Bacterial Competition Mediated by Siderophore Production Among the Human

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1 Bacterial competition mediated by siderophore production among the human

2 nasal microbiota

3 Reed M. Stubbendieck1,#, Daniel S. May1, Marc G. Chevrette1,2, Mia I. Temkin1, Evelyn

4 Wendt-Pienkowski1, Julian Cagnazzo1, Caitlin M. Carlson1, James E. Gern3,4, and

5 Cameron R. Currie1,#

6 1Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706

7 2Laboratory of Genetics, University of Wisconsin-Madison, Madison, WI 53706

8 3Department of Pediatrics, University of Wisconsin School of Medicine and Public

9 Health, Madison, WI 53792

10 4Department of Medicine, University of Wisconsin School of Medicine and Public Health,

11 Madison, WI 53792

12 Running Title: Siderophore competition among human nasal microbiota

13 Word Count: 5807

14 Keywords: Actinobacteria, competition, Corynebacterium, dehydroxynocardamine,

15 exploitation, iron, nasal microbiome, siderophore, Staphylococcus

16 Address correspondence to Reed M. Stubbendieck ([email protected]) or

17 Cameron R. Currie ([email protected])

19 ABSTRACT

20 Resources available in the human nasal cavity are limited. Therefore, to successfully

21 colonize the nasal cavity, bacteria must compete for scarce nutrients. Competition may

22 occur directly through interference (e.g., antibiotics) or indirectly by nutrient

23 sequestration. To investigate the nature of nasal bacterial competition, we performed

24 co-culture inhibition assays between nasal Actinobacteria and Staphylococcus spp. We

25 found that Staphylococcus epidermidis isolates were sensitive to growth inhibition by

26 Actinobacteria but Staphylococcus aureus isolates were resistant to inhibition. Among

27 Actinobacteria, we observed that Corynebacterium spp. were variable in their ability to

28 inhibit S. epidermidis. We sequenced the genomes of ten Corynebacterium spp.

29 isolates, including three Corynebacterium propinquum that strongly inhibited S.

30 epidermidis and seven other Corynebacterium spp. isolates that only weakly inhibited S.

31 epidermidis. Using a comparative genomics approach, we found that the C. propinquum

32 genomes were enriched in genes for iron acquisition and encoded a biosynthetic gene

33 cluster (BGC) for siderophore production, absent in the non-inhibitory Corynebacterium

34 spp. genomes. Using a chromeazurol S assay, we confirmed that C. propinquum

35 produced siderophores. We demonstrated that iron supplementation rescued S.

36 epidermidis from inhibition by C. propinquum, suggesting that inhibition was due to iron

37 restriction through siderophore production. Using comparative metabolomics, we

38 identified the siderophore produced by C. propinquum as dehydroxynocardamine.

39 Finally, we confirmed that the dehydroxynocardamine BGC is expressed in vivo by

40 analyzing human nasal metatranscriptomes from the NIH Human Microbiome Project.

41 Together, our results suggest that bacteria produce siderophores to compete for limited

42 available iron in the nasal cavity and improve their fitness.

44 IMPORTANCE

45 Within the nasal cavity, interference competition through antimicrobial production is

46 prevalent. For instance, nasal Staphylococcus spp. strains can inhibit the growth of

47 other bacteria through the production of nonribosomal peptides and ribosomally

48 synthesized and post-translationally modified peptides. In contrast, bacteria engaging in

49 exploitation competition modify the external environment to prevent competitors from

50 growing, usually by depleting access to essential nutrients. As the nasal cavity is a

51 nutrient limited environment, we hypothesized that exploitation competition occurs in

52 this system. We determined that Corynebacterium propinquum produces an iron-

53 chelating siderophore and is able to use this molecule to sequester iron and inhibit the

54 growth of Staphylococcus epidermidis. Further, we found that the genes required for

55 siderophore production are expressed in vivo. Thus, though siderophore production by

56 bacteria is often considered a virulence trait, our work indicates that bacteria may

57 produce siderophores to compete for limited iron in the human nasal cavity.

59 INTRODUCTION

60 Humans engage in symbioses with diverse sets of microbes at nearly every body site.

61 Collectively, these microbes are referred to as the human microbiota. Members of the

62 microbiota provide their human host with essential services. For example, within the

63 gastrointestinal tract, mutualistic bacteria metabolize recalcitrant nutrients to release

64 metabolites that are accessible to the human host (1, 2), synthesize many essential

65 amino acids, cofactors and vitamins (3, 4), and provide defense against pathogens (5,

66 6). However, despite providing critical services to their hosts, nutrient acquisition is often

67 a principle challenge for bacteria colonizing humans and other animals. In part, this is

68 due to host sequestration of resources as a means to control bacterial growth (7).

70 To form and maintain associations with humans and other eukaryotes, bacteria

71 have evolved mechanisms to derive nutrition from their hosts (8, 9). Both commensal

72 and pathogenic bacteria colonizing the gastrointestinal tract consume host-derived

73 compounds (10). For instance, Bacteroides acidifaciens and Akkermansia muciniphila

74 cells have been shown to incorporate carbon and nitrogen from host proteins (11) and

75 the pathogen Salmonella enterica serotype Typhimurium uses tetrathionate derived

76 from thiosulphate produced by the host inflammatory response during infection as a

77 terminal electron acceptor (12). In addition to consuming host-derived products, bacteria

78 colonizing the gastrointestinal tract can also acquire nutrients from meals that their host

79 consumes (10). However, outside of the gastrointestinal tract, bacteria have

80 increasingly limited access to nutrients. Cutibacterium (Propionibacterium) acnes

81 colonizing the sebaceous glands in human skin hydrolyzes sebum triglycerides to

82 generate free fatty acids and glycerol (13, 14), which they may metabolize

83 (15).Similarly, anaerobic oral bacteria that have spread to the lower respiratory tracts of

84 cystic fibrosis patients may ferment host mucins to generate short chain fatty acids and

85 amino acids to feed other bacteria, including Pseudomonas aeruginosa (16).

87 To colonize the upper airway, bacteria must attach to the epithelial surface,

88 accommodate or evade the host’s immune system, and cope with decreased access to

89 freely available nutrients when compared to bacteria colonizing the gastrointestinal tract

90 (reviewed in 7). Within the human nasal cavity there are minute concentrations of free

91 amino acids, carbohydrates, organic acids, and minerals (17–19). For comparison,

92 nasal secretions contain ~65-fold lower glucose concentrations than the lumen of the

93 small intestine (19, 20). Further, humans actively deplete the available glucose in the

94 airway through polarized glucose importers in the membranes of airway epithelial cells

95 (21). Similar to other environments such as the soil or the ocean, the bioavailability of

96 iron in the nasopharynx is low (17, 18). As a means to acquire scarce iron from their

97 environment, bacteria release iron-chelating molecules called siderophores that

98 scavenge ferric iron and other minerals and are resorbed by their producers (22).

