Abstract
Secondary metabolites (SMs) are compounds with relevant biological activities. Their production under laboratory conditions, especially in broth, is still challenging. An example is the pedopeptins, which are nonribosomal peptides active against some bacteria listed by the WHO for which new antibiotics are urgently needed. Their biosynthesis is inhibited by high concentrations of peptone from casein (PC) in tryptic soy broth (TSB), and we applied a RNA-seq approach to identify Pedobacter lusitanus NL19 cellular pathways modulated by this condition. Results were validated by qPCR and revealed 261 differentially expressed genes (DEGs), 46.3% of them with a predicted biological function. Specifically, high concentration of PC significantly repressed the de novo biosynthesis of biotin (− 60X) and the production of nonribosomal peptide synthetases (NRPS) of pedopeptins (about − 14X), but no effect was observed on the expression of other NRPS. Transcription of a L-Dap synthesis operon that includes a protein with a σ70-like domain was also reduced (about − 7X). High concentrations of PC led to a significant overexpression of MFS and RND efflux pumps and a ferrous iron uptake system, suggesting the redirection of cell machinery to export compounds such as amino acids, sugars and metal divalent cations, alongside with a slight increase of iron import.
Key points
• Higher concentrations of phosphate sources highly repress many operons
• High concentrations of peptone from casein (PC) cause biotin’s operon repression
• High concentrations of PC downregulate the production of peptides of unknown function
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Data availability
RNA-seq data was deposited in the Mendeley Database and can be accessed at https://data.mendeley.com/datasets/t5ryx5mk2k/1.
References
Barrios-González J (2012) Solid-state fermentation: physiology of solid medium, its molecular basis and applications. Process Biochem 47:175–185. https://doi.org/10.1016/j.procbio.2011.11.016
Barrios-González J (2018) Secondary metabolites production. In: Current developments in biotechnology and bioengineering. Elsevier, pp 257–283
Beauchene NA, Mettert EL, Moore LJ, Keleş S, Willey ER, Kiley PJ (2017) O 2 availability impacts iron homeostasis in Escherichia coli. Proc Natl Acad Sci 114:12261–12266. https://doi.org/10.1073/pnas.1707189114
Brakhage AA, Schroeckh V (2011) Fungal secondary metabolites—strategies to activate silent gene clusters. Fungal Genet Biol 48:15–22. https://doi.org/10.1016/j.fgb.2010.04.004
Chan YA, Podevels AM, Kevany BM, Thomas MG (2009) Biosynthesis of polyketide synthase extender units. Nat Prod Rep 26:90–114. https://doi.org/10.1039/b801658p
Covas C, Almeida B, Esteves AC, Lourenço J, Domingues P, Caetano T, Mendo S (2021) Peptone from casein, an antagonist of nonribosomal peptide synthesis: a case study of pedopeptins produced by Pedobacter lusitanus NL19. N Biotechnol 60:62–71. https://doi.org/10.1016/j.nbt.2020.07.006
Cronan JE, Waldrop GL (2002) Multi-subunit acetyl-CoA carboxylases. Prog Lipid Res 41:407–435
Croucher NJ, Thomson NR (2010) Studying bacterial transcriptomes using RNA-seq. Curr Opin Microbiol 13:619–624
Davati N, Habibi Najafi B MB (2013) Overproduction strategies for microbial secondary metabolites: a review. International Journal of Life Science and Pharma Research 3(1):L23–L37
