A unique histone H3 variant protein is encoded only by the CTG-clade species

Using the S. cerevisiae histone H3 (YNL031C) amino acid sequence as the query in a basic local alignment search tool (BLAST) search, a variant of the core histone H3 protein, exclusively present in a group of ascomycetes belonging to the CTG clade (Fig 1A) was identified. The sequence of the newly identified CTG-clade-specific histone H3 variant is different from the core canonical histone H3, as well as the variant histone H3 present in Basidiomycota and Zoopagomycota fungal phyla. In C. albicans, HHT2 (ORF 19.1853) and HHT21 (ORF 19.1061) code for an identical polypeptide, the canonical histone H3, whereas HHT1 (ORF 19.6791) encodes a variant protein differing at positions 31, 32, and 80 in the amino acid sequence when compared with HHT2/HHT21 (Fig 1B and S1A Fig). These changes are found to be conserved in the variant histone H3 of most CTG-clade species (S1B Fig). Moreover, variations in the nucleotide sequences of HHT1, HHT2, and HHT21 were inspected using genome sequence data available for 182 C. albicans isolates [35]. Only 2 synonymous SNPs were identified in HHT1, indicating that the amino acid sequence of Hht1 protein is invariant across the 182 tested C. albicans strains. Four synonymous and 1 nonsynonymous SNPs were found in HHT2, whereas 8 synonymous and 2 nonsynonymous SNPs were identified in HHT21 (S1 Table). Notably, these variations do not occur at the 3 positions where Hht1 differs from Hht2 or Hht21 in the primary amino acid sequence. The presence of serine (S) and threonine (T) at position 31 and 80, respectively, in the canonical histone H3 (HHT2 and HHT21) is similar to that of the mammalian histone H3.3. In addition, these 2 amino acid positions (31st and 80th) of the histone H3 polypeptide are known to tolerate variations as evident in other organisms [36]. Therefore, we propose that HHT1 is indeed encoding a variant histone H3, possibly independently evolved in the CTG clade, and we named it as H3V^CTG.

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Fig 1. H3V^CTG is a CTG-clade specific histone H3 variant.

(A) Phylogeny of fungi in the phylum Ascomycota analyzed in this study. The canonical histone H3 genes (gray box) along with the presence (black box) or absence (empty box) of the variant histone H3 of the corresponding species are shown. This tree is illustrative as the branches are not drawn to scale. The species included in this phylogeny are Aspergillus fumigatus, A. flavus, Ashbya gossypii, Alternaria alternate, Botrytis cinerea, Blastomyces dermatitidis, Bipolaris oryzae, Candida lusitaniae, C. guilliermondii, C. tenuis, C. parapsilosis, C. albicans, C. dubliniensis, C. tropicalis, C. auris, C. glabrata, Coccidioides immitis, Cordyceps militaris, Debaryomyces hansenii, Fusarium graminearum, Histoplasma capsulatum, Kluyveromyces waltii, K. lactis, Lodderomyces elongisporus, Magnaporthe oryzae, Neurospora crassa, Naumovozyma castellii, Paracoccidioides brasiliensis, Pichia stipitis, Pyronema omphalodes, Spathaspora passalidarum, Suhomyces tanzawaensis, Saccharomyces kudriavzevii, S. makatea, S. cerevisiae, S. paradoxus, Schizosaccharomyces pombe, S. japonicus, Sclerotinia sclerotiorum, Torrubiella hemipterigena, Tuber melanosporum, Uncinocarpus reesii, and Yarrowia lipolytica. (B) Amino acid sequence alignment of histone H3 proteins coded by HHT2, HHT21, and HHT1 in C. albicans. Changes in the amino acid sequence among these histone H3 variants are marked by an asterisk. Identical amino acids are shaded, and numbers above the sequence denote amino acid locations on the primary protein sequence. Secondary structures such as α helices and loops of the corresponding sequence are indicated above the alignment. (C) Sketch showing locations of histone H3 genes and histone gene clusters on various chromosomes of C. albicans. (D) Expression levels of C. albicans histone H3 proteins encoded by HHT21-V5 (H3-V5) and HHT1-V5 (H3V^CTG-V5) were monitored by western blot analysis in the planktonic (yeast and hyphae) and biofilm growth conditions. Histone H3 molecular weight is approximately 17 kDa. The parental strain SN148 was used as the untagged control, whereas PSTAIRE (approximately 34 kDa) was used as the loading control. (E) Wild-type SC5314 (HHT1/HHT1), variant histone H3 null mutant LR108 (hht1/hht1), canonical histone H3 mutants LR142 (hht21/hht21) and LR152 (hht2/hht2), and canonical histone H3 null strain LR155 (hht21/hht21 hht2/hht2) were streaked on YPD plates and grown at 30 °C for 3 days. Chr, chromosome; YPD, yeast peptone dextrose.

https://doi.org/10.1371/journal.pbio.3000422.g001

Variant histone H3 is less abundant than the canonical histone H3 in major morphological forms of C. albicans

The canonical histone H3 genes HHT2 and HHT21 in C. albicans are divergently transcribed from the histone H4 genes, HHF22 (ORF 19.1059) and HHF1 (ORF 19.1854), respectively, whereas the variant histone H3 gene, HHT1, is located outside the canonical histone gene clusters (Fig 1C). Based on genomic locations and sequence features, we considered the polypeptide coded by HHT21 or HHT2 as the canonical histone H3 and the one coded by HHT1 as the variant histone H3 (H3V^CTG) found exclusively in the CTG-clade species. Reverse transcription PCR (RT-PCR) confirmed that both the canonical histone H3 and the variant histone H3 genes are transcribed (S2A and S2B Fig). Further, epitope tagging of these genes confirmed that the corresponding proteins are translated in planktonic (yeast and hypha) as well as biofilm grown cells of C. albicans (Fig 1D). Although no significant difference in relative abundance of the 2 canonical histone H3 proteins was observed in C. albicans (S2C Fig), variant histone H3V^CTG was expressed in lower abundance than the canonical histone H3 proteins in both planktonic and biofilm mode of growth in C. albicans (Fig 1D). Finally, indirect immunolocalization assays confirmed nuclear localization of both forms of histone H3 proteins, canonical H3 (LR143), or H3V^CTG (LR144) (S2D Fig). Although the canonical histone H3 localization was found to be uniform across the entire nucleus, a distinct scattered pattern of nuclear localization of variant histone H3 was observed.

