Fluorescent method for studying the morphogenesis and viability of dermatophyte cells

The dermatophyte Microsporum canis is commonly isolated from human and animal infection. The morphogenesis of this fungus was studied during its developmental stages through the fluorescent method Fluorescein Diacetate and Ethidium Bromide. To this end, 50 l dermatophyte suspension were transferred onto cellophane wrapping esterilized discs (2.5 cm of diameter) placed over the surface of Sabouraud dextrose agar on Petri dishes and incubated at 25 °C for 30 days. Every 60 minutes during the first 24 hours and every 12 hours for next 29 days, one disc was transferred onto glass slide, covered with equal volumes of freshly prepared fluorescein diacetate (FDA) and ethidium bromide (EB) solution, mounted with a coverslip and incubated in the dark for 30 minutes, at 25 °C. Each preparation was then examined on a fluorescent microscope. M. canis presented well defined growth stages: (1) tumescence of cells; (2) germination; (3) development of hyphae; (4) production of conidia and (5) tumescence and formation of arthroconidiae. Using the fluorescent method, non viable cells showed a light bright red coloration and viable cells presented green fluorescence. The principal morphological changes have occurred between the 3rd until the 18th day of culture. The method is very useful to demonstrate the dermatophyte growth stages as well as the perfect differentiation between viable and non viable cells.This revised version was published online in October 2005 with corrections to the Cover Date.     

Isolation and phylogenetic analysis of bacteria with antimicrobial activities from the Mediterranean sponges Aplysina aerophoba and Aplysina cavernicola

The aim of this study was to isolate bacteria with antimicrobial activities from the marine sponges Aplysina aerophoba and Aplysina cavernicola. The obtained 27 isolates could be subdivided into eight phylogenetically different clusters based on comparative sequence analysis of their 16S rDNA genes. The sponge isolates were affiliated with the low (Bacillus) and high G+C Gram-positive bacteria (Arthobacter, Micrococcus), as well as the alpha-Proteobacteria (unknown isolate) and gamma-Proteobacteria (Vibrio, Pseudoalteromonas). One novel Bacillus species was identified and two species were closely related to previously uncharacterized strains. Isolates with antimicrobial activity were numerically most abundant in the genera Pseudoalteromonas and the alpha-Proteobacteria. The sponge isolates show antimicrobial activities against Gram-positive and Gram-negative reference strains but not against the fungus Candida albicans. A general pattern was observed in that Gram-positive bacteria inhibited Gram-positive strains while Gram-negative bacteria inhibited Gram-negative isolates. Antimicrobial activities were also found against clinical isolates, i.e. multi-resistant Staphylococcus aureus and Staphylococcus epidermidis strains isolated from hospital patients. The high recovery of strains with antimicrobial activity suggests that marine sponges represent an ecological niche which harbors a hitherto largely uncharacterized microbial diversity and, concomitantly, a yet untapped metabolic potential.     

Inhibition of multicellular development switches cell death of Dictyostelium discoideum towards mammalian-like unicellular apoptosis

The multicellular development of the single celled eukaryote Dictyostelium discoideum is induced by starvation and consists of initial aggregation of the isolated amoebae, followed by their differentiation into viable spores and dead stalk cells. These stalk cells retain their structural integrity inside a stalk tube that support the spores in the fruiting body. Terminal differentiation into stalk cells has been shown to share several features with programmed cell death (Cornillon et al. (1994), J. Cell Sci. 107, 2691-2704). Here we report that, in the absence of aggregation and differentiation, D. discoideum can undergo another form of programmed cell death that closely resembles apoptosis of most mammalian cells, involves loss of mitochondrial transmembrane potential, phosphatidylserine surface exposure, and engulfment of dying cells by neighboring D. discoideum cells. This death has been studied by various techniques (light microscopy and scanning or transmission electron microscopy, flow cytometry, DNA electrophoresis), in two different conditions inhibiting D. discoideum multicellular development. The first one, corresponding to an induced unicellular cell death, was obtained by starving the cells in a "conditioned" cell-free buffer, prepared by previous starvation of another D. discoideum cell population in potassium phosphate buffer (pH 6.8). The second one, corresponding to death of D. discoideum after axenic growth in suspension, was obtained by keeping stationary cells in their culture medium. In both cases of these unicellular-specific cell deaths, microscopy revealed morphological features known as hallmarks of apoptosis for higher eukaryotic cells and apoptosis was further corroborated by flow cytometry. The occurrence in D. discoideum of programmed cell death with two different phenotypes, depending on its multicellular or unicellular status, is further discussed.     

Crystal structure of an aerobic FMN-dependent azoreductase (AzoA) from Enterococcus faecalis

The initial critical step of reduction of the azo bond during the metabolism of azo dyes is catalyzed by a group of NAD(P)H dependant enzymes called azoreductases. Although several azoreductases have been identified from microorganisms and partially characterized, very little is known about the structural basis for substrate specificity and the nature of catalysis. Enterococcus faecalis azoreductase A (AzoA) is a highly active azoreductase with a broad spectrum of substrate specificity and is capable of degrading a wide variety of azo dyes. Here, we report the crystal structure of the AzoA from E. faecalis determined at 2.07 A resolution with bound FMN ligand. Phases were obtained by single wavelength anomalous scattering of selenomethionine labeled protein crystals. The asymmetric unit consisted of two dimers with one FMN molecule bound to each monomer. The AzoA monomer takes a typical NAD(P)-binding Rossmann fold with a highly conserved FMN binding pocket. A salt bridge between Arg18 and Asp184 restricts the size of the flavin binding pocket such that only FMN can bind. A putative NADH binding site could be identified and a plausible mechanism for substrate reduction is proposed. Expression studies revealed azoA gene to be expressed constitutively in E. faecalis.     

