Table 1.
DX mutants in protein kinase-related genes.
Fig 1.
Strains were stained with Calcofluor White after 4 hr growth at 30°C in YPD medium. Expression levels of core filamentation genes in mutants grown for 4 hr at 30° in YPD are expressed as fold-change to the wild type; complete data are in S2 Table. Strains: WT (DAY286), cln3 DX (CW994), cak1 DX (CW1003), kin28 DX (CW1041), gin4 DX (CW900), dbf2 DX (CW914), ctk1 DX (CW1005), ipl1 DX (CW1038), snf1 DX (CW927), sak1 DX (CW995), ire1 DX (CW906), mck1 DX (CW1006), cdc28 DX (CW991), cmk2 DX (CW999), cdc7 DX (CW993), sha3 DX (CW1010).
Fig 2.
Multiple isolates of each DX strain indicated were obtained from a single transformation of a heterozygous deletion mutant. RNA was extracted from cells grown for 4 hr at 30°C in YPD and used for nanoString expression analysis (S3 Table). Hierarchal clustering of gene expression data was performed using MeV software. Fold change values were obtained by dividing normalized expression values for each mutant strain by the wild-type strain (DAY185) for each of the probes. The color scale represents Log2 fold change compared to wild type. (Blue limit: 10-fold down; yellow limit: 10-fold up.) Strains are listed in S3 Table.
Fig 3.
A. Cell morphology. Strains were stained with Calcofluor White after 4 hr growth at 30°C in YPD medium. Strains: cak1 DX (CW1428), cak1 DX comp (CW1431), cln3 DX (CW1437), cln3 DX comp (CW1440), kin28 DX (CW1082), kin28 DX comp (CW1085), ctk1 DX (CW1076), ctk1 DX comp (CW1078), snf1 DX (CW1138), snf1 DX comp (CW1141). B. Cek1 phosphorylation. Immunoblots were performed on yeast extracts from mid-log phase cells grown for 5 hr at 30°C. Strains for lanes 1, 3, 6, 8, 11, 12, 15, and 18 are His-; strains for lanes 2, 4, 5, 7, 9, 10, 13, 14, 16, 17, and 19 are His+. The lanes show experiments with the strain in parentheses: 1 (DAY286), 2 (CW1078), 3 (CW1005), 4 (CW1075), 5 (CW1076), 6 (TA72), 7 (TA86), 8 (CW1041), 9 (CW1082), 10 (CW1085), 11 (DAY286), 12 (CW927), 13 (CW1138), 14 (CW1141), 15 (CW995), 16 (CW1111), 17 (CW1114), 18 (TA72), 19 (TA86). C. Gene expression profiles. RNA was extracted from cells grown for 4 hr at 30°C in YPD and used for nanoString expression analysis (S3 Table). Hierarchal clustering of gene expression data was performed using MeV software. Fold change values were obtained by dividing normalized expression values for each mutant or complemented strain by the wild type strain (DAY185) for each of the probes. The color scale represents Log2 fold change compared to wild type. (Blue limit: 10-fold down; yellow limit: 10-fold up.) Strains are listed in S3 Table.
Fig 4.
Apical projections of biofilm and measurements of biofilm matrix components.
A. Strains were grown under in vitro biofilm conditions for 48 hr, then visualized by confocal microscopy. Side projections of a biofilm of each strain is shown. B. Apical view projections of the same biofilms shown in panel A were obtained using maximum intensity Z-projection of 100 planes at 0.9 μm step-size at the distance indicated from the basal layer. C. Biofilms were grown for matrix isolation. Ten ml of matrix was collected for each strain, biomass collected and dried, and quantitation of matrix total protein, carbohydrate, and normalized values of β-1,3 glucan were determined. Strains: WT (DAY185), cak1 DX (CW1428), cak1 DX comp (CW1431). Triplicate samples for all but cak1 DX carbohydrate (duplicate) determinations. ** = p<0.01 for comparison of either cak1 DX or cak1 DX comp to WT.
Fig 5.
Core filamentation gene expression and phenotypes of filamentation/biofilm activator mutants in CAK1 and cak1 DX backgrounds.
A. RNA levels for environmental response genes was determined by nanoString for strains grown for 4 hr at 37°C in YPD (S4 Table). Normalized expression levels are shown for core filamentation genes in the strains indicated. Transcription activator mutant strains in a CAK1 background as well as the cak1 DX mutant background are shown. Symbols: * = p < 0.05; ** = p < 0.01 for comparison of CAK1 and cak1 DX strains carrying the same activator mutation. Strains are listed in S4 Table. B. Cell morphology. Cell cultures were grown for 4 hr at 37°C in YPD, then fixed and stained with Calcofluor White prior to visualization. Strains: WT (DAY185), bcr1Δ/Δ (CW1627), brg1Δ/Δ (CW1639), efg1Δ/Δ (CW1651), tec1Δ/Δ (CW1646), ume6Δ/Δ (CW1633), cak1 DX (CW1428), cak1 DX bcr1Δ/Δ (CW1547), cak1 DX brg1Δ/Δ (CW1460), cak1 DX efg1Δ/Δ (CW1603), cak1 DX tec1Δ/Δ (CW1579), cak1 DX ume6Δ/Δ (CW1534). C. In vitro biofilms. Strains were grown under in vitro biofilm conditions for 48 hr, then visualized by confocal microscopy. Cross-sectional views are shown. D. Gene expression profiles. RNA was extracted from cells grown for 4 hr at 37°C in YPD and used for nanoString expression analysis (S4 and S5 Tables). Hierarchal clustering of gene expression data was performed from the average of either three isolates (single and double mutants) or four isolates (triple mutants) using MeV software. Fold change values were obtained by dividing normalized expression values for each strain genotype by the wild type strain (DAY185) for each of the probes. The color scale represents Log2 fold change compared to wild type. (Blue limit: 10-fold down; yellow limit: 10-fold up) See S4 and S5 Tables for strains.
Fig 6.
Phenotype and core filamentation gene expression of cak1 DX brg1Δ/Δ ume6Δ/Δ mutant strain.
A. In vitro biofilms. Strains were grown under in vitro biofilm conditions for 48 hr, then visualized by confocal microscopy. Cross-sectional views are shown. Strains: CAK1 (DAY185), cak1 DX brg1Δ/Δ ume6Δ/Δ (CW1715), cak1 DX CAK1 brg1Δ/Δ ume6Δ/Δ (CW1720). B. Cell morphology. Cell cultures were grown for 4 hr at 37°C in YPD, then fixed and stained with Calcofluor White prior to visualization. C. Cell morphology. Cell cultures were grown for 4 hr at 30°C in YPD, then fixed and stained with Calcofluor White prior to visualization. D. RNA levels for environmental response genes were determined by nanoString for strains grown for 4 hr at 37°C in YPD (S5 Table). Normalized expression levels are shown for core filamentation genes in the strains indicated. Symbols: * = p < 0.05; ** = p < 0.01 for comparison of cak1 DX brg1Δ/Δ ume6Δ/Δ to cak1 DX CAK1 brg1Δ/Δ ume6Δ/Δ strains.