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Applied Microbiology and Biotechnology (v.67, #4)
About the nature of RNA interference by F. R. Schmidt (pp. 429-435).
In the context of yet unclarified issues of RNA interference (RNAi), it is discussed that RNAi-induced histone modification may not only have the purpose of inactivating native genes by blocking their transcription in the sense direction but may also simultaneously trigger transcription of the corresponding antisense strand to form double-stranded RNA for posttranscriptional gene-silencing in cells lacking RNA replicase activities. Invading foreign genetic traits may be posttranscriptionally silenced through complementary transcripts from specific, highly variable genomic regions, which are able to finally match any given sequence by the appropriate recombination and processing of their transcripts. The information to fight these traits may additionally become anchored in the genome, to provide at least a temporary “immunity” and may be inherited at least for a few generations. It is further proposed that: (1) RNA viruses evolved from constituents of the RNAi machinery through the capture of functions essential for their maintenance and replication and (2) viruses and RNAi are mutually interacting components of a universal and predominant genetic steering system that is involved in the modulation of gene expression on the cellular level and simultaneously constitutes a driving force for evolution, particularly in imperfect organisms. Such a model would deliver explanations for yet unresolved issues of RNAi, the clarification of which will have a significant impact on its future medical and biotechnological application.
Biosynthesis of the polyene macrolide antibiotic nystatin in Streptomyces noursei by Espen Fjærvik; Sergey B. Zotchev (pp. 436-443).
The polyene macrolide antibiotic nystatin, produced commercially by the bacterium Streptomyces noursei, is an important antifungal agent used in human therapy for treatment of certain types of mycoses. Early studies on nystatin biosynthesis in S. noursei provided important information regarding the precursors utilised in nystatin biosynthesis and factors affecting antibiotic yield. New insights into the enzymology of nystatin synthesis became available after the gene cluster governing nystatin biosynthesis in S. noursei was cloned and analysed. Six large polyketide synthase proteins were implicated in the formation of the nystatin macrolactone ring, while other enzymes, such as P450 monooxygenases and glycosyltransferase, were assumed responsible for ring “decoration”. The latter data, supported by analysis of the polyene mixture synthesised by the nystatin producer, helped elucidate the complete nystatin biosynthetic pathway. This information has proved useful for engineered biosynthesis of novel nystatin analogues, suggesting a plausible route for the generation of potentially safer and more efficient antifungal drugs.
Mannopeptimycins, a novel class of glycopeptide antibiotics active against gram-positive bacteria by Haiyin He (pp. 444-452).
Mannopeptimycins α–ɛ, novel glycopeptides with activity against methicillin-resistant staphylococci and vancomycin-resistant enterococci, are purified from the fermentation broth of a strain of Streptomyces hygroscopicus, LL-AC98, and their structures characterized using spectroscopic analyses and chemical methods. The SAR data of the natural and synthetic esters demonstrate that the presence of hydrophobic groups near the terminal mannosyl moiety is critical for antibacterial potency. Scalable syntheses of 4,6-cyclic acetals and ketals on this moiety are used to produce significant quantities of the respective mannopeptimycin derivatives. These acetal and ketal derivatives exhibit potent activities against susceptible and resistant Gram-positive bacteria in both in vitro and in vivo experiments, comparable with or exceeding the activity of vancomycin. Studies on the mechanism of action suggest that the mannopeptimycins interfere with the late stages of bacterial cell wall biosynthesis. It is believed that these antibiotics inhibit the transglycosylation by binding to the transglycosylase substrate, lipid II.
Medicinal mushroom modulators of molecular targets as cancer therapeutics by Ben-Zion Zaidman; Majed Yassin; Jamal Mahajna; Solomon P. Wasser (pp. 453-468).
Empirical approaches to discover anticancer drugs and cancer treatments have made limited progress in the past several decades in finding a cure for cancer. The expanded knowledge of the molecular basis of tumorigenesis and metastasis, together with the inherently vast structural diversity of natural compounds found in mushrooms, provided unique opportunities for discovering new drugs that rationally target the abnormal molecular and biochemical signals leading to cancer. This review focuses on mushroom low-molecular-weight secondary metabolites targeting processes such as apoptosis, angiogenesis, metastasis, cell cycle regulation, and signal transduction cascades. Also discussed in this review are high-molecular-weight polysaccharides or polysaccharide–protein complexes from mushrooms that appear to enhance innate and cell-mediated immune responses, exhibit antitumor activities in animals and humans, and demonstrate the anticancer properties of selenium compounds accumulated in mushrooms.