99 However, human hosts have evolved countermeasures to circumvent bacterial

100 siderophores. For instance, bacterial colonization of the nasopharynx triggers

101 neutrophils to produce lipocalin-2, a protein that binds to enterobactin-type siderophores

102 and prevents their uptake by bacteria (23, 24). Finally, in addition to experiencing

103 nutrient and mineral limitation, bacteria that colonize the nasal cavity are exposed to

104 oxygen stress, which may be either abiotic or produced by the action of host immune

105 cells (25, 26). Therefore the human nasal cavity is a low resource and high stress

106 environment, requiring bacteria in this environment to engage in competitive interactions

107 for survival (27, 28).

108

109 Competition is split into two modes called interference and exploitation. Bacteria

110 engaging in exploitation competition compete by preventing their competitors from

111 accessing resources, either by rapidly consuming or sequestering them. In contrast,

112 bacteria using interference competition use toxic effectors to directly inhibit their

113 competitors (29). Bacteria from the human nasal cavity are well known to use

114 specialized (secondary) metabolites with antimicrobial properties to engage in

115 interference competition. As examples, Staphylococcus lugdunensis produces a

116 thiazolidine-containing cyclic peptide called lugdunin that inhibits the growth of

117 Staphylococcus aureus (30). Corynebacterium accolens secretes a lipase that cleaves

118 human nasal triacylglycerols to produce antimicrobial free fatty acids that inhibit the

119 growth of Streptococcus pneumoniae (31). Further, under conditions of iron-limitation

120 and hydrogen peroxide induced oxidative stress, which reflect conditions experienced

121 by bacteria colonizing the nasal cavity, Staphylococcus epidermidis IVK45 increases

122 production of an antimicrobial peptide called nukacin IVK45 (32). Despite these

123 examples of interference competition and the low resource environment of the nasal

124 cavity, mechanisms of exploitation competition are not well studied among the human

125 nasal microbiota (33).

126

127 In this study, we investigated bacterial competition between Actinobacteria and

128 Staphylococcus, which are among the most abundant members of the human nasal

129 microbiota (33, 34). We found that Corynebacterium (Phylum: Actinobacteria) vary in

130 their ability to inhibit the growth of S. epidermidis. Using a comparative genomics

131 approach, we identified a gene cluster for siderophore biosynthesis that is present in the

132 genomes of Corynebacterium propinquum strains that more strongly inhibit S.

133 epidermidis. We confirmed the siderophore production and demonstrate that iron

134 sequestration as the mechanism of S. epidermidis inhibition. We identified the

135 siderophore as dehydroxynocardamine. Finally, we detected expression of the

136 dehydroxynocardamine biosynthetic gene cluster (BGC) in metatranscriptomic reads

137 from the human nasal cavity. Together, the data suggest that members of the human

138 nasal microbiota engage in exploitation competition for limiting iron, and may influence

139 the composition of the human nasal microbiota in vivo.

140

141 MATERIALS AND METHODS

142 Nasal lavage specimen collection

143 The nasal lavage samples that we used in this study were banked as part of the

144 Childhood Origins of ASThma (COAST) study (35). Informed consent was obtained

145 from the parents, and the Human Subjects Committee at the University of Wisconsin-

146 Madison approved the study (IRB approval number H-2013-1044). Briefly, lavage

147 samples were collected by spraying Deep Sea Nasal Spray (Major®) into one of the

148 participant’s nostrils. To collect the sample, the participant was then instructed to blow

149 their nose into a plastic bag. Subsequently, the samples were stored at 4 °C.

150 Approximately 3 mL of either phosphate-buffered saline or Amies transport media

151 (Copan Diagnostics) was added to each sample before storage at -80 °C.

152

153 Strain isolation and maintenance

154 For general strain propagation, we used brain heart infusion (BHI) (DOT Scientific). All

155 plates were solidified using 1.5% agar (VWR). For iron supplementation experiments,

156 we added 200 µM FeCl3 to the BHI agar after autoclaving and cooling the media to 55

157 °C before pouring the plates. The strains of bacteria used in this study are listed in

158 Table S1. To culture bacteria from the lavage samples, we spread 100 µL of each of the

159 thawed samples onto BHI plates, and incubated the plates aerobically at 37 °C for 1

160 week. We selected ≥2 colonies of each distinct morphotype per plate and passaged the

161 isolates aerobically on BHI plates at 37 °C until we obtained pure cultures. All bacterial

162 isolates were cryopreserved at -80 °C in 25% glycerol.

163

164 Bacterial isolate identification

165 To identify bacterial isolates from the nasal lavage samples, we sequenced the 16S

166 rRNA gene. We used colony PCR with the universal 27F (5′-

167 AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-CGGTTACCTTGTTACGACTT-3′)

168 primers to amplify the 16S rRNA gene (36). We sequenced PCR products using the

169 Sanger method at the University of Wisconsin-Madison Biotechnology Center. We

170 identified isolates to the genus level using the Ribosomal Database Project Classifier

171 (37). To distinguish between S. aureus and S. epidermidis isolates, we used BLAST

172 searches against the NCBI 16S ribosomal RNA sequence database with a threshold

173 sequence identity of 99%. We corroborated the identity of Staphylococcus spp. isolates

174 using colony pigmentation and hemolysis phenotypes (38–40).

175

176 16S rRNA gene phylogenetic analysis

177 We aligned the 16S rRNA gene sequences using SINA v1.2.11 (41) through the Silva

178 webserver (42) and discarded unaligned bases at the ends of the sequences. For

179 phylogenetic analysis, we imported the alignments into MEGA7 (43). We inferred

180 phylogenetic trees using the maximum likelihood method based on the general time

181 reversible model. All nucleotide positions containing gaps were removed in the final

182 analysis.

183

184 Co-culture plate inhibition assays

185 We used co-culture plate inhibition assays to assess the activity of nasal Actinobacteria

186 isolates against nasal Staphylococcus. We spread Actinobacteria from saturated

187 overnight cultures in BHI broth over one-half of a well (d = 2.4 cm) containing 3 mL of

188 BHI agar on a 12-well plate (Greiner Bio-One). We incubated the plates for one week at

189 37 °C. Subsequently, we spotted ~3 µL of a ten-fold diluted saturated overnight culture

190 of each Staphylococcus spp. isolate in BHI adjacent to the Actinobacteria and returned

191 the plates to the 37 °C incubator. After 1 week of co-culture, we removed the plates,

192 scanned them, and scored the inhibition for each interaction pair. All interactions were

193 tested with ≥2 replicates with consistent results. An inhibition score of 0 indicated no

194 inhibition, a score of 1 indicated weak inhibition, and a score of 2 indicated strong

195 inhibition (Fig. 1A). We determined significance using the chi-square test.

196

197 Whole genome sequencing and assembly

198 We cultured bacterial strains for whole genome sequencing in 3 mL of BHI broth

199 supplemented with 0.5% glycine overnight at 37 °C. We harvested the cells by

200 centrifuging the cultures at 21,330×g for 5 min. The cell pellets were washed with 10.3%

201 sucrose and resuspended in 450 µL of lysozyme solution [3 mg/mL lysozyme (Sigma),

202 0.3 M sucrose, 25 mM Tris-HCl (pH 8), 25 mM EDTA (pH 8)] for 30 min at 37 °C.

