Population heterogeneity in Mycobacterium smegmatis and Mycobacterium abscessus

Bacteria use population heterogeneity, the presence of more than one phenotypic variant in a clonal population, to endure diverse environmental challenges - a 'bet-hedging' strategy. Phenotypic variants have been described in many bacteria, but the phenomenon is not well-understood in mycobacteria, including the environmental factors that influence heterogeneity. Here, we describe three reproducible morphological variants in M. smegmatis - smooth, rough, and an intermediate morphotype that predominated under typical laboratory conditions. M. abscessus has two recognized morphotypes, smooth and rough. Interest-ingly, M. tuberculosis exists in only a rough form. The shift from smooth to rough in both M. smegmatis and M. abscessus was observed over time in extended static culture, however the frequency of the rough morphotype was high in pellicle prepara-tions compared to planktonic culture, suggesting a role for an aggregated microenvironment in the shift to the rough form. Differences in growth rate, biofilm formation, cell wall composition, and drug tolerance were noted among M. smegmatis and M. abscessus variants. Deletion of the global regulator lsr2 shifted the M. smegmatis intermediate morphotype to a smooth form but did not fully phenocopy the naturally generated smooth morphotype, indicating Lsr2 is likely downstream of the initiating regulatory cascade that controls these morphotypes. Rough forms typically correlate with higher invasiveness and worse out-comes during infection and our findings indicate the shift to this rough form is promoted by aggregation. Our findings suggest that mycobacterial population heterogeneity, reflected in colony morphotypes, is a reproducible, programmed phenomenon that plays a role in adaptation to unique environments and this heterogeneity may influence infection progression and response to treatment.

Automated summary

Bacteria use population heterogeneity to adapt to different environmental challenges. This study describes multiple morphological variants in M. smegmatis and M. abscessus, and observes a shift from smooth to rough form in extended static culture. The presence of an aggregated microenvironment plays a role in this shift. Variants show differences in growth, biofilm formation, cell wall composition, and drug tolerance. Deletion of the global regulator lsr2 shifts the intermediate morphotype of M. smegmatis to a smooth form, but does not fully replicate the naturally generated smooth morphotype. Rough forms are associated with higher invasiveness and worse infection outcomes.