Fig 1.
The A. actinomycetemcomitans iron-restricted regulon.
Cellular processes differentially expressed by iron restriction. Shaded numbers above each process indicate fold change. 1.5–2.0 fold, light shade; >2.0–4.0 fold, medium shade; >4.0 fold, dark shade. Octagon, ferritin; Q, quinone; R, respiratory reductase; TMAO, trimethylamine N-oxide; TMA, trimethylamine; PFL, pyruvate formate lyase; FHL, formate hydrogen lyase; Afu and Afe, characterized transporters; Hg, hemoglobin; Tf, transferrin; Cys, cysteine; G3P, glycerol-3-phosphate; hairpin, sRNA; TRX, thioredoxin; DspB, Dispersin B.
Fig 2.
The A. actinomycetemcomitans Fur regulon.
The metabolic network differentially expressed in the Δfur mutant. Legend: green, pathways downregulated in the Δfur mutant compared to the wild type (Fur-activated); red, pathways upregulated in the Δfur mutant compared to the wild type (Fur-repressed); blue, pathways mediated by both Fur-activated and -repressed genes. Dots and lines represent compounds and reactions, respectively. PEP, phosphoenolpyruvate; ADH, alcohol dehydrogenase; Ac-CoA, acetyl-CoA; TCA, tricarboxylic acid.
Fig 3.
Iron and Fur regulate Dispersin B.
(A) Structure of the dspB promoter. Gray, OxyR box; orange, Fur box; underlined, -35 and -10 regions; +1, transcriptional start site; bold, start codon. (B) dspB transcription in colony biofilms was measured using a dspB promoter-lacZ transcriptional fusion. Left panel: blue, A. actinomycetemcomitans strain 624; red, A. actinomycetemcomitans strain VT1169. Right panel: blue, A. actinomycetemcomitans strain 624 wild type (wt); red, A. actinomycetemcomitans strain 624 Δfur (Δfur). Chelator is 250 μM 2,2’-dipyridyl, and Fe is 250 μM FeSO4. Y axis is fold change (FC) in dspB expression relative to no chelator (-chelator) and no FeSO4 (-Fe) addition. Error bars represent standard deviation (n = 3). (C) dspB mRNA levels in colony biofilms was measured using reverse transcriptase PCR in iron-replete (+Fe) and iron-restricted (-Fe) conditions. clpX serves as a control that is not regulated by iron or Fur. Wild type (wt), Δfur (Δfur), Δfur + fur-vsv-g (Δfur genetically complemented with VSV-G tagged Fur). (D) Biofilm dispersal assay. A second, higher ring biofilm (indicated by arrow) indicates dispersal. The purple stain is crystal violet. Chelator is 250 μM 2,2’-dipyridyl; -oxygen is anaerobic growth; +oxygen is aerobic growth.
Fig 4.
The A. actinomycetemcomitans direct Fur regulon.
(A) Sequence logo representation of the Fur binding motif generated from all Fur-bound promoters (False Discovery Rate < 0.1) as outlined in Materials and Methods. The height of each base represents its frequency of occurrence. (B) Cellular processes directly regulated by Fur. Each process is encoded by a gene(s) whose promoter is bound by Fur. Shaded numbers above each process indicate fold enrichment of the ChIP to input DNA signal. 1.5–2.0 fold, light shade; >2.0–4.0 fold, medium shade; >4.0 fold, dark shade. Colored boxes below each process indicate transcriptional regulation by iron (blue or orange), Fur (purple or green), or neither (gray). Act. Is activated, and rep. is repressed. Afu, characterized transporter; 3+ in circle, free ferric iron; 3+ in square, ferric iron siderophore; Hg, hemoglobin; Tf, transferrin; octagon, ferritin; DspB, Dispersin B; hairpin, sRNA; hairpin in gray oval, CRISPR inhibiting a phage at the cell surface; G3P, glycerol-3-phosphate; cAMP, cyclic AMP; Trp, tryptophan; Phe, phenylalanine; Tyr, tyrosine.
Fig 5.
A. actinomycetemcomitans is not iron-restricted in murine abscess mono-infection.
(A) Principal component analysis of the 93 genes regulated by iron. Each dot is a single replicate. Legend: Fe+, biofilm on rich media; Fe-, biofilm on iron-chelated media; abscess, wild-type abscess infection. Axes: Percentages are the amount of variation captured by each principal component. (B) Correlation analysis of the 93 genes regulated by iron. Spearman’s rank correlation was determined by comparing gene expression in wild-type A. actinomycetemcomitans abscess infection to Fe+ and Fe- in vitro biofilms. Error bars represent standard deviation (n = 6 pairwise comparisons). Significance was determined using a 2-tailed t test. (C) Survival of the wild type (wt) and Δfur mutant in abscesses. Each dot is a single abscess (n = 2 biological replicates). Significance was determined using a Mann-Whitney U test. Y axis represents colony forming units (CFU) per abscess after 3 days post-infection. (D) Venn diagram showing the overlap between the in vitro and in vivo ChIP-seq results.
Fig 6.
A. actinomycetemcomitans is iron-restricted in murine abscess co-infection.
(A) Principal component analysis of the 93 genes regulated by iron. Each dot is a single replicate. Legend: Fe+, biofilm on rich media; Fe-, biofilm on iron-chelated media; mono, abscess mono-infection; co, abscess co-infection with S. gordonii. Axes: Percentages are the amount of variation captured by each principal component. (B) Correlation analysis of the 93 genes regulated by iron. Spearman’s rank correlation was determined by comparing Fe+ and Fe- in vitro biofilms to A. actinomycetemcomitans gene expression in mono-infection (mono vs. Fe+ and Fe-) or co-infection with S. gordonii (co vs. Fe+ and Fe-). Error bars represent standard deviation (n = 4–6 pairwise comparisons). Significance was determined using a 2-tailed t test.
Fig 7.
A. actinomycetemcomitans is iron-restricted in human periodontitis.
(A) Principal component analysis of the 93 genes regulated by iron. Legend: Fe+, biofilm on rich media; Fe-, biofilm on iron-chelated media; health, A. actinomycetemcomitans from healthy human gingival crevice; disease, A. actinomycetemcomitans from diseased human gingival crevice. Axes: Percentages are the amount of variation captured by each principal component. (B) Correlation analysis of the 93 genes regulated by iron. Spearman’s rank correlation was determined by comparing Fe+ and Fe- in vitro biofilms to A. actinomycetemcomitans gene expression in healthy human gingival crevice samples (health vs. Fe+ and Fe-) or diseased human gingival crevice samples (disease vs. Fe+ and Fe-). Error bars represent standard deviation (n = 6 pairwise comparisons). Significance was determined using a 2-tailed t test.