Effect of lipopolysaccharide mutation on oxygenation of linoleic acid by recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> expressing CYP102A2 of <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

The effects of cell wall mutation on the oxygenation of linoleic acid (M.W. 280) by recombinant Escherichia coli expressing the CYP102A2 gene encoding self-sufficient P450 monooxygenase of Bacillus subtilis was investigated. After the CYP102A2 gene was heterologously expressed in E. coli W3110 and its isogenic lipopolysaccharide (LPS) structural mutant strains, their whole-cell biotransformation activities were compared. The mutants used in this study had previously been designated as MLK53, MLK1067, and MLK986. These strains carry one or two defined mutations in the secondary acyl fatty acids of the LPS lipid A constituent. The CYP102A2 gene was overexpressed in both wild type E. coli W3110 and its mutant strains, with the specific activity ranging from 1.7 to 2.1 U/mg protein. Interestingly, the whole-cell biotransformation activity of those recombinant biocatalysts differed significantly. Indeed, MLK986 possessing the tetraacylated LPS showed a higher oxygenation activity of linoleic acid than those in wild type or other mutant strains having hexa- or penta-acylated LPSs. These results suggest that the biotransformation efficiency of E. coli-based biocatalysts, especially for medium- to large-sized lipophilic organic substrates, can be enhanced via engineering their LPS, which is known to function as a formidable barrier for hydrophobic molecules.     

Deletion of Cg-emb in corynebacterianeae leads to a novel truncated cell wall arabinogalactan, whereas inactivation of Cg-ubiA results in an arabinan-deficient mutant with a cell wall galactan core

The cell wall of Mycobacterium tuberculosis has a complex ultrastructure that consists of mycolic acids connected to peptidoglycan via arabinogalactan (AG) and abbreviated as the mAGP complex. The mAGP complex is crucial for the survival and pathogenicity of M. tuberculosis and is the target of several anti-tubercular agents. Apart from sharing a similar mAGP and the availability of the complete genome sequence, Corynebacterium glutamicum has proven useful in the study of orthologous M. tuberculosis genes essential for viability. Here we examined the effects of particular genes involved in AG polymerization by gene deletion in C. glutamicum. The anti-tuberculosis drug ethambutol is thought to target a set of arabinofuranosyltransferases (Emb) that are involved in arabinan polymerization. Deletion of emb in C. glutamicum results in a slow growing mutant with profound morphological changes. Chemical analysis revealed a dramatic reduction of arabinose resulting in a novel truncated AG structure possessing only terminal arabinofuranoside (t-Araf) residues with a corresponding loss of cell wall bound mycolic acids. Treatment of wild-type C. glutamicum with ethambutol and subsequent cell wall analyses resulted in an identical phenotype comparable to the C. glutamicum emb deletion mutant. Additionally, disruption of ubiA in C. glutamicum, the first enzyme involved in the biosynthesis of the sugar donor decaprenol phosphoarabinose (DPA), resulted in a complete loss of cell wall arabinan. Herein, we establish for the first time, (i) that in contrast to M. tuberculosis embA and embB mutants, deletion of C. glutamicum emb leads to a highly truncated AG possessing t-Araf residues, (ii) the exact site of attachment of arabinan chains in AG, and (iii) DPA is the only Araf sugar donor in AG biosynthesis suggesting the presence of a novel enzyme responsible for "priming" the galactan domain for further elaboration by Emb, resulting in the final maturation of the native AG polysaccharide.     

Myo-inositol facilitators IolT1 and IolT2 enhance d-mannitol formation from d-fructose in Corynebacterium glutamicum

