Origin of (−)-Geosmin on Grapes: On the Complementary Action of Two Fungi, <Emphasis Type="Italic">Botrytis Cinerea</Emphasis> and <Emphasis Type="Italic">Penicillium Expansum</Emphasis>

One of the consequences of rot on grapes is the development of volatile compounds giving fungal, mouldy or earthy odours. Among these compounds, (−)-geosmin (trans-1,10-dimethyl-trans-9-decalol), a powerful aromatic compound with an earthy smell is a persistent defect in grape juice and wines made with at least partially rotten grapes. A microbiota analysis of rotten grapes containing (−)-geosmin was carried out on sites from four French regions from 1999 to 2002, to clarify the involvement in geosmin appearance of Streptomyces spp. and Penicillium spp., two types of microorganisms present on grape, that are known for their ability to produce geosmin. In earthy grapes, Botrytis cinerea was largely present. Different species of Streptomyces were also isolated, but their pH sensitivity was an extremely limiting parameter for their development on grape juice, grapes or stem, and consequently for their potentiality to generate geosmin in the vineyard. Penicillium expansum, producing geosmin on a model medium, was omnipresent. Penicillium carneum, which is also a geosmin producer, was represented by a single colony during the 4 years of this study. P. expansum alone was able to produce geosmin on a model medium but not on grapes. However, after 7 days’ pre-culture of some B. cinerea strains on grape juice, this juice became favourable to geosmin production by P. expansum. We demonstrated the necessary and complementary action of B. cinerea and P. expansum in geosmin production in grape juice and in crushed grape berries.     

<Emphasis Type="Italic">Carex</Emphasis> × <Emphasis Type="Italic">involuta</Emphasis> and <Emphasis Type="Italic">Carex juncella</Emphasis> in the flora of Slovakia

The paper brings information on an isolated occurrence and morphological characters of Carex × involuta and C. juncella populations in the Vel’ká Fatra Mts. Their presence has been known neither from the territory of Slovakia nor from the whole Western Carpathians till now.     

Functional expression of the lipase gene Lip2 of <Emphasis Type="Italic">Pleurotus</Emphasis> <Emphasis Type="Italic">sapidus</Emphasis> in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

The lipase Lip2 of the edible basidiomycete, Pleurotus sapidus, is an extracellular enzyme capable of hydrolysing xanthophyll esters with high efficiency. The gene encoding Lip2 was expressed in Escherichia coli TOP10 using the gene III signal sequence to accumulate proteins in the periplasmatic space. The heterologous expression under control of the araBAD promoter led to the high level production of recombinant protein, mainly as inclusion bodies, but partially in a soluble and active form. A fusion with a C-terminal His tag was used for purification and immunochemical detection of the target protein. This is the first example of a heterologous expression and periplasmatic accumulation of a catalytically active lipase from a basidiomycete fungus.     

Brood cell size of <Emphasis Type="Italic">Apis</Emphasis> <Emphasis Type="Italic">mellifera</Emphasis> modifies the reproductive behavior of <Emphasis Type="Italic">Varroa</Emphasis> <Emphasis Type="Italic">destructor</Emphasis>

We undertook a field study to determine whether comb cell size affects the reproductive behavior of Varroa destructor under natural conditions. We examined the effect of brood cell width on the reproductive behavior of V. destructor in honey bee colonies, under natural conditions. Drone and worker brood combs were sampled from 11 colonies of Apis mellifera. A Pearson correlation test and a Tukey test were used to determine whether mite reproduction rate varied with brood cell width. Generalized additive model analysis showed that infestation rate increased positively and linearly with the width of worker and drone cells. The reproduction rate for viable mother mites was 0.96 viable female descendants per original invading female. No significant correlation was observed between brood cell width and number of offspring of V. destructor. Infertile mother mites were more frequent in narrower brood cells.     

Effect of <Emphasis Type="Italic">POX3</Emphasis> gene disruption using self-cloning <Emphasis Type="Italic">CRF1</Emphasis> cassette in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> on the γ-decalactone production

γ-Decalactone, an important flavor compound, can be produced by Yarrowia lipolytica, but the yield is poor because of lactone degradation by enzyme Aox3 (POX3 gene encoded). A yeast strain Yarrowia lipolytica TA1 of high γ-decalactone yield was constructed by integrating copper-resistance gene CRF1 from Yarrowia lipolytica into the locus of POX3 genes. After being cultured in shake-flask at 28°C for 90 h, TA1 reached a γ-decalactone yield of 0.531 g/l (highest at 63 h), being 2.9 times higher than that of Yarrowia lipolytica As2.1045 (0.194 g/l, highest at 57 h). It was free of heterologous DNA sequences and drug-resistance genes and could be safely used in γ-decalactone production. And this work may throw a new light on addressing the problems in commercial fragrance manufacture.     

