An A-factor-dependent extracytoplasmic function sigma factor (sigma(AdsA)) that is essential for morphological development in Streptomyces griseus.
A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) at an extremely low concentration triggers streptomycin production and aerial mycelium formation in Streptomyces griseus. A-factor induces the expression of an A-factor-dependent transcriptional activator, AdpA, essential for both morphological and physiological differentiation by binding to the A-factor receptor protein ArpA, which has bound and repressed the adpA promoter, and dissociating it from the promoter. Nine DNA fragments that were specifically recognized and bound by histidine-tagged AdpA were isolated by cycles of a gel mobility shift-PCR method. One of them was located in front of a gene encoding an extracytoplasmic function sigma factor belonging to a subgroup of the primary sigma(70) family. The cloned gene was named AdpA-dependent sigma factor gene (adsA), and the gene product was named sigma(AdsA). Transcription of adsA depended on A-factor and AdpA, since adsA was transcribed at a very low and constant level in an A-factor-deficient mutant strain or in an adpA-disrupted strain. Consistent with this, transcription of adsA was greatly enhanced at or near the timing of aerial hyphae formation, as determined by low-resolution S1 nuclease mapping. High-resolution S1 mapping determined the transcriptional start point 82 nucleotides upstream of the translational start codon. DNase I footprinting showed that AdpA bound both strands symmetrically between the transcriptional start point and the translational start codon; AdpA protected the antisense strand from positions +7 to +41 with respect to the transcriptional start point and the sense strand from positions +12 to +46. A weak palindrome was found in the AdpA-binding site. The unusual position bound by AdpA as a transcriptional activator, in relation to the promoter, suggested the presence of a mechanism by which AdpA activates transcription of adsA in some unknown way. Disruption of the chromosomal adsA gene resulted in loss of aerial hyphae formation but not streptomycin or yellow pigment production, indicating that sigma(AdsA) is involved only in morphological development and not in secondary metabolic function. The presence of a single copy in each of the Streptomyces species examined by Southern hybridization suggests a common role in morphogenesis in this genus.
Yamazaki
H
H
Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
Ohnishi
Y
Y
Horinouchi
S
S
D015107:4-Butyrolactone / Q000031:analogs & derivatives* / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D003850:Deoxyribonuclease I; D006133:Growth Substances / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012808:Sigma Factor / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013305:Streptomyces griseus / Q000235:genetics* / Q000254:growth & development; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
C401645:AdpA protein, Streptomyces griseus; C412466:AdpA-dependent sigma factor, Streptomyces griseus; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D006133:Growth Substances; D011994:Recombinant Proteins; D012808:Sigma Factor; D015534:Trans-Activators; C025035:A-factor (Streptomyces); D003850:Deoxyribonuclease I; D015107:4-Butyrolactone