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
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ABSTRACT |
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Present address: Centre for Microbiology & Virology, Polish Academy of Sciences, Lodowa 106, 93-231
odz, Poland.
These authors contributed equally to the work.
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INTRODUCTION |
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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.
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METHODS |
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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 amipftsZsmeg; 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 amipftsZsmeggfp). This construct was used to produce the FtsZGFP 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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ftsZ from the native ftsZ promoter.
A BLAST search of 5 kb sequence containing M. tuberculosis murCftsQftsZorf 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 ftsZpftsZsmeg; 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, ftsQftsZ 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 ScaIHindIII 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 ftsZpftsZsmeg 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 amipftsZsmeg (FZ1-78) or amipftsZtb (FZ1-20) on plates containing 0·2 % acetamide.
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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 FtsZGFP 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.
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RESULTS |
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Filamentous cells contain multiple FtsZ rings
To evaluate the fate of the overproduced FtsZsmeg in elongated cells, amipftsZsmeggfp was expressed from a replicating plasmid (pJfr79) and the fusion protein visualized by microscopy. Since both ftsZsmeggfp and ftsZsmeg are expressed from the ami promoter, we assumed that comparable levels of FtsZsmegGFP and FtsZsmeg proteins are produced under our experimental conditions. Similar to amipftsZsmeg 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 FtsZsmegGFP bands at regularly spaced intervals, indicative of Z-ring-like structures (Fig. 2B
, g and h). Prolonged overproduction of FtsZsmegGFP resulted in the formation of fluorescent ribbon-like structures (data not shown), similar to those reported with the overproduction of E. coli FtsZGFP 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 ftsZpftsZsmeg 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. 4
A, compare lanes 1 and 2).
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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 12 µm in length (Fig. 5
A) 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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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. 12 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
).
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DISCUSSION |
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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 proteinprotein 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 3050 %, 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.
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ACKNOWLEDGEMENTS |
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Received 26 September 2002;
revised 4 March 2003;
accepted 4 March 2003.
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