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The development of biodegradable nanoparticles is an important tool for the biological transport of chemical compounds. The nanoencapsulation reduces the biopharmaceutical and pharmacokinetic drawbacks of compounds and enhances their biological properties. Naturally occurring polymers such as proteins and polysaccharides have been widely applied in the development of nanostructured systems of several therapeutic agents. Among them is chitosan, a crustacean-carapace-chitin derived biopolymer. In addition to its biocompatibility and biodegradability, chitosan is known for its mucoadhesion properties. Chitosan-based nanostructured systems potentiate most of the aspects of the loaded drugs, including cellular transport and other biological effects. The use of chitosan nanoparticles enhances permeation, stability, and bioactivity of natural compounds. In this review, an overview of the main features of chitosan nanoparticles that improved in vitro and in vivo effects of bioactive natural molecules is given, emphasizing the results obtained with curcumin.

Fungal infections are diseases that are considered neglected although their infection rates have increased worldwide in the last decades. Thus, since the antifungal arsenal is restricted and many strains have shown resistance, new therapeutic alternatives are necessary. Nanoparticles are considered important alternatives to promote drug delivery. In this sense, the objective of the present study was to evaluate the contributions of newly developed nanoparticles to the treatment of fungal infections. Studies have shown that nanoparticles generally improve the biopharmaceutical and pharmacokinetic characteristics of antifungals, which is reflected in a greater pharmacodynamic potential and lower toxicity, as well as the possibility of prolonged action. It also offers the proposition of new routes of administration. Nanotechnology is known to contribute to a new drug delivery system, not only for the control of infectious diseases but for various other diseases as well. In recent years, several studies have emphasized its application in infectious diseases, presenting better alternatives for the treatment of fungal infections.

In a given habitat, bacterial cells often experience recurrent exposures to the same environmental stimulus. The ability to memorize the past event and to adjust current behaviors can lead to efficient adaptation to the recurring stimulus. Here we demonstrate that the versatile bacterium Pseudomonas aeruginosaadopts a virulence phenotype after serial passage in the invertebrate model host Galleria mellonella. The virulence phenotype was not linked to the acquisition of genetic variations and was sustained for several generations, despite cultivation of the ex vivo virulence-adapted P. aeruginosacells under rich medium conditions in vitro. Transcriptional reprogramming seemed to be induced by a host-specific food source, as reprogramming was also observed upon cultivation of P. aeruginosain rich medium supplemented with polyunsaturated long-chain fatty acids. The establishment of induced memory responses adds a time dimension and seems to fill the gap between long-term evolutionary genotypic adaptation and short-term induced individual responses. Efforts to unravel the fundamental mechanisms that underlie the carry-over effect to induce such memory responses will continue to be of importance as hysteretic behavior can serve survival of bacterial populations in changing and challenging habitats.

In a given habitat, bacterial cells often experience recurrent exposures to the same environmental stimulus. The ability to memorize the past event and to adjust current behaviors can lead to efficient adaptation to the recurring stimulus. Here we demonstrate that the versatile bacterium Pseudomonas aeruginosaadopts a virulence phenotype after serial passage in the invertebrate model host Galleria mellonella. The virulence phenotype was not linked to the acquisition of genetic variations and was sustained for several generations, despite cultivation of the ex vivo virulence-adapted P. aeruginosacells under rich medium conditions in vitro. Transcriptional reprogramming seemed to be induced by a host-specific food source, as reprogramming was also observed upon cultivation of P. aeruginosain rich medium supplemented with polyunsaturated long-chain fatty acids. The establishment of induced memory responses adds a time dimension and seems to fill the gap between long-term evolutionary genotypic adaptation and short-term induced individual responses. Efforts to unravel the fundamental mechanisms that underlie the carry-over effect to induce such memory responses will continue to be of importance as hysteretic behavior can serve survival of bacterial populations in changing and challenging habitats.

The need for effective compounds to combat antimicrobial resistance and biofilms which play important roles in human infections continues to pose a major health challenge. Seven previously undescribed acyclic diterpenes linked to isocitric acid by an ether linkage, microporenic acids A–G (1–7), were isolated from the cultures of a hitherto undescribed species of the genus Microporus(Polyporales, Basidiomycota) originating from Kenya’s Kakamega forest. Microporenic acids D and E (4and 5) showed antimicrobial activity against a panel of Gram positive bacteria and a yeast, Candida tenuis. Moreover, microporenic acids A and B (1and 2) demonstrated dose-dependent inhibition of biofilm formation by Staphylococcus aureus. Compound 1further showed significant activity against Candida albicansand Staphylococcus aureuspreformed biofilms.

