Abstract
The aim of this work is to establish the correlation between the transcriptional activator PTA1 and the expression of the penicillin genes in different penicillin-producing strains. The level of expression of the first two genes of the penicillin pathway was clearly higher in Penicillium chrysogenum than in Penicillium notatum and Penicillium nalgiovense. The divergent promoter pcbAB-pcbC region contains binding sequences for several transcriptional factors that are conserved in P. notatum and P. chrysogenum, but not in P. nalgiovense. Binding of the purified P. chrysogenum transcriptional activator PTA1 to the palindromic heptamer TTAGTAA took place when the P. chrysogenum 35 bp DNA fragment containing the heptamer was used as a probe, but not when the sequence occurring in P. nalgiovense was used. P. nalgiovense protein fractions purified by heparin agarose chromatography did not bind to the 35-bp DNA fragment either from P. nalgiovense or P. chrysogenum, although some degree of binding was observed when crude extracts were used. This finding may explain the low expression of pcbC in P. nalgiovense. All the P. chrysogenum strains, including the industrial strain E1, showed the same nucleotide sequence, including the consensus PTA1 binding site.
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References
Aharonowitz Y, Cohen G, Martín JF (1992) Penicillin and cephalosporin biosynthetic genes: structure, organization, regulation and evolution. Ann Rev Microbiol 46:461–495
Barredo JL, Álvarez E, Cantoral JM, Díez B, Martín JF (1988) Glucokinase deficient mutant of Penicillium chrysogenum is de repressed in glucose catabolite regulation of both β-galactosidase and penicillin biosynthesis. Antimicrob Agents Chemother 32:1061–1067
Barredo JL, Cantoral JM, Alvarez E, Díez B, Martín JF (1989a) Cloning, sequence analysis and transcriptional study of the isopenicillin N synthase of Penicillium chrysogenum AS-P-78. Mol Gen Genet 216:91–98
Barredo JL, van Solingen P, Díez B, Alvarez E, Cantoral JM, Kattevilder A, Smaal EB, Groenen MAM, Veenstra AE, Martín JF (1989b) Cloning and characterization of the acyl-coenzyme A:6-aminopenicillanic acid acyltransferase gene of Penicillium chrysogenum. Gene 83:291–300
Beck CF, Warren RA (1988) Divergent promoters, a common form of gene organization. Microbiol Rev 52:318–326
Brakhage AA (1998) Molecular regulation of beta-lactam biosynthesis in filamentous fungi. Microbiol Mol Biol Rev 62:547–585
Brakhage AA, Andrianopoulos A, Kato M, Steidl S, Davis MA, Tsukagoshi N, Hynes MJ (1999) HAP-like CCAAT-binding complexes in filamentous fungi: implications for biotechnology. Fungal Genet Biol 27:243–252
Bruns TD, Vilgalys R, Barns SM, Gonzalez D, Hibbett DS, Lane DJ, Simon L, Stickel S, Szaro TM, Weisburg WG et al (1992) Evolutionary relationships within the fungi: analyses of nuclear small subunit rRNA sequences. Mol Phylogenet Evol 1:231–241
Chu YW, Renno D, Saunders G (1995) Detection of a protein which binds specifically to the upstream region of the pcbAB gene in Penicillium chrysogenum. Curr Genet 27:184–189
Díez B, Gutiérrez S, Barredo JL, van Solingen P, van der Voort LH, Martín JF (1990) The cluster of penicillin biosynthetic genes. J Biol Chem 265:16358–16365
Díez B, Mellado E, Rodríguez M, Fouces R, Barredo JL (1997) Recombinant microorganisms for the industrial production of antibiotics. Biotechnol Bioeng 55:216–226
Esmahan C, Álvarez E, Montenegro E, Martín JF (1994) Catabolism of lysine in Penicillium chrysogenum leads to formation of α-aminoadipic, a precursor of penicillin biosythesis. Appl Environ Microbiol 60:1705–1710
Espeso EA, Peñalva MA (1992) Carbon catabolite repression can account for temporal pattern of expression of a penicillin biosynthetic gene in Aspergillus nidulans. Mol Microbiol 6:1457–1465
Espeso EA, Peñalva MA (1996) Three binding sites for the Aspergillus nidulans PacC zinc-finger transcription factor are necessary and sufficient for regulation by ambient pH of the isopenicillin N synthase gene promoter. J Biol Chem 271:28825–28830
Feng B, Friedlin E, Marzluf GA (1995) Nuclear DNA-binding proteins which recognize the intergenic control region of penicillin biosynthetic genes. Curr Genet 27:351–358
Fierro F, Barredo JL, Díez B, Gutiérrez S, Fernández FJ, Martín JF (1995) The penicillin gene cluster is amplified in tandem repeats linked by conserved hexanucleotide sequences. Proc Natl Acad Sci USA 92:6200–6204
Fierro F, Montenegro E, Gutiérrez S, Martín JF (1996) Mutants blocked in penicillin biosynthesis show a deletion of the entire penicillin gene cluster at a specific site within a conserved hexanucleotide sequence. Appl Microbiol Biotechnol 44:597–604
Fierro F, García-Estrada C, Castillo I, Rodríguez R, Velasco-Conde T, Martín JF (2006) Transcriptional and bioinformatic analysis of the 56.8 kb DNA region amplified in tandem repeats containing the penicillin gene cluster in Penicillium chrysogenum. Fungal Genet Biol 43:618–629
