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Since polyamines such as spermine, which are synthesized by infected macrophages, are able to inhibit the growth of\n M. tuberculosis<\/jats:italic>\n , the pathogen needs strategies to cope with these toxic metabolites. The actinomycete\n Streptomyces coelicolor<\/jats:italic>\n , a close relative of\n M. tuberculosis,<\/jats:italic>\n makes use of a gamma-glutamylation pathway to functionally neutralize spermine. We therefore considered whether a similar pathway would be functional in\n M. tuberculosis<\/jats:italic>\n . In the current study, we demonstrated that\n M. tuberculosis<\/jats:italic>\n growth was inhibited by the polyamine spermine. Using\n in vitro<\/jats:italic>\n enzymatic assays we determined that GlnA3\n \n Mt<\/jats:sub>\n <\/jats:italic>\n (Rv1878) possesses genuine gamma-glutamylspermine synthetase catalytic activity. We further showed that purified His-Strep-GlnA3\n \n Mt<\/jats:sub>\n ,\n <\/jats:italic>\n as well as native GlnA3\n \n Mt<\/jats:sub>\n ,\n <\/jats:italic>\n prefer spermine as a substrate over putrescine, cadaverine, spermidine, or other monoamines and amino acids, suggesting that GlnA3\n \n Mt<\/jats:sub>\n <\/jats:italic>\n may play a specific role in the detoxification of the polyamine spermine. However, the deletion of the\n glnA3<\/jats:italic>\n gene in\n M. tuberculosis<\/jats:italic>\n did not result in growth inhibition or enhanced sensitivity of\n M. tuberculosis<\/jats:italic>\n in the presence of high spermine concentrations. Gene expression analysis of spermine-treated\n M. tuberculosis<\/jats:italic>\n revealed no difference in the level of\n \n glnA3\n Mt<\/jats:sub>\n <\/jats:italic>\n expression relative to untreated cells, whereas a gene encoding a previously characterized efflux pump (Mmr;\n rv3065<\/jats:italic>\n ) was significantly upregulated. This suggests that bacterial survival under elevated spermine concentrations can not only be achieved by detoxification of spermine itself but also by mechanisms resulting in decreased spermine levels in the bacteria.\n <\/jats:p>\n <\/jats:sec>\n \n IMPORTANCE<\/jats:title>\n \n Upon\n Mycobacterium tuberculosis<\/jats:italic>\n infection macrophages synthesize the polyamine spermine, which at elevated concentrations is toxic for\n M. tuberculosis<\/jats:italic>\n . Based on our investigations of spermine resistance in the closely related actinomycete\n Streptomyces coelicolor<\/jats:italic>\n , we hypothesized that the glutamylspermine synthetase GlnA3 may be responsible for the resistance of\n M. tuberculosis<\/jats:italic>\n against toxic spermine. Here we show that GlnA3\n \n Mt<\/jats:italic>\n <\/jats:sub>\n can indeed covalently modify spermine via glutamylation. However, GlnA3\n \n Mt<\/jats:italic>\n <\/jats:sub>\n is probably not the only resistance mechanism since a\n glnA3<\/jats:italic>\n null mutant of\n M. tuberculosis<\/jats:italic>\n can survive under spermine stress. Gene expression studies suggest that an efflux pump may participate in resistance. Thus a combination of GlnA3\n \n Mt<\/jats:italic>\n <\/jats:sub>\n and specific efflux pumps acting as putative spermine transporters may constitute an active spermine-detoxification system in\n M. tuberculosis<\/jats:italic>\n .\n <\/jats:p>\n <\/jats:sec>","DOI":"10.1128\/jb.00439-24","type":"journal-article","created":{"date-parts":[[2025,1,30]],"date-time":"2025-01-30T14:00:31Z","timestamp":1738245631000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":4,"title":["GlnA3\n \n Mt<\/sub>\n <\/i>\n is able to glutamylate spermine but it is not essential for the detoxification of spermine in\n Mycobacterium tuberculosis<\/i>"],"prefix":"10.1128","volume":"207","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3739-5857","authenticated-orcid":true,"given":"Sergii","family":"Krysenko","sequence":"first","affiliation":[{"name":"Interfaculty Institute of Microbiology and Infection Medicine T\u00fcbingen (IMIT), 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