An A-factor-dependent extracytoplasmic function sigma factor (sigma(AdsA)) that is essential for morphological...

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) is a crucial signaling molecule in Streptomyces griseus, regulating streptomycin production and aerial mycelium formation.
• A-factor exerts its effects by inducing the transcriptional activator AdpA, which is essential for developmental processes.
• The precise regulatory mechanisms downstream of AdpA, particularly concerning morphological differentiation, remain incompletely understood.

Purpose of the Study:

• To identify and characterize novel AdpA-dependent genes involved in Streptomyces griseus development.
• To elucidate the role of AdpA in regulating genes associated with morphological differentiation.
• To investigate the function of a newly identified AdpA-regulated gene, adsA, and its product, sigma(AdsA).

Main Methods:

• Gel mobility shift-PCR was employed to isolate DNA fragments bound by histidine-tagged AdpA.
• Gene cloning and sequencing were performed to identify the novel gene.
• S1 nuclease mapping and DNase I footprinting were used to determine transcriptional start sites and AdpA binding sites.
• Gene disruption via chromosomal integration was utilized to assess gene function.

Main Results:

• Nine AdpA-binding DNA fragments were identified, including one upstream of a gene encoding an extracytoplasmic function sigma factor, named adsA.
• Transcription of adsA was found to be dependent on both A-factor and AdpA, with enhanced expression correlating with aerial hyphae formation.
• AdpA binds to a specific site between the transcriptional start and translational start codons of adsA.
• Disruption of adsA resulted in the loss of aerial mycelium formation, while streptomycin and pigment production remained unaffected.
• Southern hybridization indicated the presence of adsA in various Streptomyces species, suggesting a conserved role.

Conclusions:

• The study identifies sigma(AdsA) as a novel A-factor and AdpA-dependent sigma factor essential for aerial mycelium formation in Streptomyces griseus.
• Sigma(AdsA) plays a specific role in regulating morphological development, distinct from secondary metabolite production.
• The findings reveal a new layer of transcriptional regulation governing morphogenesis in Streptomyces, mediated by the AdpA-adsA regulatory cascade.