24h

pup dop pafA prcA

48h

prcB arc

72h

Fig. S1: RT-PCR (A) and RT-qPCR (B) analysis of transcription of the pupylation and proteasome genes.

We detected transcripts for the pupylation (pup/dop/pafA) and proteasome (prcA/prcB/arc) genes at various time

points (24, 48 and 72h).

# no significant difference was observed between the two FC (Wilcoxon rank sum tests, p-value < 0.05)

FC 24h/72h 10,31+/-4,60 2,62+/-0,58 1,64+/-0,39 3,49+/-0,75 3,35+/-1,06 1,78+/-0,63 4,08+/0,75

FC 48h/72h 2,85+/-0,56 2,13+/-0,16 1,56+/-0,24 1,74+/-0,29 1,63+/-0,29 0,95+/-0,23 1,85+/0,11

0

2

4

6

8

10

12

14

16

arc dop pup sco1645 prcB prcA pafA

Fo

ld c

han

ge (

FC

)

FC 24h/72h FC 48h/72h

# #

B

A Figure S1

M. tuberculosis H37rv M. smegmatis S. coelicolor R. erythropolis

A. SCO6042 = unknown protein

Figure S2

Figure S2: Alignment of homologous pupylated proteins in actinobacteria, with their sites of pupylation.

A. SCO6042 and its homologues. B. SCO3629 and its homologues. Pupylated sites are indicated by arrows..

B. SCO3629=PurA

C. glutamicum

J+5

J+7

J+10

Figure S3

Figure S3: Morphological differentiation on SFM solid medium. Plates on which the wild-type strain (M145),

the pup mutant (∆pup), the pup mutant with the empty vector pSET152 (∆pup+pSET152), the pup mutant with the

vector pSET-E*-His-pup (∆pup+pSET-E*His-pup) and the proteasome mutant (prcB::pOJ260) were grown are

shown after 5, 7 and 10 days of cultivation.

Figure S4: Scanning electron microscopy of cultures on SFM. The wild type strain (M145), the pup mutant

(∆pup), the pup mutant with the empty vector pSET152 (∆pup+pSET152), the pup mutant with the vector pSET-

E*-His-pup (∆pup+pSET-E*His-pup) and the proteasome mutant (prcB::pOJ260) are observed.The magnification

is the same for all images and the scale bar represents 10 µm.

Figure S4

Strains Diameter

(cm)

wt 6.8+/-0.14

pup 6.25+/-0.07

pup + pSET152 6.7+/-0.28

pup + pSET-

E*His-pup

6.9+/-0.14

prcB::pOJ260 6.35+/-0.21

B

1 cm

Figure S5

Figure S5: Calcium-dependent antibiotic production. The wild-type strain (M145), the pup mutant (∆pup),

the pup mutant with the empty vector pSET152 (∆pup+pSET152), the pup mutant with the vector pSET-E*-

His-pup (∆pup+pSET-E*His-pup) and the proteasome mutant (prcB::pOJ260) were grown on ONA medium in

the presence of Ca(NO3)2. A. Inhibition zone using Micrococcus luteus as the indicator strain (Scale bar 1 cm).

B. Diameter of the inhibition zone.

∆pup M145 ∆pup+

pSET152

∆pup+pSET-E*His-pup prcB::pOJ260

A

Supplemental information 1

2

Materials and methods 3

Reverse transcription and PCR 4

RNA was isolated from S. coelicolor M145 wild type strain grown at 30°C on cellophane 5

disks laid on NE (1% glucose, 0.2% yeast extract, 0.2% meat extract, 0.2% casamino acids, 6

pH 7) solid medium. The RNA was purified with RNeasy Mini kits (Qiagen) and treated with 7

Turbo DNase (Turbo DNA-free kit, Ambion). Reverse transcription was performed with 8

Transcriptor Reverse Transcriptase (Roche). GoTaq polymerase (Promega) was used for 9

standard PCR, with the following cycling conditions: 95 °C/3 min; 32 cycles of 95 °C/45 sec, 10

58 °C/45 sec, 72 °C/45 sec; then 72 °C/5 min. The primers used are detailed in Table S2. 11

Each primer pair was tested with purified S. coelicolor genomic DNA as a template. The 12

absence of DNA contamination in each RNA sample was assayed with each primer pair by 13

using twice the amount of RNA used to generate the corresponding cDNA. 14

For quantitative (q)PCR, specific primers (Table S2) were designed with BEACON 15

Designer 4.02 software (Premier Biosoft International, Palo Alto, CA). RNA was extracted 16

from cells cultivated at 30°C with shaking at 200 rpm in R2YE liquid medium, and 20 µg 17

aliquots were treated with 2 Units of DNase I (TURBO DNA-free reagent, Ambion) twice for 18

30 min each time at 37°C. cDNA synthesis and quantitative real-time PCR were performed 19

as described by Bellier et al. (2) with the following modifications. PCR involved an initial 20

denaturation step at 95°C for 15 min, followed by 40 cycles of amplification (95°C for 15 s, 21

