Autogenous transcriptional activation of a thiostrepton-induced gene in Streptomyces lividans.

Abstract

Although the antibiotic thiostrepton is best known as an inhibitor of protein synthesis, it also, at extremely low concentrations (< 10-9 M), induces the expression of a regulon of unknown function in certain Streptomyces species. Here, we report the purification of a Streptomyces lividans thiostrepton-induced transcriptional activator protein, TipAL, whose N-terminus is similar to a family of eubacterial regulatory proteins represented by MerR.TipAL was first purified from induced cultures of S.lividans as a factor which bound to and activated transcription from its own promoter. The tipAL gene was overexpressed in Escherichia coli and TipAL protein purifi'ed in a single step using a thiostrepton affinity column. Thiostrepton enhanced binding of TipAL to the promoter and catalysed specific transcription in vitro.TipAs, a second gene product of the same open reading frame consisting of the C-terminal domain of TipAL, is apparently translated using its own in-frame initiation site. Since it is produced in large molar excess relative to TipAL after induction and also binds thiostrepton, it may competitively modulate transcriptional activation.

Nocardithiocin, a novel thiopeptide antibiotic, produced by pathogenic Nocardia pseudobrasiliensis IFM 0757

“…4,5 During our ongoing research for new metabolites from a Nocardia strain (IFM 0757 strain) obtained from a patient at the Health Center, University of Texas, we isolated a new cyclic thiopeptide antibiotic with potent antibacterial activity against rifampicin-resistant Mycobacterium tuberculosis, called nocardithiocin (Figure 1), which was classified as a 'series d' group peptide antibiotic based on the definition by Bagley et al 6 Cyclic thiopeptides are known as activators of thiostrepton-induced protein A, which is a member of the mercury resistance regulator family, and are active against Gram-positive bacteria. 3,7 In this paper, we describe the fermentation, isolation, structure elucidation and antimicrobial activities of the antibiotic.…”

Construction of thiostrepton-inducible, high-copy-number expression vectors for use in Streptomyces spp.

“…This may reflect transcription initiating at PtipA, or elsewhere in the vector that is not completely terminated at to. Induction of PtipA requires tipA both in vitro (Holmes et al, 1993) and in vivo (T. Katoh and C.J.T., unpublished data). While tipA homologues apparently occur in many Streptomyces species, including the S. lividans and S. coelicolor strains used here, they are not present in all (E.T., unpublished data).…”

“…A 115-bp synthetic PtipA fragment (corresponding to nt 4-119 of the published tipA sequence; Holmes et al, 1993) was made that contained a NdeI site (5'-CATATG) overlapping the tipA translational start codon (ATG). This fragment was inserted into a derivative of the hcn plasmid pIJ487 (50-100 copies per chromosome; T. Kieser, personal communication; Ward et al, 1986) to give pIJ6021 (Fig.…”

“…tipA was cloned and characterised (Murakami et al, 1989) and the regulation ofpt~p A studied in detail (Holmes et al, 1993). P,,A has been used to regulate gene expression in integrative vectors (Smokvina et al, 1990), and in mcn and hcn plasmids (Murakami et al, 1989;Kuhstoss and Rao, 1991;Holt et al, 1992).…”

Overexpression of the polynucleotide phosphorylase gene (pnp) of Streptomyces antibioticus affects mRNA stability and poly(A) tail length but not ppGpp levels

“…pIJ8600 is a streptomycete expression vector based on the thiostrepton-inducible promoter tipA (Holmes et al, 1993;Sun et al, 1999). The vector also contains a ribosomebinding site downstream of tipA, the attachment site for the streptomycete phage wC31 and genes conferring resistance to apramycin and thiostrepton (Kieser et al, 2000).…”