Conditional expression of Mycobacterium smegmatis ftsZ, an essential cell division gene

Abstract

To understand the role of Mycobacterium smegmatis ftsZ (ftsZ smeg ) in the cell division process, the ftsZ gene was characterized at the genetic level. This study shows that ftsZ smeg is an essential gene in that it can only be disrupted in a merodiploid background carrying another functional copy. Expression of ftsZ smeg in M. smegmatis from a constitutively active mycobacterial promoter resulted in lethality whereas that from a chemically inducible acetamidase (ami ) promoter led to FtsZ accumulation, filamentation and cell lysis. To further understand the roles of ftsZ in cell division a conditionally complementing ftsZ smeg mutant strain was constructed in which ftsZ expression is controlled by acetamide. Growth in the presence of 0?2 % acetamide increased FtsZ levels approximately 1?4-fold, but did not decrease viability or change cell length. Withdrawal of acetamide reduced FtsZ levels, decreased viability, increased cell length and eventually lysed the cells. Finally, it is shown that ftsZ smeg function in M. smegmatis can be replaced with the Mycobacterium tuberculosis counterpart, indicating that heterologous FtsZ tb can independently initiate the formation of Z-rings and catalyse the septation process. It is concluded that optimal levels of M. smegmatis FtsZ are required to sustain cell division and that the cell division initiation mechanisms are similar in mycobacteria.

Characterization of CrgA, a New Partner of the Mycobacterium tuberculosis Peptidoglycan Polymerization Complexes

“…Therefore, all the cellular localization experiments were carried out in the nonpathogenic M. smegmatis mc 2 155 strain. Cell division proteins in various mycobacterial species have shown a high degree of homology, and M. smegmatis has therefore served as a favorite surrogate host for cell division protein localization studies (6,14,19,20,33). Bocillin-FL, a fluorescent derivative of penicillin V, was used to visualize the overall localization of penicillin-binding proteins in M. smegmatis strains expressing altered CrgA levels.…”

“…Transformants were selected in this same medium supplemented with agar containing either Km (10 g/ml), Hyg (50 g/ml), or both. When required, acetamide and/or anhydrotetracycline was supplied in the growth medium at a final concentration of 0.2% acetamide and 10 to 100 ng/ml anhydrotetracycline (19,20). Growth was followed by monitoring absorbance at 600 nm.…”

“…strains were detected by immunoblotting as previously described (19). Cell lysates were prepared from exponentially grown cultures (20).…”

“…To investigate whether the localization of CrgA to the midcell was dependent on the prior localization of FtsZ, we examined ECFP-CrgA localization under conditions where FtsZ was depleted. We expressed Ptet-ecfp-crgA from a replicating vector (Table 1) in an FtsZ conditional mutant strain, FZ-3 (M. smegmatis ⌬ftsZ Pami::ftsZ) ( Table 1) (19). Growth of FZ-3 in the absence of acetamide results in FtsZ depletion and the formation of filaments that eventually lyse (19).…”

“…We expressed Ptet-ecfp-crgA from a replicating vector (Table 1) in an FtsZ conditional mutant strain, FZ-3 (M. smegmatis ⌬ftsZ Pami::ftsZ) ( Table 1) (19). Growth of FZ-3 in the absence of acetamide results in FtsZ depletion and the formation of filaments that eventually lyse (19). When grown in 0.2% acetamide and 100 ng/ml anhydrotetracycline, M. smegmatis FZ-3 Ptet::ecfp-crgA showed a distinct ECFP-CrgA localization to the membrane, midcell sites, and poles, which was similar to the pattern in the wild type (Fig.…”