Fluorescent riboswitch-controlled biosensors for the genome scale analysis of metabolic pathways.
Green fluorescent protein
Metabolic pathway
Riboswitch
Thiamin pyrophosphate
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
31 May 2024
31 May 2024
Historique:
received:
08
01
2024
accepted:
13
05
2024
medline:
1
6
2024
pubmed:
1
6
2024
entrez:
31
5
2024
Statut:
epublish
Résumé
Fluorescent detection in cells has been tremendously developed over the years and now benefits from a large array of reporters that can provide sensitive and specific detection in real time. However, the intracellular monitoring of metabolite levels still poses great challenges due to the often complex nature of detected metabolites. Here, we provide a systematic analysis of thiamin pyrophosphate (TPP) metabolism in Escherichia coli by using a TPP-sensing riboswitch that controls the expression of the fluorescent gfp reporter. By comparing different combinations of reporter fusions and TPP-sensing riboswitches, we determine key elements that are associated with strong TPP-dependent sensing. Furthermore, by using the Keio collection as a proxy for growth conditions differing in TPP levels, we perform a high-throughput screen analysis using high-density solid agar plates. Our study reveals several genes whose deletion leads to increased or decreased TPP levels. The approach developed here could be applicable to other riboswitches and reporter genes, thus representing a framework onto which further development could lead to highly sophisticated detection platforms allowing metabolic screens and identification of orphan riboswitches.
Identifiants
pubmed: 38821978
doi: 10.1038/s41598-024-61980-w
pii: 10.1038/s41598-024-61980-w
doi:
Substances chimiques
Riboswitch
0
Thiamine Pyrophosphate
Q57971654Y
Green Fluorescent Proteins
147336-22-9
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
12555Informations de copyright
© 2024. The Author(s).
Références
Bennett, B. D. et al. Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli. Nat. Chem. Biol. 5, 593–599 (2009).
pubmed: 19561621
pmcid: 2754216
doi: 10.1038/nchembio.186
Luo, B., Groenke, K., Takors, R., Wandrey, C. & Oldiges, M. Simultaneous determination of multiple intracellular metabolites in glycolysis, pentose phosphate pathway and tricarboxylic acid cycle by liquid chromatography-mass spectrometry. J. Chromatogr. A 1147, 153–164 (2007).
pubmed: 17376459
doi: 10.1016/j.chroma.2007.02.034
Zhang, J., Jensen, M. K. & Keasling, J. D. Development of biosensors and their application in metabolic engineering. Curr. Opin. Chem. Biol. 28, 1–8 (2015).
pubmed: 26056948
doi: 10.1016/j.cbpa.2015.05.013
Salvail, H. & Breaker, R. R. Riboswitches. Curr. Biol. 33, R343–R348 (2023).
pubmed: 37160088
doi: 10.1016/j.cub.2023.03.069
Breaker, R. R. The biochemical landscape of riboswitch ligands. Biochemistry 61, 137–149 (2022).
pubmed: 35068140
doi: 10.1021/acs.biochem.1c00765
Mccown, P. J., Corbino, K. A., Stav, S., Sherlock, M. E. & Breaker, R. R. Riboswitch diversity and distribution. RNA 23, 995–1011 (2017).
pubmed: 28396576
pmcid: 5473149
doi: 10.1261/rna.061234.117
Bédard, A.-S.V., Hien, E. D. M. & Lafontaine, D. A. Riboswitch regulation mechanisms: RNA, metabolites and regulatory proteins. Biochim. Biophys. Acta Gene Regul. Mech. 1863, 194501 (2020).
pubmed: 32036061
doi: 10.1016/j.bbagrm.2020.194501
Chauvier, A. et al. Transcriptional pausing at the translation start site operates as a critical checkpoint for riboswitch regulation. Nat. Commun. 8, 13892 (2017).