<Emphasis Type="Italic">In vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> inhibition of α-amylases of stored-product mite <Emphasis Type="Italic">Acarus siro</Emphasis>

The stored-product mites are the most abundant and frequent group of pests living on the stored food products in Europe. They endanger public health since they produce allergens and transmit mycotoxin-producing fungi. Novel acaricidal compounds with inhibitory effects on the digestive enzymes of arthropods are a safe alternative to the traditional neurotoxic pesticides used for control of the stored-product pests. In this work, we explored the properties of acarbose, the low molecular weight inhibitor of -amylases (AI), as a novel acaricide candidate for protection of the stored products from infestation by Acarus siro (Acari: Acaridae). In vitro analysis revealed that AI blocked efficiently the enzymatic activity of digestive amylases of A. siro, and decreased the physiological capacity of mites gut in utilizing a starch component of grain flour. In vivo experiments showed that AI suppressed the population growth of A. siro. The mites were kept for three weeks on experimental diet enriched by AI in concentration range of 0.005 to 0.25%. Population growth of A. siro was negatively correlated with the content of AI in the treated diet with a half-population dose of 0.125%. The suppressive effect of AIs on stored-product mites is discussed in the context of their potential application in GMO crops     

Disruption of the GPI Protein-Encoding Gene <Emphasis Type="Italic">IFF4</Emphasis> of <Emphasis Type="Italic">Candida</Emphasis> <Emphasis Type="Italic">albicans</Emphasis> Results in Decreased Adherence and Virulence

Candida albicans is the most important cause of systemic fungal infection in immunocompromised humans. Candidiasis is often initiated by the adherence and the colonization of inert surfaces such as peripheral venous catheters, central catheters, prosthetic cardiac valves, and other prostheses. We have studied the early stage of adherence and have shown that the disruption of C. albicans IFF4 gene encoding a GPI-anchor protein, led to a decrease of adherence of the germ tubes to plastic. Here, we demonstrated the role of the IFF4 gene in adherence to silicone catheter, as well as in virulence using a murine model of disseminated candidiasis. The iff4 Δ null mutant showed both a decrease of adherence to silicone catheter and a reduction of virulence. This work presents evidence for the importance of the IFF4 gene in host-fungal interaction.     

Factors influencing the production of stilbenes by the knotweed, <Emphasis Type="Italic">Reynoutria</Emphasis> × <Emphasis Type="Italic">bohemica</Emphasis>

Background  

Japanese knotweed, Reynoutria japonica, is known for its high growth rate, even on adverse substrates, and for containing organic substances that are beneficial to human health. Its hybrid, Reynoutria × bohemica, was described in the Czech Republic in 1983 and has been widespread ever since. We examined whether Reynoutria × bohemica as a medicinal plant providing stilbenes and emodin, can be cultivated in spoil bank substrates and hence in the coalmine spoil banks changed into arable fields. We designed a pot experiment and a field experiment to assess the effects of various factors on the growth efficiency of Reynoutria × bohemica on clayish substrates and on the production of stilbenes and emodin in this plant.     

Butterfly diversity in Mediterranean islands and in Pentadaktylos <Emphasis Type="Italic">Pinus</Emphasis><Emphasis Type="BoldItalic"> </Emphasis><Emphasis Type="Italic">brutia</Emphasis> forests of Cyprus

We analysed the influence of contemporary geography on butterfly diversity for islands in the Mediterranean Basin. We found that island size and distance from the mainland has a significant effect on the number of species. We also used butterflies as an indicator group to identify the importance of forest habitats for biodiversity conservation in the island of Cyprus. To understand the relative importance of local vegetation characteristics of butterflies in the Pentadaktylos mountains transect counts were used to assess the abundance and butterfly diversity in two different forest types. A total of 1,602 butterflies and 23 species were recorded during this research. We observed highly significant effects of forest type on abundance and species richness of butterflies. For example, number of butterflies was significantly higher in old forest than young pine forest. Also, the abundance of endemic butterflies was highest in old forest habitats. Therefore, the survival of the majority of endemic butterflies in Cyprus may depend on conservation of old forests and their understorey plants.     

