Biogenesis of antibiotics-viewing its history and glimpses of the future.

This review aims at comparing some historical data with the current situation in the study of biogenesis of natural compounds, antibiotics in the first place. Biogenesis of tetracyclines and cycloheximide and related compounds serves as example. Examples of molecular biological and bioinformatics methods used in the study of antibiotic biogenesis are described both in terms of its historical aspects and the current knowledge.

Lipidomics as an important key for the identification of beer-spoilage bacteria.

Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) was used for characterizing intact plasmalogen phospholipid molecules in beer-spoilage bacteria. Identification of intact plasmalogens was carried out using collision-induced dissociation and the presence of suitable marker molecular species, both qualitative and quantitative, was determined in samples containing the anaerobic bacteria Megasphaera and Pectinatus. Using selected ion monitoring (SIM), this method had a limit of detection at 1 pg for the standard, i.e. 1-(1Z-octadecenyl)-2-oleoyl-sn-glycero-3-phosphoethanolamine and be linear in the range of four orders of magnitude from 2 pg to 20 ng. This technique was applied to intact plasmalogen extracts from the samples of contaminated and uncontaminated beer without derivatization and resulted in the identification of contamination of beer by Megasphaera and Pectinatus bacteria. The limit of detection was about 830 cells of anaerobic bacteria, i.e. bacteria containing natural cyclopropane plasmalogenes (c-p-19:0/15:0), which is the majority plasmalogen located in both Megasphaera and Pectinatus. The SIM ESI-MS method has been shown to be useful for the analysis of low concentration of plasmalogens in all biological samples, which were contaminated with anaerobic bacteria, e.g. juice, not only in beer. Significance and impact of the study: Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) using collision-induced dissociation was used to characterize intact plasmalogen phospholipid molecules in beer-spoilage anaerobic bacteria Megasphaera and Pectinatus. Using selected ion monitoring (SIM), this method has a detection limit of 1 pg for the standard 1-(1Z-octadecenyl)-2-oleoyl-sn-glycero-3-phosphoethanolamine and is linear within four orders of magnitude (2 pg to 20 ng). The limit of detection was about 830 cells of bacteria containing natural cyclopropane plasmalogen (c-p-19:0/15:0). SIM ESI-MS method is useful for analyzing low concentrations of plasmalogens in biological samples contaminated with anaerobic bacteria, e.g. beer or juice.

Trachydiscus minutus, a new biotechnological source of eicosapentaenoic acid.

The yellow-green alga Trachydiscus minutus (class Xanthophyta) was cultivated in a standard medium and in media without sulfur and nitrogen. Its yield after a 16-d cultivation reached 13 g dry mass per 1 L medium. The content of oligoenoic ('polyenoic') fatty acid (PUFA), i.e. eicosapentaenoic (EPA), was in excess of 35 % of total fatty acids; the productivity was thus 88 mg/L per d. This result makes the alga a very prospective organism that may serve as a new biotechnological source of single cell oil.

Cell ATP level of Saccharomyces cerevisiae sensitively responds to culture growth and drug-inflicted variations in membrane integrity and PDR pump activity.

Cellular ATP level in Saccharomyces cerevisiae was measured during culture growth of strain US50-18C overproducing all major PDR pumps and its isogenic mutants variously deleted in these pumps. It was found to be inversely proportional to the intensity of cell metabolism during different growth phases and to the activity of PDR pumps, which are thus among major ATP consumers in the cells. The ATP level was increased when membrane integrity was affected by 0.5% butanol, and further increased by compound 23.1, a semisynthetic phenol lipid derivative that acts as inhibitor of Pdr5p and Snq2p pumps. The magnitude of increase in cell ATP caused by inhibition of Pdr5p pump by compound 23.1 and the Pdr5p pump inhibitor FK506 used for comparison reflects the activity and hence the energy demand of the pump. The rise in cell ATP caused by different PDR pump inhibitors can be thus used as an indicator of pump activity and the potency of the inhibitor.

Net effect of wort osmotic pressure on fermentation course, yeast vitality, beer flavor, and haze.

