Article Figures & Data
Figures
- FIG 1
(A) Growth curves for Rothia mucilaginosa ATCC 25296 incubated under aerobic conditions in liquid M9 medium, supplemented with different carbon sources (x axis, hours; y axis, optical density [OD600]). (B) Colorimetric absorbance at 500 nm (y axis, A500) capturing catecholate derivatives in liquid R. mucilaginosa (Rmuc) and R. dentocariosa M567 (Rdent) growth cultures (x axis) using Arnow’s assay. R. mucilaginosa was grown in M9 medium supplemented with 100 mM glycerol (Glyc_Rmuc), 100 mM lactate (Lac_Rmuc), and 100 mM sucrose (Suc_Rmuc). R. dentocariosa was grown in 100 mM glycerol to see if glycerol induced siderophore production as seen for R. mucilaginosa cultures.
- FIG 2
Replicate mirror mass fragmentation patterns for enterobactin (m/z 670.152) produced by Rothia mucilaginosa ATCC 25296 (ion fragments pointing up) and enterobactin from the gold standard spectrum (ion fragments pointing down) in the Global Natural Products Social Molecular Networking (GNPS) library (36). Six major fragments (m/z 123.04, m/z 137.02, m/z 224.05, m/z 311.09 to 311.14, m/z 447.09 to 447.11, and m/z 534.14) of the query compound matched the gold standard in GNPS. (A) Purified extract derived from R. mucilaginosa ATCC 25296 growth medium (negative ionization mode). (B) Further enrichment of the siderophore using thin-layer chromatography (positive ionization mode). Both experiments confirmed the production of enterobactin.
- FIG 3
High-performance liquid chromatography (HPLC) purification traces of enterobactin from Rothia mucilaginosa ATCC 25296 crude growth extract, measured at 210 nm, 254 nm, and 280 nm using a solvent gradient from 30 to 65% buffer B. The peak at 19.141 min (red asterisk) was eluted and further analyzed by NMR. x axis, minutes; y axis, peak absorbance intensity.
- FIG 4
Rothia mucilaginosa ATCC 25296 (R. muc) inhibits pigment production in Staphylococcus aureus NR-10129 (MRSA), S. aureus TCH70 (MRSA), S. aureus TCH130, and enterotoxin H-producing S. aureus ATCC 51811, on M9 agar plates (100 mM glycerol) with 8 μg/ml catalase added. All S. aureus strains show yellow pigmentation when growing alone.
- FIG 5
Yellow pigmentation of S. aureus strains exposed to 100 μM enterobactin and 8 μg/ml catalase on M9 agar plates supplemented with 100 μM glucose. Yellow pigmentation was measured with the R package “countcolors” (51). All strains presented statistically significant reductions in pigmentation in the presence of 100 μM enterobactin purified from R. mucilaginosa ATCC 25296 and catalase (P < 0.05, two-tailed t test). Box plots from yellow pixel measurements were generated with the R Studio program (49) and ggplot2 (50).
- FIG 6
Growth curves of cariogenic and commensal Streptococcus species. Bacteria were grown aerobically at 37°C in liquid M9 medium supplemented with 100 mM glucose, 1 μM FeCl3, either with or without 100 μM enterobactin purified from R. mucilaginosa, and with or without 8 μg/ml catalase. Growth curves in black represent cultures amended with enterobactin. Statistically significant growth curves (P < 0.05) are shown with corresponding P values. Error bars reflect the standard error of the mean (calculated from triplicates). n.s., not significant. (A) Growth of cariogenic S. mutans strain B04Sm5 with enterobactin only (left panel) and with enterobactin and catalase (right panel). (B) Growth of cariogenic S. mutans strain UA159 with enterobactin only (left panel) and with enterobactin and catalase (right panel). (C) Growth of commensal S. sanguinis ATCC 49296 with enterobactin only (left panel) and with enterobactin and catalase (right panel). (D) Growth of S. gordonii ATCC 35101 with enterobactin only (left panel) and with enterobactin and catalase (right panel). (E) S. salivarius strain SHI-3 with enterobactin only (left panel) and with enterobactin and catalase (right panel). (F) Growth of S. oralis ATCC 35037 with enterobactin only (left panel) and with enterobactin and catalase (right panel). Graphs were generated and statistically validated using R Studio and the “statmod” and “ggplot2” packages (48–50).
