Characterization of thesgtR1andsgtR2genes and their role in regulating expression of thesprTgene encodingStreptomyces griseustrypsin

Abstract

The sgtR1 and sgtR2 genes encoding putative regulators similar to the Aha1 and ArsR families, respectively, were identified downstream from the sprT gene. To investigate their function, expression vectors containing various combinations of sprT, sgtR1, and sgtR2 were transformed into Streptomyces lividans and Streptomyces griseus. The trypsin activity levels produced by S. lividans harboring pWHM3-TR2 (sprT and sgtR2) or pWHM3-TR1R2 (sprT, sgtR2, and sgtR2) were, respectively, 6.6 or 8.9 times that of S. lividans transformed with pWHM3-T (sprT). In the pWHM3-TR1R2 transformant, the transcription of sprT consistently occurred during the earlier stages of growth and was maintained at a higher level throughout the 6 days of cultivation. Streptomyces griseus IFO13350 harboring pWHM3-TR1R2 also produced trypsin activity 2.1 times that of the pWHM3-T transformant. However, all S. griseus Delta adpA transformants produced lower SGT activity than the wild-type strain, and none could overcome the deficiency in AdpA transcriptional activator, suggesting that AdpA is an absolute prerequisite for sprT expression. The sprT transcript was detected at a high level only in the wild-type strain, but the sgtR1 and sgtR2 transcript levels were very similar between the S. griseus IFO13350 and Delta adpA strains. This clearly demonstrates that the transcription of the sgtR1 and sgtR2 genes is not dependent on AdpA and that they are therefore not members of the AdpA regulon.

The complex extracellular biology ofStreptomyces

“…The many targets for AdpA in S. griseus include 18 genes for proteases (Yamazaki et al, 2004;Kato et al, 2005a;Akanuma et al, 2009). One of these, sprT, encodes the target TLP for the SgiA protease inhibitor (Kato et al, 2005b;Oh et al, 2007). Another of these proteases, a metalloprotease, has been implicated in morphological differentiation (Kato et al, 2002(Kato et al, , 2005aTomono et al, 2005).…”

Identification and Characterization of a Xyloglucan-Specific Family 74 Glycosyl Hydrolase from Streptomyces coelicolor A3(2)

“…The coding region of sco6545 was amplified by PCR using the primers in Table 1 (17). The 1,106-bp fragment (NdeI/BglII) encoding the N-terminal half of Sco6545 and the 1,175-bp fragment (BglII/EcoRI) encoding the C-terminal half of Sco6545 were ligated together with pUWL201PW (4) digested with NdeI-EcoRI restriction enzymes, under the control of the strong ermE* promoter, to produce pUWL201-6545 (20).…”

Characterization of the autophosphorylating kinase, PkaF, in Streptomyces coelicolor A3(2) M130

“…Expression and purification of PkaF in E. coli From S. coelicolor A3(2) genomic DNA, the pkaF gene encoding eukaryotic Ser/Thr protein kinase was amplified by PCR with the following two synthetic oligonucleotides: sense primer, 5 0 -TATTGTGTTCGGGGATCCGGGGTGT CGGTG-3 0 with BamHI site (iltalicized); and anti-sense primer, 5 0 -CGTCAAGAGGCCAAGCTTCCCCGCCCGC CA-3 0 with HindIII (iltalicized), under previously described conditions (Oh et al 2007). The purified PCR product was then digested with BamHI-HindIII and subcloned into pET28a(?)…”