{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T01:04:13Z","timestamp":1760058253305,"version":"build-2065373602"},"reference-count":46,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2025,3,26]],"date-time":"2025-03-26T00:00:00Z","timestamp":1742947200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Japan Science and Technology Agency","award":["JPMJAL1106","S1311017","25250028","16H02485"],"award-info":[{"award-number":["JPMJAL1106","S1311017","25250028","16H02485"]}]},{"name":"MEXT-supported Program for Strategic Research Foundation at Private Universities","award":["JPMJAL1106","S1311017","25250028","16H02485"],"award-info":[{"award-number":["JPMJAL1106","S1311017","25250028","16H02485"]}]},{"name":"Japan Society for the Promotion of Science","award":["JPMJAL1106","S1311017","25250028","16H02485"],"award-info":[{"award-number":["JPMJAL1106","S1311017","25250028","16H02485"]}]},{"name":"National Research Council of Thailand","award":["JPMJAL1106","S1311017","25250028","16H02485"],"award-info":[{"award-number":["JPMJAL1106","S1311017","25250028","16H02485"]}]},{"name":"Ministry of Science and Technology in Vietnam","award":["JPMJAL1106","S1311017","25250028","16H02485"],"award-info":[{"award-number":["JPMJAL1106","S1311017","25250028","16H02485"]}]},{"name":"National Univ. of Laos","award":["JPMJAL1106","S1311017","25250028","16H02485"],"award-info":[{"award-number":["JPMJAL1106","S1311017","25250028","16H02485"]}]},{"name":"Univ. of Brawijaya","award":["JPMJAL1106","S1311017","25250028","16H02485"],"award-info":[{"award-number":["JPMJAL1106","S1311017","25250028","16H02485"]}]},{"name":"Beuth Univ. of Applied Science","award":["JPMJAL1106","S1311017","25250028","16H02485"],"award-info":[{"award-number":["JPMJAL1106","S1311017","25250028","16H02485"]}]},{"name":"Japan Society for the Promotion of Science, MEXT\/JSPS Kakenhi","award":["JPMJAL1106","S1311017","25250028","16H02485"],"award-info":[{"award-number":["JPMJAL1106","S1311017","25250028","16H02485"]}]},{"name":"NODAI Genome Research Center, Tokyo University of Agriculture","award":["JPMJAL1106","S1311017","25250028","16H02485"],"award-info":[{"award-number":["JPMJAL1106","S1311017","25250028","16H02485"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Laboratory evolution is an effective means of understanding microbial adaptation to the environment. We previously isolated four thermoadapted Zymomonas mobilis mutants, which showed a 2 \u00b0C rise in the critical high temperature (CHT), by performing multiple-parallel adaptation experiments. In the present study, the individual mutations in these mutants were intensively analyzed. Two mutations in each adapted mutant were found to primarily contribute to the increase in the upper temperature limit. RNA sequencing (RNA-seq) analysis revealed that the two mutations led to the upregulation of 79\u2013185 genes and the downregulation of 242\u2013311 genes. The findings from transcriptomic and physiological experiments suggest two common and primary mechanisms for thermal resistance: a decrease in the activity of diacylglycerol kinase, which may change the structure of lipopolysaccharide (LPS) probably to strengthen the membrane structure, and an increase in the expression of genes for GroEL\/GroES or cell wall hydrolase to repair the protein or membrane damage that occurs at such critical temperatures. Additionally, transporters including efflux pumps may contribute to intracellular homeostasis by expelling toxic compounds such as ethanol and acetate or by maintaining the K+ concentration. The results of this study on four independently thermoadapted mutants led to the conclusion that the mutants have almost the same thermal adaptation strategies and thus their molecular diversity is limited.