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Transformants
Kinds of Transformants Selected AbstractsGlucose inhibits the formation of gas vesicles in Haloferax volcanii transformantsENVIRONMENTAL MICROBIOLOGY, Issue 1 2008Torsten Hechler Summary The effect of glucose on the formation of gas vesicles was investigated in Haloferax mediterranei and Hfx.volcanii transformants containing the mc- gvp gene cluster of Hfx. mediterranei (mc-vac transformants). Increasing amounts of glucose in the medium resulted in a successive decrease in the amount of gas vesicles in both species, with a complete inhibition of their formation at glucose concentrations of > 70 mM in mc-vac transformants, and 100 mM in Hfx. mediterranei. Maltose and sucrose imposed a similar inhibitory effect, whereas xylose, arabinose, lactose, pyruvate and 2-deoxy-glucose had no influence on the gas vesicle formation in mc-vac transformants. The activities of the two mc-vac promoters were strongly reduced in mc-vac transformants grown in the presence of > 50 mM glucose. The gas vesicle overproducing ,D transformant (lacking the repressing protein GvpD) also showed a glucose-induced lack of gas vesicles, indicating that GvpD is not involved in the repression. The addition of glucose was useful to block gas vesicle formation at a certain stage during growth, and vice versa, gas vesicle synthesis could be induced when a glucose-grown culture was shifted to medium lacking glucose. Both procedures will enable the investigation of defined stages during gas vesicle formation. [source] Stable augmentation of activated sludge with foreign catabolic genes harboured by an indigenous dominant bacteriumENVIRONMENTAL MICROBIOLOGY, Issue 10 2002Kazuya Watanabe Summary Comamonas sp. rN7 is a phenol-degrading bacterium that represents the dominant catabolic population in activated sludge. The present study examined the utility of this bacterium for establishing foreign catabolic genes in phenol-digesting activated sludge. The phc genes coding for phenol hydroxylase and its transcriptional regulators of C. testosteroni R5 were integrated into the chromosome of strain rN7. The specific phenol-oxygenating activity of a resultant transformant designated rN7(R503) was three times higher than the activity of strain rN7, and the phc genes were stably inherited by rN7(R503) grown in a non-selective laboratory medium. Inoculation of phenol-acclimatized activated sludge with rN7(R503) resulted in a high phenol-oxygenating activity and improved resistance to phenol-shock loading compared to sludge inoculated with either no cells, rN7 or R5. Quantitative competitive polymerase chain reaction (PCR) showed that the phc genes were retained in the rN7(R503)-inoculated sludge at a density of more than 108 copies per ml of mixed liquor for more than 35 days, whereas those in the R5-inoculated sludge disappeared rapidly. No transfer of the phc genes to other indigenous populations was apparent in the rN7(R503)-harbouring sludge. From these results, we concluded that the phenol treatment of the activated sludge was enhanced by the phc genes harboured by the rN7(R503) population. This study suggests a possible bioaugmentation strategy for stably utilizing foreign catabolic genes in natural ecosystems. [source] Characterization of the sgtR1 and sgtR2 genes and their role in regulating expression of the sprT gene encoding Streptomyces griseus trypsinFEMS MICROBIOLOGY LETTERS, Issue 1 2007Eun A Oh Abstract The sgtR1 and sgtR2 genes encoding putative regulators similar to the Aha1 and ArsR families, respectively, were identified downstream from the sprT gene. To investigate their function, expression vectors containing various combinations of sprT, sgtR1, and sgtR2 were transformed into Streptomyces lividans and Streptomyces griseus. The trypsin activity levels produced by S. lividans harboring pWHM3-TR2 (sprT and sgtR2) or pWHM3-TR1R2 (sprT, sgtR2, and sgtR2) were, respectively, 6.6 or 8.9 times that of S. lividans transformed with pWHM3-T (sprT). In the pWHM3-TR1R2 transformant, the transcription of sprT consistently occurred during the earlier stages of growth and was maintained at a higher level throughout the 6 days of cultivation. Streptomyces griseus IFO13350 harboring pWHM3-TR1R2 also produced trypsin activity 2.1 times that of the pWHM3-T transformant. However, all S. griseus,adpA transformants produced lower SGT activity than the wild-type strain, and none could overcome the deficiency in AdpA transcriptional activator, suggesting that AdpA is an absolute prerequisite for sprT expression. The sprT transcript was detected at a high level only in the wild-type strain, but the sgtR1 and sgtR2 transcript levels were very similar between the S. griseus IFO13350 and ,adpA strains. This clearly demonstrates that the transcription of the sgtR1 and sgtR2 genes is not dependent on AdpA and that they are therefore not members of the AdpA regulon. [source] Genetic correlation between chromium resistance and reduction in Bacillus brevis isolated from tannery effluentJOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2009T. Verma Abstract Aims:, To investigate the genetic basis of Cr(VI) resistance and its reduction to Cr(III) in indigenous bacteria isolated from tannery effluent. Methods and Results:, Four bacteria resistant to high Cr(VI) levels were isolated and identified as Bacillus spp. Their Cr(VI) reduction ability was tested. To assess the genetic basis of Cr(VI) resistance and reduction, plasmid transfer and curing studies were performed. Among all, B. brevis was resistant to 180 ,g Cr(VI) ml,1 and showed the greatest degree of Cr(VI) reduction (75·8%) within 28 h and its transformant was resistant to 