This observation is consistent with the oberserved low level expression of other stress responses [14,
16]. There was no significant difference in the growth rate or physical characteristics, such as clumping or pigmentation between M. find more smegmatis and M. tuberculosis strains expressing ssd and control strains. The primary distinguishing physical feature between the M. smegmatis and M. tuberculosis ssd expressing merodiploid strains in comparison to control bacteria was increased cell lengths and a smooth ultrastructural characteristic (Figure 2ABCD). The observed smooth ultrastructure devoid of concentric rings along the bacterial filament is important because this observation is consistent with inhibition of FtsZ polymerization and Z-ring formation as previously reported [6, 7, 17, 18]. The M. smegmatis wild type control strain exhibited cell lengths of 2.1 mTOR inhibitor ± 0.11 μm (Figure 2AF) and the M. smegmatis
ssd merodiploid strain had increased cell lengths of 3.2 ± 0.42 μm (Figure 2BF). Similarly, M. tuberculosis H37Rv control cells had lengths of 1.73 ± 0.43 μm (Figure 2CF) and expression of ssd resulted in increased cell lengths of 2.53 ± 0.76 LOXO-101 cell line μm (Figure 2DF). In contrast, a ssd::Tn M. tuberculosis mutant strain had decreased cell lengths of 1.35 ± 0.51 μm (Figure 2EF). This experimental data demonstrates a causal relationship between the expression levels of ssd and altered bacterial cell lengths, confirming the bioinformatics analysis and further substantiating Ssd as a septum regulation protein as annotated (http://genolist.pasteur.fr/TubercuList[19]) and indicated by transcriptional mapping [6]. Figure 2 Ultrastructure Analysis (SEM) and Length distributions. Bacterial morphology.
(A) M. smegmatis control strain, (B) M. smegmatis ssd merodiploid (C) M. tuberculosis control, (D) M. tuberculosis ssd merodiploid and (E) ssd::Tn mutant M. tuberculosis strain were visualized by scanning electron microscopy. Images are representative of different fields of bacteria from exponentially growing cultures at 37°C. (F) Lengths of the bacterial cells were calculated from the coordinates of CYTH4 both ends of the cell as measured from representative fields as visualized by scanning electron microscopy. Multiple fields were examined and values calculated in 0.5-1 mm increments from multiple fields of over 100 cells. Whole-genome expression profiling of ssd merodiploid and mutant strains To assess the effect of ssd expression on M. tuberculosis metabolism, global gene expression profiling was performed on the ssd overexpression M. tuberculosis merodiploid strain. A total of 2,274 ORFs were transcriptionally active with 432 of these ORFs being differentially expressed 1.5-fold or greater change (p values ≤ 0.05).