The Secreted Signaling Protein Factor C Triggers the A-factor Response Regulon in Streptomyces griseus

Members of the prokaryotic genus Streptomyces produce over 60% of all known antibiotics and a wide range of industrial enzymes. A leading theme in microbiology is which signals are received and transmitted by these organisms to trigger the onset of morphological differentiation and antibiotic production. The small γ-butyrolactone A-factor is an important autoregulatory signaling molecule in streptomycetes, and A-factor mutants are blocked in development and antibiotic production. In this study we showed that heterologous expression of the 324-amino acid secreted regulatory protein Factor C resulted in restoration of development and enhanced antibiotic production of an A-factor-deficient bald mutant of Streptomyces griseus, although the parental strain lacks an facC gene. Proteome analysis showed that in the facC transformant the production of several secreted proteins that belong to the A-factor regulon was restored. HPLC-MS/MS analysis indicated that this was due to restoration of A-factor production to wild-type levels in the transformant. This indicates a connection between two highly divergent types of signaling molecules and possible interplay between their regulatory networks. Members of the prokaryotic genus Streptomyces produce over 60% of all known antibiotics and a wide range of industrial enzymes. A leading theme in microbiology is which signals are received and transmitted by these organisms to trigger the onset of morphological differentiation and antibiotic production. The small γ-butyrolactone A-factor is an important autoregulatory signaling molecule in streptomycetes, and A-factor mutants are blocked in development and antibiotic production. In this study we showed that heterologous expression of the 324-amino acid secreted regulatory protein Factor C resulted in restoration of development and enhanced antibiotic production of an A-factor-deficient bald mutant of Streptomyces griseus, although the parental strain lacks an facC gene. Proteome analysis showed that in the facC transformant the production of several secreted proteins that belong to the A-factor regulon was restored. HPLC-MS/MS analysis indicated that this was due to restoration of A-factor production to wild-type levels in the transformant. This indicates a connection between two highly divergent types of signaling molecules and possible interplay between their regulatory networks. Bacteria of the Gram-positive filamentous Streptomyces are a well known model system for the study of prokaryotic multicellular differentiation with a complex life cycle culminating in spore formation (1Chater K.F. Genetics of differentiation in Streptomyces.Annu. Rev. Microbiol. 1993; 47: 685-713Crossref PubMed Google Scholar). The onset of development is triggered by nutritional signals (2Rigali S. Nothaft H. Noens E.E. Schlicht M. Colson S. Muller M. Joris B. Koerten H.K. Hopwood D.A. Titgemeyer F. van Wezel G.P. The sugar phosphotransferase system of Streptomyces coelicolor is regulated by the GntR-family regulator DasR and links N-acetylglucosamine metabolism to the control of development.Mol. Microbiol. 2006; 61: 1237-1251Crossref PubMed Scopus (169) Google Scholar) and temporally relates to the production of antibiotics and other secondary metabolites (3Chater K.F. Streptomyces inside-out: a new perspective on the bacteria that provide us with antibiotics.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2006; 361: 761-768Crossref PubMed Scopus (158) Google Scholar). Autoregulatory molecules play a key role in controlling both the onset of cellular differentiation and secondary metabolism in these microbes. The best studied autoregulator is A-factor (2-isocapryloyl-(3R)-hydroxymethyl-γ-butyrolactone), a small microbial hormone-like molecule (243 Da) that induces both morphological and physiological differentiation in Streptomyces griseus (4Horinouchi S. Beppu T. Autoregulatory factors and communication in actinomycetes.Annu. Rev. Microbiol. 1992; 46: 377-398Crossref PubMed Google Scholar, 5Horinouchi S. Beppu T. A-factor as a microbial hormone that controls cellular differentiation and secondary metabolism in Streptomyces griseus.Mol. Microbiol. 1994; 12: 859-864Crossref PubMed Scopus (152) Google Scholar) and its direct homologue SCB1 that plays an important role in the control of antibiotic production of Streptomyces coelicolor (6Takano E. Chakraburtty R. Nihira T. Yamada Y. Bibb M.J. A complex role for the gamma-butyrolactone SCB1 in regulating antibiotic production in Streptomyces coelicolor A3(2).Mol. Microbiol. 2001; 41: 1015-1028Crossref PubMed Scopus (182) Google Scholar, 7Takano E. Kinoshita H. Mersinias V. Bucca G. Hotchkiss G. Nihira T. Smith C.P. Bibb M. Wohlleben W. Chater K. A bacterial hormone (the SCB1) directly controls the expression of a pathway-specific regulatory gene in the cryptic type I polyketide biosynthetic gene cluster of Streptomyces coelicolor.Mol. Microbiol. 