203 Subsequently, 13 µL of 20 mg/mL Proteinase K (Thermo Fisher) was added to each

204 sample, with additional incubation at 42 °C for 15 min. Cells were lysed by adding 250

205 µL of 2% SDS and rocking for 15 min. DNA was purified using standard phenol-

206 chloroform extraction and precipitated with 3 M sodium acetate and isopropanol. We

207 visually assessed the quality of genomic DNA preparations by running samples on 0.5%

208 TBE gels. Genomic libraries for Illumina MiSeq 2 × 150 bp paired-end sequencing were

209 prepared and sequenced by the University of Wisconsin-Madison Biotechnology Center.

210 Raw reads were corrected with MUSKET v1.1 (44) and paired ends were merged with

211 FLASH v1.2.7 (45). Reads were assembled into draft genome sequences with SPAdes

212 v3.11.0 (46).

213

214 The genome sequences of Actinobacteria generated during this study have

215 been deposited in the National Center for Biotechnology Information database under

216 BioProject accession PRJNA492917.

217

218 Core-genome phylogeny

219 To construct the core-genome phylogenetic tree of nasal Actinobacteria, we used the

220 core species tree pipeline (https://github.com/chevrm/core_species_tree) developed in

221 our laboratory (47). Briefly, we called genes in each genome using prodigal v2.6.0 (48)

222 and used profile Hidden Markov Models in HMMER v3.1b2 (49) to search each genome

223 for 93 full length TIGRFAM amino acid sequences in the “core bacterial protein” set

224 (GenProp0799). Each protein family was aligned using MAFFT v7.245 (50) and

225 converted to codon alignments. We used RAxML v8.1.24 (51) to generate phylogenetic

226 trees for each of the 93 codon alignments under the GTRgamma substitution model

227 with 100 bootstraps. To generate the species phylogenetic tree, we used ASTRAL-II

228 (52) from the individual phylogenetic trees with 100 bootstraps. We used FigTree v1.4.3

229 (http://tree.bio.ed.ac.uk/software/figtree/) to root the phylogeny on B. subtilis 168 and

230 display the branch length based on proportional length to the root.

231

232 Comparative genomics

233 We annotated the assembled genomes using prokka v1.13 (53). For comparative

234 genomics, we identified orthologs from the nucleotide sequences of each protein-coding

235 open reading frame (ORF) with OMA v2.2.0 (54). We applied Euclidean hierarchical

236 clustering to the ortholog presence/absence matrix generated by OMA using R. To

237 annotate orthologues with Kyoto Encyclopedia of Genes and Genomes (KEGG)

238 orthology (KO) terms, we used DIAMOND v0.9.21.122 (55) to query one sequence of

239 each ortholog group against a custom database of KEGG annotated proteins

240 sequences with an E-value threshold cutoff of 1.0e-10. Each ORF was assigned the KO

241 terms from the top BLAST hit. For KEGG KO term enrichment analysis, we used the

242 GSEABase package v1.42.0 (56) and GOstats v1.7.4 (57) in R and performed a

243 hypergeometric test with a p-value cutoff of 0.05.

244

245 Identification of BGCs

246 To identify BGCs we used antiSMASH 4.0.2 (58). Proteins from all identified BGCs

247 were aligned via an all versus all DIAMOND (query cover >75, percent identity >60).

248 BGCs that shared over 75% of proteins were called the same family. BGC families

249 correlated to inhibition were identified through presence in the high-inhibition strains and

250 absence in the weak-inhibition strains. Proteins from all identified BGCs were combined

251 with all proteins from MIBiG v1.3 (59) and subsequently aligned with DIAMOND with the

252 same parameters as listed before to identify biosynthetic gene similarities to previously

253 described BGCs.

254

255 Chrome azurol S assay

256 We used the overlay chrome azurol S (CAS) assay to test for siderophore production as

257 previously described (60). We cultured Corynebacterium spp. overnight in 3 mL BHI at

258 37 °C. We diluted the overnight cultures to OD600 = 2 and spotted 5 µL on to 10 mL BHI

259 agar plates (d = 5 cm). We incubated the plates at 37 °C for 4 days to ensure robust

260 Corynebacterium spp. growth. Subsequently, we overlaid each plate with 6 mL of CAS

261 reagent overlay [100 µM CAS, 200 µM hexadecyltrimethylammonium bromide, 10 µM

262 FeCl3⋅6 H2O, 10 mM PIPES (pH 6.8), 1% agarose] (60, 61) and incubated the plates at

263 ambient temperature in the dark overnight before scanning plates for color change from

264 blue to yellow, indicating production of a siderophore (61).

265

266 Siderophore identification and mass spectral molecular networking

267 To identify the siderophore, we used a mass spectrometry guided approach. We

268 spotted 10 µL of either Corynebacterium genitalium HSID17231 or Corynebacterium

269 propinquum HSID18034 overnight cultures onto the center of 25 mL BHI agar plates (d

270 = 8.5 cm). After incubating the plates as above, we removed agar cores (d = 0.6 cm)

271 from near the Corynebacterium spp. colony using the wide end of a P1000 pipette tip.

272 As above, we verified siderophore activity by directly placing duplicate agar cores onto

273 CAS assay plates and observing the color change from blue to yellow. We washed the

274 agar cores in 2 mL of 50% methanol and 2 mL of 100% methanol, which we combined

275 and dried with a gentle air stream under reduced pressure.

276

277 Data-dependent LC/MS2 was run on a Thermo Scientific Q Exactive Orbitrap by the

278 University of Wisconsin Analytical Instrumentation Center of the School of Pharmacy.

279 The liquid chromatography method was run on a Phenomenex XB C18, 2.1 X 100 mm,

280 2.6 µm particle size column with solvents A (water with 0.1% formic acid) and B

281 (acetonitrile with 0.1% formic acid). A gradient of 5% B for half a minute to 30% B over

282 16 minutes, then 97% B for two minutes with a flow rate of 0.35 mL/min was used to

283 separate the metabolites. We exported the files in mzXML format and uploaded them to

284 Global Natural Products Social Molecular Networking (GNPS) (62). We generated a

285 molecular network using the online workflow at GNPS. The data was filtered by

286 removing all MS/MS peaks within ± 17 Da of the precursor m/z. The MS2 spectra were

287 window filtered by choosing only the top 6 peaks in the ± 50 Da window throughout the

288 spectrum. The data was then clustered with MS-Cluster with a parent mass tolerance of

289 2.0 Da and a MS2 fragment ion tolerance of 0.5 Da to create consensus spectra.

290 Consensus spectra that contained less than 2 spectra were discarded. A network was

291 then created where edges were filtered to have a cosine score above 0.6 and more than

292 3 matched peaks. Edges between two nodes were kept in the network if each of the

293 nodes appeared in the respective top 10 most similar nodes. The spectra in the network

294 were then searched against GNPS' spectral libraries. The library spectra were filtered in

295 the same manner as the input data. All matches kept between network spectra and

296 library spectra were required to have a score above 0.6 and at least 3 matched peaks.

