# Age-Dependent Pleomorphism in _Mycobacterium monacense_ Cultures
## 4 Discussion
### Cell Shape and Possible Role of the DnaK_3 Chaperone  
Apart from MreB, many bacteria also code for DnaK and DnaJ-like heat shock proteins with MreB_Mbl protein domains in addition to the Hsp70 domain (Figure S6A). Mutations in such genes affect protein folding, temperature-sensitivity and growth rate to name a few [129; 130; 131]. Three-dimensional structure predictions and amino acid sequence motif comparisons of some functionally different proteins have shown that, despite their differences in function and low amino acid similarity, the folding of some proteins, e.g., DnaK/Hsp70, ParM, MreB and FtsA, are similar to that of actin proteins [132; 133]. We could not successfully over-express the _dnaK_3 Mmon in _MmmT_, which might indicate that increasing the number of _dnaK_3 Mmon gene copies is lethal to _MmmT_. However, we provide data that show the introduction and expression of _dnaK_3 Mmon in _MmT_ correlated with changes in cell morphology raising a possible role (direct or indirect) for _dnaK_3 in cell shape maintenance. The observed changes in cell morphology were transient with respect to coccoids and PGB cells, which was also true for the expression of _dnaK_3 Mmar_ in _MmarT_. Similar pattern of occurrence for PGB cells was observed when _dnaK_3 Mmar was over-expressed in _MmarT_; however, there seemed to be no impact on the coccoid cell shape. These changes in cell morphology were not observed when a plasmid carrying an antisense-_dnaK_3_(pBS4011 indnaK3 Mmon) gene was introduced into _MmmarT_ (Figure S6B). In contrast to _MmmonRFPHyg_, the _dnaK_3 Mmar_ mRNA (and _grpE_, _dnaJ_1 and _hspR_) level is lower in cells in stationary phases compared to exponentially growing cells (Figure 5G; however, in late stationary cells, 48 days old cultures, we observed \(\leq\)1.3 log2-fold higher levels than in exponentially growing cells) and we observed many PGB cells during the early time points (1 day) after which the frequency decreases (Figure 6A). Together, the results indicate that _dnaK_3_ may be involved in cell shape maintenance indirectly or directly albeit, _dnaK_3 Mmar_ and _dnaK_3 Mmon_ appear to be regulated differently in these two mycobacteria (discussed below). We also emphasise that the MreB inhibitor A22 [91] prevented growth of both _MmmT_ and _MthoeT_. These differences in the frequencies of occurrence of the various morphologies observed in response to _dnaK_3 Mmon_ vs. _dnaK_3 Mmar_ (behind 'Tet' inducible promoters) over-expression in _MmmarT_ suggests possible differences in the regulation of _dnaK_3_ expression. This is supported by our data suggesting that the expression of the native _dnaK_3_\(MmarT\) and _MmmonT_/_MmonRFPHyg_ genes (with their respective upstream regions) fused to the _lacZ_ gene differed when expressed in _MmarT_ (Figure 6F). In keeping with this, our genomic analysis suggested that the _dnaK_3_ regulatory regions differ with respect to predicted promoter sequences in _MmomT_/_MmonRFPHyg_ and _MmarT_; SigB, SigE, SigH, SigG and SigM promoters for _MmomT_, and SigD, SigE and SigH promoters for _MmmarT_ (Figure S7). Also, we observed variations in sigma factor mRNA levels upon ageing comparing _MmonRFPHyg_and \(Mmar^{\text{RFPHyg}}\). It could be argued that the stability of the \(dnaK\_3\) mRNA in \(Mmon^{\text{T}}\) and \(Mmar^{\text{RFPHyg}}\) differ. However, our unpublished data suggest that the half-lives are similar, 5.6 \(\pm\) 0.67 (\(Mmon^{\text{T}}\)) and 5.0 \(\pm\) 0.81 (\(Mmar^{\text{RFPHyg}}\)) min (to be published elsewhere). Together, this indicates that the difference in the \(dnaK\_3\) regulatory region in these two mycobacteria might be the reason for the observed difference in the \(dnaK\_3\) mRNA levels in \(Mmon^{\text{RFPHyg}}\) and \(Mmar^{\text{RFPHyg}}\) as well as the appearance of a transient level of \(dnaK\_3^{\text{Mmon}}\) mRNA when introduced into \(Mmar^{\text{T}}\).