Cloning of a pleiotropic gene that positively controls biosynthesis of A-factor, actinorhodin, and prodigiosin in Streptomyces coelicolor A3(2) and Streptomyces lividans.
A-factor (2S-isocapryloyl-3S-hydroxymethyl-gamma-butyrolactone), an autoregulating factor originally found in Streptomyces griseus, is involved in streptomycin biosynthesis and cell differentiation in this organism. A-factor production is widely distributed among actinomycetes, including Streptomyces coelicolor A3(2) and Streptomyces lividans. A chromosomal pleiotropic regulatory gene of S. coelicolor A3(2) controlling biosynthesis of A-factor and red pigments was cloned with a spontaneous A-factor-deficient strain of S. lividans HH21 and plasmid pIJ41 as a host-vector system. The restriction endonuclease KpnI-digested chromosomal fragments were ligated into the plasmid vector and introduced by transformation into the protoplasts of strain HH21. Three red transformants thus selected were found to produce A-factor and to carry a plasmid with the same molecular weight, and a 6.4-megadalton fragment was inserted in the KpnI site of pIJ41. By restriction endonuclease mapping and subcloning, a restriction fragment (1.2 megadaltons, approximately 2,000 base pairs) bearing the gene which causes concomitant production of A-factor and red pigments was determined. The red pigments were identified by thin-layer chromatography and spectroscopy to be actinorhodin and prodigiosin, both of which are the antibiotics produced by S. coelicolor A3(2). The cloned fragment was introduced into the A-factor-negative mutants (afs) of S. coelicolor A3(2) by using pIJ702 as the vector, where it complemented one of these mutations, afsB, characterized by simultaneous loss of A-factor and red pigment production. We conclude that the cloned gene pleiotropically and positively controls the biosynthesis of A-factor, actinorhodin, and prodigiosin.
D015107:4-Butyrolactone / Q000031:analogs & derivatives; D000880:Anthraquinones / Q000096:biosynthesis / Q000378:metabolism; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D005798:Genes, Bacterial; D005809:Genes, Regulator; D006133:Growth Substances / Q000096:biosynthesis / Q000235:genetics*; D009154:Mutation; D010860:Pigments, Biological / Q000096:biosynthesis; D010957:Plasmids; D011353:Prodigiosin / Q000096:biosynthesis / Q000235:genetics*; D013302:Streptomyces / Q000235:genetics*; D000095622:Benzoisochromanequinones
D015107:4-Butyrolactone; D000880:Anthraquinones; D004262:DNA Restriction Enzymes; D006133:Growth Substances; D010860:Pigments, Biological; D011353:Prodigiosin; C025035:A-factor (Streptomyces); C013390:actinorhodin; D000095622:Benzoisochromanequinones