Variant histone H3 assembles into nucleosomes and can support C. albicans growth in the absence of canonical histone H3

To test the essentiality of H3V^CTG for viability of C. albicans, both alleles of HHT1 were replaced with a recyclable knock-out SAT1-flipper cassette [37] in the wild-type C. albicans strain SC5314 (S3A and S3B Fig). H3V^CTG was found to be dispensable for survival of C. albicans in rich media because the homozygous null mutants lacking H3V^CTG (LR107 and LR108) and the parent wild-type strain grew at a similar rate (Fig 1E and S3C Fig). Similarly, each of the canonical histone H3 genes, HHT2 or HHT21, was deleted individually by the SAT1-flipper cassette. Null mutants of either HHT2 (LR152) or HHT21 (LR141) did not show any significant growth defects (Fig 1E). We also examined the expression levels of variant histone H3 (Hht1-V5) in the absence of HHT21 but did not observe any significant differences in the expression levels of Hht1 (S3D Fig).

To determine whether C. albicans could survive in the absence of canonical histone H3, all 4 alleles encoding the canonical histone H3 were knocked out by clustered regularly interspaced short palindromic repeats–CRISPR associated protein 9 (CRISPR-Cas9) [38, 39]. Null mutant cells (hht2/hht2 hht21/hht21) lacking the canonical histone H3 genes HHT2 and HHT21 (LR155) were viable but grew significantly slower compared with any of the single mutants of histone genes or wild type (Fig 1E). We also examined the cell morphology of canonical histone H3 null mutant. These cells showed an elongated cell morphology compared with the wild type, suggesting they are stressed [40, 41] (S3E Fig). These results suggest that variant histone H3 (HHT1) can partially fulfill the functions of canonical histone H3 and is thus probably assembled into nucleosomes to support growth of C. albicans in the absence of canonical histone H3 genes.

Variant histone H3 acts as a determinant of major growth transitions in C. albicans

To understand the biological relevance of H3V^CTG that is present only among the CTG-clade species of Ascomycota, a genome-wide transcriptome analysis of the H3V^CTG null mutant (hht1/hht1) was carried out. The gene expression profiles of 2 biological replicates of the H3V^CTG mutant strain (hht1/hht1), LR107 and LR108, as well as 2 different colonies of parental strain SC5314 (HHT1/HHT1) grown in planktonic conditions (yeast peptone dextrose supplemented with uridine [YPDU] broth at 30 °C) were analyzed. Approximately one-fifth of all C. albicans genes (1,048 genes) were found to have an altered expression (fold change > 1.5, p < 0.05) when H3V^CTG was absent (S1A Data). Out of these altered genes, 638 genes were up-regulated, whereas the remaining 410 genes were down-regulated in null mutants of H3V^CTG compared with the wild type (S1B and S1C Data). Functional categorization of up- and down-regulated genes suggested that the biofilm gene circuit was the most significantly altered pathway due to H3V^CTG deletion (Fig 2A and S4A Fig). Transcription and cell cycle were the other 2 significantly altered pathways in null mutants of H3V^CTG (p < 0.05; S1D Data). Transcription levels of a majority of biofilm-induced genes were found to be up-regulated, whereas biofilm-repressed genes were down-regulated when cells lacked H3V^CTG. Adhesins and GPI-anchored cell wall proteins, known to play a crucial role during the biofilm development, were also up-regulated in H3V^CTG null mutants. In addition, SAP5 and SAP6 genes, which are shown to be involved in biofilm formation [42], were up-regulated in the H3V^CTG mutant. Based on the transcription profiling analysis, we posit that H3V^CTG favors planktonic growth over biofilm growth in C. albicans.

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Fig 2. Loss of variant histone H3 activates the gene circuitry for biofilm development in planktonic cells.

(A) Global gene expression array analysis was performed in the wild-type and null mutants for H3V^CTG grown in YPDU (planktonic mode). Functional classification of genome-wide expression data indicates that the most significantly altered pathways in the H3V^CTG null mutants are biofilm, transcription, and cell cycle. (B) A similar pattern of gene expression was observed when the indicated strains were grown in Spider medium under conditions that favor biofilm growth. (C) Genome-wide expression data were compared for common genes that are altered both in planktonic and biofilm growth conditions (blue and red circles, respectively) in the H3V^CTG null mutants and represented as Venn diagrams. A total of 153 up-regulated and 11 down-regulated genes are common between these 2 data sets. YPDU, yeast peptone dextrose supplemented with uridine.

https://doi.org/10.1371/journal.pbio.3000422.g002

In the laboratory, C. albicans cells form biofilms under a variety of conditions on several substrates and media, including Spider medium [24, 27]. Altered expression of biofilm genes in the null mutant of H3V^CTG in the planktonic mode prompted us to perform a similar genome-wide transcriptome analysis after growing 2 independent colonies of wild-type (SC5314) and 2 biological replicates of H3V^CTG null mutant (LR107 and LR108) in Spider medium–induced biofilm conditions. In the biofilm-inducing conditions, 751 genes were found to be differentially expressed (fold change > 1.5, p < 0.05) between the wild-type and H3V^CTG null mutants (S1E Data). Among them, 651 genes were up-regulated, whereas the remaining 100 genes were down-regulated in the H3V^CTG null mutants (S1F and S1G Data) compared with the wild type (Fig 2B and S4B Fig). Again, the biofilm was the most significantly altered pathway (p < 0.05) between the null mutants of H3V^CTG and the wild type when the cells were grown in conditions that favor biofilm growth (Fig 2B and S1H Data).

As observed in the planktonic expression profile, several adhesins, GPI-linked cell wall proteins, and biofilm-induced genes were further up-regulated, whereas biofilm-repressed genes were further down-regulated in H3V^CTG null mutants compared with the wild type in the biofilm-induced condition. Among those with an altered expression, 164 genes were common to both planktonic and Spider biofilm transcriptome data sets (Fig 2C). Subsequently, we compared the altered gene-sets in the H3V^CTG mutants grown either in the planktonic (S4C and S4E Fig) or biofilm (S4D and S4F Fig) condition with genes known to be expressed differentially in planktonic and biofilm modes of growth [33] and represented in the form of a heat map and Venn diagram. These analyses revealed a significant overlap in the altered gene expression profile in the H3V^CTG mutants with the biofilm-specific genes.

