Intracellular glutamine fluctuates with nitrogen availability and regulates <i>Mycobacterium smegmatis</i> biofilm formation

Nontuberculous mycobacteria (NTM) can form biofilms during human infection and in household plumbing systems, so understanding biofilm regulation could help us better treat and prevent NTM infections. Glucose drives NTM aggregation <i>in vitro</i>, and ammonium inhibits it, but the regulatory systems controlling this early step in biofilm formation are not understood. Here, in the model NTM <i>Mycobacterium smegmatis</i>, 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 <i>M. smegmatis</i> 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 <i>purF</i> 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 <i>garA</i> 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 <i>M. smegmatis</i> aggregation, and they introduce flux-dependent sensors as key components of the NTM biofilm regulatory system.IMPORTANCEA subset of nontuberculous mycobacteria (NTM), including <i>Mycobacterium abscessus</i>, are opportunistic pathogens that can cause severe pulmonary infections. Biofilm formation renders <i>M. abscessus</i> 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 <i>Mycobacterium smegmatis</i>. 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.