DNA methylation is an epigenetic modification regulating many aspects of biological processes. DNA cytosine methylation plays mainly a regulatory role in chromatin organization, genome maintenance and gene expression in eukaryotes, while its role in prokaryotes has been less studied. Streptomyces coelicolor is a mycelial soil microorganism, producer of several antibiotics, with a complex life cycle that includes three different cell types: unigenomic spores, a compartmentalized mycelium (MI) and a multinucleated mycelium (substrate, aerial and sporulating, MII). This life cycle is finely regulated through several mechanisms: two events of programmed cell death, PCD, and three biochemical pathways (bld cascade, sky pathway and whi cascade). The importance of DNA methylation has been already described in Streptomycetes, but its biological role remains unknown. The aim of this project is to investigate the relationship between DNA cytosine methylation and morphological and physiological differentiation in S. coelicolor. Dot blot analysis of genomic DNAs extracted from S. coelicolor grown in different media (two liquid, MG and R5A, and a solid one, GYM) revealed that the global level of methylated cytosines changes along the growth during development (MI, MII and spores) both on solid and in liquid culture. To describe the extent of cytosine methylation, sequencing of bisulfite-treated genomic DNA, extracted from S. coelicolor along the growth in all the three media, was performed and the corresponding cytosine methylomes were defined. Bioinformatic analysis of DNA cytosine methylome in liquid MG revealed that 30% of S. coelicolor genes contain two cytosine methylation consensus sequences (GCCmCG and GGCmCGG) in their upstream regions. Among these, genes involved in morphological and - 7 - physiological differentiation, genes encoding putative transcriptional regulators and sigma factors, and genes related to DNA replication and chromatin condensation were identified. DNA cytosine methylome in R5A contains 28% of S. coelicolor genes methylated. These genes contain three cytosine methylation consensus sequences (GCCmCG, GGCmCGG and CmGGGC) in their regulation region; among these genes, 58.8% was methylated during the MI phase, such as genes involved in antibiotic production and morphological differentiation. DNA cytosine methylome in GYM is constituted by 28.8% of S. coelicolor genes methylated. These genes contain the same three cytosine methylation consensus sequences (GCCmCG, GGCmCGG and CmGGGC) in their regulation region as those found in R5A; out of these genes, 63.9% was methylated during the MI phase, such as genes involved in antibiotic production and morphological differentiation. To study the effect of DNA cytosine methylation, liquid and solid cultures of S. coelicolor were treated with 5-aza-2'-deoxycytidine (aza-dC, a cytidine analogous that inhibits DNA-methyltransferase activity). This treatment influenced S. coelicolor germination and antibiotic production both in liquid and on solid culture and sporulation on solid culture. Altogether these results demonstrate a strong relationship between DNA cytosine methylation and S. coelicolor growth and differentiation. Combining methylome results and transcriptomic analysis, a correlation between the cytosine methylation consensus sequences and gene expression was found in R5A and GYM media. Indeed, the GGCmCGG consensus sequence was found to be methylated in MI phase in a set of genes (25%), whose transcription was repressed in MI phase, while this consensus sequence was found twice methylated in all the phases in a set of genes (7.5%), whose transcription was constitutive. Regarding the GCCmCG consensus sequence, it was found to be methylated in MII phase in a set of genes (13.5%) that were more transcribed in MII phase, differently CmGGGC consensus sequence was found to be methylated in MII phase in a set of genes (20%) whose transcription was repressed in the same phase. - 8 - The search for DNA methyltransferase genes into S. coelicolor genome revealed that it contains 17 genes coding for putative DNA (5-cytosine)-methyltransferases; among them, SCO1731 is mainly expressed in MI phase, both on solid and in liquid culture. Thus, an independent mutant of SCO1731 gene was generated to start the study of cytosine demethylation. Phenotypic analysis of the mutant showed the same results obtained after aza-dC treatment, indeed, the morphological and physiological differentiation was delayed on solid GYM and in liquid R5A. The results of this study demonstrate that DNA cytosine methylation is related to morphological and physiological differentiation in S. coelicolor.

Pisciotta, A.Streptomyces coelicolor: DNA cytosine methylation and differentiation.