<\/jats:p>","DOI":"10.3390\/ijms26073052","type":"journal-article","created":{"date-parts":[[2025,3,28]],"date-time":"2025-03-28T04:11:40Z","timestamp":1743135100000},"page":"3052","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Limited Diversity of Thermal Adaptation to a Critical Temperature in Zymomonas mobilis: Evidence from Multiple-Parallel Laboratory Evolution Experiments"],"prefix":"10.3390","volume":"26","author":[{"given":"Sornsiri","family":"Pattanakittivorakul","sequence":"first","affiliation":[{"name":"Graduate School of Sciences and Technology for Innovation, Yamaguchi University, Yamaguchi 753-8515, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shun","family":"Kato","sequence":"additional","affiliation":[{"name":"Graduate School of Sciences and Technology for Innovation, Yamaguchi University, Yamaguchi 753-8515, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Takashi","family":"Kuga","sequence":"additional","affiliation":[{"name":"Graduate School of Sciences and Technology for Innovation, Yamaguchi University, Yamaguchi 753-8515, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8181-9127","authenticated-orcid":false,"given":"Tomoyuki","family":"Kosaka","sequence":"additional","affiliation":[{"name":"Graduate School of Sciences and Technology for Innovation, Yamaguchi University, Yamaguchi 753-8515, Japan"},{"name":"Research Center for Thermotolerant Microbial Resources, Yamaguchi University, Yamaguchi 753-8515, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Minenosuke","family":"Matsutani","sequence":"additional","affiliation":[{"name":"NODAI Genome Research Center, Tokyo University of Agriculture, Tokyo 156-8502, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Masayuki","family":"Murata","sequence":"additional","affiliation":[{"name":"Graduate School of Sciences and Technology for Innovation, Yamaguchi University, Yamaguchi 753-8515, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Morio","family":"Ishikawa","sequence":"additional","affiliation":[{"name":"Department of Bioscience, Tokyo University of Agriculture, Tokyo 156-8502, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kankanok","family":"Charoenpunthuwong","sequence":"additional","affiliation":[{"name":"Department of Biotechnology, Faculty of Technology, Khon Kaen University, Khon Kaen 40002, Thailand"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0842-9760","authenticated-orcid":false,"given":"Pornthap","family":"Thanonkeo","sequence":"additional","affiliation":[{"name":"Department of Biotechnology, Faculty of Technology, Khon Kaen University, Khon Kaen 40002, Thailand"},{"name":"Fermentation Research Center for Value Added Agricultural Products, Khon Kaen University, Khon Kaen 40002, Thailand"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4354-7324","authenticated-orcid":false,"given":"Mamoru","family":"Yamada","sequence":"additional","affiliation":[{"name":"Graduate School of Sciences and Technology for Innovation, Yamaguchi University, Yamaguchi 753-8515, Japan"},{"name":"Research Center for Thermotolerant Microbial Resources, Yamaguchi University, Yamaguchi 753-8515, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,3,26]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1921","DOI":"10.1271\/bbb.110310","article-title":"Global analysis of the genes involved in the thermotolerance mechanism of thermotolerant Acetobacter tropicalis SKU1100","volume":"75","author":"Soemphol","year":"2011","journal-title":"Biosci. Biotechnol. Biochem."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Charoensuk, K., Sukurada, T., Tokiyama, A., Murata, M., Kosaka, T., Thanonkeo, P., and Yamada, M. (2017). Thermotolerant genes essential for survival at a critical high temperature in thermotolerant ethanologenic Zymomonas mobilis TISTR 548. Biotechnol. Biofuels, 10.","DOI":"10.1186\/s13068-017-0891-0"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Murata, M., Ishii, A., Fujimoto, H., Nishimura, K., Kosaka, T., Mori, H., and Yamada, M. (2018). Update of thermotolerant genes essential for survival at a critical high temperature in Escherichia coli. PLoS ONE, 13.","DOI":"10.1371\/journal.pone.0189487"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Tan, Y.S., Zhang, R.K., Liu, Z.H., Li, B.Z., and Yuan, Y.J. (2022). Microbial adaptation to enhance stress tolerance. Front. Microbiol., 13.","DOI":"10.3389\/fmicb.2022.888746"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Wang, G., Li, Q., Zhang, Z., Yin, X., Wang, B., and