Identification of a Fungal 1,8-Cineole Synthase from Hypoxylon sp. with Specificity Determinants in Common with the Plant Synthases

Terpenes are an important and diverse class of secondary metabolites widely produced by fungi. Volatile compound screening of a fungal endophyte collection revealed a number of isolates in the family Xylariaceae, producing a series of terpene molecules, including 1,8-cineole. This compound is a commercially important component of eucalyptus oil used in pharmaceutical applications and has been explored as a potential biofuel additive. The genes that produce terpene molecules, such as 1,8-cineole, have been little explored in fungi, providing an opportunity to explore the biosynthetic origin of these compounds. Through genome sequencing of cineole-producing isolate E7406B, we were able to identify 11 new terpene synthase genes. Expressing a subset of these genes in Escherichia coli allowed identification of the hyp3 gene, responsible for 1,8-cineole biosynthesis, the first monoterpene synthase discovered in fungi. In a striking example of convergent evolution, mutational analysis of this terpene synthase revealed an active site asparagine critical for water capture and specificity during cineole synthesis, the same mechanism used in an unrelated plant homologue. These studies have provided insight into the evolutionary relationship of fungal terpene synthases to those in plants and bacteria and further established fungi as a relatively untapped source of this important and diverse class of compounds.     

番荔枝内生真菌Hypoxylon sp.B38的代谢产物研究

从番荔枝内生真菌Hypoxylon sp.B38的固体平板发酵提取物中分离到5个化合物,通过NMR和MS等方法把它们分别被鉴定为Lachnellin D(1)、D ienetriol 9(2)、Lachnellin C(3)、Lachnellin B(4)和Benzo[d][1,3]d ioxol-5-ylm ethanol(5)。     

Bradyrhizobium aeschynomenes sp. nov., a root and stem nodule microsymbiont of Aeschynomene indica

Aeschynomene indica has a distinctive symbiosis with Bradyrhizobium in which nodulation is Nod factor-independent. In this study, we characterised three Gram-negative and rod-shaped strains (83002^T, 81013 and 83012) isolated from root nodules of Aeschynomene indica in Shandong Peninsula. The major cellular fatty acids of isolates were C_16:0, C_18:0, C_18:1 ω7c 11-methyl, summed feature 3 and summed feature 8. The major polar lipids were phosphatidylethanolamine (PE), aminolipids (AL) and phosphatidylcholine (PC). Phylogenetic analysis based on the 16S rRNA locus showed that they belonged to the Bradyrhizobium genus, and shared the highest similarity to the type strains Bradyrhizobium oligotrophicum S58^T and Bradyrhizobium denitrificans LMG 8443^T. As expected, analysis of concatenated sequences of six housekeeping genes (atpD, recA, glnII, dnaK, gyrB, and rpoB) and nifH gene proposed that these three strains formed a distinct clade within the genus Bradyrhizobium. The highest average nucleotide identity and DNA-DNA hybridization values of the three strains in comparison to the closest Bradyrhizobium species were 87.5% and 65.3%, respectively, which are far below the threshold of species delineation, and thus confirmed the three strains as a new species. The genome size of strain 83002^T is 7.52 Mbp, and the DNA G+C content is 65.42 mol%. Strain 83002^T (=KCTC 82266^T=MCCC 1K04775^T) was chosen as the type strain of the new species, for which the name Bradyrhizobium aeschynomenes sp. nov. was proposed.     

Whole-Genome Sequence of Enterobacter sp. Strain SST3, an Endophyte Isolated from Jamaican Sugarcane (Saccharum sp.) Stalk Tissue

Enterobacter sp. strain SST3 is an endophytic bacterium isolated from Saccharum spp. Here we present its annotated draft genome that may shed light on its role as a bacterial endophyte of sugarcane. To our knowledge, this is the first genome announcement of a sugarcane-associated bacterium from the genus Enterobacter.     

一株高产黑色素香灰菌菌株的鉴定、筛选及培养条件的优化

为筛选一株产黑色素能力强的菌株并优化其培养条件。通过ITS测序鉴定11株供试菌株,以菌丝生长速度、平板L值等指标筛选出一株产黑能力强的香灰菌,并对其生长所需碳源、氮源、pH等培养条件进行优化。研究表明,11株供试菌株均为香灰菌（Hypoxylon sp.）,其中Hp.sp0006菌丝生长速度较快、菌球大且均匀、L值最低,并且发酵液黑色素含量最高。该菌株最优培养条件是,以葡萄糖为碳源、牛肉浸膏为氮源、碳氮比20∶1并添加10 mg维生素B₁,黑色素含量可达（1.21±0.17）g/L。香灰菌Hp.sp0006是一株产黑色素较高的菌株,优化后的培养基更有利于黑色素的合成,为香灰菌黑色素的开发利用奠定基础。     

