VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

FBV025Orphan GPCRs in Schizophyllum communeD. Freihorst*, E. KotheInstitute of Microbiology, Department of Microbial Phytopathology,Friedrich-Schiller-University, Jena, GermanyThe filamentous fungus S. commune has been a model organism for sexualdevelopment of basidiomycetes since the early 20th century. Numerousstudies revealed the importance of two gene loci, A and B, responsible formating and sexual development. While A codes homeodomain transcriptionfactors, B codes for a pheromone/receptor system. Both occur inmultiallelic, associated subloci leading to a large number of differentspecificities in nature (Aalpha 9, Abeta 32, Balpha 9, Bbeta 9), which thencontrol compatibility or abortion of mating. The B-receptors (STE3-like, 7transmembrane domains, G-protein coupled) recognize pheromones of nonselfspecificity, inducing a signal transduction pathway and specific generegulation. After sequencing of strain H4-8, new developments in researchhave occured. E.g., there are four new STE3-like GPCRs, homologous to theknown Balpha and Bbeta specific ones. Their function is unknown, becausea B-locus deletion strain without any interactions seen in B-dependentdevelopment still contains those four GPCRs, which obviously do notrespond to any pheromone. However, first results indicate their importancesince sequence identity between unrelated strains was found arguing forconservation of these genes. Overexpression is performed to give insightsinto the function of this new class of pheromone receptor-like genes.FBP001Will not be presented!FBP002Will not be presented!FBP003Protein-dependent interactions among Streptomyceshyphae and biotechnological implicationsK. Ilona, M. Kotasinka, Y. Denno*, H. SchrempfDepartment of Biology/Chemistry, Applied Genetics of Microorganisms,Osnabrück, GermanyStreptomycetes produce many metabolites with medical andbiotechnological applications. During fermentations, their hyphae buildaggregates, a process in which the newly identified protein HyaS plays animportant role. The corresponding hyaS gene is present within in allinvestigated Streptomyces species. The HyaS protein is dominantlyassociated to the substrate-hyphae. This WT strain builds cylindricallyshaped clumps of densely packed substrate-hyphae, often fusing to higheraggregates (pellets), which remain stably associated during shaking.Investigations by immuno-electronmicroscopy suggest that HyaS inducestight fusion-like contacts among substrate-hyphae. Biochemical studiesindicate that the C-terminal region of HyaS has amine oxidase activity.Furthermore, the level of undecylprodigiosin, a red pigment with antibioticactivity, is influenced by the engineered hyaS-subtype within a strain. Asecond protein named HycS and its corresponding gene were identified.Biochemical studies revealed that HyaS and HycS are strongly interacting.These data present the first molecular basis for future manipulation ofpellets, and concomitant production of secondary metabolites duringbiotechnological processes.FBP004In situ localization of a novel protein provokingaggregation among hyphaeK. Lindebaum, T. Denno*, V. Antoni, S. HildgundDepartment of Biology/Chemistry, Applied Genetics of Microorganisms,Osnabrück, GermanyThe Streptomyces protein HycS has been found to play a role in mediatinginteractions among hyphae of Streptomyces lividans as well as with selectedfungal strains. The hycS gene and designed mutated genes were cloned intoStreptomyces strains lacking a functional hycS gene and analysed as toaggregate-formation. Based on these results, functional domains andrevelant amino acid residues of the HycS protein were deduced.Furthermore, the hycS gene and its mutated variants were fused with theegfp gene. The subsequently obtained transformants of selectedStreptomyces strains were investigated by fluorescence microscopy as to thelocation of each designed fusion protein during different stages ofdevelopment. The results of the studies led to new insights on the role of theHycS protein. The biotechnological implications will be outlined.FBP005Antioxidant and antibacterial properties of the extractsfrom four Pleurotus ostreatus strainsE. Vamanu* 1 , M. Ene 2 , D. Pelinescu 3 , I. Sarbu 31 USAMVB & Biotehnol Center, Industrial Biotechnology, Bucharest,Romania2 Horia Hulubei National Institut for Physics and Nuclear Engineering,Bucharest, Romania3 Faculty of Biology, University of Bucharest, Bucharest, RomaniaThe aim of this work was focused on the obtaining of Pleurotus ostreatusmycelium by fermentation in liquid medium, in order to realise freeze-driedbiomass and extracts with high antioxidant and antimicrobial activity. Thetests were realised at 25 0 C, in 300 ml Erlenmeyer flasks. The most effectivecarbon source and the optimum agitation speed were determined, in order tocultivate the two strains in a medium containing 2% malt extract and 2%peptone.For all four strains, the optimum agitation speed was established at 150 rpm.The most effective carbon source for Pleurotus ostreatus EVFB1 wasglucose (10%), for Pleurotus ostreatus EVFB3 was glucose (15%), forPleurotus ostreatus EVFB4 was lactose (15%) and for Pleurotus ostreatusEVFB5 was sucrose (10%). The obtained biomass was freeze-dried and wassubmitted to ethanol and pure methanol extraction. It resulted that all fourPleurotus ostreatus strains had an antioxidant activity by 15% higher onaverage when ethanol was used as solvent. The phenols quantity wassimilar, notwithstanding the solvent used, for Pleurotus ostreatus EVFB1strain being by 25% higher as compared to all Pleurotus ostreatus strainsused, namely 68,6 mg/g gallic acid. The results were also comparable in thecase of reducing power, the ethanolic extracts being the most effective. Thefour Pleurotus strains proved narrow antibacterial activity against Gramnegativeand Gram-positive bacteria tested.[1] Vamanu, E. and N. S. Vamanu (2010): The obtaining of an antioxidant based on a Rosmarinusofficinalis freeze-dried extract, International Journal of Pharmacology, 6, 4, 387 - 392.FBP006Properties of the ND5 subunit of the mitochondrialcomplex I (NADH:ubiquinone oxidoreductase) fromYarrowia lipolyticaH. Grönheim*, J. SteuberDepartment of Microbiology, University of Hohenheim, Stuttgart, GermanyThe first complex in the mitochondrial phosphorylation system is theelectrogenic NADH:ubiquinone oxidoreductase (complex I). The complexconsists of 45 subunits and especially the subunit ND5 is considered to bepart of the membrane-bound transporter module. The bacterial homolog ofND5, the subunit NuoL of complex NDH-1, actually acts as transporter forNa + and K + in vesicles from the endoplasmatic reticulum in S. cerevisiae,which lacks an endogenous complex I [1]. Furthermore the expression ofhuman ND5 in S. cerevisiae leads to an increased resistance at high externalconcentrations of Na + or K + [2]. This suggests that ND5 acts as a cationtransporter independently from other complex I subunits. Here weinvestigate the properties of the ND5 homolog from the yeast Yarrowialipolytica. This very hydrophobic protein was present in ER membranes, ordirected to the mitochondrium of S. cerevisiae. Compared to human ND5,higher expression yields were observed. ND5 was solubilized withZwittergent 3-12 and enriched by a Ni + -sepharose chromatographic step.Proteolytic digestion of native ER vesicles containing ND5 showed that theN-terminus is oriented towards the external lumen of the vesicles, paving theway for cation transport studies. Mutations in ND5 observed in patientssuffering from neurodegenerative diseases were introduced in the Y.lipolytica ND5 to study their effect on the transport properties of ND5 invivo and in vitro.[1] Gemperli, A. C. et al (2007): Transport of Na(+) and K (+) by an antiporter-related subunit fromthe Escherichia coli NADH dehydrogenase I produced in Saccharomyces cerevisiae. Arch Microbiol188(5): 509-521.spektrum | Tagungsband 2011