160 ,g Cr(VI) ml,1 and reduced 69·9% chromate. It harboured a stable 18 kb plasmid DNA. Transfer and curing studies revealed that both the chromate resistance and reduction were plasmid mediated. The presence of other metal cations did not have any significant effect on Cr(VI) bioreduction. Conclusions:,Bacillus brevis was resistant to elevated Cr(VI) levels and may potentially reduce it in short time from an environment where other metal ions are also present in addition to chromium ions. The strain tested shows a positive correlation between genetic basis of Cr(VI) resistance and reduction. Significance and Impact of the Study:, To our knowledge, this is the first study on the genetic correlation between chromium resistance and reduction in bacteria. Such strains may potentially be useful in biotechnological applications and in situ Cr(VI) bioremediation. [source] STABILITY AND PROPERTIES OF A THERMOSTABLE ,-GALACTOSIDASE IMMOBILIZED ON CHTTINJOURNAL OF FOOD BIOCHEMISTRY, Issue 4 2000JADWIGA MACIU ABSTRACT Thermostable ,-galactosidase from an E. coli transformant containing the enzyme gene from P. woesei was immobilized at pH 4.0 and a glutaraldehyde concentration of 10 mM on chitin isolated from shrimp Crangon crangon shells. These preparations had a specific activity of 43,000 U/g of chitin at 85C using ONPG as substrate. The optimum pH and temperature for immobilized ,-galactosidase activity were 5.2 and 93C. Immobilization shifts the optimum pH for the activity of the enzyme by 0.2 units towards the acid side. The immobilized enzyme is stable at temperatures close to the optimal value, and the residual activity for ONPG hydrolysis of the preparations incubated 5 h in 0.1 M phosphate citrate buffer (pH 5.4) at 90C and 100C was 70% and 40% of the initial value, respectively. [source] Use of , -Glucuronidase Activity to Quantify the Growth of Fusarium oxysporum f. sp. radicis-lycopersici during Infection of TomatoJOURNAL OF PHYTOPATHOLOGY, Issue 6 2005K. K. Papadopoulou Abstract The , -glucuronidase (gus) reporter gene was integrated into the phytopathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici (FORL) in a co-transformation experiment using the hygromycin B resistance (hph) gene as selective marker, which resulted in the generation of 10 mitotically stable transformants. One transformant, F30, was selected based on the results of prior detailed characterization of the 10 transformants for growth rate, conidia production and pathogenicity in comparison with the wild-type strain. A strong positive correlation was found between GUS activity and accumulated biomass of in vitro -grown fungus and therefore GUS activity was used to study fungal growth quantitatively in two tomato lines. Although a parallel increase in lesion development and GUS activity was noted for both tomato lines, a correlation between the GUS activity and disease progression was not always possible. Interestingly, the levels of GUS activity obtained for the more resistant line were higher than those obtained for the susceptible line, indicating that disease progression in tomato caused by FORL may not be related only to the amount of fungal biomass within the root tissue. [source] Technical advance in fungal biotechnology: development of a miniaturized culture method and an automated high-throughput screeningLETTERS IN APPLIED MICROBIOLOGY, Issue 2 2009F. Alberto Abstract Aims:, The goal of the study was to develop a reliable, reproducible and rapid method of culture in order to screen a large number of fungal transformants. Methods and Results:, The method is based upon miniaturized cell cultures and automated expression screening in microwell plates. For the method development, 50 recombinant Aspergillus vadensis clones producing feruloyl esterase B (FaeB) from Aspergillus niger were screened in 6 days. Then a panel of clones showing various behaviours was checked in flasks in order to demonstrate the reproducibility of the method. Using this method, a transformant of A. vadensis producing 1·2 g l,1 of FaeB was selected (12-fold more than the A. niger overproducing strain). Conclusions:, This miniaturized culture method allows to obtain reliable and reproducible results. The procedure has the advantages of being efficient, time-saving and more efficient than conventional in-flask culture screening as it can screen 800 clones per day after a culture of 3 days. Significance and Impact of the Study:, This method could be applied to any other fungal strain culture, enzyme activity or biodiversity screening. [source] Expression of the inulinase gene from the marine-derived Pichia guilliermondii in Saccharomyces sp.MICROBIAL BIOTECHNOLOGY, Issue 5 2010ethanol production from inulin Summary It has been confirmed that Saccharomyces sp. W0 can produce high concentration of ethanol. In this study, the INU1 gene cloned from the marine-derived Pichia guilliermondii was transformed into uracil mutant of Saccharomyces sp. W0. The positive transformant Inu-66 obtained could produce 34.2 U ml,1 of extracellular inulinase within 72 h of cultivation. It was found that 15.2 U of inulinase activity per one gram of inulin was suitable for inulin hydrolysis and ethanol production by the transformant Inu-66. During the small-scale fermentation, 13.7 ml of ethanol in 100 ml of medium was produced and 99.1% of the added inulin was utilized by the transformant. During the 2 l fermentation, 14.9% (v/v) of ethanol was produced from inulin and 99.5% of the added inulin was converted into ethanol, CO2 and cell mass. [source] A metabolomic study of substantial equivalence of field-grown genetically modified wheatPLANT BIOTECHNOLOGY JOURNAL, Issue 4 2006John M. Baker Summary The ,substantial equivalence' of three transgenic wheats expressing additional high-molecular-weight subunit genes and the corresponding parental lines (two lines plus a null transformant) was examined using metabolite profiling of samples grown in replicate field trials on two UK sites (Rothamsted, Hertfordshire and Long Ashton, near Bristol) for 3 years. Multivariate comparison of the proton nuclear magnetic resonance spectra of polar metabolites extracted with deuterated methanol,water showed a stronger influence of site and year than of genotype. Nevertheless, some separation between the transgenic and parental lines was observed, notably between the transgenic line B73-6-1 (which had the highest level of transgene expression) and its parental line L88-6. Comparison of the spectra showed that this separation resulted from increased levels of maltose and/or sucrose in this transgenic line, and that differences in free amino acids were also apparent. More detailed studies of the amino acid composition of material grown in 2000 were carried out using gas chromatography-mass spectrometry. The most noticeable difference was that the samples grown at Rothamsted consistently contained larger amounts of acidic amino acids (glutamic, aspartic) and their amides (glutamine, asparagine). In addition, the related lines, L88-6 and B73-6-1, both contained larger amounts of proline and ,-aminobutyric acid when grown at Long Ashton than at Rothamsted. The results clearly demonstrate that the environment affects the metabolome and that any differences between the control and transgenic lines are generally within the same range as the differences observed between the control lines grown on different sites and in different years. [source] Glucose inhibits the formation of gas vesicles in Haloferax volcanii transformantsENVIRONMENTAL MICROBIOLOGY, Issue 1 2008Torsten Hechler Summary The effect of glucose on the formation of gas vesicles was investigated in Haloferax mediterranei and Hfx.volcanii transformants containing the mc- gvp gene cluster of Hfx. mediterranei (mc-vac transformants). Increasing amounts of glucose in the medium resulted in a successive decrease in the amount of gas vesicles in both species, with a complete inhibition of their formation at glucose concentrations of > 70 mM in mc-vac transformants, and 100 mM in Hfx. mediterranei. Maltose and sucrose imposed a similar inhibitory effect, whereas xylose, arabinose, lactose, pyruvate and 2-deoxy-glucose had no influence on the gas vesicle formation in mc-vac transformants. The activities of the two mc-vac promoters were strongly reduced in mc-vac transformants grown in the presence of > 50 mM glucose. The gas vesicle overproducing ,D transformant (lacking the repressing protein GvpD) also showed a glucose-induced lack of gas vesicles, indicating that GvpD is not involved in the repression. The addition of glucose was useful to block gas vesicle formation at a certain stage during growth, and vice versa, gas vesicle synthesis could be induced when a glucose-grown culture was shifted to medium lacking glucose. Both procedures will enable the investigation of defined stages during gas vesicle formation. [source] A designed curved DNA segment that is a remarkable activator of eukaryotic transcriptionFEBS JOURNAL, Issue 24 2006Noriyuki Sumida To identify artificial DNA segments that can stably express transgenes in the genome of host cells, we built a series of curved DNA segments that mimic a left-handed superhelical structure. Curved DNA segments of 288 bp (T32) and 180 bp (T20) were able to activate transcription from the herpes simplex virus thymidine kinase (tk) promoter by approximately 150-fold and 70-fold, respectively, compared to a control in a transient transfection assay in COS-7 cells. The T20 segment was also able to activate transcription from the human adenovirus type 2 E1A promoter with an 18-fold increase in the same assay system, and also activated transcription from the tk promoter on episomes in COS-7 cells. We also established five HeLa cell lines with genomes containing T20 upstream of the transgene promoter and control cell lines with T20 deleted from the transgene locus. Interestingly, T20 was found to activate transcription in all the stable transformants, irrespective of the locus. This suggests that the T20 segment may allow stable expression of transgenes, which is of importance in many fields, and may also be useful for the construction of nonviral vectors for gene therapy. [source] Fission yeast decaprenyl diphosphate synthase consists of Dps1 and the newly characterized Dlp1 protein in a novel heterotetrameric structureFEBS JOURNAL, Issue 20 2003Ryoichi Saiki The analysis of the structure and function of long chain-producing polyprenyl diphosphate synthase, which synthesizes the side chain of ubiquinone, has largely focused on the prokaryotic enzymes, and little is known about the eukaryotic counterparts. Here we show that decaprenyl diphosphate synthase from Schizosaccharomyces pombe is comprised of a novel protein named Dlp1 acting in partnership with Dps1. Dps1 is highly homologous to other prenyl diphosphate synthases but Dlp1 shares only weak homology with Dps1. We showed that the two proteins must be present simultaneously in Escherichia coli transformants before ubiquinone-10, which is produced by S. pombe but not by E. coli, is generated. Furthermore, the two proteins were shown to form a heterotetrameric complex. This is unlike the prokaryotic counterparts, which are homodimers. The deletion mutant of dlp1 lacked the enzymatic activity of decaprenyl diphosphate synthase, did not produce ubiquinone-10 and had the typical ubiquinone-deficient S. pombe phenotypes, namely