2005; 56: 465-479Crossref PubMed Scopus (133) Google Scholar). In S. griseus binding of A-factor to its cellular receptor ArpA derepresses expression of the transcriptional activator AdpA. Although initially identified as the activator of streptomycin production, this protein acts as a central switch, and the AdpA regulon includes several important positive regulators of development, including ssgA, amfR, and adsA (bldN) (for a review, see Ref. 8Ohnishi Y. Yamazaki H. Kato J.Y. 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: 431-439Crossref PubMed Scopus (186) Google Scholar). A-factor-deficient mutants are neither able to sporulate nor able to produce antibiotics (streptomycin).Another interesting autoregulator is the secreted signaling protein Factor C (molecular mass, 34.555 Da), originally isolated from the culture fluid of “S. griseus 45H” but recently shown to be identical to a laboratory strain known as Streptomyces flavofungini, itself a member of the Streptomyces albidoflavus species group. 1Z. Kiss, A. C. Ward, Z. Birkó, K. F. Chater, and S. Biró, unpublished data. 1Z. Kiss, A. C. Ward, Z. Birkó, K. F. Chater, and S. Biró, unpublished data. The Factor C producer strain, like S. griseus, readily sporulates in submerged culture (9Biró S. Békési I. Vitális S. Szabó G. A substance effecting differentiation in Streptomyces griseus. Purification and properties.Eur. J. Biochem. 1980; 103: 359-363Crossref PubMed Scopus (49) Google Scholar, 10Szabó G. Vályi-Nagy T. Vitális S. A new factor regulating life cycle of Streptomyces griseus.in: Timakova V.D. Genetics of Microorganisms, Proceedings of a Symposium on Heredity and Variability of Microorganisms. State Publishing House on Medical Literature, Moscow1962: 282-292Google Scholar). Similarly to A-factor, Factor C also plays a key role in cellular communication and cytodifferentiation. Expression of facC from a low copy plasmid in a spontaneous A-factor-deficient bald mutant of S. griseus NRRL B-2682 restored its aerial mycelium and spore formation on solid medium. This strain does not produce Factor C as shown by immunoblotting (11Szeszák F. Vitális S. Tóth F. Valu G. Fachet J. Szabó G. Detection and determination of factor C – a regulatory protein – in Streptomyces strains by antiserum and monoclonal antibody.Arch. Microbiol. 1990; 154: 82-84Crossref PubMed Scopus (8) Google Scholar) and by DNA hybridization studies (12Biró S. Birkó Z. van Wezel G.P. Transcriptional and functional analysis of the gene for factor C, an extracellular signal protein involved in cytodifferentiation of Streptomyces griseus.Antonie Leeuwenhoek. 2000; 78: 277-285Crossref PubMed Scopus (9) Google Scholar). This suggested that the Factor C response regulon acts independently or has a complex interaction with the A-factor regulon. In preliminary experiments comparing one-dimensional protein gels of the above strains we observed characteristic differences between the extracellular proteomes of the strains that prompted us to perform a detailed analysis. Here we compare the extracellular proteomes of S. griseus NRRL B-2682 and its A-factor-non-producing bald mutant with the same strain expressing Factor C from a low copy number vector.Note Added in Proof—Recently new insights on A-factor biosynthesis in S. griseus were provided by the Horinouchi group (Kato, J., Funa, N., Watanabe, H., Ohnishi, Y., and Horinouchi, S. (2007) Proc. Nat. Acad. Sci. U.S.A. 104, 2378–2383. Bacteria of the Gram-positive filamentous Streptomyces are a well known model system for the study of prokaryotic multicellular differentiation with a complex life cycle culminating in spore formation (1Chater K.F. Genetics of differentiation in Streptomyces.Annu. Rev. Microbiol. 1993; 47: 685-713Crossref PubMed Google Scholar). The onset of development is triggered by nutritional signals (2Rigali S. Nothaft H. Noens E.E. Schlicht M. Colson S. Muller M. Joris B. Koerten H.K. Hopwood D.A. Titgemeyer F. van Wezel G.P. The sugar phosphotransferase system of Streptomyces coelicolor is regulated by the GntR-family regulator DasR and links N-acetylglucosamine metabolism to the control of development.Mol. Microbiol. 2006; 61: 1237-1251Crossref PubMed Scopus (169) Google Scholar) and temporally relates to the production of antibiotics and other secondary metabolites (3Chater K.F. Streptomyces inside-out: a new perspective on the bacteria that provide us with antibiotics.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2006; 361: 761-768Crossref PubMed Scopus (158) Google Scholar). Autoregulatory molecules play a key role in controlling both the onset of cellular differentiation and secondary metabolism in these microbes. The best studied autoregulator is A-factor (2-isocapryloyl-(3R)-hydroxymethyl-γ-butyrolactone), a small microbial hormone-like molecule (243 Da) that induces both morphological and physiological differentiation in Streptomyces griseus (4Horinouchi S. Beppu T. Autoregulatory factors and communication in actinomycetes.Annu. Rev. Microbiol. 