Delmar JA, Su C-C, Yu EW (2015) Heavy metal transport by the CusCFBA efflux system. Protein Sci 24:1720–1736. https://doi.org/10.1002/pro.2764
Du D, Wang-Kan X, Neuberger A, van Veen HW, Pos KM, Piddock LJV, Luisi BF (2018) Multidrug efflux pumps: structure, function and regulation. Nat Rev Microbiol 16:523–539
Götz S, García-Gómez JM, Terol J, Williams TD, Nagaraj SH, Nueda MJ, Robles M, Talón M, Dopazo J, Conesa A (2008) High-throughput functional annotation and data mining with the Blast2GO suite. Nucleic Acids Res 36:3420–3435. https://doi.org/10.1093/nar/gkn176
Gray VL, Müller CT, Watkins ID, Lloyd D (2008) Peptones from diverse sources: pivotal determinants of bacterial growth dynamics. J Appl Microbiol 104:554–565. https://doi.org/10.1111/j.1365-2672.2007.03577.x
Hirota-Takahata Y, Kozuma S, Kuraya N, Fukuda D, Nakajima M, Ando O (2014) Pedopeptins, novel inhibitors of LPS: Taxonomy of producing organism, fermentation, isolation, physicochemical properties and structural elucidation. J Antibiot (tokyo) 67:243–251. https://doi.org/10.1038/ja.2013.122
Johnson M, Zaretskaya I, Raytselis Y, Merezhuk Y, McGinnis S, Madden TL (2008) NCBI BLAST: a better web interface. Nucleic Acids Res 36:5–9. https://doi.org/10.1093/nar/gkn201
Kanehisa M, Araki M, Goto S, Hattori M, Hirakawa M, Itoh M, Katayama T, Kawashima S, Okuda S, Tokimatsu T, Yamanishi Y (2007) KEGG for linking genomes to life and the environment. Nucleic Acids Res 36:D480–D484. https://doi.org/10.1093/nar/gkm882
Keller NP (2019) Fungal secondary metabolism: regulation, function and drug discovery HHS Public Access. Nat Rev Microbiol 17:167–180. https://doi.org/10.1038/s41579-018-0121-1
Kerry A, Laudenbach DE, Trick CG (1988) Influence of iron limitation and nitrogen source on growth and siderophore production by cyanobacteria. J Phycol 24:566–571. https://doi.org/10.1111/j.1529-8817.1988.tb04263.x
Klenotic PA, Moseng MA, Morgan CE, Yu EW (2020) Structural and functional diversity of resistance–nodulation–cell division transporters. Chem Rev acs.chemrev.0c00621 doi:https://doi.org/10.1021/acs.chemrev.0c00621
Kosalková K, García-Estrada C, Barreiro C, Flórez MG, Jami MS, Paniagua MA, Martín JF (2012) Casein phosphopeptides drastically increase the secretion of extracellular proteins in Aspergillus awamori. Proteomics studies reveal changes in the secretory pathway. Microbial Cell Factories 11(5). https://doi.org/10.1186/1475-2859-11-5
Kozuma S, Hirota-Takahata Y, Fukuda D, Kuraya N, Nakajima M, Ando O (2014) Screening and biological activities of pedopeptins, novel inhibitors of LPS produced by soil bacteria. J Antibiot (tokyo) 67:237–242. https://doi.org/10.1038/ja.2013.121
Lau CKY, Krewulak KD, Vogel HJ (2016) Bacterial ferrous iron transport: the Feo system. FEMS Microbiol Rev 40:273–298. https://doi.org/10.1093/femsre/fuv049
Li H, Hu P, Wang Y, Pan Y, Liu G (2018) Enhancing the production of cephalosporin C through modulating the autophagic process of Acremonium chrysogenum. Microb Cell Fact 17:175. https://doi.org/10.1186/s12934-018-1021-9
Lu S, Wang J, Chitsaz F, Derbyshire MK, Geer RC, Gonzales NR, Gwadz M, Hurwitz DI, Marchler GH, Song JS, Thanki N, Yamashita RA, Yang M, Zhang D, Zheng C, Lanczycki CJ, Marchler-Bauer A (2020) CDD/SPARCLE: the conserved domain database in 2020. Nucleic Acids Res 48(D1):D265–D268. https://doi.org/10.1093/nar/gkz991
