{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T13:15:52Z","timestamp":1785417352976,"version":"3.56.0"},"reference-count":35,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2026,5,1]],"date-time":"2026-05-01T00:00:00Z","timestamp":1777593600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"crossref","award":["322713320"],"award-info":[{"award-number":["322713320"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"crossref"}]},{"award":["322713320"],"award-info":[{"award-number":["322713320"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Creative Research Group of Zhongshan City","award":["CXTD2022011"],"award-info":[{"award-number":["CXTD2022011"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Microorganisms"],"abstract":"<jats:p>Escherichia coli, a facultative anaerobic Gram-negative member of the Enterobacteriaceae, is an increasingly important opportunistic pathogen driven in part by rising resistance to clinically important antibiotics. Regulation of multidrug efflux systems by two-component signal transduction pathways, particularly the BaeSR system, plays a central role in this process. However, the functional residues governing signal transduction through the sensor kinase BaeS remain incompletely defined. In this study, we integrated domain prediction, homology-guided site-directed mutagenesis, in vitro protein purification, autophosphorylation assays, and reverse-transcription quantitative polymerase chain reaction (RT-qPCR)-based transcriptional analysis of selected BaeSR-regulated genes to delineate key residues required for BaeS function. Sequence analysis identified His250 as a candidate autophosphorylation site and Asn364 as a conserved residue within the catalytic domain. Biochemical characterization of purified wild-type BaeS and an H250A mutant demonstrated that His250 is indispensable for autophosphorylation. Consistently, RT-qPCR analysis showed that BaeS activation markedly induced the transcription of BaeSR-regulated efflux-associated genes, whereas genetic deletion of baeS or selective disruption of kinase activity by the N364A mutation abolished this response. Together, these findings establish His250 as a key residue for BaeS autophosphorylation and identify Asn364 as essential for inducible BaeSR signaling and activation of resistance-associated target genes, thereby establishing an experimental framework for elucidating BaeSR-mediated efflux regulation and informing future studies of resistance regulatory networks and potential intervention strategies centered on key signaling nodes.<\/jats:p>","DOI":"10.3390\/microorganisms14051031","type":"journal-article","created":{"date-parts":[[2026,5,1]],"date-time":"2026-05-01T09:33:17Z","timestamp":1777627997000},"page":"1031","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Functional Characterization of the Histidine Kinase BaeS Reveals Critical Residues for BaeSR-Dependent Stress Signaling in Escherichia coli"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-5018-9155","authenticated-orcid":false,"given":"Shurong","family":"Chen","sequence":"first","affiliation":[{"name":"Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China"},{"name":"Zhongshan Institute for Drug Discovery, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Zhongshan 528400, China"},{"name":"University of Chinese Academy of Sciences, Beijing 100049, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhengfei","family":"Qi","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Taipa, Macao 999078, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lina","family":"Wang","sequence":"additional","affiliation":[{"name":"School of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing 210023, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lian","family":"Wu","sequence":"additional","affiliation":[{"name":"School of Pharmaceutical Sciences, Guizhou Medical University, Guizhou 561113, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiayi","family":"Xie","sequence":"additional","affiliation":[{"name":"School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rui","family":"Ma","sequence":"additional","affiliation":[{"name":"School of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing 210023, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kexin","family":"Zhang","sequence":"additional","affiliation":[{"name":"School of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing 210023, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tong","family":"Ji","sequence":"additional","affiliation":[{"name":"School of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing 210023, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Min","family":"Zhou","sequence":"additional","affiliation":[{"name":"Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China"},{"name":"Zhongshan Institute for Drug Discovery, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Zhongshan 528400, China"},{"name":"University of Chinese Academy of Sciences, Beijing 100049, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lingli","family":"Zheng","sequence":"additional","affiliation":[{"name":"Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China"},{"name":"Zhongshan Institute for Drug Discovery, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Zhongshan 528400, China"},{"name":"University of Chinese Academy of Sciences, Beijing 100049, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7445-2355","authenticated-orcid":false,"given":"Qingshan Bill","family":"Fu","sequence":"additional","affiliation":[{"name":"Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China"},{"name":"Zhongshan Institute for Drug Discovery, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Zhongshan 528400, China"},{"name":"University of Chinese Academy of Sciences, Beijing 100049, China"},{"name":"State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Taipa, Macao 999078, China"},{"name":"School of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing 210023, China"},{"name":"School of Pharmaceutical Sciences, Guizhou Medical University, Guizhou 561113, China"},{"name":"School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,5,1]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1007\/978-3-030-18768-2_4","article-title":"Outer Membrane Porins","volume":"Volume 92","author":"Masi","year":"2019","journal-title":"Bacterial Cell Walls and Membranes; Sub-Cellular Biochemistry"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"126","DOI":"10.1016\/j.micres.2018.07.005","article-title":"The role of two-component regulatory system in \u03b2-lactam antibiotics resistance","volume":"215","author":"Li","year":"2018","journal-title":"Microbiol. 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