University of Palermo Department of Biological Chemical and Pharmaceutical Sciences and Technology (STEBICEF) Streptomyces coelicolor: DNA cytosine methylation and differentiation Doctoral Thesis of Annalisa Pisciotta PhD in Cellular Biology XXVI cycle Scientific area code BIO 19 Supervisor: PhD coordinator: Dr. Rosa Alduina Prof. Maria Carmela Roccheri Acknowledgment I would like to express my sincere gratitude to my supervisor Dr. Rosa Alduina for the continuous support of my Ph.D study and related research, for her patience, motivation, and knowledge. Her guidance helped me in all the time of research and writing of this thesis. My sincere thank also goes to Prof. Anna Maria Puglia, for funding the BS sequencing of MG medium. My honest thank also goes to Dr. Angel Manteca, who provided me an opportunity to join his team, who gave access to the laboratory and research facilities and for funding part of this research project. I thank my lab mates in Oviedo, Dr. Maria Teresa Lopez, Dr Paula Yagüe, Beatriz Gutierrez Beatriz Rioseras, Nathaly Gonzalez and my lab mates in Palermo, Dr. Letizia Lo Grasso for the stimulating discussions and for the help in the progress of this research, and Federica Attardo for the passion with which contributed to development of this project. ABSTRACT ........................................................................................................................................... - 6 - INTRODUCTION ................................................................................................................................ - 9 - 1. DNA METHYLATION ........................................................................................................................- 10 - 1.1 DNA METHYLATION IN EUKARYOTES .............................................................................................- 10 - 1.2 DNA METHYLATION IN PROKARYOTES ...........................................................................................- 11 - 1.2.1 Restriction-modification system .................................................................................................. - 12 - 1.2.2 Gene expression .......................................................................................................................... - 14 - 1.2.3 DNA repair .................................................................................................................................. - 15 - 1.2.4 Coordination of replication and cell division ............................................................................... - 16 - 1.2.5 Cytosine methylation ................................................................................................................... - 17 - 1.3 BACTERIAL METHYLOMES ...............................................................................................................- 18 - 2. STREPTOMYCETES ...........................................................................................................................- 21 - 2.1 STREPTOMYCES COELICOLOR TRANSCRIPTOME ON SOLID GYM AND IN LIQUID R5A .......................- 25 - 3. METHYLATION IN STREPTOMYCETEAE ..........................................................................................- 27 - AIM ..................................................................................................................................................... - 29 - RESULTS ........................................................................................................................................... - 32 - 1. DNA CYTOSINE AND ADENINE METHYLATION IN THE DEFINED LIQUID MG ................................- 33 - 1.1 CHARACTERIZATION OF S. COELICOLOR CYTOSINE METHYLOME ....................................................- 34 - 1.2 OPTIMIZATION OF THE USE OF 5-AZA-2’-DEOXYCYTIDINE (AZA-DC) ..............................................- 39 - 1.3 EFFECT OF CYTOSINE DEMETHYLATION ON GROWTH (TREATMENT EVERY 24H).............................- 40 - 1.3.1 Effect of cytosine demethylation on physiological differentiation (treatment every 24h)............ - 41 - 1.4 EFFECT OF CYTOSINE DEMETHYLATION ON GROWTH (TREATMENT EVERY 12H).............................- 42 - 1.4.1 Effect of cytosine demethylation on physiological differentiation (treatment every 12h)............ - 43 - 1.5 DNA CYTOSINE METHYLATION AND GENE EXPRESSION ..................................................................- 44 - 2. DNA CYTOSINE METHYLATION IN THE RICH LIQUID R5A.............................................................- 48 - 2.1 DNA CYTOSINE METHYLATION DURING DEVELOPMENT .................................................................- 48 - 2.2 CHARACTERIZATION OF S. COELICOLOR CYTOSINE METHYLOME ....................................................