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

Jaroslaw Dziadek{dagger},{ddagger}, Stacey A. Rutherford{ddagger}, Murty V. Madiraju, Mark A. L. Atkinson and Malini Rajagopalan

Biomedical Research, The University of Texas Health Center at Tyler, 11937 US Hwy 271, Tyler, TX 75708, USA

Correspondence
Malini Rajagopalan
malini.rajagopalan{at}uthct.edu


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
To understand the role of Mycobacterium smegmatis ftsZ (ftsZsmeg) in the cell division process, the ftsZ gene was characterized at the genetic level. This study shows that ftsZsmeg is an essential gene in that it can only be disrupted in a merodiploid background carrying another functional copy. Expression of ftsZsmeg 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 ftsZsmeg 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 ftsZsmeg function in M. smegmatis can be replaced with the Mycobacterium tuberculosis counterpart, indicating that heterologous FtsZtb 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.


Abbreviations: DIC, differential interference contrast; DCO, double-crossover; SCO, single-crossover; WT, wild-type

{dagger}Present address: Centre for Microbiology & Virology, Polish Academy of Sciences, Lodowa 106, 93-231 Lodz, Poland.

{ddagger}These authors contributed equally to the work.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The molecular genetic aspects of the cell division process in Mycobacterium tuberculosis, the causative agent of tuberculosis and a slow grower, with an average doubling time of 24 h, are not well understood. A unique characteristic of the M. tuberculosis life cycle is that it maintains a dormant and non-replicative persistent state for extended periods of time under unfavourable growth conditions, only to revive, multiply and cause infection upon the return of favourable growth conditions. Culturing of the organisms either to oxygen depletion (Wayne & Hayes, 1996) or to nutrient starvation (Betts et al., 2002) results in a non-replicative persistent state. It has been suggested that persistent M. tuberculosis are blocked at the cell division stage after completing DNA replication and therefore complete a division cycle prior to initiation of new rounds of life cycle when resuspended in fresh growth medium (Wayne & Hayes, 1996). The genus Mycobacterium also includes rapid-growing species with an average doubling time of 2–3 h, e.g. Mycobacterium smegmatis. The cell division process in M. smegmatis is also not well understood (Dziadek et al., 2002; Gomez & Bishai, 2000). Although the project to determine the nucleotide sequence of M. smegmatis is not complete, its genome appears to contain homologues of all annotated M. tuberculosis cell division genes [see http://www.tigr.org/tdb/mdb/mdbinprogress.html (Dziadek et al., 2002)]. Thus, a thorough understanding of the cell division process in various members of mycobacteria could provide clues to dormancy and, possibly, growth rate differences in the mycobacterial species.

FtsZ, a structural homologue of tubulins (Lowe & Amos, 1998), functions as the initiator of the cell division process in eubacteria (reviewed by Lutkenhaus & Addinall, 1997; Margolin, 2000). In vitro, Escherichia coli FtsZ (FtsZE.coli) protomers polymerize in a GTP-dependent manner (Bramhill & Thompson, 1994; Erickson et al., 1996; Mukherjee & Lutkenhaus, 1994). Fluorescence and immuno-electron microscopy studies revealed that FtsZE.coli protein localizes to the predivision site in the form of rings called Z-rings (reviewed by Margolin, 2000). This process is followed by an ordered assembly of a host of other proteins, e.g. FtsA, ZipA, FtsK, FtsQ, FtsL, FtsW, FtsI and FtsN, all of which are believed to be essential for septum formation. FtsZ is an essential cell division protein in E. coli (Dai & Lutkenhaus, 1991), Bacillus subtilis (Beall & Lutkenhaus, 1991) and Caulobacter crescentus (Wang et al., 2001), but is not required for growth and viability in Streptomyces coelicolor, a filamentous, Gram-positive member of the actinomycetes related to mycobacteria. Interestingly, FtsZ in S. coelicolor is required for sporulation (McCormick et al., 1994) and septum formation. Intracellular levels of FtsZE.coli are rate-limiting for cell division and E. coli ftsZ conditional mutants produce filamentous cells under nonpermissive conditions (Bi & Lutkenhaus, 1990; Ward & Lutkenhaus, 1985).

Comparable studies on FtsZ and the cell division process in members of mycobacteria have not been carried out. To gain insights into the cell division process in mycobacteria, we began characterizing M. tuberculosis ftsZ (ftsZtb) expressed from heterologous promoters. We showed that in M. tuberculosis, expression of ftsZtb from the ami promoter (amip) in a replicating vector did not result in a consistent level of expression whereas that from the Mycobacterium avium dnaA promoter led to non-viability (Dziadek et al., 2002). In contrast, viable M. smegmatis transformants producing consistent levels of FtsZtb were obtained with both plasmids. A 5 h induction of M. smegmatis merodiploids expressing ftsZtb from amip resulted in the accumulation of approximately 22-fold more FtsZ as compared to controls. Furthermore, acetamide-induced cells became filamentous and devoid of any visible septa. Merodiploids expressing ftsZtb from the dnaA promoter in a self-replicating plasmid accumulated approximately sixfold more FtsZ and cells contained both normal and abnormal septa (Dziadek et al., 2002). These results led to a suggestion that FtsZsmeg is responsible for normal septa and the FtsZtb for abnormal septa, and that the latter interferes with the M. smegmatis cell division process. An alternative possibility is that the combined elevated levels of FtsZ are responsible for both normal and abnormal septa and that FtsZtb can work with the M. smegmatis cell division machinery. In the present study, we show that FtsZ is an essential cell division protein in M. smegmatis and that the cell division process in mycobacteria is sensitive to the intracellular levels of FtsZ. Importantly, we show that ftsZtb can replace ftsZsmeg function, suggesting that initiation of the cell division process in mycobacteria can proceed with heterologous FtsZ proteins.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, growth media and transformation conditions.
The following bacterial strains were used: E. coli Top10 (Invitrogen), M. smegmatis mc2155 (Snapper et al., 1990) and M. tuberculosis H37Ra (laboratory stock). E. coli strains were grown in Luria–Bertani (LB) broth or on LB agar plates containing ampicillin or kanamycin (Kan) (50 µg ml-1 each). Mycobacterial strains were grown in Middlebrook 7H9 broth or 7H10 agar plates supplemented with albumin-glucose and Kan (25 µg ml-1) or hygromycin (Hyg) (50 µg ml-1). Mycobacterial transformants were always colony purified, their plasmid DNA was recovered into E. coli following the bead-beating protocol (Madiraju et al., 2000) and the presence of cloned insert was confirmed by restriction digestion. Acetamide at a final concentration of 0·2 % was used to induce FtsZ overproduction in M. smegmatis, although the amount of FtsZ induced was found to be independent of the acetamide concentrations tested (0·2–1 %).