McCarthy DJ, Chen Y, Smyth GK (2012) Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Res 40(10):4288–4297. https://doi.org/10.1093/nar/gks042
McLean TC, Wilkinson B, Hutchings MI, Devine R, McLean TC, Wilkinson B, Hutchings MI, Devine R (2019) Dissolution of the disparate: co-ordinate regulation in antibiotic biosynthesis. Antibiotics 8:83. https://doi.org/10.3390/antibiotics8020083
Mishra NK, Chang J, Zhao PX (2014) Prediction of membrane transport proteins and their substrate specificities using primary sequence information. PLoS ONE 9:e100278. https://doi.org/10.1371/journal.pone.0100278
Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B (2008) Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 5:621–628. https://doi.org/10.1038/nmeth.1226
Mulder NJ, Apweiler R (2008) The InterPro database and tools for protein domain analysis. Curr Protoc Bioinforma 21:2.7.1–2.7.18 doi:https://doi.org/10.1002/0471250953.bi0207s21
Naik G, Shukla S, Mall R, Mishra SK (2015) Optimization of culture conditions of Streptomyces zaomyceticus Rc 2073 by shake flask method. European Journal of Medical and Pharmaceutical Sciences 2(4):620–629
Nies DH, Nies A, Chu L, Silver S (1989) Expression and nucleotide sequence of a plasmid-determined divalent cation efflux system from Alcaligenes eutrophus. Proc Natl Acad Sci 86:7351–7355. https://doi.org/10.1073/pnas.86.19.7351
ŌMura S, Tanaka Y, Mamada H, Masuma R, (1984) Effect of ammonium ion, inorganic phosphate and amino acids on the biosynthesis of protylonolide, a precursor of tylosin aglyconeü. J Antibiot (tokyo) 37:494–502. https://doi.org/10.7164/antibiotics.37.494
World Health Organization (2017) Prioritization of pathogens to guide discovery, research and development of new antibiotics for drug-resistant bacterial infections, including tuberculosis. WHO/EMP/IAU/201712 1–87. Licence: CC BY-NC-SA 3.0 IGO
Pasqua G, Zennaro F, Micheli B, Colonna P (2019) The varied role of efflux pumps of the MFS family in the interplay of bacteria with animal and plant cells. Microorganisms 7:285. https://doi.org/10.3390/microorganisms7090285
Payne GA, Hagler WM (1983) Effect of specific amino acids on growth and aflatoxin production by Aspergillus parasiticus and Aspergillus flavus in defined media. Appl Environ Microbiol 46:805–812. https://doi.org/10.1128/aem.46.4.805-812.1983
Polyak SW, Abell AD, Wilce MCJ, Zhang L, Booker GW (2012) Structure, function and selective inhibition of bacterial acetyl-coa carboxylase. Appl Microbiol Biotechnol 93:983–992
Puel O, Galtier P, Oswald IP (2010) Biosynthesis and toxicological effects of patulin. Toxins (basel) 2:613–631. https://doi.org/10.3390/toxins2040613
Raza K, Mishra A (2012) A novel anticlustering filtering algorithm for the prediction of genes as a drug target. Am J Biomed Eng 2:206–211. https://doi.org/10.5923/j.ajbe.20120205.03
Robinson MD, McCarthy DJ, Smyth GK (2010) edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26:139–140. https://doi.org/10.1093/bioinformatics/btp616
Rodionov DA, Mironov AA, Gelfand MS (2002) Conservation of the biotin regulon and the BirA regulatory signal in eubacteria and archaea. Genome Res 12:1507–1516. https://doi.org/10.1101/gr.314502
