Commensal oral Rothia mucilaginosa produces enterobactin – a metal chelating siderophore

Next-generation sequencing studies of saliva and dental plaque from subjects in both healthy and diseased states have identified bacteria belonging to the Rothia genus as ubiquitous members of the oral microbiota. To gain a deeper understanding of molecular mechanisms underlying the chemical ecology of this unexplored group, we applied a genome mining approach that targets functionally important biosynthetic gene clusters (BGCs). All 45 genomes that were mined, representing Rothia mucilaginosa, R. dentocariosa and R. aeria, harbored a catechol-siderophore-like BGC. To explore siderophore production further we grew the previously characterized R. mucilaginosa ATCC 25296 in liquid cultures, amended with glycerol, which led to the identification of the archetype siderophore enterobactin by using tandem Liquid Chromatography Mass Spectrometry (LC/MS/MS), High Performance Liquid Chromatography (HPLC), and Nuclear Magnetic Resonance (NMR) spectroscopy. Normally attributed to pathogenic gut bacteria, R. mucilaginosa is the first commensal oral bacterium found to produce enterobactin. Co-cultivation studies including R. mucilaginosa or purified enterobactin revealed that enterobactin reduced growth of certain strains of cariogenic Streptococcus mutans and pathogenic strains of Staphylococcus aureus. Commensal oral bacteria were either unaffected by, reduced in growth, or induced to grow adjacent to enterobactin producing R. mucilaginosa or the pure compound. Taken together with Rothia’s known capacity to ferment a variety of carbohydrates and amino acids, our findings of enterobactin production adds an additional level of explanation to R. mucilaginosa’s colonization success of the oral cavity. Enterobactin is the strongest Fe(III)-binding siderophore known, and its role in oral health requires further investigation. Importance The communication language of the human oral microbiota is vastly underexplored. However, a few studies have shown that specialized small molecules encoded by BGCs have critical roles such as in colonization resistance against pathogens and quorum sensing. Here, by using a genome mining approach in combination with compound screening of growth cultures, we identified that the commensal oral community member mucilaginosa harbors a catecholate-siderophore BGC, which is responsible for the biosynthesis of enterobactin. The iron-scavenging role of enterobactin is known to have positive effects on the host’s iron pool and negative effects on host immune function, however its role in oral health remains unexplored. R. mucilaginosa was previously identified as an abundant community member in cystic fibrosis, where bacterial iron cycling plays a major role in virulence development. With respect to iron’s broad biological importance, iron-chelating enterobactin may explain R. mucilaginosa’s colonization success in both health and disease.


Introduction 64
Over the past few decades of oral microbiology research, we have come to understand 65 that oral microbiota is imperative not only for our oral health but also for our overall 66 wellness. Thus far, the oral microbiology research field has focused significant efforts on 67 either describing taxonomic shifts of complex bacterial communities in healthy and 68 8 S2), which illustrates a broader ecological importance of this siderophore. AntiSMASH 161 predicted that the closest homologue to the cat-sid BGC was a mirubactin BGC (14% 162 peptide sequence similarity) (Fig. S1C). Mirubactin is a mixed catecholate and 163 hydroxamate-type NRPS siderophore. However, AntiSMASH also predicted that the 164 core building blocks for siderophore biosynthesis were serine and dihydroxy benzoic 165 acid, showing support for the biosynthesis of a catecholate siderophore (Fig. S1). 166 167