Streptomyces coelicolor: DNA cytosine methylation and differentiation

PISCIOTTA, Annalisa

Abstract

DNA methylation is an epigenetic modification regulating many aspects of biological processes. DNA cytosine methylation plays mainly a regulatory role in chromatin organization, genome maintenance and gene expression in eukaryotes, while its role in prokaryotes has been less studied. Streptomyces coelicolor is a mycelial soil microorganism, producer of several antibiotics, with a complex life cycle that includes three different cell types: unigenomic spores, a compartmentalized mycelium (MI) and a multinucleated mycelium (substrate, aerial and sporulating, MII). This life cycle is finely regulated through several mechanisms: two events of programmed cell death, PCD, and three biochemical pathways (bld cascade, sky pathway and whi cascade). The importance of DNA methylation has been already described in Streptomycetes, but its biological role remains unknown. The aim of this project is to investigate the relationship between DNA cytosine methylation and morphological and physiological differentiation in S. coelicolor. Dot blot analysis of genomic DNAs extracted from S. coelicolor grown in different media (two liquid, MG and R5A, and a solid one, GYM) revealed that the global level of methylated cytosines changes along the growth during development (MI, MII and spores) both on solid and in liquid culture. To describe the extent of cytosine methylation, sequencing of bisulfite-treated genomic DNA, extracted from S. coelicolor along the growth in all the three media, was performed and the corresponding cytosine methylomes were defined. Bioinformatic analysis of DNA cytosine methylome in liquid MG revealed that 30% of S. coelicolor genes contain two cytosine methylation consensus sequences (GCCmCG and GGCmCGG) in their upstream regions. Among these, genes involved in morphological and - 7 - physiological differentiation, genes encoding putative transcriptional regulators and sigma factors, and genes related to DNA replication and chromatin condensation were identified. DNA cytosine methylome in R5A contains 28% of S. coelicolor genes methylated. These genes contain three cytosine methylation consensus sequences (GCCmCG, GGCmCGG and CmGGGC) in their regulation region; among these genes, 58.8% was methylated during the MI phase, such as genes involved in antibiotic production and morphological differentiation. DNA cytosine methylome in GYM is constituted by 28.8% of S. coelicolor genes methylated. These genes contain the same three cytosine methylation consensus sequences (GCCmCG, GGCmCGG and CmGGGC) in their regulation region as those found in R5A; out of these genes, 63.9% was methylated during the MI phase, such as genes involved in antibiotic production and morphological differentiation. To study the effect of DNA cytosine methylation, liquid and solid cultures of S. coelicolor were treated with 5-aza-2'-deoxycytidine (aza-dC, a cytidine analogous that inhibits DNA-methyltransferase activity). This treatment influenced S. coelicolor germination and antibiotic production both in liquid and on solid culture and sporulation on solid culture. Altogether these results demonstrate a strong relationship between DNA cytosine methylation and S. coelicolor growth and differentiation. Combining methylome results and transcriptomic analysis, a correlation between the cytosine methylation consensus sequences and gene expression was found in R5A and GYM media. Indeed, the GGCmCGG consensus sequence was found to be methylated in MI phase in a set of genes (25%), whose transcription was repressed in MI phase, while this consensus sequence was found twice methylated in all the phases in a set of genes (7.5%), whose transcription was constitutive. Regarding the GCCmCG consensus sequence, it was found to be methylated in MII phase in a set of genes (13.5%) that were more transcribed in MII phase, differently CmGGGC consensus sequence was found to be methylated in MII phase in a set of genes (20%) whose transcription was repressed in the same phase. - 8 - The search for DNA methyltransferase genes into S. coelicolor genome revealed that it contains 17 genes coding for putative DNA (5-cytosine)-methyltransferases; among them, SCO1731 is mainly expressed in MI phase, both on solid and in liquid culture. Thus, an independent mutant of SCO1731 gene was generated to start the study of cytosine demethylation. Phenotypic analysis of the mutant showed the same results obtained after aza-dC treatment, indeed, the morphological and physiological differentiation was delayed on solid GYM and in liquid R5A. The results of this study demonstrate that DNA cytosine methylation is related to morphological and physiological differentiation in S. coelicolor.
Cytosine methylation, Streptomyces, differentiation, DNA methylation, morphological differentiation, physiological differentiation, DNA, methylation
Pisciotta, A.Streptomyces coelicolor: DNA cytosine methylation and differentiation.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10447/159635
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