pubmed: 28071751
pmcid: 5234074
doi: 10.1038/ncomms13892
Tomsic, J., McDaniel, B. A., Grundy, F. J. & Henkin, T. M. Natural variability in S-adenosylmethionine (SAM)-dependent riboswitches: S-box elements in Bacillus subtilis exhibit differential sensitivity to SAM In vivo and in vitro. J. Bacteriol. 190, 823–833 (2008).
pubmed: 18039762
doi: 10.1128/JB.01034-07
Kavita, K. & Breaker, R. R. Discovering riboswitches: The past and the future. Trends Biochem. Sci. 48, 119–141 (2023).
pubmed: 36150954
doi: 10.1016/j.tibs.2022.08.009
Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. https://doi.org/10.1038/msb4100050 (2006).
doi: 10.1038/msb4100050
pubmed: 16738554
pmcid: 1681482
Zaslaver, A. et al. A comprehensive library of fluorescent transcriptional reporters for Escherichia coli. Nat. Methods 3, 623–628 (2006).
pubmed: 16862137
doi: 10.1038/nmeth895
Fowler, C. C., Brown, E. D. & Li, Y. Using a riboswitch sensor to examine coenzyme B(12) metabolism and transport in E. coli. Chem. Biol. 17, 756–765 (2010).
pubmed: 20659688
doi: 10.1016/j.chembiol.2010.05.025
Cai, Y. et al. Engineering a vitamin B12 high-throughput screening system by riboswitch sensor in Sinorhizobium meliloti. BMC Biotechnol. 18, 27 (2018).
pubmed: 29751749
pmcid: 5948670
doi: 10.1186/s12896-018-0441-2
Winkler, W., Nahvi, A. & Breaker, R. R. Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression. Nature 419, 952–956 (2002).
pubmed: 12410317
doi: 10.1038/nature01145
Winkler, W. C., Nahvi, A., Sudarsan, N., Barrick, J. E. & Breaker, R. R. An mRNA structure that controls gene expression by binding S-adenosylmethionine. Nat. Struct. Biol. 10, 701–707 (2003).
pubmed: 12910260
doi: 10.1038/nsb967
Chauvier, A., Nadon, J.-F., Grondin, J. P., Lamontagne, A.-M. & Lafontaine, D. A. Role of a hairpin-stabilized pause in the Escherichia coli thiC riboswitch function. RNA Biol. 16, 1066–1073 (2019).
pubmed: 31081713
pmcid: 6602414
doi: 10.1080/15476286.2019.1616354
Serganov, A., Polonskaia, A., Phan, A. T., Breaker, R. R. & Patel, D. J. Structural basis for gene regulation by a thiamine pyrophosphate-sensing riboswitch. Nature 441, 1167–1171 (2006).
pubmed: 16728979
pmcid: 4689313
doi: 10.1038/nature04740
Edwards, T. E. & Ferre-D’Amare, A. R. Crystal structures of the Thi-Box riboswitch bound to thiamine pyrophosphate analogs reveal adaptive RNA-small molecule recognition. Structure 14, 1459–1468 (2006).
pubmed: 16962976
doi: 10.1016/j.str.2006.07.008
Thore, S., Leibundgut, M. & Ban, N. Structure of the eukaryotic thiamine pyrophosphate riboswitch with its regulatory ligand. Science 312, 1208–1211 (2006).
pubmed: 16675665
doi: 10.1126/science.1128451
Ontiveros-Palacios, N. et al. Molecular basis of gene regulation by the THI-box riboswitch. Mol. Microbiol. 67, 793–803 (2008).
pubmed: 18179415
doi: 10.1111/j.1365-2958.2007.06088.x
Schyns, G. et al. Isolation and characterization of new thiamine-deregulated mutants of Bacillus subtilis. J. Bacteriol. 187, 8127–8136 (2005).