Loss of variant histone H3 induces biofilm gene circuitry during planktonic growth in vitro

Analysis of the transcriptome data revealed that gene expression profiles previously linked to biofilm formation were enhanced in the H3V^CTG mutants compared with the wild type both in planktonic and biofilm conditions. Altered expression of a subset of critical biofilm-related genes (Fig 3A) was verified by quantitative PCR (qPCR) analysis (Fig 3B). Interestingly, YWP1, a biofilm-repressed gene, was down-regulated in H3V^CTG null mutants. Thus, the microarray data together with qPCR analysis established that H3V^CTG contributes to the maintenance of the planktonic growth by repressing the biofilm growth promoting gene circuitry when cells are grown in planktonic conditions. This finding led us to investigate the biofilm-forming ability of the null mutant of H3V^CTG compared with the wild type. To test this, wild-type SC5314 (HHT1/HHT1), H3V^CTG mutants LR107 and LR108 (hht1/hht1), and the H3V^CTG complemented strain LR109 (hht1/hht1::HHT1) were allowed to form biofilm in 6-well polystyrene plates in the Spider medium at 37 °C (Fig 3C, upper panel). A significant enhancement in biofilm growth of H3V^CTG mutants compared with the wild-type and H3V^CTG complemented strains was observed (Fig 3D). Together the results obtained thus far suggest that the gene circuit for biofilm development is activated in the H3V^CTG mutants even when these cells are grown in planktonic conditions. To verify this possibility, we performed the biofilm assay at 30 °C in YPDU medium on silicone squares; biofilm formation was found to be enhanced in H3V^CTG null mutants compared with the wild-type or the H3V^CTG complemented strain (Fig 3C lower panel and 3E), confirming that deletion of H3V^CTG promotes biofilm formation in YPDU medium at 30 °C.

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Fig 3. Variant histone H3 null mutants form more robust biofilm than wild type.

(A) A heat map was generated for biofilm-related genes with altered levels of expression in the H3V^CTG null mutants compared with wild type. Yellow color represents up-regulation, whereas blue color represents down-regulation. Affected genes are arranged according to the step of biofilm development they are involved in. The arrowhead indicates the direction of maturation of biofilm. (B) qPCR analysis was performed for biofilm-related genes with wild-type and H3V^CTG null cells grown in YPDU under planktonic conditions. ΔCt values were derived after normalization of expression of biofilm genes with that of actin, whereas ΔΔCt values were calculated for relative expression of biofilm-related genes in the H3V^CTG null mutants compared with the wild type. The data underlying this figure can be found in S2 Data. (C) Biofilms were grown using the wild type (SC5314), 2 independent transformants of H3V^CTG null mutant (LR107 and LR108), and the complemented strain (LR109) in 6-well polystyrene plates in Spider medium at 37 °C. Biofilms were allowed to form for 24 hours at 37 °C (upper panel) or on silicone squares in YPDU medium for 24 hours at 30 °C (lower panel). The wells were washed to remove the nonadherent cells and photographed. (D) Biomass dry weights of the wild-type, H3V^CTG null mutants, and H3V^CTG complemented strains grown in Spider media at 37 °C are shown. The data underlying this figure can be found in S2 Data. (E) Biomass dry weights of the wild-type, H3V^CTG null mutant, and H3V^CTG complemented strains after growth in YPDU for 24 hours at 30 °C are shown. The data underlying this figure can be found in S2 Data. (F) Wild-type, H3V^CTG null mutant, and the H3V^CTG complemented strain were adhered to silicone squares in a 12-well polystyrene plate in YPD medium at 37 °C. Biofilms were allowed to form for 48 hours. Biofilms were stained with concanavalin A-Alexa 594 and imaged by CLSM. Images are projections of the top and side views. False color depth views were constructed, in which the blue color represents cells the closest to the silicone (bottom of the biofilm) and the red color represents cells the farthest from the silicone (top of the biofilm). Representative images of at least 3 replicates are shown. Scale bars: 50 μm. (G) The biofilm assay was performed in vivo using the rat catheter model. Strains were inoculated in the rat intravenous catheter and were allowed to form biofilms. After 24 hours of incubation, biofilms were visualized using SEM. The images are 80× and 800× magnification views of the catheter lumens. CLSM, confocal laser scanning microscopy; ns, not significant; qPCR, quantitative PCR; SEM, scanning electron microscopy; YPD, yeast peptone dextrose; YPDU, yeast peptone dextrose supplemented with uridine; ΔCt, difference in Ct values of a gene of interest and normalization control (in this case, actin); ΔΔCt, difference in Ct (threshold cycle) values of a gene of interest in a mutant strain compared to the wild type.

https://doi.org/10.1371/journal.pbio.3000422.g003

In order to determine the thickness of biofilms formed by the hht1 null mutant strain compared with wild type, confocal laser scanning microscopy (CLSM) was performed in vitro on silicone squares grown biofilms (see Materials and methods). Our CLSM results suggested that the hht1 null mutant strain formed a thicker biofilm compared with either the wild-type or the complemented strain (Fig 3F). Finally, to examine the ultrastructure of biofilms formed by the wild-type and H3V^CTG null mutant, in vitro biofilms were allowed to grow on human urinary catheters for 48 hours (see Materials and methods). The catheter luminal surfaces were visualized by scanning electron microscopy (SEM). No significant differences were observed in the H3V^CTG null mutant compared with the wild type (S5 Fig).

Absence of variant histone H3 enhances in vivo biofilm growth as well

Because of the presence of many uncharacterized host factors, in vivo biofilms may substantially differ from those formed in vitro. To examine whether absence of H3V^CTG enhanced biofilm formation in vivo, a well-established rat venous catheter model [43] was used. We inoculated catheters intraluminally with wild type (SC5314), H3V^CTG null mutants LR107 and LR108 (hht1/hht1), or H3V^CTG complemented strain LR109 (hht1/hht1::HHT1) of C. albicans. After 24 hours of biofilm growth, the catheters were removed, and catheter luminal surfaces were imaged by SEM (Fig 3G). Similar to in vitro results, significantly thicker biofilms were formed by the H3V^CTG null mutants (hht1/hht1) compared with the wild-type strain SC5314 or the H3V^CTG complemented strain LR109. Therefore, these in vivo as well as in vitro observations together support the conclusion that deletion of H3V^CTG results in the formation of more robust biofilms than those formed by wild-type C. albicans.

