Gene inactivation mediated by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> in the filamentous fungi <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis>

The list of fungal species with known complete genome and/or expressed sequence tag collections is extending rapidly during the last couple of years. Postgenomic gene function assignment is an obvious follow-up and depends on methodologies to test gene function in vivo. One of such methods is the generation of null mutants via homologous recombination at the wild–type loci by using inactivation cassettes. In this paper, the ability of Agrobacterium tumefaciens to genetically transform filamentous fungi was exploited to drive homologous recombination at the trp1 locus of the enthomopathogenic fungus Metarhizium anisopliae. The trp1 disruptants exhibited a clearly distinguishable phenotype from wild-type cells and were recovered with high efficiency of homologous recombination (22%). The complementation of such mutants with the wild-type gene generates only transformants with homologous integration.     

Do increasingly depleted δ<Superscript>15</Superscript>N values of atmospheric N<Subscript>2</Subscript>O indicate a decline in soil N<Subscript>2</Subscript>O reduction?

Growing concentrations of N₂O within the atmosphere have been accompanied by decreasing δ¹⁵N values, provoking the hypothesis of a global decline in the rate of N₂O reduction relative to its production in soil. We estimate that the ratio of N₂O produced to N₂O reduced within the soil profile has declined by about 10–25% relative to its pre-industrial value. To a smaller extent, a reduction in the uptake of atmospheric N₂O at the soil surface relative to its emission could also have contributed to the reported isotopic signal. This calls for a greater consideration of the process of N₂O reduction in soil and its role in the global turnover of N₂O.     

Enhanced crude oil biodegradability of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> ZJU after preservation in crude oil-containing medium

This study investigated the enhanced crude oil biodegradability of Pseudomonas aeruginosa ZJU, a strain isolated from the Shengli oil field (Shandong Province, China), after preservation in a crude oil-containing medium. This strain previously could not emulsify crude oil during preservation, but after switching to a subculture in a glycerol medium for passages, it expressed increased biodegradation of crude oil within the first six passages and this biodegradation sharply decreased after the seventh passage. It is noticed that about 70% of crude oil was degraded by Pseudomonas aeruginosa ZJU in the third passage while this biodegradability was less than 19% in the seventh passage. Similar to the trend on biodegradation of crude oil, rhamnolipid production increased during the first six passages and later sharply decreased. Thus, it seems that biodegradability was proportionally related to the rhamnolipid productivity in each passage in glycerol medium. Interestingly, both rhamnolipid production and crude oil biodegradation were maintained if this strain was continuously preserved in crude oil and could be retrieved if this strain was then re-preserved in crude oil-containing medium for seven days after the significant decline in these two characteristics previously observed in the seventh passage.     

Impact of sulfur on density of <Emphasis Type="Italic">Tetranychus pacificus</Emphasis> (Acari: Tetranychidae) and <Emphasis Type="Italic">Galendromus occidentalis</Emphasis> (Acari: Phytoseiidae) in a central California vineyard

Sulfur is the oldest and most widely used fungicide in the vineyards of California, where it is used for control of powdery mildew (Uncinula necator [Schw.] Burr). For decades, sulfur use has been associated with outbreaks of Tetranychus pacificus McGregor (Acari: Tetranychidae) on cultivated grapes in the San Joaquin Valley. I undertook large-scale field studies to test this association, to evaluate the impact of sulfur on Galendromus occidentalis (Nesbit) (Acari: Phytoseiidae), a major predator of T. pacificus, and to determine if timing of sulfur applications with respect to grape bloom has an impact on T. pacificus density. The studies took place in a 32 ha vineyard in Fresno County, and all fungicide applications were made with commercial-scale equipment. In 1998 a ‘high sulfur’ treatment, a combination of wettable sulfur and sulfur dust, was compared to ‘low sulfur,’ in which demethylation inhibitor (DMI) fungicides partially substituted for sulfur. In 1999 treatments were ‘sulfur,’ ‘DMI,’ ‘sulfur pre-bloom’ (here sulfur was applied prior to grape bloom, in late May, and then DMIs were applied until mid-season) and ‘sulfur post-bloom’ (the reverse of ‘sulfur pre-bloom’). In each year, the T. pacificus population increase came after the end of fungicide applications, and results clearly show a relationship between sulfur use and T. pacificus density. In 1998, mean T. pacificus density was 2.7 times higher and mean G. occidentalis density 2.5 times higher in ‘high sulfur’ compared to ‘low sulfur.’ In 1999, the highest T. pacificus counts were in the ‘sulfur’ and ‘sulfur pre-bloom’ treatments, 4.8 times higher than ‘sulfur post-bloom’ and 2 times higher than ‘DMIs.’ Density of G. occidentalis was 2.3 times as high in ‘sulfur’ or ‘sulfur pre-bloom’ than ‘DMIs.’ The predator/prey ratio was not significantly different among treatments in 1998, but in 1999 it was highest in the ‘sulfur pre-bloom’ treatment. In 1999, density of Homeopronematus anconai (Baker) (Acari: Tydeidae) was 2.7 times higher in ‘sulfur pre-bloom’ compared to ‘sulfur,’ and higher by 2.7 times in ‘DMI’ compared to ‘sulfur post-bloom,’ suggesting a negative effect of sulfur on this tydeid. These results do not support the hypotheses that the cause of the increase in T. pacificus density is due to negative effects of sulfur on phytoseiids or tydeids. Rather, it appears that a plant-based explanation is likely, first, because of the differences in pre-bloom versus post-bloom sulfuring, and second, because of the long lag time between the end of the sulfur applications and the corresponding increase in spider mite density.     