Yang, X. (2023). Recent progress in adaptive laboratory evolution of industrial microorganisms. J. Ind. Microbiol. Biotechnol., 50.","DOI":"10.1093\/jimb\/kuac023"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"92","DOI":"10.3390\/microorganisms11010092","article-title":"Adaptive laboratory evolution of microorganisms: Methodology and application for bioproduction","volume":"11","author":"Hirasawa","year":"2023","journal-title":"Microorganisms"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"457","DOI":"10.1126\/science.1212986","article-title":"The molecular diversity of adaptive convergence","volume":"335","author":"Tenaillon","year":"2012","journal-title":"Science"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2647","DOI":"10.1093\/molbev\/msu209","article-title":"Evolution of Escherichia coli to 42 \u00b0C and subsequent genetic engineering reveals adaptive mechanisms and novel mutations","volume":"31","author":"Sandberg","year":"2014","journal-title":"Mol. Biol. Evol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1038\/nature18959","article-title":"Tempo and mode of genome evolution in a 50,000-generation experiment","volume":"536","author":"Tenaillon","year":"2016","journal-title":"Nature"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"E1904","DOI":"10.1073\/pnas.1616132114","article-title":"Specificity of genome evolution in experimental populations of Escherichia coli evolved at different temperatures","volume":"114","author":"Deatherage","year":"2017","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Kishimoto, T., Lijima, L., Tatsumi, M., Ono, N., Oyake, A., Hashimoto, T., Matsuo, M., Okubo, M., Suzuki, S., and Mori, K. (2010). Transition from positive to neutral in mutation fixation along with continuing rising fitness in thermal adaptive evolution. PLoS Genet., 6.","DOI":"10.1371\/journal.pgen.1001164"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"19029","DOI":"10.1074\/jbc.M110.103374","article-title":"Evolution of Escherichia coli for growth at high temperatures","volume":"285","author":"Rudolph","year":"2010","journal-title":"J Biol Chem."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"144","DOI":"10.1128\/AEM.05773-11","article-title":"Experimental evolution of a facultative thermophile from a mesophilic ancestor","volume":"78","author":"Blaby","year":"2012","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Kosaka, T., Nakajima, Y., Ishii, A., Yamashita, M., Yoshida, S., Murata, M., Kat, K., Shiromaru, Y., Kato, S., and Kanasaki, Y. (2019). Capacity for survival in global warming: Adaptation of mesophiles to the temperature upper limit. PLoS ONE, 14.","DOI":"10.1371\/journal.pone.0215614"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Chen, J., Shen, J., Hellgren, L.H., Jensen, P.R., and Solem, C. (2015). Adaptation of Lactococcus lactis to high growth temperature leads to a dramatic increase in acidification rate. Sci. Rep., 5.","DOI":"10.1038\/srep14199"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Garc\u00eda-R\u00edos, E., Lair\u00f3n-Peris, M., Mu\u00f1iz-Calvo, S., Heras, J.M., Ortiz-Julien, A., Poirot, P., Roz\u00e8s, N., Querol, A., and Guillam\u00f3n, J.M. (2021). Thermo-adaptive evolution to generate improved Saccharomyces cerevisiae strains for cocoa pulp fermentations. Int. J. Food Microbiol., 342.","DOI":"10.1016\/j.ijfoodmicro.2021.109077"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Liu, M., and Cheng, H. (2023). Discovery and functional evaluation of heat tolerance genes in the nonconventional yeast Yarrowia lipolytica. Fermentation, 9.","DOI":"10.3390\/fermentation9060509"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"294","DOI":"10.1186\/s12934-024-02568-x","article-title":"Thermo-adaptive evolution of Corynebacterium glutamicum reveals the regulatory functions of fasR and hrcA in heat tolerance","volume":"23","author":"Li","year":"2024","journal-title":"Microb. Cell Fact."