Overexpressing <Emphasis Type="Italic">GLT1</Emphasis> in <Emphasis Type="Italic">gpd1</Emphasis>Δ mutant to improve the production of ethanol of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

To improve ethanol production in Saccharomyces cerevisiae, two yeast strains were constructed. In the mutant, KAM-4, the GPD1 gene, which encodes a glycerol 3-phosphate dehydrogenase of S. cerevisiae to synthesize glycerol, was deleted. The mutant KAM-12 had the GLT1 gene (encodes glutamate synthase) placed under the PGK1 promoter while harboring the GPD1 deletion. Notably, overexpression of GLT1 by the PGK1 promoter along with GPD1 deletion resulted in a 10.8% higher ethanol production and a 25.0% lower glycerol formation compared to the wild type in anaerobic fermentations. The growth rate of KAM-4 was slightly lower than that of the wild type under the exponential phase whereas KAM-12 and the wild type were indistinguishable in the biomass concentration at the end of growth period. Meanwhile, dramatic reduction of formation of acetate and pyruvic acid was observed in all the mutants compared to the wild type.     

Efficient production of α-ketoglutarate in the <Emphasis Type="Italic">gdh</Emphasis> deleted <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> by novel double-phase pH and biotin control strategy

Production of l-glutamate using a biotin-deficient strain of Corynebacterium glutamicum has a long history. The process is achieved by controlling biotin at suboptimal dose in the initial fermentation medium, meanwhile feeding NH₄OH to adjust pH so that α-ketoglutarate (α-KG) can be converted to l-glutamate. In this study, we deleted glutamate dehydrogenase (gdh1 and gdh2) of C. glutamicum GKG-047, an l-glutamate overproducing strain, to produce α-KG that is the direct precursor of l-glutamate. Based on the method of l-glutamate fermentation, we developed a novel double-phase pH and biotin control strategy for α-KG production. Specifically, NH₄OH was added to adjust the pH at the bacterial growth stage and NaOH was used when the cells began to produce acid; besides adding an appropriate amount of biotin in the initial medium, certain amount of additional biotin was supplemented at the middle stage of fermentation to maintain a high cell viability and promote the carbon fixation to the flux of α-KG production. Under this control strategy, 45.6 g/L α-KG accumulated after 30-h fermentation in a 7.5-L fermentor and the productivity and yield achieved were 1.52 g/L/h and 0.42 g/g, respectively.     

Characteristics of β-lactamases and their genes (<Emphasis Type="Italic">blaA</Emphasis> and <Emphasis Type="Italic">blaB</Emphasis>) in <Emphasis Type="Italic">Yersinia intermedia</Emphasis> and <Emphasis Type="Italic">Y. frederiksenii</Emphasis>

Background  

The presence of β-lactamases in Y. enterocolitica has been reported to vary with serovars, biovars and geographical origin of the isolates. An understanding of the β-lactamases in other related species is important for an overall perception of antibiotic resistance in yersiniae. The objective of this work was to study the characteristics of β-lactamases and their genes in strains of Y. intermedia and Y. frederiksenii, isolated from clinical and non-clinical sources in India.     

Infection by <Emphasis Type="Italic">Rickettsia bellii</Emphasis> and <Emphasis Type="Italic">Candidatus “Rickettsia amblyommii”</Emphasis> in <Emphasis Type="Italic">Amblyomma neumanni</Emphasis> Ticks from Argentina

The tick species, Amblyomma neumanni (Acari: Ixodidae) is the most frequent tick parasitizing humans in northwestern Argentina. The present study evaluated the rickettsial infection among 55 A. neumanni adult free-living ticks collected in Dean Funes, Córdoba Province. Ticks were individually processed by the hemolymph test with Gimenez staining, isolation of rickettsia in Vero cell culture by the shell vial technique, and polymerase chain reaction (PCR) targeting the citrate synthase rickettsial gene. Through the shell vial technique, rickettsiae were successfully isolated and established in Vero cell culture from two ticks (ticks 4 and 13), which previously showed to contain Rickettsia-like organisms by the hemolymph test. These two Rickettsia isolates were designated as An4 and An13. Molecular characterization (partial DNA sequences of two to three rickettsial genes were determined) of these two isolates and phylogenetic analyses identified them as Rickettsia bellii (isolate An4) and Candidatus “Rickettsia amblyommii” (isolate An13). After testing all A. neumanni ticks by PCR, the prevalence of Candidatus R. amblyommii and R. bellii was 23.6% (13/55) and 3.6% (2/55), respectively. These two rickettsiae have been considered of unknown pathogenicity and appropriate studies to test their pathogenicity to humans or animals need to be conducted. This is the first report of Rickettsia in ticks from Argentina, and also in the species A. neumanni. The results reinforce previous findings that R. bellii (and probably Candidatus R. amblyommii) are widespread among some Neotropical Amblyomma species, suggesting that these ticks gained these bacterial agents from a common ancestor and/or by recent horizontal transmission of rickettsiae between ticks.     