pubmed: 16291685
pmcid: 1291275
doi: 10.1128/JB.187.23.8127-8136.2005
Bastet, L. et al. Translational control and Rho-dependent transcription termination are intimately linked in riboswitch regulation. Nucleic Acids Res. 45, 7474–7486 (2017).
pubmed: 28520932
pmcid: 5499598
doi: 10.1093/nar/gkx434
Fraikin, N., Rousseau, C. J., Goeders, N. & Van Melderen, L. Reassessing the role of the type II MqsRA toxin-antitoxin system in stress response and biofilm formation: mqsA is transcriptionally uncoupled from mqsR. mBio 10, e0267819 (2019).
doi: 10.1128/mBio.02678-19
Weiss, C. A. & Winkler, W. C. Riboswitch-mediated detection of metabolite fluctuations during live cell imaging of bacteria. Methods Mol. Biol. 2323, 153–170 (2021).
pubmed: 34086280
doi: 10.1007/978-1-0716-1499-0_12
French, S. et al. A robust platform for chemical genomics in bacterial systems. Mol. Biol. Cell 27, 1015–1025 (2016).
pubmed: 26792836
pmcid: 4791123
doi: 10.1091/mbc.E15-08-0573
Côté, J.-P. et al. The genome-wide interaction network of nutrient stress genes in Escherichia coli. mBio 7, e01714-e1716 (2016).
pubmed: 27879333
pmcid: 5120140
Lu, Y.-H., Guan, Z., Zhao, J. & Raetz, C. R. H. Three phosphatidylglycerol-phosphate phosphatases in the inner membrane of Escherichia coli. J. Biol. Chem. 286, 5506–5518 (2011).
pubmed: 21148555
doi: 10.1074/jbc.M110.199265
Nakayama, H. & Hayashi, R. Biosynthesis of thiamine pyrophosphate in Escherichia coli. J. Bacteriol. 109, 936–938 (1972).
pubmed: 4550824
pmcid: 285243
doi: 10.1128/jb.109.2.936-938.1972
Kitagawa, M. et al. Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): Unique resources for biological research. DNA Res. 12, 291–299 (2005).
pubmed: 16769691
doi: 10.1093/dnares/dsi012
Yan, X. et al. The twin-arginine translocation system is important for stress resistance and virulence of Brucella melitensis. Infect. Immun. 88, e00389-e420 (2020).
pubmed: 32778612
pmcid: 7573438
doi: 10.1128/IAI.00389-20
Chowdhury, N., Kwan, B. W. & Wood, T. K. Persistence increases in the absence of the alarmone guanosine tetraphosphate by reducing cell growth. Sci. Rep. 6, 20519 (2016).
pubmed: 26837570
pmcid: 4738310
doi: 10.1038/srep20519
Krulwich, T. A., Sachs, G. & Padan, E. Molecular aspects of bacterial pH sensing and homeostasis. Nat. Rev. Microbiol. 9, 330–343 (2011).
pubmed: 21464825
pmcid: 3247762
doi: 10.1038/nrmicro2549
Yakovleva, G. M., Kim, S. K. & Wanner, B. L. Phosphate-independent expression of the carbon-phosphorus lyase activity of Escherichia coli. Appl. Microbiol. Biotechnol. 49, 573–578 (1998).
pubmed: 9650256
doi: 10.1007/s002530051215
Su, H. & Newman, E. B. A novel L-serine deaminase activity in Escherichia coli K-12. J. Bacteriol. 173, 2473–2480 (1991).
pubmed: 2013569
pmcid: 207810
doi: 10.1128/jb.173.8.2473-2480.1991
Hederstedt, L. & Rutberg, L. Succinate dehydrogenase–a comparative review. Microbiol. Rev. 45, 542–555 (1981).
pubmed: 6799760
pmcid: 281527
doi: 10.1128/mr.45.4.542-555.1981
Enos-Berlage, J. L. & Downs, D. M. Mutations in sdh (succinate dehydrogenase genes) alter the thiamine requirement of Salmonella typhimurium. J. Bacteriol. 179, 3989–3996 (1997).