Screening for the siderophore in Rothia mucilaginosa growth extracts 168
To test if Rothia mucilaginosa ATCC 25296 secreted a siderophore while growing in 169 liquid growth cultures, we tested growth in multiple conditions. We specifically optimized 170 for growth in minimal media to reduce the number of metabolites that could interfere 171 with our downstream mass spectrometric analysis and purification of the siderophore. 172 Plateauing growth curves of R. mucilaginosa ATCC 25296 were obtained in liquid 173 minimal medium M9 cultures supplemented either with 100 mM sucrose or glucose 174 during aerobic incubation in 37° C (Fig. 1A). However, when incubated with other 175 carbon sources (glycerol, lactose, arabinose, or galactose) growth was reduced (Fig.  176 1A). To explore if a siderophore was produced in any of the above culture conditions, 177 we screened liquid growth extracts using two different assays; a hydroxamate assay 178 that targets carboxylate siderophores (31) and the Arnow's assay, which targets 179 catecholate siderophores (32). Arnow's assay showed clear colorimetric changes as the 180 normally colorless M9 media turned ruby red in cultures incubated with sucrose and 181 glycerol, while the hydroxamate assay showed no color change. The presence of a 182 catecholate siderophore in these cultures was also confirmed using absorbance 183 measurements at 500 nm, which is known to capture catecholate derivatives (33). Not 184 only did these results illustrate that R. mucilaginosa ATCC 25296 can produce a 185 catechol siderophore but also that the AntiSMASH program could accurately predict the 186 correct core building blocks (serine and dihydroxy benzoic acid). To facilitate compound 187 isolation and purification, we further explored if glycerol, which is known to elicit 188 secondary metabolite production in other Actinobacteria (34), could increase 189 siderophore yields. This was indeed the case as enterobactin production increased 190 significantly in the glycerol amended cultures (Absorbance at 500nm: ~0.25) ( Fig 1B). 191 However, under the same conditions, growth decreased (Fig. 1A). We also tested if 192 liquid cultures of Rothia dentocariosa M567, which also harbors a cat-sid BGC ( Figure  193 S2), can produce a siderophore when subjected to glycerol cultivation (Fig. 1B). 194 Production was observed for this species as well (Absorbance at 500nm: ~0.15) but not 195 to the same extent as for R. mucilaginosa (Fig.1B) Fig. 2A). To verify 218 our findings, we performed additional LC-MS/MS on purified extracts from thin layer 219 chromatography, which again resulted in the identification of enterobactin or a close 220 homolog using the GNPS infrastructure, this time in positive ionization mode (Fig. 2B). 221 Further purification of this compound using HPLC showed a well-separated peak that 222 tested positive in the Arnow's assay (Fig. 3). This peak was eluted and analyzed by 1 H 223 NMR to confirm that R. mucilaginosa ATCC 25296 produces enterobactin (Table S1). 224 All observed chemical shift values in Table S1 (obtained from R. mucilaginosa's 225 enterobactin) were also reported previously in an Infrared (IR) and NMR spectroscopic 226 study of the archetypal enterobactin molecule (40). Proton and 2D NMR spectra 227 (HSQC, HMBC, and COSY) for the R. mucilaginosa-derived compound confirmed its 228 identity ( Figure S5). 229

Enterobactin activity screening using co-cultivation assays 231
Interactions between R. mucilaginosa ATCC 25296 or the purified enterobactin 232 compound and other bacterial species were studied by employing co-cultivation agar 233 assays and liquid growth assays. On agar plates, growth of R. mucilaginosa was 234 established prior to spotting the challenging species. Interactions were initially studied 235 on both rich (BHI) and minimal M9 agar media to investigate under which conditions R. 236 mucilaginosa could modulate growth of the competitor test strain, potentially via iron 237 competition (visualized as clearing zones surrounding R. mucilaginosa). In the liquid 238 mono-cultures, the purified siderophore was added at the same time as the cultures 239 were seeded with the test strain. We found that while growing on minimal M9 agar, of catalase (Fig. 4, Fig. S6). We added catalase to the agar plates to prevent growth 244 inhibition by eventual reactive oxygen species (ROS) produced by R. mucilaginosa. The 245 results suggest that inhibition of pigment production is not due to ROS but eventually to 246 enterobactin. To further elucidate this interaction, we conducted mono-culture 247 experiments on agar plates where we added the purified enterobactin to S. aureus agar 248 plates (Fig. 5). With catalase added, statistically significant differences in pigmentation 249 were observed for all S. aureus strains amended with enterobactin, including MRSA 250 strains TCH70/MRSA and NR10129 (p<0.05, two-tailed t-test) (Fig. 5), confirming an 251 important role of enterobactin in the inhibition of S. aureus virulence and growth (37,38). 252 For all the tested oral commensal and pathogenic Streptococcus species, no growth 253 inhibition was observed on agar plates. However, changes in growth were confirmed for 254 some of the species when adding the purified enterobactin compound to liquid cultures 255 (with or without catalase) (Fig. 6). Of the pathogenic Streptococcus strains tested in 256 liquid growth cultures amended with enterobactin and catalase, we observed that 257 growth of both S. mutans UA159 and S. mutans B04Sm5 were significantly reduced 258 To evaluate if enterobactin was actively chelating free iron throughout the liquid 298 cultivation experiments, we incubated 500 µM of the purified compound in sterile M9 299 liquid media as described for bacterial liquid cultures. By using Arnow's assay we 300 observed that its binding capacity to molybdenum (which in Arnow's assay substitutes 301 for iron) was reduced by 14% after six hours of incubation. Assuming that this response 302 is linear we estimated that 56% of enterobactin's activity was lost after 24 hours. This 303 suggests that the compound became inactive in our co-culture experiments over time 304 for physiochemical reasons and that a more dramatic effect of enterobactin likely would 305 have been observed if its activity had remained stable. 306