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Breland, E.J., and Eberly, A.R. (2017). An overview of two-component signal transduction systems implicated in extra-intestinal pathogenic E. coli infections. Front. Cell. Infect. Microbiol., 7.","DOI":"10.3389\/fcimb.2017.00162"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"e00980-20","DOI":"10.1128\/mSystems.00980-20","article-title":"Elucidation of regulatory modes for five two-component systems in Escherichia coli reveals novel relationships","volume":"5","author":"Choudhary","year":"2020","journal-title":"mSystems"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"6114","DOI":"10.1073\/pnas.89.13.6114","article-title":"Structures of apo and complexed Escherichia coli glycinamide ribonucleotide transformylase","volume":"89","author":"Almassy","year":"1992","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_22","first-page":"5","article-title":"Histidine biosynthesis, its regulation and biotechnological application in Corynebacterium glutamicum","volume":"7","author":"Persicke","year":"2013","journal-title":"Microb. Biotechnol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"24176","DOI":"10.1074\/jbc.272.39.24176","article-title":"Escherichia coli diacylglycerol kinase is an evolutionarily optimized membrane enzyme and catalyzes direct phosphoryl transfer","volume":"272","author":"Badola","year":"1997","journal-title":"J. Biol. Chem."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"e0049821","DOI":"10.1128\/jb.00498-21","article-title":"Diacylglycerol kinase A is essential for polymyxin pesistance provided by EptA, MCR-1, and other lipid A phosphoethanolamine transferases","volume":"204","author":"Purcell","year":"2022","journal-title":"J. Bacteriol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"286","DOI":"10.1074\/jbc.M113.518373","article-title":"Chaperones GroEL\/GroES accelerate the refolding of a multidomain protein through modulating on-pathway intermediates","volume":"289","author":"Dahiya","year":"2014","journal-title":"J. Biol. Chem."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Illingworth, M., Ellis, H., and Chen, L. (2017). Creating the functional single-ring GroEL-GroES chaperonin systems via modulating GroEL-GroES Interaction. Sci. Rep., 7.","DOI":"10.1038\/s41598-017-10499-4"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Vermassen, A., Leroy, S., Talon, R., Provot, C., Popowska, M., and Desvaux, M. (2019). Cell wall hydrolases in bacteria: Insight on the diversity of cell wall amidases, glycosidases and peptidases toward peptidoglycan. Front. Microbiol., 10.","DOI":"10.3389\/fmicb.2019.00331"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Sakunda, A., Murata, M., Keisuke, K., Kosaka, T., Sootsuwan, K., Thanonkeo, P., and Yamada, M. (2020). Improvement of thermotolerance of Zymomonas mobilis by genes for reactive oxygen species-scavenging enzymes and heat-shock proteins. Front. Microbiol., 10.","DOI":"10.3389\/fmicb.2019.03073"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"e02456-20","DOI":"10.1128\/mBio.02456-20","article-title":"Bacterial cell wall quality control during environmental stress","volume":"11","author":"Mueller","year":"2020","journal-title":"mBio"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"626","DOI":"10.1038\/s41467-020-14441-7","article-title":"Structural basis of proton-coupled potassium transport in the KUP family","volume":"11","author":"Sousa","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Kosaka, T., Nishioka, A., Sakurada, T., Miura, K., Sakunda, A., and Yamada, M. (2020). Enhancement of thermal resistance by metal ions in the thermotolerant Zymomonas mobilis TISTR 548. Front. Microbiol., 11.","DOI":"10.3389\/fmicb.2020.00502"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Symmons, M.F., Marshall, R.L., and Bavro, V.N. (2015). Architecture and roles of periplasmic adaptor proteins in tripartite efflux assemblies. Front. Microbiol., 6.","DOI":"10.3389\/fmicb.2015.00513"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"14","DOI":"10.12703\/P7-14","article-title":"Structure and mechanism of ABC transporters","volume":"7","author":"Wilkens","year":"2015","journal-title":"F1000Prime Rep."