Yeast sequencing reports. Isolation and sequence analysis of a gene from the linear DNA plasmid pPAC1–2 of Pichia acaciae that shows similarity to a killer toxin gene of Kluyveromyces lactis

The toxin-encoding linear plasmid systems found in Pichia acaciae and Kluyveromyces lactis yeasts appear to be quite similar, both in function and structural organization. By Southern hybridization, a linear plasmid of P. acaciae, pPac1–2, was found to hybridize to the second open reading frame (ORF2) of K. lactis plasmid pGKL1, known to encode the α and β subunits of the K. lactis toxin. A 1·7 kbp segment of pPac1–2 DNA was cloned, sequenced and shown to contain four regions of strong homology to four similarly oriented regions of K. lactis ORF2. This 1·7 kbp fragment also contained an ORF of 1473 bp that could encode a protein of ～ 55·8 kDa. Like the α subunit gene of K. lactis ORF2, a very hydrophobic region occurs at the N-terminus, perhaps representing a signal sequence for transport out of the cell. Unlike K. lactis ORF2, however, the encoded polypeptide is much smaller and lacks a recognizable domain common to chitinases. The structure of a toxin that includes the translation product of this P. acaciae ORF would likely be quite different from that of the K. lactis toxin. Analysis of the upstream region of the P. acaciae ORF revealed an upstream conserved sequence identical to that found before ORFs 8 and 9 of pGKL2. A possible hairpin loop structure, as has been described for each of the four K. lactis pGKL1 ORFs, was found just upstream of the presumed start codon. The similarity of the promoter-like elements found in the linear plasmid genes of these diverse yeasts reinforces the idea of the existence of a unique, but highly conserved, expression system for these novel plasmids. The sequence has been deposited in the GenBank data library under Accession Number U02596.     

Fire performance of wood (Pinus radiata) treated with fire retardants and a wood preservative

In this work, we co‐formulated an oil‐borne copper naphthenate/permethrin wood preservative system with synthetic polymer‐based fire‐retardant additives prior to the impregnation of Pinus radiata sapwood. We evaluated what effect, if any, the preservative had upon the fire performance properties of the fire retardants and whether the fire retardants impacted on the fungicidal and termiticidal efficacy of the preservative. The fire retardants included halogenated and phosphorus‐based systems. A mass loss calorimeter, in conjunction with a thermopile, was used to measure the time to ignition and the peak heat release rate (PHRR) from which the fire performance index (FPI) was determined. The preservative properties were evaluated using termite and soil‐block decay bioassays. In summary, we found that the rate of fire growth was reduced when the fire retardants were used in combination with the wood preservative. We also found that the PHRR was a better determinant of fire performance than the FPI. The performance of the wood preservative was enhanced against fungal decay and termite attack when used in combination with the fire retardants. The fire retardants also demonstrated some wood preservative properties of their own. Copyright © 2008 John Wiley & Sons, Ltd.     

丝状真菌纤维素酶合成机制的研究

丝状真菌产生的纤维素酶被认为是最有应用前景的,但就目前酶生产效率来看,离实际应用还有很大的差距,需要对酶的合成调节机制有更为全面和深刻的了解。纤维素酶的生物合成受诱导和阻遏双重控制,酶的生产既有赖于低廉的保持一定浓度诱导物的存在,又必须清除分解代谢产物对酶合成的阻遏,其中的详细机制有待进一步的阐明。真菌纤维素酶的分泌也是相当复杂的,在酶的分泌过程中,酶会发生例如糖基化等一系列变化。提高纤维素酶的活力测定方法的准确性,使其进一步规范化,是研究纤维素酶工作的另外一个重要的领域。随着现代生物学朝着分子水平的不断发展,分子生物学的许多方法也越来越多地被应用于纤维素酶的研究中,并取得了重要进展。对真菌纤维素酶合成调节机制的深入研究,将为提高纤维素酶产量,推动应用工作的发展打下理论基础。     

