Journal: PLoS Pathogens

Article Title: SREB, a GATA Transcription Factor That Directs Disparate Fates in Blastomyces dermatitidis Including Morphogenesis and Siderophore Biosynthesis

doi: 10.1371/journal.ppat.1000846

Figure Lengend Snippet: ( A ) Under iron-replete conditions (10 µM FeSO 4 ), mutant 3-15-1 and empty vector controls fail to suppress siderophore biosynthesis as demonstrated by a 2.5–3.0 fold higher absorbance at 425 nm compared to parental strain T53-19 and complemented strains A5 and D5 (p<0.01 as indicated by asterisk). Data from three independent experiments were analyzed. Siderophore production was measured using the ferric perchlorate assay. ( B ) Mutant 3-15-1 and empty vector control A4 were pigmented yellow and discolored the surrounding media when grown on 7H10 medium, which contains 150 µM FeSO 4 . In contrast, parental control T53-19 and complemented strains A5 and D5 were colored white and did not pigment the medium. All strains were incubated for 14 days at 37°C. ( C ) Mutant 3-15-1 and empty vector control A4 fail to convert to mycelia upon shifting the incubation temperature from 37°C to 22°C. Similar to the wild-type isolate, complemented strains A5 and D5 grew as mycelia at 22°C incubation. ( D ) Northern blot hybridization demonstrated reduction in transcript abundance in the insertional mutant when compared to the parent strain. Complemented strains A5 and D5 overexpress SREB . Transformation of 3-15-1 with an empty vector failed to restore transcript abundance in strain A4.

Article Snippet: Retention times were compared to siderophore standards (HPLC calibration kit – coprogens and fusarinines; EMC microcollections, Tubingen, Germany).

Techniques: Mutagenesis, Plasmid Preparation, Control, Incubation, Northern Blot, Hybridization, Transformation Assay

Journal: PLoS Pathogens

Article Title: SREB, a GATA Transcription Factor That Directs Disparate Fates in Blastomyces dermatitidis Including Morphogenesis and Siderophore Biosynthesis

doi: 10.1371/journal.ppat.1000846

Figure Lengend Snippet: ( A-C ) Culture supernatant harvested from wild-type B. dermatitidis grown under iron-poor conditions was applied to a column packed with XAD-2 resin. Following a water wash, compounds bound to the resin were eluted with methanol and analyzed by LC/MS. The chromatogram revealed the presence of several compounds in the eluate (A). Analysis of these compounds using mass spectroscopy revealed that two of the peaks (4.16 and 7.26 minutes) had masses consistent with dimerum acid (538.2) and coprogen (822.2), respectively (B, C). ( D ) Culture supernatants harvested from wild-type B. dermatitidis grown under iron-poor and iron-replete conditions were compared to siderophore standards using HPLC. During conditions of iron-limitation, the wild-type isolate produced and secreted dimerum acid and coprogen. When iron was abundant, no siderophores were detected. In contrast, the null mutant SREB Δ produced and secreted dimerum acid and coprogen under iron-replete conditions.

Article Snippet: Retention times were compared to siderophore standards (HPLC calibration kit – coprogens and fusarinines; EMC microcollections, Tubingen, Germany).

Techniques: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Produced, Mutagenesis