Journal: Eukaryotic Cell

Article Title: The High-Osmolarity Glycerol Response Pathway in the Human Fungal Pathogen Candida glabrata Strain ATCC 2001 Lacks a Signaling Branch That Operates in Baker's Yeast

doi: 10.1128/ec.00106-07

Figure Lengend Snippet: FIG. 1. Osmostress sensitivities of C. glabrata pbs2 and sho1 strains. (A) Cultures of the C. glabrata strains ATCC 2001, Cg2633, and BG2, as well as their isogenic Cgpbs2 and Cgsho1 deletion strains, were grown to an OD600 of 1, diluted to an OD600 of 0.1, 0.01, or 0.001, and spotted onto YPD and YPD agar plates containing the indicated amounts of NaCl. Plates were incubated at 30°C for 2 days. WT, wild type. (B) Genomic DNA was isolated from ATCC 2001, BG2, Cg2633, and their isogenic Cgsho1 deletion strains, followed by digestion with HindIII and NotI. After agarose separation of DNA fragments and transfer to nitrocellulose membranes, radiolabeled probes A and B were used for detection of the SHO1 WT gene and the disrupted allele. Restriction sites and expected fragment sizes for the WT and Cgsho1 disruption strains are illustrated schematically (not drawn to scale).

Article Snippet: C. glabrata sho1 (Cgsho1 ) deletion strains from the sequenced ATCC 2001 strain display severe growth defects under hyperosmotic conditions, a phenotype not observed for yeast sho1 mutants.

Techniques: Incubation, Isolation, Disruption

Journal: Eukaryotic Cell

Article Title: The High-Osmolarity Glycerol Response Pathway in the Human Fungal Pathogen Candida glabrata Strain ATCC 2001 Lacks a Signaling Branch That Operates in Baker's Yeast

doi: 10.1128/ec.00106-07

Figure Lengend Snippet: FIG. 4. Expression of CgSho1 in yeast can complement the loss of ScSho1. (A) S. cerevisiae strains W303-1B, YCG9A (sho1 ssk2 ssk22), and YCG9A transformed with either pRS-CgSHO1 or pYEp- CgSHO1 were diluted to an OD600 of 0.1, 0.01, 0.001, or 0.0001 and spotted onto YPD and YPD agar plates containing NaCl. Plates were incubated at 30°C for 2 days. (B) Alignment of the protein kinase domains of C. glabrata (ATCC 2001) and S. cerevisiae Ssk2. CgSSK2 encodes a translational stop within a region highly conserved between S. cerevisiae and C. glabrata (indicated). Below the alignment are dia- grams of the proteins encoded by the SSK2 ORFs in the two distinct C. glabrata strains, ATCC 2001 and BG2, as well as S. cerevisiae Ssk2. PKD, protein kinase domain.

Article Snippet: C. glabrata sho1 (Cgsho1 ) deletion strains from the sequenced ATCC 2001 strain display severe growth defects under hyperosmotic conditions, a phenotype not observed for yeast sho1 mutants.

Techniques: Expressing, Transformation Assay, Incubation

Journal: Eukaryotic Cell

Article Title: The High-Osmolarity Glycerol Response Pathway in the Human Fungal Pathogen Candida glabrata Strain ATCC 2001 Lacks a Signaling Branch That Operates in Baker's Yeast