Universal markers for comparative mapping and phylogenetic analysis in the Asteraceae (Compositae)

The development of universal markers that can be assayed across taxa, but which are polymorphic within taxa, can facilitate both comparative map-based studies and phylogenetic analyses. Here we describe the development of such markers for use in the Asteraceae, which includes the crops lettuce, sunflower, and safflower as well as dozens of locally important crop and weed species. Using alignments of a conserved orthologous set (COS) of ESTs from lettuce and sunflower and genomic sequences of Arabidopsis, we designed a suite of primer pairs that are conserved across species, but which are predicted to flank introns. We then tested 192 such primer pairs in 8 species from across the family. Of these, 163 produced an amplicon in at least 1 taxon, and 125 amplified in at least half of the taxa surveyed. Thirty-nine amplified in all 8 species. Comparisons amongst sequences within the lettuce and sunflower EST databases indicate that the vast majority of these loci will be polymorphic. As a direct test of the utility of these markers outside the lettuce and sunflower subfamilies, we sequenced a subset of ten loci from a panel of cultivated safflower individuals. All 10 loci proved to be single-locus, and nine of the 10 loci were polymorphic with an average of 12.8 SNPs per kb. Taken together, these loci will provide an initial backbone for comparative genetic analyses within the Asteraceae. Moreover, our results indicate that these loci are phylogenetically informative, and hence can be used to resolve evolutionary relationships between taxa within the family as well as within species. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Mark A. Chapman and JianCheng Chang have contributed equally to this work.     

Intensity of mineralization in the hyporheic zone of the prealpine river Bača (West Slovenia)

Respiratory electron transport system (ETS) activity and oxygen consumption in the interstitial water, and in the fine (i.e. silt) and coarse (sand) sediment fractions from the hyporheic zone of the prealpine river Bača (W Slovenia) have been measured in order to estimate the intensity of potential and actual carbon mineralization through microbial communities. Hyporheic samples from the river bed (RB) and gravel bars (GB) were compared. ETS activity and oxygen consumption of all fractions from the RB did not differ significantly from those from the GB. ETS activity and oxygen consumption of biofilm attached to 1 g of the silt were higher than of that attached to the same mass of the sand. A significant correlation between ETS activity and oxygen consumption indicated that the former should be a good indicator of intensity of bioactivity in hyporheic sediments. The ratio of ETS activity to oxygen consumption (ETS/R ratio) revealed that the oxygen consumption of microorganisms is responsible for approximately 60% of the metabolic potential in the hyporheic sediments. The contributions of different fractions of sediment to the total ETS activity differed between RB and GB. The contribution of microorganisms in the interstitial water and silt was higher in GB than in the RB, but the sand fraction contributed less to potential carbon loss in GB than in the RB. Average total respiratory carbon loss per volume through the hyporheic zone was higher in the RB than in GB. The main reasons suggested are the different intensity of exchange of surface water with the hyporheic zone, and the rate of consolidation of sediments, which is primarily a function of river hydrology and geomorphology. Handling editor: J. Padisak     

Spatio-temporal variation of photosynthetic pigments in the CAM tree <Emphasis Type="Italic">Clusia hilariana</Emphasis> Schlechtendal associated with dry spells during the rainy season in southeastern Brazil

In this work we assessed spatial and temporal variation of leaf pigment content of Clusia hilariana, a dominant CAM tree on the sandy coastal plains of SE Brazil. Chlorophyll a + b concentration, chlorophyll a/b ratio, and total carotenoid concentration were measured three times during the wet season. Chlorophyll a + b and total carotenoids decreased while the chlorophyll a/b ratio increased after a series of dry spells. We discuss our results in the context of Clusia literature, and argue that leaf-level pigmentation changes are an important source of phenotypic flexibility during acclimation to fluctuating light levels and groundwater availability.     