Ruiz B, Chávez A, Forero A, García-Huante Y, Romero A, Sánchez M, Rocha D, Sánchez B, Rodríguez-Sanoja R, Sánchez S, Langley E, Snchez M, Rocha D, Snchez B, Rodríguez-Sanoja R, Sánchez S, Langley E, Sánchez M, Rocha D, Sánchez B, Rodríguez-Sanoja R, Sánchez S, Langley E, Snchez M, Rocha D, Snchez B, Rodríguez-Sanoja R, Sánchez S, Langley E (2010) Production of microbial secondary metabolites: regulation by the carbon source. Crit Rev Microbiol 36:146–167. https://doi.org/10.3109/10408410903489576
Sánchez S, Chávez A, Forero A, García-Huante Y, Romero A, Sánchez M, Rocha D, Sánchez B, Avalos M, Guzmán-Trampe S, Rodríguez-Sanoja R, Langley E, Ruiz B (2010) Carbon source regulation of antibiotic production. J Antibiot (tokyo) 63:442–459. https://doi.org/10.1038/ja.2010.78
Sanchez S, Demain AL (2002) Metabolic regulation of fermentation processes. Enzyme Microb Technol 31:895–906
Santana MP, Aburjaile FF, Parise MTD, Tiwari S, Silva A, Azevedo V, Pinto AC (2016) RNA-seq—revealing biological insights in bacteria. In: Next generation sequencing—advances, applications and challenges. InTechOpen. https://doi.org/10.5772/61669
Santos T, Cruz A, Caetano T, Covas C, Mendo S (2015) Draft genome sequence of Pedobacter sp. strain NL19, a producer of potent antibacterial compounds. Genome Announc 3:e00184-e215. https://doi.org/10.1128/genomeA.00184-15
Schulz-Bohm K, Martín-Sánchez L, Garbeva P (2017) Microbial volatiles: small molecules with an important role in intra- and inter-kingdom interactions. Front Microbiol 8. https://doi.org/10.3389/fmicb.2017.02484
Sestok AE, Linkous RO, Smith AT (2018) Toward a mechanistic understanding of feo-mediated ferrous iron uptake graphical abstract HHS public access. Metallomics 10:887–898. https://doi.org/10.1039/c8mt00097b
Sinha R, Stanley G, Gulati GS, Ezran C, Travaglini KJ, Wei E, Chan CKF, Nabhan AN, Su T, Morganti RM, Conley SD, Chaib H, Red-Horse K, Longaker MT, Snyder MP, Krasnow MA, Weissman IL (2017) Index switching causes “spreading-of-signal” among multiplexed samples in Illumina HiSeq 4000 DNA sequencing. bioRxiv 125724. https://doi.org/10.1101/125724
Solovyev V, Salamov A (2011) Automatic annotation of microbial genomes and metagenomic sequences. In: Metagenomics and its applications in agriculture, biomedicine and environmental studies. Nova Science Publishers, p 61–78
Sorek R, Cossart P (2010) Prokaryotic transcriptomics: a new view on regulation, physiology and pathogenicity. Nat Rev Genet 11:9–16
Streit WR, Entcheva P (2003) Biotin in microbes, the genes involved in its biosynthesis, its biochemical role and perspectives for biotechnological production. Appl Microbiol Biotechnol 61:21–31
Tacconelli E, Carrara E, Savoldi A, Harbarth S, Mendelson M, Monnet DL, Pulcini C, Kahlmeter G, Kluytmans J, Carmeli Y, Ouellette M, Outterson K, Patel J, Cavaleri M, Cox EM, Houchens CR, Grayson ML, Hansen P, Singh N, Theuretzbacher U, Magrini N, Aboderin AO, Al-Abri SS, Awang Jalil N, Benzonana N, Bhattacharya S, Brink AJ, Burkert FR, Cars O, Cornaglia G, Dyar OJ, Friedrich AW, Gales AC, Gandra S, Giske CG, Goff DA, Goossens H, Gottlieb T, Guzman Blanco M, Hryniewicz W, Kattula D, Jinks T, Kanj SS, Kerr L, Kieny M-P, Kim YS, Kozlov RS, Labarca J, Laxminarayan R, Leder K, Leibovici L, Levy-Hara G, Littman J, Malhotra-Kumar S, Manchanda V, Moja L, Ndoye B, Pan A, Paterson DL, Paul M, Qiu H, Ramon-Pardo P, Rodríguez-Baño J, Sanguinetti M, Sengupta S, Sharland M, Si-Mehand M, Silver LL, Song W, Steinbakk M, Thomsen J, Thwaites GE, van der Meer JW, Van Kinh N, Vega S, Villegas MV, Wechsler-Fördös A, Wertheim HFL, Wesangula E, Woodford N, Yilmaz FO, Zorzet A (2018) Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis. Lancet Infect Dis 18:318–327. https://doi.org/10.1016/S1473-3099(17)30753-3
