Quantitative proteomics revealed the nature and cause of the different metabolic features underpinning weak and strong antibiotic producing abilities of two model Streptomyces species. SFEAP 201

David, Michelle

Henry, Celine

Lejeune, Clara

Millan Oropeza, Aaron

Virolle, Marie-Jöelle

English

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Quantitative proteomics revealed the nature and cause
of the different metabolic features underpinning weak
and strong antibiotic producing abilities of two model
Streptomyces species
Aaron Millan Oropeza, Celine Henry, Clara Lejeune, Michelle David,
Marie-Jöelle Virolle
To cite this version:
Aaron Millan Oropeza, Celine Henry, Clara Lejeune, Michelle David, Marie-Jöelle Virolle. Quantita-
tive proteomics revealed the nature and cause of the different metabolic features underpinning weak
and strong antibiotic producing abilities of two model Streptomyces species. SFEAP 2019, Société
Française d’Electrophorèse et d’Analyse Protéomique (SFEAP). FRA., Sep 2019, Strasbourg, France.
pp.1. ￿hal-02342396￿
Aaron Millan-Oropeza1, Céline Henry1, Clara Lejeune2, Michelle David2, Marie-Jöelle Virolle2
1 PAPPSO, Micalis Institute, Institut National de la Recherche Agronomique, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, France.
2 Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, F-91198, Gif-sur-Yvette cedex, France.
Polymeric solid phase 
extraction  (Strata-X)
Orbitrap Fusion™ Lumos™ Tribrid™ 
(Thermo Fischer Scientific)
- 50 cm column, 216 min analysis 
- HCD mode
Experimental design (48 samples): 
- 2 strains (S. coelicolor, S. lividans)
- 2 main carbon sources (Glucose, Glycerol) 
- 3 time points (36h, 48h, 72h)
- 4 independent replicates
[1] Le Marechal P, Decottignies P, Marchand CH, Degrouard J, Jaillard D, Dulermo T, Froissard M, Smirnov A, Chapuis V, Virolle M. (2013). Comparative proteomic analysis of Streptomyces lividans wild-type and ppk mutant strains reveals the 
importance of storage lipids for antibiotic biosynthesis. Appl Environ Microbiol, 79(19): 5907-17.
[2] Millán-Oropeza A. (2017). Comparative study of the proteome of S. coelicolor M145 and S. lividans TK24, two phylogenetically closely related strains with very different abilities to accumulate TAG and produce antibiotics. Université Paris-Saclay. NNT : 
2017SACLS160.
[3] Langella O, Valot B, Balliau T, Blein-Nicolas M, Bonhomme L, Zivy M. (2017). X!TandemPipeline: a tool to manage sequence redundancy for protein inference and phosphosite identification. J Proteome Res. 16(2): 494–503.
[4] Valot B, Langella O, Nano E, Zivy M. (2011). MassChroQ: A versatile tool for mass spectrometry quantification. Proteomics. 11(17): 3572–7
Total proteins
extract 
X!Tandempipeline* [3] was used for 
identification and quantification of 
number of MS2 spectra (Spectral 
counting)
Spectral counting : suitable for detecting presence/absence of a given protein. 
XIC : This method provides high sensitivity to detect small abundance variations. 
Quantitative proteomics revealed the nature and cause of the different metabolic features 
underpinning weak and strong antibiotic producing abilities of two model Streptomyces species
Contact: aaron.millan-oropeza@inra.fr
Double digestion 
(LysC / Trypsin) of 
80µg of proteins
1 µg peptides
LC-MS/MS 
Proteins 
identification 
Proteins 
quantification MassChroQ* [4] was used for peptide 
quantification by area integration on 
eXtracted Ion Chromatogram (XIC)
Statistical and 
data analysis 
were carried 
out on R*. 
Differential 
analysis
* FREE and OPEN Source software
CONTEXT OF STUDY
METHODOLOGY
CONCLUSION
RESULTS
The Streptomyces genus is well known for its ability to produce numerous and diverse bio-active molecules useful to human health. The biosynthesis of these specialized metabolites usually occurs when growth 
slows down or stops and is triggered by nutritional limitations, especially by phosphate. 
Two model species commonly used to understand the biosynthesis of these bio-active metabolites are S. coelicolor M145 and S. lividans TK24. These species are closely related (95% of orthologous genes) but 
exert different abilities to produce three well characterized secondary metabolites (CDA, RED, ACT). In presence of glucose as main carbon source, the high antibiotic production of S. coelicolor was 
correlated with a low lipid content whereas S. lividans showed a poor ability to synthetize antibiotics that was correlated with higher lipid content [1]. 
Despite numerous important scientific contributions over the past 40 years, a systemic understanding of the biosynthesis of these bio-active metabolites and the metabolic feature characterizing the producing 
bacteria remains incomplete.  To progress on the field, a label-free shotgun comparative proteomic analysis was carried out in  S. coelicolor M145 and S. lividans TK24 cultivated under different carbon sources 
and studied at different times of culture [2]. 
Cultures on solid media showed, in most cases, a reverse correlation 
between the ability to accumulate lipids (FAMES) and that to 
synthesize antibiotics (ACT). The sole exception was S. coelicolor 
cultivated on glycerol, that produced both lipids and antibiotics.
Statistical analysis of spectral counts and XICs allowed 
to detect 1040 proteins with significant abundance 
variation according to strain, medium and/or time 
(ANOVA, adjusted p-value < 0.05).  These proteins 
belonged to 13 functional categories according to their 
annotation in the databases.
A total of 4372 proteins were identified. This represents the 
largest dataset for S. coelicolor and S. lividans with >52% of 
their theoretical proteomes.
The profile of 30 proteins involved in phosphate metabolism showed higher 
abundance in S. lividans than in S. coelicolor. These proteins included the 
two component system (TCS) PhoR/PhoP, responsible for the positive and 
negative regulation of phosphate and nitrogen assimilation, respectively.
A total of 80 proteins related to 
antibiotics biosynthesis showed 
significant abundance change 
(ANOVA, adjusted p-value 
<0.05). 
These proteins corresponded to 
17 different secondary 
metabolite biosynthetic clusters. 
IDENTIFIED PROTEINS STATISTICALLY SIGNIFICANT 
PROTEINS
ANTIBIOTICS BIOSYNTHESIS
PHOSPHATE METABOLISM
PROPOSED MODEL 
295  350  395Spectralcounting
XIC
S. coelicolor
Glucose
S. coelicolor
Glycerol
S. lividans
Glucose
S. lividans
Glycerol
S. lividans
S. coelicolor
Glucose
Glycerol
73 71
  86 219 32 182    31
   87        162
  
