The life cycle of streptomycetes is remarkably complex for a prokaryote, encompassing a number of structurally differentiated states and physiological changes. Growth initiates with spore germination and continues through a mycelial outgrowth to form a mat of branched hyphae. In response to an as yet undetermined signal(s), probably the sensing of nutritional deficiency, vegetative growth ceases and aerial hyphae begin to form. Maturation of these aerial filaments proceeds through a synchronous septation event, leading to the formation of unigenomic spores. Coincident with the onset of aerial hyphae formation is a shift to secondary metabolism, which results in the production of antibiotics and a variety of other compounds. Both the morphological and the physiological changes are tightly regulated and require the integration of environmental stimuli and extracellular signalling. The events that lead up to and allow differentiation to proceed are slowly being pieced together; however, the connections between the many signals and responses are not well understood, and it is likely that many of the genes involved have yet to be identified.
Studies of sporulation in Streptomyces coelicolor have revealed a class of genes, the whi genes (named for the white colony colour, which stems from a lack of characteristic grey spore pigment when mutant), to be essential for the formation of mature spores. Understanding of the whi gene regulatory cascade is advancing rapidly, and it has been shown to involve at least two different sigma factors, σWhiG (whiG;Chater et al., 1989) and σF (sigF;Potúčkováet al., 1995), and a number of proposed DNA-binding transcription factors (reviewed by Chater, 1998). A separate group of genes appears to be required for the erection of aerial hyphae. The bld genes were initially identified by mutations that caused the loss of white fuzzy aerial hyphae, resulting in colonies with a smooth, shiny, ‘bald’ appearance. The bld genes encode a diverse range of products, including a tRNA (bldA;Lawlor et al., 1987), an ATP-binding cassette (ABC) membrane-spanning transporter (bldK;Nodwell et al., 1996), a sigma factor (bldN;Bibb et al. 2000) and an unrelated anti-anti-sigma factor (bldG;Bignell et al., 2000), as well as numerous transcription factors [bldB (Pope et al., 1998), bldD (Elliot et al., 1998) and bldM (Molle and Buttner, 2000)]. Interestingly, a number of the bld genes also appear to influence antibiotic production, and mutations in some bld genes have been found to affect the regulation of carbon utilization (Pope et al., 1996), suggesting a global regulatory role for the bld genes in colony development. Metabolic defects have also been implicated in blocking differentiation through studies on the adenylate cyclase gene, cya, which catalyses the formation of cAMP. Disruption of cya results in a classical ‘bald’ phenotype, with the mutants showing deficiencies in both aerial hyphae formation and pigmented antibiotic production when grown on unbuffered media (Susstrunk et al., 1998). As neutralization of the medium or growth on buffered media allowed the developmental defects to be overcome, this conditional bald phenotype appears to result from acidification of the medium, perhaps suggesting a connection between metabolism, the resulting generation of organic acids and development.
Assessment of environmental conditions and communication between different colony compartments is accomplished, at least in part, by extracellular signalling molecules, such as γ-butyrolactones and oligopeptides, which are involved in the control of development in Streptomyces. In S. coelicolor, bldK has been implicated in signal uptake. bldK is a complex locus that encodes an ABC transporter, and bldK mutants are resistant to the toxic tripeptide bialaphos, implying that BldK is an oligopeptide importer (Nodwell et al., 1996). A small extracellular signalling peptide has been identified as a potential import target for BldK (Nodwell and Losick, 1998). BldK is one of the early players in an apparent bld gene extracellular signalling cascade, which is thought to culminate in the formation of SapB, a surfactant that contributes to the erection of aerial hyphae on rich media by reducing the surface tension at the colony surface (Tillotson et al., 1998). Studies carried out by Willey et al. (1993) revealed that, when certain pairs of bld mutants were grown in close proximity, aerial hyphae formation was restored to one member of each pair. By examining all possible pairings of the bld mutants, a hierarchy of genes was constructed, based upon the ability of each bld mutant to restore aerial hyphae production to all others (Willey et al., 1993; Nodwell et al., 1999; Molle and Buttner, 2000).
bldJ (formerly bld261) < bldK/bldL < bldA/bldH
< bldG < bldC < bldD/bldM
Each mutant strain is able to complement all those shown to the left, presumably through the provision of some signal that the others lack or through the inactivation of an extracellular inhibitor, and is complemented by all those to the right. Those strains that share complementation groups show the same complementation profile, yet have no effect on the hyphal production of each other. The bld gene effect is not likely to be as simple as a linear cascade, however, as bldB and bldN do not conform to any one complementation group. Despite these anomalies, the number of genes in each defined complementation group has recently been expanded through a comprehensive bld gene screen by Nodwell et al. (1999). A large number of these bld genes have been found to fall into the bldD/bldM complementation class and, in some cases, may represent BldD targets.
BldD was predicted to be a DNA-binding protein (Elliot et al., 1998) and was shown to bind to its own promoter, consistent with the marked upregulation of bldD transcription in a bldD mutant (Elliot and Leskiw, 1999). Here, we identify three additional chromosomal targets for BldD, which show that BldD is a regulator of key genes associated with development.