297 Analog search was enabled against the library with a maximum mass shift of 100.0 Da.

298 Metatranscriptome analysis for dehydroxynocardamine BGC expression

299 As part of the National Institutes of Health (NIH) Integrative Human Microbiome Project

300 (iHMP), RNA from the anterior nares of 16 prediabetic subjects was isolated and

301 depleted of 16S and 23S rRNAs. The RNA was converted to cDNA with random primers

302 and paired end sequenced using an Illumina platform (63). We downloaded the raw

303 reads from the iHMP using the hmp_client (https://github.com/ihmpdcc/hmp_client) from

304 the Human Microbiome Project Data Portal (https://portal.hmpdacc.org). We removed

305 Illumina adapter sequences, low quality reads, and unpaired reads using trimmomatic

306 v0.36 (64) with the following parameter: SLIDINGWINDOW:5:20. RNA was isolated

307 from each of the 16 subjects between 1 and 11 times for a total of 95 nasal

308 metatranscriptomes (mean = 5.9/subject, median = 6.5/subject). For our analyses, we

309 treated each individual metatranscriptome as a single sample. To determine expression

310 of the dehydroxynocardamine BGC, we used kallisto version 0.44.0 (65) and

311 pseudoaligned each of the metatranscriptomes onto the indexed open reading frames

312 (ORFs) onto the Corynebacterium propinquum HSID18034 genome. We extracted the

313 expression data for the combined dehydroxynocardamine BGC (dnoA-G) and four

314 housekeeping genes: rpoB, gyrB, sigA, and rpsL. We removed genes with <1 estimated

315 count from the dataset for a total of 148 expression counts (18 siderophore BGC, 130

316 housekeeping genes).

317

318 Plot generation and data visualization

319 We generated all plots in R v3.5.0 (66) using the ggplot2 package v2.2.1 (67). We

320 visualized phylogenetic trees in R using the ggtree v1.12.0 package (68).

321 RESULTS

322 Interaction assays reveal variation in staphylococcal inhibition by nasal

323 Actinobacteria.

324 The scarcity of nutrients and minerals in the human nasal cavity results in a stressful

325 environment where the microbiota must compete to survive. To pursue mechanisms of

326 competition that occur among the nasal microbiota, we chose to use a culture-based

327 approach and investigate interactions between Actinobacteria and Staphylococcus spp.,

328 which are major bacterial colonizers of the human nasal cavity. For this study, we

329 isolated bacteria from frozen nasal lavage samples donated by healthy children as part

330 of the COAST study (35, 69), which we identified by colony morphology, pigmentation,

331 hemolysis, and 16S rRNA gene sequence (see Methods).

332

333 To identify differences in patterns among interactions between Actinobacteria

334 and Staphylococcus spp., we assessed the ability of a subset of Actinobacteria isolates

335 to inhibit the growth of a subset of Staphylococcus spp. isolates. In total, we tested 21

336 Actinobacteria isolates (10 Corynebacterium, 1 Curtobacterium, 1 Dermabacter, 3

337 Kocuria, 1 Microbacterium, 3 Micrococcus, and 2 Rothia) against 39 Staphylococcus

338 spp. isolates (15 S. aureus, 19 S. epidermidis, and 5 S. warneri/pasteuri) (Table S1) for

339 a total of 812 pairwise combinations with ≥2 replicates. In the case of 7 pairwise

340 combinations, the replicates were not in agreement or the Actinobacteria isolate

341 overgrew its well and prohibited inoculation of the Staphylococcus spp. isolate. We

342 removed these 7 points from further analysis. We scored inhibition by visual inspection

343 of the Staphylococcus spp. colonies after 1 week of co-incubation. Strong inhibition

344 corresponded to colonies with severe growth defects or total growth inhibition, whereas

345 weak inhibition corresponded to colonies with moderate inhibition resulting in diminished

346 growth. Staphylococcus spp. colonies that were indistinguishable to a monoculture

347 control were considered uninhibited (Fig. 1A). To visualize patterns in the inhibition

348 assays, we plotted the inhibition scores as a heat map that was clustered based on the

349 phylogenetic relationships of the Actinobacteria and Staphylococcus spp. isolates on

350 the horizontal and vertical dimensions, respectively. The Staphylococcus spp.

351 phylogenetic tree was built from 16S rRNA gene sequence alignments, whereas the

352 Actinobacteria phylogenetic species tree was built from alignments of 93 single copy

353 core bacterial genes that were extracted after whole genome sequencing of the

354 Actinobacteria isolates (Fig. 1B).

355

356 By clustering the inhibition scores with respect to phylogeny, we immediately

357 observed significant differences in the inhibition patterns between S. aureus and S.

358 epidermidis. Broadly, most S. epidermidis isolates were inhibited by nasal

359 Actinobacteria but the S. aureus isolates were insensitive [휒2 = 371.73, df = 2, p < 2.2e-

360 16] (Fig. 1B). However, the inhibition pattern was not solely dominated by the identity of

361 the Staphylococcus species; we observed variation among Corynebacterium spp.

362 isolates to inhibit S. epidermidis. In particular, we noticed 3 Corynebacterium spp.

363 isolates forming a monophyletic clade (highlighted in yellow in Fig. 1B) that more

364 strongly inhibited the growth of S. epidermidis than the other Corynebacterium spp.

365 isolates [휒2 = 89.74, df = 2, p < 2.2e-16] (Fig. 1B). Given the limited variation in

366 staphylococcal inhibition by other genera of Actinobacteria, we focused on

367 Corynebacterium spp. to determine underlying factors responsible for the differences in

368 inhibition patterns.

369

370 Corynebacterium propinquum strains strongly inhibit S. epidermidis

371 To identify the Corynebacterium strains, we constructed a core genome phylogeny from

372 the ten Corynebacterium spp. that we isolated and 45 human-associated strains whose

373 sequences were available from GenBank. All three of the strong inhibitors of S.

374 epidermidis were mostly closely related to Corynebacterium propinquum, while the

375 weak inhibitors of S. epidermidis were either closely related to Corynebacterium

376 pseudodiphtheriticum (5 strains) or Corynebacterium genitalium (1 strain) (Fig. S1). For

377 clarity, we will refer to the Corynebacterium strong inhibitors of S. epidermidis as C.

378 propinquum and the other isolates as C. pseudodiphtheriticum or C. genitalium.

379

380 Iron acquisition by Corynebacterium spp. is related to increased S. epidermidis

381 inhibition

382 To identify differences among the Corynebacterium spp. strains that corresponded to

383 differential S. epidermidis inhibition, we took a comparative genomics approach. We

384 generated draft genome sequences for each of the ten Corynebacterium spp. isolates.

385 Using the OMA algorithm, we predicted orthologs across the ten genomes. In total, we

386 predicted 2702 ortholog groups, with 1198 groups (44% total) universally shared across

387 the ten Corynebacterium spp. strains and 1504 ortholog groups (56% total) that were

388 absent in at least one strain. In particular, the gene content in Corynebacterium

389 genitalium HSID17239 was more divergent than the other isolates (Fig. 2A, top row).

