Accumulation of bldA-specified tRNA is temporally regulated in Streptomyces coelicolor A3(2)

Abstract

Deletion of the bldA gene of Streptomyces coelicolor A3(2), which encodes the only tRNA for the rare UUA codon, had no obvious effects on primary growth but interfered with aerial mycelium formation and antibiotic production. To investigate the possible regulatory role of bldA, its transcription start point was identified, and time courses were determined for the appearance of its primary transcript, the processing of the primary transcript to give a mature 5' end, and the apparent efficiency of translation of ampC mRNA, which contains multiple UUA codons. The bldA promoter was active at all times, but processing of the 5' end of the primary transcript was comparatively inefficient in young cultures. This may perhaps involve an antisense RNA, evidence of which was provided by promoter probing and in vitro transcription. The presence of low levels of the processed form of the tRNA in young cultures followed by increased abundance in older cultures contrasted with the pattern observed for accumulation of a different, presumably typical tRNA which was approximately equally abundant throughout growth. The increased accumulation of the 5' processed form of bldA tRNA coincided with more-efficient translation of ampC mRNA in older cultures, supporting the hypothesis that in at least some physiological conditions, bldA may have a regulatory influence on events late in growth, such as morphological differentiation and antibiotic production.

A relA/spoT Homologous Gene from Streptomyces coelicolor A3(2) Controls Antibiotic Biosynthetic Genes

“…For actII-ORF4 (9), the actII-ORF4 promoter region included in a 635-base pair fragment (nucleotides 4824 -5458) was uniquely labeled at the 5Ј-end of the XhoI site (nucleotide 5458) within the actII-ORF4 coding region and was used as probe. RNA was extracted as described (61) from 3-day-old mycelium grown on the surface of cellophane discs on R5 agar plates as described (72).…”

The positive activator of cephamycin C and clavulanic acid production in Streptomyces clavuligerus is mistranslated in a bldA mutant The GenBank accession number for the sequence reported in this paper is AF436078.

“…This inability of young Streptomyces cultures to efficiently translate UUAcontaining mRNAs suggests that the bldA tRNA may function to modulate the expression of key genes involved in sporulation and antibiotic production, as well as genes involved in other late developmental and metabolic processes. However, in contrast, studies by Gramajo et al (1993) found that functional bldA tRNA was present throughout growth in liquid cultures and did not show the temporal pattern of accumulation observed in both solid and in liquid grown cultures by Leskiw et al (1993). Therefore, there is still no firm evidence that bldA ' regulates ' translation of UUAcontaining mRNAs.…”

“…RNA was extracted from surfacegrown or liquid cultures as described elsewhere (Leskiw et al, 1993). Northern blot analysis of bldA and leuU transcripts was performed as previously described (Trepanier et al, 1997) [the probes for bldA, leuU and the 5S rRNA control were the oligonucleotides BKL5, BKL42 and BKL53 (Table 2), respectively, except that the hybridization temperature for the bldA probe was 37 mC].…”

“…Although the S. coelicolor bldA tRNA is transcribed from a vegetative promoter and expressed throughout growth, it appears to be regulated at the post-transcriptional level. In contrast to a tRNA for the abundant leucine codon CUC, the mature, 5h-processed bldA tRNA shows growth-phase dependent accumulation, being expressed at low levels until the onset of secondary metabolism and morphological differentiation and reaching maximal levels of accumulation late in growth (Leskiw et al, 1993 ;Trepanier et al, 1997). Consistent with the low level of the tRNA Leu UUA in vegetative cultures, a TTA-containing reporter gene, ampC, showed delayed production of β-lactamase in S. lividans (Leskiw et al, 1991b), and a subtilisin-inhibitor-encoding ssi gene, that had been engineered to contain two TTA codons, showed reduced expression, particularly during early growth, in S. lividans (Ueda et al, 1993).…”

Biosynthesis of rapamycin and its regulation: past achievements and recent progress

“…13 A sequence analysis also revealed that both RapH and RapG contain the rare leucine codon TTA, which has been proposed to serve as part of the regulatory mechanism of secondary metabolites in Streptomyces. 60 Recently, the Biotica group showed that RapH and RapG have important roles as positive regulators in rapamycin biosynthesis. 23 Overexpression of rapH and rapG under the control of the ActII-ORF4/PactI activator/promoter in S. hygroscopicus enhanced the production of rapamycin by approximately 27-55% and 20-32%, respectively.…”