The net effect of increased wort osmolarity on fermentation time, bottom yeast vitality and sedimentation, beer flavor compounds, and haze was determined in fermentations with 12 degrees all-malt wort supplemented with sorbitol to reach osmolarity equal to 16 degrees and 20 degrees. Three pitchings were performed in 12 degrees/12 degrees/12 degrees, 16 degrees/16 degrees/12 degrees, and 20 degrees/20 degrees/12 degrees worts. Fermentations in 16 degrees and 20 degrees worts decreased yeast vitality measured as acidification power (AP) by a maximum of 10%, lowered yeast proliferation, and increased fermentation time. Repitching aggravated these effects. The 3rd "back to normal" pitching into 12 degrees wort restored the yeast AP and reproductive abilities while the extended fermentation time remained. Yeast sedimentation in 16 degrees and 20 degrees worts was delayed but increased about two times at fermentation end relative to that in 12 degrees wort. Third "back-to-normal" pitching abolished the delay in sedimentation and reduced its extent, which became nearly equal in all variants. Beer brewed at increased osmolarity was characterized by increased levels of diacetyl and pentanedione and lower levels of dimethylsulfide and acetaldehyde. Esters and higher alcohols displayed small variations irrespective of wort osmolarity or repitching. Increased wort osmolarity had no appreciable effect on the haze of green beer and accelerated beer clarification during maturation. In all variants, chill haze increased with repitching.

Control and prediction of the course of brewery fermentations by gravimetric analysis.

A simple, fast and cheap test suitable for predicting the course of brewery fermentations based on mass analysis is described and its efficiency is evaluated. Compared to commonly used yeast vitality tests, this analysis takes into account wort composition and other factors that influence fermentation performance. It can be used to predict the shape of the fermentation curve in brewery fermentations and in research and development projects concerning yeast vitality, fermentation conditions and wort composition. It can also be a useful tool for homebrewers to control their fermentations.

Identification of odorous compounds from nine fermentor-cultivated Streptomyces strains.

The chemical composition was determined of odors produced by nine strains of streptomycetes (Streptomyces aureofaciens, S. avermitilis, S. cinnamonensis, S. coelicolor, S. griseus, S. lividans, S. rimosus, S. spectabilis, S. virginiae) cultivated in a fermentor under similar cultivation conditions. GC-MS analysis identified more than twenty noteworthy volatile chemical individuals. The main components of the odor spectrum were geosmin and unique homologues of oxolones (dihydrofuranones), minor compounds included, e.g., pyrazine derivatives, acetoin and its homologues, aromatic esters, furan derivatives.

A new method of optical detection of yeast acidification power.

We describe here a newly developed method for a contact-free optical pH measurement in yeast suspensions supplemented with glucose, and containing the pH sensitive triphenylmethane dye bromocresol green. It is suitable for performing the acidification power test (based on measuring the rate of pH drop of yeast suspension caused by active extrusion of acidity from cells after glucose addition) used for assessing yeast vitality in fermentation industries. Using this methodology we monitored the pH in yeast suspensions in the course of acidification in the pH range of 3.5-5.3. Optical pH measurement allows simultaneous testing of several samples, minimizes the sample volume, simplifies sample handling and reduces the hands-on time in sample processing.

Structural analysis of a polysaccharide from Chlorella kessleri by means of gas chromatography-mass spectrometry of its saccharide alditols.

Interpretation of gas chromatographic-mass spectrometric data of oligosaccharide alditols was used to determine their structures and to derive the structure of a water soluble polysaccharide isolated from Chlorella kessleri. 1H- and 13C-NMR was employed to assess the configuration of glycosidic bonds and individual monosaccharides were assigned to the L or D series by means of gas chromatography of the acetylated (S)-2-butyl glycosides.

Relationship between volatile odorous substances and production of avermectins by Streptomyces avermitilis.

The time course of production of odorous compounds, i.e. geosmin and oxolones, and of avermectins was determined during the cultivation of S. avermitilis in flasks, 1- and 50-L fermentors. The amount of the antibiotics increased with increasing cultivation time up to more than 2 g/L while the concentration of geosmin rose to more than 4 mg/L. Cultivation without reflux condenser resulted in a lower product formation due to the higher stripping of geosmin. A relatively tight correlation was found between the production of geosmin and the production of avermectins. The production of oxolones peaked on cultivation days 3-5, the sum of oxolones being 60 microg/L. Subsequently, the production dropped below a measurable level. This can be explained as being due to the inhibition of oxolone production by geosmin.

