Characterization of Sgr3394 Produced only by the A-Factor-Producing Streptomyces griseus IFO 13350, not by the A-Factor Deficient Mutant HH1

Protein D (9.7 kDa) is an extracellular protein detected in the culture broth of A-factor-producing Streptomyces griseus IFO 13350, but not of the A-factor-deficient mutant strain S. griseus HH1. Comparison of the N-terminal amino acid sequence with the genomic sequencing data of S. griseus IFO 13350 identified protein D as Sgr3394, which encodes a putative secretory protein with unknown function. The premature Sgr3394 consisted of 128 amino acids (13.5 kDa), showed 87.5% identity with SACT1DRAFT-0503, from Streptomyces sp. ACT-1, and 68.8% identity with SrosN15-18634, from S. roseosporus NRRL15998, and was confirmed to be matured for secretion by a peptide cleavage between the Ala-38 and Ala-39 bond. RT-PCR anaylsis of Sgr3394 clearly showed that it can be transcribed in the wild-type strain, but not in the A-factor-deficient strain. However, a gel-mobility shift assay of the promoter region of sgr3394 with A-factor-dependent transcriptional regulator (AdpA) showed that AdpA could not specifically recognize the putative AdpA-binding site (5'-TCCCCCGAAT-3'). All of these data strongly suggest that the expression of sgr3394 is not directly induced by AdpA but is regulated indirectly by an A-factor dependent protein. Introduction of sgr3394 on a high-copy-numbered plasmid (pWHM3-sgr3394) into S. lividans TK21 induced massive production of actinorhodin (blue pigment) and undecylprodigiosin (red pigment). Compared to the control, production of each pigment increased by 6.1 and 2.6 times, respectively, on R2YE agar, and 3.1 and 1.4 times, respectively, in R2YE broth; there was little influence on morphogenesis. In S. coelicolor A3(2)/pWHM3-sgr3394, actinorhodin and undecylprodigiosin productions were enhanced to 1.8 and 1.1 times those observed in the control, respectively, suggesting that overexpression of sgr3394 can stimulate secondary metabolism, especially actinorhodin biosynthesis, in S. lividans and S. coelicolor.