Comparison of Thraustochytrids Aurantiochytrium sp., Schizochytrium sp., Thraustochytrium sp., and Ulkenia sp. for Production of Biodiesel,Long-Chain Omega-3 Oils,and Exopolysaccharide

Heterotrophic growth of thraustochytrids has potential in coproducing biodiesel for transportation, as well as producing a feedstock for omega-3 long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA), especially docosahexaenoic acid (DHA) for use in nutraceuticals. In this study, we compared eight new endemic Australian thraustochytrid strains from the genera Aurantiochytrium, Schizochytrium, Thraustochytrium, and Ulkenia for the synthesis of exopolysaccharide (EPS), in addition to biodiesel and LC-PUFA. Aurantiochytrium sp. strains readily utilized glucose for biomass production, and increasing glucose from 2 to 4 % w/v of the culture medium resulted in increased biomass yield by an average factor of 1.7. Ulkenia sp. strain TC 010 and Thraustochytrium sp. strain TC 033 did not utilize glucose, while Schizochytrium sp. strain TC 002 utilized less than half the glucose available by day 14, and Thraustochytrium sp. strain TC 004 utilized glucose at 4 % w/v but not 2 % w/v of the culture suggesting a threshold requirement between these values. Across all strains, increasing glucose from 2 to 4 % w/v of the culture medium resulted in increased total fatty acid methyl ester content by an average factor of 1.9. Despite an increasing literature demonstrating the capacity of thraustochytrids for DHA synthesis, the production of EPS from these organisms is not well documented. A broad range of EPS yields was observed. The maximum yield of EPS was observed for Schizochytrium sp. strain TC 002 (299 mg/L). High biomass-producing strains that also have high lipid and high EPS yield may be better candidates for commercial production of biofuels and other coproducts.     

Bhargavaea indica sp. nov., a member of the phylum Firmicutes, isolated from Arabian Sea sediment

A Gram-positive, aerobic, coccoid-rod shaped, non-motile, catalase- and oxidase-positive bacterium, designated strain KJW98^T, was isolated from the marine sediment of Karwar jetty, west coast of India. The strain was β-haemolytic, non-endospore-forming and grew with 0–8.5% (w/v) NaCl, at 15–48°C and at pH 6.5–9.0, with optimum growth with 0.5% (w/v) NaCl, at 42°C and at pH 7.0–8.0. Phylogenetic analyses based on 16S rRNA and gyrB gene sequences showed that strain KJW98^T forms a lineage within the genus Bhargavaea. The G+C content of the genomic DNA was 55 mol%. The DNA-DNA relatedness values of strain KJW98^T with B. beijingensis DSM 19037^T, B. cecembensis LMG 24411^T and B. ginsengi DSM 19038^T were 43.2, 39 and 26.5%, respectively. The major fatty acids were anteiso-C_15:0 (37.7%), iso-C_15:0 (19.7%), anteiso-C_17:0 (17.0%) and iso-C_16:0 (11.1%). The predominant menaquinone was MK-8 and the cell-wall peptidoglycan was of A4α type with L-lysine as the diagnostic diamino acid. The major polar lipids were diphosphatidylglycerol and phosphatidylglycerol. The phenotypic, genotypic and DNA-DNA relatedness data indicate that strain KJW98^T should be distinguished from the members of the genus Bhargavaea, for which the name Bhargavaea indica sp. nov. is proposed with the type strain KJW98^T (=KCTC 13583^T =LMG 25219^T).     

Verticase： a Fibrinolytic Enzyme Produced by Verticillium sp, Tj33, an Endophyte of Trachelospermum jasminoides

Plant endophytes are among the most important resources of biologically active metabolites. Twenty-three endophyte strains residing in Trachelospermum jasminoides were cultivated in vitro with the cultures assayed for the fibrinolytic substance production. As a result, the culture of VerticUlium sp. Tj33 was shown to be the most active. A fibrinolytic enzyme designated as verticase was subsequently purified from the supernatant of Verticillium sp. culture broth by a combination of DEAE-52, Sephadex G-75 and hydrophobic column chromatographies. Verticase, with its molecular mass of 31 kDa and pl of 8.5, was demonstrated to be homogeneous by sodium dodecyl suIfate-polyacrylamide gel electrophoresis and isoelectric focusing electrophoresis. Verticase is an enzyme that hydrolyzes fibrin directly without activation of plaminogen. It was stable in a broad pH range from 4 through to 11 with the optimal reaction pH value and temperature shown to be around 9-10 and 50-60℃, respectively. The fibrinolytic activity of verticase was severely inhibited by phenylmethylsulfony fluoride, indicating that verticase was a serine protease.     

