SummaryBldD is a transcriptional regulator essential for morphological development and antibiotic production in

In Streptomyces coelicolor A3(2) and the related species Streptomyces lividans 66, aerial mycelium formation and antibiotic production are blocked by mutations in bidA, which specifies a tRNAL'-like gene product which would recognize the UUA codon. Here we show that phenotypic expression of three disparate genes (carB, lacZ, and ampC) containing TTA codons depends strongly on bWd. Site-directed mutagenesis of carB, changing its two TTA codons to CTC (leucine) codons, resulted in bld4-independent expression; hence the bi4 product is the principal tRNA for the UUA codon. Two other genes (hyg and aad) containing TTA codons show a medium-dependent reduction in phenotypic expression (hygromycin resistance and spectinomycin resistance, respectively) in bM4 mutants. For hyg, evidence is presented that the UUA codon is probably being translated by a tRNA with an imperfectly matched anticodon, giving very low levels of gene product but relatively high resistance to hygromycin. It is proposed that TTA codons may be generally absent from genes expressed during vegetative growth and from the structural genes for differentiation and antibiotic production but present in some regulatory and resistance genes associated with the latter processes. The codon may therefore play a role in developmental regulation.

BldD is a transcription factor required for aerial hyphae formation in the filamentous bacterium Streptomyces coelicolor. Three targets of BldD regulation were discovered by a number of means, including examination of bld gene interdependence, selective enrichment of chromosomal DNA fragments bound by BldD and searching the promoter regions of known developmental genes for matches to a previously characterized BldD binding site. The three BldD targets identified were the developmental sigma factor genes, whiG and bldN, and a previously uncharacterized gene, designated bdtA, encoding a putative transcription factor. In each target gene, the sequences bound by BldD were characterized by electrophoretic mobility shift and DNase I footprinting assays, and their alignment suggested AGTgA (n)m TCACc as a consensus BldD operator. The in vivo effect of mutation in bldD on the expression of these three target genes was assessed using S1 nuclease protection assays. In each case, target gene expression was upregulated during early colony development in the bldD background, suggesting that, in the wild type, BldD acts to repress premature expression of whiG, bldN and bdtA during vegetative growth.

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.

,B-Lactam compounds account for more than 60% of worldwide consumption of antibiotics as a result of the high selectivity, low toxicity, and versatility of these compounds. However, the chemotherapeutic application of ,-lactam antibiotics has been continually threatened by the development of bacterial resistance. The most common mechanism of resistance among both gram-positive and gram-negative bacteria involves the production of 3-lactamases, enzymes which cleave the p-lactam ring. The fact that some ,Blactamases are encoded on transferable plasmids or transposable elements has contributed substantially to the incidence of clinically significant P-lactam resistance (12). While chemical modification has helped to improve the resistance of the naturally occurring P-lactam antibiotics to cleavage, the need for further improvement resulted in an effort to find useful ,-lactamase inhibitors. Several effective, mechanismbased, low-molecular-weight P-lactamase inhibitors have been developed, but only clavulanic acid, a clavam-type P-lactam compound, has been used clinically (33). Clavulanic acid is one of several P-lactam compounds produced by Streptomyces clavuligerus, a filamentous gram-positive bacterium which also produces penicillin N, desacetoxycephalosporin C, and cephamycin C (28). The significance of the production of ,-lactamase inhibitors to S. clavuligerus is unclear, but this is also true of the production of ,-lactam antibiotics. No consensus exists as to how antibiotic-producing organisms benefit from the production of antibiotics (1,14). Although the majority of Streptomyces species produce ,-lactamases (43) (Preliminary results of this study were presented at the