Three tuf-like genes in the kirromycin producer Streptomyces ramocissimus

Abstract

We have identified, cloned and sequenced three tuf-like genes from Streptomyces ramocissimus (Sr.), the producer of the antibiotic kirromycin which inhibits protein synthesis by binding the polypeptide chain elongation factor EF-Tu. The tuf-7 gene encodes a protein with 71 O/ O amino acid residues identical to the well characterized elongation factor T u of Escherichia coli (Ec.EF-Tu). The genetic location of tuf-7 downstream of a fus homologue and the in vitro activity of Sr.EF-Tul show that tuf-7 encodes a genuine EF-Tu. The putative Sr.EF-Tu2 and Sr.EF-Tu3 proteins are 69% and 63% identical to Ec.EFTu. Homologues of tuf-7 and tuf-3 were detected in all five Streptomyces strains investigated, but tuf-2 was found in S. ramocissimus only. The three tuf genes were expressed in E. coli and used to produce polyclonal antibodies. Western blot analysis showed that Sr.EF-Tul was present at all times under kirromycin production conditions in submerged and surface-grown cultures of 5. ramocissimus and in germinating spores. The expression of tuf-2 and tuf-3 was, however, below the detection level. Surprisingly, Sr.EF-Tul was kirromycin sensitive, which excludes the possibility that EF-Tu is involved in the kirromycin resistance of 5. ramocissimus.

Substrate induction and glucose repression of maltose utilization by Streptomyces coelicolor A3(2) is controlled by malR, a member of the lacI–galR family of regulatory genes

“…Western analyses were conducted as described by Vijgenboom et al (1994) using a 1:1000 dilution of antibodies raised against CcpA of B. megaterium.…”

ssgA Is Essential for Sporulation of Streptomyces coelicolor A3(2) and Affects Hyphal Development by Stimulating Septum Formation

“…The insert of pGWS1 was cloned behind P ermE in pWHM3-E, and the P ermE -ssgA cassette was transferred to pSET152, resulting in pGWS4 (Table 2). From earlier work we know that the ribosome binding site of S. ramocissimus tuf1 is efficiently recognized by ribosomes and hence typically results in high expression (40). We therefore replaced the upstream region of S. griseus ssgA in some of the constructs by that of S. ramocissimus tuf1, to allow higher expression of the gene.…”

The variant tuf3 gene of Streptomyces coelicolor A3(2) encodes a real elongation factor Tu, as shown in a novel Streptomyces in vitro translation system

“…Previous attempts to detect EF-Tu3 in either S. coelicolor [16] or S. ramocissimus [13] were unsuccessful with standard Western blotting techniques. However, here we have shown low levels of this peculiar factor in S. coelicolor J1501 and M145 with the more sensitive chemiluminescence immunodetection method, thus excluding the idea Fig.…”

“…This tuf gene duplication is widespread among Gram-negative bacteria, while most Gram-positive microorganisms contain a single tuf gene [11,12]. However, many members of the Gram-positive genus Streptomyces possess at least two highly divergent tuf genes ( [13] and L. N. Olsthoorn-Tieleman, unpublished results). In the genetically well-characterized strain Streptomyces coelicolor M145 two unlinked genes for an EF-Tu and an EF-Tu-like protein have been identified [14], designated tuf1 and tuf3 by analogy to their homologues in Streptomyces ramocissimus, which contains three tuf genes [13].…”