Studies toward the comprehension of fungal-macroalgae interaction in cold marine regions from a biotechnological perspective

In marine ecosystems, macroalgae are the habitat for several microorganisms, fungi being among them. In the Antarctic benthic coastal ecosystem, macroalgae play a key role in organic matter cycling. In this study, 13 different macroalgae from Potter Cove and surrounding areas were sampled and 48 fungal isolates were obtained from six species, four Rhodophyta Ballia callitricha, Gigartina skottsbergii, Neuroglossum delesseriae and Palmaria decipiens, and two Phaeophyceae: Adenocystis utricularis and Ascoseira mirabilis. Fungal isolates mostly belonged to the Ascomycota phylum (Antarctomyces, Cadophora, Cladosporium, Penicillium, Phialocephala, and Pseudogymnoascus) and only one to the phylum Mucoromycota. Two of the isolates could not be identified to genus level, implying that Antarctica is a source of probable novel fungal taxa with enormous bioprospecting and biotechnological potential. 73% of the fungal isolates were moderate eurypsychrophilic (they grew at 5–25 °C), 12.5% were eurypsychrophilic and grew in the whole range, 12.5% of the isolates were narrow eurypsychrophilic (growth at 15–25 °C), and Mucoromycota AUe4 was classified as stenopsychrophilic as it grew at 5–15 °C. Organic extracts of seven macroalgae from which no fungal growth was obtained (three red algae Georgiella confluens, Gymnogongrus turquetii, Plocamium cartlagineum, and four brown algae Desmarestia anceps, D. Antarctica, Desmarestia menziesii, Himantothallus grandifolius) were tested against representative fungi of the genera isolated in this work. All extracts presented fungal inhibition, those from Plocamium cartilagineum and G. turquetii showed the best results, and for most of these macroalgae, this represents the first report of antifungal activity and constitute a promising source of compounds for future evaluation.     

Novel Hydrophobins from <Emphasis Type="Italic">Trichoderma</Emphasis> Define a New Hydrophobin Subclass: Protein Properties,Evolution, Regulation and Processing

Hydrophobins are small proteins, characterised by the presence of eight positionally conserved cysteine residues, and are present in all filamentous asco- and basidiomycetes. They are found on the outer surfaces of cell walls of hyphae and conidia, where they mediate interactions between the fungus and the environment. Hydrophobins are conventionally grouped into two classes (class I and II) according to their solubility in solvents, hydropathy profiles and spacing between the conserved cysteines. Here we describe a novel set of hydrophobins from Trichoderma spp. that deviate from this classification in their hydropathy, cysteine spacing and protein surface pattern. Phylogenetic analysis shows that they form separate clades within ascomycete class I hydrophobins. Using T. atroviride as a model, the novel hydrophobins were found to be expressed under conditions of glucose limitation and to be regulated by differential splicing.     

Antimicrobial resistance and virulence determinants in European Salmonella genomic island 1-positive Salmonella enterica isolates from different origins

Salmonella genomic island 1 (SGI1) contains a multidrug resistance region conferring the ampicillin-chloramphenicol-streptomycin-sulfamethoxazole-tetracycline resistance phenotype encoded by bla(PSE-1), floR, aadA2, sul1, and tet(G). Its increasing spread via interbacterial transfer and the emergence of new variants are important public health concerns. We investigated the molecular properties of SGI1-carrying Salmonella enterica serovars selected from a European strain collection. A total of 38 strains belonging to S. enterica serovar Agona, S. enterica serovar Albany, S. enterica serovar Derby, S. enterica serovar Kentucky, S. enterica serovar Newport, S. enterica serovar Paratyphi B dT+, and S. enterica serovar Typhimurium, isolated between 2002 and 2006 in eight European countries from humans, animals, and food, were subjected to antimicrobial susceptibility testing, molecular typing methods (XbaI pulsed-field gel electrophoresis [PFGE], plasmid analysis, and multilocus variable-number tandem-repeat analysis [MLVA]), as well as detection of resistance and virulence determinants (PCR/sequencing and DNA microarray analysis). Typing experiments revealed wide heterogeneity inside the strain collection and even within serovars. PFGE analysis distinguished a total of 26 different patterns. In contrast, the characterization of the phenotypic and genotypic antimicrobial resistance revealed serovar-specific features. Apart from the classical SGI1 organization found in 61% of the strains, seven different variants were identified with antimicrobial resistance properties associated with SGI1-A (S. Derby), SGI1-C (S. Derby), SGI1-F (S. Albany), SGI1-L (S. Newport), SGI1-K (S. Kentucky), SGI1-M (S. Typhimurium), and, eventually, a novel variant similar to SGI1-C with additional gentamicin resistance encoded by aadB. Only minor serovar-specific differences among virulence patterns were detected. In conclusion, the SGI1 carriers exhibited pathogenetic backgrounds comparable to the ones published for susceptible isolates. However, because of their multidrug resistance, they may be more relevant in clinical settings.     