Cloning of the <Emphasis Type="Italic">Penicillium canescens</Emphasis> endo-1,4-β-glucanase gene <Emphasis Type="Italic">egl3</Emphasis> and the characterization of the recombinant enzyme

The gene egl3 of the filamentous fungus Penicillium canescens endo-1,4-β-glucanase, belonging to family 12 glycosyl hydrolases, was cloned and sequenced. The gene was expressed in P. canescens under the control of the strong promoter of gene bgaS, coding for β-galactosidase of this fungus, and efficient endoglucanase producer strains were obtained. The recombinant protein was isolated from the culture liquid of the producer strain EGL3-13 and purified to homogeneity; its specific activity was 31.7 IU; molecular weight, 26 kDa; and pH and temperature optimums, 3.2 and 54°C, respectively. The K _m and V _m values for CMC hydrolysis were determined; they amounted to 17.1 g/l and 0.31 μM/(mg s), respectively.     

Biosynthesis of ω-alicyclic fatty acids induced by cyclic precursors and change of membrane fluidity in thermophilic bacteria <Emphasis Type="Italic">Geobacillus stearothermophilus</Emphasis> and <Emphasis Type="Italic">Meiothermus</Emphasis><Emphasis Type="Italic">ruber</Emphasis>

Two thermophilic strains belonging to Geobacillus stearothermophilus and Meiothermus ruber, which naturally do not synthesize ω-alicyclic fatty acids (ω-FAs) were cultivated with cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl carboxylic acids. Gas chromatography–mass spectrometry analysis of fatty acid methyl and picolinyl esters showed that both strains are able to synthesize ω-FAs when cultivated with the appropriate precursor. The incorporation of cyclic acids influenced the whole FA composition as well as membrane fluidity. Membrane fluidity of intact cells was studied by measuring the fluorescence polarisation of the probe l,6-diphenyl-1,3,5-hexatriene incorporated into membrane lipid bilayers. Cytoplasmic membrane became more fluid with increasing content of ω-FAs. This is caused by considerable changes in lipid packing within the membrane induced by the presence of ω-FAs not found in the natural environment of Geobacillus and Meiothermus strains.     

Effect of extracts from in vitro-grown shoots of <Emphasis Type="Italic">Quillaja saponaria</Emphasis> Mol. on <Emphasis Type="Italic">Botrytis cinerea</Emphasis> Pers.

The ethanolic and aqueous extracts from in vitro shoots of Quillaja saponaria Mol. (Quillay) were studied for their antifungal activity against the phytopathogenic fungus Botrytis cinerea Pers. These extracts reduced conidial germination and mycelial growth of B. cinerea, ethanolic extracts being more active than aqueous extracts. In addition, the damage areas produced by this fungus on tomato leaves and strawberry fruits pre-treated with quillay extracts were diminished. The fungitoxic effect of in vitro-grown quillay extract was similar to those obtained with commercial fungicides of both natural (BC-1000) and synthetic (iprodione–dicarboximide) origin. On the other hand, the antifungal action of quillay extracts obtained from adult trees naturally grown was only slightly superior to the fungitoxic activity of the extract from in vitro plants. HPLC analysis of the extract showed that it contained saponins and some phenolic compounds such as chlorogenic, caffeic, vanillic, and salicylic acids, and scopoletin, which have been identified as antifungal agents on phytopathogenic fungi. The results obtained in this work, suggests that extracts of in vitro-grown quillay have an important protective effect against B. cinerea and support the use of an in vitro culture system as a biotechnological alternative to obtain environmental safe antifungal quillay extracts to control B. cinerea, contributing to the preservation of this indigenous Chilean species.     

<Emphasis Type="Italic">Astyanax</Emphasis> <Emphasis Type="Italic">bockmanni</Emphasis> Vari and Castro, 2007: an ambiguous karyotype in the <Emphasis Type="Italic">Astyanax</Emphasis> genus

Despite the widespread distribution of Astyanax bockmanni in streams from Upper Paraná River system in central, southeastern, and southern Brazil, just recently, it has been identified as a distinct Astyanax species. Cytogenetic studies were performed in two populations of this species, revealing conservative features. A. bockmanni shows 2n = 50 chromosomes, a karyotypic formula composed of 10 M + 12SM + 12ST + 16A and multiple Ag-NORs. Eight positive signals in subtelocentric/acrocentric chromosomes were identified by fluorescent in situ hybridization (FISH) with 18S rDNA probes. After FISH with 5S rDNA probes, four sites were detected, comprising the interstitial region of a metacentric pair and the terminal region on long arms of another metracentric pair. Little amounts of constitutive heterochromatin were observed, mainly distributed at distal region in two chromosomal pairs. Additionally, heterochromatin was also located close to the centromeres in some chromosomes. No positive signals were detected in the chromosomes of A. bockmanni by FISH with the As-51 satellite DNA probe. The studied species combines a set of characteristics previously identified in two different Astyanax groups. The chromosomal evolution in the genus Astyanax is discussed.