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  • 1
    Online Resource
    Online Resource
    Versatile Enzyme Expression and Characterization System for Aspergillus nidulans, with the Penicillium brevicompactum Polyketide Synthase Gene from the Mycophenolic Acid Gene Cluster as a Test Case
    American Society for Microbiology ; 2011
    In:  Applied and Environmental Microbiology Vol. 77, No. 9 ( 2011-05), p. 3044-3051
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 77, No. 9 ( 2011-05), p. 3044-3051
    Abstract: Assigning functions to newly discovered genes constitutes one of the major challenges en route to fully exploiting the data becoming available from the genome sequencing initiatives. Heterologous expression in an appropriate host is central in functional genomics studies. In this context, filamentous fungi offer many advantages over bacterial and yeast systems. To facilitate the use of filamentous fungi in functional genomics, we present a versatile cloning system that allows a gene of interest to be expressed from a defined genomic location of Aspergillus nidulans . By a single USER cloning step, genes are easily inserted into a combined targeting-expression cassette ready for rapid integration and analysis. The system comprises a vector set that allows genes to be expressed either from the constitutive PgpdA promoter or from the inducible PalcA promoter. Moreover, by using the vector set, protein variants can easily be made and expressed from the same locus, which is mandatory for proper comparative analyses. Lastly, all individual elements of the vectors can easily be substituted for other similar elements, ensuring the flexibility of the system. We have demonstrated the potential of the system by transferring the 7,745-bp large mpaC gene from Penicillium brevicompactum to A. nidulans . In parallel, we produced defined mutant derivatives of mpaC , and the combined analysis of A. nidulans strains expressing mpaC or mutated mpaC genes unequivocally demonstrated that mpaC indeed encodes a polyketide synthase that produces the first intermediate in the production of the medically important immunosuppressant mycophenolic acid.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.01768-10
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2011
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
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  • 2
    Online Resource
    Online Resource
    Method for Screening Compounds That Influence Virulence Gene Expression in Staphylococcus aureus
    American Society for Microbiology ; 2010
    In:  Antimicrobial Agents and Chemotherapy Vol. 54, No. 1 ( 2010-01), p. 509-512
    Details
    In: Antimicrobial Agents and Chemotherapy, American Society for Microbiology, Vol. 54, No. 1 ( 2010-01), p. 509-512
    Abstract: We present a simple assay to examine effects of compounds on virulence gene expression in the human pathogen Staphylococcus aureus . The assay employs transcriptional reporter strains carrying lacZ fused to central virulence genes. Compounds affecting virulence gene expression and activity of the agr locus are scored based on color change in the presence of a chromogenic β-galactosidase substrate. The assay can be used to screen for novel antivirulence compounds from many different sources, such as fungi, as demonstrated here.
    Type of Medium: Online Resource
    ISSN: 0066-4804 , 1098-6596
    URL: Article
    DOI: 10.1128/AAC.00940-09
    RVK:
    XA 24552
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2010
    detail.hit.zdb_id: 1496156-8
    SSG: 12
    SSG: 15,3
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  • 3
    Online Resource
    Online Resource
    Submerged Conidiation and Product Formation by Aspergillus niger at Low Specific Growth Rates Are Affected in Aerial Developmental Mutants
    American Society for Microbiology ; 2011
    In:  Applied and Environmental Microbiology Vol. 77, No. 15 ( 2011-08), p. 5270-5277
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 77, No. 15 ( 2011-08), p. 5270-5277