hypersensitivity to hydrogen peroxide, the need for antioxidants for growth on minimal medium and an elevated production of H2S. Both the dps1 (formerly dps) and dlp1 mutants could generate ubiquinone when they were transformed with a bacterial decaprenyl diphosphate synthase, which functions in its host as a homodimer. This indicates that both dps1 and dlp1 are required for the S. pombe enzymatic activity. Thus, decaprenyl diphosphate from a eukaryotic origin has a heterotetrameric structure that is not found in prokaryotes. [source] Microbial interactions affecting the natural transformation of Bacillus subtilis in a model aquatic ecosystemFEMS MICROBIOLOGY ECOLOGY, Issue 3 2003Kazuaki Matsui Abstract The involvement of microbial interactions in natural transformation of bacteria was evaluated using an aquatic model system. For this purpose, the naturally transformable Bacillus subtilis was used as the model bacterium which was co-cultivated with the protist Tetrahymena thermophila (a consumer) and/or the photosynthetic alga Euglena gracilis (a producer). Co-cultivation with as few as 102 individuals ml,1 of T. thermophila lowered the number of transformants to less than the detectable level (<1×100 ml,1), while co-cultivation with E. gracilis did not. Metabolites from co-cultures of T. thermophila and B. subtilis also decreased the number of transformants to less than the detectable level, while metabolites from co-culture of T. thermophila and B. subtilis with E. gracilis did not. Thus, the introduction of transformation inhibitory factor(s) by the grazing of T. thermophila and the attenuation of this inhibitory factor(s) by E. gracilis is indicated. These observations suggest that biological components do affect the natural transformation of B. subtilis. The study described is the first to suggest that ecological interactions are responsible not only for the carbon and energy cycles, but also for the processes governing horizontal transfer of genes, in microbial ecosystems. [source] Characterization of the sgtR1 and sgtR2 genes and their role in regulating expression of the sprT gene encoding Streptomyces griseus trypsinFEMS MICROBIOLOGY LETTERS, Issue 1 2007Eun A Oh Abstract The sgtR1 and sgtR2 genes encoding putative regulators similar to the Aha1 and ArsR families, respectively, were identified downstream from the sprT gene. To investigate their function, expression vectors containing various combinations of sprT, sgtR1, and sgtR2 were transformed into Streptomyces lividans and Streptomyces griseus. The trypsin activity levels produced by S. lividans harboring pWHM3-TR2 (sprT and sgtR2) or pWHM3-TR1R2 (sprT, sgtR2, and sgtR2) were, respectively, 6.6 or 8.9 times that of S. lividans transformed with pWHM3-T (sprT). In the pWHM3-TR1R2 transformant, the transcription of sprT consistently occurred during the earlier stages of growth and was maintained at a higher level throughout the 6 days of cultivation. Streptomyces griseus IFO13350 harboring pWHM3-TR1R2 also produced trypsin activity 2.1 times that of the pWHM3-T transformant. However, all S. griseus,adpA transformants produced lower SGT activity than the wild-type strain, and none could overcome the deficiency in AdpA transcriptional activator, suggesting that AdpA is an absolute prerequisite for sprT expression. The sprT transcript was detected at a high level only in the wild-type strain, but the sgtR1 and sgtR2 transcript levels were very similar between the S. griseus IFO13350 and ,adpA strains. This clearly demonstrates that the transcription of the sgtR1 and sgtR2 genes is not dependent on AdpA and that they are therefore not members of the AdpA regulon. [source] Efficient downregulation of alb1 gene using an AMA1-based episomal expression of RNAi construct in Aspergillus fumigatusFEMS MICROBIOLOGY LETTERS, Issue 2 2007Vahid Khalaj Abstract An episomal RNAi silencing construct containing the inducible cbhB promoter and a hairpin structure has been made to downregulate the alb1 gene in the human pathogen Aspergillus fumigatus. Transformation of fungal protoplasts resulted in a high number of transformants with an inducible silenced phenotype (white spores). Efficient downregulation of the alb1 gene using this system suggests that this approach may overcome the variable downregulation observed with integrative constructs. [source] Folic acid utilisation related to sulfa drug resistance in Saccharomyces cerevisiaeFEMS MICROBIOLOGY LETTERS, Issue 2 2001Ann M. Bayly Abstract Saccharomyces cerevisiae mutants deficient in folate synthesis have been constructed and employed to study the utilisation of exogenous folates in yeast. One mutant specifically lacked dihydropteroate synthase while the second lacked dihydrofolate synthase. Exogenous folinic acid restored optimal growth to both strains. Folic acid did not generally rescue growth but spontaneous isolates capable of utilising folic acid were selected. The folic acid synthesis pathway in the folate utilising isolates was restored via transformation with FOL1 or FOL3 expression plasmids and transformants were tested for resistance to sulfamethoxazole (SMX). The presence of elevated levels of folic acid led to greatly reduced SMX sensitivity regardless of whether strains were folate utilisers or not. [source] Metabolic engineering of Saccharomyces cerevisiae for the synthesis of the wine-related antioxidant resveratrolFEMS YEAST RESEARCH, Issue 1 2003John V.W. Becker Abstract The stilbene resveratrol is a stress metabolite produced by Vitis vinifera grapevines during fungal infection, wounding or UV radiation. Resveratrol is synthesised particularly in the skins of grape berries and only trace amounts are present in the