Flavonoids released by carrot (Daucus carota) seedlings stimulate hyphal development of vesicular-arbuscular mycorrhizal fungi in the presence of optimal CO2 enrichment

Carbon dioxide has been previously identified as a critical volatile factor that stimulates hyphal growth ofGigaspora margarita, a vesiculararbuscular mycorrhizal fungus, and we determined the optimal concentration at 2.0%. The beneficial effect of CO₂ on fungal development is also visible in the presence of stimulatory (quercetin, myricetin) or inhibitory (naringenin) flavonoids. Sterile root exudates from carrot seedlings stimulate the hyphal development ofG. margarita in the presence of optimal CO₂ enrichment. Three flavonols (quercetin, kaempferol, rutin or quercetin 3-rutinoside) and two flavones (apigenin, luteolin) were identified in carrot root exudates by means of HPLC retention time. Flavonols like quercetin and kaempferol are known to have stimulatory effects on hyphal growth ofG. margarita.     

Influence of Phenolic acids on microbial populations in the rhizosphere of cucumber

Experiments were conducted to determine whether changes in soil microbial populations that occur in response to additions of certain allelopathic phenolic acids to bulk soil also occur in the rhizosphere. Cucumber seedlings were transplanted into cups containing a nutrient-enriched mixture of Portsmouth B₁, soil and sand and were watered five times (once every 48 hr) with aqueous solutions of ferulic,p-coumaric, or vanillic acid (each at 0, 0.25, or 0.50ol/g soil material). Nutrient solution was applied on alternate days. Leaf growth was suppressed by up to 42% by phenolic acids, but changes in root growth varied with the compound and concentration in solution. Significant increases (over 600% relative to controls) in populations of fast-growing bacteria in the rhizosphere were detected after two but not after five treatments, and increases (400% relative to controls) in numbers of fungal propagules were detected after five treatments. Such increases suggested that chronic exposure to a phenolic acid might resuit in high populations of rhizosphere microorganisms that could metabolize the compounds and thus alter observable responses by the plant. To test this, plants were watered repeatedly with a low-concentration solution of ferulic acid (chronic treatments; 0.0 or 0.1mol/g soil material in one experiment, 0.000 or 0.025imol/g soil material in a second) and then once with a highconcentration solution (acute treatment; 0.0, 0.5, or 1.0mol/g soil material in the first experiment; 0.000, 0.125, or 0.250mol/g soil material in the second).Paper No. 12385 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service or the United States Department of Agriculture of the products named, nor criticism of similar ones not mentioned. Acute treatments and some chronic treatments suppressed leaf growth, but results were inconsistent for root growth. Acute treatments increased numbers of several types of bacteria in the rhizosphere but had inconsistent effects on fungi. Chronic treatments had no effect on numbers of bacteria or fungal propagules in the rhizosphere. Furthermore, chronic treatments did not alter responses of plants or microbial populations to the subsequent acute treatment. Results demonstrated that phenolic acids in soil, which must pass through the rhizosphere before interaction with plant roots can occur, alter the microbial ecology of the rhizosphere. However, microbially mediated acclimation of plants to relatively high concentrations of ferulic acid was not observed.     

Hydroxylation of Progesterone and Its Derivatives by the Entomopathogenic Strain Isaria farinosa KCh KW1.1

Progesterone biotransformation is worth studying because of the high industrial value of its derivatives. This study investigated the catalytic ability of the entomopathogenic filamentous fungus strain Isaria farinosa KCh KW1.1 to transform progesterone derivatives: 11α-hydroxyprogesterone, 17α-hydroxyprogesterone, 16α,17α-epoxyprogesterone and pregnenolone. In the culture of Isaria farinosa KCh KW1.1, 11α-hydroxyprogesterone was effectively transformed into only one product: 6β,11α-dihydroxyprogesterone. Transformation of 17α-hydroxyprogesterone gave three hydroxy derivatives: 6β,17α-dihydroxyprogesterone, 12β,17α-dihydroxyprogesterone and 6β,12β,17α-trihydroxyprogesterone. Two products: 6β-hydroxy-16α,17α-epoxyprogesterone and 6β,11α-dihydroxy-16α,17α-epoxyprogesterone, were obtained from the 16α,17α-epoxyprogesterone transformation. We isolated two compounds from the biotransformation medium with pregnenolone: 11α-hydroxy-7-oxopregnenolone and 5α,6α-epoxy-3β,11α-dihydroxypregnan-7,20-dione. In this study, we observed only mono- and dihydroxy derivatives of the tested substrates, and the number of obtained products for each biotransformation did not exceed three.     