390 Indeed, when C. genitalium HSID17239 was excluded from the analysis, 1801 of the

391 2702 (67% total) ortholog groups were shared universally across the remaining nine

392 strains. To observe differences in the patterns of gene content within the

393 Corynebacterium spp. genomes, we applied hierarchical clustering to the presence-

394 absence matrix of the 2702 ortholog groups. There were 337 orthologs (12% total) that

395 were uniquely shared among C. propinquum (Fig. 2A). Though 223 of the orthologues

396 were annotated as encoding hypothetical or putative proteins, a manual investigation of

397 the 114 annotated orthologues revealed that 31 (27.2% of subset) were annotated with

398 functions related to iron acquisition and siderophore biosynthesis. This result represents

399 a 6.4-fold enrichment over the 4.3% abundance (68/1596 orthologs) of the

400 corresponding terms in all ten Corynebacterium genomes (hypergeometric test:

401 expected = 4.9%, p = 1.3e-19). As a second test, we performed an enrichment analysis

402 for KEGG orthology (KO) terms. We annotated 169 of the 337 orthologues (50% of

403 subset) with 117 KO terms. When compared to the total set of 357 KO terms, the 169

404 orthologues were enriched in 12 KO terms. In particular, we noted that among the KO

405 terms, 2 KO terms were associated with iron acquisition and siderophore biosynthesis:

406 1) iron complex transport system permeases and 2) bifunctional isochorismate

407 lyase/aryl carrier proteins (Fig. 2B).

408

409 The results of each set of enrichment analyses led us to hypothesize that

410 Corynebacterium spp. that weakly inhibit S. epidermidis would have growth defects

411 under conditions of iron limitation. To directly test for iron-related phenotypic differences

412 between the two groups of Corynebacterium, we cultured strains on standard BHI

413 media and on synthetic nasal medium 3 (SNM3), a medium that is formulated to

414 replicate the low concentrations of nutrients and mineral content found in human nasal

415 secretions (19). We observed no difference in the growth of Corynebacterium spp.

416 strains on standard BHI media, but found that C. genitalium HSID17239 had a growth

417 defect when cultured on SNM3 and was unable to form a colony (Fig. 2C). Together,

418 these results demonstrate that different strains of Corynebacterium spp. isolated from

419 the human nasal cavity possess differential ability to survive under iron-limited

420 conditions, which correlates with the ability to inhibit the growth of S. epidermidis.

421

422 Corynebacterium propinquum strains produce siderophores

423 The results of the gene and KO term enrichment analyses and iron-limitation phenotype

424 assay (Fig. 2) led us to hypothesize that the observed variation in inhibition of S.

425 epidermidis by Corynebacterium spp. (Fig. 1B) may be due to siderophore production.

426 To directly detect siderophore production we used the CAS assay, which detects

427 siderophore production by a color change from blue to yellow as siderophores remove

428 iron from the CAS dye (61). Consistent with our previous results, we confirmed

429 siderophore production by all three C. propinquum strains, whereas none of the other

430 seven Corynebacterium spp. produced detectable siderophore activity (Fig. 3A).

431 432 Iron supplementation rescues S. epidermidis from siderophore-mediated

433 inhibition.

434 The results from the CAS assay (Fig. 3A), indicated that siderophore production is

435 correlated with strong inhibition of S. epidermidis. To directly test if iron sequestration by

436 C. propinquum was responsible for S. epidermidis inhibition, we repeated the co-culture

437 plate inhibition assays on BHI and on BHI supplemented with 200 µM FeCl3. We found

438 that iron supplementation rescued S. epidermidis from inhibition by Corynebacterium

439 propinquum (Fig. 3B). We performed the assay with two C. propinquum siderophore

440 producers and five non-siderophore producers (1 strain of C. genitalium and 4 strains of

441 C. pseudodiphtheriticum) against 22 strains of S. epidermidis isolated from different

442 nasal specimens. On normal BHI medium, C. propinquum universally inhibited isolates

443 of S. epidermidis and the inhibition was completely reversed when the medium was

444 supplemented with FeCl3 (Fig. 3B-C), whereas under the conditions of this assay, there

445 was little-to-no inhibition of S. epidermidis by either C. genitalium or C.

446 pseudodiphtheriticum under either culture condition (Fig. 3C) [휒2 = 291.16, df = 3, p <

447 2.2e-16]. This result indicates that inhibition of S. epidermidis in vitro by C. propinquum

448 is due to iron sequestration.

449

450 The C. propinquum-produced siderophore is dehydroxynocardamine

451 As a first step to identify the siderophore produced by C. propinquum, we used

452 antiSMASH to BGCs for siderophore biosynthesis in the Corynebacterium spp.

453 genomes that we sequenced for this study. We identified a single 12553 bp siderophore

454 BGC that was present within all three C. propinquum genomes but absent in the other

455 seven Corynebacterium spp. genomes. This siderophore BGC was composed of seven

456 open reading frames (ORFs), which were annotated with functions consistent with iron

457 acquisition and siderophore biosynthesis (70–72) (Fig. 4A). Specifically, in addition to

458 transport-associated genes, this BGC encodes a PLP-dependent decarboxylase, an L-

459 lysine N6-monooxygenase, and a siderophore synthetase that contains acyl-CoA N-

460 acyltransferase (IPR016181), aerobactin siderophore biosynthesis, IucA/IucC, N-

461 terminal (IPR007310), and ferric iron reductase FhuF (IPR022770) domains. Though

462 there were no experimentally characterized BGCs that were identical to the C.

463 propinquum siderophore BGC, the order of the biosynthetic genes was identical to the

464 desferrioxamine E dfo BGC from Pantoea agglomerans strain B025670 (MIBiG

465 BGC0001572). The biosynthesis enzymes encoded by both siderophore BGCs are

466 highly similar (Table 1) and both synthetases belong to the type C’ nonribosomal

467 peptide synthetase-independent family (71, 72), which indicated that the siderophore

468 produced by C. propinquum is likely macrocyclic.

469

470 To identify the siderophore produced by C. propinquum, we used comparative

471 mass spectrometry-based metabolomics. We cultured C. propinquum HSID18034 and

472 C. genitalium HSID17239 on BHI agar plates. We sampled agar cores near the bacterial

473 colonies, which we extracted using methanol, and analyzed using data dependent liquid

474 chromatography-tandem mass spectrometry. To visualize differences in the

475 metabolomic profiles of the two strains, we used mass spectral molecular networking

476 (73). Surprisingly, in a network containing 222 clusters and 1531 singletons (Fig. 4B),

477 we observed only a single cluster that was unique to C. propinquum HSID18034 (Fig.

+ 478 4C). The central node in this cluster corresponded to m/z 585.358 [M+H] (C27H48N6O8,

479 ∆ppm = 3.44). The molecular weight and tandem mass spectrometry fragmentation

480 pattern matched that of the siderophore dehydroxynocardamine (m/z 585.361 [M+H]+,

481 C27H49N6O8), which is also called terragine E (Fig. 4D). This siderophore has been

482 previously reported from marine sponge-associated Streptomyces (74) and produced by

483 heterologous expression of soil DNA libraries in Streptomyces lividans (75), but has not

484 been reported from Corynebacterium or any other human-associated bacteria. The

485 other nodes in the cluster correspond to two dehydroxynocardamine analogs with

+ + 486 protonated ions of m/z 569.364 [M+H] and 553.368 [M+H] , as well as the doubly

487 charged states of each molecule (Fig. 4C). The high similarity in fragmentation patterns

488 as well as the differences of m/z -16 and -32 suggested these compounds contained

489 one and two fewer N-hydroxy groups than dehydroxynocardamine (Figs. 4D, S3).

490 Based on the product, we chose to name the C. propinquum siderophore BGC dno for

491 dehydroxynocardamine.