Potential Role of a Novel Psychrotolerant Member of the Family Geobacteraceae, Geopsychrobacter electrodiphilus gen. nov., sp. nov., in Electricity Production by a Marine Sediment Fuel Cell

Previous studies have shown that members of the family Geobacteraceae that attach to the anodes of sediment fuel cells are directly involved in harvesting electricity by oxidizing organic compounds to carbon dioxide and transferring the electrons to the anode. In order to learn more about this process, microorganisms from the anode surface of a marine sediment fuel cell were enriched and isolated with Fe(III) oxide. Two unique marine isolates were recovered, strains A1^T and A2. They are gram-negative, nonmotile rods, with abundant c-type cytochromes. Phylogenetic analysis of the 16S rRNA, recA, gyrB, fusA, rpoB, and nifD genes indicated that strains A1^T and A2 represent a unique phylogenetic cluster within the Geobacteraceae. Both strains were able to grow with an electrode serving as the sole electron acceptor and transferred ca. 90% of the electrons available in their organic electron donors to the electrodes. These organisms are the first psychrotolerant members of the Geobacteraceae reported thus far and can grow at temperatures between 4 and 30°C, with an optimum temperature of 22°C. Strains A1^T and A2 can utilize a wide range of traditional electron acceptors, including all forms of soluble and insoluble Fe(III) tested, anthraquinone 2,6-disulfonate, and S⁰. In addition to acetate, both strains can utilize a number of other organic acids, amino acids, long-chain fatty acids, and aromatic compounds to support growth with Fe(III) nitrilotriacetic acid as an electron acceptor. The metabolism of these organisms differs in that only strain A1^T can use acetoin, ethanol, and hydrogen as electron donors, whereas only strain A2 can use lactate, propionate, and butyrate. The name Geopsychrobacter electrodiphilus gen. nov., sp. nov., is proposed for strains A1^T and A2, with strain A1^T (ATCC BAA-880^T; DSM 16401^T; JCM 12469) as the type strain. Strains A1^T and A2 (ATCC BAA-770; JCM 12470) represent the first organisms recovered from anodes that can effectively couple the oxidation of organic compounds to an electrode. Thus, they may serve as important model organisms for further elucidation of the mechanisms of microbe-electrode electron transfer in sediment fuel cells.     

Shivajiella indica gen. nov., sp. nov., a marine bacterium of the family "Cyclobacteriaceae" with nitrate reducing activity

Novel orange pigmented, Gram-negative-staining, rod-shaped, non-motile, strictly aerobic strains designated NIO-S1(T) and NIO-S2 were isolated from the water sample of a pond adjacent to the coast and an algal mat from a fish pond, respectively, at Kakinada, India. Both strains were positive for oxidase, catalase and β-galactosidase activities. The predominant fatty acids in NIO-S1(T) were iso-C(15:0) (39.6%), anteiso-C(15:0) (9.9%), iso-C(17:0) 3OH (10.9%) and C(16:1)ω7c/C(16:1)ω6c (summed feature 3) (5.7%). The strains contained MK-7 as the major respiratory quinine, and diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and three unidentified lipids as the polar lipids. Phylogenetic analysis indicated that strain NIO-S1(T) was a member of the family "Cyclobacteriaceae" of the class "Sphingobacteriia" and it clustered with the genera Fontibacter, Cecembia and Aquiflexum with phylogenetic distances of 6.8, 9.0 and 12.2% (93.2, 91.0 and 87.8% similarity), respectively. DNA-DNA hybridization between strains NIO-S1(T) and NIO-S2 showed a relatedness of 93% and rep-PCR banding patterns were similar. Based on data from the current polyphasic study, it is proposed that the new isolates be placed in a new genus and species with the name Shivajiella indica gen. nov., sp. nov. The type strain of Shivajiella indica is NIO-S1(T) (= KCTC 19812(T)=MTCC 11065(T)).     

