Histoplasma Requires SID1, a Member of an Iron-Regulated Siderophore Gene Cluster, for Host Colonization
A. Southern analysis of SID1 disruption. Genomic DNA from parental strain (WU8-Wild-type) and potential sid1Δ::hph strains (HcLH25, HcLH26, and HcLH27) was digested by EcoRI or SalI, separated by gel electrophoresis, blotted and hybridized to probes containing either the open reading frame of SID1 or the hph gene. For the SID1 probe, fragments of the expected sizes of 6.4 kb (EcoRI) and 12.2 kb (SalI) are seen in the DNA from the parental strain. For the hph probe, fragments of the expected sizes of 3.6 kb and 2.3 kb (EcoRI) and 6.8 kb (SalI) are seen in the DNA from sid1Δ strains. B. Growth curves of HcLH95 (WT+vector), HcLH97 (sid1Δ+vector), HcLH103 (WT+SID1), and HcLH106 (sid1Δ+SID1). Yeast cells were grown in mRPMI medium (pH 6.5) without or with 5 µM FeSO4 added (+Fe). Growth and siderophore production experiments were repeated at least three times with two independent sid1Δ strains and four independent complementation strains. Samples for OD600 were taken in triplicate. The mean+/−standard deviation is indicated. A representative experiment is presented. C. Siderophore production of strains. Yeast cells (same strains used in B) were grown in mRPMI medium (pH 6.5) for 24 hours and culture supernatant was examined for presence of siderophores using chrome azurol S assay as described in Materials and Methods. Samples were taken in triplicate. The mean+/−standard deviation is indicated. Results are given as percentage of siderophore production compared with WT+vector.