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Glucose drives NTM aggregation\n                      <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                      , and ammonium inhibits it, but the regulatory systems controlling this early step in biofilm formation are not understood. Here, in the model NTM\n                      <jats:italic toggle=\"yes\">Mycobacterium smegmatis<\/jats:italic>\n                      , we show that multiple carbon and nitrogen sources have similar impacts on aggregation as glucose and ammonium , suggesting that the response to these nutrients is general and likely sensed through downstream, integrated signals. Next, we performed a transposon screen in\n                      <jats:italic toggle=\"yes\">M. smegmatis<\/jats:italic>\n                      to uncover these putative regulatory nodes. Our screen revealed that mutating specific genes in the purine and pyrimidine biosynthesis pathways caused an aggregation defect, but supplementing with adenosine and guanosine had no impact on aggregation either in a\n                      <jats:italic toggle=\"yes\">purF<\/jats:italic>\n                      mutant or WT. Realizing that the only genes we hit in purine or pyrimidine biosynthesis were those that utilized glutamine as a nitrogen donor, we pivoted to the hypothesis that intracellular glutamine could be a nitrogen-responsive node affecting aggregation. We tested this hypothesis in a defined M63 medium using targeted mass spectrometry. Indeed, intracellular glutamine increased with nitrogen availability and correlated with planktonic growth. Furthermore, a\n                      <jats:italic toggle=\"yes\">garA<\/jats:italic>\n                      mutant, which has an artificially expanded glutamine pool in the growth phase, grew solely as planktonic cells even without nitrogen supplementation. Altogether, these results establish that intracellular glutamine controls\n                      <jats:italic toggle=\"yes\">M. smegmatis<\/jats:italic>\n                      aggregation, and they introduce flux-dependent sensors as key components of the NTM biofilm regulatory system.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        A subset of nontuberculous mycobacteria (NTM), including\n                        <jats:italic toggle=\"yes\">Mycobacterium abscessus<\/jats:italic>\n                        , are opportunistic pathogens that can cause severe pulmonary infections. Biofilm formation renders\n                        <jats:italic toggle=\"yes\">M. abscessus<\/jats:italic>\n                        more tolerant to antibiotics; hence, the ability to inhibit NTM biofilm formation could help us better prevent and treat NTM infections. However, the regulatory systems controlling NTM biofilm formation, which could include targets for anti-biofilm therapeutics, are poorly understood. The significance of this work is that it reveals intracellular glutamine as an important node controlling the initiation of biofilm formation in the model NTM\n                        <jats:italic toggle=\"yes\">Mycobacterium smegmatis<\/jats:italic>\n                        . Building on this foundation, future studies will investigate how NTM biofilms can be dispersed by altering glutamine levels and will describe how NTM translates intracellular glutamine to the alteration of surface adhesins.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/jb.00252-25","type":"journal-article","created":{"date-parts":[[2025,10,8]],"date-time":"2025-10-08T13:03:16Z","timestamp":1759928596000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":6,"title":["Intracellular glutamine fluctuates with nitrogen availability and regulates\n                    <i>Mycobacterium smegmatis<\/i>\n                    biofilm 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