# Age-Dependent Pleomorphism in _Mycobacterium monacense_ Cultures
## 1 Introduction  
Ever since the identification of _Mycobacterium tuberculosis_ (_Mtb_) and _Mycobacterium leprrae_ as the etiological agents of tuberculosis and [PERSON]'s disease (leprosy), respectively, different species of mycobacteria have been the subject of extensive investigations into their growth, metabolism, morphology, pathogenicity and response to stress. The ability of rod-shaped mycobacteria to grow as coccoids, branched and spore-like cells has been noticed in early studies, and it appeared that mycobacterial cells undergo morphologicalchanges upon ageing [1; 2; 3; 4; 5; 6; 7; 8; 9; 10]. Recent studies using modern technologies such as atomic force microscopy confirm that the slow-growing mycobacteria (SGM) _Mtb_ shows variation in cell shape at a late stage of its growth [11]. Moreover, the SGM _Mycobacterium marinum_ (_Mmar_) and _Mycobacterium avium_ subsp. _paratuberculosis_s (MAP) were reported to form spores while spore-like structures have been detected in _Mycobacterium smgematis_ mc\({}^{2}\)155 (_Msmeg_), _Mtb_ and _Mycobacterium bovis_ BCG and _Mycobacterium phlei_ cultures [7; 12; 13; 14; 15; 16]. Our understanding of the underlying molecular mechanism dictating changes and variations in cell shape among mycobacteria is, however, limited. To identify the general genetic machinery that might be involved in regulating changes in cell shape in mycobacterial cultures, we examined different mycobacteria that appeared to demonstrate pleomorphic variations at different stages of their growth and/or under stress. Here, we focus on the non-tuberculous (NTM) _Mycobacterium monacense_ (_Mmon_). _M. monacense_ was first isolated in 1998 in Germany from the bronchial lavage of an 80-year-old patient suffering from multifocal lung carcinoma and insulin-dependent diabetes mellitus. It forms smooth, yellow, scotochromogenic colonies within 5-7 days after inoculation when grown on solid media and _Mmon_ belongs to the rapidly growing mycobacteria (RGM) [17]. _Mmon_ has also been isolated from patients suffering from lung infections, nodular lesions or open wound infections [18; 19; 20; 21; 22]. During a microscopic survey of various mycobacterial species, we noted that the _Mmon_ type strain DSM44395, referred to as _Mmon_\({}^{\rm T}\), changed its cell shape in an ageing culture. Hence, we decided to investigate growth, morphological characteristics and search for genes that might influence the cell shape of this RGM. Here, we present data that _Mmon_\({}^{\rm T}\), and _Mycobacterium boenickei (Mboe)_, cultures in liquid medium showing bi-phasic exponential growth curves obtained from absorbance. We determined a roughly four-fold difference in generation time comparing \"early\" and \"late\" exponential phases. However, for _Mmon_ the generation times obtained by counting colony-forming units (CFU) did not differ at early or late phases of growth. Microscopy analysis of cells from different stages of growth showed changes in cell morphology from rod-shaped cells to coccoid and spore-like PGB cells upon ageing. This offered a probable explanation for the apparent bi-phasic nature of the growth curve plotted using absorbance data as cylindrical and smaller spherical shapes would scatter light differently [23]. To examine if there were any changes in the expression of genes associated with the growth-dependent changes in cell shapes, we identified genes predicted to be involved in the maintenance of cell shape and cell division using the complete _Mmon_\({}^{\rm T}\) genome. Their mRNA levels were monitored by RNASeq over time and the _dnaK_3 mRNA level increased significantly upon ageing. DnaK_3 is a known heat shock chaperone and analysis of its protein architecture identified a domain of _Mmon_\({}^{\rm T}\) (and _Mmar_) DnaK_3 as an MreB-like homolog. Overexpression of _dnaK_3\({}^{\rm Mmon}\)_ appeared to be lethal in _Mmon_\({}^{\rm T}\) but its exogenous expression in _Mmar_ resulted in transient changes in cell morphology.