Pleading for the use of biodegradable polymers in favor of marine environments and to avoid an asbestos-like problem for the future by Masahisa Kubota; Katsumi Takayama; Daisuke Namimoto (pp. 469-476).
Research results about the movement and accumulation of floating marine debris drifting throughout the world’s oceans are reviewed in this paper. A mechanism for this accumulation and movement is strongly associated with surface currents consisting of the Ekman drift and the geostrophic current, because all floating marine debris is passive to surface currents. The basic published mechanism for the North Pacific is common across the world’s ocean. After marine debris accumulates in the narrow Ekman convergence zone, it is moved to the east by geostrophic currents. The most important thing is that floating marine debris concentrates in some specific regions, independent of the initial quantity of marine debris. In order to resolve this problem and to avoid an asbestos-like problem, the use of biodegradable polymers is important in our daily life.
Bioconversion of (+)-valencene in submerged cultures of the ascomycete Chaetomium globosum by Rüdiger Kaspera; Ulrich Krings; Tsevegsuren Nanzad; Ralf G. Berger (pp. 477-483).
Submerged cultures of the ascomycete Chaetomium globosum oxidised the exogenous sesquiterpene (+)-valencene to nootkatone via the stereoselective generation of α-nootkatol. Inhibition experiments suggested that the first introduction of oxygen, the rate-limiting step of the bioconversion, may have been catalysed by a cytochrome-P450-monooxygenase. However, nootkatone was not the final metabolite: further flavour-active and inactive, non-volatile oxidation products were identified. (+)-Valencene and the flavour-active mono-oxyfunctionalised transformation products, α-nootkatol, nootkatone, and valencene-11,12-epoxide accumulated preferably inside the fungal cells. Di- and poly-oxygenated products, such as nootkatone-11,12-epoxide, were found solely in the culture medium, indicating an active transport of these metabolites into the extracellular compartment during (+)-valencene detoxification. These metabolic properties may have contributed to the high tolerance of the fungus towards the exogenous hydrocarbon.
Influence of substrate surface loading on the kinetic behaviour of aerobic granules by Yu Liu; Yong-Qiang Liu; Zhi-Wu Wang; Shu-Fang Yang; Joo-Hwa Tay (pp. 484-488).
In the aerobic granular sludge reactor, the substrate loading is related to the size of the aerobic granules cultivated. This study investigated the influence of substrate surface loading on the growth and substrate-utilization kinetics of aerobic granules. Results showed that microbial surface growth rate and surface biodegradation rate are fairly related to the substrate surface loading by the Monod-type equation. In this study, both the theoretical maximum growth yield and the Pirt maintenance coefficient were determined. It was found that the estimated theoretical maximum growth yield of aerobic granules was as low as 0.2 g biomass g−1 chemical oxygen demand (COD) and 10–40% of input substrate-COD was consumed through the maintenance metabolism, while experimental results further showed that the unit oxygen uptake by aerobic granules was 0.68 g oxygen g−1 COD, which was much higher than that reported in activated sludge processes. Based on the growth yield and unit oxygen uptake determined, an oxidative assimilation equation of acetate-fed aerobic granules was derived; and this was confirmed by respirometric tests. In aerobic granular culture, about 74% of the input substrate-carbon was converted to carbon dioxide. The growth yield of aerobic granules was three times lower than that of activated sludge. It is likely that high carbon dioxide production is the main cause of the low growth yield of aerobic granules, indicating a possible energy uncoupling in aerobic granular culture.
Oxidation of milled wood lignin with laccase, tyrosinase and horseradish peroxidase by S. Grönqvist; L. Viikari; M.-L. Niku-Paavola; M. Orlandi; C. Canevali; J. Buchert (pp. 489-494).
