Microbiology (2003), 149, 1593–1603
DOI 10.1099/mic.0.26023-0
Conditional expression of Mycobacterium
smegmatis ftsZ, an essential cell division gene
Jaroslaw Dziadek,34 Stacey A. Rutherford,4 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
[email protected]
Received 26 September 2002
Revised
4 March 2003
Accepted 4 March 2003
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.
INTRODUCTION
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 rapidgrowing species with an average doubling time of 2–3 h, e.g.
Mycobacterium smegmatis. The cell division process in
3Present address: Centre for Microbiology & Virology, Polish Academy
of Sciences, Lodowa 106, 93-231 Łodz, Poland.
4These authors contributed equally to the work.
Abbreviations: DIC, differential interference contrast; DCO, doublecrossover; SCO, single-crossover; WT, wild-type.
0002-6023 G 2003 SGM
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 immunoelectron 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.,
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J. Dziadek and others
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).
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 PCRamplified products was confirmed by sequencing.
Construction of ftsZ expression plasmids
ftsZ from the ami promoter. The ftsZsmeg coding region was
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
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 mg ml21 each). Mycobacterial strains were
grown in Middlebrook 7H9 broth or 7H10 agar plates supplemented
with albumin-glucose and Kan (25 mg ml21) or hygromycin (Hyg)
(50 mg ml21). 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
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amplified using primers MVM240 (59-GCTCTAGAGTGCCGCATGAAGGGCGGC-39) and MVM174 (59-GCGGATCCATGACCCCCCCGCAC-39) 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 (59-GCTCTAGAAACAACAACCTGCAGATGAGTAAAGGAGAAG-39) and MVM189 (59-GCTCTAGAAACAACAACCTGCAGATGAGTAAAGGAGAAG-39) 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).
ftsZ from the dnaA promoter. The ftsZ coding region was amplified using MVM240 and MVM124 (59-GCGGATCCAATGACCCCCCCGCACAA-39), 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–ftsZ–orf 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 (59-CCCAAGCTTCCGCGCAACACGATCCG-39) and MVM305 (59-TGCTCTAGATCATCAGTGCCGCATGAAGGG-39) 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 39
end of the ftsZ gene (30 bp) and the downstream region was amplified
using the primers MVM270 (59-GACGATGTCGAAGCTTCGCCCTTCATG-39) and MVM271 (59-CGTAGTCATGGATCCGCGCCATGCC-39) 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 59 end of ftsZ was amplified using
MVM286 (59-GCTGGCAAAAGTACTCCCGCCGCGAGGCCAAGC-39)
and MVM287 (59-ACGGCCCACGTCAAGCTTGACGTCGGCG-39)
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 cm22 (UV Stratalinker 2400), and integrated into the
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Microbiology 149
Conditional expression of M. smegmatis ftsZ
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 59 to 39. (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 39 end of ftsZ.
Table 1. Plasmids
Plasmid
Description
Cloning vectors
pUC18
Used for cloning PCR products, Ampr
p2NIL*
Non-replicating recombination vector, Kanr
pGOAL17
Plasmid carrying PacI cassette, Kanr
pMV306d
L5 integration vector, Hygr
pJAM2D
Shuttle vector carrying inducible ami promoter, Kanr
Vectors used for M. smegmatis ftsZ gene replacement
pSAR29
1?7 kb HindIII/BamHI fragment including 39 end of ftsZ
and its downstream region in p2NIL, Kanr
pSAR30
1?2 kb ScaI/HindIII fragment carrying 170 bp of 59 ftsZ,
ftsQ–ftsZ intergenic region and ftsQ in pSAR29, Kanr
pSAR33*
pSAR30 with PacI cassette from pGOAL17, Kanr
Vectors used for FtsZ overproduction
pSAR16D
ftsZtb gene under the ami promoter, Kanr
pFR32D
ftsZtb gene under the dnaA promoter, Kanr
pSAR20d
amip–ftsZtb cloned in pMV306, Hygr
pSAR37d
ftsZp–ftsZsmeg and its 920 bp upstream region in pMV306, Hygr
pJfr39D
ftsZsmeg cloned into BamHI/HindIII sites of
pFR32 replacing the ftsZtb gene, Kanr
pJfr72D
ftsZsmeg cloned in pJAM2, Kanr
pJfr78d
HindIII/XbaI fragment from pJfr72 cloned in pMV306, Hygr
pJfr79D
gfp gene cloned into XbaI site of pJfr72 to make the
ftsZsmeg : : gfp C-terminal fusion, Kanr
Source
Lab. stock
Parish & Stoker (2000)
Parish & Stoker (2000)
MedImmune Inc.
