Mechanisms of bacterial resistance

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Table of contents

1. General context
2. Secondary metabolism and microbial natural products
2.1 Interconnection between primary and secondary metabolism
2.2 The antibiotics
2.3 Mechanisms of bacterial resistance
2.3.1 Active-efflux pumps
2.3.2 Enzymatic inactivation of the antibiotic
2.3.3 Modification of the target site
3. Strategies to discover novel natural bioactive molecules
3.1 Genome mining
3.1.1 OSMAC approach
3.1.2 Genomisotopic approach
3.1.3 Gene inactivation and comparative metabolic profiling approach
3.1.4 Heterologous expression
3.1.5 Modification of the regulatory network
3.2 Metagenomics
3.3 Combinatorial biosynthesis
3.4 Screening of rare genera of actinomycetes and other untapped sources
4. Insights in the regulation of secondary metabolism
4.1 Nutritional regulation
4.2 Global regulation
4.3Extracellular signals
4.3.1 γ-butyrolactones
4.3.2 Furans
4.3.3 PI factor
4.4 Pathway-specific regulators
4.4.1 SARP regulators
4.4.2 LAL regulators
5. Polyketide synthases versus non-ribosomal peptide synthases
5.1 The core domains
5.2 The auxiliary domains
5.3 The initiation and termination domains
5.3.1 Type II thioesterase
6. Polyketide synthases
6.1 Typology of polyketide synthases
6.1.1 Modular type I PKS
6.1.2 Iterative type I PKS
6.1.3 Type II PKS
6.1.4 Type III PKS
6.2 Polyketide-tailoring genes
6.2.1 Glycosyltransferases
6.2.2 Methyltransferases and oxygenases
7. Streptomyces, a prolific producing genus
7.1 General characteristics
7.2 Streptomyces ambofaciens
8. Objectives of the thesis
Chapter 1 In silico characterization of a large type I PKS cluster
1.1 Sequence analysis of the enzymatic domains
1.1.1 Ketosynthase domains
1.1.2 Acyl transferase domains
1.1.3 Acyl carrier protein domains
1.1.4 Ketoreductase domains
1.1.5 Dehydratase domains
1.1.6 Enoyl reductase domains
1.1.7 Thioesterase domain
1.2 Prediction of the linear polyketide structure
1.3 Analysis of the other genes putatively involved in the biosynthesis
1.3.1 Glycosyltransferase and sugar genes
1.3.2 Cytochrome P450
1.3.3 Thioesterase type II
1.3.4 Additional genes
1.4 The resistance genes
1.5 The regulatory genes
1.5.1 A two component system
1.5.2 A LAL regulator
Chapter 2 Activation of a silent cluster and isolation of a novel bioactive macrolide of Streptomyces ambofaciens ATCC2387771
2.1 Activation of a silent cluster
2.2 Detection and isolation of a novel metabolite
2.3 The detected metabolites are synthetised by the type I PKS cluster
2.4 Structural elucidation of the novel compounds
2.5 Biological properties of the macrolide sambomycin
Article: Discovery of a novel macrolide in Streptomyces ambofaciens by awaking a sleeping giant
Chapter 3 Characterization of sambomycin biosynthesis
3.1 Limits of the sambomycin cluster
3.2 The phosphopantetheinyl transferase
3.3 The cytochromes P450
3.4 The acyl-CoA synthetase and the acyl-CoA carboxylase
3.5 Secretion of sambomycin
3.6 The resistance genes
Chapter 4 Regulation of the sambomycin cluster
4.1 The LAL regulator is an activator of the sambomycin gene cluster
4.2 Searching for the targets of the LAL regulator
4.3 In vitro characterization of the LAL regulator
4.4 Sambomycin production in R2 medium
4.5 Effect of SAMR0484 on spiramycin production
4.6 The two component system
CONCLUSION AND PERSPECTIVES
1. Sambomycin, a peculiar 50-membered macrolide
1.1 A new cyclization mechanism for polyketides
1.2 Biosynthesis of an unusual extender unit
1.3 Glycosylation of the sambomycin aglycones
2. Macrolides: biological properties and resistance mechanisms
2.1 Biological properties
2.2 Resistance mechanisms
3. The LAL regulator is a positive pathway-specific regulator of the sambomycin cluster
4. Combinatorial biosynthesis of the sambomycin gene cluster
5. The role of sambomycin in nature
6. Streptomyces ambofaciens and its fascinating secondary metabolites
APPENDIX
REFERENCES