No abstract

The antibiotic kirromycin inhibits prokaryotic protein synthesis by immobilizing elongation factor Tu (EF-Tu) on the elongating ribosome. Streptomyces ramocissimus, the producer of kirromycin, contains three tuf genes. While tuf1 and tuf2 encode kirromycin-sensitive EF-Tu species, the function of tuf3 is unknown. Here we demonstrate that EF-Tu3, in contrast to EF-Tu1 and EF-Tu2, is resistant to three classes of EF-Tu-targeted antibiotics: kirromycin, pulvomycin, and GE2270A. A mixture of EF-Tu1 and EF-Tu3 was sensitive to kirromycin and resistant to GE2270A, in agreement with the described modes of action of these antibiotics. Transcription of tuf3 was observed during exponential growth and ceased upon entry into stationary phase and therefore did not correlate with the appearance of kirromycin in stationary phase; thus, it is unlikely that EF-Tu3 functions as a resistant alternative for EF-Tu1. EF-Tu3 from Streptomyces coelicolor A3(2) was also resistant to kirromycin and GE2270A, suggesting that multiple antibiotic resistance is an intrinsic feature of EF-Tu3 species. The GE2270A-resistant character of EF-Tu3 demonstrated that this divergent elongation factor is capable of substituting for EF-Tu1 in vivo.

In Streptomyces coelicolor, the regular and abundant elongation factor (EF)-Tu1 is encoded by tuf1, while the actual function of the highly divergent tuf3 gene product is not yet known. Expression of the latter could so far only be detected on the transcriptional level under stress conditions. In this paper we demonstrate the presence of low levels of EF-Tu3 in strains of the J1501 lineage. Enhanced expression was observed for J1501 glkA and relA deletion mutants, which lack glucose kinase and ribosome-bound ppGpp synthetase, respectively.To assess the putative translational capacities of EF-Tu3, a novel Streptomyces in vitro translation assay was designed, based on the full elimination by Ni 21 affinity adsorption of chromosomally encoded (His) 6 -tagged EF-Tu1 from a S. coelicolor cell-free extract. Translational activity of this system is totally dependent on the addition of purified EFTu species or on the presence of an additional elongation factor Tu in the extract, e.g. encoded by a plasmid-borne tuf gene. Using this EF-Tu-dependent translation system, we have established that S. coelicolor EF-Tu3 has translational capacities despite its striking deviation from the common prokaryotic EF-Tu sequence at positions involved in either aminoacyl-tRNA binding or interaction with the guaninenucleotide exchange factor EF-Ts.

No abstract

Streptomyces ramocissimus, the producer of elongation factor Tu (EF-Tu)-targeted antibiotic kirromycin, contains three divergent tuf-like genes, with tuf1 encoding regular kirromycin-sensitive EF-Tu1; the functions of tuf2 and tuf3 are unknown. Analysis of the tuf gene organization in nine producers of kirromycin-type antibiotics revealed that they all contain homologues of tuf1 and sometimes of tuf3 but that tuf2 was found in S. ramocissimus only. The tuf2-flanking regions were sequenced, and the two tuf2-surrounding open reading frames were shown to be oriented in opposite directions. In vivo transcription analysis of the tuf2 gene displayed an upstream region with bidirectional promoter activity. The transcription start site of tuf2 was located approximately 290 nucleotides upstream of the coding sequence. Very small amounts of tuf2 transcripts were detected in both liquid-and surface-grown cultures of S. ramocissimus, consistent with the apparent absence of EF-Tu2 in total protein extracts. The tuf2 transcript level was not influenced by the addition of kirromycin to exponentially growing cultures. To assess the function of S. ramocissimus EF-Tu2, the protein was overexpressed in Streptomyces coelicolor LT2. This strain is a J1501 derivative containing His 6 -tagged EF-Tu1 as the sole EF-Tu species, which facilitated the separation of EF-Tu2 from the interfering EF-Tu1. S. ramocissimus EF-Tu1 and EF-Tu2 were indistinguishable in their ability to stimulate protein synthesis in vitro and exhibited the same kirromycin sensitivity, which excludes the possibility that EF-Tu2 is directly involved in the kirromycin resistance mechanism of S. ramocissimus.