Plasmid constructions.
Most of the oligonucleotide primers used for PCR included restriction enzyme recognition sites to facilitate cloning; these are underlined (see below). The sequence of all PCR-amplified products was confirmed by sequencing.

Construction of ftsZ expression plasmids
ftsZ from the ami promoter.
The ftsZsmeg coding region was amplified using primers MVM240 (5'-GCTCTAGAGTGCCGCATGAAGGGCGGC-3') and MVM174 (5'-GCGGATCCATGACCCCCCCGCAC-3') and cloned downstream from amip in the self-replicating pJAM2 vector to create pJfr72 (referred to as amip–ftsZsmeg; see Fig. 1). The gfp gene from pJfr11 (Dziadek et al., 2002) was amplified using primers MVM188 (5'-GCTCTAGAAACAACAACCTGCAGATGAGTAAAGGAGAAG-3') and MVM189 (5'-GCTCTAGAAACAACAACCTGCAGATGAGTAAAGGAGAAG-3') and cloned downstream of the ftsZ gene at the XbaI site of pJfr72 to create pJfr79 (referred to as amip–ftsZsmeg–gfp). This construct was used to produce the FtsZ–GFP fusion protein. For some experiments, ftsZsmeg and ftsZtb were cloned under the control of amip in the mycobacteriophage L5-based integration-proficient vector, pMV306, to create pJfr78 and pSAR20, respectively (see Table 1).



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Fig. 1. Construction of ftsZ expression plasmids. (A) Physical map of the ftsZ region of M. smegmatis. The coding regions of ftsQ and ftsZ are marked. Various primers used for amplification are indicated. Arrows indicate the orientation of the primers from 5' to 3'. (B) The ftsZ expression plasmids. The ami, dnaA and ftsZ promoters used to drive the expression of ftsZ gene are boxed. Note that the pJfr79 construct contains the gfp gene fused to the 3' end of ftsZ.

 

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Table 1. Plasmids

 
ftsZ from the dnaA promoter.
The ftsZ coding region was amplified using MVM240 and MVM124 (5'-GCGGATCCAATGACCCCCCCGCACAA-3'), and exchanged with the corresponding fragment in pFR32 (Dziadek et al., 2002) to create pJfr39 (referred to as dnaAp–ftsZsmeg).

ftsZ from the native ftsZ promoter.
A BLAST search of 5 kb sequence containing M. tuberculosis murC–ftsQ–ftsZorf against the unannotated M. smegmatis genome sequence revealed a significant sequence identity, indicating that the region is conserved in both species (data not shown) and that the region upstream of ftsZsmeg could function as the ftsZ promoter. Accordingly, a 2·1 kb fragment containing the M. smegmatis ftsZ coding region and its 920 bp upstream region was amplified using primers MVM304 (5'-CCCAAGCTTCCGCGCAACACGATCCG-3') and MVM305 (5'-TGCTCTAGATCATCAGTGCCGCATGAAGGG-3') and cloned into integrating pMV306 vector to create pSAR37 (referred to as ftsZp–ftsZsmeg; see Fig. 1, Table 1).

Construction of ftsZ gene replacement vectors.
A suicidal recombination delivery vector, designated as pSAR33, was constructed in three steps. First, a 1·7 kb fragment containing the 3' end of the ftsZ gene (30 bp) and the downstream region was amplified using the primers MVM270 (5'-GACGATGTCGAAGCTTCGCCCTTCATG-3') and MVM271 (5'-CGTAGTCATGGATCCGCGCCATGCC-3') and cloned into p2NIL to create pSAR29. Next, a 1·2 kb fragment spanning the putative ftsQ, ftsQ–ftsZ intergenic region and 55 codons from the 5' end of ftsZ was amplified using MVM286 (5'-GCTGGCAAAAGTACTCCCGCCGCGAGGCCAAGC-3') and MVM287 (5'-ACGGCCCACGTCAAGCTTGACGTCGGCG-3') and cloned into the ScaI–HindIII sites of pSAR29 to create pSAR30. This construct contains 2·9 kb ftsZ region lacking most of the ftsZ coding region. Finally, a 6 kb PacI marker cassette from pGOAL17 carrying lacZ and sacB genes was cloned into the PacI site of pSAR30 to create pSAR33.