Background: Fumitremorgins are mycotoxins but can also inhibit cancer cells and reverse their drug resistance. Objective: The bioactivity of prenylated cyclo-Trp-Pro dipeptides and their derivatives concerning their application in anti-cancer therapies will be discussed. Methods: Reports on the discovery and assessment of this class of fungal compounds are compiled from literature using Google Scholar and PubMed. The bioactivities of the natural compounds are discussed with the aim of their improvement for cancer therapy. Results: Although a number of compounds of this class have been found, only a minority of them showed bioactivity in the applied bioassays. Fumitremorgins and related compounds are active against various cancer cells but they are also mycotoxins. Some of these natural compounds can arrest cancer cells in their cell cycle and some can block ABC-transporters and reverse resistance in chemotherapy. Structure activity relationships have been deduced leading to the prediction of highly active compounds. Several easily accessible derivatives of these natural products have been discovered being highly selective and non-toxic. Conclusion: Sophisticated screening methods, high throughput screening, metabolic engineering, and synthetic biology are novel and promising technologies for the search for highly active drugs. Rapid gene sequencing in combination with engineered biosynthetic pathways should contribute substantially to novel pharmaceutics.

Background: Fumitremorgins are mycotoxins but can also inhibit cancer cells and reverse their drug resistance. Objective: The bioactivity of prenylated cyclo-Trp-Pro dipeptides and their derivatives concerning their application in anti-cancer therapies will be discussed. Methods: Reports on the discovery and assessment of this class of fungal compounds are compiled from literature using Google Scholar and PubMed. The bioactivities of the natural compounds are discussed with the aim of their improvement for cancer therapy. Results: Although a number of compounds of this class have been found, only a minority of them showed bioactivity in the applied bioassays. Fumitremorgins and related compounds are active against various cancer cells but they are also mycotoxins. Some of these natural compounds can arrest cancer cells in their cell cycle and some can block ABC-transporters and reverse resistance in chemotherapy. Structure activity relationships have been deduced leading to the prediction of highly active compounds. Several easily accessible derivatives of these natural products have been discovered being highly selective and non-toxic. Conclusion: Sophisticated screening methods, high throughput screening, metabolic engineering, and synthetic biology are novel and promising technologies for the search for highly active drugs. Rapid gene sequencing in combination with engineered biosynthetic pathways should contribute substantially to novel pharmaceutics.

Trichothecenes are sesquiterpenoid metabolites produced by fungi and species of the plant genus Baccharis, family Asteraceae. They comprise a tricyclic core with an epoxide at C-12 and C-13 and can be grouped into non-macrocyclic and macrocyclic compounds. While many of these compounds are of concern in agriculture, the macrocyclic metabolites have been evaluated as antiviral, anti-cancer, antimalarial and antifungal compounds. Some known cytotoxic responses on eukaryotic cells include inhibition of protein, DNA and RNA syntheses, interference with mitochondrial function, effects on cell division and membranes. These targets however have been elucidated essentially employing non-macrocyclic trichothecenes and only one or two closely related macrocyclic compounds. For several macrocyclic trichothecenes high selectivity against fungal species and against cancer cell lines have been reported suggesting that the macrocycle and its stereochemistry are of crucial importance regarding biological activity and selectivity. This review is focused on compounds belonging to the macrocyclic type, where a cyclic diester or triester ring binds to the trichothecane moiety at C-4 and C- 15 leading to natural products belonging to the groups of satratoxins, verrucarins, roridins, myrotoxins and baccharinoids. Their biological activities, cytotoxic mechanisms and structure-activity relationships (SAR) are discussed. From the reported data it becomes evident that even small changes in the molecules can lead to pronounced effects on biological activity or selectivity against cancer cells lines. Understanding the underlying mechanisms may help to design highly specific drugs for cancer therapy.

ABSTRACTThe respiratory mycobiome is an important but understudied component of the human microbiota. Like bacteria, fungi can cause severe lung diseases, but their infection rates are much lower. This study compared the bacterial and fungal communities of sputum samples from a large cohort of 56 adult patients with cystic fibrosis (CF) during nonexacerbation periods and under continuous antibiotic treatment. Molecular fingerprinting based on single-strand conformation polymorphism (SSCP) analysis revealed fundamental differences between bacterial and fungal communities. Both groups of microorganisms were taxonomically classified by identification of gene sequences (16S rRNA and internal transcript spacer), and prevalences of single taxa were determined for the entire cohort. Major bacterial pathogens were frequently observed, whereas fungi of known pathogenicity in CF were detected only in low numbers. Fungal species richness increased without reaching a constant level (saturation), whereas bacterial richness showed saturation after 50 patients were analyzed. In contrast to bacteria, a large number of fungal species were observed together with high fluctuations over time and among patients. These findings demonstrated that the mycobiome was dominated by transient species, which strongly suggested that the main driving force was their presence in inhaled air rather than colonization. Considering the high exposure of human airways to fungal spores, we concluded that fungi have low colonization abilities in CF, and colonization by pathogenic fungal species may be considered a rare event. A comprehensive understanding of the conditions promoting fungal colonization may offer the opportunity to prevent colonization and substantially reduce or even eliminate fungus-related disease progression in CF.