    3225
33      76          55    26
   14
* This work is pioneering in the elucidation of the basis of the metabolic differences underlying the drastically different 
abilities of S. coelicolor  and S. lividans to produce antibiotics. 
* The low abundance of the TCS PhoR/PhoP in S. coelicolor compared to S. lividans led to an alleviation of its regulatory role 
likely to be responsible of the specific metabolic features of this strain (see model). 
* A novel view of the role of the antibiotics in the physiology of the producing bacteria was proposed. These molecules would 
play an important role in the regulation of the energetic metabolism of the bacteria in condition of phosphate scarcity.
PHENOTYPES
S. lividans   S. coelicolor
The low P and high N availability in S. coelicolor supported the activation of 
the Krebs cycle and thus the oxidative metabolism, resulting in high ATP. 
The limitation of P altered respiratory chain resulting into electrons leaking 
towards alternative acceptors, the induced response is the production of the 
specialized metabolite ACT (‘antibiotic’) in order to capture the electrons of 
the respiratory chain and adjust ATP. This would reduce oxidative stress.
1 2 3
4
5
6
PhoP
regulator
PhoP
regulator
Phosphate uptake 
and scavenging
Phosphate 
availability
Nitrogen 
assimilation
Nitrogen 
limitation
Slow-down of 
Krebs cycle
Glycolytic  
metabolism
- acetylCoA 
- Gly3P
+ Lipids accumulation
- NO antibiotics
- Antibiotics biosynthesis
- NO Lipids 
Phosphate uptake 
And scavenging  
Phosphate 
limitation
Nitrogen 
assimilation
Nitrogen 
availability
Activation of 
Krebs cycle
Oxidative  
metabolism
- ATP 
- oxidative stress


