Study of the spiramycin biosynthesis and its regulation in streptomyces ambofaciens

N°D’ORDRE UNIVERSITE DE PARIS-SUD XI UFR SCIENTIFQUE D’ORSAY THESE Présentée pour obtenir LE GRADE DE DOCTEUR EN SCIENCE DE L’UNIVERSITE DE PARIS-SUD XI PAR HOANG CHUONG NGUYEN SUJET : Study of the spiramycin biosynthesis and its regulation in Streptomyces ambofaciens DIRECTEURS DE THESE Pr Thuy Duong HO HUYNH Dr Jean-Luc PERNODET Soutenue publiquement le 29 Septembre 2009 JURY Pr Armel GUYONVARCH Pr Pierre LEBLOND Pr Florence MATHIEU Pr Philippe JACQUES Dr Jean-Luc PERNODET Pr Thuy Duong HO HUYNH Président Rapporteur Rapporteur Examinateur Examinateur Examinateur N°D’ORDRE UNIVERSITE DE PARIS-SUD XI UFR SCIENTIFQUE D’ORSAY THESE Présentée pour obtenir LE GRADE DE DOCTEUR EN SCIENCE DE L’UNIVERSITE DE PARIS-SUD XI PAR HOANG CHUONG NGUYEN SUJET : Study of the spiramycin biosynthesis and its regulation in Streptomyces ambofaciens DIRECTEURS DE THESE Pr Thuy Duong HO HUYNH Dr Jean-Luc PERNODET Soutenue publiquement le 29 Septembre 2009 JURY Pr Armel GUYONVARCH Pr Pierre LEBLOND Pr Florence MATHIEU Pr Philippe JACQUES Dr Jean-Luc PERNODET Pr Thuy Duong HO HUYNH Président Rapporteur Rapporteur Examinateur Examinateur Examinateur Remerciements J’ecris ces remerciements en Français parce que, simplement, les gens auxquels je tiens exprimer ma gratitude sont français ou bien qu’ils connaissent bien la langue française En plus, je trouve que le français est une langue qui convient bien pour exprimer des sentiments La plupart de ce travail a été réalisé dans le Laboratoire de Microbiologie Moléculaire des Actinomycètes de l’Institut de Génétique et Microbiologie, UMR CNRS 8621 en collaboration avec le Laboratoire de Génétique de l’Université des Sciences Naturelles Ho Chi Minh Ville dans le cadre d’une thèse en co-tutelle Tout d’abord, je suis très reconnaissant mes directeurs de thèse, Madame Thuy Duong Ho Huynh et Monsieur Jean-Luc Pernodet Madame Ho Huynh a initié cette collaboration, m’a donné la possibilité de venir travailler en France et m’a aidé pour toutes les démarches administratives Elle a toujours suivi mon travail et m’a conseillé Je tiens remercier tout particulièrement Monsieur Pernodet pour la façon dont il a dirigé ma thèse : sa confiance en moi, sa grande disponibilité, sa générosité, son enthousiasme et ses connaissances qu’il a partagées avec moi Grâce lui, j’ai pu également découvrir pleinement la vie ‘‘à la française’’ et passer des moments mémorables un peu partout en France, en Angleterre et en Allemagne Je remercie le Professeur Florence Mathieu et le Professeur Pierre Leblond de me faire l’honneur d’accepter d’être rapporteurs de cette thèse Je remercie le Professeur Philippe Jacques et le Professeur Armel Guyonvarch d’avoir accepté de participer ce jury d’examen Je tiens remercier mes deux tuteurs de thèse, le Professeur Armel Guyonvarch et le Docteur Philippe Mazodier pour avoir suivi ce travail et pour leurs conseils Je remercie aussi le conseiller aux thèses, le Docteur Marie-José Daboussi et le directeur de l’école doctorale GGC, le Professeur Giuseppe Baldacci pour leurs encouragements, le suivi de mon travail et leur soutien J’exprime mes profonds remerciements tous les membres passés ou présents du Laboratoire de Microbiologie Moléculaire des Actinomycètes, du Laboratoire de Métabolisme Energétique des Streptomyces et de l’institut de Génétique et Microbiologie : Fatma Karray pour m’avoir guidé quand j’ai débuté mon travail de thèse, Sylvie Lautru pour sa grande aide aussi bien pour les expériences, en particulier