Abstract
Geobacillus kaustophilus is a thermophilic bacterium that grows at temperatures ranging between 42 and 74 °C. Here, we modified this organism to produce the thermolabile protein (PyrFA) or its thermostable variant (PyrFV) and analyzed the transcriptome and growth efficiency profiles of the resultant strains. In the producer of PyrFA, the transcriptome profile was changed to facilitate ATP synthesis from NADH without pooling reduced quinones. This change implies that PyrFA production at elevated temperatures places an energy burden on cells potentially to maintain protein homeostasis. This was consistent with the observation that the PyrFA producer grew slower than the PyrFV producer at > 45 °C and had a lower cellular fitness. Similar growth profiles were also observed in the PyrFA and PyrFV producers derived from another thermophile (Geobacillus thermodenitrificans) but not in those from Escherichia coli at 30 °C. Thus, we suggest that the production of thermolabile proteins impairs host survival at higher temperatures; therefore, thermophiles are under evolutionary selection for thermostable proteins regardless of whether their functions are associated with survival advantages. This hypothesis provides new insights into evolutionary protein selection in thermophiles and suggests an engineering approach to select thermostable protein variants generated via random gene mutagenesis.
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References
Amartey SA, Leak DJ, Hartley BS (1991) Development and optimization of a defined medium for aerobic growth of Bacillus stearothermophilus LLD-15. Biotechnol Lett 13:621–626. https://doi.org/10.1007/bf01086315
Asial I, Cheng YX, Engman H, Dollhopf M, Wu B, Nordlund P, Cornvik T (2013) Engineering protein thermostability using a generic activity-independent biophysical screen inside the cell. Nat Commun 4:2901. https://doi.org/10.1038/ncomms3901
Baker TA, Sauer RT (2006) ATP-dependent proteases of bacteria: recognition logic and operating principles. Trends Biochem Sci 31:647–653. https://doi.org/10.1016/j.tibs.2006.10.006
Bednarska NG, Schymkowitz J, Rousseau F, Van Eldere J (2013) Protein aggregation in bacteria: the thin boundary between functionality and toxicity. Microbiology 159:1795–1806. https://doi.org/10.1099/mic.0.069575-0
Brouns SJJ, Wu H, Akerboom J, Turnbull AP, de Vos WM, van der Oost J (2005) Engineering a selectable marker for hyperthermophiles. J Biol Chem 280:11422–11431. https://doi.org/10.1074/jbc.M413623200
Chautard H, Blas-Galindo E, Menguy T, Grand’Moursel L, Cava F, Berenguer J, Delcourt M (2007) An activity-independent selection system of thermostable protein variants. Nat Methods 4:919–921. https://doi.org/10.1038/nmeth1090
Foit L, Morgan GJ, Kern MJ, Steimer LR, von Hacht AA, Titchmarsh J, Warriner SL, Radford SE, Bardwell JCA (2009) Optimizing protein stability in vivo. Mol Cell 36:861–871. https://doi.org/10.1016/j.molcel.2009.11.022
Fridjonsson O, Watzlawick H, Mattes R (2002) Thermoadaptation of α-galactosidase AgaB1 in Thermus thermophilus. J Bacteriol 184:4326–4326. https://doi.org/10.1128/JB.184.15.4326.2002
Hoseki J, Yano T, Koyama Y, Kuramitsu S, Kagamiyama H (1999) Directed evolution of thermostable kanamycin-resistance gene: a convenient selection marker for Thermus thermophilus. J Biochem 126:951–956. https://doi.org/10.1093/oxfordjournals.jbchem.a022539
Ibba M, Söll D (2001) The renaissance of aminoacyl-tRNA synthesis. EMBO Rep 2:382–387. https://doi.org/10.1093/embo-reports/kve095
Kobayashi J, Furukawa M, Ohshiro T, Suzuki H (2015a) Thermoadaptation-directed evolution of chloramphenicol acetyltransferase in an error-prone thermophile using improved procedures. Appl Microbiol Biotechnol 99:5563–5572. https://doi.org/10.1007/s00253-015-6522-4
Kobayashi J, Tanabiki M, Doi S, Kondo A, Ohshiro T, Suzuki H (2015b) Unique plasmids generated via pUC replicon mutagenesis in an error-prone thermophile derived from Geobacillus kaustophilus HTA426. Appl Environ Microbiol 81:7625–7632. https://doi.org/10.1128/aem.01574-15
Kumar S, Dangi AK, Shukla P, Baishya D, Khare SK (2019) Thermozymes: adaptive strategies and tools for their biotechnological applications. Bioresour Technol 278:372–382. https://doi.org/10.1016/j.biortech.2019.01.088
Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359. https://doi.org/10.1038/nmeth.1923
Lau CKY, Krewulak KD, Vogel HJ (2015) Bacterial ferrous iron transport: the Feo system. FEMS Microbiol Rev 40:273–298. https://doi.org/10.1093/femsre/fuv049
Liao H, McKenzie T, Hageman R (1986) Isolation of a thermostable enzyme variant by cloning and selection in a thermophile. Proc Natl Acad Sci USA 83:576–580. https://doi.org/10.1073/pnas.83.3.576
Liao Y, Smyth GK, Shi W (2014) featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30:923–930. https://doi.org/10.1093/bioinformatics/btt656
Matsumura M, Aiba S (1985) Screening for thermostable mutant of kanamycin nucleotidyltransferase by the use of a transformation system for a thermophile, Bacillus stearothermophilus. J Biol Chem 260:5298–5303