Disruption of ftsZ and construction of conditional expression strains.
The two-step recombination protocol of Parish & Stoker (2000) was used to disrupt the ftsZsmeg gene at its native locus on the chromosome. The pSAR33 plasmid DNA was exposed to UV at 100 mJ cm-2 (UV Stratalinker 2400), and integrated into the M. smegmatis mc2155 ftsZ region by homologous recombination generating single-crossovers (SCOs) that were blue, Kanr and sensitive to sucrose. The site of integration was confirmed by PCR and Southern hybridization. In the next step, one SCO strain, FZ1 (Table 2), was further processed to select for double-crossover (DCO) strains that were white, Kans but resistant to sucrose on 7H10 plates containing 2 % sucrose and X-Gal (100 µg ml-1). DCOs were also selected in ftsZp–ftsZsmeg merodiploid background (FZ1-37). PCR and Southern analyses revealed wild-type (WT) and mutant patterns in a 9 : 1 ratio. For the construction of an ftsZ conditional strain, DCOs were selected in the ftsZ merodiploids expressing either amip–ftsZsmeg (FZ1-78) or amip–ftsZtb (FZ1-20) on plates containing 0·2 % acetamide.


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Table 2. M. smegmatis strains

 
Analysis of SCOs and DCOs.
Both PCR and Southern hybridization reactions were carried out to characterize SCOs and DCOs. Primers MVM302 (5'-GCGGATCCATGACCCCCCCGCATAAC-3', which binds to a region 500 bp upstream of ftsZ (see Fig. 1) and MVM271 were used to detect either a WT (2·9 kb) or mutant (1·9 kb) copy of ftsZ in DCOs and both mutant and WT copies in SCOs. An integrated copy of ftsZ was confirmed by PCR using MVM315 (5'-CCGCAGCCGAACGACCGAGC-3'), which binds to the pMV306 vector sequences and MVM304. A DNA fragment bearing the ftsQ–ftsZ region was used as a probe in Southern hybridization experiments.

Characterization of ftsZ strains: growth and viability experiments.
Experimental conditions for the determination of growth and viability were as described earlier (Dziadek et al., 2002). The conditionally complemented ftsZ mutant strains grown in the presence of 0·2 % acetamide were washed with acetamide-free medium, resuspended in the same medium, diluted, and spread on agar plates with and without acetamide; colonies that appeared after 72 h were counted. Viability experiments were performed with clump-free cell suspensions as judged by microscopy. In some experiments, cultures after resuspension in acetamide-free medium were grown for different periods and samples were processed for microscopy and protein analysis.

Protein methods.
Preparation of mycobacterial cell lysates by bead-beating using 0·1 mm Zirconia beads and detection of FtsZ by Western blotting using affinity-purified anti-M. tuberculosis FtsZ antibodies were essentially as described previously (Dziadek et al., 2002). For quantitative immunoblotting, known amounts of cell lysates based on equivalent protein concentrations were loaded on SDS-polyacrylamide gels (Dziadek et al., 2002). Purified recombinant FtsZtb protein was used as standard (Dziadek et al., 2002). Western blots were processed using the Amersham Pharmacia ECF chemifluorescence kit and protocol, and FtsZ bands were visualized by scanning the nitrocellulose blots in a Bio-Rad Molecular Imager and quantified using the Quantity One software. Although the predicted sizes of both M. smegmatis and M. tuberculosis FtsZ proteins are ~40 kDa, consistent with our earlier results (Dziadek et al., 2002), Western analyses often revealed the presence of two bands: one corresponding to an intact, full-length 40 kDa product and another to a smaller proteolytic product of ~38 kDa. The overproduced FtsZtb appeared sensitive to proteolysis, accumulating ~38 kDa protein (Dziadek et al., 2002). When present, both bands were included for the determination of FtsZ levels.

Microscopy methods.
M. smegmatis cells were resuspended in a buffer containing 10 mM Tris/HCl, pH 7·5, 10 mM MgCl2 and 0·02 % (v/v) Tween 80, processed as described previously (Dziadek et al., 2002) and examined by conventional microscopy on a Nikon TS 100 inverted microscope with a 100x Nikon Plan fluor DIC oil immersion objective with a numerical aperture of 1·3 and working distance of 0·17 (Greendyke et al., 2002). Images were acquired using a Sensicam 12-bit monochromatic CCD camera and SlideBook 3.0 software from 3I Imaging. For some experiments, mycobacterial cells were permeabilized by exposure to 2 % (v/v) toluene for 2 min prior to staining for DNA with a combination of ethidium bromide (40 µg ml-1) and mithramycin A (180 µg ml-1) for 30 min on ice (Dziadek et al., 2002). The ethidium bromide/mithramycin A-stained nucleoids imaged using a 100 W mercury lamp and a Chroma filter set (excitation from 540 to 565 nm and emission from 560 to 623 nm) appear as fluorescent red globular structures (Dziadek et al., 2002; Greendyke et al., 2002). For acquiring FtsZ–GFP images, a standard FITC filter set (excitation from 484 to 499 nm and emission from 459 to 509 nm) from Chroma was used. All images were optimized using Adobe Photoshop 7.0.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
FtsZ levels and filamentation
To test whether the cell division process in M. smegmatis is sensitive to the intracellular levels of FtsZ, we investigated the consequences of overproduction of FtsZsmeg in M. smegmatis. Transformation of M. smegmatis with self-replicating plasmid pJfr39, expressing dnaAp–ftsZsmeg, did not result in any viable transformants. In one case, a few transformants were obtained, but all recovered plasmids had deletions in the ftsZ gene (data not shown). Since M. smegmatis merodiploids expressing dnaAp–ftsZtb, which produce approximately sixfold higher levels of FtsZ than plasmid-free cells, are viable (Dziadek et al., 2002), these results indicated that comparable intracellular levels of homologous FtsZsmeg are toxic to M. smegmatis. To test if lower levels of homologous FtsZ are tolerated, we created three constructs producing different amounts of FtsZsmeg protein. The first construct, designated as pSAR37, expresses ftsZsmeg from its native promoter in an integrating plasmid (see Fig. 1). If this construct were to function like the ftsZp–ftsZ present at the native chromosomal location, then we would expect to detect twice the amount of FtsZ in the lysates of merodiploids, as compared to controls. Quantification of the Western blots did not reveal any significant increase in the FtsZ levels in the merodiploid strain (Fig. 2a, compare lane 6 with lane 5), but our experiments described below (see Fig. 3B) indicate that FtsZ amounts sufficient for cell survival were produced from this construct. Presumably, FtsZsmeg levels are regulated such that large amounts of FtsZ are not accumulated in vivo. Growth, viability and morphology of the pSAR37 merodiploid strain were indistinguishable from those of the control strain (data not shown).



