Control of Ammonium Permease Expression and Filamentous Growth by the GATA Transcription Factors GLN3 and GAT1 in Candida albicans

(A) Growth of mep1Δ mep2Δ double mutants expressing a GFP-tagged MEP2 gene from the wild-type MEP2 promoter and derivatives containing the indicated deletions or mutations. The strains were grown for 4 days at 30°C on SD plates containing 1 mM ammonium. The following strains were used: MEP12M6A and -B (negative [neg.] control), MEP12MG6A and -B (positive [pos.] control), MEP12MG6ΔP1A and -B (ΔP1), MEP12MG6ΔP2A and -B (ΔP2), MEP12MG6ΔP3A and -B (ΔP3), MEP12MG6ΔP4A and -B (ΔP4), MEP12MG6ΔP5A and -B (ΔP5), MEP12MG6ΔP6A and -B (ΔP6), MEP12MG6ΔP7A and -B (ΔP7), MEP12MG6MP1A and -B (MP1), MEP12MG6MP2A and -B (MP2), and MEP12MG6MP3A and -B (MP3). The two independently constructed reporter strains behaved identically, and only one of them is shown in each case. (B) Filamentation of mep2Δ mutants expressing MEP2 from the wild-type MEP2 promoter and derivatives containing the indicated deletions or mutations. The strains were grown for 6 days at 37°C on SLAD plates, and individual representative colonies were photographed. The following strains were used: MEP2MK13A and -B (pos. control), MEP2MK13ΔP5A and -B (ΔP5), MEP2MK13ΔP6A and -B (ΔP6), MEP2MK13MP1A and -B (MP1), MEP2MK13MP2A and -B (MP2), and MEP2MK13MP3A and -B (MP3). Independently constructed strains behaved identically, and only one of them is shown in each case.

Construction of gln3Δ mutants and complemented strains. (A) Structure of the deletion cassette from plasmid pGLN3M2 (top), which was used to delete the GLN3 open reading frame, and genomic structure of the GLN3 locus in strain SC5314 (bottom). The GLN3 coding region is represented by the white arrow and the upstream and downstream regions by the solid lines. Details of the SAT1 flipper (gray rectangle bordered by FRT sites [black arrows]) have been presented elsewhere (22). The 34-bp FRT sites are not drawn to scale. The probes used for Southern hybridization analysis of the mutants are indicated by the black bars. (B) Structure of the DNA fragment from pGLN3K1 (top), which was used for reintegration of an intact GLN3 copy into one of the disrupted GLN3 loci in the gln3Δ single and gln3Δ gat1Δ double mutants (bottom). Only relevant restriction sites are given in panels A and B, as follows: A, ApaI; B, BamHI; Bg, BglII; C, ClaI; ScI, SacI; ScII, SacII; Sl, SalI; and Xh, XhoI. Sites shown in parentheses were destroyed by the cloning procedure. The ClaI site marked in italics is present only in the GLN3-1 allele. (C) Southern hybridization of ClaI-digested genomic DNA of the wild-type strain SC5314 and gln3Δ mutants and complemented strains with the GLN3-specific probe 1. The sizes of the hybridizing fragments (in kb) are given on the left side of the blot, and their identities are indicated on the right. Insertion of the SAT1 flipper into either of the two GLN3 alleles of the parental strain SC5314 (lane 1) and subsequent FLP-mediated excision of the cassette produced the heterozygous mutants GLN3M2A and GLN3M2B (lanes 2 and 3). Insertion of the SAT1 flipper into the remaining wild-type GLN3 alleles, followed by recycling of the SAT1 flipper cassette, gave rise to the homozygous gln3Δ mutants GLN3M4A and GLN3M4B (lanes 4 and 5). An intact GLN3 copy was reintroduced into the gln3Δ mutants with the help of the SAT1 flipper, which was subsequently excised again to produce the complemented strains GLN3MK2A and GLN3MK2B (lanes 6 and 7).

GLN3 and GAT1 control ammonium permease expression in C. albicans. (A and B) Expression of GFP-tagged Mep2p (A) and Mep1p (B) in the wild type, gln3Δ and gat1Δ single mutants, and gln3Δ gat1Δ double mutants in liquid media containing limiting concentrations (100 μM) of the indicated nitrogen sources. Overnight cultures of the reporter strains in SD-Pro medium were diluted 50-fold in the test media and grown for 6 h at 30°C. The fluorescence of the cells was measured by flow cytometry. The first columns show the results obtained with the A series, and the second columns show the results obtained with the B series of the reporter strains (see Table 1). Note that the scales of the y axis are different for Mep2p-GFP and Mep1p-GFP. (C) Detection of MEP2 mRNA by Northern hybridization with a MEP2-specific probe. Overnight cultures of the wild-type strain SC5314 (lane 1), the gln3Δ mutants GLN3M4A (lane 2) and GLN3M4B (lane 5), the gat1Δ mutants GAT1M4A (lane 3) and GAT1M4B (lane 6), and the gln3Δ gat1Δ double mutants Δgln3GAT1M4A (lane 4) and Δgln3GAT1M4B (lane 7) in SD-Pro medium were diluted 50-fold in liquid SLAD medium, and RNA was isolated after 6 h of growth at 30°C. The MEP2 transcript is absent from the gln3Δ gat1Δ double mutants; the faint band of slightly higher molecular weight than that of MEP2 seen in these and all other strains represents a cross-hybridizing transcript.

(A) GLN3 is required for the normal filamentous growth of C. albicans in response to nitrogen limitation. YPD precultures of the strains were appropriately diluted and spread on SD agar plates containing the indicated nitrogen sources at a concentration of 100 μM or on agar plates containing serum as an inducer of filamentous growth. Individual colonies were photographed after 6 days of incubation at 37°C. The following strains were used: SC5314 (wild type), GLN3M4A and -B (gln3Δ), GLN3MK2A and -B (gln3Δ + GLN3), GAT1M4A and -B (gat1Δ), Δgln3GAT1M4A and -B (gln3Δ gat1Δ), and Δgln3GAT1MK2A and -B (gln3Δ gat1Δ + GLN3). Independently constructed mutants and complemented strains behaved identically, and only one of them is shown in each case. (B) Expression of GFP-tagged Mep2p in the wild-type (strains SCMEP2G7A and -B), gat1Δ (strains Δgat1MEP2G7A and -B), gln3Δ (strains Δgln3MEP2G7A and -B), and gln3Δ gat1Δ (strains Δgln3Δgat1MEP2G7A and -B) backgrounds on SLAD plates. The pictures show fluorescence and corresponding phase-contrast micrographs of cells taken from colonies of the reporter strains grown for 6 days at 37°C.

Inactivation of GAT1 results in MEP2-independent filamentation. The indicated strains were grown for 6 days at 37°C on SLAD plates, and individual colonies were photographed. Independently constructed mutants behaved identically, and only one of them is shown in each case.