- FIG 7
Reduced growth of Staphylococcus aureus incubated in liquid M9 growth medium (100 μM enterobactin, 8 μg/ml catalase, 100 mM glucose) at 37°C for 24 h. No enterobactin was added to the control samples. Growth curves in black represent cultures amended with enterobactin. Statistically significant growth curves (P < 0.05) are shown with corresponding P values. Error bars reflect the standard error of the mean (calculated from triplicates). (A) Growth of S. aureus strain 51811 with enterobactin only (left panel) and with enterobactin and catalase (right panel). (B) Growth of S. aureus strain TCH130/ST-72 with enterobactin only (left panel) and with enterobactin and catalase (right panel). (C) Growth of S. aureus TCH70/MRSA with enterobactin only (left panel) and with enterobactin and catalase (right panel). (D) Growth of S. aureus NR-10129/MRSA with enterobactin only (left panel) and with enterobactin and catalase (right panel). Graphs were generated and statistically validated using R Studio and the “statmod” and “ggplot2” packages (48–50).
Supplemental Material
FIG S1
(A and B) Overview of peptide sequence alignments showing the closest homologues to the biosynthetic gene clusters (BGCs) encoding enterobactin produced by Rothia mucilaginosa ATCC 25296 (A) and enterobactin produced by Escherichia coli K-12 (B). Alignments were obtained using the antiSMASH v. 5.0 program (bacterial version). Adenylation domains in both pathways were predicted to select 2,3-dihydroxybenzoic acid (dhb) and serine (ser) as the substrates, respectively. (C) Closeup view of peptide alignment of R. mucilaginosa ATCC 25296 BGC and its closest homologue pathways (i.e., mirubactin [14%]; perquinoline A, B, and C [15%]; and steffimycin D [5%]). (D) Closeup view of peptide alignment of E. coli strain K-12 BGC to its closest homologue pathways (i.e., turnerbactin [13%], enterobactin [12%], streptobactin [23%], etc.). The R. mucilaginosa BGC could not be aligned with the E. coli BGC due to nonexisting peptide sequence homology in any of the genes except the NRPS genes, which showed 41% homology (Fig. S4). Download FIG S1, TIF file, 2.9 MB.
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FIG S2
Catecholate siderophore encoding biosynthetic gene clusters identified by the antiSMASH software in genomes of Rothia mucilaginosa ATCC 25296 (I), R. dentocariosa M567 (II), and R. aeria F0184 (III). Predicted core biosynthetic genes, iron-transporting genes, and genes encoding protein with putative species-specific functions (A to F) are highlighted. Download FIG S2, TIF file, 2.3 MB.
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FIG S3
Results from peptide sequence alignment analysis of the NRPS gene in the Rothia mucilaginosa ATCC 25296 cat-sid BGC using the Phyre2 protein structure prediction tool showed 41% sequence homology to the EntE/EntB fusion protein harbored by the enterobactin BGC from Escherichia coli JM109. Download FIG S3, TIF file, 2.5 MB.
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FIG S4
(A) 1H NMR spectrum of enterobactin purified from R. mucilaginosa ATCC 25296. (B) 2D heteronuclear single quantum correlation (HSQC) NMR spectrum of enterobactin purified from R. mucilaginosa ATCC 25296. (C) 2D heteronuclear multiple bond coherence (HMBC) NMR spectrum of enterobactin purified from R. mucilaginosa ATCC 25296. (D) 2D proton correlation spectroscopy (H COSY) NMR spectrum of enterobactin purified from R. mucilaginosa ATCC 25296. Download FIG S4, TIF file, 2.8 MB.
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TABLE S1
Experimental 13C and 1H chemical shifts (ppm) of enterobactin produced by Rothia mucilaginosa ATCC 25296 in DMSO-d6. All chemical shifts in this work are identical to the already-characterized enterobactin compound (37). Download Table S1, PDF file, 0.03 MB.
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FIG S5
(A) Rothia mucilaginosa ATCC 25296 growth was established first on M9 agar (100 mM sucrose) (required for growth of the challenging species Actinomyces timonensis DSM 23838 under aerobic conditions). A. timonensis was spotted adjacent to R. mucilaginosa, and its growth was inhibited. (B) Streptococcus salivarius SHI-3 presents a growth boost and forms growth on top of R. mucilaginosa when plated adjacent to R. mucilaginosa on M9 minimal agar medium (100 mM sucrose). Download FIG S5, TIFF file, 2.9 MB.
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FIG S6
Rothia mucilaginosa ATCC 25296 inhibits pigment production in Staphylococcus aureus enterotoxin H-producing strain ATCC 51811 and MRSA strain TCH70 growing on M9 agar plates with no catalase added (100 mM glycerol). Download FIG S6, TIF file, 2.8 MB.
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FIG S7
Standard curves for the calmagite compleximetric assay for siderophore activity. (A) A 25 μM concentration of MgSO4 complexed with calmagite. (B) A 25 μM concentration of ZnSO4 complexed with calmagite. Both curves arise from dilutions of EDTA from 0 to 500 μM added to the calmagite-metal complex at pH 10 and a color change from red to blue monitored at 650 nm (41, 42). Enterobactin at 100 μM bound metal ions equal to the amount bound by 40 μM EDTA. Download FIG S7, TIF file, 2.9 MB.
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