# Age-Dependent Pleomorphism in _Mycobacterium monacense_ Cultures
## 4 Discussion
### Variation in Cell Shape and Growth Phase  
The possibility of reduced growth rate due to depletion of nutrients or accumulation of growth inhibitors in ageing cultures was discounted from resuspension experiments (see Section 3). But cell density in the culture appeared to be critical since growth rates could be adjusted by the cell density levels. Since growth on solid media, as measured by CFU, did not appear to suggest a bi-phasic growth pattern, changes in cell shape and/or size with age causing a shift in absorbance resulting in the observed bi-phasic growth in liquid media seemed plausible. Since coccoid shaped cells scatter light differently compared to rods [23] an increasing population of coccoid cells may manifest as apparent reduction in turbidity measurements and hence bi-phasic growth. Genotypically clonal mycobacterial cells are known to exhibit phenotypic heterogeneity that is reflected in cell size, among other characteristics [25; 61]. Transition from rod to coccoid or to smaller rods has been observed in mycobacteria isolated from infected cells or organs [107; 108]. Nutrient starvation or microaerobic conditions have been reported to result in rod to coccoid transitions in laboratory cultures (liquid media/agar plates) [109]. Such a change in morphology has been associated with rise in the intracellular level of (p)ppGpp in _M. smegmatis_[110]. Also, reduction in size in stationary phase cells has often been attributed to division without growth undertaken by the bacterial cell as an adaptive measure against nutritional deprivation [111; 112]. In the present study, however, \(Mmon^{\mathrm{T}}/Mmon^{\mathrm{RFPHyg}}\) cells showed a heterogenous population of cells with different cell shapes. A shift in the frequency of occurrence from rods to coccoids as early as 3 days was observed, while the cells were still in their exponential growth phase (Figures 2 and S3B), suggesting factors other than stress or ageing as the cause for such natural pleomorphism. The cause for such a change from rod-shaped cells to coccoids (Figure 2) in \(Mmon^{\mathrm{T}}/Mmon^{\mathrm{RFPHyg}}\) with increasing cell density is however, not clear. Moreover, the rod to coccoid transition in \(Mmon^{\mathrm{RFPHyg}}\) with increasing cell density was reversed by dilution to the original cell density with either 'fresh' or'spent media' (Table S3) apparently without any help from factors released into media with ageing. How higher cell density generates this signal is not clear and whether such signalling is mediated through stringent control remains to be investigated. The distribution of diverse cell types at different stages of growth from 2 to 30 days in \(Mmon^{\mathrm{T}}\) and \(Mmon^{\mathrm{RFPHyg}}\) are shown in Figure 2 [_Mboe_T (Figure S4D)]. Despite their appearance in phase microscopy, the PGB cells did not demonstrate heat resistance or DPA-content. However, use of membrane-specific stains (FM4-64 and MTG) and TEM revealed internal membrane structures and compartmentalisation (Figure 3). In addition, endospore staining (Schaefer-Fulton stain) of cells from old cultures also indicated the possibility that these refractive structures could be endospore-like structures (Figures S11 and S12), supporting the possibility that mycobacteria such as \(Mmon\) might have evolved a pathway to undergo a transition from standard rods to more spherical/cocoid and/or spore-like PGB cell shapes. This pathway would be different from the well-known endosporulation process in Firmicutes, spore formation in Actinobacteria such as _Streptomyces_ spp., and in the SGM \(Mmur^{\mathrm{T}}\) and \(M\). _avium_ subsp. _paratuberculosis_ in response to various stress/growth conditions [14; 16; 113; 114; 115; 116].