55°C for 30 s and 72°C for 30 s). Each assay was performed in triplicate at least and 22

repeated with at least two independent RNA samples. The critical threshold cycle (CT) was 23

defined for each sample. The expression levels of the tested genes were normalized using 24

the hrdB gene encoding the housekeeping RNA polymerase principal sigma factor, a gene 25

classically used as reference in RT-PCR experiments in Streptomyces. The change (n-fold) 26

in the amount of transcript was calculated using the following equations: : CT = CT(test DNA) – 27

CT(hrdB) and ratio = 2CT (8). The significance of fold changes was tested using Wilcoxon rank 28

sum statistical tests. 29

30

Exploitation of RNAseq datasets 31

We used different S. coelicolor M145 RNAseq datasets for available in the GEO NCBI 32

database (1). Moody and collaborators used RNA extracted from mycelium grown on 33

maltose, yeast extract, malt extract media (MYM) and isolated at various time points (from 34

vegetative growth to sporulation, datasets GSM1121652 and GSM1121655) (10). Nasser 35

and collaborators used RNA extracted from mycelium grown on R2YE solid medium after 1 36

or 3 days of growth (datasets GSM1378112, GSM1378113 and GSM1378114) (12). The 37

SRA data were converted to BAM format by using Sratoolkit.2.5.1 38

(https://github.com/ncbi/sra-tools) and bowtie2 (6). Mapping of reads was done on the region 39

from SCO1649 to SCO1638 with Tablet 1.14.10.20 (9). 40

41

42

Table S1: bacterial strains and plasmids used 43

Strain or

plasmid

Description Reference

E. coli

ET12567

pUZ8002

strain used for intergeneric conjugation between E.

coli and S. coelicolor

(5)

DH5α host strain for cloning experiments Promega

S. coelicolor

M145 SCP1− SCP2− (5)

pup M145 with the pup deletion. This work

pup+

pSET152

pup deletion mutant harboring the empty vector

pSET152.

This work

pup+ pSET-

E*-His-pup

pup deletion mutant harboring the vector pSET-E*-

His-pup.

This work

prcB::pOJ260 disruption of prcB transcription by insertion of

pOJ260-prcB vector (aprar).

This work

Plasmids

pGEM-T

Easy

High copy number (ampir) vector with a polycloning

site within lacZ gene.

Promega

pGEM-T

Easy-pup-BG

pGEM-T Easy (ampir) with 2kb DNA fragment

corresponding to the upstream region of pup (PCR

product, primers used HBP32-HBP59).

This work

pGEM-T

Easy-pup-BD

pGEM-T Easy with 2kb DNA fragment corresponding

to the downstream region of pup (PCR product,

primers used HBP30-HBP58).

This work

pGEM-T

Easy-prcB

pGEM-T Easy with internal fragment of prcB gene

(PCR product, primers used HB88-HB89).

This work

pOSV234 Source of the excisable apramycin cassette (att3

aacC4) used for the pup deletion.

(13)

pOSV236 Plasmid containing the xis and int genes for site

specific excision of the att3 aacC4 cassette (ampir,

thior).

(13)

pOSV400 Shuttle E.coli/Streptomyces (OriT, hygror) used as

suicide vector in Streptomyces.

F. Lorieux,

unpublished

pOSV400-

pup

Insertion into the HindIII-BamHI sites of pOSV400 of

the apramycin cassette (att3 aacC4 from pOSV234)

flanked by the pup upstream and downstream regions

(respectively from pGEM-T Easy-pup-BD and pGEM-T

Easy-pup-BG).

This work

pHM11a E. coli/Streptomyces integrative shuttle vector. (11)

pSET152 E.coli/Streptomyces integrative shuttle vector. (3)

pSET-E*-His-

pup

pSET152 with a modified pup gene encoding a protein

with a His6 N-terminal tag. pup transcription is under

the control of the strong promoter ermE*.

This work

pOJ260 Shuttle E.coli/Streptomyces (OriT, aprar) used as

suicide vector in Streptomyces.

(3)

pOJ260-prcB pOJ260 with an internal fragment of the prcB gene

(557nt, primers HBP88-HBP89).

This work

ampir: ampicillin resistance; aprar: apramycin resistance, hygror: hygromycin resistance, thior: 44

thiostrepton resistance 45

46

Table S2: primers used 47

Primers Sequence 5' to 3’ Used for

HBP88 CGGGTCCTCATCCTTCATGG amplification of a sequence internal to

the prcB gene HBP89 CACGGAACAGCTTCTTCATC

HBP32 AAGCTTACACGGTGGCAAAC amplification of the upstream region of

pup with HindIII site in forward

HBP59 GATATCCCCTCACCCCGCTCGGT amplification of the upstream region of

pup with EcoRV site in reverse

HBP58 GGATCCCGACGACGACAAACTG amplification of the downstream

region of pup with BamHI site in

reverse

HBP30 GATATCAAGGGCGGCGAGTAG amplification of the downstream

region of pup with EcoRV site in

forward

D5 GGATGTGCTGCAAGGCGATT Verification of the disruption of prcB

by amplification and sequencing.