Variant histone H3 acts as a molecular switch that favors planktonic growth over biofilm growth

Enhancement of both in vitro and in vivo biofilm formation in the H3V^CTG mutant compared with the wild type motivated us to investigate the mechanism by which H3V^CTG negatively regulates biofilm growth in C. albicans. To understand this mechanism at the molecular level, the V5 epitope was used to tag either the variant histone H3 (H3V^CTG) or the canonical histone H3 (encoded by HHT21 as described above). Based on the complementation assay, the V5-tagged H3V^CTG (LR145 and LR146) was proved to be functional when expressed as the only copy in the cell (S6A Fig). Chromatin immunoprecipitation–quantitative PCR (ChIP-qPCR) analysis was performed either in the planktonic or biofilm growth conditions to determine the binding of both canonical and variant histone H3 to the promoters of a set of biofilm-induced and biofilm-repressed genes. The promoter of ORF19.874, which was unaltered in the genome-wide study above as well as in the previous report [33] during the planktonic to biofilm growth transition, was used to normalize the binding efficiency of histone H3 molecules in all ChIP-qPCR studies. The untagged parent strain (SN148) was used as a control to calculate the background enrichment. The promoters of biofilm-induced genes (BMT7, CAN1, ECE1, HWP1, HGT2, JEN2, and SAP5), biofilm-repressed genes (NRG1 and YWP1), and also of an uncharacterized gene (ORF19.7380), to which 5 of the 6 biofilm master regulators bind, were examined to study the occupancy of either canonical (Hht21-V5) or variant histone H3 (H3V^CTG-V5). The occupancy of H3V^CTG was significantly higher at the promoters of biofilm-related genes compared with that of the canonical histone H3 when the cells were grown in the planktonic mode of growth (Fig 4A and S1I Data). Therefore, we posit that a higher occupancy of the variant histone H3 at the promoters of biofilm genes may prevent access of gene expression modulators (transcription activators or repressors) required for induction of the biofilm gene circuitry under planktonic growth conditions. Further, the occupancy of the H3V^CTG was compared in both planktonic and biofilm growth conditions. A significant drop in the binding of H3V^CTG to the promoters of biofilm genes was observed during the transition from planktonic to biofilm growth, except at SAP5 and NRG1 promoters (Fig 4B). We also examined the occupancy of variant histone H3 to the gene body of biofilm-related genes in cells grown in planktonic and biofilm conditions. Similar to the promoter regions, we also observed a drop in the level of variant histone H3 in biofilm condition compared with the planktonic condition (S6B Fig). Further, we did not observe a similar trend of changes in the occupancy of canonical H3 to the promoters of biofilm genes between planktonic and biofilm conditions (Fig 4C). These analyses reveal that the binding of H3V^CTG to the promoter of each of the biofilm genes was more efficient in the planktonic than in the biofilm condition. Micrococcal nuclease (MNase) digestion was performed to determine the occupancy of these 2 histone H3 on the BMT7 gene. Both total DNA MNase digestion and ChIP results show that both histone H3 can occupy the same regions (S6C Fig). This also suggests the possibility of formation of heterotypic nucleosomes, which will be addressed in future studies. Moreover, we examined the total pool of histone H3 molecules bound to the promoters of biofilm genes in both planktonic and biofilm growth by ChIP assays using antihistone H3 antibodies. Our results show a drop in the binding of total histone H3 to the promoters of BMT7, CAN1, ECE1, HGT12, JEN1, SAP5, and NRG1 in biofilm conditions (Fig 4D). To assess the occupancy of one of the biofilm master regulators, namely, Bcr1, to the promoters of biofilm-related genes, we performed ChIP-qPCR analysis in the wild-type or the H3V^CTG null mutant expressing Bcr1-myc. Binding of Bcr1-myc was analyzed on promoters of a set of genes that showed an altered (up-regulation or down- regulation) expression in the H3V^CTG mutant by ChIP-qPCR (Fig 4E). The results of this analysis indeed confirmed that absence of H3V^CTG enhances the binding of the biofilm master regulator Bcr1 to the promoters of these genes in the planktonic conditions. These results were in accordance with the gene expression profile further strengthening the fact that H3V^CTG indeed plays a major role in repressing the biofilm promoting gene network during planktonic growth in C. albicans. Taken together with all the results presented so far, we propose a general model (Fig 4F) that suggests H3V^CTG is involved in modulating chromatin in a way that limits access of biofilm gene-specific transcription factors to the promoters of biofilm-related genes.

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Fig 4. Variant histone H3 limits access of transcription modulators to promoters of biofilm-related genes.

(A) ChIP assays with anti-V5 antibodies were performed in cells of LR143 (HHT21/HHT21-V5) and LR144 (HHT1/HHT1-V5) expressing a V5-tagged canonical histone H3 or variant histone H3 grown in planktonic conditions. IP DNA fractions were analyzed by qPCR with gene-specific promoter primer pairs (see S3 Table) for binding of either canonical histone H3 or H3V^CTG. Quantitative PCR (qPCR) was also performed with untagged strain to detect the background DNA elution in the ChIP assay. The enrichment of canonical histone H3 or H3V^CTG to the promoters of biofilm-related genes is represented as a normalized percent input IP with SEM. The values from 3 independent ChIP experiments were plotted. A two-way ANOVA test was performed to determine statistical significance. The data underlying this figure can be found in S2 Data. (B) Similarly, ChIP assays with anti-V5 antibodies were performed in LR144 cells grown as a biofilm. The enrichment of H3V^CTG to the promoters of biofilm genes was compared in both planktonic and biofilm conditions. The data underlying this figure can be found in S2 Data. (C) ChIP assays with anti-V5 antibodies were performed in LR143 cells grown in biofilm conditions. The enrichment of H3-V5 to the promoters of biofilm genes was compared in both planktonic and biofilm conditions. The data underlying this figure can be found in S2 Data. (D) ChIP assays with anti-H3 antibodies were performed in SN148 cells grown in planktonic and biofilm conditions. The enrichment of H3 to the promoters of biofilm-related genes was compared in both planktonic and biofilm conditions. The data underlying this figure can be found in S2 Data. (E) The extent of binding of a biofilm master regulator, namely, Bcr1-myc, was examined either in presence or in absence of H3V^CTG in the planktonic mode of growth. Bcr1-myc ChIP assays were performed with anti-myc antibodies in both the parental strain (CJN1785) and the H3V^CTG mutant (LR133) expressing Bcr1-myc. IP DNA fractions were analyzed by qPCR with promoter specific primer pairs for the binding of Bcr1-myc. The enrichment of Bcr1-myc was calculated and normalized with ORF19.874. A two-way ANOVA test was performed to determine statistical significance. The data underlying this figure can be found in S2 Data. (F) A proposed model to depict the role of the H3V^CTG in creating repressive chromatin for the biofilm genes. In this model, during planktonic growth, H3V^CTG is more efficiently bound to the promoters of biofilm genes compared with the canonical H3 (Hht21-V5), although they can form heterotypic nucleosomes. Possibly H3V^CTG restricts binding of biofilm regulators to the promoters of target genes in the planktonic state, resulting in a tight regulation of their expression when cells grow in planktonic conditions. However, in the biofilm state, when nucleosomes containing the variant histone H3 are reduced, or in the absence of H3V^CTG, an open chromatin structure is established that allows a more efficient access of biofilm regulators to the promoters of these genes. This causes either up-regulation of biofilm-induced genes (by transcription inducers) or down-regulation (by transcription repressors) of biofilm-repressed genes. Bcr1, biofilm and cell wall regulator 1; ChIP, chromatin immunoprecipitation; IP, immunoprecipitated; qPCR, quantitative PCR; RT-qPCR, real time quantitative PCR; SEM, standard error of the mean.