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Fig. 2. Characterization of M. smegmatis ftsZ merodiploids. (A) Western analysis of FtsZ levels. M. smegmatis strains overexpressing ftsZ to various levels were grown in the absence (-) or presence (+) of 0·2 % acetamide for 6 h, then lysed by bead-beating; lysates were resolved by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were probed with FtsZtb-specific antibodies as described previously (Dziadek et al., 2002). Lanes: 1 and 2, control vector pJAM2; 3 and 4, pJfr72; 5, no plasmid; 6, pSAR37; 7 and 8, pJfr78; 9 and 10, pSAR20. (B) Microscopy of FtsZsmeg-overproducing cells. M. smegmatis cells containing pJAM2 (a, b), pJfr72 (c, d) or pJfr79 (e–h) were grown in the absence (-) or presence (+) of 0·2 % acetamide for 6 h and examined by DIC and fluorescence microscopy. (a) pJAM2 (-); (b) pJAM2 (+); (c), pJfr72 (-); (d), pJfr72 (+); (e–h), pJfr79 (+). Panels (a)–(f) are DIC images; panels (g) and (h) are fluorescence images corresponding to (e) and (f).

 


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Fig. 3. Verification and characterization of ftsZ DCO strains. (A) Verification of M. smegmatis ftsZ complementing mutant DCOs: Southern blot of SCO strain FZ1-37 and the respective derived ftsZ complementing mutant and WT DCOs. Genomic DNA was prepared, digested with NotI, separated on agarose gels, transferred to nitrocelluose membranes and probed with ftsZ-specific probe (see Methods). Lanes: WT, wild-type M. smegmatis; SCO, SCO strain FZ1-37; WT DCO, wild-type DCO; Mut DCO, mutant DCO strains. The bands corresponding to the chromosomal copy of ftsZ (WT ftsZ), integrated copy of ftsZ (Int ftsZ) and the mutated copy of ftsZ (Mut ftsZ) are indicated. (B) Western analysis of FtsZ levels in SCO and complemented mutant and WT DCO strains. Lanes: 1, WT M. smegmatis; 2, FZ1-37; 3, FZ2-37; 4, FZ3-37. (C) Selection of conditionally complementing mutant DCOs. The Kans, sucrose-resistant DCOs derived from FZ1-78 were propagated on plates with or without acetamide to distinguish the conditionally complementing ftsZ mutant strains from the WT DCO strains.

 
Another construct, designated as pJfr72, maintained as an extrachromosomal plasmid, expresses ftsZsmeg from the chemically inducible amip (see Fig. 1). Six hours after the addition of acetamide, the induced cells accumulated approximately sixfold more FtsZ than uninduced cultures (Fig. 2A, compare lane 4 with lane 3) and the control strain (Fig. 2A, lanes 1 and 2). Under similar induction conditions, approximately 22-fold more FtsZtb was accumulated in M. smegmatis as compared to control strains (Dziadek et al., 2002). Continuous growth in the presence of acetamide resulted in clumping and eventual cell lysis, indicating that FtsZsmeg levels beyond sixfold are toxic to M. smegmatis. Examination of the FtsZ-overproducing cells by DIC microscopy revealed that approximately 60 % cells were two times or more longer than the uninduced and control cells (Fig. 2B, compare d with a–c). Some elongated cells also contained buds and branch-like structures (data not shown). Nuclear staining revealed the presence of well-segregated nucleoids, indicating that DNA replication and possibly segregation were not affected under FtsZ-overproducing conditions (data not shown). When cultures induced with acetamide for 6 h were spread on acetamide-free plates, approximately 50 % loss in viability was noted. It should be noted that these cultures were not visibly clumpy as revealed by microscopy. The colonies that appeared on acetamide-free plates were fully capable of overproducing FtsZ when induced with acetamide (data not shown). Presumably, filamentous cells produced upon FtsZ overproduction do not recover and eventually lyse. Finally, M. smegmatis merodiploids expressing integrated amip–ftsZsmeg accumulated approximately twofold more FtsZ when grown with acetamide (see Fig. 2A, compare lane 8 with lane 7). These cultures were fully viable and showed no significant changes in morphology (data not shown), indicating that FtsZsmeg accumulation up to twofold is not toxic. Mini-cell phenotype, as is seen with the limited FtsZE. coli overproduction in E. coli, was not observed under our experimental conditions.

Filamentous cells contain multiple FtsZ rings
To evaluate the fate of the overproduced FtsZsmeg in elongated cells, amip–ftsZsmeg–gfp was expressed from a replicating plasmid (pJfr79) and the fusion protein visualized by microscopy. Since both ftsZsmeg–gfp and ftsZsmeg are expressed from the ami promoter, we assumed that comparable levels of FtsZsmeg–GFP and FtsZsmeg proteins are produced under our experimental conditions. Similar to amip–ftsZsmeg merodiploids, growth in the presence of acetamide led to cell length increase of pJfr79 cells (Fig. 2B, e and f). Furthermore, the elongated cells showed distinct FtsZsmeg–GFP bands at regularly spaced intervals, indicative of Z-ring-like structures (Fig. 2B, g and h). Prolonged overproduction of FtsZsmeg–GFP resulted in the formation of fluorescent ribbon-like structures (data not shown), similar to those reported with the overproduction of E. coli FtsZ–GFP fusion protein (Ma et al., 1996).

