Fejlődési stádiumtól függően expresszálódó gének izolálása és jellemzése mikotoxin termelő Gibberella fujikuroi törzsekben = Isolation and characterisation of developmentally regulated genes from mycotoxin producing Gibberella fujikuroi isolates

Munkánk elsődleges célja fejlődésspecifikus gének izolálása és jellemzése volt F. proliferatum törzsben. A cDNS-AFLP módszert alkalmaztuk és a kapott 310 differenciáló fragmentumból hét mutatott szignifikánsan eltérő expressziót a fiatal és az idős tenyészet között. Génbanki adatok alapján megállapítottuk, hogy az egyik klón neutrális aminosav transzportert kódoló fehérjékkel mutatnak nagy hasonlóságot. A Fpmtr-nek nevezett gén teljes kópiáját izoláltuk, mely egy kópiában van jelen és 462 aminósavat kódol. A gén funkciójának megismeréséhez ∆Fpmtr mutánsokat hoztunk létre irányított génrontással. A transzformánsok hím-fertilitása nem változott, női fertilitásuk azonban jelentősen gyengült. A vad típusú, ITEM 2287 törzs vegetatíve ön-inkompatibilis, azaz komplementer, nitrátot nem hasznosító mutánsai (nit1, nitM és nit3) nem hoznak létre életképes heterokarióta sejteket. A ∆Fpmtr nit-mutánsai azonban képesek voltak komplementálni a vad típusú törzs megfelelő auxotróf mutánsait, azaz ennek az érdekes és szokatlan aminosav transzporter génnek az inaktiválásával sikerült megszüntetni a vegetatív ön-inkompatibilitást. Mindezek arra utalnak, hogy a F. proliferatum Fpmtr génje mind a szexuális, mind a paraszexuális folyamatokban kitüntetett szerepet játszik, és az FpMtr géntermék nem tipikus aminosav transzporter, hanem inkább szenzor/receptor funkciót ellátó fehérje. | To identify growth stage specific genes in F. proliferatum a comparative analysis of gene expression was performed by cDNA-AFLP. Altogether 310 fragments showed strikingly different intensities depending on growth stage. One of these fragments, which was strongly expressed in the early stage of conidial germination and repressed in the late stationary phase, gave significant sequence homology to a neutral aromatic and aliphatic amino acid transporter gene. We isolated the Fpmtr from the genomic library of F. proliferatum. Fpmtr is a single copy gene and encodes a polypeptide containing 462 amino acids. To find the specific function of Fpmtr, a transformation vector was constructed by using the hygromicin B phosphotransferase gene (hph). Male fertility of the ΔFpmtr mutants was not affected, however their female fertility became strongly retarded. Strain ITEM 2287 is a vegetatively self-incompatible strain, i.e. complementary nitrate non-utilizing mutants (nit1, nitM and nit3) of this fungus are unable to form viable heterokaryons. Our data suggest that Fpmtr is involved in multiple developmental processes related to both sexual and parasexual recombination events in F. proliferatum and FpMtr functions as a sensor/receptor protein rather than a typical amino acid transporter

Cloning and Heterologous Expression of a β-d-Mannosidase (EC 3.2.1.25)-Encoding Gene from Thermobifida fusca TM51

Thermobifida fusca TM51, a thermophilic actinomycete isolated from composted horse manure, was found to produce a number of lignocellulose-degrading hydrolases, including endoglucanases, exoglucanases, endoxylanases, β-xylosidases, endomannanases, and β-mannosidases, when grown on cellulose or hemicellulose as carbon sources. β-Mannosidases (EC 3.2.1.25), although contributing to the hydrolysis of hemicellulose fractions, such as galacto-mannans, constitute a lesser-known group of the lytic enzyme systems due to their low representation in the proteins secreted by hemicellulolytic microorganisms. An expression library of T. fusca, prepared in Streptomyces lividans TK24, was screened for β-mannosidase activity to clone genes coding for mannosidases. One positive clone was identified, and a β-mannosidase-encoding gene (manB) was isolated. Sequence analysis of the deduced amino acid sequence of the putative ManB protein revealed substantial similarity to known mannosidases in family 2 of the glycosyl hydrolase enzymes. The calculated molecular mass of the predicted protein was 94 kDa, with an estimated pI of 4.87. S. lividans was used as heterologous expression host for the putative β-mannosidase gene of T. fusca. The purified gene product obtained from the culture filtrate of S. lividans was then subjected to more-detailed biochemical analysis. Temperature and pH optima of the recombinant enzyme were 53°C and 7.17, respectively. Substrate specificity tests revealed that the enzyme exerts only β-d-mannosidase activity. Its kinetic parameters, determined on para-nitrophenyl β-d-mannopyranoside (pNP-βM) substrate were as follows: K(m) = 180 μM and V(max) = 5.96 μmol min(−1) mg(−1); the inhibition constant for mannose was K(i) = 5.5 mM. Glucono-lacton had no effect on the enzyme activity. A moderate trans-glycosidase activity was also observed when the enzyme was incubated in the presence of pNP-αM and pNP-βM; under these conditions mannosyl groups were transferred by the enzyme from pNP-βM to pNP-αM resulting in the synthesis of small amounts (1 to 2%) of disaccharides