Background/Aims: A-factor, a γ-butyrolactone autoregulator, in Streptomyces griseus is involved in the regulation of differentiation and antibiotic production. Here we studied the S. griseus B2682-AFN (A-factor negative) bald mutant that harbors a nonsense mutation in the afsR gene encoding a pleiotropic regulator. Our aim was to prove that this mutation is the cause of the A-factor deficiency in AFN. We also studied whether AfsR regulates A-factor production by AfsA, which is supposed to be the only specific key enzyme in A-factor biosynthesis. Methods: Wild afsR was cloned to the pHJL401 shuttle vector and was transformed to the S. griseus AFN and B2682 strains. During phenotypic characterization, sporulation, antibiotic, protease, A-factor, and AfsA protein production were studied. Results: Transformation of AFN by a wild afsR restored its phenotype including sporulation, antibiotic, extracellular protease, and A-factor production. Introduction of afsR to the B2682 wild-type strain resulted in antibiotic and extracellular protease overproduction that was accompanied with an elevated A-factor level. AfsA was detected both in AFN and B2682. Conclusions: AfsR has an effect on the regulation of A-factor production in S. griseus. The presence of AfsA is not sufficient for normal A-factor production. AfsR regulates A-factor biosynthesis independently of AfsA.

1.
Ando N, Matsumori N, Sakuda S, Beppu T, Horinouchi S: Involvement of afsA in A-factor biosynthesis as a key enzyme. J Antibiot 1997; 50: 847–852.
2.
Birkó Z, Bialek S, Buzás K, Szájli E, Traag BA, Medzihradszky KF, Rigali S, Vijgenboom E, Penyige A, Kele Z, van Wezel GP, Biró S: The secreted signaling protein factor C triggers the A-factor response regulon in Streptomyces griseus: overlapping signaling routes. Mol Cell Proteomics 2007; 6: 1248–1256.
3.
Birkó Z, Swiatek M, Szájli E, Medzihradszky KF, Vijgenboom E: Lack of A-factor production induces the expression of nutrient scavenging and stress-related proteins in Streptomyces griseus. Mol Cell Proteomics 2009; 8: 2396–403.
4.
Chaudhary AK, Dhakal D, Sohng JK: An insight into the “-omics” based engineering of streptomycetes for secondary metabolite overproduction. Biomed Res Int 2013; 2013: 968518.
5.
Daza A, Martín JF, Dominguez A, Gil JA: Sporulation of several species of Streptomyces in submerged cultures after nutritional downshift. J Gen Microbiol 1989; 135: 2483–2491.
6.
Flärdh K, Buttner MJ: Streptomyces morphogenetics: dissecting differentiation in a filamentous bacterium. Nat Rev Microbiol 2009; 7: 36–49.
7.
Floriano B, Bibb M: afsR is a pleiotropic but conditionally required regulatory gene for antibiotic production in Streptomyces coelicolor A3(2). Mol Microbiol 1996; 21: 385–396.
8.
Hara O, Horinouchi S, Uozumi T, Beppu T: Genetic analysis of A-factor synthesis in Streptomyces coelicolor A3(2) and Streptomyces griseus. J Gen Microbiol 1983; 129: 2939–2944.
9.
Horinouchi S: A microbial hormone, A-factor, as a master switch for morphological differentiation and secondary metabolism in Streptomyces griseus. Front Biosci 2002; 7: 2045–2057.
10.
Horinouchi S: Mining and polishing the treasure trove in the bacterial genus Streptomyces. Biosci Biotechnol Biochem 2007; 71: 283–299.
11.
Horinouchi S, Kumada Y, Beppu T: Unstable genetic determinant of A-factor biosynthesis in streptomycin-producing organisms: cloning and characterization. J Bacteriol 1984; 158: 481–487.
12.
Jin Q, Jin Z, Zhang L, Yao S, Li F: Probing the molecular mechanisms for pristinamycin yield enhancement in Streptomyces pristinaespiralis. Curr Microbiol 2012; 65: 792–798.
13.
Kato JY, Funa N, Watanabe H, Ohnishi Y, Horinouchi S: Biosynthesis of γ-butyrolactone autoregulators that switch on secondary metabolism and morphological development in Streptomyces. Proc Natl Acad Sci USA 2007; 104: 2378–2383.
14.
Kato JY, Miyahisa I, Mashiko M, Ohnishi Y, Horinouchi S: A single target is sufficient to account for the biological effects of the A factor receptor protein of Streptomyces griseus. J Bacteriol 2004; 186: 2206–2211.
15.
Kato JY, Suzuki A, Yamazaki H, Ohnishi Y, Horinouchi S: Control by A-factor of a metalloendopeptidase gene involved in aerial mycelium formation in Streptomyces griseus. J Bacteriol. 2002; 184: 6016–25.
16.
Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA: Practical Streptomyces Genetics. Norwich, John Innes Foundation, 2000.
17.
Kim DW, Hesketh A, Kim ES, Song JY, Lee DH, Kim IS, Chater KF, Lee KJ: Complex extracellular interactions of proteases and a protease inhibitor influence multicellular development of Streptomyces coelicolor. Mol Microbiol 2008; 70: 1180–1193.
18.
