Aggregation of nontuberculous mycobacteria is regulated by carbon:nitrogen balance

Nontuberculous mycobacteria (NTM) are emerging opportunistic pathogens that form biofilms in environmental reservoirs such as household water systems and aggregate into phagocytosis-resistant clusters during infection. NTM constitutively aggregate in vitro, a phenotype typically considered to be a by-product of the mycolic-acid-rich cell wall. While culturing a model NTM, Mycobacterium smegmatis, in rich medium, we fortuitously discovered that planktonic cells accumulated in the culture after [~]3 days. By providing selective pressure for bacteria that disperse earlier, we isolated a strain with two mutations in the oligopeptide permease operon (opp). A mutant lacking the opp operon ({Delta}opp) dispersed earlier and more completely than wildtype (WT). We show that {Delta}opps aggregation defect was nutrient related; aggregation was restored by non-peptide carbon sources. Experiments with WT M. smegmatis revealed that growth as aggregates is favored when carbon is replete, while dispersal can be induced by carbon starvation. In addition, under conditions of low available carbon relative to available nitrogen, M. smegmatis grows as planktonic cells. By adjusting carbon and nitrogen sources in defined medium, we tuned the cellular C:N ratio such that M. smegmatis grows either as aggregates or planktonic cells. Lastly, we tested the effect of C:N balance on aggregation in clinically relevant NTM. Altogether, we show that NTM aggregation is a controlled process that is regulated by the relative availability of carbon and nitrogen for metabolism. Because NTM aggregation is correlated with increased virulence, these results may contribute to targeted anti-biofilm therapeutics.nnImportanceFree-living bacteria can assemble into multicellular aggregates called biofilms. Biofilms help bacteria tolerate multiple stresses, including antibiotics and the host immune system. Differing environmental pressures have resulted in biofilm architecture and regulation varying among bacterial species and strains. Nontuberculous mycobacteria are a group of emerging opportunistic pathogens that utilize biofilms to adhere to household plumbing and showerheads and to avoid phagocytosis by host immune cells. Mycobacteria harbor a unique cell wall built chiefly of long chain mycolic acids that confers hydrophobicity and has been thought to cause constitutive aggregation in liquid media. Here we show that aggregation is instead a regulated process dictated by the balance of available carbon and nitrogen. Understanding that mycobacteria utilize metabolic cues to regulate the transition between planktonic and aggregated cells reveals an inroad to controlling aggregation through targeted therapeutics.