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"8310","DOI":"10.1073\/pnas.1819682116","article-title":"Genome-wide effects on Escherichia coli transcription from ppGpp binding to its two sites on RNA polymerase","volume":"116","author":"Dewey","year":"2019","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Spira, B., and Ospino, K. (2020). Diversity in E.coli (p)ppGpp levels and its consequences. Front. Microbiol., 11.","DOI":"10.3389\/fmicb.2020.01759"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1243","DOI":"10.1093\/bbb\/zbab009","article-title":"Thermal adaptation of acetic acid bacteria for practical high-temperature vinegar fermentation","volume":"85","author":"Matsumoto","year":"2021","journal-title":"Biosci. Biotechnol. Biochem."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"e0010123","DOI":"10.1128\/jb.00101-23","article-title":"Implication of amino acid metabolism and cell surface integrity for the thermotolerance mechanism in the thermally adapted acetic acid bacterium Acetobacter pasteurianus TH-3","volume":"205","author":"Matsumoto","year":"2023","journal-title":"J. Bacteriol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"2551","DOI":"10.1038\/s41467-019-10429-0","article-title":"A mechanism to prevent production of reactive oxygen species by Escherichia coli respiratory complex I","volume":"10","author":"Schulte","year":"2019","journal-title":"Nat. Commun."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"832","DOI":"10.1080\/09168451.2019.1703638","article-title":"In vitro thermal adaptation of mesophilic Acetobacter pasteurianus NBRC 3283 generates thermotolerant strains with evolutionary trade-offs","volume":"84","author":"Matsumoto","year":"2020","journal-title":"Biosci. Biotechnol. Biochem."},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Shin, J., and Noireaux, V. (2010). Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70. J. Biol. Eng., 4.","DOI":"10.1186\/1754-1611-4-8"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"784","DOI":"10.1038\/nbt1183-784","article-title":"A broad host range mobilization system for in vivo genetic engineering: Transposon mutagenesis in Gram-negative bacteria","volume":"1","author":"Simon","year":"1983","journal-title":"Bio\/Technol."},{"key":"ref_42","unstructured":"Sambrook, J., and Russell, D.W. (2001). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press. [3rd ed.]."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"278","DOI":"10.1128\/AEM.65.1.278-282.1999","article-title":"New mobilizable vectors suitable for gene replacement in Gram-negative bacteria and their use in mapping of the 3\u2032 end of the Xanthomonas campestris pv. campestris gum operon","volume":"65","author":"Katzen","year":"1999","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"210","DOI":"10.1007\/s10059-013-2258-0","article-title":"Saccharomyces cerevisiae KNU5377 stress response during high-temperature ethanol fermentation","volume":"35","author":"Kim","year":"2013","journal-title":"Mol. Cells"},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Tomczak, A., Mortensen, J.M., Winnenburg, R., Liu, C., Alessi, D.T., Swamy, V., Vallania, F., Lofgren, S., Haynes, W., and Shah, N.H. (2018). Interpretation of biological experiments changes with evolution of the Gene Ontology and its annotations. Sci. Rep., 8.","DOI":"10.1038\/s41598-018-23395-2"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"2628","DOI":"10.1093\/bioinformatics\/btz931","article-title":"ShinyGO: A graphical gene-set enrichment tool for animals and plants","volume":"36","author":"Ge","year":"2020","journal-title":"Bioinformatics"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/7\/3052\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T17:02:40Z","timestamp":1760029360000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/7\/3052"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,3,26]]},"references-count":46,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2025,4]]}},"alternative-id":["ijms26073052"],"URL":"https:\/\/doi.org\/10.3390\/ijms26073052","relation":{},"ISSN":["1422-0067"],"issn-type":[{"type":"electronic","value":"1422-0067"}],"subject":[],"published":{"date-parts":[[2025,3,26]]}}}