Three tuf-like genes in the kirromycin producer Streptomyces ramocissimus.
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-1 gene encodes a protein with 71% amino acid residues identical to the well characterized elongation factor Tu of Escherichia coli (Ec.EF-Tu). The genetic location of tuf-1 downstream of a fus homologue and the in vitro activity of Sr.EF-Tu1 show that tuf-1 encodes a genuine EF-Tu. The putative Sr.EF-Tu2 and Sr.EF-Tu3 proteins are 69% and 63% identical to Ec.EF-Tu. Homologues of tuf-1 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-Tu1 was present at all times under kirromycin production conditions in submerged and surface-grown cultures of S. ramocissimus and in germinating spores. The expression of tuf-2 and tuf-3 was, however, below the detection level. Surprisingly, Sr.EF-Tu1 was kirromycin sensitive, which excludes the possibility that EF-Tu is involved in the kirromycin resistance of S. ramocissimus.
Department of Biochemistry, Leiden University, Gorlaeus Laboratories, The Netherlands.
E
E
Vijgenboom
L P
LP
Woudt
P W
PW
Heinstra
K
K
Rietveld
J
J
van Haarlem
G P
GP
van Wezel
S
S
Shochat
L
L
Bosch
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010444:Peptide Elongation Factor Tu / Q000096:biosynthesis / Q000235:genetics*; D011728:Pyridones / Q000378:metabolism / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D017385:Sequence Homology; D013045:Species Specificity; D013171:Spores, Bacterial; D013302:Streptomyces / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism / Q000502:physiology
D001426:Bacterial Proteins; D011728:Pyridones; D011993:Recombinant Fusion Proteins; D010444:Peptide Elongation Factor Tu; C008536:mocimycin