Screening and characterization of in vitro biofilm-defective transcription regulator mutants. Biofilm biomass (dry weight) determinations of the entire transcription regulator (TR) mutant library (165 strains) are shown in panel A. The average total biomass +/− standard deviation for each TR mutant strain grown under standard biofilm conditions (Experimental Procedures) was calculated from five independent samples of each strain. Statistical significance (P values) was calculated with a Student’s one-tailed paired t test, and is represented by the red asterisk under the nine regulator strains (TF022, TF091, TF095, TF103, TF110, TF115, TF117, TF137, and TF156) with biomasses significantly deviating (P<0.0005) from the reference strain (SN250). Phenotypic characterization of the mutants is shown in panels B–O. Panels B–H show the visual appearance after 48 h of growth on polystyrene plates. Panels I–O are CSLM side view images of the wild-type and six biofilm-defective mutant strains. Scale bars represent 20 μm. See also Dataset S1, Figure S1 and Figure S2.

Biofilm formation in two in vivo rat models – a catheter model and a denture model. For the catheter model, the wild-type reference strain SN250 (panel A), and the six transcription regulator mutant strains (panels B–G) were inoculated into rat intravenous catheters; resulting biofilms were visualized after 24 h of growth by scanning electron microscopy (SEM). SEM catheter Images show the catheter luminal surfaces at magnifications of 1000X (see also Figure S4A). For the denture model, the wild-type reference strain SN425 (panel H), and the six transcription regulator mutant strains (panels I–N) were inoculated into rat dentures, and the resulting biofilms were visualized after 24 h of growth by SEM. SEM denture images show the denture surfaces at magnifications of 2000X. See also Figure S4B for SEM images of denture surfaces containing extensive bacterial biofilms, which formed in the presence of the mutant C. albicans strains.

The biofilm regulatory network. The six master biofilm regulators are represented by the six large circular hubs. Smaller circles represent target genes, which are connected to their respective regulators by dashed lines, indicating a direct interaction as determined by genome-wide ChIP-chip. Genes that are differentially regulated as determined by expression data (using a 2-fold cutoff) in biofilm compared to planktonic cells are shown in blue for those genes upregulated in biofilms, in yellow for those downregulated, and in grey for those with no change. Grey boxes are drawn around the 23 target genes bound by all six regulators, and are connected to their respective regulators by red dashed lines (panel A). The identity of these 23 genes are indicated as the colored ovals in panel B (blue ovals are genes that are upregulated, and yellow ovals are genes that are downregulated in biofilm compared to planktonic cells). Overall, 23 genes are bound by all six, 77 are bound by five or more, 165 are bound by four or more, 265 are bound by three or more, and 458 are bound by two or more of the biofilm regulators. See also Dataset S4.

Chromatin immunoprecipitation (ChIP) mapping and motif identification of the six master biofilm regulators. All six regulators bind to each another’s upstream promoter regions (panels A–F). Immunoprecipitation (IP) binding data for Bcr1-Myc (orange line), Tec1-custom antibody (light blue line), Efg1-Myc (magenta line), Ndt80-Myc (dark blue line), Rob1-Myc (red line), Brg1-Myc (green line), untagged wild-type/control IP (grey line), and tec1Δ/Δ (yellow line) strains are shown. The ChIP-chip microarray binding data was mapped and plotted onto the chromosomes containing BCR1 (panel A), TEC1 (panel B), EFG1 (panel C), NDT80 (panel D), ROB1 (panel E), and BRG1 (panel F) using MochiView. The promoters of these genes show significant peak enrichments for the binding of the indicated biofilm regulators. The X-axis represents ORF chromosomal locations. The Y-axis gives the Agilent normalized enrichment value (log2) post-smoothing for the binding of each regulator. Genes (pink boxes) plotted above the bold line read in the sense direction; genes plotted below the bold line read in the antisense direction. Using de novo motif-finding based on our ChIP-chip data, we identified significantly enriched core binding motifs for all six of our biofilm regulators (panel G). Motifs were identified using MochiView, independently verified using MEME, and motif graphics were generated with MochiView. See Datasets S5 and S2 for details on motif analysis and bound motif locations, respectively, for each regulator. Colored stars corresponding to the colors of the regulators indicate the location of strong instances of the indicated biofilm regulator motifs under the enrichment peaks in panels A–F. Panel H shows the evolutionary age of target genes in the biofilm network. Genes were divided into three categories based on when they arose during evolution, with the numbers in each bar giving the number of C. albicans genes that fall into that age category. The enrichment of each age category in biofilm-regulated genes is log₁₀ of the observed divided by the expected (for all age categories, P<1.23E-9). Panel I shows a histogram of the length of the intergenic regions between tandem and divergent gene pairs targeted by the biofilm regulators. Each category was normalized to the total number of intergenic regions in that category. See also Dataset S7.

