Identification and Characterization of a Developmentally Regulated Protein, EshA, Required for Sporogenic Hyphal Branches in Streptomyces griseus

Abstract

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 ...

Molecular and Functional Analyses of the Gene ( eshA ) Encoding the 52-Kilodalton Protein of Streptomyces coelicolor A3(2) Required for Antibiotic Production

“…Therefore, it is likely that EshA is involved in the response to environmental stress, as represented by nutritional deficiency. Importantly, recent studies indicate that the EshA protein (and also the MMPI protein of M. leprae [35,36]) are recovered in the membrane fraction and exist in a multimer form in cells (15). Our preliminary experiments (unpublished data) have also demonstrated that EshA exists as a multimer in its native state.…”

“…5B and C), we believe that the A site mapped 111 bp upstream of the translation start codon would be the transcription start point because (i) the transcript at this site was the major one in either case, and other shorter or longer transcripts were detected only in S1 nuclease protection experiments or primer extension analysis, and (ii) the Ϫ10 (TAGCTT) and Ϫ35 (TTGGTG) regions in the putative promoter (Fig. 5A) show extensive similarity to those for S. griseus eshA (TAGTGT for Ϫ10 and TTGGTC for Ϫ35) as previously assigned by Kwak et al (15). Thus, the longer signal detected in S1 nuclease protection analysis (Fig.…”

Differential Regulation of ftsZ Transcription during Septation of Streptomyces griseus

“…A 1.0% agarose gel was used for low-resolution S1 nuclease protection experiments. A 6% polyacrylamide gel was used for fractionation of the protected fragments for high resolution (31). The relative intensity of transcripts was compared by densitometry (ChemiImager; Alpha Innotech Corporation) with developed films.…”

“…R2YE medium was used for S. lividans transformation (21). Sporulation was induced by phosphate starvation or nutritional downshift (31).…”

“…Phase-contrast microscopy and transmission electron microscopy were performed according to methods used in the previous experiment (31). TMax 400 film was used for the photographs.…”