celles de chimie analytique, que pour la rédaction, Muriel Decraene pour toutes les formalités françaises compliquées pour lesquelles je ne sais pas me débrouiller sans elle, et aussi tous les autres pour leurs conseils, leur sympathie et leur soutien, Karine Tuphile, Annick Friedmann, Alain Raynal, Michel Cassan, Claude Gerbaud, Emmanuelle Darbon-Rongère, Marie-Hélène Blondelet-Rouault, Josette Gagnat, Catherine Esnault, Marie-Joëlle Virolle, Sylvain Pendino, Cécile Martel, Jean-Denis Le Manach, Thierry Locatelli Je tiens également remercier les jeunes des labos, Sarka, Maud, Nicolas, Céline, Aleksandra, Noriyasu, Aleksei, Hanane, Amélie, Fabien, Emilie, Florence, Hasna, Delin qui ont suivi au fil de ma thèse mes espoirs aussi bien que mes découragements et m’ont fait bénéficier de leur aide, leur soutien constant et leur bonne humeur Ces quatre dernières années ont été riches en moments de convivialité Les nombreux barbecues, les sorties (culturelles et/ou gastronomiques) et les soirées de fêtes resteront des souvenirs inoubliables REMERCIEMENTS Chapter I: INTRODUCTION The genus Streptomyces 1.1.Taxonomy 1.2 The genome of Streptomyces 1.2.1 Streptomyces chromosomes 1.2.2 Streptomyces extrachromosomal elements 1.2.3 Streptomyces phages 1.3 Morphological development of Streptomyces 1.4 Ecology 6 8 12 13 13 18 2.The biosynthesis of secondary metabolites by Streptomyces and related actinobacteria 2.1 The importance of secondary metabolites for medicine and agriculture 2.2 Biological activities of secondary metabolites from Streptomyces and related actinobacteria 2.2.1 Antibacterial and antifungal agents 2.2.2 Antitumor agents 2.2.3 Enzymes inhibitors 2.2.4 Immunosuppressants 2.2.5 Insecticides and antiparasitic drugs 2.2.6 Herbicides 2.3 The genetics of secondary metabolism in Streptomyces 2.3.1 Initial genetic studies of secondary metabolism in S coelicolor 2.3.2 The isolation of secondary metabolites biosynthetic gene clusters in Streptomyces 2.4 The biosynthesis of polyketides 2.4.1 The assembly-line enzymology of polyketide synthases 2.4.2 The different types of PKSs 2.4.2.1 Type I PKS 2.4.2.2 Type II PKS 2.4.2.3 Type III PKS 2.5 The biosynthesis of nonribosomal peptides 2.6 The regulation of secondary metabolism in Streptomyces 2.6.1 Multiple factors influence the onset of secondary metabolism 2.6.2 Gowth rate and nutriment limitation 2.6.3 Diffusible signalling molecules 2.6.4 Regulatory proteins controlling secondary metabolism in Streptomyces 23 23 25 25 26 27 28 29 29 29 31 31 33 33 36 37 38 40 40 41 43 45 Macrolide antibiotics and their biosynthesis in Actinobacteria 3.1 Definition and classification of macrolide antibiotics 3.2 The mode of action of macrolide antibiotics 3.3 Resistance to macrolide antibiotics 3.3.1 Target modification 3.3.2 Efflux of macrolides 3.3.3 Inactivation of macrolides 3.4 Biosynthesis of macrolides 3.4.1 Biosynthesis of the macrolactone ring 3.4.2 Biosynthesis of the sugars 3.4.3 Tailoring reactions 3.4.4 Enzymes involved in glycosylation during macrolide biosynthesis 3.5 Combinatorial biosynthesis of new macrolide antibiotics 3.5.1 Combinatorial biosynthesis of the macrolactone ring 3.5.2 Modification of the glycosylation pattern through combinatorial biosynthesis 3.6 The regulation of macrolide biosynthesis 3.6.1 Regulation of erythromycin biosynthesis 3.6.2 Regulation of methymycin/pikromycin biosynthesis 3.6.3 Regulation of tylosin biosynthesis 3.7 Spiramycin biosynthesis in S ambofaciens 47 47 50 52 53 54 54 55 55 57 58 60 62 63 66 68 68 69 70 72 Chapter II: GLYCOSYLATION