1992; 46: 377-398Crossref PubMed Google Scholar, 5Horinouchi S. Beppu T. A-factor as a microbial hormone that controls cellular differentiation and secondary metabolism in Streptomyces griseus.Mol. Microbiol. 1994; 12: 859-864Crossref PubMed Scopus (152) Google Scholar) and its direct homologue SCB1 that plays an important role in the control of antibiotic production of Streptomyces coelicolor (6Takano E. Chakraburtty R. Nihira T. Yamada Y. Bibb M.J. A complex role for the gamma-butyrolactone SCB1 in regulating antibiotic production in Streptomyces coelicolor A3(2).Mol. Microbiol. 2001; 41: 1015-1028Crossref PubMed Scopus (182) Google Scholar, 7Takano E. Kinoshita H. Mersinias V. Bucca G. Hotchkiss G. Nihira T. Smith C.P. Bibb M. Wohlleben W. Chater K. A bacterial hormone (the SCB1) directly controls the expression of a pathway-specific regulatory gene in the cryptic type I polyketide biosynthetic gene cluster of Streptomyces coelicolor.Mol. Microbiol. 2005; 56: 465-479Crossref PubMed Scopus (133) Google Scholar). In S. griseus binding of A-factor to its cellular receptor ArpA derepresses expression of the transcriptional activator AdpA. Although initially identified as the activator of streptomycin production, this protein acts as a central switch, and the AdpA regulon includes several important positive regulators of development, including ssgA, amfR, and adsA (bldN) (for a review, see Ref. 8Ohnishi Y. Yamazaki H. Kato J.Y. 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: 431-439Crossref PubMed Scopus (186) Google Scholar). A-factor-deficient mutants are neither able to sporulate nor able to produce antibiotics (streptomycin). Another interesting autoregulator is the secreted signaling protein Factor C (molecular mass, 34.555 Da), originally isolated from the culture fluid of “S. griseus 45H” but recently shown to be identical to a laboratory strain known as Streptomyces flavofungini, itself a member of the Streptomyces albidoflavus species group. 1Z. Kiss, A. C. Ward, Z. Birkó, K. F. Chater, and S. Biró, unpublished data. 1Z. Kiss, A. C. Ward, Z. Birkó, K. F. Chater, and S. Biró, unpublished data. The Factor C producer strain, like S. griseus, readily sporulates in submerged culture (9Biró S. Békési I. Vitális S. Szabó G. A substance effecting differentiation in Streptomyces griseus. Purification and properties.Eur. J. Biochem. 1980; 103: 359-363Crossref PubMed Scopus (49) Google Scholar, 10Szabó G. Vályi-Nagy T. Vitális S. A new factor regulating life cycle of Streptomyces griseus.in: Timakova V.D. Genetics of Microorganisms, Proceedings of a Symposium on Heredity and Variability of Microorganisms. State Publishing House on Medical Literature, Moscow1962: 282-292Google Scholar). Similarly to A-factor, Factor C also plays a key role in cellular communication and cytodifferentiation. Expression of facC from a low copy plasmid in a spontaneous A-factor-deficient bald mutant of S. griseus NRRL B-2682 restored its aerial mycelium and spore formation on solid medium. This strain does not produce Factor C as shown by immunoblotting (11Szeszák F. Vitális S. Tóth F. Valu G. Fachet J. Szabó G. Detection and determination of factor C – a regulatory protein – in Streptomyces strains by antiserum and monoclonal antibody.Arch. Microbiol. 1990; 154: 82-84Crossref PubMed Scopus (8) Google Scholar) and by DNA hybridization studies (12Biró S. Birkó Z. van Wezel G.P. Transcriptional and functional analysis of the gene for factor C, an extracellular signal protein involved in cytodifferentiation of Streptomyces griseus.Antonie Leeuwenhoek. 2000; 78: 277-285Crossref PubMed Scopus (9) Google Scholar). This suggested that the Factor C response regulon acts independently or has a complex interaction with the A-factor regulon. In preliminary experiments comparing one-dimensional protein gels of the above strains we observed characteristic differences between the extracellular proteomes of the strains that prompted us to perform a detailed analysis. Here we compare the extracellular proteomes of S. griseus NRRL B-2682 and its A-factor-non-producing bald mutant with the same strain expressing Factor C from a low copy number vector. Note Added in Proof—Recently new insights on A-factor biosynthesis in S. griseus were provided by the Horinouchi group (Kato, J., Funa, N., Watanabe, H., Ohnishi, Y., and Horinouchi, S. (2007) Proc. Nat. Acad. Sci. U.S.A. 104, 2378–2383. Recently new insights on A-factor biosynthesis in S. griseus were provided by the Horinouchi group (Kato, J., Funa, N., Watanabe, H., Ohnishi, Y., and Horinouchi, S. (2007) Proc. Nat. Acad. Sci. U.S.A. 104, 2378–2383. We thank Anikó Szilágyi for the excellent technical support in A-factor extraction. Supplementary Material Download .pdf (.94 MB) Help with pdf files Download .pdf (.94 MB) Help with pdf files