In this paper the oxidation of milled wood lignin (MWL), catalysed by three enzymes, i.e. laccase, tyrosinase and horseradish peroxidase (HRP) was studied. The oxidation was followed by measuring the consumption of O2 during laccase and tyrosinase treatment and of H2O2 during HRP treatment. Both laccase and HRP were found to oxidise lignin effectively, whereas the effect of tyrosinase was negligible. The changes in MWL molecular-weight distributions caused in the reactions were analysed by gel permeation chromatography. Both laccase and HRP treatments were found to polymerise MWL. Peroxidase treatment was found to decrease the amount of phenolic hydroxyls in MWL, whereas no such effect could be detected in the laccase-treated sample. Both laccase and HRP treatments were, however, found to increase the amount of conjugated structures in MWL. The formation of phenoxy radicals during the treatments was studied by electron paramagnetic resonance spectroscopy. Phenoxy radicals were detected in both laccase and HRP-treated samples. The amount of the formed phenoxy radicals was found to be essentially constant during the detected time (i.e. 20–120 min after the addition of enzyme).
Thermostable xylanases, Xyn10A and Xyn11A, from the actinomycete Nonomuraea flexuosa: isolation of the genes and characterization of recombinant Xyn11A polypeptides produced in Trichoderma reesei by S. Leskinen; A. Mäntylä; R. Fagerström; J. Vehmaanperä; R. Lantto; M. Paloheimo; P. Suominen (pp. 495-505).
Two endoxylanases, Nf Xyn11A and Nf Xyn10A, were cloned from a Nonomuraea flexuosa (previously Actinomadura flexuosa) DSM43186 genomic expression library in Escherichia coli. The coding sequences of xyn11A and xyn10A consist of 344 and 492 amino acids, respectively. The catalytic domains belong to family 11 and family 10 of glycoside hydrolases. The C-termini share strong amino acid sequence similarity to carbohydrate-binding module (CBM) families CBM2 and CBM13, respectively. Native Nf Xyn11A, and recombinant Xyn11A expressed in the filamentous fungus Trichoderma reesei, were purified from cultivation media and characterized. The molecular masses of the full-length enzymes determined by mass spectrometry were 32.9 kDa and 33.4 kDa, the recombinant enzyme having higher molecular mass due to glycosylation. In addition, shorter polypeptides with molecular masses of 23.8 kDa and 22.0 kDa were characterized from the T. reesei culture medium, both lacking the C-terminal CBM and the 22.0 kDa polypeptide also lacking most of the linker region. The recombinant polypeptides were similar to each other in terms of specific activity, pH and temperature dependence. However, the 23.8 kDa and 22.0 kDa polypeptides were more thermostable at 80°C than the full-length enzyme. All polypeptide forms were effective in pretreatment of softwood kraft pulp at 80°C.
Metabolism of dibenzofuran and dibenzo-p-dioxin by the biphenyl dioxygenase of Burkholderia xenovorans LB400 and Comamonas testosteroni B-356 by José-Bruno L’Abbée; Diane Barriault; Michel Sylvestre (pp. 506-514).
We examined the metabolism of dibenzofuran (DF) and dibenzo-p-dioxin (DD) by the biphenyl dioxygenase (BPDO) of Comamonas testosteroni B-356 and compared it with that of Burkholderia xenovorans LB400. Data showed that both enzymes oxygenated DF at a low rate, but Escherichia coli cells expressing LB400 BPDO degraded DF at higher rate (30 nmol in 18 h) compared with cells expressing B-356 BPDO (2 nmol in 18 h). Furthermore, both BPDOs produced dihydro-dihydroxy-dibenzofuran as a major metabolite, which resulted from the lateral oxygenation of DF. 2,2′,3-Trihydroxybiphenyl (resulting from angular oxygenation of DF) was a minor metabolite produced by both enzymes. Deuterated DF was used to demonstrate the production of 2,2′,3-dihydroxybiphenyl through angular oxygenation of DF. When tested for their ability to oxygenate DD, both enzymes produced as sole metabolite, 2,2′,3-trihydroxybiphenyl ether at about the same rate, indicating similar catalytic properties toward this substrate. Altogether, although LB400 and B-356 BPDOs oxygenate a different range of chlorobiphenyls, their metabolite profiles toward DF and DD are similar. This suggests that co-planarity influences the regiospecificity of BPDO toward DF and DD to a higher extent than the presence of an ortho substituent on the molecule.
