Cloning and Analysis of the Cypemycin Biosynthetic Gene Cluster Jan
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Cloning and analysis of the cypemycin biosynthetic gene cluster Jan Claesen September 2010 Thesis submitted to the University of East Anglia for the degree of Doctor of Philosophy © This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that no quotation from the thesis, nor any information derived therefrom may be published without the author’s prior, written consent. i Acknowledgements I would like to start by saying that it has been a wonderful experience to work on this project in such a productive environment as the John Innes Centre. I could not have managed this on my own, so via this way, I would like to thank all the people that have turned the past four years into a really pleasant time. First of all, I would like to thank my supervisor Prof. Mervyn Bibb for his support, optimism, guidance and for giving me the freedom to explore this project. For many useful discussions on biochemical subjects and lantibiotic genetics, I would like to thank my supervisory committee Dr. Stephen Bornemann and Dr. Arjan Narbad. A big thank you goes to Dr. Gerhard Saalbach and Dr. Mike Naldrett for the mass spectrometric analysis of many samples and the help in the interpretation of the resulting data. I am also very grateful to Prof. Wilfred van der Donk, Dr. Yuki Goto and Dr. Bo Li for their collaboration on the venezuelin project. For day-to-day advice and guidance, I would like to thank everyone in Molecular Microbiology. I am especially grateful to Nick Bird and Sean O’Rourke for making me feel at home in the lab from day one and teaching me the tricks of Streptomyces biology. I would like to thank the ‘lantibiotics crew’ Robert Bell, Lucy Foulston, Emma Sherwood and Diane Hatziioanou for many useful discussions. For helping me out in the lab, I would also like to thank Maureen Bibb, Govind Chandra, Juan-Pa Gomez- Escribano, Jennifer Parker, Richard Little, Steve Pullan, Nick Tucker, Andy Hesketh, Kay Fowler and Anyarat Thanapipatsiri. A special thanks goes to the staff at the Bateson and Chatt media kitchens and the stores facility. Their support has made it possible for me to focus on the science. Finally, I would like to thank my family and friends for their support. Especially my parents and brother for believing in me and showing interest in my work. Last but not least, I would like to thank Marij for experiencing this project together with me, keeping me motivated on bad days and sharing the joys of successful experiments. ii Abstract Lantibiotics are post-translationally modified peptide antibiotics produced by Gram- positive bacteria that contain characteristic ‘lanthionine’ residues and sometimes other unusual modifications. Cypemycin was identified as an anti-leukemia compound produced by Streptomyces sp. OH-4156. It contains two unique modifications ( N,N-dimethylalanine and allo -isoleucine) and shares two further modifications with the lantibiotic family, namely 2,3-didehydrobutyrine and a S-[(Z)- 2-aminovinyl]-D-cysteine. Streptomycetes are filamentous soil-dwelling Gram- positive bacteria that have a complex developmental cycle and are well known as producers of many antibiotics, immunosuppressants and anti-tumour compounds. The cypemycin biosynthetic gene cluster was identified, expressed in a heterologous host and a reduced gene set constructed that identified the genes sufficient for production. Mutational analysis of the individual genes within this set revealed that even the previously described modifications are carried out by unusual enzymes or via a modification pathway unrelated to lantibiotic biosynthesis. In vitro enzyme assays unambiguously confirmed the involvement of the two biosynthetic enzymes that are responsible for the modification of the N- and C-terminus of cypemycin. Bioinformatic analysis revealed the widespread occurrence of cypemycin-like gene clusters within the bacterial kingdom and in the Archaea . Cypemycin is the founding member of an unusual class of post-translationally modified peptides, the linaridins. Genetic analysis of a linaridin cluster in Streptomyces griseus resulted in the identification of grisemycin. Analysis of cryptic lantipeptide gene clusters (clusters for which the product has not yet been identified) in the genome of Streptomyces venezuelae identified a novel type of lanthionine-synthetase. Various genetic approaches were employed to identify the products from these clusters. Our collaborators reconstituted the activity of the unusual synthetase in vitro , revealing novel mechanistic and evolutionary insights. iii General abbreviations ABC ATP-binding cassette Act actinorhodin AIP auto-inducing peptide Apra apramycin ATP adenosine triphosphate AviCys S-[(Z)-2-aminovinyl]-D-cysteine BSA bovine