STEPS DURING SPIRAMYCIN BIOSYNTHESIS IN STREPTOMYCES AMBOFACIENS: INVOLVEMENT OF THREE GLYCOSYLTRANSFERASES AND TWO AUXILIARY PROTEINS 75 Chapter III: A POST-PKS PLATENOLIDE KETOREDUCTASE IS INVOLVED IN SPIRAMYCIN BIOSYNTHESIS IN STREPTOMYCES AMBOFACIENS 103 Chapter IV: REGULATION OF SPIRAMYCIN BIOSYNTHESIS IN STREPTOMYCES AMBOFACIENS 122 22 22 Chapter V: TRANSCRIPTIONAL ORGANIZATION OF THE SPIRAMYCIN CLUSTER AND ACTIVITIES OF THE TRANSCRIPTIONAL ACTIVATORS SRMR AND SRMS 151 GENERAL CONCLUSIONS 176 RESUME DE LA THESE EN FRANÇAIS 182 REFERENCES 200 Lederberg, J (2000) Infectious history Science 288, 287-293 Lee, Y J., Kitani, S., Kinoshita, H & Nihira, T (2008) Identification by gene deletion analysis of barS2, a gene involved in the biosynthesis of gamma-butyrolactone autoregulator in Streptomyces virginiae Arch Microbiol 189, 367-374 Lin, Y S., Kieser, H M., Hopwood, D A & Chen, C W 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le gène codant cette céto-réductase été identifié parmi trois candidats Enfin parmi les quatre régulateurs putatifs de la biosynthèse de spiramycine, deux, SrmR et SrmS, sont réellement impliqués dans la régulation SrmR est un activateur transcriptionnel nécessaire l’expression du gène srmS SrmS active la transcription des gènes de biosynthèse L’organisation transcriptionnelle de l’ensemble du groupe de gènes a été établie, permettant l’identification de 16 régions promotrices sur lesquelles l’effet de SrmR et SrmS a été étudié Abstract Spiramycin, a macrolide antibiotic synthesized by the bacterium Streptomyces ambofaciens, consists in a lactone macrocycle, the platenolide, on which three sugars are attached A group of about forty genes directing the biosynthesis of spiramycin had been identified Based on bioinformatic analysis, a function in biosynthesis, resistance to spiramycin or regulation of the expression of biosynthetic genes had been proposed for most proteins encoded by this gene cluster But for several biosynthetic steps, the genes involved had to be identified and characterized For the three glycosylation reactions, among the four putative glycosyltransferases encoded in the cluster, only three are necessary for spiramycin biosynthesis Sugars transferred by each of these three glycosyltransferases have been identified Two of these glycosyltransferases require an auxiliary protein for their activity and two auxiliary proteins have been identified Another important step in the biosynthesis is the reduction of a keto group on platenolide; the gene encoding the ketoreductase has been identified among three candidates Finally, among the four putative regulators of spiramycin biosynthesis, two, SrmR and SrmS are really involved in regulation SrmR is a transcriptional activator required for expression of the gene srmS SrmS activates the transcription of biosynthetic genes The transcriptional organization of the entire gene cluster has been established, allowing the identification of 16 promoter regions on which the effect of SrmR and SrmS has been studied [...]