Effect of Sorghum vulgare phosphoenolpyruvate carboxylase and Lactococcus lactis pyruvate carboxylase coexpression on succinate production in mutant strains of Escherichia coli by Henry Lin; Ka-Yiu San; George N. Bennett (pp. 515-523).
Sorghum vulgare phosphoenolpyruvate carboxylase (PEPC) and Lactococcus lactis pyruvate carboxylase (PYC) were overexpressed in Escherichia coli concurrently to improve the production of succinate, a valuable industrial specialty chemical. This coexpression system was also applied to E. coli mutant strains strategically designed by inactivating the competing pathways of succinate formation. The highest level of succinate production was observed in E. coli strains coexpressing both PEPC and PYC when compared with E. coli strains individually overexpressing either PEPC or PYC. Lactate production was also significantly reduced with PEPC and PYC coexpression. Lactate and acetate pathways were inactivated to eliminate the competing pathways of succinate formation. Results showed that inactivation of both the lactate and acetate pathways with the coexpression of PEPC and PYC was most effective in improving succinate production. Inactivating the lactate or acetate pathway alone only caused a majority of the carbon flux to shift to other metabolites rather than succinate. Coexpression of PEPC and PYC was also applied to an E. coli mutant strain deficient in lactate dehydrogenase and pyruvate:formate lyase that accumulated a substantial amount of the intermediate metabolite pyruvate during growth. Results showed that PEPC and PYC coexpression was effective in depleting pyruvate accumulation and increasing the production of metabolites.
A PCR method for the detection and differentiation of Lentinus edodes and Trametes versicolor in defined-mixed cultures used for wastewater treatment by Jaime García-Mena; Claudia Cano-Ramirez; Claudio Garibay-Orijel; Sergio Ramirez-Canseco; Héctor M. Poggi-Varaldo (pp. 524-531).
A PCR-based method for the quantitative detection of Lentinus edodes and Trametes versicolor, two ligninolytic fungi applied for wastewater treatment and bioremediation, was developed. Genomic DNA was used to optimize a PCR method targeting the conserved copper-binding sequence of laccase genes. The method allowed the quantitative detection and differentiation of these fungi in single and defined-mixed cultures after fractionation of the PCR products by electrophoresis in agarose gels. Amplified products of about 150 bp for L. edodes, and about 200 bp for T. versicolor were purified and cloned. The PCR method showed a linear detection response in the 1.0 μg–1 ng range. The same method was tested with genomic DNA from a third fungus (Phanerochaete chrysosporium), yielding a fragment of about 400 bp. Southern-blot and DNA sequence analysis indicated that a specific PCR product was amplified from each genome, and that these corresponded to sequences of laccase genes. This PCR protocol permits the detection and differentiation of three ligninolytic fungi by amplifying DNA fragments of different sizes using a single pair of primers, without further enzymatic restriction of the PCR products. This method has potential use in the monitoring, evaluation, and improvement of fungal cultures used in wastewater treatment processes.
Heterologous expression of enterocin A, a bacteriocin from Enterococcus faecium, fused to a cellulose-binding domain in Escherichia coli results in a functional protein with inhibitory activity against Listeria by Michael Klocke; Kerstin Mundt; Frank Idler; Sabrina Jung; Jan E. Backhausen (pp. 532-538).
The genes for the bacteriocins enterocin A and B were isolated from Enterococcus faecium ATB 197a. Using the pET37b(+) vector, the enterocin genes were fused to an Escherichia coli specific export signal sequence, a cellulose-binding domain (CBDcenA) and a S-tag under the control of a T7lac promotor. The constructs were subsequently cloned into E. coli host cells. The expression of the recombinant enterocins had different effects on both the host cells and other Gram-positive bacteria. The expression of entA in Esc. coli led to the synthesis and secretion of functional active enterocin A fusion proteins, which were active against some Gram-positive indicator bacteria, but did not influence the viability of the host cells. In contrast, the expression of enterocin B fusion proteins led to a reduced viability of the host cells, indicating a misfolding of the protein or interference with the cellular metabolism of Esc. coli. Indicator strains of Gram-positive bacteria were not inhibited by purified enterocin B fusion proteins. However, recombinant enterocin B displayed inhibitory activity after the proteolytic cleavage of the fused peptides.