Staphylococcus epidermidis, a commensal microorganism at the human skin and mucosae, is nowadays considered an important opportunistic pathogen related to nosocomial infections on indwelling medical devices due biofilm formation. Bacterial biofilms are the worst aspect in the treatment of infections and now efforts have been made in the search for new molecular entities to overcome this situation. In this work, a compound isolated from marine associated fungi was capable to interfere with the adherence and biofilm formation of S. epidermidis. This compound, identified as mevalonolactone, showed significant inhibition of S. epidermidis ATCC 35984 biofilm formation, without antibacterial activity, evaluated by crystal violet assay, turbidimetric assay and scanning electron microscopy. When assayed against 12 clinical isolates of S. epidermidis, this compound exhibited both biofilm inhibition and antimicrobial activity, but no activity against gram-negative bacteria was observed. Therefore, when this constitutive molecule is added in the antibiofilm and antibacterial assays, it might act as an important agent against this pathogen, contributing to the arsenal of antibiofilm compounds.

ABSTRACTHuman β-defensins are host defense peptides performing antimicrobial as well as immunomodulatory functions. The present study investigated whether treatment of Escherichia coliwith human β-defensin 2 could generate extracellular molecules of relevance for immune regulation. Mass spectrometry analysis of bacterial supernatants detected the accumulation of purine nucleosides triggered by β-defensin 2 treatment. Other cationic antimicrobial peptides tested presented variable outcomes with regard to extracellular adenosine accumulation; human β-defensin 2 was the most efficient at inducing this response. Structural and biochemical evidence indicated that a mechanism other than plain lysis was involved in the observed phenomenon. By use of isotope (13C) labeling, extracellular adenosine was found to be derived from preexistent RNA, and a direct interaction between the peptide and bacterial nucleic acid was documented for the first time for β-defensin 2. Taken together, the data suggest that defensin activity on a bacterial target may alter local levels of adenosine, a well-known immunomodulator influencing inflammatory processes.

Many bacteria grow on surfaces forming biofilms but often high dosages of antibiotics can not clear infectious biofilms. Biofilms are controlled by a process called quorum-sensing triggered by species-specific, small, diffusible autoinducers (N-acyl homoserine lactones (AHL), small peptides or a furanosyl borate diester). Several herbal, animal and microbial extracts possess quorum-quenching activity but only few active compounds and synthetic analogues are known. However, quorum-sensing seems not to be the only way to control biofilms. Bacteria have mechanisms to dissolve their biofilms and return to planktonic lifestyle. Only few compounds responsible for the signalling of these processes are known but may open a completely novel line of biofilm control. Furthermore, controlling the attachment of bacterial by inhibiting the formation of pili is another option and some inhibitors have already been characterized. In this article different approaches to control bacterial biofilms are discussed together with the balance between biofilm formation and virulence.

Biofilms are matrix-enclosed microbial aggregations that adhere to biological or non-biological surfaces. They represent a significant and incompletely understood mode of growth for bacteria and fungi. Biofilm infections cause many deaths and high health costs worldwide. Biofilm infections on indwelling devices or implants are difficult to eradicate because of their much better protection against macrophages and antibiotics, compared to free living cells, leading to severe clinical complications often with lethal outcome. One promising approach to combat biofilm infections independent from the conventional control by antibiotics is the generation of functional surfaces preventing the attachment of bacteria. Another aim is the communication machinery used by bacteria to establish a biofilm, the so called quorum-sensing. Here, small diffusible compounds are produced and sensed by the producing cells to measure their concentration and hence cell density. Natural compounds and synthetic analogues have been used successfully to prevent biofilm formation by quorum- quenching. These compounds are still in the preclinical phase, often struggling with toxicity. A principal problem of quorum-quenchers is their high species specificity, resulting in the control of only some pathogenic strains leaving other pathogens untouched. A field still in its infancy is the control of virulence factors expression not preventing the biofilm but suppressing its virulence. This review will give an overview over the pros and cons of the individual targets and an outlook of future developments.

Many bacteria can form aggregates on interfaces, called biofilms, where they are much more protected against toxic agents such as antibiotics or antibodies. Bacteria organized in biofilms are therefore very difficult to control and often even high dosages of antibiotics cannot clear infectious biofilms. To form biofilms bacteria have to start a complex genetic program to switch from planktonic to sessile lifestyle. This starts with the determination of their cell density, a process called quorum sensing, triggered by small, water soluble molecules, so called autoinducers. From Gram-positive bacteria several small peptides are known, many of them thiolactones. More recently another group of compounds was discovered probably formed from ribose-homocysteine and the first autoinducer-II identified is a furanosyl borate diester. While small peptides are found to trigger the intraspecies communication autoinducer-II is assumed to be used for communication at the interspecies level. Taking the lead structure from these peptides several derivatives have been developed which prevent biofilm formation in many Gram-positive bacteria, including Staphylococcus aureus. Some of these compounds are already in clinical studies. In this review the different approaches to control bacterial biofilms are discussed together with the difficulties arising from the species-specificity of the autoinducers.