Assaying the antioxidant and radical scavenging properties of aliphatic mono- and di-N-oxides in superoxide dismutase-deficient yeast and in a chemiluminescence test.

The antioxidative action of amphiphilic mono-(alkanoylamino) ethyldimethylamine-N-oxides (EDA), di-N-oxides 1,1-bis {[2-(N,N-dimethylamino)ethyl]amido}alkane-di-N-oxides (MEDA) and 1,1-bis {[3-(N,N-dimethylamino)propyl]amido}alkane-di-N-oxides (MPDA) with a 12- and 14-membered acyl chain against tert-butylhydroperoxide (TBHP)-produced peroxyl and paraquat (PQ)-generated superoxide radicals was determined in superoxide dismutase-deficient mutants of Saccharomyces cerevisiae, and, in parallel, in a chemical assay based on chemiluminescence changes caused in a luminol system by peroxyl radicals generated from the azo-compound 2,2'-azobis(2-amidinopropane dihydrochloride) (AAPH). At 30 micromol/L, the shorter-chain compounds did not affect strain survival while longer-chain ones, in some cases, lowered the survival of sod2 and sod1 sod2 cells. Whether nontoxic or medium-toxic, all N-oxides protected the sod strains against the toxic effect of PQ and TBHP, the protection being stronger with the di-N-oxides. The survival was lowered only by 14-MPDA in the TBHP-exposed sod2 mutant. Membrane lipids isolated from all strains were protected against TBHP-induced peroxidation by both mono- and di-N-oxides, the protection being dependent on the alkyl chain length. Mono-N-oxides were again less active than di-N-oxides with the same alkyl chains, the antiperoxidative activity being also dependent on lipids isolated from the individual mutants. In the chemiluminescence assay, the IC50 value of the N-oxides for scavenging of radicals generated from AAPH generally decreased (i.e. the scavenging efficiency increased) with increasing chain length and was the highest in MEDA.

Protective role of mitochondrial superoxide dismutase against high osmolarity, heat and metalloid stress in saccharomyces cerevisiae.

Superoxide dismutases, both cytosolic Cu, Zn-SOD encoded by SOD1 and mitochondrial Mn-SOD encoded by SOD2, serve Saccharomyces cerevisiae cells for defense against the superoxide radical but the phenotypes of sod1A and sod2delta mutant strains are different. Compared with the parent strain and the sod1delta mutant, the sod2delta mutant shows a much more severe growth defect at elevated salt concentrations, which is partially rescued by 2 mmol/L glutathione. The growth of all three strains is reduced at 37 degrees C, the sod2delta showing the highest sensitivity, especially when cultured in air. Addition of 1 mmol/L glutathione to the medium restores aerobic growth of the sod1delta mutant but has only a minor effect on the growth of the sod2delta strain at 37 degrees C. The sod2delta strain is also sensitive to AsIIl and AsV and its sensitivity is much more pronounced under aerobic conditions. These results suggest that, unlike the Sodlp protein, whose major role is oxidative stress defense, Sod2p also plays a role in protecting S. cerevisiae cells against other stresses--high osmolarity, heat and metalloid stress.

Cell-protective and antioxidant activity of two groups of synthetic amphiphilic compounds--phenolics and amine N-oxides.

Two classes of newly synthesized amphiphilic compounds, phenolic antioxidants ("phenolics") and N-oxides exert in vivo antioxidant effects on live S. cerevisiae cells. Both groups have low toxicity, phenolics being more toxic than N-oxides and compounds with a longer alkyl chain having higher toxicity than those with a shorter alkyl chain. Phenolic antioxidants protect yeast cells exposed to the superoxide producer paraquat and peroxyl generator tert-butylhydroperoxide better than N-oxides at 3-fold higher concentration. Both types of antioxidants enhance the survival of pro-oxidant-exposed cells of S. cerevisiae mutants deficient in cytosolic and/or mitochondrial superoxide dismutase and could be good compounds which mimic the role of superoxide dismutases. The results of measurement of antioxidant activity in an in vitro chemiluminescence test differ from the results obtained in vivo with S. cerevisiae superoxide dismutase mutants. In contrast to their action on live cells, phenolics are less effective than N-oxides in preventing lipid peroxidation of an emulsion of lipids isolated from S. cerevisiae membranes.