serum albumin Carb carbenicillin Cl-Trp 5-chlorotryptophan Cm chloramphenicol DAB deoxyactagardine B Dha 2,3-didehydroalanine Dhb (Z)-2,3-didehydrobutyrine DMSO dimethylsulfoxide DNA gluc Difco Nutrient agar with supplemented glucose EDTA ethylenediaminetetraacetic acid EF-Tu elongation factor Tu FAB-MS fast atom bombardment mass spectrometry GlcNAc N-acetylglucosamine HFCD homo-oligomeric flavin-containing cysteine-decarboxylases Hyg hygromycin B IC50 half maximum inhibitory concentration IPTG isopropyl β-D-1-thiogalactopyranoside Kan kanamycin Lab (2 S,4 S,8 R)-labionin L-allo-Ile L-allo -Isoleucine Lan lanthionine MALDI-TOF matrix-assisted laser desorption/ionisation time-of-flight mass MS spectrometry Mcc microcin Me 2-Ala N,N-dimethylalanine MeAviCys S-[(Z)-2-aminovinyl]-3-methyl-D-cysteine MIC minimal inhibitory concentration MRSA methicillin resistant Staphylococcus aureus Nal nalidixic acid NMR nuclear magnetic resonance NRP non-ribosomal peptide NRPS non-ribosomal peptide synthase ORF open reading frame PAPA p-aminophenylalanine PCR polymerase chain reaction PE phosphatadylethanolamine PFGE pulsed-field gel electrophoresis PG peptidoglycan PPC 4'-phosphopanthothenoylcysteine QS quorum sensing Q-TOF quadrupole time-of-flight iv RBS ribosome binding site Red undecylprodigiosin SAM S-adenosyl methionine SDS sodium dodecyl sulphate SNA soft nutrient agar sp . species Spec spectinomycin SRP signal recognition particle Strep streptomycin TEMED N,N,N',N'-tetramethylethylenediamine Tet tetracycline Thio thiostrepton Vio viomycin VRE vancomycin resistant enterococci WT wild-type v Table of contents Chapter I - Introduction 1 I.1. Post-translationally modified peptide natural products of bacterial origin 1 I.2. Bacteriocins that only require leader cleavage 4 I.3. Circular bacteriocins 5 I.4. Lantipeptides 7 I.4.1. Introduction 7 I.4.2. Lantibiotic gene clusters 8 I.4.2.a. Structural gene, lanA 9 I.4.2.b. lanB and lanC 10 I.4.2.c. lanM 11 I.4.2.d. lanP 11 I.4.2.e. lanD 12 I.4.2.f. Other modifications 13 I.4.2.g. Regulatory genes 13 I.4.2.h. Transport genes 15 I.4.2.i. Immunity genes 15 I.4.3. The influence of recent developments in actinomycete research on classification of lantipeptides 16 I.4.3.a. Class I - LanBC (microbisporicin and planomonosporin) 21 I.4.3.b. Class II - LanM (cinnamycin and the duramycins, actagardine and michiganin A) 23 I.4.3.c. Class III - Lantionine synthetase containing a Ser/Thr kinase domain 25 I.4.4. Mechanisms of action 28 I.4.5. Engineering lantipeptides 30 I.4.6. Genome mining to identify lantipeptide compounds 32 I.4.6.a. Biochemical approaches to genome mining 33 I.4.6.b. Molecular genetic approaches to genome mining 34 I.5. Microcins 35 I.5.1. Microcin B17 contains thiazole and oxazole heterocycles 35 vi I.5.2. Lassopeptides 36 I.5.3. Siderophore-peptides, a marriage between the ribosomal and non-ribosomal worlds 37 I.5.4. Microcin C7 38 I.6. Modified peptides produced by cyanobacteria 38 I.6.1. Cyanobactins 38 I.6.2. Microviridins 39 I.7. Other five-membered heterocycle-containing peptides 40 I.7.1. Linear toxins 40 I.7.2. Thiopeptides 41 I.8. Linaridins 42 I.9. Modified quorum sensing peptides 43 I.9.1. Cyclic (thio)lactones 43 I.9.2. Bacillus pheromones 44 I.9.3. Pep1357C 44 I.10. Aims of this project 45 Chapter II – Materials and methods 46 II.1. Bacterial plasmids and strains 46 II.2. Culture media and antibiotics 53 II.2.1. Antibiotics 53 II.2.2. Culture media 53 II.3. Growth conditions and genetic manipulations 55 II.3.1. Growth and storage of E. coli 55 II.3.2. Growth and storage of Streptomyces 55 II.3.3. Plasmid isolation from E. coli 56 II.3.4. Cosmid isolation from E. coli 56 II.3.5. Genomic DNA extractions from Streptomyces 57 II.3.6. Digestion of DNA with restriction enzymes 57 II.3.7. Agarose gel electrophoresis 58 II.3.8. Extraction of DNA fragments from agarose gels 58 II.3.9. Preparation and transformation of electro-competent E. coli 58 vii II.3.10. Preparation and transformation of chemically competent E. coli 59 II.3.11. Ligation of DNA 59 II.3.12. Conjugation of DNA into Streptomyces 59 II.3.13. Construction of a Streptomyces sp . OH-4156 cosmid library 60 II.3.14. Generation of protein fusion constructs 60 II.3.15. Cypemycin bio-assays 61 II.4. Polymerase chain reaction (PCR) methods and DNA sequencing 61 II.4.1. General PCR 61 II.4.2. E. coli and Streptomyces colony PCR 62 II.4.3. Purification of PCR products 62 II.4.4. DNA sequencing 62 II.5. DNA hybridisation methods 70 II.5.1. Non-radioactive Southern hybridisation 70 II.5.2. Radioactive Southern hybridisation 72 II.6. PCR targeting 72 II.6.1. PCR amplification of disruption cassette 72 II.6.2. PCR targeting of a cosmid 74 II.6.3. Transfer of mutant cosmids into Streptomyces 74 II.7. Protein methods 75 II.7.1. Purification of His-tagged proteins 75 II.7.2.