A trypsin inhibitor from <Emphasis Type="Italic">Cassia obtusifolia</Emphasis> seeds: isolation,characterization and activity against <Emphasis Type="Italic">Pieris rapae</Emphasis>

A trypsin inhibitor was isolated from Cassia obtusifolia by ammonium sulfate precipitation, Sepharose 4B-trypsin affinity and Sephadex G-75 chromatography. The inhibitor consisted of a single polypeptide chain with a molecular mass of 19, 812.55 Da. It was stable from pH 2 to 12 for 24 h, whereas it was unstable either above 70°C for 10 min or under reduced conditions. The inhibitor, which inhibited trypsin activity with an apparent Ki of 0.3 μM, had one reactive site involving a lysine residue. The native inhibitor was resistant to pepsin digestion, whereas the heated inhibitor produced 40% degree of susceptibility. The disulfide linkage and lysine residue were important in maintaining its conformation. Partial amino acid sequence of the purified protein showed a high degree of homology with various members of the Kunitz inhibitor family. Moreover, the inhibitor showed significant inhibitory activity against trypsin-like proteases present in the larval midgut on Pieris rapae and could suppress the growth of larvae.     

Evidence for statistical epistasis between catechol-<Emphasis Type="Italic">O</Emphasis>-methyltransferase (COMT) and polymorphisms in RGS4, G72 (DAOA), GRM3, and DISC1: influence on risk of schizophrenia

Catechol-O-methyltransferase (COMT) regulates dopamine degradation and is located in a genomic region that is deleted in a syndrome associated with psychosis, making it a promising candidate gene for schizophrenia. COMT also has been shown to influence prefrontal cortex processing efficiency. Prefrontal processing dysfunction is a common finding in schizophrenia, and a background of inefficient processing may modulate the effect of other candidate genes. Using the NIMH sibling study (SS), a non-independent case-control set, and an independent German (G) case-control set, we performed conditional/unconditional logistic regression to test for epistasis between SNPs in COMT (rs2097603, Val158Met (rs4680), rs165599) and polymorphisms in other schizophrenia susceptibility genes. Evidence for interaction was evaluated using a likelihood ratio test (LRT) between nested models. SNPs in RGS4, G72, GRM3, and DISC1 showed evidence for significant statistical epistasis with COMT. A striking result was found in RGS4: three of five SNPs showed a significant increase in risk [LRT P-values: 90387 = 0.05 (SS); SNP4 = 0.02 (SS), 0.02 (G); SNP18 = 0.04 (SS), 0.008 (G)] in interaction with COMT; main effects for RGS4 SNPs were null. Significant results for SNP4 and SNP18 were also found in the German study. We were able to detect statistical interaction between COMT and polymorphisms in candidate genes for schizophrenia, many of which had no significant main effect. In addition, we were able to replicate other studies, including allelic directionality. The use of epistatic models may improve replication of psychiatric candidate gene studies.     

Purification and Characterization of an Alkaline Protease from the Marine Yeast <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> for Bioactive Peptide Production from Different Sources

The extracellular alkaline protease in the supernatant of cell culture of the marine yeast Aureobasidium pullulans 10 was purified to homogeneity with a 2.1-fold increase in specific protease activity as compared to that in the supernatant by ammonium sulfate fractionation, gel filtration chromatography (Sephadex™ G-75), and anion-exchange chromatography (DEAE Sepharose Fast Flow). According to the sodium dodecyl sulfate-polyacrylamide gel electrophoresis data, the molecular mass of the purified enzyme was estimated to be 32.0 kDa. The optimal pH and temperature of the purified enzyme were 9.0 and 45°C, respectively. The enzyme was activated by Cu²⁺ (at a concentration of 1.0 mM) and Mn²⁺ and inhibited by Hg²⁺, Fe²⁺, Fe³⁺, Zn²⁺, and Co²⁺. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride, but weakly inhibited by EDTA, 1–10-phenanthroline, and iodoacetic acid. The K _m and V _max values of the purified enzyme for casein were 0.25 mg/ml and 0.0286 μmol/min/mg of protein, respectively. After digestion of shrimp protein, spirulina (Arthospira platensis) protein, proteins of marine yeast strains N3C (Yarrowia lipolytica) and YA03a (Hanseniaspora uvarum), milk protein, and casein with the purified alkaline protease, angiotensin I converting enzyme (ACE) inhibitory activities of the resulting peptides reached 85.3%, 12.1%, 29.8%, 22.8%, 14.1%, and 15.5%, respectively, while the antioxidant activities of these were 52.1%. 54.6%, 25.1%, 35%, 12.5%, and 24.2%, respectively, indicating that ACE inhibitory activity of the resulting peptides from the shrimp protein and antioxidant activity of those produced from the spirulina protein were the highest, respectively. These results suggest that the bioactive peptides produced by digestion of the shrimp protein with the purified alkaline protease have potential applications in the food and pharmaceutical industries.