Abstract The flow of new and native plant-derived C in the rhizosphere of an agricultural field during one growing season was tracked, the ratios in different soil C pools were quantified, and the residence times (ts) were estimated. For this the natural differences in 13C abundances of: (1) C4 soil (with a history of C4 plant, Miscanthus sinensis, cultivation), (2) C3 soil (history of C3 plant cultivation), and (3) C4/3 soil (C4 soil, planted with a C3 plant, Triticum aestivum) were used. Total amounts and d13C values of total soil C, non-hydrolysable C, light fraction C, water-soluble C, microbial biomass C, and phospholipid fatty acids (PLFA) were determined. Using the d13C values of soil C in a mixing and a 1-box model enabled the quantification of relative contributions of C3 plant and C4 plant C to the total amount of the respective C pools in the C4/3 soil and their ts. Compared to early spring (March), the percentage of C3 plant C increased in all pools in June and August, showing the addition of new C to the different soil C fractions. In August the contribution of new C to microbial biomass C and water-soluble C reached 64 and 89%, respectively. The ts of these pools were 115 and 147 days. The d13C values of the dominant soil PLFA, 18:1?7c, cy19:0, 18:1?9c, 16:0, and 10Me16:0, showed wide ranges (-35.1 to -13.0‰) suggesting that the microbial community utilized different pools as C sources during the season. The d13C values of PLFA, therefore, enabled the analysis of the metabolically active populations. The majority of d13C values of PLFA from the C4/3 soil were closely related to those of PLFA from the C3 soil when T. aestivum biomass contributions to the soil were high in June and August. Specific populations reacted differently to changes in environmental conditions and supplies of C sources, which reflect the high functional diversity of soil microorganisms.

Many bacteria start production of pathogenicity factors when organised in aggregates, so-called biofilms, where they are much more protected against toxic agents. To form biofilms bacteria monitor their cell density by a process called quorum sensing using small, water soluble molecules which act as autoinducers. N-acyl homoserine lactones (AHL) where the acyl side chains vary in length and degree of oxidation and 4,5-dihydroxy-2,3-pentanedion or furanosyl borate diester formed from ribose-homocysteine have been identified as autoinducers but more must exist. Plants and animals exposed to a wet environment developed several strategies to control biofilms and embedded pathogenic bacteria in particular. The red macroalgae Delisea pulchra is devoid of biofilms at its surface and produces a number of secondary metabolites with a furanone ring interfering with the action of AHL. Taking the lead structure from these metabolites and from AHL several smaller molecules have been developed, which prevent biofilm formation in many bacteria. In the review the different approaches to control bacterial biofilms will be described focussing on the progress made in the development of autoinducer antagonists and problems caused by their specificity and toxicity for the host. The combination with antibiotics and first applications in animal models will be discussed.

Polar lipid-derived fatty acids (PLFA) commonly found in sulfate-reducing bacteria were detected in high abundance in the sediment harvested from a monitoring well of a petroleum-hydrocarbon (PHC)-contaminated aquifer. Aquifer microcosms were incubated under sulfate-reducing conditions with [methyl-14C]toluene to determine the 14C-mass balances and with [methyl-13C]toluene to follow the flow of carbon from toluene into biomarker fatty acids. An aliquot was used to establish an aquifer-derived toluene-degrading sulfate-reducing consortium, which grew well in liquid medium. Whole-cell hybridization using 16S rRNA-targeted oligonucleotide probes specific for different phylogenetic levels within the sulfate-reducing bacteria was applied in order to characterize the sulfate-reducing populations in the original sediment, the aquifer microcosms, and the aquifer-derived consortium. In the aquifer microcosms, the 14C quantification revealed that 61.6% of the [methyl-14C]toluene was mineralized and 2.7% was assimilated. Following [methyl-13C]toluene depletion (<1 μM), the highest 13C-enrichment was found in PLFA 16:1ω5c. In addition, biomarker fatty acids characteristic for the genera Desulfobacterand Desulfobacula(cy17:0 and 10Me16:0) were also 13C-enriched, contrary to those of other sulfate-reducing genera, e.g. Desulfovibrioand Synthrophobacter(i17:1ω7c), Desulfobulbusand Desulforhabdus(15:1ω6c and 17:1ω6c). Although hybridization detection rates remained low, indicating low bacterial activities, 43% (aquifer sediment) and 30% (aquifer microcosm) of the total active bacteria belonged to the Desulfobacteriaceae thus supporting the PLFA-based results. Desulfobacter-species (42%), which belong to the Desulfobacteriaceae, dominated the community of the consortium. Our study showed that carbon stable isotope analysis in combination with whole-cell hybridization could link toluene degradation in aquifer microcosms to the metabolic activity of the Desulfobacter-like populations. These populations could play an important role in the clean up of aromatic PHC-contaminated aquifers.