Peptaibol production by sepedonium strains parasitizing boletales

Fungi of the genus Sepedonium (anamorphic ascomycetes) are known to infect fruiting bodies of Basidiomycetes of the order Boletales. We have characterized twelve Sepedonium isolates by intact-cell mass spectrometry (IC-MS) with the help of respective biomarkers and their metabolite spectra focusing on peptaibol production. A strain of mycoparasitic S. chalcipori was grown in solid-state fermentation, and tylopeptin production was found, suggesting an ascomycete origin of these peptaibols, which were first described in the basidiomycete Tylopilus neofelleus. In addition, the structures of two new peptaibols, chalciporin A (=Ac-Trp-Val-Aib-Val-Ala-Gln-Ala-Aib-Ser-Leu-Ala-Leu-Aib-Gln-Leuol) and chalciporin B (=Ac-Trp-Val-Aib-Val-Ala-Gln-Ala-Aib-Gln-Aib-Ala-Leu-Aib-Gln-Leuol) are presented. The IC-MS technique was applied for in situ peptaibol analysis of Sepedonium strains growing on Boletales, in particular S. chrysospermum infecting Xerocomus cf. badius. We found chrysospermins at the surface and within basidiomycete tissue, as well as in the cultivated parasite.     

Phylogenetic analysis supports horizontal transfer of P transposable elements

Nucleotide sequence comparisons were used to investigate the evolution of P transposable elements and the possibility that horizontal transfer has played a role in their occurrence in natural populations of Drosophila and other Diptera. The phylogeny of P elements was examined using published sequences from eight dipteran taxa and a new, partial sequence from Scaptomyza elmoi. The results from a number of different analyses are highly consistent and reveal a P-element phylogeny that contradicts the phylogeny of the species. At least three instances of horizontal transfer are necessary to explain this incongruence, but other explanations cannot be ruled out at this time.      

Effect of bleeding site on clinical laboratory testing of rats: orbital venous plexus versus posterior vena cava.

We sought to determine if there were any differences in the results of clinical laboratory tests between blood samples collected from the orbital venous plexus and the posterior vena cava of adult male rats. Thirty healthy adult male Sprague Dawley rats were anesthetized by ether inhalation, and blood samples were collected successively from the orbital venous plexus (OVP) and the posterior vena cava (PVC) for hematologic (n = 10), serum chemistry (n = 10), and coagulation (n = 10) analyses. The prothrombin and partial thromboplastin times of samples from the OVP were prolonged (17% and 288%, respectively) when compared with samples from the PVC. Respective hematologic biases were as follows: red blood cell count (7%), hemoglobin (6%), hematocrit (5%), mean corpuscular volume (-3%), mean corpuscular hemoglobin (-1%), mean corpuscular hemoglobin content (1%), white blood cell count (13%), and platelet count (-7%). Respective serum chemistry biases were as follows: sorbitol dehydrogenase (-7%), glucose (-7%), blood urea nitrogen (-10%), creatinine (-2%), total protein (4%), albumin (2%), globulin (9%), alkaline phosphatase (5%), lactate dehydrogenase (-6%), aspartate aminotransferase (-5%), alanine aminotransferase (-2%), total bilirubin (0%), direct bilirubin (0%), magnesium (-17%), sodium (4%), potassium (0), chloride (4%), calcium (-2%), phosphorous (-17%), cholesterol (3%), triglycerides (24%), creatinine kinase (-8%), 5'nucleotidase (0%), and total bile acids (4%). For hematologic testing, there were no biologically significant differences between samples collected from the OVP and PVC. The coagulation times and serum Mg and P showed biologically significant differences between samples collected from the OVP and PVC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential Regulation and Posttranslational Processing of the Class II Hydrophobin Genes from the Biocontrol Fungus Hypocrea atroviridis