Matzanke BF, Anemüller S, Schünemann V, Trautwein AX, Hantke K (2004) FhuF, part of a siderophore-reductase system. Biochemistry 43:1386–1392. https://doi.org/10.1021/bi0357661
Nakamura A, Takakura Y, Kobayashi H, Hoshino T (2005) In vivo directed evolution for thermostabilization of Escherichia coli hygromycin B phosphotransferase and the use of the gene as a selection marker in the host-vector system of Thermus thermophilus. J Biosci Bioeng 100:158–163. https://doi.org/10.1263/jbb.100.158
Rodnina MV, Peske F, Peng B-Z, Belardinelli R, Wintermeyer W (2020) Converting GTP hydrolysis into motion: versatile translational elongation factor G. Biol Chem 401:131–142. https://doi.org/10.1515/hsz-2019-0313
Ryabova NA, Marchenkov VV, Marchenkova SY, Kotova NV, Semisotnov GV (2013) Molecular chaperone GroEL/ES: unfolding and refolding processes. Biochemistry (mosc) 78:1405–1414. https://doi.org/10.1134/s0006297913130038
Schneider R, Hantke K (1993) Iron-hydroxamate uptake systems in Bacillus subtilis: identification of a lipoprotein as part of a binding protein-dependent transport system. Mol Microbiol 8:111–121. https://doi.org/10.1111/j.1365-2958.1993.tb01208.x
Severance S, Chakraborty S, Kosman DJ (2004) The Ftr1p iron permease in the yeast plasma membrane: orientation, topology and structure-function relationships. Biochem J 380:487–496. https://doi.org/10.1042/bj20031921
Sheppard K, Yuan J, Hohn MJ, Jester B, Devine KM, Söll D (2008) From one amino acid to another: tRNA-dependent amino acid biosynthesis. Nucleic Acids Res 36:1813–1825. https://doi.org/10.1093/nar/gkn015
Song Y, Zhu Z, Zhou W, Zhang YPJ (2021) High-efficiency transformation of archaea by direct PCR products with its application to directed evolution of a thermostable enzyme. Microb Biotechnol 14:453–464. https://doi.org/10.1111/1751-7915.13613
Suzuki H (2018) Peculiarities and biotechnological potential of environmental adaptation by Geobacillus species. Appl Microbiol Biotechnol 102:10425–10437. https://doi.org/10.1007/s00253-018-9422-6
Suzuki H, Wada K, Furukawa M, Doi K, Ohshima T (2013a) A ternary conjugation system for the construction of DNA libraries for Geobacillus kaustophilus HTA426. Biosci Biotechnol Biochem 77:2316–2318. https://doi.org/10.1271/bbb.130492
Suzuki H, Yoshida K, Ohshima T (2013b) Polysaccharide-degrading thermophiles generated by heterologous gene expression in Geobacillus kaustophilus HTA426. Appl Environ Microbiol 79:5151–5158. https://doi.org/10.1128/aem.01506-13
Suzuki H, Kobayashi J, Wada K, Furukawa M, Doi K (2015) Thermoadaptation-directed enzyme evolution in an error-prone thermophile derived from Geobacillus kaustophilus HTA426. Appl Environ Microbiol 81:149–158. https://doi.org/10.1128/aem.02577-14
Suzuki H, Taketani T, Kobayashi J, Ohshiro T (2018) Antibiotic resistance mutations induced in growing cells of Bacillus-related thermophiles. J Antibiot 71:382–389. https://doi.org/10.1038/s41429-017-0003-1
Takami H, Takaki Y, Chee G, Nishi S, Shimamura S, Suzuki H, Matsui S, Uchiyama I (2004) Thermoadaptation trait revealed by the genome sequence of thermophilic Geobacillus kaustophilus. Nucleic Acids Res 32:6292–6303. https://doi.org/10.1093/nar/gkh970
Tamakoshi M, Nakano Y, Kakizawa S, Yamagishi A, Oshima T (2001) Selection of stabilized 3-isopropylmalate dehydrogenase of Saccharomyces cerevisiae using the host-vector system of an extreme thermophile, Thermus thermophilus. Extremophiles 5:17–22. https://doi.org/10.1007/s007920000168
Wada K, Kobayashi J, Furukawa M, Doi K, Ohshiro T, Suzuki H (2016) A thiostrepton resistance gene and its mutants serve as selectable markers in Geobacillus kaustophilus HTA426. Biosci Biotechnol Biochem 80:368–375. https://doi.org/10.1080/09168451.2015.1079478
Winstedt L, von Wachenfeldt C (2000) Terminal oxidases of Bacillus subtilis strain 168: one quinol oxidase, cytochrome aa3 or cytochrome bd, is required for aerobic growth. J Bacteriol 182:6557–6564. https://doi.org/10.1128/jb.182.23.6557-6564.2000
Yu H, Rao X, Zhang K (2017) Nucleoside diphosphate kinase (Ndk): a pleiotropic effector manipulating bacterial virulence and adaptive responses. Microbiol Res 205:125–134. https://doi.org/10.1016/j.micres.2017.09.001
Acknowledgements
This work was supported by the following organizations: JSPS KAKENHI Grant Number 17K06925, the Institute for Fermentation, Osaka (IFO), and the Nagase Science and Technology Foundation.
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Suzuki, H., Okumura, Y., Mikawa, Y. et al. Transcriptome and growth efficiency comparisons of recombinant thermophiles that produce thermolabile and thermostable proteins: implications for burden-based selection of thermostable proteins. Extremophiles 25, 403–412 (2021). https://doi.org/10.1007/s00792-021-01237-w
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DOI: https://doi.org/10.1007/s00792-021-01237-w