Kim ES, Hong HJ, Choi CY, Cohen SN: Modulation of actinorhodin biosynthesis in Streptomyces lividans by glucose repression of afsR2 gene transcription. J Bacteriol 2001; 183: 2198–2203.
19.
Kiss Z, Ward AC, Birkó Z, Chater KF, Biró S: Streptomyces griseus 45H, a producer of the extracellular autoregulator protein factor C, is a member of the species Streptomyces albidoflavus. Int J Syst Evol Microbiol 2008; 58: 1029–1031.
20.
Lee J, Hwang Y, Kim S, Kim E, Choi C: Effect of a global regulatory gene, afsR2, from Streptomyces lividans on avermectin production in Streptomyces avermitilis. J Biosci Bioeng 2000; 89: 606–608.
21.
Lee PC, Umeyama T, Horinouchi S: afsS is a target of AfsR, a transcriptional factor with ATPase activity that globally controls secondary metabolism in Streptomyces coelicolor A3(2). Mol Microbiol 2002; 43: 1413–1430.
22.
Lian W, Jayapal KP, Charaniya S, Mehra S, Glod F, Kyung YS, Sherman DH, Hu WS: Genome-wide transcriptome analysis reveals that a pleiotropic antibiotic regulator, AfsS, modulates nutritional stress response in Streptomyces coelicolor A3(2). BMC Genomics 2008; 9: 56.
23.
MacNeil DJ, Gewain KM, Ruby CL, Dezeny G, Gibbons PH, MacNeil T: Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector. Gene 1992; 111: 61–68.
24.
Maharjan S, Oh TJ, Lee HC, Sohng JK: Identification and functional characterization of an afsR homolog regulatory gene from Streptomyces venezuelae ATCC 15439. J Microbiol Biotechnol 2009; 19: 121–127.
25.
Matsumoto A, Ishizuka H, Beppu T, Horinouchi S: Involvement of a small ORF downstream of the afsR gene in the regulation of secondary metabolism in Streptomyces coelicolor A3(2). Actinomycetologica 1995; 9: 37–43.
26.
Ohnishi Y, Ishikawa J, Hara H, Suzuki H, Ikenoya M, Ikeda H, Yamashita A, Hattori M, Horinouchi S: Genome sequence of the streptomycin-producing microorganism Streptomyces griseus IFO 13350. J Bacteriol 2008; 190: 4050–4060.
27.
Ohnishi Y, Yamazaki H, Kato JY, Tomono A, Horinouchi S: AdpA, a central transcriptional regulator in the A-factor regulatory cascade that leads to morphological development and secondary metabolism in Streptomyces griseus. Biosci Biotechnol Biochem 2005; 69: 431–439.
28.
Parajuli N, Viet HT, Ishida K, Tong HT, Lee HC, Liou K, Sohng JK: Identification and characterization of the afsR homologue regulatory gene from Streptomyces peucetius ATCC 27952. Res Microbiol 2005; 156: 707–712.
29.
Romero-Rodríguez A, Maldonado-Carmona N, Ruiz-Villafán B, Koirala N, Rocha D, Sánchez S: Interplay between carbon, nitrogen and phosphate utilization in the control of secondary metabolite production in Streptomyces. Antonie Van Leeuwenhoek 2018; 111: 761–781.
30.
Seipke RF, Song L, Bicz J, Laskaris P, Yaxley AM, Challis GL, Loria R: The plant pathogen Streptomyces scabies 87–22 has a functional pyochelin biosynthetic pathway that is regulated by TetR- and AfsR-family proteins. Microbiology 2011; 157(Pt 9): 2681–2693.
31.
Sekurova O, Sletta H, Ellingsen TE, Valla S, Zotchev S: Molecular cloning and analysis of a pleiotropic regulatory gene locus from the nystatin producer Streptomyces noursei ATCC11455. FEMS Microbiol Lett 1999; 177: 297–304.
32.
Solovyev V, Salamov A: Automatic annotation of microbial genomes and metagenomic sequences; in Li RW (ed): Metagenomics and Its Applications in Agriculture, Biomedicine and Environmental Studies. Hauppauge, Nova Science, 2011, pp 61–78.
33.
Szilágyi M, Kwon NJ, Bakti F, M-Hamvas M, Jámbrik K, Park HS, Pócsi I, Yu JH, Emri T: Extracellular proteinase formation in carbon starving Aspergillus nidulans cultures – physiological function and regulation. J Basic Microbiol 2011; 51: 625–634.
34.
Umeyama T, Lee PC, Ueda K, Horinouchi S: An AfsK/AfsR system involved in the response of aerial mycelium formation to glucose in Streptomyces griseus. Microbiology 1999; 145: 2281–2292.
35.
Vogtli M, Chang PC, Cohen SN: afsR2: a previously undetected gene encoding a 63-amino-acid protein that stimulates antibiotic production in Streptomyces lividans. Mol Microbiol 1994; 14: 643–653.
36.
Zhang L: Identification and characterization of developmental genes in Streptomyces; thesis Leiden University, 2015.
37.
Zhu H, Sandiford SK, van Wezel GP: Triggers and cues that activate antibiotic production by actinomycetes. J Ind Microbiol Biotechnol 2014; 41: 371–386.
You do not currently have access to this content.