Reduction of d -fructose to d -mannitol by whole-cell biotransformation with recombinant resting cells of Corynebacterium glutamicum ATCC13032 requires the coexpression of mdh and fdh , which encode mannitol and formate dehydrogenases, respectively. However, d -mannitol formation is limited by the uptake of d -fructose in its unphosphorylated form, because additional expression of the sugar facilitator from Zymomonas mobilis resulted in a significantly increased productivity. Here we identified similarities of the myo -inositol transporters IolT1 and IolT2 of C. glutamicum to the sugar facilitator of Z. mobilis . The myo -inositol transporter genes were both individually overexpressed and deleted in recombinants expressing mdh and fdh . Biotransformation experiments showed that the presence and absence, respectively, of IolT1 and IolT2 significantly influenced d -mannitol formation, indicating a d -fructose transport capability of these transporters. For further evidence, a C. glutamicum Δ ptsF mutant unable to grow with d -fructose was complemented with a heterologous fructokinase gene. This resulted in restoration of growth with d -fructose. Using overexpressed iolT1, mdh and fdh , d -mannitol formation obtained with C. glutamicum was 34.2 g L⁻¹, as opposed to 16 g L⁻¹ formed by the strain overexpressing only mdh and fdh , showing the suitability of myo -inositol transporters for d -fructose uptake to obtain d -mannitol formation by whole-cell biotransformation with C. glutamicum .     

Fluorescent phospholipid analogs as microscopic probes for detection of the mycolic acid-containing layer in Corynebacterium glutamicum: detecting alterations in the mycolic acid-containing layer following ethambutol treatment

Corynebacterium glutamicum belongs to the mycolic acid-containing actinomycetes, which also include Mycobacterium, Nocardia, and Rhodococcus. The cells of this group possess a cell wall with a thick outer layer composed primarily of mycolic acid, which functions as a permeability barrier. To investigate the mechanism of mycolic acid-containing layer (mycolate layer) formation, we have developed a fluorescence microscopic technique detecting the mycolate layer in situ. The staining specificity of fluorescence-labeled phospholipid analogs was determined by simultaneous staining with the hydrophobic fluorescent dye Nile Red and peptidoglycan-staining fluorescence-conjugated vancomycin. We found that fluorescence-labeled phospholipid analogs preferentially stain the mycolate layer. Using this technique, we observed the effect of the anti-mycobacterial drug ethambutol on C. glutamicum mycolate-layer formation. Ethambutol interfered specifically with mycolate-layer formation on the division planes and cell poles, while the side-wall mycolate layer was not severely affected. This indicates that mycolate-layer formation occurs mainly on division planes and cell poles in C. glutamicum, where the peptidoglycan layer is actively synthesized.     

Oxidative biotransformations by microorganisms: production of chiral synthons by cyclopentanone monooxygenase fromPseudomonas sp. NCIMB 9872

Cyclopentanone monooxygenase, an NADPH- plus FAD-dependent enzyme induced by the growth ofPseudomonas sp. NCIMB 9872 on cyclopentanol, has been utilised as a biocatalyst in Baeyer-Villiger oxidations. Washed whole-cell preparations of the microorganism oxidised 3-hexylcyclopentanone in a regio- but not enantioselective manner to give predominantly the racemic γ-hexyl valerolactone. similar preparations biotransformed 5-hexylcyclopent-2-enone exclusively by regio- plus enantioselective oxidation to the equivalent , β-unsaturated (S)-(+)-δ-hexyl valerolactone (ee = 78%), with no reductive biotransformations catalysed by either EC 1.1.x.x- or EC 1.3.x.x-type dehydrogenases.

An equivalent biotransformation of 5-hexylcyclopent-2-enone was catalysed by highly-purified NADPH- plus FAD-dependent cyclopentanone monooxygenase from the bacterium. The regio- plus enantioselective biotransformation by the pure enzyme of 2-(2′-acetoxyethyl)cyclohexanone yielded optically-enriched (S)-(+ )-7-(2′-acetoxyethyl)-2-oxepanone (ee = 72%). The same biotransformation when scaled up again provided optically-enriched (S)-(+)-ε-caprolactone which was converted, using methoxide, to (S)-(−)-methyl 6,8-dihydroxyoctanoate (ee = 42%). thereby providing a two-step access from the substituted cyclohexanone to this important chiron for the subsequent synthesis of (R-(+)-lipoic acid.

Some characteristics of pure NADPH- plus FAD-dependent cyclopentanone monooxygenase were determined including the molecular weight of the monomeric subunit (50000) of this homotetrameric enzyme, and the N-terminal amino acid sequence up to residue 29, which includes a putative flavin nucleotide-binding site.     