Quantitative proteomics revealed the nature and cause of the different metabolic features underpinning weak and strong antibiotic producing abilities of two model Streptomyces species

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Southern Illinois University Edwardsville

Southern Illinois University EdwardsvilleSPARKSIUE "Bulletin" University Archives and Special Collections12-4-1995Edwardsville Bulletin: December 4, 1995Southern Illinois University EdwardsvilleFollow this and additional works at: http://spark.siue.edu/bulletinThis Article is brought to you for free and open access by the University Archives and Special Collections at SPARK. It has been accepted for inclusionin SIUE "Bulletin" by an authorized administrator of SPARK. For more information, please contact gpark@siue.edu.Recommended CitationSouthern Illinois University Edwardsville, "Edwardsville Bulletin: December 4, 1995" (1995). SIUE "Bulletin". 565.http://spark.siue.edu/bulletin/565  X b■ ■  ■SIUE BULLETo th e  Faculty, Staff a n d  Students of Southern Illinois University a t  EdwardsvilleVol. 26, No. 19 December 4, 1995MEMO TO: The University Community FROM: Nancy BelckSUBJECT: Appointment of Vice C h ^ ce llo r for Student AffairsIt is a pleasure to announce the appointment of Narbeth R. Emmanuel to the office of Vice Chancellor for Student Affairs, effective January 1, 1996. Please come meet him this afternoon from 3:30 to 5:30 at the Faculty and Staff Holiday Open House in theMeridian Ballroom. The appointment has been approved by the Chancellor and issubject to formal ratification by the Board of Trustees. Dr. Emmanuel will succeed Harvey Welch, Vice President for Student Affairs at Southern Illinois University at Carbondale, who has been on loan to us on a part-time basis.Dr. Emmanuel has served with distinction as vice president at Ferris State for the past five years. Of special note is his selection as the 1996 Outstanding Performance Award given by the National Association of Student Personnel Administrators, Region IV-East. Recently, he was recognized by the Ferris State student newspaper for his innovative residence halls program and his leadership in promoting harmonious race relations.Previously, Dr. Emmanuel was assistant vice president for student affairs at the University of Vermont, after serving seven years as associate dean of students and one year as interim dean of students. At SIUE, he will be responsible for Campus Recreation, the Carol Kimmel Student Leadership Development Center, Counseling Service,Disability Support Services, Early Childhood Center, Health Service, Information and ID Card Center, International Student Services, Office of Campus Life, Student Publications, University Center, and University Housing.He completed his baccalaureate and master's of education degrees and a doctorate in educational administration and supervision at Bowling Green, Ohio, State University. There he held a number of positions, including assistant to the vice provost for educational development, assistant program director for the Upward Bound Program, a doctoral fellow in the Office of the Vice Provost for Student Affairs and a special assistant to the dean of the College of Education, a doctoral intern in the Office of Student Activities, and an international fellow in the Office of International Student Programs.Dr. Emmanuel held faculty rank and taught numerous courses as an assistant professor in the College of Education and Social Services at The University of Vermont and as an assistant professor in the College of Education at Bowling Green State University. He has published extensively and has been a presenter at many conferences on issues concerning student affairs. A leader on numerous committees and task forces at the institutions where he has served, he is active in professional and community organizations.Please join me in welcoming Narbeth Emmanuel, his wife Ann, and their children, Jason and Chandra, to the University community. Nobby, as he is called, will bring outstanding leadership in helping us continue to pursue excellence at SIUE.be: Chronou47leNB/SS/lm