Amphiphilic amine-N-oxides with aliphatic alkyl chain act as efficient superoxide dismutase mimics, antioxidants and lipid peroxidation blockers in yeast.

Amphiphilic 3-(alkanoylamino)propyldimethylamine-N-oxides with different length of the alkyl chain, i.e. different hydrophilic-lipophilic balance, act in micromolar concentrations as SOD mimics by lifting the inhibition of aerobic growth caused by SOD deletions in Saccharomyces cerevisiae. They also enhance the survival of sod mutants of S. cerevisiae exposed to the hydrophilic superoxide-generating prooxidant paraquat and the amphiphilic hydroperoxide-producing tert-butylhydroperoxide (TBHP), and largely prevent TBHP-induced peroxidation of isolated yeast plasma membrane lipids. Unlike the SOD-mimicking effect, the magnitude of these effects depends on the alkyl chain length of the amine-N-oxides, which incorporate into S. cerevisiae membranes, causing fluidity changes in both the hydrophilic surface part of the membrane and the membrane lipid matrix. Unlike wild-type strains, the membranes of sod mutants were found to contain polyunsaturated fatty acids; the sensitivity of the mutants to lipophilic pro-oxidants was found to increase with increasing content of these acids. sod mutants are useful in assessing pro- and antioxidant properties of different compounds.

Factors affecting the outcome of the acidification power test of yeast quality: critical reappraisal.

Brewery bottom yeast strain 95 from the Pilsner Urquell propagation unit was used to reappraise the efficiency of the acidification power (AP) test consisting in determining the spontaneous (oxygen-induced) and glucose-induced medium acidification caused by yeast and lactic acid bacteria under standard conditions, and used widely for assessing and predicting the vitality of industrial strains. AP was evaluated in yeast stored for different periods of time (0-28 d) at 4 degrees C, at different temperatures before and during the test (0-55 degrees C), and at different concentrations of cells and glucose and different cells-to-glucose ratios. All these factors had a strong effect on acidification kinetics and the AP value. By contrast, the duration of the lag period between yeast collection and the test (0-6 h) had no perceptible effect on the AP value. The best results were achieved at saturation concentrations of cells (> 10 g pressed yeast or approximately 14 g yeast slurry per 100 mL) and glucose (approximately 3 %) and at 25 degrees C. Since an exact evaluation of acidification characteristics depends strongly on the kinetics of the process, the AP test should include monitoring the time course of the acidification.

Process-independent quantitative assessment of residual biological contamination of medical devices reprocessed in washer-disinfectors.

A method based on measuring a soil-induced fluorescence intensity response of 1,8-anilinonaphthalene sulfonate at two fixed wavelengths (460 and 510 nm) was used for determining residual contamination on test soil carriers simulating medical devices after passage through a hospital washer-disinfector. The fluorescence response can be satisfactorily calibrated to soil levels as low as approximately 1 microg/L. Practical tests were performed in two hospitals with washer-disinfectors of 3 types and with several chemical or enzymic cleansers-disinfectants. In combination with the previously developed system of standardized test soil carriers simulating both easily and poorly accessible parts of soiled medical devices, the liver-lactose-oil test soil and an efficient sonication procedure for stripping the residual soil off the carriers, this soil detection method permits the detection of very low contamination levels.

Glucose-induced MDR pump resynthesis in respiring yeast cells depends on nutrient level.

Glucose addition to a stationary culture of wild-type Saccharomyces cerevisiae BY4742 cells with zero activity of MDR pumps resuspended in a fresh medium causes pump resynthesis (measured as pump-effected diS-C3(3) efflux). In a stationary culture in its original growth medium, this glucose-induced pump resynthesis fails to occur due to depletion of essential nutrients or to extracellular metabolites produced by cells during growth. Direct pump inactivation by metabolites is excluded since exponential cells with high MDR pump activity cultured in a medium with high concentration of extracellular metabolites retain this activity for at least 2 h. The metabolites also do not affect pump synthesis on the level of gene expression as addition of concentrated growth medium or an amino acid mixture to stationary cells in spent growth medium restores glucose-induced pump synthesis. The block of MDR pump synthesis is therefore due to the lack of essential nutrients in spent medium.