https://doi.org/10.1371/journal.pbio.3000422.g004

Loss of variant histone H3 rescues biofilm-forming defects of mutants lacking master biofilm regulators

Previously, it has been shown that the overexpression of ALS3 in the bcr1/bcr1 mutant rescues the defects in biofilm formation [44]. On the other hand, overexpression of Bcr1 target genes, such as ALS1, ECE1 or HWP1, partially restores biofilm formation of the bcr1/bcr1 mutant. Because several biofilm-related target genes, including ALS3, ECE1 and HWP1, were up-regulated in the H3V^CTG null mutants and H3V^CTG binds to the promoters of many biofilm-related genes possibly to inhibit biofilm growth and to promote planktonic growth, we next examined the extent of biofilm formation in the absence of both a biofilm master regulator and the possible biofilm repressor H3V^CTG. Deletion of each of the 6 master regulators, namely, BCR1, BRG1, EFG1, NDT80, ROB1, and TEC1, has been shown to severely hamper biofilm formation [33]. To test whether the deletion of H3V^CTG can rescue biofilm defects associated with the absence of biofilm master regulators, we deleted both copies of HHT1 (H3V^CTG) in each of the following mutant strains—bcr1/bcr1, brg1/brg1, efg1/efg1, ndt80/ndt80, rob1/rob1, and tec1/tec1—and examined the biofilm-forming ability of these double mutants by growing biofilms in YPD medium. Strikingly, biofilm growth was found to be significantly enhanced in the bcr1/bcr1 hht1/hht1 and tec1/tec1 hht1/hht1 double-mutant strains and also partially in ndt80/ndt80 hht1/hht1 compared with bcr1/bcr1, tec1/tec1, or ndt80/ndt80, respectively (Fig 5A). The remaining 3 double mutants (brg1/brg1 hht1/hht1, efg1/efg1 hht1/hht1, and rob1/rob1 hht1/hht1) did not show any significant change in biofilm formation compared with the mutants lacking only the master regulator (Fig 5A). Next, the enhancement of biofilm formation observed in these double-mutant strains was quantified by standard optical density measurement as well as dry biomass of biofilm formed (Fig 5B and 5C). These results strongly suggest that variant histone H3 is a major regulator of biofilm formation in C. albicans. We measured by CLSM the thickness of the biofilm formed by the tec1 hht1 double-mutant on silicone squares compared to the tec1 mutant alone (Fig 5D). Finally, we examined in vivo biofilm formation of tec1/tec1 as well as tec1/tec1 hht1/hht1 mutant using the rat catheter model (Fig 5E). The double-mutant strain tec1/tec1 hht1/hht1 formed enhanced biofilms compared with those formed by the tec1/tec1 single mutant in both in vitro and in vivo conditions. Our results thus confirm that in the absence of H3V^CTG, biofilm defects in the tec1/tec1 mutant can be rescued both in vitro and in vivo. We speculate that the genes that are regulated by Bcr1, Ndt80, and Tec1 are derepressed at the chromatin level in the absence of H3V^CTG. These results together confirm that H3V^CTG negatively regulates biofilm growth in C. albicans.

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Fig 5. Deletion of the variant histone H3 rescues defects in biofilm formation associated with mutants of 3 biofilm master regulators.

(A) In vitro biofilm formation assay was performed using single mutants of each of the 6 master regulators of biofilm formation, (Bcr1, Tec1, Ndt80, Brg1, Efg1, and Rob1) and the corresponding double-mutant strains in which H3V^CTG was deleted in each mutant background. Biofilms were grown in YPD medium in 24-well polystyrene plates for 24 hours at 37 °C. Biofilm formation defects were significantly rescued in bcr1, tec1, and ndt80 null mutants in the absence of H3V^CTG. (B) Biofilm formation was quantified using the Standard Optical Density Assay by measuring OD₆₀₀ of the cells adhered to the bottom of the plates. In vitro biofilm assay was performed in YPD using indicated single- and corresponding double-mutant strains. Biofilms were grown in 24-well plates for 24 hours at 37 °C. Data are the mean of 3 independent wells per condition. Error bars represent the standard deviation. The data underlying this figure can be found in S2 Data. (C) Biomass of the wild type and each single- and double-mutant strains grown in YPD in 24-well plates for 24 hours at 37 °C. The data underlying this figure can be found in S2 Data. (D) tec1 single mutant and tec1 hht1 double mutant were adhered to silicone squares in a 12-well polystyrene plate in YPD at 37 °C, and biofilms were allowed to form for 48 hours at 37 °C. Biofilms were stained with concanavalin A-Alexa Fluor 594 and imaged by CLSM. Images represent projections of the top and side views. Representative images of at least 3 replicates are shown. Scale bars: 50 μm. (E) Biofilm assay was performed in vivo by using a rat catheter model. CJN308 (tec1/tec1) or LR131 (tec1/tec1 hht1/hht1) were inoculated in the rat intravenous catheter and were allowed to form a biofilm. After 24 hours of incubation, biofilms were visualized using SEM. The images are 80× and 800× magnification views of the catheter lumens. Bcr1, biofilm and cell wall regulator 1; Brg1, biofilm regulator 1; CLSM, confocal laser scanning microscopy; Efg1, enhanced filamentous growth protein 1; Ndt80, non-dityrosine 80; OD, optical density; Rob1, regulator of biofilm 1; SEM, scanning electron microscopy; Tec1, transposon enhancement control 1; YPD, yeast peptone dextrose.

https://doi.org/10.1371/journal.pbio.3000422.g005

Co-occurrence of amino acid residues at position 31 and 32 is essential for the function of variant histone H3