Polar lipid‐derived fatty acids (PLFA) commonly found in sulfate‐reducing bacteria were detected in high abundance in the sediment harvested from a monitoring well of a petroleum‐hydrocarbon (PHC)‐contaminated aquifer. Aquifer microcosms were incubated under sulfate‐reducing conditions with [methyl‐14C]toluene to determine the 14C‐mass balances and with [methyl‐13C]toluene to follow the flow of carbon from toluene into biomarker fatty acids. An aliquot was used to establish an aquifer‐derived toluene‐degrading sulfate‐reducing consortium, which grew well in liquid medium. Whole‐cell hybridization using 16S rRNA‐targeted oligonucleotide probes specific for different phylogenetic levels within the sulfate‐reducing bacteria was applied in order to characterize the sulfate‐reducing populations in the original sediment, the aquifer microcosms, and the aquifer‐derived consortium. In the aquifer microcosms, the 14C quantification revealed that 61.6% of the [methyl‐14C]toluene was mineralized and 2.7% was assimilated. Following [methyl‐13C]toluene depletion (<1 μM), the highest 13C‐enrichment was found in PLFA 16:1ω5c. In addition, biomarker fatty acids characteristic for the genera Desulfobacterand Desulfobacula(cy17:0 and 10Me16:0) were also 13C‐enriched, contrary to those of other sulfate‐reducing genera, e.g. Desulfovibrioand Synthrophobacter(i17:1ω7c), Desulfobulbusand Desulforhabdus(15:1ω6c and 17:1ω6c). Although hybridization detection rates remained low, indicating low bacterial activities, 43% (aquifer sediment) and 30% (aquifer microcosm) of the total active bacteria belonged to the Desulfobacteriaceae thus supporting the PLFA‐based results. Desulfobacter‐species (42%), which belong to the Desulfobacteriaceae, dominated the community of the consortium. Our study showed that carbon stable isotope analysis in combination with whole‐cell hybridization could link toluene degradation in aquifer microcosms to the metabolic activity of the Desulfobacter‐like populations. These populations could play an important role in the clean up of aromatic PHC‐contaminated aquifers.

Higher fungi are characterised by the production of macroscopic fruiting bodies to generate and to distribute their spores. These fruiting bodies are under constant threat of other organisms feeding on them. As a consequence these organisms developed a number of strategies for protection, one of them is the production of toxins. The fungal subdivision Basidiomycotina produce toxic sesquiterpenes many of them are derived from the protoilludane skeleton. This skeleton is transformed and rearranged to a large number of compounds. Some of these sesquiterpenes show interesting biological properties which may be attractive for medicinal chemistry. The overview describes the different types of bioactive fungal sesquiterpenes derived from humulene known to date in Basidiomycotina and their formation. The metabolites are discussed according to their sesquiterpene skeleton and the different metabolites are compared. Where available biological activities concerning antifungal, antibacterial, cytotoxic and enzyme inhibition data are given. Special attention was paid for the different activities of these metabolites and the attempts made to use them in medicinal chemistry. The question whether metabolites produced for the self-protection of fungi can be used for pharmaceutical applications for humans will be adressed and discussed.

While biotransformations of nortricyclanol did not give detectable products, microbial hydroxylation could be achieved by using its benzoate ester or its phenylcarbamate. Here eight fungi were found to hydroxylate these substrates in moderate yield. All strains formed optically active exo, endo-5-hydroxy-nortricyclanol-3-benzoate or -phenylcarbamate as the main product. Rhizopus arrhizus ATCC 11145 and Mortierella isabellina DSM 63355 were the best strains. Only the fermentation of the benzoate gave additional products, namely the corresponding endo, exo- and exo, endo-isomers in low yield. The structures of the metabolites were elucidated spectroscopically, particularly by using various NMR techniques. The hydroxylated nortricyclanol esters are compounds which are difficult to produce chemically. The presence of the ester function makes them chiral compounds and the biotransformations were found to be enantioselective. Therefore, microbial oxidation of these nortricyclanol esters is a valuable tool to produce chiral hydroxylated nortricyclanol derivatives which can serve as intermediates in syntheses.

Various mono- and disubstituted cinnamic acid derivatives and aromatic carboxylic acids with saturated side chains were incubated mainly with Bacillus, Candida, Hansenula, and Saccharomyces strains. The cinnamic acids carrying a hydroxy- and/or a methoxy group at the 3- and/or 4-position of the benzene ring were decarboxylated with high yields. Most of the reactions were terminated within 24 to 48 h. Substitution at other ring positions afforded also decarboxylation, but at much lower yields. Derivatives with other residues like methyl, chloride, or bromide were not transform ed to the respective styrene. None of the saturated aromatic carboxylic acids could be decarboxylated by the strains used.

Biotransformation of isopinocampheol with 100 bacterial and fungal strains yielded 1-, 2-, 4-, 5-, 7-, 8 - and 9-hydroxy-isopinocampheol besides three rearranged monoterpenes, one of them bearing the novel isocarane skeleton. A pronounced enantioselectivity between (+)- and (-)-isopinocampheol was observed. The phylogenetic position of the individual strains could be seen in their ability to form the products from (+)-isopinocampheol. The formation of 1,3-dihydroxypinane is a domain of bacteria, while 3,5- or 3,7-dihydroxypinane was mainly formed by fungi, especially those of the phylum Zygomycotina. The activity of Basidiomycotina towards oxidation of isopinocampheol was rather low. Such informations can be used in a more effective selection of strains for screening.

