# Age-Dependent Pleomorphism in _Mycobacterium monacense_ Cultures
## 3 Results
### Analysis of Selected M. monacense Gene Transcripts at Different Growth Stages  
Of the 3557 functionally classified genes in \(Mmon^{\text{T}}\approx\)3.7% were categorised into the subsystems \"Cell Wall and Capsule\" (104 genes) and \"Cell Division and Cell Cycle\" (27 genes; Figure 4B). This is comparable to other mycobacteria, see, e.g., Refs [29; 33; 67; 68]. To analyse the mRNA levels at different growth stages we focused on selected genes with relevance to cell shape and as such might be involved in various stages of the formation of the peptidoglycan, septum, divisome complex and arabinolactan (Table S4) [69; 70; 71; 72; 73; 74; 75]. The time points were chosen based on the frequency of occurrence of the different morphologies observed in order to have a more homogenous population (Figures S3B and S4F). Albeit coccoids occurred at all growth phases, the majority of cells were rods at early time points (exponential, i.e., 2 to 3 days), coccoids between 5 and 14 days and PGB structures at \(\approx\)1.5 months (48 days; for viability details of old cells see Figure S4G). Hence, we isolated total RNA from \(Mmon^{\text{RFHPs}}\) cells at different growth stages, determined mRNA levels by RNASeq (see Section 2) and mapped the transcripts to the \(Mmon^{\text{T}}\) complete genome. On the basis of the log\({}_{2}\)-fold change (Table S3), mRNA levels for the majority of genes involved in formation of cell wall components were higher in exponentially growing cells compared to mRNA levels detected in cells from old cultures (14 and 48 days; Figure 5A-C; Table S4): cell envelope genes, _ftsW_1_, _ppbB_, _ppbI_, _ripA_1_, _ripA_3_, _fbpA_1_, _fbpA_2_, _fbpC_3 and _dprE1\(2\). The mRNA levels for some gene transcripts, _cvsA_, _fbpC_4_ and _fbpC_2\(1\), were higher in cells from older cultures while those for _ponA1\(1\) and _dprE1\(1\) increased initially with age followed by a reduced level in 48 days old cells (Figure 5A-C; Table S4). For _ftsZ_ and _wag31_ (a _divIVA_ homologue [71]) we detected lower mRNA levels in old cells compared to cells growing exponentially as would be expected (Table S4). Noteworthy, we cannot distinguish whether the change in mRNA levels is due to transcription/expression or degradation (but see Section 4). Mycobacteria are equipped with several serine threonine protein kinases, STPKs, and among these, _pknA_ and _pknB_ influence cell morphology [70; 76]. For _Mmon_[7], 18 were annotated as STPK genes and four among these lack the kinase domain (Table S4); the mRNA levels for most of these were either lower in cells in older cultures or did not vary significantly between the growth phases of \(Mmon^{\text{RFHPs}}\) (6 d, 14 d, 48 d; Figure S5 and Table S4). For _pknA_, the mRNA level is higher in exponential cells in keeping with what has been reported for _Mtb_[70], while for _pknB_1 (positioned adjacent and downstream of _pknA_), we did not detect any significant change in the mRNA level. Moreover, the transcripts of _pknK_ and one of the annotated _pknH_ copies, _pknH_5 (which lacks the kinase domain) were higher in the stationary phase than in the exponential phase (Figure S5; Table S4). The function of \(pknK\) is not well-established although it has been shown to be higher during stationary phase and may be linked to secondary metabolite metabolism [77; 78]. The \(pknH\) is known to be involved in the regulation of the \(embCAB\) operon, which encodes the membrane-associated ethambutol target arabinosyl transferase (EMB), and might control intracellular bacterial growth during infection [79; 80; 81; 82; 83; 84]. Finally, we detected a significant increase in \(dnaK\_3\) mRNA levels, from 0.31-fold (log\({}_{2}\)) in exponential phase (6 vs. 3 days) to 2.5- and 4.4-fold (log\({}_{2}\)), in 14 and 48 days old \(Mmon^{\rm RFPHy8}\) cells (Figure 5D; see below). Figure 5: Analysis of mRNA levels of selected genes and \(dnaK\) paralogs in \(Mmon^{\rm T}\).