We were also interested to know the contribution of each of the 3 amino acids residues that are different between variant and canonical histone H3. To understand this, we generated single-point mutant strains of Hht1 at each of the positions 31, 32, and 80 similar to that of the canonical histone H3 sequence (Fig 6A). In addition, we were curious to know whether the enhancement in biofilm formation was specifically associated with the loss of the variant histone H3 or if it could be due to the loss of overall histone H3 levels in the cell. We examined the extent of biofilm formation in the null mutant of HHT21 as well and did not observe any increase in biofilm formation. (Fig 6B and 6C). This confirms that the enhancement of biofilm growth is specifically associated with the loss of H3V^CTG. In addition, we did not observe any significant differences in biofilm formation between wild-type and point-mutant strains (Fig 6B and 6D). We also generated a double mutant at positions 31 and 32 of Hht1 to change the amino acid residues similar to those of the canonical histone H3 at the corresponding positions. The double mutant yielded a phenotype similar to that of the null mutants of variant histone H3 (Fig 6B and 6D). This suggests that co-occurrence of amino acid residues at positions 31 and 32 is essential for variant histone H3 function as a biofilm repressor during planktonic growth. This further confirms that the phenotype observed in null mutants of variant histone H3 is specifically associated with variant histone H3 and not due to the global levels of histone H3.

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Fig 6. Amino acid residues 31 and 32 are essential for the function of H3V^CTG.

(A) Schematics represent various point-mutant strains constructed by changing amino acid residues at positions 31, 32, and/or 80 of variant histone H3 to that of the canonical histone H3. (B) SC5314, H3V^CTG null mutant LR108, H3V^CTG complemented strain LR109, canonical histone H3 mutant LR142 (hht21/hht21), point-mutant strains at position 31 (RS103), 32 (RS105), 80 (RS107), and point-mutant strain at positions 31 and 32 (RS109) were allowed to form biofilms in YPDU for 48 hours at 37 °C; wells were washed to remove the nonadherent cells and photographed. (C) Biomass dry weights of the wild-type, H3V^CTG null mutant, H3V^CTG complemented, and hht21/hht21 strains grown in YPDU at 37 °C. The data underlying this figure can be found in S2 Data. (D) Biomass dry weights of the wild-type, H3V^CTG null mutant, H3V^CTG complemented, and point-mutant strains grown in YPDU at 37 °C. The data underlying this figure can be found in S2 Data. (E) SC5314, H3V^CTG null mutant LR108, H3V^CTG complemented strain LR109, canonical histone H3 mutant LR142 (hht21/hht21), point-mutant strain at position 31 (RS103), 32 (RS105), 80 (RS107), and point-mutant strain at position 31 and 32 (RS109) were spotted on synthetic dextrose medium complemented with essential amino acids (CM) agar plates, and CM+NAG (synthetic dextrose with 1 mM N-acetyl glucosamine) agar plates, and incubated for 3 days at 37 °C. (F) The extent of filamentation was monitored for the indicated strains by growing colonies from single cells on CM medium at 37 °C. (G) SC5314, H3V^CTG H3 null mutant LR107, H3V^CTG complemented strain LR109, point-mutant strains at position 31 (RS103), 32 (RS105), 80 (RS107), point-mutant strain at positions 31 and 32 (RS109), and canonical histone H3 mutant LR142 (hht21/hht21) were allowed to form filaments in liquid YPD with 10% FBS at 37 °C. The proportion of hyphal cells formed by these strains are plotted. The data underlying this figure can be found in S2 Data. CM, complete media; CM+NAG, complete media supplemented with N-acetyl glucosamine; FBS, fetal bovine serum; ns, not significant; YPDU, yeast peptone dextrose supplemented with uridine.

https://doi.org/10.1371/journal.pbio.3000422.g006

Variant histone H3 null mutant is hyperfilamentous

To test whether variant histone H3 has an effect on filamentation, we performed filamentation assays with wild-type SC5314 (HHT1/HHT1), H3V^CTG (hht1/hht1) mutants LR107 and LR108, and the H3V^CTG complemented strain LR109 (hht1/hht1::HHT1) on solid media. Colony wrinkling was enhanced in the mutant strains (hht1/hht1) compared with the wild-type (HHT1/HHT1) and the H3V^CTG complemented strain LR109 (hht1/hht1::HHT1) at both 30 °C and 37 °C (S7A Fig). This hyperfilamentation phenotype was specific to H3V^CTG mutants because null mutants of neither canonical histone H3 genes produced this phenotype (Fig 6E and S7B Fig). We also examined the extent of filamentation in the point-mutant strains. Our results confirm that the double mutant at positions 31 and 32 yields a phenotype similar to that of the null mutant of variant histone H3 (Fig 6E). The extent of filamentation was also found to be enhanced at the level of colonies derived from a single cell (Fig 6F and S7C Fig). Further, to test whether the increase in filamentation observed during growth on solid media can be found in liquid culture, hyphal growth was triggered in YPD containing fetal bovine serum at 37 °C. We did not observe any significant differences in the filamentation patterns between the wild type, the hht1/hht1 mutant strains, the H3V^CTG complemented strain LR109 (hht1/hht1::HHT1), and the hht21/hht21 null mutant or point-mutant strains (Fig 6G).

Finally, we performed a comparative transcriptome analysis of filamentation-specific genes between the wild type in filamentation inducing conditions [45] and H3V^CTG null mutant in planktonic and biofilm conditions. Our analysis does not show a significant overlap of genes with altered expression between hht1 null mutants and existing filamentation-specific expression profile (S7D Fig). Taken together, our results suggest that the enhancement in filamentation leading to a more robust biofilm formation in the H3V^CTG null mutants is only associated with the solid surface media.

Ethics statement

All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Wisconsin according to the guidelines of the Animal Welfare Act, the Institute of Laboratory Animal Resources Guide for the Care and Use of Laboratory Animals, and Public Health Service Policy under protocol MV1947. Ketamine and xyalazine were used for anesthesia. CO₂ asphyxiation was used for euthanasia at the end of study.

Media and growth conditions

C. albicans strains were grown in YPDU (1% yeast extract, 2% peptone, 2% dextrose, and 100 μg/ml uridine) at 30 °C unless stated otherwise. For biofilm formation, YPDU, Spider media (1% peptone, 1% yeast extract, 1% manitol, 0.5% NaCl, and 0.2% K₂HPO₄) at 37 °C [51] were used. The filamentation assays were performed with complete medium, Spider medium, and complete media containing 1 mM N-acetylglucosamine [52, 53]. Each strain was diluted to an OD₆₀₀ of 0.080, spotted on a plate, and incubated for 2 or 3 days at 30 °C and photographed [52]. Serum (10%) was used for induction of hyphae in YPDU media at 37 °C. C. albicans cells were transformed by the standard lithium acetate method as described previously by Sanyal and colleagues [54]. Nourseothricin was used at a concentration of 100 μg/ml.