    Abstract: Exposure to an aerial environment or severe nutrient limitation induces asexual differentiation in filamentous fungi. Submerged cultivation of Aspergillus niger in carbon- and energy-limited retentostat cultures both induces and fuels conidiation. Physiological and transcriptomic analyses have revealed that this differentiation strongly affects product formation. Since conidiation is inherent in the aerial environment, we hypothesized that product formation near zero growth can be influenced by affecting differentiation or development of aerial hyphae in general. To investigate this idea, three developmental mutants (Δ fwnA , scl-1 , and scl-2 mutants) that have no apparent vegetative growth defects were cultured in maltose-limited retentostat cultures. The secondary-metabolite profile of the wild-type strain defined flavasperone, aurasperone B, tensidol B, and two so far uncharacterized compounds as associated with conidium formation, while fumonisins B 2 , B 4 , and B 6 were characteristic of early response to nutrient limitation by the vegetative mycelium. The developmental mutants responded differently to the severe substrate limitation, which resulted in distinct profiles of growth and product formation. fwnA encodes the polyketide synthase responsible for melanin biosynthesis during aerial differentiation, and we show that conidial melanin synthesis in submerged retentostat cultures and aurasperone B production are fwnA dependent. The scl-1 and scl-2 strains are two UV mutants generated in the Δ fwnA background that displayed reduced asexual conidiation and formed sclerotium-like structures on agar plates. The reduced conidiation phenotypes of the scl-1 and scl-2 strains are reflected in the retentostat cultivation and are accompanied by elimination or severely reduced accumulation of secondary metabolites and distinctly enhanced accumulation of extracellular protein. This investigation shows that submerged conidiation and product formation of a mitosporic fungus cultured at low specific growth rates can be fundamentally affected by interfering with the genetic program for differentiation of aerial hyphae, opening new perspectives for tailoring industrial performance.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.00118-11
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2011
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
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  • 4
    Online Resource
    Online Resource
    Methylenetetrahydrofolate Reductase Activity Is Involved in the Plasma Membrane Redox System Required for Pigment Biosynthesis in Filamentous Fungi
    American Society for Microbiology ; 2010
    In:  Eukaryotic Cell Vol. 9, No. 8 ( 2010-08), p. 1225-1235
    Details
    In: Eukaryotic Cell, American Society for Microbiology, Vol. 9, No. 8 ( 2010-08), p. 1225-1235
    Abstract: Methylenetetrahydrofolate reductases (MTHFRs) play a key role in biosynthesis of methionine and S -adenosyl- l -methionine (SAM) via the recharging methionine biosynthetic pathway. Analysis of 32 complete fungal genomes showed that fungi were unique among eukaryotes by having two MTHFRs, MET12 and MET13. The MET12 type contained an additional conserved sequence motif compared to the sequences of MET13 and MTHFRs from other eukaryotes and bacteria. Targeted gene replacement of either of the two MTHFR encoding genes in Fusarium graminearum showed that they were essential for survival but could be rescued by exogenous methionine. The F. graminearum strain with a mutation of MET12 (FgΔ MET12 ) displayed a delay in the production of the mycelium pigment aurofusarin and instead accumulated nor-rubrofusarin and rubrofusarin. High methionine concentrations or prolonged incubation eventually led to production of aurofusarin in the MET12 mutant. This suggested that the chemotype was caused by a lack of SAM units for the methylation of nor-rubrofusarin to yield rubrofusarin, thereby imposing a rate-limiting step in aurofusarin biosynthesis. The FgΔ MET13 mutant, however, remained aurofusarin deficient at all tested methionine concentrations and instead accumulated nor-rubrofusarin and rubrofusarin. Analysis of MET13 mutants in F. graminearum and Aspergillus nidulans showed that both lacked extracellular reduction potential and were unable to complete mycelium pigment biosynthesis. These results are the first to show that MET13, in addition to its function in methionine biosynthesis, is required for the generation of the extracellular reduction potential necessary for pigment production in filamentous fungi.