492

493 The dehydroxynocardamine BGC is expressed in vivo.

494 We identified a 19 bp putative Diphtheria Toxin Repressor (DtxR) iron box operator

495 sequence (5′-TTATGCAAGGTTTTCCTAT-3′) (76) situated upstream of dnoB (Fig. 4A).

496 When iron is abundant, DtxR forms a complex with ferrous iron and binds iron box

497 operator sequences to repress the transcription of downstream genes, but this

498 repression is relieved when iron is scarce (77). Transcriptional profiling of S. aureus

499 from the nasal cavity has indicated that bacteria colonizing the nose are iron-starved in

500 vivo (78). Therefore, we wanted to determine if the dehydroxynocardamine BGC is

501 expressed in vivo. As part of the human microbiome project (HMP) prediabetes study

502 (63), nasal swabs from the anterior nares were collected from 16 individuals over

503 multiple visits and processed for metatranscriptome sequencing. We analyzed the nasal

504 metatranscriptomes for expression of the dehydroxynocardamine BGC that we

505 identified and several housekeeping genes (rpoB, gyrB, sigA, and rpsL). We detected

506 dno expression in 7/16 individuals from at least one visit (mean = 2.5 visits/subject,

507 median = 1 visit/subject) with expression values ranging from 0.01 - 154.69 transcripts

508 per kilobase million (TPM) (mean = 26.94 TPM, median = 3.44 TPM). For comparison,

509 we detected expression of housekeeping genes at similar levels (Fig. 5), with the

510 exception of rpsL, which is known to be more highly expressed than the other selected

511 housekeeping genes in the model Gram-positive organism B. subtilis (79). Together,

512 these results indicate that the dehydroxynocardamine BGC is expressed in vivo.

513

514 DISCUSSION

515 In this study we investigated inhibition of nasal Staphylococcus spp. isolates by strains

516 of sympatric Actinobacteria. We uncovered two distinct patterns of interactions: 1) S.

517 epidermidis was significantly more sensitive than S. aureus to inhibition by nasal

518 Actinobacteria, and 2) there was variability among Corynebacterium spp. in their ability

519 to inhibit the growth of S. epidermidis (Fig. 1). Specifically, we noted that C. propinquum

520 more strongly inhibited S. epidermidis when compared to inhibition by other

521 Corynebacterium spp. Through a combination of comparative genomics (Fig. 2) and

522 culture-based approaches (Fig. 3), we determined that the difference in inhibitory ability

523 was due to production of a siderophore, dehydroxynocardamine (Fig. 4). By analyzing

524 metatranscriptome samples from the anterior nares of human subjects, we established

525 that the dehydroxynocardamine BGC is expressed in vivo (Fig. 5), indicating that iron-

526 mediated exploitation competition is potentially relevant to competition and organismal

527 fitness within the human nasal cavity.

528

529 One broad explanation for the difference in inhibition patterns between S. aureus

530 and S. epidermidis strains (Fig. 1B) is that S. epidermidis and other coagulase-negative

531 staphylococci (CoNS) lack the mechanisms that S. aureus uses to obtain iron from the

532 hosts and their external environment. Strains of S. epidermidis and other CoNS do not

533 grow well on iron-limited media (19) and, unlike S. aureus, are unable to scavenge iron

534 from human transferrin (80). Our finding that iron supplementation rescues S.

535 epidermidis from inhibition by C. propinquum (Fig. 3B-C) is consistent with previous

536 results showing that S. epidermidis and other CoNS are sensitive to inhibition by

537 desferrioxamine siderophores and other iron-chelating agents in vitro (81, 82). In

538 contrast to S. epidermidis, strains of S. aureus may survive siderophore-mediated

539 inhibition through production of their own carboxylate siderophores called staphyloferrin

540 A (83) and B (84). Though the genomes of some strains of S. epidermidis and other

541 CoNS encode BGCs for staphyloferrin, their siderophore activity is markedly decreased,

542 indicating lower levels of siderophore production (85). As S. epidermidis strains lack the

543 same mechanisms of iron acquisition and their siderophore production is under different

544 regulatory control than S. aureus (85), they are susceptible to iron limitation imposed by

545 C. propinquum. In addition, it has been previously reported that Staphylococcus spp.

546 are able to use catecholate and hydroxamate siderophores produced by

547 Corynebacterium spp. and other organisms (86). Perhaps in addition to higher levels of

548 siderophore production, S. aureus is also better able to pirate dehydroxynocardamine

549 produced by C. propinquum and circumvent inhibition.

550

551 Here, we note that siderophore-mediated inhibition does not fully explain the

552 contrast between the results of interactions of S. aureus and S. epidermidis with nasal

553 Actinobacteria. In addition to the three siderophore-producing strains of

554 Corynebacterium, only five of the eleven other Actinobacteria encoded siderophore

555 BGCs in their genomes (Fig. 1B). Therefore, these Actinobacteria likely use alternative

556 mechanisms to inhibit the growth of S. epidermidis, such as interference competition

557 mediated by antibiotic production, which is common among nasal bacteria (30–32).

558 Subsequent work will be required to identify the molecules responsible for inhibition of

559 S. epidermidis that are produced by non-siderophore producing nasal Actinobacteria.

560

561 Exploitation competition mediated by siderophore production is common among

562 soil and marine bacteria (87–89), but within the context of host-associated systems,

563 siderophore production is considered as a pathogen virulence factor (reviewed in 90)

564 because mutants that are unable to produce siderophores are often defective in

565 colonizing hosts and causing disease (91, 92). The view of siderophores solely as

566 pathogen-produced molecules is strengthened by lipocalin-2, a protein component of

567 the innate immune response that binds to catechol siderophores to prevent their uptake

568 by bacteria and whose production is induced by bacterial colonization of mucosal

569 surfaces (23, 24). However, thus far, little work has considered siderophore-mediated

570 competition among the microbiota and between the microbiota and pathogenic bacteria

571 (93). For instance, siderophore piracy is reported to occur between pathogens and

572 beneficial or commensal bacteria in the human gastrointestinal tract (94, 95), but

573 hitherto no such mechanisms have been reported within the human nasal cavity.

574

575 In conclusion, as 1) dehydroxynocardamine is not a known virulence factor, 2),

576 C. propinquum is considered a normal part of the nasal microbiota (33, 96, 97), and 3)

577 expression of dnoA-G was detected in vivo (Fig. 5), we propose that C. propinquum

578 produces dehydroxynocardamine as a means to mediate exploitation competition for

579 iron with other bacteria within the human nasal cavity.

580

581 ACKNOWLEDGMENTS

582 We thank members of the Cameron Currie, James Gern, and Paul Straight

583 (Texas A&M University) laboratories for feedback on this manuscript and project. We

584 thank Aaron Stubbendieck for 3D printing stamps for rapid inoculation of co-culture

585 inhibition assays. We thank Samuel McInturf for thoughtful discussion and assistance in

586 R scripting. We thank Drs. Robert Lemanske Jr. and Daniel Jackson (University of

587 Wisconsin-Madison), who are the principal investigators of the COAST birth cohort

588 study, for providing nasal samples from which bacteria were isolated.

