# Age-Dependent Pleomorphism in _Mycobacterium monacense_ Cultures
## 3 Results
### Expression of dnaK_3 Mmon in M. marinum Resulted in a Transient Change in Cell Morphology  
Morphological changes correlated with increased _dnaK_3_ mRNA levels upon ageing of the cell culture. Hence, to examine whether overexpression of _dnaK_3_ affect cell morphology we cloned _dnaK_3 Mmon_ in plasmid pB8401 behind the inducible tetracycline (tet) promoter. The resulting plasmid (pB5401-_dnaK_3 Mmon_) was introduced in _MmonT_. However, we were unable to obtain any transformantsants after several trials, suggesting that an increase in copy number of _dnaK_3 Mmon_ is probably lethal to _Mmon_T. Since we could not overexpress _dnaK_3 Mmon_ in _Mmon_T, we tried to introduce the pBS401-_dnaK_3 Mmon_ construct into the rod-shaped _M. marinum_ CCUG20998 type strain (_Mmar_T, see Section 2) to investigate whether a higher _dnaK_3 Mmon_ expression affected the _Mmar_T cell morphology. The _Mmon_T DnaK_3 protein is 94% identical to _Mmar_T DnaK_3 (gene identity CCUG20998_00587 [66]) and the gene synteny of the _dnaK_3 transcriptional units in these two mycobacteria are identical pertaining to _grpE_, _dnal_ and _lspR_ (Figure 5F). First, the natural frequencies of appearance of the various cell morphotypes in _Mmar_T was compared with that of _Mmon_T (see below) as control. For this purpose, a red fluorescence-tagged _Mmar_T strain [41], similar to _Mmon_RFPHy8, was used and is referred to as _Mmar_RFPHy8 (see Table S2). The transformation of _Mmar_RFPHy8 with pBS401-_dnaK_3 Mmon_ was successful. The resulting strain, _Mmar_RB401-dnaK_3 Mmon, was examined for patterns of morphotype variations with growth, which appeared to be opposite to the pattern for _Mmon_RFPHy8: _Mmar_RFPHy8 showed higher frequencies of PGB cells during the early time points (Figure 6A) contrary to the pattern observed in _Mmon_RFPHy8 (Figure 2). Consistently, the _dnaK_3 mRNA transcript levels in the two species with respect to growth phase differed, with an increase in ageing _Mmon_RFPHy8 cells while a decrease was detected in _Mmar_RFPHy8 (Figure 5G). Microscopy and statistics of the different cell morphotypes showed a decrease in the frequency of PGB structures after 24 h incubations (Figure 6B, un-induced) compared to the control (_Mmar_RFPHy8; Figure 6A bottom panel). Upon \"tet-induction\" however, there seemed to be a transient increase in the frequency of PGB cells (up \(\approx 15\%\)) compared to the un-induced condition (Figure 6B). Compared to _Mmar_RFPHy8 the overall frequency of PGB cells decreased in _Mmar_RB401-dnaK3 Mmon_ irrespective of induced or un-induced conditions. Thus, the increased transcription of _dnaK_3 Mmon_ in _Mmar_RFPHy8 resulted in a decrease in the frequency of PGB cells. As shown in Figure 6C, phase contrast microscopy (Figure 6C, left panel) and MTG staining of cells from these cultures (Figure 6C, middle panel) indicated formation of internal membrane structures. Examination of the _Mmar_RB401-dnaK3 Mmon cells with TEM also showed and confirmed the appearance of internal membrane structures (Figure 6C, right panel). Introduction of an \"antisense-_dnaK_3 Mmon_\" (_Mmar_RB401-antidnK3 Mmon) targeting the chromosomal _dnaK_3 Mmar_ gene in _Mmar_T did not result in such changes in cell morphology (Figure S6B) and appeared similar to _Mmar_RFPHy8 (Figure 6A). Moreover, a plasmid carrying _dnaK_3 Mmar_ behind a \"Tet'-inducible promoter (pBS401-_dnaK3 Mmar_) in _Mmar_T (_Mmar_RB401-dnaK3 Mmar; Figure 6D), showed similar patterns of morphological variations as seen for _Mmar_RB401-dnaK3 Mmon under tet-induced/un-induced condition with a decrease in the frequency of PGB cells (cf. Figure 6B,D). Thus, a reduction in the frequency of PGB cells in response to over-expression of either _dnaK_3 Mmar_ or _dnaK_3 Mmon (induced condition) in _Mmar_T was consistent. The _dnaK_3 Mmon_ and _dnaK_3 Mmar_ transcript levels in _Mmar_RB401-dnaK3 Mmon and _Mmar_RB401-dnaK3 Mmar, respectively, were verified by qRT-PCR (see Section 2).