fruit flesh. Red wine contains a much higher resveratrol concentration than white wine, due to skin contact during fermentation. Apart from its antifungal characteristics, resveratrol has also been shown to have cancer chemopreventive activity and to reduce the risk of coronary heart disease. It acts as an antioxidant and anti-mutagen and has the ability to induce specific enzymes that metabolise carcinogenic substances. The objective of this pilot study was to investigate the feasibility of developing wine yeasts with the ability to produce resveratrol during fermentation in both red and white wines, thereby increasing the wholesomeness of the product. To achieve this goal, the phenylpropanoid pathway in Saccharomyces cerevisiae would have to be introduced to produce p -coumaroyl-CoA, one of the substrates required for resveratrol synthesis. The other substrate for resveratrol synthase, malonyl-CoA, is already found in yeast and is involved in de novo fatty-acid biosynthesis. We hypothesised that production of p -coumaroyl-CoA and resveratrol can be achieved by co-expressing the coenzyme-A ligase-encoding gene (4CL216) from a hybrid poplar and the grapevine resveratrol synthase gene (vst1) in laboratory strains of S. cerevisiae. This yeast has the ability to metabolise p -coumaric acid, a substance already present in grape must. This compound was therefore added to the synthetic media used for the growth of laboratory cultures. Transformants expressing both the 4CL216 and vst1 genes were obtained and tested for production of resveratrol. Following ,-glucosidase treatment of organic extracts for removal of glucose moieties that are typically bound to resveratrol, the results showed that the yeast transformants had produced the resveratrol ,-glucoside, piceid. This is the first report of the reconstruction of a biochemical pathway in a heterologous host to produce resveratrol. [source] The use of the green fluorescent protein as a biomarker for sapstain fungiFOREST PATHOLOGY, Issue 3 2002S. LEE To understand wood colonization by sapstain fungi and their potential biocontrol agents, it is necessary to differentiate these organisms directly on their natural substrates. In the present study the feasibility of transforming with the green fluorescent protein (GFP), the sapstain fungus Ophiostoma piceae and a potential biocontrol agent Cartapip®, an Ophiostoma piliferum albino strain was assessed. Transformants of the two fungal species were screened by polymerase chain reaction and Southern blot analyses. The GFP was expressed in spores, synnemata and mycelia of the transformants grown in artificial media or wood. The growth, pigmentation and wood colonization of the transformants were similar to that of the non-transformants, suggesting that the presence of the gfp gene had no negative effect on the biology of the transformants. Using fluorescence and confocal microscopy, the GFP-expressing fungi were easily differentiated from the wild-type strains and other fungal species in wood, even 4 months after inoculation. The results show that the use of the GFP system is feasible to monitor Ophiostoma fungi in wood. Utilisation de la protéine fluorescente verte (GFP) comme marqueur biologique des champignons de bleuissement du bois Pour comprendre la colonisation du bois par les champignons de bleuissement et par les agents de lutte biologique potentiels, il est nécessaire de distinguer ces organismes directement dans leur substrat naturel. Nous avons évalué la possibilité de transformation par la protéine fluorescente verte (GFP) du champignon de bleuissement Ophiostoma piceae et d'une souche albinos de Ophiostoma piliferum, agent de lutte biologique potentiel Cartapip®. Des transformants des deux espèces fongiques ont été triés par analyses PCR et Southern blot. La GFP a été exprimée dans les spores, les synnemas et le mycélium des transformants cultivés sur milieux artificiels et sur bois. Avec les transformants, la croissance, la pigmentation et la colonisation du bois étaient semblables à celles des non transformants, ce qui suggère que la présence du gène gfp n'a pas d'effet négatif sur la biologie des transformants. Par microscopie confocale à fluorescence, les champignons exprimant la GFP ont été facilement distingués des souches de type sauvage et d'autres espèces fongiques dans le bois, même 4 mois après inoculation. Nos résultats montrent que l'utilisation de la GFP est possible pour suivre les Ophiostoma dans le bois. Verwendung des Grünen Fluoreszenzproteins als Biomarker für Bläuepilze Um die Besiedelung von Holz durch Bläuepilze und ihre möglichen Antagonisten zu verstehen, muss man diese Organismen direkt auf ihrem natürlichen Substrat unterscheiden können. Es wurde überprüft, ob sich der Bläuepilze Ophiostoma piceae und der mögliche Antagonist Cartapip®, ein Albinostamm von Ophiostoma piliferum, mit dem Grünen Fluoreszenzprotein (GFP) transformieren lassen. Transformierte Stämme der beiden Pilzarten wurden mit PCR und Southern Blot Analysen untersucht. Das GFP wurde in Sporen, Synnemata und Myzelien der transformierten Stämme exprimiert. Dies war auf künstlichen Medien ebenso wie auf Holz der Fall. Wachstum, Pigmentierung und Holzbesiedelung waren bei den transformierten Stämmen ähnlich wie bei den nichttransformierten; somit dürfte die Präsenz des gfpGens keine negativen Auswirkungen auf die Biologie der transformierten Stämme haben. Mit Hilfe der Fluoreszenz- und Konfokal-Mikroskopie konnten die GFP exprimierenden Pilze leicht von den Wildtyp-Stämmen und anderen Pilzarten auf Holz unterschieden werden. Dies war auch noch vier Monate nach der Inokulation der Fall. Die Ergebnisse zeigen, dass das GFP-System zur Beobachtung von Ophiostoma -Arten im Holz geeignet ist. [source] Large-scale screening of intracellular protein localization in living fission yeast cells by the use of a GFP-fusion genomic DNA libraryGENES TO CELLS, Issue 3 2000Da-Qiao Ding Background Intracellular localization is an important part of the characterization of a gene product. In an attempt to search for genes based on the intracellular localization of their products, we constructed a green fluorescent protein (GFP)-fusion genomic DNA library of S. pombe. Results We constructed the S. pombe GFP-fusion genomic DNA library by fusing, in all three reading frames, random fragments of genomic DNA to the 5, end of the GFP gene in such a way that expression of potential GFP-fusion proteins would be under the control of the own promoters contained in the genomic DNA fragments. Fission yeast cells were transformed with this plasmid library, and microscopic screening of 49 845 transformants yielded 6954 transformants which exhibited GFP fluorescence, of which 728 transformants showed fluorescence localized to distinct intracellular structures such as the nucleus, the nuclear membrane, and cytoskeletal structures. Plasmids were isolated from 516 of these transformants, and a determination of their DNA sequences identified 250 independent genes. The intracellular localizations of the 250 GFP-fusion constructs was categorized as an image database; using this database, DNA sequences can be searched for based on the localizations of their products. Conclusions A number of new intracellular structural components were found in this library. The library of GFP-fusion constructs also provides useful fluorescent markers for various intracellular structures and cellular activities, which can be readily used for microscopic observation in living cells. [source] New Approaches for Validation of Lethal Phenotypes and Genetic Reversion in Helicobacter pyloriHELICOBACTER, Issue 1 2001Timothy K. McDaniel Background. Because of limited genetic tools for use in Helicobacter pylori, tests routinely applied in other bacteria for demonstrating a gene's role in viability and other phenotypes have not been applied to this organism. In a mutational study of putative response regulator genes, we aimed to develop such tools for H. pylori. Materials and Methods. We attempted to mutate five response regulator genes by allelic exchange insertional mutagenesis. For genes that yielded no viable mutants, a second copy of the gene was inserted into the chromosome via a suicide vector, and it was seen if providing the second copy would permit the gene's disruption. For genes that yielded mutants with selectable phenotypes, a strategy was developed for reversion whereby an intact copy of the gene is introduced to the organism by transformation with PCR products. Following this procedure, revertants were selected by phenotypic tests then tested for genetic reversion. Results. After failure to attain transformants upon attempted mutation of genes HP0166 and HP1365, we inserted a second copy of each gene within the H. pylori chromosome. In each case the second copy relieved the block of transformation. Mutation of genes HP0703 and HP1021 gave non-motile and small-colony phenotypes, respectively. Following transformation with PCR products containing intact copies of the genes, both phenotype and genotype had reverted following phenotypic selections. Conclusions. The methods used in this study provide new approaches for confirming suspected genotype/phenotype associations and should be widely applicable in the study of H. pylori. [source] High efficiency site-specific genetic engineering of the mosquito genomeINSECT MOLECULAR BIOLOGY, Issue 2 2006D. D. Nimmo Abstract Current techniques for the genetic engineering of insect genomes utilize transposable genetic elements, which are inefficient, have limited carrying capacity and give rise to position effects and insertional mutagenesis. As an alternative, we investigated two site-specific integration mechanisms in the yellow fever mosquito, Aedes aegypti. One was a modified CRE/lox system from phage P1 and the other a viral integrase system from Streptomyces phage phi C31. The modified CRE/lox system consistently failed to produce stable germline transformants but the phi C31 system was highly successful, increasing integration efficiency by up to 7.9-fold. The ability to efficiently target transgenes to specific chromosomal locations and the potential to integrate very large transgenes has broad applicability to research on many medically and economically important species. [source] Transgene expression from the Tribolium castaneum Polyubiquitin promoterINSECT MOLECULAR BIOLOGY, Issue 5 2002M. D. Lorenzen Abstract The highly conserved Ubiquitin proteins are expressed from genes with strong, constitutively active promoters in many species, making these promoters attractive candidates for use in driving transgene expression. Here we report the cloning and characterization of the Tribolium castaneum Polyubiquitin (TcPUb) gene. We placed the TcPUb promoter upstream of the coding region of the T. castaneum eye-colour gene Tc vermilion (Tcv) and injected this construct into embryos from a Tcv -deficient strain. Transient expression of Tcv during embryogenesis resulted in complete rescue of the larval mutant phenotype. We then incorporated the TcPUb-Tcv chimera into a piggyBac donor. Resulting germline transformants were easily recognized by rescue of eye pigmentation, illustrating the potential of the TcPUb promoter for use in driving transgene expression. [source] Construction and evaluation of food-grade vectors for Lactococcus lactis using aspartate aminotransferase and , -galactosidase as selectable markersJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2006V.R. Sridhar Abstract Aims:, We report development of two food-grade cloning vectors for Lactococcus lactis, which utilize either a lactococcal aspartate aminotransferase gene (aspC), or Bifidobacterium longum, -galactosidase gene (aglL) as selectable markers. Methods and Results:, The theta-replicon of lactococcal plasmid, pW563, was combined with aspC and a multiple cloning site. When electroporated into L. lactis JLS400 (AspC,), the resulting vector, pSUW611 (3·9 kbp), restores ability of the mutant to grow in milk thus allowing for selection of the transformants. The vector is stable during 100 generations of nonselective growth (0·2% loss per generation). The second vector, pSUW711 (5·1 kbp), was constructed by exchanging aspC with aglL under the control of usp45 promoter. Lactococcus lactis transformed with pSUW711 produced distinctive colonies within 48,72 h on melibiose-containing plates. Expression of two Lactobacillus helveticus peptidases was attempted using these new vehicles. Introduction of pepN on pSUW611 and pSUW711 into L. lactis led to a sixfold, or 27-fold increase in aminopeptidase activity, respectively. However, no changes in endopeptidase activity were recorded upon transformation with pSUW611 carrying pepO2 under control of three different promoters. Attempts were also made to construct high copy variants of pSUW711. Conclusions:, The aspC and aglL can be employed as food-grade genetic markers for L. lactis. The vectors, pSUW611 and pSUW711, were successfully used to express Lact. helveticus PepN in L. lactis. Significance and Impact of the Study:, Two novel food-grade vectors were developed which provide simple and convenient selection and maintenance in L. lactis. [source] pH Control of the production of recombinant glucose oxidase in Aspergillus nidulansJOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2004R. Luque Abstract Aims:, Recombinant Aspergillus nidulans sVAL040, capable of synthesizing and secreting glucose oxidase derived from Aspergillus niger was used to study the influence of pH and carbon source on enzyme production. Methods and Results:, Glucose oxidase gene (goxC) was expressed under transcriptional regulation by using the promoter of A. nidulans xlnB gene (encoding an acidic xylanase). A maximum specific glucose oxidase activity of approx. 10 U mg,1 protein and a maximum volumetric productivity of 29·9 U l,1 h,1 were obtained at pH 5·5, after 80 h of growth by using xylose as inducer. Enzyme volumetric productivity increased when xylans were used instead of xylose; however, specific glucose oxidase activity did not differ significantly. Conclusions:, Specific GOX activity obtained at pH 5·5 are two to three times more than those previously described for goxC multicopy transformants of A. nidulans. Xylans were a more powerful inducer than xylose although fungal growth was lower when the polymers were used. Significance and Impact of the Study:, The obtained results by using xlnB promoter in A. nidulans could be useful in improving heterologous enzyme production by using genetic- and process-engineering strategies. [source] Expression of 2 Lipomyces kononenkoae,-Amylase Genes in Selected Whisky Yeast StrainsJOURNAL OF FOOD SCIENCE, Issue 7 2004K. la Grange-Nel ABSTRACT: Nineteen whisky yeasts were evaluated according to their fermentation performance and ability to produce a palatable spirit. Four of these strains were selected and, together with a commercial wine yeast strain (control), were transformed with integration plasmids containing the LKA1 and LKA2 , -amylase genes from the yeast Lipomyces kononenkoae. Fermentation trials with starch-containing media indicated that the transformants produced between 47% and 66% of the theoretical ethanol yield. This study has resulted in progress toward the development of whisky yeast that could ultimately be used in a process during which production of amylases, hydrolysis of starch, and fermentation of resulting sugars to grain whisky occur in a single step. [source] MOLECULAR GENETIC MANIPULATION OF THE DIATOM THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE),JOURNAL OF PHYCOLOGY, Issue 5 2006Nicole Poulsen Here, we describe the first system for genetic transformation of Thalassiosira pseudonana (Hustedt) Hasle et Heimdal, the only diatom for which a complete genome sequence is presently available. This method is based on microparticle bombardment followed by selection of transformants using the antibiotic nourseothricin. It exhibits the highest transformation efficiency compared with transformation systems for other diatom species. To achieve the high transformation efficiency, it is important to allow recovery of the bombarded T. pseudonana cells in non-selective suspension culture before spreading on nourseothricin containing agar plates. It is demonstrated that T. pseudonana is readily susceptible to co-transformation allowing for the simultaneous introduction of a non-selective gene together with the selection marker gene. Both introduced genes are stably inherited even in the absence of the antibiotic selection pressure. We have developed two T. pseudonana -specific expression vectors that can drive constitutive expression (vector pTpfcp) and inducible expression (vector pTpNR) of introduced genes. In combination with the available genome data the T. pseudonana transformation system is expected to provide a powerful tool for functional genomics in diatoms. [source] Use of , -Glucuronidase Activity to Quantify the Growth of Fusarium oxysporum f. sp. radicis-lycopersici during Infection of TomatoJOURNAL OF PHYTOPATHOLOGY, Issue 6 2005K. K. Papadopoulou Abstract The , -glucuronidase (gus) reporter gene was integrated into the phytopathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici (FORL) in a co-transformation experiment using the hygromycin B resistance (hph) gene as selective marker, which resulted in the generation of 10 mitotically stable transformants. One transformant, F30, was selected based on the results of prior detailed characterization of the 10 transformants for growth rate, conidia production and pathogenicity in comparison with the wild-type strain. A strong positive correlation was found between GUS activity and accumulated biomass of in vitro -grown fungus and therefore GUS activity was used to study fungal growth quantitatively in two tomato lines. Although a parallel increase in lesion development and GUS activity was noted for both tomato lines, a correlation between the GUS activity and disease progression was not always possible. Interestingly, the levels of GUS activity obtained for the more resistant line were higher than those obtained for the susceptible line, indicating that disease progression in tomato caused by FORL may not be related only to the amount of fungal biomass within the root tissue. [source] Gluconic acid production by Aspergillus terreusLETTERS IN APPLIED MICROBIOLOGY, Issue 3 2010C. Dowdells Abstract Aim:,Aspergillus terreus produces itaconic acid at low pH but lovastatin and other secondary metabolites at higher pH in the fermentation. The utilization of glucose as a carbon substrate was investigated for secondary metabolite production by A. terreus. Methods and Results:, With a starting pH of 6·5, glucose was rapidly metabolized to gluconic acid by the wild-type strain and by transformants harbouring Aspergillus niger genes encoding 6-phosphofructo-1-kinases with superior kinetic and regulatory properties for bioproduction of metabolites from glucose. On exhaustion of the glucose in batch fermentations, the accumulated gluconic acid was utilized as a carbon source. Conclusions:, A novel pathway of glucose catabolism was demonstrated in A. terreus, a species whose wild type is, without any strain development, capable of producing gluconic acid at high molar conversion efficiency (up to 0·7 mol mol,1 glucose consumed). Significance and Impact of the Study:,Aspergillus terreus is a potential novel producer organism for gluconic acid, a compound with many uses as a bulk chemical. With a new knowledge of glucose catabolism by A. terreus, fermentation strategies for secondary metabolite production can be devised with glucose feeding using feedback regulation by pH. [source] A food-grade site-directed mutagenesis system for Streptococcus thermophilus LMG 18311LETTERS IN APPLIED MICROBIOLOGY, Issue 3 2010T. Blomqvist Abstract Aims:, To develop a general method for site-directed mutagenesis in the dairy starter strain Streptococcus thermophilus LMG 18311 which does not depend on antibiotic-resistance genes or other selection markers for the identification of transformants. Methods and Results:, In a previous study, we demonstrated that Strep. thermophilus LMG 18311 can be made competent for natural genetic transformation by overexpression of the alternative sigma factor ComX. In the present study, we wanted to investigate whether the natural transformation mechanism of Strep. thermophilus LMG 18311 is efficient enough to make it feasible to perform site-directed mutagenesis in this strain without the use of a selection marker. Competent bacteria were mixed with a DNA fragment engineered to contain a nonsense and a frameshift mutation in the middle of the target gene (lacZ) and subsequently seeded on agar plates. By performing colony-lift hybridization using a digoxigenin-labelled oligonucleotide probe, we succeeded in identifying transformants containing the sought after mutation. Conclusions:, By exploiting the natural transformability of Strep. thermophilus LMG 18311 and standard molecular methods, we have demonstrated that the genome of this bacterium can be altered at preselected sites without introduction of any foreign DNA. Significance and Impact of the Study:, A food-grade site-directed mutagenesis system has been developed for Strep. thermophilus LMG 18311 that can be used by the dairy industry to construct starter strains with novel and/or improved properties. [source] Technical advance in fungal biotechnology: development of a miniaturized culture method and an automated high-throughput screeningLETTERS IN APPLIED MICROBIOLOGY, Issue 2 2009F. Alberto Abstract Aims:, The goal of the study was to develop a reliable, reproducible and rapid method of culture in order to screen a large number of fungal transformants. Methods and Results:, The method is based upon miniaturized cell cultures and automated expression screening in microwell plates. For the method development, 50 recombinant Aspergillus vadensis clones producing feruloyl esterase B (FaeB) from Aspergillus niger were screened in 6 days. Then a panel of clones showing various behaviours was checked in flasks in order to demonstrate the reproducibility of the method. Using this method, a transformant of A. vadensis producing 1·2 g l,1 of FaeB was selected (12-fold more than the A. niger overproducing strain). Conclusions:, This miniaturized culture method allows to obtain reliable and reproducible results. The procedure has the advantages of being efficient, time-saving and more efficient than conventional in-flask culture screening as it can screen 800 clones per day after a culture of 3 days. Significance and Impact of the Study:, This method could be applied to any other fungal strain culture, enzyme activity or biodiversity screening. [source] | ||||||||||||||||||||||||||||||||||||||||