Corynespora cassiicola DSM 62474 and Diplodia gossypina ATCC 10936 were found to cleave some acyclic terminal terpendiols and prolongate them later by addition of a C2-unit to afford 1,2-dihydroxy-propyl-compounds. The enzymes involved in this reaction from both microorganisms displayed a strong stereoselectivity which is completely different in both strains. The stereochemi­cal requirements of the substrates for both strains and the mechanism were elucidated by using closely related substrates. The absolute configuration of the diols were solved by correlation with com pounds of known absolute configuration.

The accumulation of volatile metabolites in liquid cultures of the basidiomycete Gloeophyllum odoratum CBS 444.61 was studied after cultivation on defined synthetic liquid culture medium containing glucose (2%), asparagine (0.15%), and mineral salts. The composition of the steam volatile compounds differed distinctly from that obtained with another, previously investigated strain of this species. Two major constituents, the bicyclic sesquiterpene alcohol drimenol and a linalool derivative, 3,7-dimethyl-3-hydroxy-6-octenic acid methyl ester, could be identified by mass spectral data and 1H and 13C NMR spectroscopy. Besides several monoterpene alcohols, other minor constituents were trans-linalool oxide and 1-octen-3-ol. 3,7-Dimethyl-3-hydroxy-6-octenic acid methyl ester is described for the first time as a natural product.

Monoterpenoids with 1-p-menthene-structure can be transformed by Corynespora cassiicola DSM 62 474, DSM 62 475, and Diplodia gossypina ATCC 10 936 to chiral 1,2-trans-diols in yields of more than 60% with only minor amounts of side-products. Whereas the substrates (S)-(-)-limonene, a-terpinene, ?-terpinene, and terpinolene are converted to the (1R,2R)-p-menthene-1,2-diols, (R)-(+)-limonene and (R)-(-)-phellandrene yield the (1S,2S)-1,2-diols. For the transformation of (S)-a-terpineol and (S)-a-terpinene-4-ol to the (1S,2S)-1,2-diols Gibberella cyanea DSM 62 719 can be used, which however oxidizes parallel at the 6- and 7-position.

Microorganisms which efficiently oxidize limonene 1, do not attack 3,3,5,5-tetramethyllimonene 4. Gibberella cyanea which converts limonene 1 into 8-p-menthene-1,2-diol 2, transforms 4 into 8,9-epoxy-3,3,5,5-tetramethyl-1-menthenol-6 5 as major product. Although epoxidation is not completely stereoselective, the substituents at C-4 and C-6 are always trans to each other.

Fusarium solani strains DSM 62416 and 62413 were found to hydrate trans-nerolidol or geranylacetone at the inner double bond. The stereochemical requirements for the substrate are rather narrow. Only E-configurated double bonds were accepted by the hydratases and no hydration was observed at the terminal isoprenyl unit of various substrates. The hydratases differ in their substrate specificity. While trans-nerolidol is accepted by the hydratase of strain DSM 62416 and geranylacetone is not, the contrary is found for the hydratase of strain DSM 62413. Higher yields of hydrated products under anaerobic conditions revealed that oxygen is not required as a cofactor.

Several benzoic, cinnamic and phenylacetic acid derivatives were screened with 20 microorganisms, mainly fungi, for the reduction of their carboxylic function. For all organisms several compounds were reduced in fairly good yields up to 80% to the corresponding alcohol. No general rule could be established, concerning the substitution pattern, as to which compounds were transformed to the alcohol. Generally the reactions were accomplished within 48-70h. Only minor, if any, side products were detected. Dicarboxylic acids, such as phthalic or phenylglutaric acids and similar compounds could not be reduced by the microorganisms tested.

In order to produce the important flavour compound sinensal microbial ?-hydroxylation of farnesene and its sulfone derivative were investigated. While farnesene proved to be a poor substrate, its sulfone could be hydroxylated to the ?-hydroxyfamesene sulfones in up to 27% yield. Some strains could discriminate between the E- and Z-configurated substrate. In low yields products hycroxylated at different positions and two monocyclofarnesane derivatives could also be isolated

Trans-nerolidol and other related terpenoid compounds varying in configuration and chain length, cis-nerolidol, farnesol, and nerylacetone, were used as substrates in biotransformation reactions. These transformations afforded, among the various metabolites obtained, at least one ω-hydroxylated compound of each respective terpenoid. Aspergillus niger ATCC 9142 and Rhodococcus rubropertinctus DSM 43197, incubated with trans-nerolidol, both gave the 12-hydroxy-trans-nerolidol and the latter, in addition, was further oxidized to the 12-carboxylic acid of trans-nerolidol in fairly good yield.

