Characterization of ssfR and ssgA, two genes involved in sporulation of Streptomyces griseus.

Streptomycetes are aerobic gram-positive soil bacteria that grow as multinucleoidal, multicellular, branched filaments and that undergo morphological and physiological differentiation in response to environmental factors (4–6, 20). The first sign of morphological differentiation is the formation of aerial sporogenic hyphae, which give a fuzzy, white appearance to the colonies. When these specialized hyphae have stopped growing, DNA segregates and sporulation septa form to generate the uninucleoidal compartments that become the spores. Sporulation septation occurs relatively synchronously throughout a single sporogenic hypha (22, 32). Most studies of Streptomyces sporulation have been accomplished by the characterization of nonsporulating mutants of Streptomyces coelicolor. These mutants have been divided into bald (bld) strains, which appear not to form aerial hyphae, and white (whi) strains, which form aerial hyphae but which are blocked at various stages of sporulation. In S. coelicolor, 10 classes of bald mutants have been characterized: bld-261, bldA, bldB, bldC, bldD, bldF, bldG, bldH, bldI, and bldK (3, 8, 26, 27, 34, 35), and other constructed null mutants have displayed a bald phenotype (e.g., relA mutants [2]). In addition to their inability to produce aerial hyphae, many bald mutants are also defective in antibiotic production (3, 4), catabolite repression (29), and cell-cell signaling (34, 35). The highly pleiotropic phenotype of these mutants suggests that they identify genes involved at an early stage in the initiation of development and that the synthesis of antibiotics and the formation of aerial hyphae share some important regulatory links. Aerial mycelium formation and sporulation are partially restored to several bld mutants when they are grown on mannitol minimal medium (MM+M) rather than on complex or glucose minimal medium (MM+G) (3, 4, 6, 26). Several of the whi genes encode likely regulatory proteins (5). For example, whiG encodes a sigma factor (7, 25, 33) and whiH encodes a protein with similarity to members of a large family of bacterial regulatory proteins. Many proteins in this family are repressors of some genes involved in carbon metabolism (30). We studied the sporulation of Streptomyces griseus, a species that can be induced to sporulate in submerged culture in response to phosphate or nutritional downshift (20, 21). With the submerged sporulation system of S. griseus, physiological and ultrastructural studies of rapidly sporulating cultures have been conducted (13, 22) and the time course of gene expression during sporulation has been determined (9). In previous studies, it was shown that cefoxitin completely prevented sporulation without affecting vegetative growth (13). Electron-microscopic examination of sporulating cultures that had been exposed to cefoxitin during 12 h of induction confirmed that this antibiotic inhibited sporulation septation (13). These results suggest that a sporulation-specific, penicillin-binding protein (PBP) is a target of cefoxitin during sporulation of S. griseus. To investigate this cefoxitin-binding protein, a cefoxitin-resistant mutant was isolated by a sonication enrichment method. This mutant showed not only cefoxitin resistance but also some other phenotypes that are different from those of the wild-type strain. To study this mutant further, a complementing DNA fragment has been cloned and characterized in the present study. It is shown to contain two genes (ssfR and ssgA) required for sporulation.