Molecular mechanism of mureidomycin biosynthesis activated by introduction of an exogenous regulatory gene ssaA into Streptomyces roseosporus.

Mureidomycins (MRDs), a group of unique uridyl-peptide antibiotics, exhibit antibacterial activity against the highly refractory pathogen Pseudomonas aeruginosa. Our previous study showed that the cryptic MRD biosynthetic gene cluster (BGC) mrd in Streptomyces roseosporus NRRL 15998 could not be activated by its endogenous regulator 02995 but activated by an exogenous activator SsaA from sansanmycin's BGC ssa of Streptomyces sp. strain SS. Here we report the molecular mechanism for this inexplicable regulation. EMSAs and footprinting experiments revealed that SsaA could directly bind to a 14-nt palindrome sequence of 5'-CTGRCNNNNGTCAG-3' within six promoter regions of mrd. Disruption of three representative target genes (SSGG-02981, SSGG-02987 and SSGG-02994) showed that the target genes directly controlled by SsaA were essential for MRD production. The regulatory function was further investigated by replacing six regions of SSGG-02995 with those of ssaA. Surprisingly, only the replacement of 343-450 nt fragment encoding the 115-150 amino acids (AA) of SsaA could activate MRD biosynthesis. Further bioinformatics analysis showed that the 115-150 AA situated between two conserved domains of SsaA. Our findings significantly demonstrate that constitutive expression of a homologous exogenous regulatory gene is an effective strategy to awaken cryptic biosynthetic pathways in Streptomyces.