The brown-rot fungus Lentinus lepideus (Fr.: Fr.) Fr. FPRL 7B (Basidiomycotina) was cultivated on a defined synthetic liquid medium containing glucose (2%), isoleucine (0.15%), and mineral salts for 105 days. The steam distillate was separated by column chromatography. Three novel sesquiterpene ethers with muurolane skeleton (lentideusether, isolentideusether, and 10-hydroxylentideusether) were isolated and their structures elucidated by spectroscopic methods, in particular by 1H NMR spectra and by two-dimensional 1H/1H- and 13C/1H-chemical shift correlation. These compounds are described for the first time as natural products. As a further metabolite, the acyclic sesquiterpene alcohol terrestrol was identified

Biotransformation of cedrol with 5 different strains afforded 8 previously undescribed hydroxy-cedrols. The attack came from a hemisphere centered at C-12 approximately. At two positions epimeric pairs were formed, which could be avoided by using different strains. Contrary to previous studies of other groups we found the 2-and 12-hydroxylations as main reactions. Cedrene needed prolonged fermentation and gave lower yields than cedrol. Apart from allylic hydroxylations the oxidation pattern by Corynespora cassiicola DSM 62474 of cedrene and cedrol was the same. The structures of the metabolites were elucidated by 2D NMR techniques.

Humulene was transform ed by strains of Chaetomium cochlioides and Diplodia gossypina ATCC 10936 affording 33 new humulanes. The first step is a non-enantioselective epoxidation of the 1,2-double bond, whereas the subsequent hydroxylations were performed preferably at the 1R ,2R -epoxide. To proof these results the anti-1,2;8,9-diepoxide was synthesized and used as substrate. While the 1R ,2R ,8S,9S-antipode of this diepoxide was hydroxylated the other one was recovered unchanged. Diplodia gossypina ATCC 10936 gave the same products but differing in relative and absolute configurations.

The basidiomycete Lentinellus cochleatus CBS 201.47 was cultivated on a defined synthetic liquid culture medium containing glucose (2%), asparagine (0.15%), malt extract (0.2%), and mineral salts. 12 weeks old cultures produced a “sweet” odour with an anise-or cinnamon-like note. 3 major volatiles could be identified from the steam distillates by their GC/MS-and 1H NMR data. Predominant constituents were the acyclic sesquiterpene alcohols trans-nerolidol (41.4%) and fokienol (14.8%). 2,2-Dimethyl-6-formylchromene (6.1%) is described for the first time as a natural product. Besides these, 15 minor constituents could be identified or chemically charac­terized, respectively, the majority of them being sesquiterpene hydrocarbons and alcohols.

The trimethylsilylalkyl acetoacetates 1 b and 2 b as well as their carba analogues 1 a and 2 a have been reduced microbiologically by Kloeckera corticis (ATCC 20109), leading to the corresponding ( + )-3(S)-hydroxybutanoates 3b, 4b, 3a, and 4a. The enantiomeric purity was found to be 80% (3a, 3b, 4b) and 65% (4a), respectively. The reduction of lb and 2b is - to our knowledge - the first example for a controlled microbiological transformation of organosilicon substrates.

Fermentation of a mixture of 3,17a,21-triacetoxy-3,5-pregna-diene-20-one and 17a ,21- diacetoxy-4-pregnene-3,20-dione with Flavobacterium dehydrogenans ATCC 13930 led to 17a-acetoxy-21-hydroxy-4-pregnene-3,20-dione as the main product and the novel 17 aacetoxy- 6ß,21-dihydroxy-4-pregnene-3,20-dione and 17a-acetoxy- 6a ,21-dihydroxy-4-pregnene- 3,20-dione as the minor ones. 6 -Hydroxylation was not found so far with this strain. The finding is in good accordance with the model of 6 -hydroxylation of steroids proposed for fungi by Holland et al

In a comparative taxonomic study, fatty acid contents of whole-cell hydrolysates and phospholipid fractions of strains belonging to the pseudomonads were analyzed. Major fatty acids in all taxa were 16:0 and various isomers of 16:1 and 18:1. Fatty acid patterns of phospholipids were easily differentiated from whole-cell profiles by their absence of hydroxylated fatty acids. The latter constituents proved to be diagnostic markers for differentiation of the main phylogenetic lineages within the pseudomonads (rRNA groups). Based on the presence or absence of three 3-hydroxy fatty acids, 10:0 30H, 12:0 30H, and 14:0 30H, the majority of the strains could be allocated to one of the four rRNA groups (I-IV) studied. In general, rRNA group I organisms were characterized by the presence of 10:0 30H and 12:0 30H, rRNA group II strains contained 14:0 30H, group III strains contained 10:0 30H and group IV strains 12:0 30H. A numerical analysis of the whole-cell fatty acid data yielded separate entities for all four rRNA groups. When using the fatty acid contents of phospholipids, no clear differentiation between rRNA group II and III taxa and rRNA group I organisms was obtained. Both approaches, however, yielded an analogous subgrouping of rRNA group I organisms which reflected their phylogenetic relationships.