Conditional expression of M. smegmatis ftsZ
Gene replacement experiments revealed that M. smegmatis ftsZ is an essential gene in that it can only be disrupted in a merodiploid background carrying another functional copy of ftsZ (Fig. 3A). Western analysis revealed that both WT (FZ2-37, Fig. 3B, lane 3) and complemented mutant (FZ3-37, Fig. 3B, lane 4) DCOs had FtsZ levels similar to the WT M. smegmatis (Fig. 3B, lane 1) and merodiploid SCO (FZ1-37, Fig. 3B, lane 2). Furthermore, these strains showed no differences in viability and cell size as compared to the controls (data not shown). These results also confirm that integrated ftsZp–ftsZsmeg construct produces enough FtsZ for normal cell division in M. smegmatis.

An ftsZ conditional expression strain was constructed by selecting DCOs in merodiploid strains expressing ftsZ from amip following similar strategies (Fig. 3C). Of the 21 DCOs patched on acetamide-free plates, only 9 survived (Fig. 3C), indicating that the remaining 12 DCOs could be conditionally complementing mutants requiring acetamide for growth. One such strain, FZ3-78, was characterized and found to produce approximately 1·4 (1·38±0·14)-fold more FtsZ in the presence of acetamide than the WT M. smegmatis (Fig. 4A, compare lanes 1 and 2).



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Fig. 4. Characterization of conditionally complementing mutant DCO strains FZ3-78 and FZ3-20. (A) Western analysis of FtsZ levels in the DCO strains. The various M. smegmatis strains were grown, lysed and analysed by SDS-PAGE and Western blotting as described under Fig. 1. Lanes: 1, M. smegmatis mc2155; 2, FZ3-78; 3, WT M. smegmatis; 4, FZ3-20. (B) Western analysis of FtsZ levels in the conditionally complementing mutant strains grown in the absence of acetamide. Exponentially growing cultures of FZ3-78 (top panel) and FZ3-20 (bottom panel) were washed, transferred to medium lacking acetamide and grown for various periods of time. At the indicated time points, the cultures were harvested, lysed, and analysed by SDS-PAGE and Western blotting. Lanes: M, WT M. smegmatis; 0, 3, 6, 9 and 12 h, FZ3-78 or FZ3-20 grown in the absence of acetamide for the respective time periods. (C) Morphology of FZ3-78. Cultures grown in the absence of acetamide for the above time periods were stained for nucleoids as described in the text and examined by DIC and fluorescence microscopy. (a, b) 0 h; (c, d) 3 h; (e, f) 6 h; (g, h) 9 h; (I, j) 12 h. Panels (a), (c), (e), (g) and (i) are DIC images of FZ3-78 and panels (b), (d), (f), (h) and (j) are the respective fluorescence images. Black arrowheads indicate bulb-like structures (1). White arrowheads indicate stained nucleoids (D) Morphology of FZ3-20. All experimental conditions were as described for (C) except that 12 h time point cells are not shown because of the clumping problem. Black arrowheads indicate branch-like structures (2).

 
Consequences of blocked FtsZ production
To determine the physiological consequences associated with decreasing FtsZ levels below those present in the conditionally complemented ftsZ mutant strain, FZ3-78, we attempted to block ftsZ expression by growing the latter in broth lacking acetamide for various time periods, viz. 3, 6, 9 and 12 h. Continuous growth beyond 12 h resulted in severe clumping and eventual cell lysis (data not shown). Growth in the absence of acetamide led to a gradual decrease in FtsZ levels (see Fig. 4B, top panel). Furthermore, a FtsZ band corresponding to 38 kDa, in addition to the 40 kDa band, was evident with increasing times of depletion, i.e. 6, 9 and 12 h. Approximately 72, 55 and 41 % of initial FtsZ levels remained in cultures grown for 3, 6 and 12 h respectively, in the absence of acetamide (data not shown).

Acetamide-starved FZ3-78 cells showed a gradual increase in cell length coincident with the reduction in FtsZ levels (Fig. 4C, compare panel a with c, e, g and i). Nuclear staining followed by epifluorescence microscopy showed the presence of well-separated nucleoids (seen as distinct red globular structures), indicating that FtsZ depletion did not noticeably affect DNA replication and nucleoid segregation (see Fig. 4C, compare panel b with d, f, h and j). The majority of cells grown in the presence of acetamide were 1–2 µm in length (Fig. 5A) whereas 70 and 38 % of those grown in the absence of acetamide for 3 and 6 h, respectively, remained 2 µm in length while others increased in length (Fig. 5A). These results indicated that reduction in FtsZ levels interfered with cell division and led to filamentation. Cultures grown for 9 and 12 h continued to elongate (Fig. 4C, panels g and i), but viability and precise cell length measurements could not be made due to the difficulty in obtaining clump-free cell suspensions. Some of the elongated cells showed buds (data not shown) and bulb-like structures (Fig. 4C) indicative of cell division defects. Similar structures were also seen under FtsZtb overproduction conditions (Dziadek et al., 2002). When 6 h acetamide-starved cultures of FZ3-78 were spread on agar plates containing acetamide, a marked reduction in viability was noted as compared to those grown in the presence of acetamide (see FZ3-78 in Fig. 5B). In contrast, the WT DCO strain (FZ2-78) did not show any decrease in viability in the absence of acetamide (Fig. 5B). Together, these results indicate that inhibition of cell division due to blockage of FtsZ production leads to irreversible loss of viability of M. smegmatis.



