipitation, was able to hydrolyse delignified OPEFB fibres, converting about 68 % of the cellulosics to reducing sugars after 5 days. Received: 17 June 1996 / Received revision: 18 November 1996 / Accepted: 23 November 1996     

Poly(hydroxyalkanoate) biosynthesis from triglyceride substrates

The biosynthesis of poly(hydroxyalkanoates) (PHA) by Pseudomonas resinovorans from triglyceride substrates was investigated. Each triglyceride, whether animal fat or vegetable oil, supported cellular growth to relatively high average cell yields (3.3 ± 0.2 g/l). PHA yields ranged from 1.1 g/l to 2.1 g/l, representing approximately 45% of the bacterial cell dry weight. The repeat-unit composition of the polymers was determined by gas chromatography (GC) and GC/mass spectrometry of the β-hydroxyalkanoate methyl esters from the hydrolyzed polymers. With the exception of PHA from soybean oil (PHA-soy), each polyester was composed of β-hydroxyacyl moieties with chain lengths ranging from C₄ to C₁₄, with C₈ and C₁₀ being the predominant species. PHA-soy contained an additional fraction (2%) of C₁₆ monomers. The alkyl side-chains of the PHA contained varying degrees of unsaturation. PHA from coconut oil was composed entirely of saturated side-chains, whereas PHA-soy contained 4.2 mol% olefinic groups in its side-chains. The increase in the degree of side-chain unsaturation caused decreased melting temperatures, enthalpies of fusion, and glass transition temperatures. The molar masses of the polymers were relatively constant and ranged from 6.5 × 10⁴ to 10.1 × 10⁴ g/mol. Received: 2 September 1997 / Received revision: 21 November 1997 / Accepted: 2 January 1998     

Metabolism of EDTA and its metal chelates by whole cells and cell-free extracts of strain BNC1

The influence of metal ions on the metabolism of ethylenediaminetetraacetate (EDTA) by whole cells and cell-free extracts of strain BNC1 was investigated. Metal-EDTA chelates with thermodynamic stability constants below 10¹² were readily mineralized by whole cells with maximum specific turnover rates of 15 (MnEDTA) to 20 (Ca-, Mg-, and BaEDTA) μmol g protein⁻¹ min⁻¹. With the exception of ZnEDTA, chelates with stability constants greater than 10¹² were not oxidized at a significant rate. However, it was shown for Fe(III)EDTA that even strong complexes can be degraded after pretreatment by addition of calcium and magnesium salts in the pH range 9–11. The range of EDTA chelates converted by cell-free extracts of strain BNC1 did not depend on their thermodynamic stabilities. The EDTA chelates of Ba²⁺, Co²⁺, Mg²⁺, Mn²⁺, and Zn²⁺ were oxidized whereas Ca-, Cd-, Cu-, Fe-, Pb-, and SnEDTA were not. The first catabolic enzyme appears to be an EDTA monooxygenase since it requires O₂, NADH, and FMN for its activity and yields glyoxylate and ethylenediaminetriacetate as products. The latter is further degraded via N,N′-ethylenediaminediacetate. The maximum specific turnover rate with MgEDTA, the favoured EDTA species, was 50–130 μmol g protein⁻¹ min⁻¹, and the K _m value was 120 μmol/l (K _s for whole cells = 8 μmol/l). Whole cells as well as cell-free extracts of strain BNC1 also converted several structural analogues of EDTA. Received: 4 July 1997 / Received revision: 25 September 1997 / Accepted: 29 September 1997     

Production of ketocarotenoids by microalgae

Among the highly valued ketocarotenoids employed for food coloration, astaxanthin is probably the most important. This carotenoid may be produced biotechnologically by a number of microorganisms, and the most promising seems to be the freshwater flagellate Haematococcus pluvialis (Chlorophyceae), which accumulate astaxanthin in their aplanospores. Many physiological aspects of the transition of the flagellate into aplanospores have been described. Mixotrophic cultivation and suitable irradiance may result in fairly good yields (up to 40 mg/l; 43 mg/g cell dry weight) within a reasonable time, under laboratory conditions. In order to compete with synthetic astaxanthin, suitable scaling-up is required. However, large-scale production in open ponds has proved unsatisfactory because of severe contamination problems. A selective medium might overcome this difficulty. Further research for the development of suitable strains is thus warranted. Received: 8 July 1998 / Received revision: 12 November 1998 / Accepted: 14 November 1998     

Biochemical basis for carbon monoxide tolerance and butanol production by Butyribacterium methylotrophicum