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We also investigated if enterobactin can bind other metal ions by using the calmagite 308 assay, which is normally used for testing the hardness of water by detecting ions such 309 as magnesium (Mg), calcium (Ca) and zinc (Zn) that bind to ethylenediaminetetraacetic 310 acid (EDTA) (40,41). EDTA is used as a reference compound due to its well-known 311 chelation of divalent cations. Results from this test revealed that enterobactin can 312 chelate both Mg 2+ and Zn 2+ ions. Enterobactin showed a chelation activity of 40 µM 313 EDTA-equivalents at a concentration of 100 µM, which is 2.5 times less than EDTA 314 affinity for Mg 2+ and Zn 2+ (Fig. S7). These results illustrate that enterobactin can indeed 315 chelate additional ions and not only ferrous iron (Fe 3+ ), which could have critical impacts 316 on other microbial community members as well as the human host. It is well known that 317 Zn 2+ is a critical component of the ubiquitous protein zinc fingers that are able to interact 318 directly with DNA, RNA, and proteins. Magnesium (Mg 2+ ) is involved in nervous system 319 signaling, immune system function and bone formation. Our findings show that R. 320 mucilaginosa has the potential to compete with the host not only for Fe 3+ but also for 321 Mg 2+ and Zn 2+ , which suggest that R. mucilaginosa could play an important role in 322 health and disease outcomes. 323

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In conclusion, our study shows that R. mucilaginosa ATCC 25296 produces the

Mining for biosynthetic gene clusters (BGCs) in Rothia genomes 370
The BGC prediction program antiSMASH, bacterial version (30), which is able to predict 371 core secondary metabolite structures from BGC sequences, was used to identify BGCs

Co-cultivation agar assays 387
For bacterial interaction screening studies, all bacterial isolates were seeded from 388 glycerol stocks in BHI (Oxoid, Thermo Scientific) media and incubated for 24 h before 389 placing on either BHI agar or M9 minimal agar media supplemented with either 100 mM 390 sucrose (Acros Organics, Pittsburgh, PA, USA), glucose (Millipore-Sigma), or glycerol 391 (Honeywell, Mexico City, Mexico). Plates were incubated either in a 5% CO2 incubator 392 at 37°C or anaerobically at 37°C in an anaerobic chamber (Coy Laboratory Products, 393 Grass Lake, MI) with a gas mix of 5% H2, 5%CO2, and 90% N2. All tested strains were 394 grown similarly except A. timonensis, which was incubated for 48 h in BHI in an 395 anaerobic chamber at 37°C before plating due to its slower growth rate in M9 minimal 396 media. R. mucilaginosa or R. dentocariosa were plated by dropping 20-30 µL, in three 397 to six replicates, onto plates, allowing the drops to air-dry before incubation. Control preparations, but without the siderophore. All plates were incubated at 37ºC for 48 h in a 475 Tecan Infinite M Nano spectrophotometer and growth was monitored every hour by 476 absorbance measurements at 600 nm. Statistical analysis was done with the R package 477 "statmod" using the "compareTwoGrowthCurves" function with an nsim parameter value 478 of 100000 (48) based on a meanT calculation. All graphical figures were generated 479 using R Studio, version 1.2.5001 (49) and the package ggplot2 (50). 480 were detected and counted by the R program "countcolors". The yellow pixels were 504 automatically replaced with blue by the program to better visualize the effect of 505 enterobactin on S. aureus pigmentation. Three images per condition and strain were 506 measured for yellow pigmentation, and a two-tailed T-test performed for each strain 507 using the percentage values obtained by the "countcolors" program, comparing each 508 strain with a control strain as described above. Enterobactin is best known for its ability to bind the insoluble trivalent iron (Fe 3+ ). In 526 order to assess the ability of enterobactin purified from R. mucilaginosa to bind 527 magnesium and zinc, the compleximetric dye calmagite was used in an assay format 528 (40,41). Calmagite forms colored complexes with magnesium and zinc as well as other 529 metals and was developed to quantify magnesium in biological samples (40). A 530 chelating compound such as EDTA or enterobactin, is able to break this complex and 531 elicit a color change from red to blue that can be measured spectrophotometrically. This