Gutiérrez S, Díez B, Álvarez E, Barredo JL, Martín JF (1991) Expression of the penDE gene of Penicillium chrysogenum encoding isopenicillin N acyltransferase in Cephalosporium acremonium: production of benzylpenicillin by the transformants. Mol Gen Genet 225:56–64
Gutiérrez S, Marcos AT, Casqueiro J, Kosalková K, Fernández FJ, Velasco J, Martín JF (1999) Transcription of the pcbAB, pcbC and penDE genes of Penicillin chrysogenum AS-P-78 is repressed by glucose and the repression is not reversed by alkaline pHs. Microbiology 145:317–324
Haas H, Marzluf GA (1995) NRE, the major nitrogen regulatory protein of Penicillium chrysogenum, binds specifically to elements in the intergenic promoter regions of nitrate assimilation and penicillin biosynthetic gene clusters. Curr Genet 28:177–183
Haas H, Bauer B, Redl B, Stoffler G, Marzluf GA (1995) Molecular cloning and analysis of nre, the major nitrogen regulatory gene of Penicillium chrysogenum. Curr Genet 27:150–158
Ishida C, Aranda C, Valenzuela L, Riego L, DeLuna A, Recillas-Targa F, Filetici P, López-Revilla R, González A (2006) The UGA3-GLT1 intergenic region constitutes a promoter whose bidirectional nature is determined by chromatin organization in Saccharomyces cerevisiae. Mol Microbiol 59:1790–1806
Kosalková K, Marcos AT, Fierro F, Hernando-Rico V, Gutiérrez S, Martín JF (2000) A novel heptameric sequence (TTAGTAA) is the binding site for a protein required for high level expression of pcbAB, the first gene of the penicillin biosynthesis in Penicillium chrysogenum. J Biol Chem 275:2423–2430
Laich F, Fierro F, Cardoza RE, Martín JF (1999) Organization of the gene cluster for biosynthesis of penicillin in Penicillium nalgiovense and antibiotic production in cured dry sausages. Appl Environ Microbiol 65:1236–1240
Laich F, Fierro F, Martín JF (2003) Isolation of Penicillium nalgiovense strains impaired in penicillin production by disruption of the pcbAB gene and application as starters on cured meat products. Mycol Res 107:717–726
Lamas-Maceiras M, Vaca I, Rodríguez E, Casqueiro J, Martín JF (2005) Amplification and disruption of the phenylacetyl-CoA ligase gene of Penicillium chrysogenum encoding an aryl-capping enzyme that supplies phenylacetic acid to the isopenicillin N acyltransferase. Biochem J 395:147–155
Martín JF (1998) New aspects of genes and enzymes for ß-lactam antibiotic biosynthesis. Appl Microbiol Biotechnol 50:1–15
Martín JF (2000) Molecular control of expression of penicillin biosynthesis genes in fungi: regulatory proteins interact with a bidirectional promoter region. J Bacteriol 182:2355–2362
Martín JF, Casqueiro J, Liras P (2005) Secretion systems for secondary metabolites: how producer cells send out messages of intercellular communication. Curr Opin Microbiol 8:282–293
Newbert RW, Barton B, Greaves P, Harper J, Turner G (1997) Analysis of a commercially improved Penicillium chrysogenum strain series: involvement of recombinogenic regions in amplification and deletion of the penicillin biosynthesis gene cluster. J Ind Microbiol Biotechnol 19:18–27
Revilla G, Ramos FR, López-Nieto MJ, Álvarez E, Martín JF (1986) Glucose represses formation of δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine and isopenicillin N synthase but not penicillin acyltransferase in Penicillium chrysogenum. J Bacteriol 168:947–952
Rodríguez-Sáiz M, Díez B, Barredo JL (2005) Why did the Fleming strain fail in penicillin industry? Fungal Genet Biol 42:464–470
Sambrook KJ, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual. 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Stauffer JF, Backus MP (1954) Spontaneous and induced variation in selected stocks of the Penicillium chrysogenum series. Ann N T Acad Sci 60:34–49
Suárez T, Peñalva MA (1996) Characterization of a Penicillium chrysogenum gene encoding a PacC transcription factor and its binding sites in the divergent pcbAB-pcbC promoter of the penicillin biosynthetic cluster. Mol Microbiol 20:529–540
Tag A, Hicks J, Garifullina G, Ake C Jr, Phillips TD, Beremand M, Keller N (2000) G-protein signalling mediates differential production of toxic secondary metabolites. Mol Microbiol 38:658–665
Tilburn J, Sarkar S, Widdick DA, Espeso EA, Orejas M, Mungroo J, Peñalva MA, Arst HN Jr (1995) The Aspergillus PacC zinc finger transcription factor mediates regulation of both acid- and alkaline-expressed genes by ambient pH. EMBO J 14:779–790
Acknowledgments
This work was supported by a grant of the European Union (Eurofung II) to J.F. Martín. We thank Antibióticos S.A. for technical support and B. Martín, J. Merino, A. Casenave and B. Aguado for their excellent technical assistance.
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Communicated by J. Heitman.
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Kosalková, K., Rodríguez-Sáiz, M., Barredo, J.L. et al. Binding of the PTA1 transcriptional activator to the divergent promoter region of the first two genes of the penicillin pathway in different Penicillium species . Curr Genet 52, 229–237 (2007). https://doi.org/10.1007/s00294-007-0157-7
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DOI: https://doi.org/10.1007/s00294-007-0157-7