Rapid screening and dereplication of bacterial isolates from marine sponges of the Sula Ridge by Intact-Cell-MALDI-TOF mass spectrometry (ICM-MS) by R. Dieckmann; I. Graeber; I. Kaesler; U. Szewzyk; H. von Döhren (pp. 539-548).
Rapid grouping of bacterial isolates is critical in comprehensive microbial studies of environmental samples or screening programmes e.g. in unknown marine environments where large numbers of strains have to be isolated on different growth media. Sets of bacteria have been cultured from the marine sponges Isops phlegraei, Haliclona sp. 1, Phakellia ventilabrum and Plakortis sp. growing at a depth of about 300 m on the Sula Ridge close to the Norwegian coast. We employed Intact-Cell MALDI-TOF (ICM) mass spectrometry to achieve a rapid proteometric clustering of a subset of the strain collection including 456 isolates. Cluster analysis of mass spectra resolved the strains into 11 groups corresponding to species of Alteromonas (15), Bacillus (3), Colwellia (31), Erythrobacter (19), Marinobacter (14), Marinococcus (6), Pseudoalteromonas (297), Pseudomonas (56), Roseobacter (3), Sphingomonas (2) and Vibrio (10) as verified by 16 S rDNA analysis. A further discrimination into subgroups was demonstrated for different isolates from the genus Pseudoalteromonas. The approach described here permits the rapid identification of isolates for dereplication, and the selection of strains representing rare species for subsequent characterization.
Analysis of mating-dependent transcription of Blakeslea trispora carotenoid biosynthesis genes carB and carRA by quantitative real-time PCR by André D. Schmidt; Thorsten Heinekamp; Markus Matuschek; Burghard Liebmann; Claus Bollschweiler; Axel A. Brakhage (pp. 549-555).
The zygomycete fungus Blakeslea trispora is used commercially as natural source of β-carotene. β-Carotene production is strongly induced during mating of two strains of the opposite sex and results in the production of the pheromone trisporic acid, which in turn stimulates enhanced β-carotene biosynthesis. β-Carotene production is due to the enzymatic activity of phytoene synthase, lycopene cyclase and phytoene dehydrogenase. The corresponding genes, carRA and carB, were isolated from a cosmid library generated from B. trispora strain ATCC14272. The steady state level of carB and carRA mRNA transcripts under different mating conditions was monitored by both northern blot analysis and quantitative real-time PCR. The steady state levels of carRA and carB mRNA of non-mated and mated B. trispora were quantified relative to transcript levels of the translation elongation factor 1α-encoding tef1 gene, since tef1 is transcribed independently of mating. Transcription levels of both carB and carRA were strongly induced only under mating conditions. These data suggest that β-carotene production in B. trispora is due to increased transcription of the biosynthesis genes carB and carRA.
Hydroxylation activity of P450 BM-3 mutant F87V towards aromatic compounds and its application to the synthesis of hydroquinone derivatives from phenolic compounds by Woro Triarsi Sulistyaningdyah; Jun Ogawa; Qing-Shan Li; Chiharu Maeda; Yuki Yano; Rolf D Schmid; Sakayu Shimizu (pp. 556-562).
Cytochrome P450 BM-3 from Bacillus megaterium is a fatty acid hydroxylase exhibiting selectivity for long-chain substrates (12–20 carbons). Replacement of Phe87 in P450 BM-3 by Val (F87V) greatly increased its activity towards a variety of aromatic and phenolic compounds. The apparent initial reaction rates of F87V as to benzothiophene, indan, 2,6-dichlorophenol, and 2-(benzyloxy)phenol were 227, 204, 129, and 385 nmol min−1 nmol−1 P450, which are 220-, 66-, 99-, and 963-fold those of the wild type, respectively. These results indicate that Phe87 plays a critical role in the control of the substrate specificity of P450 BM-3. Furthermore, F87V catalyzed regioselective hydroxylation at the para position of various phenolic compounds. In particular, F87V showed high activity as to the hydroxylation of 2-(benzyloxy)phenol to 2-(benzyloxy)hydroquinone. With F87V as the catalyst, 0.71 mg ml−1 2-(benzyloxy)hydroquinone was produced from 1.0 mg ml−1 2-(benzyloxy)phenol in 4 h, with a molar yield of 66%.
