Possible involvement of cAMP in aerial mycelium formation and secondary metabolism in Streptomyces griseus.
In Streptomyces griseus, A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) triggers secondary metabolism and morphogenesis by binding a repressor protein (ArpA) and dissociating it from DNA. UV-mutagenesis of the A-factor-deficient mutant HH1 generated strain HO2, defective in the synthesis of ArpA and therefore able to form aerial mycelium, spores and streptomycin. Shotgun cloning of chromosomal DNA from wild-type S. griseus in strain HO2 yielded a gene that suppressed aerial mycelium formation and streptomycin production. Nucleotide sequencing and subcloning revealed that the gene encoded a eukaryotic-type adenylate cyclase (CyaA). In mutant HO2 production of cAMP was growth-dependent until the middle of the exponential growth stage; the production profile was the same as in the wild-type strain. However, the amount of cAMP produced was five times larger when mutant HO2 harboured cyaA on the high-copy-number plasmid pIJ486. Consistent with this, supplying cAMP exogenously at a high concentration to mutant HO2 suppressed formation of both aerial mycelium and streptomycin. On the other hand, some lower concentrations of cAMP stimulated or accelerated aerial mycelium formation. No effects of exogenous cAMP on morphogenesis and secondary metabolism were apparent in the wild-type strain. In addition, disruption of the chromosomal cyaA gene in the wild-type strain had almost no effect. Introducing cyaA cloned in either a low- or a high-copy-number plasmid suppressed morphogenesis and secondary metabolism not only in mutant HO2 but also in other arpA mutants, implying that the effects of cAMP became apparent in the arpA-defective background. When mutant HO2 carried cyaA on a plasmid, synthesis of the stringent response factor ppGpp was greatly reduced; this may account for the observed suppression by cAMP of morphogenesis and secondary metabolism. cAMP also affected protein tyrosine phosphorylation, as determined with antiphosphotyrosine antibody.
Kang
Dae-Kyung
DK
Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
Li
Xin-Ming
XM
Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
Ochi
Kozo
K
National Food Research Institute, Tsukuba, Ibaraki 305-8642, Japan.
Horinouchi
Sueharu
S
Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
D015107:4-Butyrolactone / Q000031:analogs & derivatives* / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000235:genetics; D001426:Bacterial Proteins; D015153:Blotting, Western; D000242:Cyclic AMP / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D017353:Gene Deletion; D006159:Guanosine Tetraphosphate / Q000378:metabolism; D008969:Molecular Sequence Data; D019000:Phosphotyrosine / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D013305:Streptomyces griseus / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D013307:Streptomycin / Q000378:metabolism
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D019000:Phosphotyrosine; D006159:Guanosine Tetraphosphate; C025035:A-factor (Streptomyces); D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D015107:4-Butyrolactone; D013307:Streptomycin