A antibiotic modulates human skin microbiota composition in hair follicles.

Supplementary Material
Figure S1Competition assay between C. acnes and S. aureus under conditions mimicking the natural follicle environment.

Figure S2HPLC chromatogram of cutimycin purification.

Figure S3Low energy mass spectrum for cutimycin (calculated ([M+H]+ 1131.3374 for C51H51N14O15S+) isolated from A) the heterologous C. glutamicum producer ([M+H]+ 1131.3411) and B) from C. acnes HL030PA1 ([M+H]+ 1131.3364).

Figure S4High energy mass spectrum for cutimycin labeled with predicted fragments by Unifi.

Figure S51H NMR Spectrum of cutimycin taken in DMSO-d6 at 600 MHz.

Figure S613C NMR Spectrum of cutimycin taken in DMSO-d6 at 151 MHz.

Figure S71H-1H COSY NMR in DMSO-d6.

Figure S81H-13C HSQC NMR in DMSO-d6.

Figure S91H-13C HMBC NMR in DMSO-d6.

Figure S10Zoom in of 1H-13C HMBC NMR in DMSO-d6 identifying dehydroalanine residues through presence of diastereotopic terminal alkene protons (position 20, δH 5.74, 5.75 and position 24 δH 5.73, 6.35) with HMBC correlations to carbon atoms 19 and 23 respectively.

Figure S11Zoom in of 1H-13C HMBC NMR in DMSO-d6 showing the weak four-bond HMBC correlation from position 13 to quaternary carbon 16 at δC 139. 13.

Figure S13Marfey’s analysis of cutimycin. Individual amino acid Marfey’s analysis derivatives, cutimycin hydrolysis product derivatives, and coinjections.

Figure S12The subunits of cutimycin with key HMBC (arrows) and COSY (bold bonds) correlations.

Figure S14Structure of cutimycin

Figure S15The structures of A) cutimycin, B) berninamycin A, and C) LFF-571.

Figure S16Mass spectrometric detection of cutimycin from pooled human follicular content (n=60) for one of the tested samples.

Figure S18Box plots of the impact of the cutimycin BGC presence/absence on C. acnes, C. granulosum and S. epidermidis CFUs in human skin follicles.

Figure S19Linear depiction of cutimycin with the key mass spec fragments labeled (Table S9).

Figure S17Metagenomic analysis of C. acnes BGCs at 17 skin sites from sites from 12 healthy participants.

Table S9The key fragments confirming the order of the amino acids in cutimycin.

Table S1Cutimycin BGC presence/absence in 219 Cutibacterium genomes

Table S2NMR Data of cutimycin taken in DMSO-d6 at 600 MHz and 151 MHz.

Table S3Minimal inhibitory concentrations for cutimycin and bernamycin

Table S4Cutimycin in human follicular samples has been detected in 28% of all samples across two separate experiments.

Table S5Locus tags for the C. acnes BGCs assayed for in skin metagenomic data.

Table S6Cutimycin BGC presence/absence; the CFUs of C. acnes, C. granulosum and S. epidermidis; and the C. acnes/S. epidermidis CFU ratio in the content of individual human skin follicles.

Table S7Bacterial strains and plasmids used in this study.

Table S8Primers used in this study.

Supplementary