... from Streptomyces are used in the treatment of cancer: actinomycin D, anthracyclines (e.g daunorubicin, doxorubicin, epirubicin, pirirubicin and valrubicin) bleomycin, mitomycin C These compounds bind to DNA Among them, actinomycin D is the oldest metabolite used in cancer therapy It is produced by Streptomyces antibioticus, isolated by Waksman and Woodruff (Waksman & Woodruff, 1941) Actinomycin D binds... more copies of chromosome Then, the sporogenic cell stops its extension and begins synchronous, multiple cell division, under the effect of the whiA and whiB gene products The next step is the formation of septum, directed by the bacterial tubulin homologue FtsZ In virtually all bacteria, FtsZ assembles into a cytokinetic ring, the Z ring that defines the site of division and recruits other cell-division... blindness) and in veterinary medicine for the prevention and treatment of various parasitic infections Avermectins and spinosyns both belong to the macrolide family and their structure are presented in figure 15 27 Avermectins (Streptomyces avermitilis) Spinosyn A (Saccharopolyspora spinosa) Figure 15 Structures of avermectins and spinosyn A 2.2.6 Herbicides Herbicides inhibit normal plant growth and. .. groups of organisms, and have diverse, unusual, and often complex chemical structures Secondary metabolites are nonessential for the growth of the producing organism, at least under the conditions studied, and are often made after the phase of active vegetative growth They present a wide range of biological activities, including the inhibition or killing of other microorganisms (the narrow definition of. .. Actinobacteria synthesize about two thirds of the known antibiotics, the genus Streptomyces being at the origin of more than half of the known antibiotics The proportions are roughly the same for the compounds that are use in human and animal medicine Streptomyces and closely related genera produced compounds belonging to all major families of antibiotics: beta-lactams, cyclines, macrolides, aminoglycosides,... polyenes… These antibiotics are used as antibacterial and antifungal agents in medicine and agriculture The chemical diversity of these compounds is illustrated in figure 11 23 Cephamycin C (beta-lactam, Streptomyces clavuligerus) Tetracycline (cycline, Streptomyces rimosus) Spiramycin I (macrolide, Streptomyces ambofaciens) Kanamycin (aminoglycoside, Streptomyces kanamyceticus) Vancomycin (glycopeptide,... recombination AICEs represent a special class of ICEs, because unlike other ICEs, they have the ability to replicate autonomously like a plasmid For some of the AICEs, the integrated and replicative forms can even coexist Most of the AICEs integrate in a specific tRNA gene in the host chromosome and this gene is not inactivated after integration because the AICE and the chromosome share a segment of identity... cyclosporin A It was approved by FDA in 1994 for use 26 as an immunosupressant in liver transplantation (Demain & Sanchez, 2009) Its structure is presented in figure 14 Figure 14 Structure of tacrolimus 2.2.5 Insecticides and antiparasitic drugs These compounds are used in agriculture, medicine, industry, and households The use of insecticides is believed to be one of the major factors behind the increase in. .. mating pair via cell-to-cell junctions DNA transfer is initiated by the relaxase that nicks DNA at the origin of transfer (oriT) to generate a single-stranded molecule The resulting structure, the relaxosome, is then coupled to the transferosome (related to type IV secretion systems) by the intermediate of a coupling protein In Streptomyces, the conjugation machinery is actually much simpler with a single... to minimize the number of gene acquisition and gene loss events From (Ventura et al., 2007) 1.4 Ecology Streptomyces are mostly living in soil, where they represent a large proportion of the cultivable bacteria In soil, they can exist in the form of spores or vegetative mycelium In soils, Streptomyces are present mostly as dormant spores, providing a large proportion of the Streptomyces colony forming