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The global regulator MtrA controls development and primary and secondary metabolism in Streptomyces species. However, residues critical for its function have not yet been characterized. In this study, we identified residue D53 as the potential phosphorylation site of MtrA from Streptomyces venezuelae, a model Streptomyces strain. MtrA variants with amino acid substitutions at the D53 site were generated, and the effects of these substitutions were evaluated in vitro and in vivo . We showed that, although substitutions at D53 did not alter MtrA's secondary structure, the MtrA D53 protein variants lost the ability to bind known MtrA recognition sequences (MtrA sites) in electrophoretic mobility shift assays. Complementation of the Δ mtrA strain with MtrA D53 protein variants did not affect overall strain growth. However, in comparison to the wild-type strain, chloramphenicol and jadomycin production were aberrant in the D53 variant strains, with levels similar to the levels in the Δ mtrA strain. Transcriptional analysis showed that the expression patterns of genes were also similar in the Δ mtrA strain and the D53 variant strains. Although the D53 protein variants and wild-type MtrA were produced at similar levels in S. venezuelae, chromatin immunoprecipitation-quantitative PCR results indicated that replacing the D53 residue rendered the altered proteins unable to bind MtrA sites in vivo , including MtrA sites that regulate genes involved in nitrogen metabolism and in chloramphenicol and jadomycin biosynthesis. In conclusion, our study demonstrates that the predicted phosphorylation site D53 is critical for the role of MtrA in regulation and suggests that MtrA functions in a phosphorylated form in the genus Streptomyces. IMPORTANCE Although phosphorylation has been shown to be essential for the activation of many response regulator proteins of two-component systems, the role of the phosphorylation site in the function of the global regulator MtrA in the genus Streptomyces has not been reported. In this study, we generated Streptomyces mutants that had amino acid substitutions at the predicted phosphorylation site of MtrA, and the effects of the substitutions were investigated by comparing the phenotypes of the resulting strains and their gene expression patterns with those of the wild-type strain and an MtrA deletion mutant. The ability of the altered proteins to bind known promoter targets in vitro was also evaluated. Our analyses showed that the predicted phosphorylation site D53 is critical for MtrA binding in vitro and for the normal functioning of MtrA in vivo . These studies further demonstrate the importance of MtrA as a global regulator in the genus Streptomyces.