Hydrophobins are small extracellular proteins, unique to and ubiquitous in filamentous fungi, which mediate interactions between the fungus and environment. The mycoparasitic fungus Hypocrea atroviridis has recently been shown to possess 10 different class II hydrophobin genes, which is a much higher number than that of any other ascomycete investigated so far. In order to learn the potential advantage of this hydrophobin multiplicity for the fungus, we have investigated their expression patterns under different physiological conditions (e.g., vegetative growth), various conditions inducing sporulation (light, carbon starvation, and mechanical injury-induced stress), and confrontation with potential hosts for mycoparasitism. The results show that the 10 hydrophobins display different patterns of response to these conditions: one hydrophobin (encoded by hfb-2b) is constitutively induced under all conditions, whereas other hydrophobins were formed only under conditions of carbon starvation (encoded by hfb-1c and hfb-6c) or light plus carbon starvation (encoded by hfb-2c, hfb-6a, and hfb-6b). The hydrophobins encoded by hfb-1b and hfb-5a were primarily formed during vegetative growth and under mechanical injury-provoked stress. hfb-22a was not expressed under any conditions and is likely a pseudogene. None of the 10 genes showed a specific expression pattern during mycoparasitic interaction. Most, but not all, of the expression patterns under the three different conditions of sporulation were dependent on one or both of the two blue-light regulator proteins BLR1 and BLR2, as shown by the use of respective loss-of-function mutants. Matrix-assisted laser desorption ionization-time of flight mass spectrometry of mycelial solvent extracts provided sets of molecular ions corresponding to HFB-1b, HFB-2a, HFB-2b, and HFB-5a in their oxidized and processed forms. These in silico-deduced sequences of the hydrophobins indicate cleavages at known signal peptide sites as well as additional N- and C-terminal processing. Mass peaks observed during confrontation with plant-pathogenic fungi indicate further proteolytic attack on the hydrophobins. Our study illustrates both divergent and redundant functions of the 10 hydrophobins of H. atroviridis.Hydrophobins are unique and ubiquitous small proteins, characterized by the presence of eight positionally conserved cysteine residues, and present in all multicellular asco- and basidiomycetes. According to their hydropathy profiles and spacing between the conserved cysteines (37), they are divided into two classes (class I and class II). Hydrophobins are secreted proteins, found on the outer surfaces of the cell walls of hyphae and conidia, where they mediate interactions between the fungus and the environment (18, 24, 37), such as surface recognition during pathogenic interaction with plants, insects, or other fungi, but also in symbiosis (38). In addition, they also influence cell wall composition (33). Because of these manifold roles, it is less surprising that the expression of hydrophobin genes is subject to complex patterns of signals, including those that are related to the triggering of conidiogenesis or indicating the presence of a plant host.Many species of the fungal genus Hypocrea/Trichoderma are known as mycoparasites, and several of them are therefore applied as biocontrol agents (6, 7, 36). In addition, Trichoderma spp. have recently been reported to occur as endophytes and to be able to elicit positive plant responses against potential pathogens (17). Because of the reasons given above, hydrophobins would be candidate proteins playing a role in this process, and in fact a class I hydrophobin gene has recently been reported to be overproduced during endophytic interaction of Trichoderma asperellum and cucumber roots (35). In addition, other hydrophobins may be involved in the mechanism of mycoparasitism itself as well as the colonization of decaying wood.Our information about the roles of hydrophobins in the physiology of Trichoderma as well as other ascomycetous fungi is mostly derived from reversed genetics of a few major members (3, 4, 19-22). In Hypocrea jecorina (= Trichoderma reesei), two major class II hydrophobins (HFB-1 and HFB-2) have been studied in detail (4) and shown to be formed under different physiological conditions (29). However, the genome sequence of H. jecorina contains six class II hfb genes (27), and the roles of HFB-3, HFB-4, HFB-5, and HFB-6 are yet unknown. In the biocontrol fungus Hypocrea atroviridis (formerly called “Trichoderma harzianum”), only a single hydrophobin gene has been characterized so far (srh1 [28]) and shown to be expressed mainly under conditions of sporulation. Consequently, very little is known about hydrophobins and their regulation in Trichoderma.We have recently reported that two species of the Trichoderma/Hypocrea genus, Hypocrea virens and Hypocrea atroviridis, have an exceptional high number of class II hydrophobin genes (i.e., 11 and 10 phylogenetically different genes, respectively [22]). Therefore, the objective of this work was to investigate whether all of them are in fact expressed and, if so, under which conditions. We thereby put emphasis on vegetative growth, mycoparasitic interaction, and different triggers of sporulation and on learning whether the sporulation- and stress-regulating proteins BLR1 and BLR2 (10, 15) play a role in this process.In addition, we used matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry to detect the respective proteins and to learn their mode of processing. It has previously been shown that direct solvent extraction of mycelia and spores of Ascomycetes in the process of sample preparation provides a small set of protein peaks in the range of 5,000 to 10,000 Da representing the hydrophobin inventory (27). Structural studies of hydrophobins from H. jecorina (2, 20, 30, 31), Schizophyllum commune (13), and Agaricus bisporus (26) have shown expected signal peptide cleavage but also unusual processing patterns, including cleavage after Arg and Pro, as well as C-terminal modification.     