FIELD FUNGI AND β-GLUCAN SOLUBILASE IN BARLEY KERNELS*

Significant amounts of β-glucan solubilase activity have been found in barleys harvested from a number of test sites. Enzyme activity appeared to be related to the climatic conditions at crop maturity, indicating that β-glucan solubilase was generated, possibly, by microflora on the barley grain. Species of the most common field fungi genera, Alternaria, Cladosporium, Epicoccum and Helminthosporium and two bacterial cultures were isolated from barley kernels and incubated on autoclaved barley for solubilase examinations. All the fungal isolates studied showed abilities to reduce the viscosity of carboxymethyl cellulose and to solubilise barley β-glucan. The molecular size distribution of the solubilised β-glucan products resembled that obtained for products formed by a partially purified preparation of solubilase from barley. It has been concluded, therefore, that the common field fungi associated with the hull and seed cot of barley may be the source of β-glucan solubilase.     

Cloning of Candida albicans SEC14 gene homologue coding for a putative essential function

The yeast SEC14 gene product is required for the transport of proteins from the Golgi complex. We have cloned the homologous Candida albicans SEC14 gene (CaSEC14) by functional complementation of a Saccharomyces cerevisiae thermosensitive mutant, sec14. Some putative TATA boxes have been identified in CaSEC14 and, contrary to S. cerevisiae SEC14, no introns were found in the Candida homologue. Sequence analysis revealed that CaSec14p is a 301 amino acid protein, 67% identical to S. cerevisiae and Kluyveromyces lactis Sec14p, and 61% identical to the 300 amino-terminal residues of Yarrowia lipolytica Sec14p. Hydrophatic profile analysis of CaSec14p suggests a soluble protein without transmembrane domains, as has been described for the S. cerevisiae counterpart. While it was easy to disrupt one allele of SEC14 in C. albicans, repeated attempts to disrupt the second allele were unsuccessful, thus suggesting that the gene could be essential for vegetative growth in C. albicans. The sequence has been deposited in the EMBL data library under Accession Number X81937.     

Combination Strategy of Genetic Dereplication and Manipulation of Epigenetic Regulators Reveals a Novel Compound from Plant Endophytic Fungus

The strategies of genetic dereplication and manipulation of epigenetic regulators to activate the cryptic gene clusters are effective to discover natural products with novel structure in filamentous fungi. In this study, a combination of genetic dereplication (deletion of pesthetic acid biosynthetic gene, PfptaA) and manipulation of epigenetic regulators (deletion of histone methyltransferase gene PfcclA and histone deacetylase gene PfhdaA) was developed in plant endophytic fungus Pestalotiopsis fici. The deletion of PfptaA with PfcclA and/or PfhdaA led to isolation of 1 novel compound, pestaloficiol X (1), as well as another 11 known compounds with obvious yield changes. The proposed biosynthesis pathway of pestaloficiol X was speculated using comparative analysis of homologous biosynthetic gene clusters. Moreover, phenotypic effects on the conidial development and response to oxidative stressors in the mutants were explored. Our results revealed that the new strain with deletion of PfcclA or PfhdaA in ΔPfptaA background host can neutralise the hyperformation of conidia in the PfptaA mutant, and that the ΔPfptaA ΔPfhdaA mutant was generally not sensitive to oxidative stressors as much as the ΔPfptaA ΔcclA mutant in comparison with the single mutant ΔPfptaA or the parental strains. This combinatorial approach can be applied to discover new natural products in filamentous fungi.