589 This work, including the efforts of Cameron R. Currie and James E. Gern, was

590 funded by the NIH Centers for Excellence for Translational Research (U19-AI109673-

591 01) and the NIH National Heart, Lung, and Blood Institute (P01 HL070831),

592 respectively. The funders had no role in study design, data collection and interpretation,

593 or the decision to submit the work for publication.

594

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909 FIGURE LEGENDS AND TABLES 910 911 Table 1. Corynebacterium propinquum siderophore BGC annotation.

ORF Size KO Annotation Desferrioxamine E (nt) BGC Homologue (E-Value*)

dnoA 1074 02016 Iron-siderophore n/a ABC transporter substrate-binding protein

dnoB 1539 13745 PLP-dependent dfoJ (4e-116) decarboxylase

dnoC 1152 03897 L-lysine N6- dfoA (1e-119) monooxygenase

dnoD 2553 n/a siderophore dfoC (2e-153) biosynthetic enzyme

dnoE 1062 02015 Ferric siderophore n/a ABC transporter permease

donF 1110 02015 Siderophore ABC n/a transport system permease

dnoG 840 02013 Siderophore n/a transport system ATP-binding protein 912 *E-value from blastp protein sequence alignment.

Staphylococcus Monoculture

Actinobacteria + Staphylococcus Corynebacterium spp.

B. subtilis S. epidermidis

S. aureus

B. subtilis bioRxiv preprint doi: https://doi.org/10.1101/432948; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Figure 1. Inhibition of nasal Staphylococcus spp. by nasal Actinobacteria. (A)

Representative images of monocultures of Staphylococcus and co-cultures of

Actinobacteria (left) with Staphylococcus spp. isolates (right). The inhibition scoring

system is depicted below the co-culture images. (B) 21 nasal Actinobacteria isolates

(horizontal) were monocultured on BHI agar wells for 1 week before 39 Staphylococcus

spp. isolates (vertical) were spotted adjacent to the Actinobacteria colony. The colonies

were cultured together for one week before inhibition of the Staphylococcus spp. colony

was scored. The heat map displays the inhibition scores of each Staphylococcus spp.

isolate when paired with the corresponding Actinobacteria isolate. Each interaction was

technically replicated at least twice. The gray cells indicate interactions where replicates

were in disagreement or the Actinobacteria colony overgrew the well before

Staphylococcus inoculation. (left) Phylogenetic tree of Staphylococcus spp. isolates built

from the 16S rRNA gene. (top) Core-genome phylogenetic tree of Actinobacteria built

from 93 conserved, single copy genes. Both phylogenies are rooted on B. subtilis 168

and nodes with ≥50% bootstrap support are indicated. The dashed yellow lines highlight

strong inhibition of S. epidermidis by one clade of Corynebacterium spp. Actinobacteria

taxa marked with the ● symbol encode a siderophore BGC within their genome. bioRxiv preprint doi: https://doi.org/10.1101/432948; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

C. propinquum C. genitalium

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v C GM L) v) B) bioRxiv preprint doi: https://doi.org/10.1101/432948; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Figure 2. Comparative genomics of nasal Corynebacterium spp. (A) (left) The core-

genome phylogeny of nasal Corynebacterium from Fig. 1. The node corresponding to

other Actinobacteria is collapsed and represented by a triangle. The C. propinquum

clade is shaded in orange. (right) Clustered presence-absence matrix of 1504

orthologues predicted across the 10 Corynebacterium spp. genomes. The 1198

orthologues that were conversed across all 10 genomes were excluded. Orthologs

encoding functions pertaining to iron metabolism, iron transport, or siderophore

biosynthesis are depicted in red. (B) KO term enrichment analysis of the orthologues

from highlighted box in panel A, relative to the other orthologues. The odds ratios of all

12 significantly enriched (p < 0.05) KO terms are plotted. Note, an odds ratios of infinity

(Inf) correspond to KO terms where all orthologues that were annotated with the KO

term were present in the enrichment set. KO terms in bold are related to iron transport

or siderophore biosynthesis. The color of each point indicates significance level and the

size of the point indicates how many of the orthologues were annotated with the KO

term. (C) Growth assay of Corynebacterium spp. strains that either weakly (C.

genitalium) or strongly (C. propinquum) inhibited S. epidermidis on BHI and iron-limited

SNM3.

C. propinquum C. genitalium

−

S. epidermidis C. propinquum Corynebacterium bioRxiv preprint doi: https://doi.org/10.1101/432948; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Figure 3. Siderophore production by C. propinquum inhibits S. epidermidis. (A)

CAS assay results from C. propinquum HSID18034 (left) and C. genitalium HSID17239

(right), which strongly and weakly inhibit S. epidermidis, respectively. A shift in the color

of the CAS overlay from blue to yellow indicates siderophore production. (B) Co-culture

plate inhibition between the strains in A and S. epidermidis on BHI (−Iron) and BHI

supplemented with 200 µM FeCl3 (−Iron). (C) Co-culture plate inhibition assays were

performed between C. propinquum strains (HSID18034 and HSID18036) and five

Corynebacterium spp. non-siderophore-producers (HSID17231, HSID17239,

HSID17260, HSID17564, HSID17575) against 22 strains of S. epidermidis on BHI

(−Iron) and BHI supplemented with 200 µM FeCl3 (+Iron). Each pairing was duplicated

with consistent results. The error bars represent the standard error of the mean. The

photographs in A and B are representative of duplicate samples. bioRxiv preprint doi: https://doi.org/10.1101/432948; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

′′

dnoA dnoB dnoC dnoD dnoE dnoF dnoG

k k bioRxiv preprint doi: https://doi.org/10.1101/432948; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Figure 4. Corynebacterium propinquum produces the siderophore

dehydroxynocardamine. (A) The dehydroxynocardamine BGC from C. propinquum.

ORFs encoding biosynthetic and transport-related functions are filled with gray and

white, respectively. See Table 1 for specific ORF annotation. The DtxR sequence motif

is shown upstream of dnoB. (B) Subset of the molecular network of C. propinquum and

C. genitalium agar core extracts. Grey nodes are metabolites shared by both strains

and black nodes are metabolites unique to C. propinquum. There were no detected

metabolites that were unique to C. genitalium. The edges are weighted to the cosine

score between the two features. A single cluster unique to C. propinquum is outlined in

a dashed box. See Fig. S2 for the full molecular network (C) Zoomed in view of the

single cluster unique to C. propinquum with the nodes labeled by their corresponding

m/z. This cluster contains single and double charged states of dehydroxynocardamine,

dehydroxynocardamine B, and dehydroxynocardamine C. (D) Structures of

dehydroxynocardamine, dehydroxynocardamine B, and dehydroxynocardamine C. certified bypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmadeavailableunder bioRxiv preprint doi: https://doi.org/10.1101/432948 pB yBsigA rpoBgyrB a CC-BY-NC-ND 4.0Internationallicense ; this versionpostedOctober2,2018. rpsL The copyrightholderforthispreprint(whichwasnot . bioRxiv preprint doi: https://doi.org/10.1101/432948; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Figure 5. The dehydroxynocardamine BGC is expressed in vivo. 95 nasal

metatranscriptomes from 16 subjects in the NIH HMP were pseudoaligned to the

genome of C. propinquum HSID18034 to detect expression of the

dehydroxynocardamine BGC (donA-G, white) and housekeeping genes gyrB, rpoB,

sigA, and rpsL (gray). The upper and lower bounds of the notched box plot indicate the

75th and 25th percentiles, respectively. The bars indicate the median and the notches

represent the 95% confidence interval of the median. Each dot indicates the expression

of a single gene from a metatranscriptome in transcripts per kilobase million (TPM).

