Streptomycetes are Gram-positive mycelial bacteria that have an extensive secondary metabolism and undergo complex morphological differentiation to form a sporulating aerial mycelium. In the model organism Streptomyces coelicolor A3(2), and in other species tested, development and many secondary metabolism are pleiotropically defective in mutants of bldA, which encodes the only tRNA that can efficiently translate the rare leucine codon UUA (Leskiw et al., 1991b; Chater, 2006; Chater & Chandra, 2006). Thus, although bldA mutants show apparently normal vegetative growth, they are defective in the production of at least four known antibiotics and in the formation of aerial mycelium on most media (Merrick, 1976; Champness, 1988). The pleiotropic effects of bldA mutations in S. coelicolor are at least partially attributable to the presence of UUA codons in the mRNA of critical regulatory genes (Chater, 2006). For example, actII-4, which encodes the pathway-specific regulator of actinorhodin production, contains a TTA codon (Fernandez-Moreno et al., 1991), as does redZ, a regulatory gene required for undecylprodigiosin production (White & Bibb, 1997; Guthrie et al., 1998). Another TTA-containing transcriptional regulatory gene, adpA (also termed bldH), is the main route by which bldA affects morphological differentiation (Nguyen et al., 2003; Takano et al., 2003). Recent proteomic analyses showed that a bldA-deleted mutant had impaired production of several extracellular proteins, including a potentially developmentally significant trypsin-like protease inhibitor SCO0762 (Kim et al., 2005b); and two hypothetical proteins, SCO4244 and SCO4252, were absent (Kim et al., 2005a). SCO0762 does not contain a TTA codon, and its disruption mutant differentiates normally, but transcription of SCO0762 depends on the TTA-containing gene adpA (Kim et al., 2005b). SCO4244 and SCO4252 are in two operons that are located close to each other and the transcription of the operons were inactivated by disruption of the nearby TTA-containing regulatory gene, SCO4263, although disruption of SCO4263 had no obvious phenotype with respect to antibiotic production or morphological differentiation (Kim et al., 2005a; Hesketh et al., in preparation). Expression analysis indicated that the abundance of the bldA-encoded tRNA is at its highest in stationary phase, in contrast to what is expected for most tRNA species (Trepanier et al., 1997). In agreement with this, expression of TTA-containing genes was found to be delayed during early differentiation (Kataoka et al., 1999).
All Streptomyces ssp. have a very high G+C content (typically more than 70%), making the TTA codon rare. Many known TTA-containing genes have been found to be associated with morphological and physiological differentiation, and expression of these genes may be limited even in wild-type streptomycetes (Leskiw et al., 1991b; Chater, 2006). The genome sequences of two Streptomyces species –S. coelicolor (Bentley et al., 2002) and Streptomyces avermitilis (Ikeda et al., 2003) – are now available. Analysis of these genomes has shown that the position in mRNAs of UUA codons is biased towards the start of coding sequences, implying that translational selection of codon usage occurs in streptomycetes (Fuglsang, 2005; Chater & Chandra, 2006). In S. coelicolor, knowledge of the roles of TTA-containing genes has mostly resulted from investigations of mutants with obvious phenotypic defects. Here, we have attempted to approach this problem using different approaches, a bioinformatics analysis coupled with targeted mutagenesis of 21 of the 145 TTA-containing chromosomal genes.