Strain construction

All C. albicans strains and primer sequences used for the construction of strains in this study are listed in S2 and S3 Tables, respectively. Detailed information about the strain construction is available in the S1 Text.

RNA extraction and cDNA synthesis

RNA was isolated from C. albicans strains with the mirVana RNA isolation kit (Ambion, AM1560). Cells were grown in YPDU to an OD₆₀₀ = 0.5 or in biofilm condition from 48 hours grown biofilms. Approximately 4 × 10⁷ cells were taken for spheroplasting. Cells were pelleted down at 4,000 rpm, were washed with 1 ml of Y1 buffer (2.5 M sorbitol, 0.5 M EDTA [pH 8]) and finally were resuspended in 2 ml of Y1 buffer. Approximately 20 μl of lyticase and 2 μl of β-Mercaptoethanol were added and spheroplasting was done at 30 °C at 70 rpm. After 90% spheroplasting was achieved, spheroplasts were isolated by centrifugation at 1,800 rpm for 5 minutes, and RNA was isolated with the mirVana RNA kit. A total of 500 ng of purified RNA was used to make cDNA. The reaction mixture contained 4 μl of RNA, 4 μl of 5× RT buffer, 2 μl of 10 mM dNTPs, 2 μl of 0.1 M DTT, 2 μl of 10 pMol gene-specific reverse primer (H3-4), 1 μl of Superscript Reverse Transcriptase (Invitrogen, 18064–022) added in a final volume of 20 μl. Reactions were carried out at 37 °C for 60 minutes followed by heat inactivation at 85 °C for 5 minutes.

qPCR

The C. albicans wild-type SC5314 and H3V^CTG null mutant strains were grown in YPD medium till OD = 0.5. RNA was isolated as described before treated with DNase I (NEB, M03032), and the quality of RNA was examined on agarose gel. To ensure the absence of genomic DNA, a control PCR reaction was performed before the reverse transcription step. cDNA was synthesized by reverse transcription using M-MuLV reverse transcriptase (Fermentas, EP0732) and Oligo(dT) primers (Sigma, O-4387). Primers designed for RT-PCR reactions were between 100 and 110 bp long (listed on S3 Table). Analysis of melting curves was also performed to ensure specific amplification without any secondary nonspecific amplicons (melting curve temperatures, used are 78°C (ACT1), 76 °C (ALD6), 79 °C (ECE1), 80.5 °C (HWP1), 78 °C (GCA1), and 79.5 °C (YWP1)). PCR was carried out in a final volume of 20 μl using iQSYBR Green supermix (BIO-RAD, 170-8880AP). The RT-PCR analysis was carried out in i-Cycler (BIO-RAD) using the following reaction conditions: 95 °C for 2 minutes, then 40 cycles of 95 °C for 30 seconds, 55° C for 30 seconds, 72 °C for 30 seconds. Fold difference in expression of mRNA was calculated by the ΔΔCt method (RT-PCR applications guide BIO-RAD) [55]. Actin was used as normalization control.

Cell lysate and western blot analysis

Western blot analysis was performed as described before by Chatterjee and colleagues [56]. C. albicans strains were grown in YPDU till OD₆₀₀ = 1. For hyphal induction, cells were grown in the presence of 10% serum, and to obtain biofilms, C. albicans was grown in 6-well polystyrene plates in YPDU. Cells were harvested, washed with lysis buffer (0.2 M Tris, 1 mM EDTA, 0.39 M (NH₄)₂SO₄, 4.9 mM MgSO₄, 20% glycerol, 0.95% acetic acid [pH 7.8]), resuspended in 0.5 ml of the same buffer, and disrupted using acid-washed glass beads (Sigma, G8772) by vortexing 5 minutes (1 minute vortexing followed by 1 minute cooling on ice) at 4 °C. C. albicans cell lysates were electrophoresed on a 12% SDS-PAGE gel and blotted onto a nitrocellulose membrane in a semidry apparatus (BIO-RAD). The blotted membranes were blocked with 5% skim milk containing 1× phosphate buffered saline (PBS; pH 7.4) for 1 hour at room temperature and then incubated with the following dilutions of primary antibodies: anti-V5 antibodies at a dilution of 1:5,000 (Invitrogen, R96025), anti-PSTAIRE antibodies (Abcam, ab10345) at a dilution 1:3,000. Anti-PSTAIRE antibodies recognize Cdc28 and are widely used as a protein loading control in C. albicans. Next, membranes were washed 3 times with PBST (0.1% Tween-20 in 1× PBS) solution. Anti-rabbit HRP conjugated antibodies (Bangalore Genei, 105499) and anti-mouse IgG-HRP antibodies (Bangalore Genei, 105502) were added at a dilution of 1: 1,000 and incubated for 1 hour at room temperature followed by 3 to 4 washes with PBST solution. Signals were detected using the chemiluminescence method (Super Signal West Pico Chemiluminescent substrate, Thermo scientific, 34080).

Indirect immunofluorescence

Indirect immunofluorescence was performed as described before by Sanyal and Carbon and Chatterjee and colleagues [54, 56]. Asynchronously grown C. albicans cells were fixed with a one-tenth volume of formaldehyde (37%) for 15 minutes at room temperature. Antibodies used were diluted as follows: 1:100 for mouse anti-V5 antibodies (Invitrogen, R96025). The dilutions for secondary antibodies used were Alexa Fluor 488 goat anti-mouse IgG (Invitrogen, A-11001) diluted 1:1,000. DAPI (4, 6-Diamino-2-phenylindole) (Sigma, D9542) was used to stain the nuclei of the cells. Cells were examined under 100× magnifications using a GE Delta vision microscope. The digital images were processed with Adobe Photoshop.