    Type of Medium: Online Resource
    ISSN: 1535-9778 , 1535-9786
    URL: Article
    DOI: 10.1128/EC.00031-10
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2010
    detail.hit.zdb_id: 2071564-X
    SSG: 12
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  • 5
    Online Resource
    Online Resource
    Genetic Dissection of Tropodithietic Acid Biosynthesis by Marine Roseobacters
    American Society for Microbiology ; 2008
    In:  Applied and Environmental Microbiology Vol. 74, No. 5 ( 2008-03), p. 1535-1545
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 74, No. 5 ( 2008-03), p. 1535-1545
    Abstract: The symbiotic association between the roseobacter Silicibacter sp. strain TM1040 and the dinoflagellate Pfiesteria piscicida involves bacterial chemotaxis to dinoflagellate-produced dimethylsulfoniopropionate (DMSP), DMSP demethylation, and ultimately a biofilm on the surface of the host. Biofilm formation is coincident with the production of an antibiotic and a yellow-brown pigment. In this report, we demonstrate that the antibiotic is a sulfur-containing compound, tropodithietic acid (TDA). Using random transposon insertion mutagenesis, 12 genes were identified as critical for TDA biosynthesis by the bacteria, and mutation in any one of these results in a loss of antibiotic activity (Tda − ) and pigment production. Unexpectedly, six of the genes, referred to as tdaA-F , could not be found on the annotated TM1040 genome and were instead located on a previously unidentified plasmid (ca. 130 kb; pSTM3) that exhibited a low frequency of spontaneous loss. Homologs of tdaA and tdaB from Silicibacter sp. strain TM1040 were identified by mutagenesis in another TDA-producing roseobacter, Phaeobacter sp. strain 27-4, which also possesses two large plasmids (ca. 60 and ca. 70 kb, respectively), and tda genes were found by DNA-DNA hybridization in 88% of a diverse collection of nine roseobacters with known antibiotic activity. These data suggest that roseobacters may use a common pathway for TDA biosynthesis that involves plasmid-encoded proteins. Using metagenomic library databases and a bioinformatics approach, differences in the biogeographical distribution between the critical TDA synthesis genes were observed. The implications of these results to roseobacter survival and the interaction between TM1040 and its dinoflagellate host are discussed.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.02339-07
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2008
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
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  • 6
    Online Resource
    Online Resource
    Inactivation of Vibrio anguillarum by Attached and Planktonic Roseobacter Cells
    American Society for Microbiology ; 2010
    In:  Applied and Environmental Microbiology Vol. 76, No. 7 ( 2010-04), p. 2366-2370
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 76, No. 7 ( 2010-04), p. 2366-2370
    Abstract: The purpose of the present study was to investigate the inhibition of Vibrio by Roseobacter in a combined liquid-surface system. Exposure of Vibrio anguillarum to surface-attached roseobacters (10 7 CFU/cm 2 ) resulted in significant reduction or complete killing of the pathogen inoculated at 10 2 to 10 4 CFU/ml. The effect was likely associated with the production of tropodithietic acid (TDA), as a TDA-negative mutant did not affect survival or growth of V. anguillarum .
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.02717-09
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2010
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
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  • 7
    Online Resource
    Online Resource
    Influence of Iron on Production of the Antibacterial Compound Tropodithietic Acid and Its Noninhibitory Analog in Phaeobacter inhibens
    American Society for Microbiology ; 2016
    In:  Applied and Environmental Microbiology Vol. 82, No. 2 ( 2016-01-15), p. 502-509
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 82, No. 2 ( 2016-01-15), p. 502-509
    Abstract: Tropodithietic acid (TDA) is an antibacterial compound produced by some Phaeobacter and Ruegeria spp. of the Roseobacter clade. TDA production is studied in marine broth or agar since antibacterial activity in other media is not observed. The purpose of this study was to determine how TDA production is influenced by substrate components. High concentrations of ferric citrate, as present in marine broth, or other iron sources were required for production of antibacterially active TDA. However, when supernatants of noninhibitory, low-iron cultures of Phaeobacter inhibens were acidified, antibacterial activity was detected in a bioassay. The absence of TDA in nonacidified cultures and the presence of TDA in acidified cultures were verified by liquid chromatography–high-resolution mass spectrometry. A noninhibitory TDA analog (pre-TDA) was produced by P. inhibens , Ruegeria mobilis F1926, and Phaeobacter sp. strain 27-4 under low-iron concentrations and was instantaneously converted to TDA when pH was lowered. Production of TDA in the presence of Fe 3+ coincides with formation of a dark brown substance, which could be precipitated by acid addition. From this brown pigment TDA could be liberated slowly with aqueous ammonia, and both direct-infusion mass spectrometry and elemental analysis indicated a [Fe III (TDA) 2 ] x complex. The pigment could also be produced by precipitation of pure TDA with FeCl 3 . Our results raise questions about how biologically active TDA is produced in natural marine settings where iron is typically limited and whether the affinity of TDA to iron points to a physiological or ecological function of TDA other than as an antibacterial compound.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.02992-15
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2016
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
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