pubmed: 9190816
pmcid: 179209
doi: 10.1128/jb.179.12.3989-3996.1997
Bucurenci, N. et al. CMP kinase from Escherichia coli is structurally related to other nucleoside monophosphate kinases. J. Biol. Chem. 271, 2856–2862 (1996).
pubmed: 8576266
doi: 10.1074/jbc.271.5.2856
Leif, H., Sled, V. D., Ohnishi, T., Weiss, H. & Friedrich, T. Isolation and characterization of the proton-translocating NADH: Ubiquinone oxidoreductase from Escherichia coli. Eur. J. Biochem. 230, 538–548 (1995).
pubmed: 7607227
doi: 10.1111/j.1432-1033.1995.tb20594.x
Alyahya, S. A. et al. RodZ, a component of the bacterial core morphogenic apparatus. Proc. Natl Acad. Sci. USA 106, 1239–1244 (2009).
pubmed: 19164570
pmcid: 2633561
doi: 10.1073/pnas.0810794106
Hobbs, E. C., Astarita, J. L. & Storz, G. Small RNAs and small proteins involved in resistance to cell envelope stress and acid shock in Escherichia coli: Analysis of a bar-coded mutant collection. J. Bacteriol. 192, 59–67 (2010).
pubmed: 19734312
doi: 10.1128/JB.00873-09
Artsimovitch, I., Svetlov, V., Anthony, L., Burgess, R. R. & Landick, R. RNA polymerases from Bacillus subtilis and Escherichia coli differ in recognition of regulatory signals in vitro. J. Bacteriol. 182, 6027–6035 (2000).
pubmed: 11029421
pmcid: 94735
doi: 10.1128/JB.182.21.6027-6035.2000
Billinton, N. & Knight, A. W. Seeing the wood through the trees: A review of techniques for distinguishing green fluorescent protein from endogenous autofluorescence. Anal. Biochem. 291, 175–197 (2001).
pubmed: 11401292
doi: 10.1006/abio.2000.5006
Wickiser, J. K., Winkler, W. C., Breaker, R. R. & Crothers, D. M. The speed of RNA transcription and metabolite binding kinetics operate an FMN riboswitch. Mol. Cell 18, 49–60 (2005).
pubmed: 15808508
doi: 10.1016/j.molcel.2005.02.032
Wickiser, J. K., Cheah, M. T., Breaker, R. R. & Crothers, D. M. The kinetics of ligand binding by an adenine-sensing riboswitch. Biochemistry 44, 13404–13414 (2005).
pubmed: 16201765
doi: 10.1021/bi051008u
Lemay, J.-F. et al. Comparative study between transcriptionally- and translationally-acting adenine riboswitches reveals key differences in riboswitch regulatory mechanisms. PLoS Genet. 7, e1001278 (2011).
pubmed: 21283784
pmcid: 3024265
doi: 10.1371/journal.pgen.1001278
Chauvier, A. et al. Monitoring RNA dynamics in native transcriptional complexes. Proc. Natl Acad. Sci. USA 118, e2106564118 (2021).
pubmed: 34740970
pmcid: 8609307
doi: 10.1073/pnas.2106564118
Guedich, S. et al. Quantitative and predictive model of kinetic regulation by E. coli TPP riboswitches. RNA Biol. 13, 373–390 (2016).
pubmed: 26932506
pmcid: 4841613
doi: 10.1080/15476286.2016.1142040
Larson, M. H. et al. A pause sequence enriched at translation start sites drives transcription dynamics in vivo. Science 344, 1042–1047 (2014).
pubmed: 24789973
pmcid: 4108260
doi: 10.1126/science.1251871
Heller, R. C. & Marians, K. J. The disposition of nascent strands at stalled replication forks dictates the pathway of replisome loading during restart. Mol. Cell 17, 733–743 (2005).
