To identify sporulation-specific proteins that might serve as targets of developmental regulatory factors in Streptomyces, we examined total proteins of Streptomyces griseus by two-dimensional gel electrophoresis. Among five proteins that were present at high levels during sporulation but absent from vegetative cells, two of the proteins, P3 and P4, were absent from developmental mutants that undergo aberrant morphogenesis. The deduced amino acid sequence of the gene that encodes P3 (EshA) showed extensive similarity to proteins from mycobacteria and a cyanobacterium, Synechococcus, that are abundant during nutritional stress but whose functions are unknown. Uniquely among these proteins, EshA contains a cyclic nucleotide-binding domain, suggesting that the activity of EshA may be modulated by a cyclic nucleotide. The eshA gene was strongly expressed from a single transcription start site only during sporulation, and accumulation of the eshA transcript depended on a developmental gene, bldA. During submerged sporulation, a null mutant strain that produced no EshA could not extend sporogenic hyphae from new branch points but instead accelerated septation and spore maturation at the preexisting vegetative filaments. These results indicated that EshA is required for the growth of sporogenic hyphae and localization of septation and spore maturation but not for spore viability.Streptomyces is a gram-positive bacterium that undergoes morphological differentiation. In a nutritionally favorable condition, Streptomyces spores germinate to give rise to vegetative hyphae, which are characterized by filamentous, multigenomic cells called mycelia. Sporulation begins by the growth of aerial hyphae (or sporogenic hyphae when the sporulation process is induced in liquid culture; 34), which subsequently undergo septation to form chains of unicellular spores. We previously identified two types of sporogenic hyphae during sporulation of Streptomyces griseus (22): we suggest the term "distal" sporogenic hyphae for those that grow from the tips of preexisting vegetative filaments and the term "proximal" sporogenic hyphae for those that grow de novo within the vegetative hyphae at the onset of sporulation. Since both vegetative growth and spore formation are inherently polar processes similar to those observed in yeast and filamentous fungi (18), formation of sporogenic hyphae in Streptomyces is likely to require proteins that direct the location of sporogenic hyphae, establishment of polarity, and emergence of the sporogenic hyphae.To understand the molecular details of Streptomyces sporulation, researchers have identified developmental genes primarily by complementation of nonsporulating mutants. Most of the sporulation-specific genes that have been characterized appear to encode regulatory functions. Some, such as bldA, which encodes tRNA (38), are required for the production of aerial hyphae (35, 43) and antibiotics (3, 34, 43). Genes that specifically regulate spore formation include the whi genes, so named because mutations in ...