The ultrastructure and ATPase nature of polar membrane in Campylobacter jejuni

Polar membrane in Campylobacter jejuni has been visualized on membrane vesicles. It was composed of doughnut-shaped particles 5-6 nm in diameter, with stalks, arranged in a hexagonal array. This structure was stabilized on the membrane by a high ionic strength buffer in the presence of 2-mercaptoethanol. Histochemical staining indicated localized ATPase activity at the poles of the cells. An ATPase with distinctive properties has been isolated and purified from this organism; it gives a specific activity of approximately 0.3 units/mg of protein. Electron microscopy showed doughnut-shaped particles 5-6 nm in diameter. Nondissociating and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed, respectively, a single band with ATPase activity and a molecular weight of ca. 75,000 Da. The enzyme was cold labile and activity was abolished by trypsin. Dicyclohexylcarbodiimide inhibited the membrane-bound form of the enzyme, but did not inhibit the soluble form. Oligomycin had no inhibitory activity on either form of the enzyme. The enzyme specifically hydrolysed ATP, but other nucleotide substrates were not degraded. The enzyme was activated by Mg2+ and inhibited by Ca2+, whereas other ions had no effect on activity. Antibodies prepared to this enzyme bound to the polar regions of whole cells as shown by protein A - colloidal gold immunoelectron microscopy. The antibodies to this ATPase cross reacted (shown by Western blotting) with four proteins from a whole-cell extract of this organism, two proteins in Aquaspirillum serpens MW5, and three proteins from Escherichia coli K12. They did not cross-react with any proteins from Spirillum volutans, Methanococcus voltae, Vibrio cholerae, or rat liver mitochondria. Antibodies raised against the F1-ATPase of E. coli K12 cross reacted with six proteins in a whole-cell extract of this organism, and one protein species in each of the whole-cell extracts of V. cholera, A. serpens MW5, S. volutans, and rat liver mitochondria. These antibodies did not recognize any whole cell proteins from either C. jejuni or M. voltae. These results along with the ATPase activity localized by histochemical staining suggest that polar membrane is an assembly of ATPase molecules at the poles of the cell and that the ATPase isolated from C. jejuni is serologically and structurally unusual.     

Enhanced biotransformation of sitosterol to androstenedione by <Emphasis Type="Italic">Mycobacterium</Emphasis> sp. using cell wall permeabilizing antibiotics

Mycobacterial cell wall is rigid and offers a high resistance to the transport of sitosterol into cytosol. The effect of ethambutol, penicillin, polymixin and bacitracin on biotransformation of sitosterol to androstenedione by modification of cell wall permeability was examined. Drug sensitivity assay results established that bacitracin increased the permeability of the cell wall to hydrophobic compounds. Growth inhibitory study of bacitracin and rifamycin, individually as well as in combination showed that these two antibiotics act synergistically to reduce cell growth. A comparison of transmission electron micrograph results of the bacitracin-treated cells with untreated cells, revealed deformities caused in the cell wall structure by bacitracin treatment. These deformities increased the cell wall permeability and transport of sitosterol inside the cell, and thus enhanced androstenedione (AD) production. A maximum of 1.37, 1.44, 1.65 and 1.76 g AD per gram dry cell weight of mycobacterial cells was produced in the presence of ethambutol, penicillin, polymixin and bacitracin, respectively. Below the minimum inhibitory concentration, bacitracin can be used as potent enhancer of permeability of hydrophobic substances across the mycobacterial cell wall.     

Expression of alr gene from Corynebacterium glutamicum ATCC 13032 in Escherichia coli and molecular characterization of the recombinant alanine racemase