pubmed: 15749022
doi: 10.1016/j.molcel.2005.01.019
Hong, K.-Q., Zhang, J., Jin, B., Chen, T. & Wang, Z.-W. Development and characterization of a glycine biosensor system for fine-tuned metabolic regulation in Escherichia coli. Microb. Cell Fact. 21, 56 (2022).
pubmed: 35392910
pmcid: 8991567
doi: 10.1186/s12934-022-01779-4
Robinson, C. J. et al. Modular riboswitch toolsets for synthetic genetic control in diverse bacterial species. J. Am. Chem. Soc. 136, 10615–10624 (2014).
pubmed: 24971878
doi: 10.1021/ja502873j
Kennedy, K. J. et al. Cobalamin riboswitches are broadly sensitive to corrinoid cofactors to enable an efficient gene regulatory strategy. mBio 13, e0112122 (2022).
pubmed: 35993747
doi: 10.1128/mbio.01121-22
Hwang, Y., Kim, S. G., Jang, S., Kim, J. & Jung, G. Y. Signal amplification and optimization of riboswitch-based hybrid inputs by modular and titratable toehold switches. J. Biol. Eng. 15, 11 (2021).
pubmed: 33741029
pmcid: 7977183
doi: 10.1186/s13036-021-00261-w
Polaski, J. T., Webster, S. M., Johnson, J. E. & Batey, R. T. Cobalamin riboswitches exhibit a broad range of ability to discriminate between methylcobalamin and adenosylcobalamin. J. Biol. Chem. 292, 11650–11658 (2017).
pubmed: 28483920
pmcid: 5512062
doi: 10.1074/jbc.M117.787176
Ghazi, Z., Jahanshahi, S. & Li, Y. RiboFACSeq: A new method for investigating metabolic and transport pathways in bacterial cells by combining a riboswitch-based sensor, fluorescence-activated cell sorting and next-generation sequencing. PLoS One 12, e0188399 (2017).
pubmed: 29211762
pmcid: 5718407
doi: 10.1371/journal.pone.0188399
Dutta, D., Belashov, I. A. & Wedekind, J. E. Coupling green fluorescent protein expression with chemical modification to probe functionally relevant riboswitch conformations in live bacteria. Biochemistry 57, 4620–4628 (2018).
pubmed: 29897738
doi: 10.1021/acs.biochem.8b00316
Wu, L., Chen, D., Ding, J. & Liu, Y. A transient conformation facilitates ligand binding to the adenine riboswitch. iScience 24, 103512 (2021).
pubmed: 34927032
pmcid: 8652005
doi: 10.1016/j.isci.2021.103512
Wang, X., Wei, W. & Zhao, J. Using a riboswitch sensor to detect Co
pubmed: 33659237
pmcid: 7917058
doi: 10.3389/fchem.2021.631909
Lim, H. G., Jang, S., Jang, S., Seo, S. W. & Jung, G. Y. Design and optimization of genetically encoded biosensors for high-throughput screening of chemicals. Curr. Opin. Biotechnol. 54, 18–25 (2018).
pubmed: 29413747
doi: 10.1016/j.copbio.2018.01.011
Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).
pubmed: 19363495
doi: 10.1038/nmeth.1318
Chung, C. T., Niemela, S. L. & Miller, R. H. One-step preparation of competent Escherichia coli: Transformation and storage of bacterial cells in the same solution. Proc. Natl Acad. Sci. USA 86, 2172–2175 (1989).
pubmed: 2648393
pmcid: 286873
doi: 10.1073/pnas.86.7.2172
Carpenter, A. E. et al. Cell profiler: Image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 7, R100 (2006).
pubmed: 17076895
pmcid: 1794559
doi: 10.1186/gb-2006-7-10-r100
Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).
pubmed: 22743772
doi: 10.1038/nmeth.2019