Gene expression design and data analysis

The concentration of the extracted RNA was evaluated using Bioanalyzer (Agilent), whereas purity of the extracted RNA was determined by the standard procedure for the same by measuring A₂₆₀ and A₂₈₀ using a Nanodrop Spectrophotometer (Thermo Scientific). The samples were labeled using Agilent Quick Amp labeling kit (Part number 5190–0442). A total of 500 ng RNA was reverse transcribed using Oligo(dT) primer tagged to the T7 promoter sequence. The cDNA obtained was converted to double stranded cDNA in the same reaction. Further, cDNA was converted to cRNA in the in vitro transcription step using T7 RNA polymerase, and Cy3 dye was added into the reaction mix. During cRNA synthesis, Cy3 dye was incorporated to the newly synthesized strands. cRNA obtained was cleaned up using Qiagen RNeasy columns (Qiagen, 74106). Concentration and the amount of dye incorporated in each sample were determined using a Nanodrop. Samples that passed the QC for the specific activity (minimum RNA concentration 500 ng/μl and absorbance 260/280 = 2) were taken for hybridization. A total of 600 ng of labeled cRNA was hybridized on the array (AMADID 29460) using the Gene Expression Hybridization kit (part number 5190–0404; Agilent) in Sure hybridization Chambers (Agilent) at 65 °C for 16 hours. Hybridized slides were washed using Agilent Gene Expression wash buffers (part no. 5188–5327). The hybridized, washed microarray slides were then scanned on a G 2600 D scanner (Agilent). Data extraction from images was done using Feature Extraction software version 10.7 of Agilent. Microarray data was preprocessed using Limma package (Smyth and colleagues, 2005) of statistical R language. Briefly, preprocessing includes (a) background correction (b) within array normalization and (c) fitting data to linear model. Finally, an empirical Bayes moderated statistics was applied to find significant changes in the expression levels. We used a cut-off of p < 0.05 and |fold change| > 1.5 for the differential expression of mutant over wild type to define differentially expressed genes. Differentially regulated genes were clustered using hierarchical clustering to identify significant gene expression patterns. All genes represented on the array are annotated using CGD database. The differentially regulated genes (up or down) were categorized under biofilm, cell cycle, transcription, and others based on the gene ontology annotations. Chi-square test was applied to know the significance of the categorized functions. Gene expression data have been deposited into the NCBI Gene Expression Omnibus (GEO) portal under the accession number GSE72824.

In vitro biofilm growth and biomass determination

In vitro biofilm growth in YPD medium at 30 °C and biomass determination

To examine the biofilm formation ability of H3V^CTG mutant strains in YPDU medium at 30 °C, in vitro biofilm growth assays were carried out by growing biofilms on either the silicone squares substrate as previously described by Nobile and Mitchell [49]. Biofilms were developed as described before, except in this case, YPDU was the medium of choice and the temperature was 30 °C. Finally, medium was removed, and the silicone squares gently washed with 1× PBS prior to being photographed. For dry biomass measurements, the medium was removed and 2 ml of 1× PBS was added to each well to remove unadhered cells. Biofilms were then scrapped, and the content of each well was transferred to preweighed nitrocellulose filter paper, dried, and weighed as described before.

CLSM for biofilm imaging

Biofilms were imaged as described before by Nobile and Mitchell [49]. Biofilms were grown on silicone squares for 48 hours, gently washed with 1× PBS, and stained with 50 μg ml⁻¹ of concanavalin A-Alexa Fluor 594 (Invitrogen, C-11253) for 1 hour at 110 rpm. CLSM was performed at PPMS facility of Institut Pasteur using an upright LSM700 microscope equipped with a Zeiss 40X/ 1.0 W plan-Apochromat immersion objective. Silicone squares were placed in a petri dish, and the biofilms were covered with 1× PBS. Images were acquired and assembled into maximum intensity Z-stack projection using ZEN software (Zeiss).

In vivo rat catheter biofilm model

To form biofilm in vivo, the rat central-venous catheter infection model [43] was used, as described previously Nobile and colleagues, Andes and colleagues, Nobile and colleagues, and Dalal and colleagues [33, 43, 44, 57]. For this specific pathogen free Sprague-Dawley rats weighing 400 g each were used. A heparinized (100 U/ml) polyethylene catheter with 0.76 mm inner and 1.52 mm outer diameters was inserted into the external jugular vein. The catheter was secured to the vein with the proximal end tunneled subcutaneously to the midscapular space and externalized through the skin. The catheters were inserted 24 hours prior to infection to permit a conditioning period for a deposition of host protein on the catheter surface. Infection was achieved by intraluminal instillation of 500 μl C. albicans cells (10⁶ cells/ml). After a 4 hour dwelling period, the catheter volume was withdrawn, and the catheter was flushed with heparinized 0.15 M NaCl. Catheters were removed after 24 hours of C. albicans infection to assay biofilm development on the intraluminal surface by SEM. Catheter segments were washed with 0.1 M phosphate buffer (pH 7.2) fixed in 1% glutaraldehyde/4% formaldehyde, washed again with phosphate buffer for 5 minutes, and placed in 1% osmium tetraoxide for 30 minutes. The samples were dehydrated in a series of 10 min ethanol washes (30%, 50%, 85%, 95%, and 100%) followed by critical point drying. Specimens were mounted on aluminium stubs, sputter coated with gold, and imaged using a Hitachi S-5700 or JEOL JSM-6100 SEM in the high-vacuum mode at 10kV. Images were processed using Adobe Photoshop software.

ChIP

The ChIP assays were performed as described previously by Chatterjee and colleagues and Mitra and colleagues [56, 58]. Briefly, each strain was grown until exponential phase (approximately 2 × 10⁷ cells/ml) or grown in biofilm condition for 24 hours, and cells were cross-linked with 1% final concentration of formaldehyde for 13 minutes. Chromatin was isolated and sonicated to yield an average fragment size of 300 to 500 bp. The DNA was immunoprecipitated with anti-V5 antibodies (final concentration 20 μg/ml) or anti H3 antibodies (Abcam, ab1791) or anti myc antibodies (Cal Biochem, OP10) and purified. The total, immunoprecipitated (IP) DNA, and beads only material were used to determine the binding of H3V^CTG to the promoters of biofilm genes by qPCR, as described before. The template used was as follows: 1 μl of 1:100 dilution for input and 1 μl of 1:2 dilution for IP for H3-V5 ChIP and undiluted for H3 and myc ChIP. The conditions used in qPCR were as follows: 94 °C for 2 minutes; the 40 cycles of 94 °C for 30 seconds, 58 °C for 30 seconds, 72 °C for 45 seconds. The results were analyzed using CFX Manager Software. The graph was plotted according to the percentage input method [59], and the formula for calculation is 100 × 2^{(adjusted Ct input − adjusted Ct of IP)}. Here, the adjusted value is the dilution factor (log2 of dilution factor) subtracted from the C_t value of diluted input/IP.