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Fig. 5. Effect of FtsZ depletion on cell size and viability of M. smegmatis conditionally complementing ftsZ mutant strains. (A) Cell length measurements. M. smegmatis strains FZ3-78 and FZ3-20 were grown in the presence of 0·2 % acetamide (+) and then transferred to acetamide-free medium (-) for 3 and 6 h. The cells were examined by DIC microscopy and the sizes of 100 bacteria from each set were measured. (B) Viability of conditionally complementing ftsZ mutants. FZ2-78, FZ3-78, FZ2-20 and FZ3-20 were grown in the presence or absence of acetamide for 6 h as in (A) and then plated on 7H10 agar plates containing 0·2 % acetamide. Colonies that appeared after 72 h were counted. Means and error bars (representing SD) from three separate experiments are shown.

 
M. tuberculosis FtsZ protein can substitute for M. smegmatis FtsZ protein
The M. smegmatis and M. tuberculosis FtsZ proteins are ~92 % identical, with most differences found in the C-terminal region (data not shown). It is unknown whether these differences are important for mycobacterial FtsZ function. If they are not important, then we would expect that ftsZsmeg function can be substituted with ftsZtb and vice versa. It is pertinent to note that the C-terminal region of FtsZE.coli has been shown to be involved in interactions with FtsA and ZipA proteins (Din et al., 1998; Hale & de Boer, 1997; Hale et al., 2000; Ma et al., 1996, 1997; Wang et al., 1997). To address this question, amip–ftsZtb (pSAR20) was integrated into M. smegmatis SCO FZ1 to create a merodiploid FZ1-20. DCOs were then selected on media containing 0·2 % acetamide as described above. When tested, 8 of 20 DCOs did not grow in the absence of acetamide, indicating that they could be conditionally complementing mutant DCOs (data not shown). The DCOs were confirmed by PCR and Southern hybridization (data not shown). One conditionally complementing mutant DCO was designated as FZ3-20 (integrated amip–ftsZtb serves as the sole source for FtsZ) and a WT DCO as FZ2-20 (contains both a native copy of ftsZsmeg and an integrated copy of amip–ftsZtb). Our ability to obtain mutant DCOs in merodiploids expressing ftsZtb indicates that the latter can substitute for ftsZsmeg function. The WT DCO strain showed similar viability in the presence and absence of acetamide (Fig. 5B).

Western analysis revealed that the FZ3-20 cells produced twofold more FtsZ than WT M. smegmatis (Fig. 4A, compare lane 4 with 3). This may not be a precise estimate as the antibody response to the FtsZtb and FtsZsmeg proteins may not be similar. Consistent with our earlier results, most of the detectable FtsZtb corresponded to a 38 kDa band (Dziadek et al., 2002). Cell length measurements revealed that ~60 % of the FZ3-20 cells were normal in length, i.e. 1–2 µm, and the remainder were elongated (see Fig. 5A, compare FZ3-20+ with FZ3-78+). When grown in broth, the ftsZtb conditionally complementing mutant strain, FZ3-20, unlike its ftsZsmeg counterpart, FZ3-78, showed a general tendency to clump. However, as with FZ3-78, the withdrawal of acetamide resulted in a decrease in FtsZ levels (Fig. 4B, bottom panel), an increase in cell length (Fig. 4D) and a decrease in viability (Fig. 5B). A small amount of FtsZtb corresponding to the intact FtsZ size of 40 kDa was detected with increasing times of acetamide starvation, i.e. 6, 9 and 12 h. Acetamide-starved cells of FZ3-20 were generally more elongated than FZ3-78 cells grown under similar conditions (see Fig. 5A).


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The key findings of this study are the following: (1) M. smegmatis ftsZ is an essential cell division gene; (2) M. tuberculosis ftsZ can substitute for the M. smegmatis counterpart; and (3) conditions that result either in blocked FtsZ production or in elevated levels of FtsZ interfere with the cell division process, produce filamentous cells and decrease viability. Collectively these results indicate that the cell division process in mycobacteria is sensitive to the intracellular levels of FtsZ and that the M. tuberculosis FtsZ protein can work with the M. smegmatis cell division machinery.

Functional replacement of ftsZsmeg with its M. tuberculosis counterpart has implications for understanding ftsZ function in the cell division process of mycobacteria. The genetic and biochemical aspects of the cell division process in mycobacteria are just beginning to be understood. Our earlier studies on the characterization of M. smegmatis ftsZ merodiploids expressing ftsZtb suggest that the M. smegmatis cell division machinery can accommodate heterologous FtsZtb (see Dziadek et al., 2002). Results presented in this study expand these initial observations and demonstrate that ftsZtb can ‘truly’ replace the function of ftsZsmeg and suggest that it localizes to the putative division septa of M. smegmatis either directly, or possibly by its interactions with ZipA-like membrane-anchoring proteins, recruits and coordinates the ordered localization of other cell division proteins and thereby orchestrates the cell division process. It is pertinent to note that very recently, using biochemical approaches, physical interactions between M. tuberculosis FtsZ and FtsW proteins have been demonstrated (Datta et al., 2002). Assuming that these interactions also occur in vivo, then the FtsZtb-initiated cell division process in M. smegmatis could involve its physical and functional interactions with the M. smegmatis FtsW protein. The conditionally complementing ftsZtb mutant strain FZ3-20, although viable, showed an increased tendency to clump and was more elongated than that of its ftsZsmeg counterpart FZ3-78 (see Fig. 5A). These results indicate that the replacement of ftsZsmeg by ftsZtb does not lead to a total restoration of the WT phenotype. Presumably, the observed differences in the C-terminal region between the two mycobacterial FtsZ proteins, while not important for protein–protein interactions required for the cell division process, could be important for specific regulation of the cell division process, if any. This could, in turn, affect coordination between the cell division and the cell wall synthesis processes in FZ3-20, thereby resulting in increased cell length and clumping.

