Abstract
Streptomyces coelicolor is a Gram-positive microorganism often used as a model of physiological and morphological differentiation in streptomycetes, prolific producers of secondary metabolites with important biological activities. In the present study, we analysed Streptomyces coelicolor growth and differentiation in the presence of the hypo-methylating agent 5′-aza-2′-deoxycytidine (5-aza-dC) in order to investigate whether cytosine methylation has a role in differentiation. We found that cytosine demethylation caused a delay in spore germination, aerial mycelium development, sporulation, as well as a massive impairment of actinorhodin production. Thus, we searched for putative DNA methyltransferase genes in the genome and constructed a mutant of the SCO1731 gene. The analysis of the SCO1731::Tn5062 mutant strain demonstrated that inactivation of SCO1731 leads to a strong decrease of cytosine methylation and almost to the same phenotype obtained after 5-aza-dC treatment. Altogether, our data demonstrate that cytosine methylation influences morphological differentiation and actinorhodin production in S. coelicolor and expand our knowledge on this model bacterial system.
Highlights
Base methylation is a DNA modification present in all kingdoms of life, including bacteria
Genomic DNA was extracted from different developmental stages of S. coelicolor M145, S. avermitilis ATCC 31267, S. griseus NBRC 102592 and S. lividans 1326 and analyzed by dot blot assay using the antibody against 5-MeC (Fig. 1)
DNA cytosine methylation during development is comparable in all the Streptomyces species in all the growth phases analyzed, indicating that (i) the cytosine methylation is conserved in the Streptomycetaceae family, (ii) has a broader role on Streptomyces differentiation and (iii) is a tag to recognize and degrade exogenous DNA
Summary
Base methylation is a DNA modification present in all kingdoms of life, including bacteria. Additional roles in regulating several important cellular processes, such as initiation of DNA replication, DNA repair and gene regulation, were proposed for bacterial adenine methyltransferases[3,4,5,6,7,8]. Roles in regulating gene expression were given to orphan cytosine methyltransferases of Helicobacter pylori and E. coli. 5-azacytidine (5-azaC) and 5-aza-2′-deoxycytidine (5-aza-dC) are cytosine DNA methylation inhibitors routinely used to demethylate DNA in a variety of eukaryotes to assess the consequences of cytosine DNA methylation loss[17,18] They are nucleoside analogs that are converted intracellularly to the corresponding 5′-triphosphates upon cell entry; 5-azaC is incorporated into both RNA and DNA, whereas 5-aza-dC only into DNA18–22. The role of DNA methyltransferases in Streptomyces antibioticus and S. coelicolor was investigated by treating the cultures with demethylating agents and it was found that methylation could influence development and differentiation[35,36,37]
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