We constructed the high-expression system of the alr gene from Corynebacterium glutamicum ATCC 13032 in Escherichia coli BL 21 (DE3) to characterize the enzymological and structural properties of the gene product, Alr. The Alr was expressed in the soluble fractions of the cell extract of the E. coli clone and showed alanine racemase activity. The purified Alr was a dimer with a molecular mass of 78 kDa. The Alr required pyridoxal 5'-phosphate (PLP) as a coenzyme and contained 2 mol of PLP per mol of the enzyme. The holoenzyme showed maximum absorption at 420 nm, while the reduced form of the enzyme showed it at 310 nm. The Alr was specific for alanine, and the optimum pH was observed at about nine. The Alr was relatively thermostable, and its half-life time at 60 degrees C was estimated to be 26 min. The K(m) and V(max) values were determined as follows: l-alanine to d-alanine, K(m) (l-alanine) 5.01 mM and V(max) 306 U/mg; d-alanine to l-alanine, K(m) (d-alanine) 5.24 mM and V(max) 345 U/mg. The K(eq) value was calculated to be 1.07 and showed good agreement with the theoretical value for the racemization reaction. The high substrate specificity of the Alr from C. glutamicum ATCC 13032 is expected to be a biocatalyst for d-alanine production from the l-counter part.     

Arabinan-deficient mutants of Corynebacterium glutamicum and the consequent flux in decaprenylmonophosphoryl-D-arabinose metabolism

The arabinogalactan (AG) of Corynebacterianeae is a critical macromolecule that tethers mycolic acids to peptidoglycan, thus forming a highly impermeable cell wall matrix termed the mycolyl-arabinogalactan peptidoglycan complex (mAGP). The front line anti-tuberculosis drug, ethambutol (Emb), targets the Mycobacterium tuberculosis and Corynebacterium glutamicum arabinofuranosyltransferase Mt-EmbA, Mt-EmbB and Cg-Emb enzymes, respectively, which are responsible for the biosynthesis of the arabinan domain of AG. The substrate utilized by these important glycosyltransferases, decaprenylmonophosphoryl-D-arabinose (DPA), is synthesized via a decaprenylphosphoryl-5-phosphoribose (DPPR) synthase (UbiA), which catalyzes the transfer of 5-phospho-ribofuranose-pyrophosphate (pRpp) to decaprenol phosphate to form DPPR. Glycosyl compositional analysis of cell walls extracted from a C. glutamicum::ubiA mutant revealed a galactan core consisting of alternating beta(1-->5)-Galf and beta(1-->6)-Galf residues, completely devoid of arabinan and a concomitant loss of cell-wall-bound mycolic acids. In addition, in vitro assays demonstrated a complete loss of arabinofuranosyltransferase activity and DPA biosynthesis in the C. glutamicum::ubiA mutant when supplemented with p[14C]Rpp, the precursor of DPA. Interestingly, in vitro arabinofuranosyltransferase activity was restored in the C. glutamicum::ubiA mutant when supplemented with exogenous DP[14C]A substrate, and C. glutamicum strains deficient in ubiA, emb, and aftA all exhibited different levels of DPA biosynthesis.     

Identification of a novel arabinofuranosyltransferase (AftA) involved in cell wall arabinan biosynthesis in Mycobacterium tuberculosis

The cell wall mycolyl-arabinogalactan-peptidoglycan complex is essential in mycobacterial species, such as Mycobacterium tuberculosis, and is the target of several anti-tubercular drugs. For instance, ethambutol targets arabinogalactan biosynthesis through inhibition of the arabinofuranosyltransferases Mt-EmbA and Mt-EmbB. Following a detailed bioinformatics analysis of genes surrounding the conserved emb locus, we present the identification and characterization of a novel arabinofuranosyltransferase AftA (Rv3792). The enzyme catalyzes the addition of the first key arabinofuranosyl residue from the sugar donor beta-D-arabinofuranosyl-1-monophosphoryldecaprenol to the galactan domain of the cell wall, thus "priming" the galactan for further elaboration by the arabinofuranosyltransferases. Because aftA is an essential gene in M. tuberculosis, we deleted its orthologue in Corynebacterium glutamicum to produce a slow growing but viable mutant. Analysis of its cell wall revealed the complete absence of arabinose resulting in a truncated cell wall structure possessing only a galactan core with a concomitant loss of cell wall-bound mycolates. Complementation of the mutant was fully restored to the wild type phenotype by Cg-aftA. In addition, by developing an in vitro assay using recombinant Escherichia coli expressing Mt-aftA and use of cell wall galactan as an acceptor, we demonstrated the transfer of arabinose from beta-D-arabinofuranosyl-1-monophosphoryldecaprenol to galactan, and unlike the Mt-Emb proteins, Mt-AftA was not inhibited by ethambutol. This newly discovered glycosyltransferase represents an attractive drug target for further exploitation by chemotherapeutic intervention.     

