Overexpression of a Gene That Modulates Cyclic-di-GMP Enhances Granulation in Mycobacterium smegmatis

Granulation is a complex microbial-aggregation process essential for forming aerobic granular sludge (AGS) and other microbial granules used in wastewater treatment. However, the biological mechanisms that drive granule formation remain poorly understood. Cyclic-di-GMP (c-di-GMP) is a well-established second messenger that regulates biofilm formation, suggesting it may be used to enhance microbial granulation. Mycobacterium smegmatis mc²155, a nonpathogenic model bacterium for Mycobacterium tuberculosis, naturally forms granules. Unlike other bacteria, M. smegmatis carries a single c-di-GMP modulating gene, dcpA, that encodes an enzyme with both diguanylate cyclase (DGC) and phosphodiesterase (PDE) activities; this genetic simplicity offers a unique opportunity to examine the role of c-di-GMP in granulation. Here, we generated and studied three engineered M. smegmatis strains overexpressing dcpA, dcpA∆EAL, or dcpA D192A/E193A, the latter two of which are defective in either PDE or DGC activity, respectively. Using these engineered strains, we examined different forms of biofilm growth, cell morphology, plastic surface adhesion, granulation, and settleability. Results of sludge volume index and microscopy indicated that the aggregates of M. smegmatis were granules rather than flocs, and the settleability of the granules was particularly robust when the cells were grown in a carbon rich medium known to promote granulation. Engineered strains sustained stable granulation more effectively than the wildtype under a low concentration Tween-80 treatment, which was used to induce dispersion. These results suggest that overproduction of DcpA and thus the modulated level of intracellular c-di-GMP enhances granulation and promotes granule persistence in M. smegmatis. Our study further demonstrates that M. smegmatis is a useful model for elucidating biological mechanisms underlying granulation, which could be leveraged to improve granular technologies for wastewater treatment.