The biochemical mechanisms for growth tolerance to a 100% CO headspace in cultures, and butanol plus ethanol production from CO by Butyribacterium methylotrophicum were assessed in the wild-type and CO-adapted strains. The CO-adapted strain grew on glucose or CO under a 100% CO headspace, whereas, the growth of the wild-type strain was severely inhibited by 100% CO. The CO-adapted strain, unlike the wild-type, also produced butyrate, from either pyruvate or CO. The CO-adapted strain was a metabolic mutant having higher levels of ferredoxin–NAD oxidoreductase activity, which was not inhibited by NADH. Consequently, only the CO-adapted strain can grow on CO because CO oxidation generates reduced ferredoxin which, via the mutated ferredoxin–NAD reductase activity, forms reduced NADH required for catabolism. When the CO-adapted strain was grown at pH 6.0 it produced butanol (0.33 g/l) and ethanol (0.5 g/l) from CO and the cells contained the following NAD-linked enzyme activities (μmol min⁻¹ mg protein⁻¹): butyraldehyde dehydrogenase (227), butanol dehydrogenase (686), acetaldehyde dehydrogenase (82) and ethanol dehydrogenase (129). Received: 15 September 1998 / Received revision: 12 February 1999 / Accepted: 19 February 1999     

A novel method for characterisation of microbial growth kinetics on volatile organic compounds

A novel method for the determination of microbial growth kinetics on hydrophobic volatile organic compounds (VOC) has been developed. A stirred tank reactor was operated as a fed-batch system to which the VOC was continuously fed via the gas phase, assuring a constant VOC concentration in the mineral medium. A flow of air was saturated with the VOC, and then mixed with a further flow of air, to obtain a predetermined VOC concentration. Thus, different VOC concentrations in the mineral medium could be obtained by altering the VOC concentration in the feed gas. The growth kinetics of Xanthobacter autotrophicus GJ10 on 1,2-dichloroethane (DCE) and of Pseudomonas sp. strain JS150 on MonoChloroBenzene (MCB) were assessed using this method. The growth of strain JS150 was strongly inhibited at MCB concentrations higher than 160 mg l⁻¹, and the results were fitted using a piecewise function. The growth kinetics of strain GJ10 were described by the Luong model where maximum growth rate μ_max = 0.12 h⁻¹, substrate saturation constant K _S = 7.8 mg l⁻¹, and maximum substrate concentration S _m (above which growth is completely inhibited) = 1080 mg l⁻¹. Varying nitrogen and oxygen flows enabled the effect of oxygen concentration on the growth kinetics of Pseudomonas JS150 to be determined. Received: 30 November 1998 / Received revision: 19 March 1999 / Accepted: 20 March 1999     

Auxin augments conductance of K+ inward rectifier in maize coleoptile protoplasts

Potassium is taken up by maize (Zea mays L.) coleoptile cells via a typical plant inward rectifier (K _ir ). Sufficient conductance of this channel is essential in order to maintain auxin-stimulated cell elongation. It was therefore investigated whether the activity of this channel is subject to direct or indirect control by this growth hormone. Patch-clamp measurements of whole coleoptile protoplasts revealed no appreciable effect of externally applied 10 μM or 100 μM α-naphthaleneacetic acid (NAA) on the activity of K _ir over test periods of ≥ 18 or ≥ 8 min, respectively. When, however, K _ir was recorded in the cell-attached configiuration and 10 μM NAA administered to the bath medium, the conductance of K _ir increased significantly in 13 out of 18 protoplasts over the control. This rise occurred at a fixed protoplast voltage after a lag period of less than 10 min and exhibited no voltage dependency. The absence of response to NAA of protoplasts in the whole-cell configuration indicates that auxin perception and channel control is linked via a soluble cytoplasmic factor and that this mediator is washed out or modified upon perfusion of the cytoplasm with pipette solution. To search for this expected diffusible factor the K _ir current was recorded before and after elevation of Ca²⁺ and H⁺ in the cytoplasm. In the whole-cell configuration the increase in Ca²⁺ from a nanomolar value to >1 μM by means of Ca²⁺-release from the caged precursor Na₂-DM-nitrophen left K _ir unaffected. The whole-cell K _ir conductance was also not affected upon addition of 10 mM Na⁺-acetate to the bath medium, an operation used to lower the cytoplasmic pH. This excludes a primary role for the known auxin-evoked rise in cytoplasmic Ca²⁺ and H⁺ in K _ir activity. We postulate that another, as yet unknown, mechanism mediates the auxin-evoked stimulation of the number of active K _ir channels in the plasma membrane. Received: 13 May 1998 / Accepted: 9 November 1998