FtsZ is an abundant protein, accounting for ~1·2 % of the total soluble cellular protein in actively growing cells. This corresponds to approximately 15 000 molecules per cell in mycobacteria (Dziadek et al., 2002). Mycobacteria are rod-shaped bacteria with mean sizes ranging from 1 to 1·5 µm in length and 0·5 to 0·8 µm in width (Brennan & Nikaido, 1995). Thus, one would expect that the amount of FtsZ present is more than sufficient to encircle the cell a few times (Bramhill & Thompson, 1994). We showed that up to twofold FtsZ accumulation (Fig. 4) does not significantly affect cell morphology and viability whereas sixfold FtsZ overproduction does (Fig. 2). No intermediate levels of overproduction could be obtained with any of our constructs. The mini-cell phenotype, as reported with the limited FtsZE.coli overproduction (Bi & Lutkenhaus, 1990; Ward & Lutkenhaus, 1985), was not observed under our experimental conditions. Nonetheless, these results suggest that M. smegmatis cells have adopted mechanisms to tolerate a limited increase in FtsZ levels (see below). On the other hand, blocked FtsZ production, which led to a reduction in FtsZ levels by 30–50 %, resulted in an increase in cell length and a significant decrease in viability (see Figs 4C and 5A). Together, these results suggest that the cell division process in mycobacteria is sensitive to the intracellular levels of FtsZ, as is seen with other bacteria (Dai & Lutkenhaus, 1991; Quardokus et al., 1996; Ward & Lutkenhaus, 1985).

Western analyses revealed that FtsZ depletion was associated with the appearance of a 38 kDa polypeptide (Fig. 4B). However, polypeptides of smaller sizes were not detected under these conditions. We speculate that in vivo, the mycobacterial FtsZ may be stabilized by being in polymers and oligomers, and become unstable and subject to proteolytic processing when effective FtsZ concentrations decrease below a certain level. It is pertinent to note in this regard that an ftsH orthologue from M. tuberculosis has recently been cloned and overexpressed in E. coli (Anilkumar et al., 1998) and FtsH protease activity has been implicated in the regulation of FtsZ levels (Anilkumar et al., 2001). Studies with Caulobacter crescentus ftsZ have also indicated proteolysis as one of the important determinants in regulation of FtsZ levels (Kelly et al., 1998). Further studies are required to evaluate the relationship between proteolysis and FtsZ levels, and the regulation of FtsZ during the mycobacterial cell cycle.

A 40 kDa band corresponding to intact FtsZtb was also detected in the lysates of FZ3-20 cells grown in the absence of acetamide (Fig. 5B, bottom panel). Our other experiments on the expression of dnaA (Greendyke et al., 2002) and ftsZ (Dziadek et al., 2002) indicate that amip is leaky in M. smegmatis. Thus, presumably the 40 kDa protein corresponds to small amounts of the newly synthesized FtsZtb.

The above studies raise a question as to why the majority of FtsZtb produced from amip is truncated whereas FtsZsmeg is not. We propose that proteolytic processing mechanisms, if any, in M. smegmatis could be more efficient with homologous FtsZsmeg than those with heterologous FtsZtb. This could, in turn, result in lower levels of truncated FtsZsmeg and might explain why higher levels of truncated FtsZtb are accumulated in FZ3-20 (Fig. 4B) and in merodiploids (Fig. 2A, compare lanes 10 and 8). Further studies are required to test this hypothesis.

Finally, our results showing that the FtsZtb can work with the M. smegmatis cell division machinery will enable us to understand the functional roles played by the putative M. tuberculosis cell division proteins in M. smegmatis. This is important because efficient regulatory promoter systems to attain conditional expression in M. tuberculosis are not available. The best-characterized ami promoter is somewhat leaky in M. tuberculosis, thus making it difficult to evaluate the consequences of both blocked FtsZ production and elevated FtsZ levels on cell morphology and cell division in M. tuberculosis. With the conditional ftsZ expression strains at hand, we can now begin to understand the molecular details involved in the regulation of the cell division process in mycobacteria.


   ACKNOWLEDGEMENTS
 
We thank Dr Zissis Chroneos for help with microscopy and Dr Tanya Parish for the recombination vectors. We thank Kimberly Calloway and Aurora Rosillo for technical help. This work is supported in part from Public Health Service Grant AI48417.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Anilkumar, G., Chauhan, M. M. & Ajitkumar, P. (1998). Cloning and expression of the gene coding for FtsH protease from Mycobacterium tuberculosis H37Rv. Gene 214, 7–11.[CrossRef][Medline]

Anilkumar, G., Srinivasan, R., Anand, S. P. & Ajitkumar, P. (2001). Bacterial cell division protein FtsZ is a specific substrate for the AAA family protease FtsH. Microbiology 147, 516–517.[Free Full Text]

Beall, B. & Lutkenhaus, J. (1991). FtsZ in Bacillus subtilis is required for vegetative septation and for asymmetric septation during sporulation. Genes Dev 5, 447–455.[Abstract]

Betts, J. C., Lukey, P. T., Robb, L. C., McAdam, R. A. & Duncan, K. (2002). Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression profiling. Mol Microbiol 43, 717–731.[CrossRef][Medline]

Bi, E. & Lutkenhaus, J. (1990). FtsZ regulates frequency of cell division in Escherichia coli. J Bacteriol 172, 2765–2768.[Medline]

Bramhill, D. & Thompson, C. M. (1994). GTP-dependent polymerization of Escherichia coli FtsZ protein to form tubules. Proc Natl Acad Sci U S A 91, 5813–5817.[Abstract]

Brennan, P. J. & Nikaido, H. (1995). The envelope of mycobacteria. Annu Rev Biochem 64, 29–63.[CrossRef][Medline]

Dai, K. & Lutkenhaus, J. (1991). ftsZ is an essential cell division gene in Escherichia coli. J Bacteriol 173, 3500–3506.[Medline]

Datta, P., Dasgupta, A., Bhakta, S. & Basu, J. (2002). Interaction between FtsZ and FtsW of Mycobacterium tuberculosis. J Biol Chem 277, 24983–24987.[Abstract/Free Full Text]

