The Unique tuf2 Gene from the Kirromycin Producer Streptomyces ramocissimus Encodes a Minor and Kirromycin-Sensitive Elongation Factor Tu

Elongation factor Tu (EF-Tu), a member of the family of GTPase switch proteins, plays a pivotal role in the elongation process of bacterial protein synthesis (for a review see reference 15). The GTP-bound form of EF-Tu is responsible for delivery of aminoacyl-tRNA to the mRNA-programmed ribosomal A site. Cognate codon-anticodon recognition triggers the GTPase center on EF-Tu, causing the dissociation of inactive EF-Tu·GDP from the ribosome. Reactivation of the factor occurs via a nucleotide exchange reaction catalyzed by EF-Ts. EF-Tu is specifically affected by four different types of antibiotics of which kirromycin is the first identified and best studied (for references see reference 25). The binding of this antibiotic to EF-Tu still allows the factor to interact sequentially with aminoacyl-tRNA and the ribosomal A site. However, after GTP hydrolysis, EF-Tu·GDP is no longer ejected from the ribosome, thus immobilizing this and all following ribosomes on the mRNA, which explains the recessive character of kirromycin resistance in a mixed population of resistant and sensitive EF-Tu species. Certain error-restrictive mutations in ribosomal protein S12 (encoded by rpsL) overcome this recessivity; the mutant ribosomes will preferentially use the kirromycin-resistant EF-Tu for translation (33). Polyketide antibiotic kirromycin and related compounds, called elfamycins, are produced by actinomycetes. These gram-positive mycelial soil bacteria undergo a complex process of morphological differentiation and produce a wide variety of secondary metabolites (12). Antibiotic production is generally confined to stationary phase in liquid culture and usually coincides with the onset of morphological differentiation in surface-grown cultures. Various mechanisms are exploited by antibiotic-producing microorganisms to protect themselves from the toxic action of their own products (5); these include the use of an efficient drug efflux system, intracellular storage of the antibiotic in an inactive form, modification of an otherwise sensitive target, and (temporary) expression of a resistant target. The mechanism used by producers of kirromycin-type antibiotics to protect themselves against their own products is only partially known; some producers contain an intrinsically kirromycin-resistant EF-Tu (4, 9). Kirromycin producer Streptomyces ramocissimus contains three divergent tuf genes, which are designated tuf1, tuf2, and tuf3 and which code for EF-Tus that are surprisingly heterogeneous: EF-Tu2 displays 88% amino acid identity with EF-Tu1, and EF-Tu3 shows only about 65% amino acid identity with both EF-Tu1 and EF-Tu2 (37). The tuf1 gene encodes the major, kirromycin-sensitive EF-Tu (37) and is the promoter-distal gene in the rpsL operon, which also includes the genes for ribosomal proteins S12 (rpsL) and S7 (rpsG) and EF-G (fus). tuf1 is transcribed at a very high level during exponential growth from both the rpsL operon promoter and a tuf1-specific promoter (32). The roles of S. ramocissimus tuf2 and tuf3 are not yet clear; the gene products could not be detected under normal growth conditions, and overexpression in Escherichia coli yielded inactive products, deposited in inclusion bodies (37). Studies of the genetically well-characterized Streptomyces coelicolor revealed that this strain contains both tuf1 and tuf3 homologues (35) but lacks a tuf2 equivalent. Transcription of S. coelicolor tuf3 is subject to positive stringent control (36), and the tuf3 gene product can function as a real EF-Tu in a Streptomyces in vitro translation system (24). The lack of tuf2 homologues in all Streptomyces species studied so far (35, 37; L. N. Olsthoorn-Tieleman, unpublished results) and the apparent absence of a tuf2 gene product in S. ramocissimus (37) raised the question of whether tuf2 encodes an EF-Tu with a general or specialized function. In this paper we provide the sequences of the flanking genes of S. ramocissimus tuf2 and perform a transcriptional analysis of tuf2 and describe the overexpression and purification of its gene product. The actual functioning of EF-Tu2 as an EF-Tu and its interaction with kirromycin were studied by using a recently developed Streptomyces in vitro translation system (24).