A novel species of Xenorhabdus, family Enterobacteriaceae: Xenorhabdus indica sp. nov., symbiotically associated with entomopathogenic nematode Steinernema thermophilum Ganguly and Singh, 2000

In the search for novel Xenorhabdus strains in a recently described nematode species, Steinernema thermophilum, three strains (strain 28(T) = DSM 17382(T), strain 42 = DSM 17383 and strain 43 = DSM 17384) were isolated from three independent isolation approaches from crushed mixture of infective juveniles. 16S rRNA gene sequence comparison of strains 28(T) and DSM 17383 indicated identity and the phylogenetic position pointed towards an individual taxon within the phylogenetic dendrogram of Xenorhabdus type strains. The nearest phylogenetic relatives of strain 28(T) were Xenorhabdus poinarii and Xenorhabdus szentirmaii (97.7% each). The three isolates were almost identical in reaction towards the API and BIOLOG substrate panels but differed in their reactions from those of the established type strains of the genus Xenorhabdus. These clear genomic and metabolic differences let us propose a new species, Xenorhabdus indica sp. nov. for the three clones. The type strain is strain 28(T), DSM 17382(T), CIP 108830(T).     

Effects of Polyenes, Detergents, and Other Potential Membrane Perturbants on an Osmotolerant Yeast, Saccharomyces rouxii

The osmotolerance of Saccharomyces rouxii 48-28 was confirmed with both NaCl- and KCl-fortified growth media, with more tolerance being exhibited for the potassium salt. Washed and buffered cells from unfortified medium were challenged with a variety of compounds (and also with physical treatments) that potentially would elicit membrane perturbations. The efficacy of these brief treatments was judged primarily by monitoring subsequent viability. Change in the degree of expression of beta-fructofuranosidase (EC 3.2.1.26), which is cryptic in young cells of S. rouxii, was a second criterion. There was a linear correlation between cell death and enzyme expression for treatments with polyenes, detergents, some organic solvents which did not denature the enzyme, and various freeze-thaw regimens in graded amounts of glycerol. The species is relatively insensitive to polyene antimycotics, the order of decreasing effect being filipin, nystatin, and amphotericin B. S. rouxii was found to be less sensitive to osmotic shock than is Saccharomyces cerevisiae, but in neither species is beta-fructofuranosidase released to the medium. The sensitivity of S. rouxii to ionic detergents, but not to nonionic detergents, was rationalized as being due to cell wall discrimination against larger micelles for the nonionic examples. This was confirmed by showing that protoplasts were sensitive to both classes. In cultures older than 5 days the normal agreement between colony-forming units and methylene blue exclusion (another test of viability) no longer held. Delayed fermentation of sucrose by S. rouxii, which is a diagnostic feature of the species, is explained by death of some cells, expression of their beta-fructofuranosidase, and utilization of the monosaccharides by the surviving cells.     

胡椒、芒果和黄皮的化感作用潜力

以胡椒、芒果和黄皮作为供体,玉米、黄豆、南瓜、花生、萝卜、稗草、马唐、柱花草为受体,通过种子萌发法和根生长法测定胡椒、芒果和黄皮的化感作用潜力.结果表明,玉米、黄豆、南瓜、稗草和马唐的萌发和根生长都受到3种供体水浸提液的影响,3种供体水浸提液在高浓度下抑制而在低浓度下则促进受体的萌发和根生长;胡椒和芒果根系周围的土壤对玉米萌发和根生长有促进作用而对花生的萌发和根生长则有抑制作用.3种供体的水浸提液通过乙酸乙酯、正丁醇萃取分离成不同极性的三相后分别对玉米、萝卜和柱花草进行处理.结果表明,胡椒和芒果浸提液的水相与正丁醇相对受体的抑制作用明显强于乙酸乙酯相,表明胡椒和芒果的化感物质具有较强的极性.     

Isolation, Identification and Study of Antimicrobial Property of a Bioactive Compound in an Indian Medicinal Plant Acalypha indica (Indian-Nettle)

Summary Fresh, dried and powdered samples of leaf, stem and root of Acalypha indica were subjected to fractional distillation in a soxhlet apparatus using solvents such as hexane, chloroform, acetone and methanol. The plant extracts and a synthetic antifungal compound, Clotrimazole (authentic standard) were subjected to TLC and HPLC analyses. The R_f (relative front) value of Clotrimazole was 0.371. The plant’s leaf, root and stem extracts also gave distinct spots respectively at R_f value of 0.371 ± 0.0009. In HPLC, the TLC-separated active compound and Clotrimazole resolved at 1.90 ± 0.2 min (retention time). The amounts of active compound present in root, leaf and stem extracts were 538, 415 and 171 μg/g respectively. From the results of our study, we infer that the active compound isolated from Acalypha indica is more potent in controlling Candida albicans, Aspergillus niger and Escherichia coli. The Active compound present in the plant had more than 100% activity when compared to standard Clotrimazole.