Din, N., Quardokus, E. M., Sackett, M. J. & Brun, Y. V. (1998). Dominant C-terminal deletions of FtsZ that affect its ability to localize in Caulobacter and its interaction with FtsA. Mol Microbiol 27, 1051–1063.[CrossRef][Medline]

Dziadek, J., Madiraju, M. V., Rutherford, S. A., Atkinson, M. A. & Rajagopalan, M. (2002). Physiological consequences associated with overproduction of Mycobacterium tuberculosis FtsZ in mycobacterial hosts. Microbiology 148, 961–971.[Abstract/Free Full Text]

Erickson, H. P., Taylor, D. W., Taylor, K. A. & Bramhill, D. (1996). Bacterial cell division protein FtsZ assembles into protofilament sheets and minirings, structural homologs of tubulin polymers. Proc Natl Acad Sci U S A 93, 519–523.[Abstract/Free Full Text]

Gomez, J. E. & Bishai, W. R. (2000). whmD is an essential mycobacterial gene required for proper septation and cell division. Proc Natl Acad Sci U S A 97, 8554–8559.[Abstract/Free Full Text]

Greendyke, R., Rajagopalan, M., Parish, T. & Madiraju, M. V. (2002). Conditional expression of Mycobacterium smegmatis dnaA, an essential DNA replication gene. Microbiology 148, 3887–3900.[Abstract/Free Full Text]

Hale, C. A. & de Boer, P. A. (1997). Direct binding of FtsZ to ZipA, an essential component of the septal ring structure that mediates cell division in E. coli. Cell 88, 175–185.[Medline]

Hale, C. A., Rhee, A. C. & de Boer, P. A. (2000). ZipA-induced bundling of FtsZ polymers mediated by an interaction between C-terminal domains. J Bacteriol 182, 5153–5166.[Abstract/Free Full Text]

Kelly, A. J., Sackett, M. J., Din, N., Quardokus, E. & Brun, Y. V. (1998). Cell cycle-dependent transcriptional and proteolytic regulation of FtsZ in Caulobacter. Genes Dev 12, 880–893.[Abstract/Free Full Text]

Lowe, J. & Amos, L. A. (1998). Crystal structure of the bacterial cell-division protein FtsZ. Nature 391, 203–206.[CrossRef][Medline]

Lutkenhaus, J. & Addinall, S. G. (1997). Bacterial cell division and the Z ring. Annu Rev Biochem 66, 93–116.[CrossRef][Medline]

Ma, X., Ehrhardt, D. W. & Margolin, W. (1996). Colocalization of cell division proteins FtsZ and FtsA to cytoskeletal structures in living Escherichia coli cells by using green fluorescent protein. Proc Natl Acad Sci U S A 93, 12998–13003.[Abstract/Free Full Text]

Ma, X., Sun, Q., Wang, R., Singh, G., Jonietz, E. L. & Margolin, W. (1997). Interactions between heterologous FtsA and FtsZ proteins at the FtsZ ring. J Bacteriol 179, 6788–6797.[Abstract]

Madiraju, M. V., Qin, M. H. & Rajagopalan, M. (2000). Development of simple and efficient protocol for isolation of plasmids from mycobacteria using zirconia beads. Lett Appl Microbiol 30, 38–41.[CrossRef][Medline]

Margolin, W. (2000). Themes and variations in prokaryotic cell division. FEMS Microbiol Rev 24, 531–548.[CrossRef][Medline]

McCormick, J. R., Su, E. P., Driks, A. & Losick, R. (1994). Growth and viability of Streptomyces coelicolor mutant for the cell division gene ftsZ. Mol Microbiol 14, 243–254.[Medline]

Mukherjee, A. & Lutkenhaus, J. (1994). Guanine nucleotide-dependent assembly of FtsZ into filaments. J Bacteriol 176, 2754–2758.[Abstract]

Parish, T. & Stoker, N. G. (2000). Use of a flexible cassette method to generate a double unmarked Mycobacterium tuberculosis tlyA plcABC mutant by gene replacement. Microbiology 146, 1969–1975.[Abstract/Free Full Text]

Quardokus, E., Din, N. & Brun, Y. V. (1996). Cell cycle regulation and cell type-specific localization of the FtsZ division initiation protein in Caulobacter. Proc Natl Acad Sci U S A 93, 6314–6319.[Abstract/Free Full Text]

Snapper, S. B., Melton, R. E., Mustafa, S., Kieser, T. & Jacobs, W. R., Jr (1990). Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Mol Microbiol 4, 1911–1919.[Medline]

Triccas, J. A., Parish, T., Britton, W. J. & Gicquel, B. (1998). An inducible expression system permitting the efficient purification of a recombinant antigen from Mycobacterium smegmatis. FEMS Microbiol Lett 167, 151–156.[CrossRef][Medline]

Wang, X., Huang, J., Mukherjee, A., Cao, C. & Lutkenhaus, J. (1997). Analysis of the interaction of FtsZ with itself, GTP, and FtsA. J Bacteriol 179, 5551–5559.[Abstract]

Wang, Y., Jones, B. D. & Brun, Y. V. (2001). A set of ftsZ mutants blocked at different stages of cell division in Caulobacter. Mol Microbiol 40, 347–360.[CrossRef][Medline]

Ward, J. E., Jr & Lutkenhaus, J. (1985). Overproduction of FtsZ induces minicell formation in E. coli. Cell 42, 941–949.[Medline]

Wayne, L. G. & Hayes, L. G. (1996). An in vitro model for sequential study of shiftdown of Mycobacterium tuberculosis through two stages of nonreplicating persistence. Infect Immun 64, 2062–2069.[Abstract]

Received 26 September 2002; revised 4 March 2003; accepted 4 March 2003.



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