Tudzynski B, Homann V, Feng B, Marzluf GA (1999) Isolation, characterization and disruption of the area nitrogen regulatory gene of Gibberella fujikuroi. Mol Gen Genet 261:106–114. https://doi.org/10.1007/s004380050947
van Iterson M, Boer JM, Menezes RX (2010) Filtering, FDR and power. BMC Bioinformatics 11:450. https://doi.org/10.1186/1471-2105-11-450
Vandermolen KM, Raja HA, El-Elimat T, Oberlies NH (2013) Evaluation of culture media for the production of secondary metabolites in a natural products screening program. AMB Express 3(1):713. https://doi.org/10.1186/2191-0855-3-71
Yin W, Keller NP (2011) Transcriptional regulatory elements in fungal secondary metabolism. J Microbiol 49:329–339
Yu NY, Wagner JR, Laird MR, Melli G, Rey S, Lo R, Dao P, Sahinalp SC, Ester M, Foster LJ, Brinkman FSL (2010) PSORTb 3.0: improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes. Bioinformatics 26:1608–1615. https://doi.org/10.1093/bioinformatics/btq249
Zhang J, you, Banko G, Wolfe S, Demain AL, (1987) Methionine induction of ACV synthetase in Cephalosporium acremonium. J Ind Microbiol 2:251–255. https://doi.org/10.1007/BF01569547
Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415. https://doi.org/10.1093/nar/gkg595
Funding
Cláudia Covas was supported by Fundação para a Ciência e Tecnologia (FCT), POPH and European Union grant SFRH/BD/98446/2013. Tânia Caetano (CEECIND/01463/2017) and Joana Lourenço (SFRH/BPD/92554/2013) were supported by POPH and national funds (OE), through FCT—Fundação para a Ciência e a Tecnologia, I.P., in the scope of the framework contract foreseen in the numbers 4, 5 and 6 of the article 23, of the Decree-Law 57/2016, of August 29, changed by Law 57/2017, of July 19. We acknowledge financial support to CESAM by FCT/MCTES (UIDP/50017/2020 + UIDB/50017/2020 + LA/P/0094/2020), through national funds. This work supported by Fundação de Apoio à Pesquisa do Distrito Federal (FAPDF), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (PVE 390876-2014), Conselho Nacional de Desenvolvimento e Tecnológico (CNPq) and Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado de Mato Grosso do Sul (FUNDECT). Sónia Mendo received support under the MEC/MCTI/CAPES/CNPQ/FAPS project (390876-2014), through a Special Visiting Researcher scholarship - PVE 2014.
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CCJF, VA and AB performed the experiments. CCJF analyzed the data and wrote the manuscript. LJ, FG and FO analyzed the data. MS supervised the work and wrote the manuscript. CT designed and supervised the work, analyzed data and wrote the manuscript. All authors participated in the preparation and revision of the manuscript.
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C., C., A., V., B., A. et al. Nitrogen source as a modulator of the metabolic activity of Pedobacter lusitanus NL19: a transcriptomic approach. Appl Microbiol Biotechnol 106, 1583–1597 (2022). https://doi.org/10.1007/s00253-022-11796-3
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DOI: https://doi.org/10.1007/s00253-022-11796-3