# Age-Dependent Pleomorphism in _Mycobacterium monacense_ Cultures
## 3 Results  
Growth of M. monacense in Liquid Media: Influence of \(\mathit{hyg}^{R}\) and the Red Fluorescence Protein (rfp) Gene  
The type of strain \(\mathit{Mmon}\) DSM44395 (\(\mathit{Mmon}^{\mathrm{T}}\)) forms round, smooth and pale yellow colonies after 5-7 days of growth on MiddleBrook 7H10 (solid; Fisher Scientific, Gothenburg, Sweden) medium at 37 \({}^{\circ}\)C [17]. \(\mathit{Mmon}^{\mathrm{T}}\) was also grown on other solid media, viz., Arret-Kirshbaum (AK), modified G (mG; a modified medium based on one known to promote sporulation in _Bacillus subtilis_[26]), LA and Potato Dextrose Agar (PDA; for details, see Section 2). Growth on LA, PDA and mG plates was slower and poorer, but similar pale yellow to yellow colonies did appear after 15 days at 37 \({}^{\circ}\)C (Table 1; Figure S1). Moreover, \(\mathit{Mmon}^{\mathrm{T}}\) appeared to grow poorly (if at all) in liquid media (7H9, LB or mG). Some growth was, however, apparent in liquid LB and mG cultures after five days of incubation but with heavy clumping that made estimation of growth rates by measuring OD\({}_{600}\) unreliable (Table 2). To track \(\mathit{Mmon}^{\mathrm{T}}\) (and ensure absence of contamination), we introduced the hygromycin and kanamycin resistance genes linked to the red fluorescent protein (RFP) gene into the \(\mathit{attB}\) site on the \(\mathit{Mmon}^{\mathrm{T}}\) chromosome (see below and Section 2). These derivatives are referred to as \(\mathit{Mmon}^{\mathrm{RFPHyg}}\) and \(\mathit{Mmon}^{\mathrm{RFPKan}}\), respectively (Table S2). The \(\mathit{Mmon}^{\mathrm{RFPHyg}}\) grew in 7H9 liquid media supplemented with hygromycin while the \(\mathit{Mmon}^{\mathrm{RFPKan}}\) did not show any detectable growth in 7H9 containing kanamycin (Table 2). Since \(\mathit{Mmon}^{\mathrm{T}}\) transformed with the non-integrative plasmid pBS401 carrying \(\mathit{hyg}^{\mathrm{R}}\) (referred to as \(\mathit{Mmon}^{\mathrm{pBS401}}\); Table S2) grew in 7H9 media supplemented with hygromycin (Table 2) it seemed that the growth was promoted by the presence of hygromycin in 7H9 liquid media and not a consequence of the chromosomal integration of pDEAM5 at the \(\mathit{attB}\) site (see below). Also, growth of \(\mathit{Mmon}^{\mathrm{RFPHyg}}\) in liquid 7H9 medium without hygromycin resulted in heavy clumping. This \begin{table}
\begin{tabular}{l l l} \hline \hline
**Growth in Liquid Media** & & \\ \hline
**Strain** & **Media** & **Colony Morphology After 15 Days of Growth** \\ \hline  & 7H10 *** & pale yellow and smooth \\  & AK *** & pale yellow and smooth \\ \(\mathit{Mmon}^{\mathrm{T}}\) & mG *** & yellow and smooth \\  & LA ** & yellow and smooth \\  & PDA * & smooth and yellow \\ \hline \hline \end{tabular}  
* good growth, **
* moderate growth,
* very poor or no growth.  
\end{table} 
Table 1: Compilation of the colony morphology characteristics of \(\mathit{Mmon}^{\mathrm{T}}\) grown on different media. Observations were made after 15 days of growth at 37 \({}^{\circ}\)C. \begin{table}
\begin{tabular}{l l l} \hline \hline
**Growth in Liquid Media** & & \\ \hline
**Strain** & **Media** & **OD\({}_{600}\) After 5 Days** \\ \hline  & 7H9 & 0.0–0.004 \\ \(\mathit{Mmon}^{\mathrm{T}}\) & LB * & 1.0–1.63 \\  & mG * & 2.4–3.0 \\ \hline \(\mathit{Mmon}^{\mathrm{RFPHyg}}\) & 7H9 + hyg & 0.72–1.5 \\ \(\mathit{Mmon}^{\mathrm{RFPKan}}\) & 7H9 + kan & 0.0 \\ \(\mathit{Mmon}^{\mathrm{pBS401}}\) & 7H9 + hyg & 3.5–4.0 \\ \hline \hline \end{tabular}  
* growth with clumping making the OD\({}_{600}\) measurements unreliable.  
\end{table} 
Table 2: Compilation of OD\({}_{600}\) measurements for \(\mathit{Mmon}^{\mathrm{T}}\) derivatives in various liquid media supplemented with different antibiotics as indicated. The measurements were made after 5 days of growth at 37 \({}^{\circ}\)C.  
would be consistent with earlier observations where hygromycin resistance appeared to confer a growth advantage (compared to kanamycin resistance) upon mycobacteria in the presence of respective antibiotics [51].