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Colistin heteroresistance is common among\n                  <jats:italic>Enterobacter cloacae<\/jats:italic>\n                  clinical isolates, threatens its utility as a last-line therapeutic, and has become a model with which to understand the fundamental bases of heteroresistance. Despite numerous insights, the mechanism by which phenotypic heterogeneity is generated within the population and leads to colistin heteroresistance has been unclear. Here, using a transposon-based mutagenesis screen, we identify the sigma factor \u03c3\n                  <jats:sup>E<\/jats:sup>\n                  as the source of heterogeneity in population-wide colistin resistance levels. Single-cell tracking experiments revealed that \u03c3\n                  <jats:sup>E<\/jats:sup>\n                  is active in only one percent of the population at baseline, and only those cells with active \u03c3\n                  <jats:sup>E<\/jats:sup>\n                  survive colistin exposure. However, \u03c3\n                  <jats:sup>E<\/jats:sup>\n                  expression and population heterogeneity are insufficient for survival, as a mutant lacking the PhoPQ two-component system controlling lipid A modifications necessary for colistin resistance retains heterogeneity but loses colistin resistance. These findings lead to a new paradigm in heteroresistance, where the combinatorial action of multiple regulatory systems, encompassing a heterogeneity generator and a distinct resistance generator, are required to give rise to colistin heteroresistance.\n                <\/jats:p>","DOI":"10.1101\/2025.08.11.668767","type":"posted-content","created":{"date-parts":[[2025,8,12]],"date-time":"2025-08-12T15:55:18Z","timestamp":1755014118000},"source":"Crossref","is-referenced-by-count":1,"title":["Combinatorial action of regulatory systems generates colistin heteroresistance"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3077-0730","authenticated-orcid":false,"given":"Jacob E.","family":"Choby","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emily K.","family":"Crispell","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Muqing","family":"Ma","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Linda M.","family":"Vu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mary-Kate","family":"Key","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5016-8694","authenticated-orcid":false,"given":"Robert K.","family":"Ernst","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1594-4971","authenticated-orcid":false,"given":"Minsu","family":"Kim","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0980-7866","authenticated-orcid":false,"given":"David S.","family":"Weiss","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025081512323132000_2025.08.11.668767v1.1","doi-asserted-by":"crossref","unstructured":"A. Sabnis et al., Colistin kills bacteria by targeting lipopolysaccharide in the cytoplasmic membrane. Elife 10, (2021).","DOI":"10.7554\/eLife.65836"},{"key":"2025081512323132000_2025.08.11.668767v1.2","doi-asserted-by":"publisher","DOI":"10.1038\/s41579-019-0201-x"},{"key":"2025081512323132000_2025.08.11.668767v1.3","unstructured":"World Health Organization, \u201cWHO Bacterial Priority Pathogens List 2024: bacterial pathogens of public health importance to guide research, development and strategies to prevent and control antimicrobial resistance,\u201d (Geneva, 2024)."},{"key":"2025081512323132000_2025.08.11.668767v1.4","unstructured":"CDC, \u201cAntibiotic Resistance Threats in the United States,\u201d (U.S. Department of Health and Human Services, CDC, Atlanta, GA, 2019)."},{"key":"2025081512323132000_2025.08.11.668767v1.5","doi-asserted-by":"crossref","unstructured":"V. I. 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