Corynebacterium glucuronolyticum Corynebacterium glucuronolyticum Corynebacterium glucuronolyticum Corynebacterium jeikeium Corynebacterium jeikeium Corynebacterium amycolatum Corynebacterium jeikeium Corynebacterium mycolatum Corynebacterium mycolatum Corynebacterium glutamicum Corynebacterium glutamicum Corynebacterium glutamicum Corynebacterium matruchotii Corynebacterium matruchotii Corynebacterium pseudotuberculosis Corynebacterium pseudotuberculosis Corynebacterium pseudotuberculosis Corynebacterium pseudotuberculosis Corynebacterium diphtheriae Corynebacterium diphtheriae Corynebacterium diphtheriae Corynebacterium diphtheriae e Corynebacterium diphtheriae Corynebacterium genitalium Corynebacterium accolens Corynebacterium accolens Corynebacterium aurimucosum e Corynebacterium tuberculostearicum Corynebacterium pseudogenitalium Corynebacterium aurimucosum ATCC7 Corynebacterium aurimucosum Corynebacterium accolens Corynebacterium striatum Corynebacterium striatum Corynebacterium striatum Corynebacterium propinquumee Corynebacterium propinquumee Corynebacterium propinquumee Corynebacterium propinquumee Corynebacterium pseudodiphtheriticume Corynebacterium pseudodiphtheriticume Bacillus subtilis bioRxiv preprint doi: https://doi.org/10.1101/432948; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Figure S1. Core-genome cladogram of human-associated Corynebacterium spp.

Core-genome cladogram of 55 human-associated Corynebacterium spp. built from 93

conserved, single copy genes. The strains of Corynebacterium spp. used in this study

are in bold. The cladogram is rooted on B. subtilis 168 and nodes with less than 50%

bootstrap support were collapsed. Nodes with ≥50% bootstrap support are indicated.

Figure S2. Full molecular network of C. propinquum and C. genitalium agar core

extracts. Grey nodes are metabolites shared by both strains and black nodes are

metabolites unique to C. propinquum. The edges are weighted to the cosine score

between the two features. bioRxiv preprint doi: https://doi.org/10.1101/432948; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Figure S3. Tandem mass spectra of dehydroxynocardamine variants. The tandem

mass spectra of (A) dehydroxynocardamine, (B), dehydroxynocardamine B, and (C)

dehydroxynocardamine C are shown. The insets show the structure of the

dehydroxynocardamines and their molecular weights. bioRxiv preprint doi: https://doi.org/10.1101/432948; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Table S1 Strains of nasal Actinobacteria and Staphylococcus used in this study. Bolded strains indicate Actinobacteria with sequenced genomes.

Strain Host Genus Species

HSID16896 HID3263 Staphylococcus warneri/pasteuri HSID16901 HID3258 Kocuria

HSID17227 HID3266 Micrococcus

HSID17228 HID3263 Micrococcus HSID17231 HID3232 Corynebacterium pseudodiphtheriticum HSID17239 HID3265 Corynebacterium genitalium HSID17241 HID3232 Corynebacterium pseudodiphtheriticum HSID17244 HID3229 Staphylococcus warneri/pasteuri HSID17245 HID3232 Micrococcus HSID17249 HID3262 Staphylococcus warneri/pasteuri HSID17250 HID3263 Staphylococcus epidermidis HSID17251 HID3263 Staphylococcus epidermidis HSID17252 HID3266 Staphylococcus epidermidis HSID17253 HID3266 Staphylococcus aureus HSID17254 HID3266 Microbacterium

HSID17255 HID3232 Staphylococcus warneri/pasteuri HSID17257 HID3229 Curtobacterium

HSID17260 HID3229 Corynebacterium pseudodiphtheriticum HSID17554 HID3235 Dermabacter

HSID17556 HID3266 Staphylococcus aureus HSID17557 HID3263 Staphylococcus epidermidis HSID17560 HID3264 Staphylococcus epidermidis HSID17561 HID3261 Staphylococcus aureus HSID17562 HID3263 Staphylococcus epidermidis HSID17564 HID3264 Corynebacterium pseudodiphtheriticum HSID17565 HID3261 Staphylococcus aureus HSID17567 HID3583 Staphylococcus epidermidis HSID17574 HID3242 Staphylococcus aureus HSID17575 HID3230 Corynebacterium pseudodiphtheriticum HSID17576 HID3232 Corynebacterium pseudodiphtheriticum HSID17578 HID3584 Staphylococcus warneri/pasteuri bioRxiv preprint doi: https://doi.org/10.1101/432948; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

HSID17579 HID3242 Staphylococcus aureus HSID17580 HID3233 Staphylococcus aureus HSID17581 HID3266 Staphylococcus aureus HSID17582 HID3232 Kocuria

HSID17585 HID3235 Staphylococcus aureus HSID17586 HID3243 Staphylococcus aureus HSID17587 HID3242 Staphylococcus aureus HSID17589 HID3233 Staphylococcus aureus HSID17590 HID3232 Kocuria

HSID17591 HID3261 Staphylococcus aureus HSID17593 HID3242 Staphylococcus aureus HSID17596 HID3266 Staphylococcus aureus HSID17597 HID3266 Staphylococcus epidermidis HSID18025 HID3583 Staphylococcus epidermidis HSID18026 HID3583 Staphylococcus epidermidis HSID18027 HID3583 Staphylococcus epidermidis HSID18028 HID3583 Staphylococcus epidermidis HSID18029 HID3583 Staphylococcus epidermidis HSID18030 HID3583 Staphylococcus epidermidis HSID18034 HID3583 Corynebacterium propinquum HSID18035 HID3583 Corynebacterium propinquum HSID18036 HID3583 Corynebacterium propinquum HSID18037 HID3583 Staphylococcus epidermidis HSID18038 HID3583 Staphylococcus epidermidis HSID18039 HID3583 Staphylococcus epidermidis HSID18040 HID3583 Staphylococcus epidermidis HSID18041 HID3583 Staphylococcus epidermidis HSID18067 HID3240 Rothia

HSID18069 HID3238 Rothia

HSID18541 HID3780 Staphylococcus epidermidis HSID18545 HID3779 Staphylococcus epidermidis HSID18588 HID3767 Staphylococcus epidermidis HSID18590 HID3769 Staphylococcus epidermidis HSID18603 HID3780 Staphylococcus epidermidis bioRxiv preprint doi: https://doi.org/10.1101/432948; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

HSID18614 HID3757 Staphylococcus epidermidis HSID18654 HID3758 Staphylococcus epidermidis HSID18993 HID3777 Staphylococcus epidermidis HSID19050 HID3773 Staphylococcus epidermidis HSID19074 HID3790 Staphylococcus epidermidis HSID19079 HID3803 Staphylococcus epidermidis