A nonribosomal peptide synthetase involved in the biosynthesis of ampullosporins in Sepedonium ampullosporum.

Recently, the saprophytic ascomycete Sepedonium ampullosporum strain HKI-0053 was isolated from a basidiomycete on account of its premature induction of pigment formation in Phoma destructiva, a process often related to the neuroleptic activity of the inducing compound. The active substance was identified as the 15-membered peptaibol type peptide Ampullosporin. Although to date more than 300 peptaibols have been discovered, their biosynthetic machinery has not been characterized yet. By improving the culture conditions it was possible to grow S. ampullosporum in a submerged culture and to increase Ampullosporin production by more than three times to 33 mg/l at reduced fermentation times. The appearance of two high molecular weight proteins, HMWP1 (1.5 MDa) and HMWP2 (350 kDa) was closely related to the production of Ampullosporin during the course of fermentation. Both proteins showed a cross-reaction with antibodies against a core fragment of nonribosomal peptide synthetases (NRPSs). Biochemical characterization of the partially purified enzymes exhibited selectivity for the substrate amino acid alpha-aminoisobutyric acid (Aib). substantiating their involvement in Ampullosporin biosynthesis. Our data suggest that Ampullosporin synthetase has been isolated, and provides the basis for the characterization of the entire biosynthetic gene cluster. Furthermore, this knowledge will enable the manipulation of its NRPS template, in order to engineer mutant strains of Sepedonium ampullosporum which could produce more potent analogues of Ampullosporin.     

Enzymatic characterisation of the multifunctional enzyme delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Streptomyces clavuligerus.

delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase, the multienzyme catalyzing the formation of ACV from the constituent amino acids and ATP in the presence of Mg2+ and dithioerythritol, was purified about 2700-fold from Streptomyces clavuligerus. The molecular mass of the native enzyme as determined by gel filtration chromatography is 560 kDa, while that determined by denaturing gel electrophoresis is 500 kDa. The enzyme is able to catalyze pyrophosphate exchange in dependence on L-cysteine and L-valine, but no L-alpha-aminoadipic-acid-dependent ATP/PPi exchange could be detected. Other L-cysteine- and L-valine-activating enzymes present in crude extracts were identified as aminoacyl-tRNA synthetases which could be separated from ACV synthetase. The molecular mass of these enzymes is 140 kDa for L-valine ligase and 50 kDa for L-cysteine ligase. The dissociation constants have been estimated, assuming three independent activation sites, to be 1.25 mM and 1.5 mM for cysteine and ATP, and 2.4 mM and 0.25 mM for valine and ATP, respectively. The enzyme forms a thioester with alpha-aminoadipic acid and with valine in a molar ratio of 0.6:1 (amino acid/enzyme). Thus, the bacterial ACV synthetase is a multifunctional peptide synthetase, differing from fungal ACV synthetases in its mechanism of activation of the non-protein amino acid.     

Effects of N-cadherin overexpression on the adhesion properties of embryonic stem cells

Constitutive overexpression of N-cadherin in mouse embryonic stem cells led to marked changes in the phenotype and adhesion properties of these cells. The changes included the formation of smaller embryonic bodies, elevated mRNA and total protein levels of N-cadherin, and increased amounts of p120 catenin and connexin-43. N-cadherin cells exhibited decreased attachment to non-cell surfaces, while their adhesiveness to each other and to rat neonatal cardiomyocytes was significantly elevated. The findings suggest that N-cadherin overexpression can facilitate electromechanical integration of stem cells into excitable tissues with endogenously high levels of N-cadherin, such as the heart and brain.Key words: stem cells, cardiomyocytes, N-cadherin, connexin 43, gap junctions