Impact of Cell Wall Structure on the Behavior of Bacterial Cells as Sorbents of Cadmium and Lead

Batch metal sorption studies were conducted to compare the behavior of Gram-positive Bacillus subtilis and Gram-negative Escherichia coli as sorbents of Cd 2+ and Pb 2+ . A pH range from 3.0 to 6.5 was investigated at total metal concentrations of 1 2 10 -4.0 and 3.2 2 10 -5 M. Concentration apparent equilibrium sorption constants (K s n M ) and sorption capacity (S max n ) values were determined for the bacteria by fitting experimental data to one- ( n = 1) and two-site ( n = 2) Langmuir sorption isotherms. The sorption data for each of the bacteria were described well by a one-site model (r 2 > 0.9), Cd 2+ exhibited somewhat lower sorption affinities (log K s M =- 1.5 for B. subtilis , and -0.7 for E. coli ) than Pb 2+ (log K s M =-0.6 for B. subtilis and -0.8 for E. coli ). Corresponding S max values for Cd 2+ and Pb 2+ on B. subtilis were 0.36 mmole/g and 0.27 mmole/g, respectively. For E. coli Cd 2+ and Pb 2+ S max values were lower at 0.10 mmole/g and 0.21 mmole/g. A two-site sorption model yielded an improved fit for only the E. coli data with several orders of magnitude difference evident between high (Cd 2+ log K s1 M = 0.9; Pb 2+ log K s1 M = 1.5) and low (Cd 2+ log K s2 M =- 1.1; Pb 2+ log K s2 M = -1.6) affinity sorption sites. In addition, allowing for the presence of low affinity sorption (i.e., S max2 ) sites further increased the total E. coli metal sorption capacity closer to that of B. subtilis . As expected, the sorption of Cd 2+ and Pb 2+ by the bacteria exhibited a strong dependence on pH with sorption edges in the range of pH 4.2 to 5.6. The results of this study show that, despite differences in cell wall structure and composition, B. subtilis and E. coli exhibit remarkably similar sorption behavior toward Cd 2+ and Pb 2+ , respectively. These similarities can be attributed to the specific chemical reactivity of acidic functional groups (e.g., carboxyl, phosphoryl) that occur in the cell walls of both bacteria.     

Preparative scale Baeyer-Villiger biooxidation at high concentration using recombinant Escherichia coli and in situ substrate feeding and product removal process

An efficient biocatalytic process based on the use of adsorbent resin (in situ substrate feeding and product removal) makes experiments at high substrate concentration possible by overcoming limitations due to substrate and product inhibition. This process was successfully applied to the preparative scale Baeyer-Villiger biooxidation of (-)-(1S,5R)-bicyclo[3.2.0]hept-2-en-6-one (25 g). Whole cells of recombinant E. coli (1 liter) overexpressing cyclohexanone monooxygenase were used as a biocatalyst and the substrate was preloaded onto the adsorbent resin. The corresponding lactone was obtained in 75-80% yield. Time for cell growth and biotransformation is about 24 h each and oxygen supply can be improved by using a tailor-made bubble column.     

Rapid structural characterization of the arabinogalactan and lipoarabinomannan in live mycobacterial cells using 2D and 3D HR-MAS NMR: structural changes in the arabinan due to ethambutol treatment and gene mutation are observed

Mycobacteria possess a unique, highly evolved, carbohydrate- and lipid-rich cell wall that is believed to be important for their survival in hostile environments. Until now, our understanding of mycobacterial cell wall structure has been based upon destructive isolation and fragmentation of individual cell wall components. This study describes the observation of the major cell wall structures in live, intact mycobacteria using 2D and 3D high-resolution magic-angle spinning (HR-MAS) nuclear magnetic resonance (NMR). As little as 20 mg (wet weight) of [13C]-enriched cells were required to produce a whole-cell spectra in which discrete cross-peaks corresponding to specific cell wall components could be identified. The most abundant signals of the arabinogalactan (AG) and lipoarabinomannan (LAM) were assigned in the HR-MAS NMR spectra by comparing the 2D and 3D NMR whole-cell spectra with the spectra of purified cellular components. This study confirmed that the structures of the AG and LAM moieties in the cell wall of live mycobacteria are consistent with structural reports in the literature, which were obtained via degradative analysis. Most important, by using intact cells it was possible to directly demonstrate the effects of ethambutol on the mycobacterial cell wall polysaccharides, characterize the effects of embB gene knockout in the M. smegmatis DeltaembB mutant, and observe differences in the cell wall structures of two mycobacterial species (M. bovis BCG and M. smegmatis.) Herein, we show that HR-MAS NMR is a powerful, rapid, nondestructive technique to monitor changes in the complex, carbohydrate-rich cell wall of live mycobacterial cells.     