Core candidate biofilm target genes. Using hierarchical cluster analysis of our gene expression microarray data, we identified a set of nineteen candidate target genes (IHD1, PGA54, FAV2, ORF19.3337, ALS1, TPO4, ORF19.4000, EHT1, HYR1, HWP1, CAN2/ORF19.111, IDP2, MDH1, PCK1, PGK1, AOX2, ORF19.4653, ORF19.4080, and ORF19.2220) that were differentially regulated (log₂ > 0.58, and log₂ < −0.58) in all gene expression array experiments that compared each biofilm regulator mutant to a reference strain under biofilm conditions (panel A). Eight of these targets were differentially regulated in the same direction (all down in the mutants), and were chosen for further functional analyses (panel A, as indicated by the blue square). ChIP-chip enrichment data for the binding of the six biofilm regulators in the promoters of these eight candidate target genes (panels B–I). IP binding data for Bcr1-Myc (orange line), Tec1-custom antibody (light blue line), Efg1-Myc (magenta line), Ndt80-Myc (dark blue line), Rob1-Myc (red line), Brg1-Myc (green line), untagged wild-type/control IP (grey line), and tec1Δ/Δ (yellow line) strains are shown. The ChIP-chip microarray binding data was mapped and plotted onto the chromosomes containing ORF19.3337 (panel B), ALS1 (panel C), TPO4 (panel D), ORF19.4000 (panel E), EHT1 (panel F), HYR1 (panel G), HWP1 (panel H), and CAN2 (panel I) using MochiView. The promoters of these genes show significant peak enrichments for the binding of the indicated biofilm regulators: ORF19.3337 by Bcr1, Efg1, Ndt80, and Rob1 (panel B); ALS1 by Bcr1, Tec1, Efg1, Ndt80, and Brg1 (panel C); TPO4 by Tec1, and Ndt80 (panel D); ORF19.4000 by Bcr1, Tec1, Efg1, Ndt80, and Brg1 (panel E); EHT1 by Ndt80 (panel F); HYR1 by Efg1 (panel G); HWP1 by Ndt80 (panel H); and CAN2 by Efg1 (panel I). The X-axis represents ORF chromosomal locations. The Y-axis is the Agilent normalized enrichment value (log2) post-smoothing for the binding of each regulator. Genes (pink boxes) plotted above the bold line read in the sense direction; genes plotted below the bold line read in the antisense direction. Colored stars corresponding to the colors of the regulators indicate the location of strong instances of the indicated biofilm regulator motifs under the enrichment peaks.

Functionally relevant biofilm target genes. Biofilm biomass (dry weight) determinations were measured for the eight core candidate biofilm target gene deletion mutants (panel A), and the strains in which each of the eight target genes was ectopically expressed in the background of each regulator mutant (panel B). The average total biomass +/− standard deviation for each strain grown under standard biofilm conditions was calculated from five independent samples of each strain. Statistical significance (P values) was calculated with a Student’s one-tailed paired t test, and is represented by the red asterisks above the strains with biomasses significantly deviating (P<0.0005) from either the reference strain (WT) for panel A or the corresponding mutant strain for panel B.

Regulatory network model for biofilm formation. Panel A shows the biofilm network model based on our ChIP-chip and expression data. Solid arrows indicate direct binding interactions determined by our ChIP-chip analysis. Solid black arrows indicate experimentally validated regulatory interactions (as determined by expression profiling data and validated by qPCR) in addition to direct binding interactions (as determined by ChIP-chip data), and solid grey arrows indicate direct binding interactions only. The dashed black arrow indicates an indirect regulatory interaction only.