Biofiltration of waste gases with the fungi Exophiala oligosperma and Paecilomyces variotii by Elena Estévez; María C. Veiga; Christian Kennes (pp. 563-568).
Two biofilters fed toluene-polluted air were inoculated with new fungal isolates of either Exophiala oligosperma or Paecilomyces variotii, while a third bioreactor was inoculated with a defined consortium composed of both fungi and a co-culture of a Pseudomonas strain and a Bacillus strain. Elimination capacities of 77 g m−3 h−1 and 55 g m−3 h−1 were reached in the fungal biofilters (with removal efficiencies exceeding 99%) in the case of, respectively, E. oligosperma and Paecilomyces variotii when feeding air with a relative humidity (RH) of 85%. The inoculated fungal strains remained the single dominant populations throughout the experiment. Conversely, in the biofilter inoculated with the bacterial–fungal consortium, the bacteria were gradually overgrown by the fungi, reaching a maximum elimination capacity around 77 g m−3 h−1. Determination of carbon dioxide concentrations both in batch assays and in biofiltration studies suggested the near complete mineralization of toluene. The non-linear toluene removal along the height of the biofilters resulted in local elimination capacities of up to 170 g m−3 h−1 and 94 g m−3 h−1 in the reactors inoculated, respectively, with E. oligosperma and P. variotii. Further studies with the most efficient strain, E. oligosperma, showed that the performance was highly dependent on the RH of the air and the pH of the nutrient solution. At a constant 85% RH, the maximum elimination capacity either dropped to 48.7 g m−3 h−1 or increased to 95.6 g m−3 h−1, respectively, when modifying the pH of the nutrient solution from 5.9 to either 4.5 or 7.5. The optimal conditions were 100% RH and pH 7.5, which allowed a maximum elimination capacity of 164.4 g m−3 h−1 under steady-state conditions, with near-complete toluene degradation.
Degradation of anthracene and pyrene supplied by microcrystals and non-aqueous-phase liquids by Srikanth Mutnuri; N. Vasudevan; Matthias Kaestner (pp. 569-576).
Polycyclic aromatic hydrocarbons (PAHs) are worldwide environmental pollutants. Their bioavailability is limited by a low aqueous solubility, which causes specific adaptations in degrading bacteria. To compare bacterial degrading behavior, a study was conducted on the mineralization, metabolization and formation of biomass from 14C-anthracene by Sphingomonas sp. BA2 compared with those from 14C-pyrene by Gordonia-like strain BP9 and Mycobacterium gilvum VF1. Different conditions of PAH supply were used in the medium: crystals <0.5 mm, microcrystals <<0.1 mm formed by sonication, or PAH solubilized in 2,2,4,4,6,8,8-heptamethylnonane (HMN) or silicone oil. Anthracene supply by crystals and silicone oil led to similar maximum mineralization rates 33 ng ml−1 h−1 and the same amount of mineralization (24%) after 168 h. Microcrystals increased the rates and amounts only slightly. HMN decreased the values to less than one-third. In comparison with crystals, microcrystals increased overall pyrene mineralization by strain BP9 from 53% to 58%, with maximum mineralization rates of 160 ng ml−1 h−1 and 166 ng ml−1 h−1. Silicone oil heavily increased the rate to 292 ng ml−1 h−1 and the amount mineralized to 71%, whereas HMN inhibited the degradation by one order of magnitude. A similar degradation behavior showing lower mineralization rates and extent was observed with strain VF1. However, inhibition by HMN was less pronounced. Sonication, leading to decreased PAH crystal size, increased the mass transfer and mineralization rates. PAH supply by silicone oil led to a much higher mass transfer, which may be due to emulsification of the oil, whereas such effects were not observed with HMN.