Identification of a novel arabinofuranosyltransferase AftB involved in a terminal step of cell wall arabinan biosynthesis in Corynebacterianeae, such as Corynebacterium glutamicum and Mycobacterium tuberculosis

Arabinofuranosyltransferase enzymes, such as EmbA, EmbB, and AftA, play pivotal roles in the biosynthesis of arabinogalactan, and the anti-tuberculosis agent ethambutol (EMB) targets arabinogalactan biosynthesis through inhibition of Mt-EmbA and Mt-EmbB. Herein, we describe the identification and characterization of a novel arabinofuranosyltransferase, now termed AftB (Rv3805c), which is essential in Mycobacterium tuberculosis. Deletion of its orthologue NCgl2780 in the closely related species Corynebacterium glutamicum resulted in a viable mutant. Analysis of the cell wall-associated lipids from the deletion mutant revealed a decreased abundance of cell wall-bound mycolic acids, consistent with a partial loss of mycolylation sites. Subsequent glycosyl linkage analysis of arabinogalactan also revealed the complete absence of terminal beta(1 --> 2)-linked arabinofuranosyl residues. The deletion mutant biochemical phenotype was fully complemented by either Mt-AftB or Cg-AftB, but not with muteins of Mt-AftB, where the two adjacent aspartic acid residues, which have been suggested to be involved in glycosyltransferase activity, were replaced by alanine. In addition, the use of C. glutamicum and C. glutamicumDeltaaftB in an in vitro assay utilizing the sugar donor beta-D-arabinofuranosyl-1-monophosphoryl-decaprenol together with the neoglycolipid acceptor alpha-D-Araf-(1 --> 5)-alpha-D-Araf-O-C(8) as a substrate confirmed AftB as a terminal beta(1 --> 2) arabinofuranosyltransferase, which was also insensitive to EMB. Altogether, these studies have shed further light on the complexities of Corynebacterianeae cell wall biosynthesis, and Mt-AftB represents a potential new drug target.     

Protein organization of rat synaptic plasma membranes and synaptic vesicles: A one- and two-dimensional study

The protein organization of rat brain synaptic plasma membranes (SPM) and synaptic vesicles (SV) was investigated by surface iodination and one- and two-dimensional electrophoresis. Polypeptides of molecular weights (MWs, in Kilodaltons) 170 K, 135 K, 96-86 K, 68-64-61 K, 56 K, 52 K, 38 K, 35-33 K, and 18 K are predominantly or exclusively exposed on the extracellular side of synaptosomes. Several polypeptides of MW between 70 K and 40 K are exclusively exposed on the cytoplasmic side of SPM. The use of two-dimensional electrophoresis allowed to recognize that, for some classes of MW, there are polypeptides of nearly the same MW and different isoelectric points exposed on both sides of SPM. The synaptosomal membrane shows a predominance of acidic proteins on the extracellular side and more neutral and basic proteins on the cytoplasmic side. With respect to SPM, SV are particularly enriched with polypeptides of MW 71 K, 56 K, 39-38 K, 32 K, 16 K, and 15 K. One of them, a doublet of MW 39-38 K, is the most highly labeled species upon surface iodination and is similar, but not identical, with a doublet located on the cytoplasmic side of SPM.     