Differential distribution of the endoplasmic reticulum network in filamentous fungi

Filamentous fungi are composed of hyphal compartments divided by septa, which communicate via septal pores. Apical compartments can elongate to over 100 microm without septum formation and possess a polarized distribution of organelles. In Aspergillus, subapical compartments are arrested in interphase but can reinitiate mitosis and growth by branching. Recent reports using green fluorescent protein (GFP) technology have demonstrated the highly differentiated localization of the endoplasmic reticulum (ER) network in various regions of the hyphae: the gradient distribution from the apical region, the localization along the septum, differential distributions in adjacent compartments, and the dynamic morphological change during septum formation. In this review the spatial regulation of the ER network in multicellular filamentous fungi is discussed.     

Anthraquinones as defensive compounds in eggs of Galerucini leaf beetles: Biosynthesis by the beetles?

Eggs of leaf beetles of the tribe Galerucini, subfamily Galerucinae, contain polyketides that are unusual in insects: 1,8-dihydroxylated anthraquinones (chrysazin, chrysophanol) and anthrones (dithranol, chrysarobin) deterring predators. The host plants do not contain these compounds. In the present study, we tested the hypothesis that the beetles, but not bacterial or fungal microorganisms living as endosymbionts within the beetles, produce the anthraquinones. The tansy leaf beetle Galeruca tanaceti was used as Galerucini model organism. It was treated with antimicrobial substances to eradicate the microorganisms and inhibit the hypothesised endosymbiotic anthraquinone production. Despite treatment, female G. tanaceti laid eggs containing anthraquinones. Although broad spectrum antimicrobials were used, it cannot be excluded that the potential endosymbiotic microorganisms are resistant. Given that the hypothesised endosymbionts are transferred via the eggs from one generation to the next, bacterial or fungal DNA was expected to be present in the eggs. With the exception of Wolbachia pipientis, however, no further 16S rDNA from bacteria responsible for anthraquinone biosynthesis was detected in eggs of untreated beetles. Because Wolbachia were also found in closely related anthraquinone-free insects, we exclude these bacteria as producers of the defensive polyketides. Nor was any 18S rDNA from fungi with anthraquinone biosynthetic abilities detected. Our results indicate that anthraquinones and anthrones in eggs of Galerucini are produced by beetle enzymes and not by endosymbiotic microorganisms within the eggs.     

厌氧甲烷氧化微生物物质代谢与能量代谢研究进展

甲烷既是一种温室气体,也是一种潜在的能源物质,其源与汇的平衡对地球化学循环及工程应用均有重要意义。厌氧甲烷氧化(anaerobic oxidation of methane,AOM)过程是深海、湿地和农田等自然生境中重要的甲烷汇,在缓解温室气体排放方面发挥了巨大作用。AOM微生物的中枢代谢机制及其能量转化途径则是介导厌氧甲烷氧化耦合其他物质还原的关键所在。因此,本文从电子受体多样性的视角,主要分析了硫酸盐型,硝酸盐/亚硝酸盐型,金属还原型厌氧甲烷氧化微生物的生理生化过程及环境分布,并对近些年发现的新型厌氧甲烷氧化进行了梳理;重点总结了厌氧甲烷氧化微生物细胞内电子传递路径以及胞外电子传递方式;根据厌氧甲烷氧化微生物环境分布及反应特征,就其生态学意义及在污染治理与能源回收方面的潜在应用价值进行了展望。本综述以期深化对厌氧甲烷氧化过程的微生物学认知,并为其潜在的工程应用方向提供新的思路。     

<i>Azospirillum brasilense</i> and <i>Saccharomyces cerevisiae</i> as Alternative for Decrease the Effect of Salinity Stress in Tomato (<i>Lycopersicon esculentum</i>) Growth

The salinity stress is one of the most relevant abiotic stresses that affects the agricultural production. The present study was performed to study the improvement of the salt tolerance of tomato plants which is known for their susceptibility to salt stress. The present study aimed to assess to what extent strain Azospirillum brasilense (N040) and Saccharomyces cerevisiae improve the salt tolerance to tomato plants treated with different salt concentration. The inoculant strain A. brasilense (N040) was previously adapted to survive up to 7% NaCl in the basal media. A greenhouse experiment was conducted to evaluate the effect of this inoculation on growth parameter such as: plant height, root length, fresh and dry weight, fruits fresh weight, chlorophyll content, proline and total soluble sugar in tomato plants under salt stress condition. The results revealed that co-inoculation of Azospirillum brasilense (N040) and Saccharomyces cerevisiae significantly increased the level of proline (8.63 mg/g FW) and total soluble sugar (120 mg/g FW) of leaves under salinity condition comparing to non-inoculated plants (2.3 mg/g FW and 70 mg/g FW, respectively). Plants co-inoculated with adapted strain of A. brasilense and S. cerevisiae showed the highest significant (p < 0.01) increase in fruit yield (1166.6 g/plant), plant high (115 cm) and roots length (52.6) compared whit un-inoculated control plants (42 g/pant, 43.3 cm and 29.6 cm, respectively). In contrast, Na⁺ ion content was significantly decreased in the leaves of salt stressed plants treated with the A. brasilense (N040) and S. cerevisiae. Finally, the results showed that dual benefits provided by both A. brasilense (N040) and S. cerevisiae can provide a major way to improve tomato yields in saline soils.