doi: 10.1128/ec.00106-07

Figure Lengend Snippet: FIG. 5. The ATCC 2001 genome carries a nonfunctional truncated ssk2-1 allele. (A) Cultures of the C. glabrata strains ATCC 2001 and BG2 as well as their isogenic Cgpbs2 and Cgsho1 deletion strains were diluted to an OD600 of 0.1, 0.01, or 0.001 and spotted onto YPD and YPD agar plates containing the indicated amounts of methylglyoxal (MG) or onto YPD (pH 4.5) and YPD (pH 4.5) agar plates containing the indicated amounts of acetate. Plates were incubated at 30°C for 2 days. (B) Cultures of the C. glabrata strain ATCC 2001 and the isogenic Cgpbs2 and Cgsho1 deletion strains, as well as three independent clones of ATCC 2001 sho1 SSK2 (C1 to C3), were grown to the exponential-growth phase, diluted to an OD600 of 0.1, and spotted along with serial 1:10 dilutions onto plates containing the indicated amounts of NaCl, MG, or acetate. Plates were incubated at 30°C for 2 days or at 42°C for 1 day. (C) Cultures of ATCC 2001, BG2, and BG2 Cgssk2 were diluted to an OD600 of 0.1, 0.01, or 0.001; serial dilutions were spotted onto YPD and YPD agar plates containing 50 mM MG or onto YPD (pH 4.5) and YPD (pH 4.5) agar plates containing 80 mM acetate. Plates were incubated at 30°C for 2 days. (D) Cultures of BG2 and ATCC 2001 and the indicated isogenic deletion strains, as well as three clones of ATCC 2001 sho1 complemented for SSK2 (C1 to C3), were grown to the early-exponential-growth phase before 0.5 M NaCl was added. Samples were taken at the indicated time points and crude trichloroacetic acid extracts prepared. Aliquots corresponding to 0.4 OD600 equivalent per lane were fractionated through a 10% SDS-PAGE gel. Immunoblotting was carried out using polyclonal anti-phospho- p38 MAPK or anti-Pgk1 antibodies. Extracts of ATCC 2001 sho1 SSK2 C2 and C3 were detected on different immunoblots, as indicated by the separation of these gels.

Article Snippet: C. glabrata sho1 (Cgsho1 ) deletion strains from the sequenced ATCC 2001 strain display severe growth defects under hyperosmotic conditions, a phenotype not observed for yeast sho1 mutants.

Techniques: Incubation, Clone Assay, SDS Page, Western Blot

Journal: Eukaryotic Cell

Article Title: The High-Osmolarity Glycerol Response Pathway in the Human Fungal Pathogen Candida glabrata Strain ATCC 2001 Lacks a Signaling Branch That Operates in Baker's Yeast

doi: 10.1128/ec.00106-07

Figure Lengend Snippet: FIG. 6. HOG pathways operating in S. cerevisiae and C. glabrata. Activation of the MAPKK Pbs2 can occur through at least two distinct upstream osmosensing mechanisms. One branch links the osmosensing protein Sln1 via Ypd1, Ssk1, and the MAPKKKs Ssk2 and Ssk22 to Pbs2. In the other branch, Sho1 functions to link an as yet unidentified osmosensor to the downstream components Cdc42, Ste20, Ste50, and the MAPKKK Ste11. Activated Pbs2 phosphorylates the MAPK Hog1, which in turn activates a variety of transcription factors. As indicated, Ssk22 does not exist in C. glabrata, and ATCC 2001 contains the ssk2-1 allele, encoding a truncated and nonfunctional Ssk2 version.

Article Snippet: C. glabrata sho1 (Cgsho1 ) deletion strains from the sequenced ATCC 2001 strain display severe growth defects under hyperosmotic conditions, a phenotype not observed for yeast sho1 mutants.

Techniques: Activation Assay

Journal: Eukaryotic Cell

Article Title: The High-Osmolarity Glycerol Response Pathway in the Human Fungal Pathogen Candida glabrata Strain ATCC 2001 Lacks a Signaling Branch That Operates in Baker's Yeast

doi: 10.1128/ec.00106-07

Figure Lengend Snippet: FIG. 7. C. glabrata HOG pathway mutants display sensitivity to weak acids. (A) Cultures of the C. glabrata strains ATCC 2001 and BG2, as well as their isogenic Cgpbs2 and Cgsho1 deletion strains, were diluted to an OD600 of 0.1, 0.01, or 0.001 and spotted onto YPD (pH 4.5) and YPD (pH 4.5) agar plates containing the indicated amounts of sorbate, propionate, or benzoate. Plates were incubated at 30°C for 2 days. WT, wild type. (B) Cultures of WT S. cerevisiae W303-1A and its isogenic sho1, ssk1, pbs2, and ssk1 sho1 de-

Article Snippet: C. glabrata sho1 (Cgsho1 ) deletion strains from the sequenced ATCC 2001 strain display severe growth defects under hyperosmotic conditions, a phenotype not observed for yeast sho1 mutants.

Techniques: Incubation