Regulation of the pH sensitivity of human P2X receptors by N-linked glycosylation

The human (h) P2X(3) receptor and its mutants deficient in one out of four N-glycosylation sites were expressed in HEK293 cells. Concentration-response curves were generated by whole-cell recordings of alpha,beta-methylene ATP (alpha,beta-meATP)-induced currents. A gradual change of external pH from the alkaline 8.0 to the acidic 5.0 successively decreased the maximum current amplitude (E(max)) without affecting the EC(50) value. The replacement of Asn-139 and -170 by Asp (N139D, N170D) abolished the pH sensitivity of the wild-type (WT) hP2X(3) receptor. In the case of N194D, the E(max) was again the highest at the alkaline pH value with no change from 7.4 to 6.5, whereas in the case of N290D, there was an inverse pH sensitivity, with an increase of E(max) in the acidic range. However, this effect appeared to be due to enhanced protonation by the insertion of Asp into the receptor, because replacement of Asn by the neutral Thr resulted in a comparable potency of alpha,beta-meATP at any of the pH values investigated. In accordance with the reported finding that His-206 is involved in the modulation of WT P2X(3) receptors by protons, we showed that the normal change of E(max) by an acidic, but not alkaline pH was abolished after substitution of this His by Ala. However, the double mutant H206A + N290D did not react to acidification or alkalinization with any change in E(max). In conclusion, only fully N-glycosylated P2X(3) receptors recognize external pH with a modified sensitivity towards alpha,beta-meATP.     

Ethambutol-induced alterations in Mycobacterium bovis BCG imaged by atomic force microscopy

Progress in understanding the structure-function relationships of the mycobacterial cell wall has been hampered by its complex architecture as well as by the lack of sensitive, high-resolution probing techniques. For the first time, we used atomic force microscopy (AFM) to image the surface topography of hydrated Mycobacterium bovis bacillus Calmette Guérin cells and to investigate the influence of the antimycobacterial drug ethambutol on the cell wall architecture. While untreated cells showed a very smooth and homogeneous surface morphology, incubation of cells in the presence of ethambutol caused dramatic changes of the fine surface structure. At 4 micro g mL(-1), the drug created concentric striations at the cell surface and disrupted a approximately 8 nm thick cell wall layer, attributed to the outer electron-opaque layer usually seen by electron microscopy, while at 10 micro g mL(-1) an underlying approximately 12 nm thick layer reflecting the thick electron-transparent layer was also altered. These noninvasive ultrastructural investigations provide novel information on the macromolecular architecture of the mycobacterial envelope as well as into the destructuring effects of ethambutol.     

Kinetic studies of killer toxin K1 binding to yeast cells indicate two receptor populations

A recently described new method for determination of killer toxin activity was used for kinetic measurenments of K1 toxin binding. The cells of the killer sensitive strain Saccharomyces cerevisiae S6 were shown to carry two classes of toxin binding sites differing widely in their half-saturation constants and maximum binding rates. The low-affinity and high-velocity binding component (K _T1=2.6x10⁹ L.U./ml, V _max1=0.19 s^-1) probably reflects diffusion-limited binding to cell wall receptors; the high-affinity and low-velocity component (K _T2=3.2x10⁷ L.U./ml, V _max2=0.03 s^-1) presumably indicates the binding of the toxin to plasma membrane receptors. Adsorption of most of the killer toxin K1 to the surface of sensitive cells occured within 1 min and was virtually complete within 5 min. The amount of toxin that saturated practically all cell receptors was about 600 lethal units (L.U.) per cell of S. cerevisiae S6.     

产朊假丝酵母全细胞转化合成谷胱甘肽的营养条件

为了进一步提高产朊假丝酵母全细胞转化生物合成谷胱甘肽(GSH)的能力,利用响应面分析方法对酵母培养的发酵培养基进行优化。在单因素实验的基础上,通过Plackett-Burman设计筛选出显著影响GSH转化力的2个主要因素:葡萄糖和KH2PO4。采用最陡爬坡试验和响应面设计预测了葡萄糖和KH2PO4的最佳质量浓度分别为58.5和17.2 g/L。验证实验结果表明,在该优化培养基条件下,酵母细胞的GSH转化力为1.54 mg/(g·h),比优化前提高了1倍。该结果为类似的采用全细胞转化法高效合成有用化学品的研究与开发提供了可行的优化思路。