Activity of yeast multidrug resistance pumps during growth is controlled by carbon source and the composition of growth-depleted medium: DiS-C3(3) fluorescence assay.

Like other tested wild-type strains (DTXII and IL-125-2B), exponential glucose- and/or fructose-grown cells of Saccharomyces cerevisiae BY4742 exhibit the previously described high activity of Pdr5p and Snq2p pumps (measured as export of the potentiometric fluorescent probe diS-C3(3)). Upon saccharide depletion from the medium the pump activity in these cells, which differ from other strains in having a lower membrane potential, sharply drops to a very low level similar to that found in cells grown on ethanol or glycerol. This negligible pump activity in respiring cells thus appears to have a universal character. Addition of glucose or fructose to respiring BY4742 cells grown to low culture densities restores multidrug resistance pump activity due partly to pump synthesis in pre-existing cells and partly to the high pump activity of newly grown cells; no such pump activity boost occurs when the sugar is added to high-density cultures of ethanol-grown or post-diauxic glucose-grown cells, even if these cultures are diluted to low density by their original growth-depleted medium. A strong sugar-induced increase in pump activity is found solely if respiring cells from high-density cultures are resuspended in fresh YPD or YPE medium before sugar addition. Its absence in respiring cells suspended in growth-depleted medium reflects an as yet unidentified effect of the composition of the growth-exhausted medium (depletion of some components and/or accumulation of extracellular metabolites during yeast growth) on sugar-induced pump activity rise.

Impact of the growth phase on the activity of multidrug resistance pumps and membrane potential of S. cerevisiae: effect of pump overproduction and carbon source.

The potentiometric fluorescence probe diS-C3(3) is expelled from S. cerevisiae by ABC pumps Pdr5 and Snq2 and can conveniently be used for studying their performance. The activity of these pumps in a strain with wild-type PDR1 allele was shown to drop sharply on glucose depletion from the medium and then again at the end of the diauxic shift when the cells are adapted to growth on respiratory substrates. The presence of the PDR1-3 allele causing pump overproduction prevented this second drop and the pump activity typical for diauxic cells was largely retained. Growth phase-dependent changes of membrane potential measured by the same probe in pump-free mutants included a Deltapsi drop in the late exponential and diauxic growth phase, indicating lowered activity of H+ -ATPase. Suppression of activity of both ABC pumps and H+ -ATPase obviously signifies cell transition to an energy-saving mode. Challenging respiration-adapted cells with glucose showed a novel feature of yeast ABC pumps--a strong dependence of pump activity on the type of the carbon source.

Effect of biocides on S. cerevisiae: relationship between short-term membrane affliction and long-term cell killing.

The long-term action of recommended (RC) and near-recommended concentrations of several commercial biocides (Lonzabac 12.100, Genamin CS302D, benzalkonium chloride and 2-phenoxyethanol) on cells of S. cerevisiae wild-type strain DTXII was described using plating tests while short-term effects were determined using the potentiometric fluorescent probe diS-C3(3) that detects both changes in membrane potential and impairment of membrane integrity. A 2-d plating of cells exposed to 0.5xRC of benzalkonium chloride and Genamin CS302D for 15 min showed a complete long-term cell killing, with 2-phenoxyethanol the killing was complete only at 2xRC and Lonzabac caused complete killing at RC but not at 0.5xRC. The diS-C3(3) fluorescence assay performed immediately after a 10-min biocide exposure revealed several concentration-dependent modes of action: Lonzabac at 0.5xRC caused a mere depolarization, higher concentrations causing gradually increasing cell damage; benzalkonium chloride and Genamin CS302D rapidly damaged the membrane of some cells and depolarized the rest whereas 2-phenoxyethanol, which had the lowest effect in the plating test, produced a concentration-dependent fraction of cells with impaired membranes. Cell staining slightly increased during the diS-C3(3) assay; addition of a protonophore showed that part of the remaining undamaged cells retained their membrane potential. Comparison of short-term and long-term data implies that membrane depolarization alone is not sufficient for complete long-term killing of yeast cells under the action of a biocide unless it is accompanied by perceptible impairment of membrane integrity. The results show that the diS-C3(3) fluorescence assay, which reflects the short-term effects of a biocide on cell membranes, can be successfully used to assess the microbicidal efficiency of biocides.