Phospholipids of type strains and isolates of Caulobacter, Brevundimonas, Hyphomonasand Mycoplana bullatawere studied by chromatographic and spectroscopic methods. Using tandem mass spectrometry individual phospholipids were analyzed and their structures elucidated. From the 69 strains included in this study 26 new phosphoglucolipids and two new sulfoquinovosyl-diacylglycerols could be identified. While more than 30 different fatty acids were found in the phospholipid fraction of Caulobacterand Brevundimonasonly seven different fatty acids occur within the 26 different phosphoglucolipids identified. The distribution of the different types of phospholipids, especially of the sulfoquinovosyl-, glucoronotaurine amide- and phosphatidyl-glucopyranosyl-diacylglycerols, leads to the identification of four different phenotypic groups of these bacteria in agreement with the phylogeny published by Stahlet al. in 1992. One comprises Caulobacter sensu strictowith the type strain C. vibrioides, the second the type strains of Brevundimonastogether with a considerable number of Caulobacterspecies and the third the halophilic Caulobacter spp. around C. maris. The latter group lacks the phosphatidyl-glucopyranosyl-diacylglycerols but contains sulfoquinovosyl- and glucuronotaurine amide diacylgly- cerols. They can be discerned from Hyphomonasby the occurrence of sulfoquinovosyl-diacylglycerols which are absent in strains of the genus Hyphomonas.

Main metabolites of the microbial oxidation of (6R)-aristolenepoxide are 5α-, 9β-, 10β-, and 15-hydroxy-aristolenepoxide. Aristolene-7,14-oxide-6α-ol was also formed. Some of them showed modest phytotoxic activity in the cress test, where 9β-hydroxy-aristolenepoxide is the most active one. The actvity of the strains mirrored their phylogenetic positions. Bacteria were most active in oxidizing 15-hydroxy-aristolenepoxide to the aldehyde, while fungi, especially Basidiomycotina, were more active in the hydroxylation of the substrate than bacteria.

The basidiomycete Lepista irina (F r.) Bigelow (strains CBS 366.47 and CBS 458.79) was cultivated on a biomalt (2% )-liquid medium for 28 days. The steam distillate was analyzed by GLC and GLC-S. The structure elucidation of the predominant constituent resulted from spectroscopic methods, particularly from its 13CNMR spectrum, nuclear Overhauser enhancement 1HNMR data, and from 2d 1H homonuclear shift correlation. The bisabolane derivative was nam ed lepistirone and is described for the first time as a natural product. Two minor related com pounds could be characterized by their MS data.

Actinobacillus pleuropneumoniaeis a capnophilic pathogen of the porcine respiratory tract lacking enzymes of the oxidative branch of the tricarboxylic acid (TCA) cycle. We previously claimed that A. pleuropneumoniaeinstead uses the reductive branch in order to generate energy and metabolites. Here, we show that bicarbonate and oxaloacetate supported anaerobic growth of A. pleuropneumoniae.

ABSTRACTRecombinant attenuated Salmonella entericaserovar Typhimurium strains are believed to act as powerful live vaccine carriers that are able to elicit protection against various pathogens. Auxotrophic mutations, such as a deletion of aroA, are commonly introduced into such bacteria for attenuation without incapacitating immunostimulation. In this study, we describe the surprising finding that deletion of aroAdramatically increased the virulence of attenuated Salmonellain mouse models. Mutant bacteria lacking aroAelicited increased levels of the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) after systemic application.A detailed genetic and phenotypic characterization in combination with transcriptomic and metabolic profiling demonstrated that ΔaroAmutants display pleiotropic alterations in cellular physiology and lipid and amino acid metabolism, as well as increased sensitivity to penicillin, complement, and phagocytic uptake. In concert with other immunomodulating mutations, deletion of aroAaffected flagellin phase variation and gene expression of the virulence-associated genes arnTand ansB. Finally, ΔaroAstrains displayed significantly improved tumor therapeutic activity. These results highlight the importance of a functional shikimate pathway to control homeostatic bacterial physiology. They further highlight the great potential of ΔaroA-attenuated Salmonellafor the development of vaccines and cancer therapies with important implications for host-pathogen interactions and translational medicine.IMPORTANCERecombinant attenuated bacterial vector systems based on genetically engineered Salmonellahave been developed as highly potent vaccines. Due to the pathogenic properties of Salmonella, efficient attenuation is required for clinical applications. Since the hallmark study by Hoiseth and Stocker in 1981 (S. K. Hoiseth and B. A. D. Stocker, Nature 291:238–239, 1981, http://dx.doi.org/10.1038/291238a0), the auxotrophic ΔaroAmutation has been generally considered safe and universally used to attenuate bacterial strains. Here, we are presenting the remarkable finding that a deletion of aroAleads to pronounced alterations of gene expression, metabolism, and cellular physiology, which resulted in increased immunogenicity, virulence, and adjuvant potential of Salmonella.These results suggest that the enhanced immunogenicity of aroA-deficient Salmonellastrains might be advantageous for optimizing bacterial vaccine carriers and immunotherapy. Accordingly, we demonstrate a superior performance of ΔaroA Salmonellain bacterium-mediated tumor therapy. In addition, the present study highlights the importance of a functional shikimate pathway to sustain bacterial physiology and metabolism.

Changes in the fatty acid composition of the plasma membrane in an opportunistic pathogen promote biofilm formation.

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