- 49 - 2.3 EFFECT OF CYTOSINE DEMETHYLATION ON GROWTH ......................................................................- 53 - 2.4 EFFECT OF CYTOSINE DEMETHYLATION ON PHYSIOLOGICAL DIFFERENTIATION .............................- 53 - 2.5 DNA CYTOSINE METHYLATION AND GENE EXPRESSION ..................................................................- 54 - 2.5.1 GGCmCGG consensus sequence .................................................................................................. - 54 - 2.5.2 GCCmCG consensus sequence ..................................................................................................... - 56 - 2.5.3 CmGGGC consensus sequence .................................................................................................... - 58 - 3. DNA CYTOSINE METHYLATION ON SOLID RICH GYM .......................................................................- 60 - 3.1 DNA CYTOSINE METHYLATION DURING DEVELOPMENT .................................................................- 60 - 3.2 CHARACTERIZATION OF S. COELICOLOR CYTOSINE METHYLOME ....................................................- 62 - 3.3 EFFECT OF CYTOSINE DEMETHYLATION ON GROWTH ......................................................................- 66 - 3.4 EFFECT OF CYTOSINE DEMETHYLATION ON MORPHOLOGICAL DIFFERENTIATION ...........................- 66 - 3.5 EFFECT OF DEMETHYLATION ON PHYSIOLOGICAL DIFFERENTIATION ..............................................- 69 - 3.6 DNA CYTOSINE METHYLATION AND GENE EXPRESSION ..................................................................- 69 - 3.6.1 GGCmCGG consensus sequence .................................................................................................. - 69 - 3.6.2 GCCmCG consensus sequence ..................................................................................................... - 72 - 3.6.3 CmGGGC consensus sequence .................................................................................................... - 74 - 4. ANALYSIS OF S. COELICOLOR CYTOSINE METHYLOME ...................................................................- 76 - 4.1 SIMILARITIES AND DIFFERENCES BETWEEN CYTOSINE METHYLOME IN LIQUID AND SOLID MEDIUM- 76 - 5. ROLE OF THE DNA METHYLTRANSFERASE SCO1731 ....................................................................- 79 - 5.1 CONSTRUCTION OF A KNOCK OUT MUTANT IN SCO1731 GENE .......................................................- 79 - 5.2 EFFECT OF THE INACTIVATION OF SCO1731 GENE ..........................................................................- 81 - 5.3 COMPLEMENTATION OF MUTANT ΔSCO1731 AND CONSTRUCTION OF A STRAIN EXPRESSING TWO COPIES OF THE SCO1731 GENE ..............................................................................................................- 83 - 6. ROLE OF THE DNA METHYLTRANSFERASE SCO0995 ....................................................................- 88 - 6.1 CONSTRUCTION OF A KNOCK OUT MUTANT IN SC0995 GENE ...........................................................- 88 - 6.2 EFFECT OF THE INACTIVATION OF SCO0995 GENE ..........................................................................- 90 - 6.3 COMPLEMENTATION OF MUTANT ΔSCO0995 ..................................................................................- 90 - 7. DNA CYTOSINE METHYLATION IN SEVERAL STREPTOMYCETES ...................................................- 93 - DISCUSSION ..................................................................................................................................... - 96 - 1. DNA CYTOSINE METHYLATION ALONG THE GROWTH ...................................................................- 97 - 2. CYTOSINE METHYLOMES .................................................................................................................- 98 - 3. CORRELATION BETWEEN CYTOSINE METHYLOME AND TRANSCRIPTOMIC ANALYSIS ...............- 100 - 4. EFFECT OF CYTOSINE DEMETHYLATION BY ADDING AZA-DC .....................................................- 101 - 5. ROLE OF A PUTATIVE DNA(5-CYTOSINE)-METHYLTRANSFERASE ..............................................- 102 - MATERIAL AND METHODS ....................................................................................................... - 105 - 1. STRAINS AND MEDIA.......................................................................................................................- 106 - 2. PLASMIDS AND COSMIDS ................................................................................................................- 107 - 3. TREATMENT OF S. COELICOLOR WITH AZA-DC .............................................................................- 107 - 4. DOT BLOT ASSAY ...........................................................................................................................-