Primer in lacZ of pOJ260

HBP27 TGCGACGACGACAAACTG Verification of the disruption of prcB

by amplification. Primer in prcA in

reverse orientation

HBP6 AGCCCGTCACGCATCGCCTTGTTTC Verification of the scar obtained before

and after cassette excision for deletion

in pup. In reverse orientation

HBP5 AAGCAGCCGGACAGGGTGGAATATC Verification of the scar obtained before

and after cassette excision for deletion

in pup. In forward orientation

L-acc2R CCTGTCAGTCATGCGGGCAAC Verification of the scar obtained before

cassette excision for deletion in pup. In

reverse orientation

R-acc2F GGCCGTGACTGAGGAGGTCTAC Verification of the scar obtained before

cassette excision for deletion in pup. In

forward orientation

NS9 GGAATTCCATATGCACCACCACCACCA

CCACATGGCGACCAAGGACACCGGCG

amplification of his6 pup with NdeI site

in forward orientation

NS12 CGCGGATCCTTGTGGTCCTTACACCGC

GA

amplification of his6 pup with BamHI

site in reverse orientation

MG183 CCTCGCGGAGCACGAGAAGGACAA RT PCR experiment to probe the

transcription of arc MG184 CAGTCGTCGGGGTTGGTGGTGTT

MG156 TCGAGACGGAGTACGGAAT RT PCR experiment to probe the

transcription of dop MG157 GTGGTCGACGTAGAGCCGTG

MG158 CTACTCGCCGCCCTTTTGAA RT PCR experiment to probe the

transcription of pup NS11 ATGGCGACCAAGGACACCGG

MG185 TGGAGCACTTCGAGAAGGTCGA RT PCR experiment to probe the

transcription of prcB MG186 TGATCACAGTGATGATCGGATAGA

MG152 GTTCTATGTATCTCCTCAGC RT PCR experiment to probe the

transcription of prcA MG153 CCGATCCGCAGGTTCTCGTA

MG150 TTCGGGCTGGAGAACGAGTA RT PCR experiment to probe the

transcription of pafA MG151 CTTGTCGTGGGTGACGAGTT

MG192 CTCTTCCTGGACCTCATC qRT PCR experiment to probe the

transcription of hrdB MG193 TTGTAGCCCTTGGTGTAG

MG194 AGTACAACGAGTACGAGAAC qRT PCR experiment to probe the

transcription of prcA MG195 CACGGTCGTAGGTGTAAC

MG196 AGATGGTGAAGCTGTTC qRT PCR experiment to probe the

transcription of prcB MG197 TTGCCCTCCAAGGAC

MG198 GCGTGATTTGTTTGAAGG qRT PCR experiment to probe the

transcription of SCO1645 MG199 GCAGTTGAAGCACAGTAC

MG200 TCGGAGGACCTCAAGGAA qRT PCR experiment to probe the

transcription of pup MG201 AGTACATCGTCGATCTCGTC

MG202 CACCTCTACAAGAACAACAC qRT PCR experiment to probe

transcription of dop MG203 GGTTTCCCGCTTCATCAG

MG204 TCGACGTGAAGATCAAGAT qRT PCR experiment to probe the

transcription of arc MG205 CGTTCGGTGAGGTACTTG

MG206 ATGAAGTACAAGCTGCTC qRT PCR experiment to probe the

transcription of pafA MG207 TAGGCGAGGTCTATCTGC

48

49

50

Table S3: Predictions about the pupylated lysine identified in S. coelicolor by different 51

pupylation site prediction softwares (4, 7, 14) 52

Gene locus Protein name

Pupylated lysine(s)

GPS-Pup iPUP Pup-pred

SCO1814 InhA/ FabI K177 High No Low

SCO2090 FtsI K582 No No No

SCO2318 - K293 High Medium Low

SCO2389 AcpP K31 No High Low

SCO2390 FabF K333 No High Low

SCO2950 HupA K38 K42

Low No

High No

No No

SCO3373 ClpC K431 No High No

SCO3581 - K134 No No Low

SCO3629 PurA K139 No No No

SCO3878 DnaN K182 No No No

SCO4296 GroEL2 K41 No No No

SCO4496 - K147 No High Low

SCO4662 Tuf1 K397 High Medium Medium

SCO4762 GroEL1 K338 K490

No Low

Low No

Low No

SCO5032 AhpC K31 No No No

SCO5679 - K351 No High No

SCO5748 OsaA K813 High Medium Low

SCO6042 - K62 No High Medium

SCO7343 HemC1 K113 No No No

SCO7442 - K22 No No No

Total - 22 6 11 10 high, medium and low prediction scores are accounted in the total. 53

54

55

Supplemental References: 56

57

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10. Moody, M. J., R. A. Young, S. E. Jones, and M. A. Elliot. 2013. Comparative analysis of non-80 coding RNAs in the antibiotic-producing Streptomyces bacteria. BMC Genomics 14:558. 81

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14. Tung, C. W. 2013. Prediction of pupylation sites using the composition of k-spaced amino 89 acid pairs. J Theor Biol 336:11-7. 90

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93