Triccas et al. (1998)
This study
This study
This study
Dziadek et al. (2002)
Dziadek et al. (2002)
This study
This study
This study
This study
This study
This study
*Non-replicating plasmid.
DSelf-replicating plasmid.
dIntegrating plasmid.
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J. Dziadek and others
Table 2. M. smegmatis strains
Strain
Description*
mc2155
FZ1
FZ1-37
FZ1-20
FZ1-78
FZ2-37
FZ3-37
FZ2-20
FZ3-20
FZ2-78
FZ3-78
Laboratory strain
SCO strain
SCO strain integrated with pSAR37
SCO strain integrated with pSAR20
SCO strain integrated with pJfr78
DCO strain (WT) with pSAR37 integrated
DCO strain (mutant) with pSAR37 integrated
DCO strain (WT) with pSAR20 integrated
DCO strain (mutant) with pSAR20 integrated
DCO strain (WT) with pJfr78 integrated
DCO strain (mutant) with pJfr78 integrated
SCO, single-crossover; DCO, double-crossover.
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 mg ml21).
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.
Analysis of SCOs and DCOs. Both PCR and Southern hybridiza-
tion reactions were carried out to characterize SCOs and DCOs.
Primers MVM302 (59-GCGGATCCATGACCCCCCCGCATAAC-39,
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 (59-CCGCAGCCGAACGACCGAGC-39), 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).
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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 1006 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 mg ml21) and mithramycin A (180 mg ml21)
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
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 selfreplicating 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
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Conditional expression of M. smegmatis ftsZ
A
From plasmid
Integrated
_ +
_ +
_
_
_ +
_ +
1
3 4
5
6
7 8
9 10
2
Acetamide
B
(e)
(f)
(a)
(g)
(b)
(c)
(h)
(d)
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).
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).
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 %
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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 FtsZoverproducing conditions (data not shown). When cultures
induced with acetamide for 6 h were spread on acetamidefree 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
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DC
O
_
ftsZp ftsZsmeg
Mut
WT
DC
O
WT
B
SCO
A
1
2
3
4
C
Int ftsZ
WT ftsZ
Mut ftsZ
0.2% acetamide
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 (FZ337, Fig. 3B, lane 4) DCOs had FtsZ levels similar to the
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No acetamide
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, wildtype 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, sucroseresistant 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.
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).
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
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Microbiology 149
Conditional expression of M. smegmatis ftsZ
A
B
amip_ftsZsmeg
2
1
M
0
3
6
9
12
h
amip_ftsZtb
3
4
C
(a)
(b)
(g)
(e)
(i)
(c)
(f)
(d)
(h)
(j)
D
(a)
(c)
(e)
(d)
(f)
(b)
(g)
(h)
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).
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1599
J. Dziadek and others
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 mm in length (Fig. 5A) whereas 70 and
38 % of those grown in the absence of acetamide for 3
and 6 h, respectively, remained 2 mm 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 bulblike structures (Fig. 4C) indicative of cell division defects.
Similar structures were also seen under FtsZtb overproduction conditions (Dziadek et al., 2002). When 6 h acetamidestarved 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.
M. tuberculosis FtsZ protein can substitute for M. smegmatis
FtsZ protein. The M. smegmatis and M. tuberculosis FtsZ
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.
1600
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 FZ320 (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 mm, and the remainder were elongated (see Fig. 5A,
compare FZ3-20+ with FZ3-78+). When grown in broth,
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Microbiology 149
Conditional expression of M. smegmatis ftsZ
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
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 FtsZtbinitiated 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
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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 mm in length and 0?5 to 0?8 mm 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
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J. Dziadek and others
corresponds to small amounts of the newly synthesized
FtsZtb.
Brennan, P. J. & Nikaido, H. (1995). The envelope of mycobacteria.
Annu Rev Biochem 64, 29–63.
Dai, K. & Lutkenhaus, J. (1991). ftsZ is an essential cell division gene
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.
in Escherichia coli. J Bacteriol 173, 3500–3506.
Datta, P., Dasgupta, A., Bhakta, S. & Basu, J. (2002). Interaction
between FtsZ and FtsW of Mycobacterium tuberculosis. J Biol Chem
277, 24983–24987.
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.
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.
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.
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.
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.
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.
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.
Kelly, A. J., Sackett, M. J., Din, N., Quardokus, E. & Brun, Y. V.
(1998). Cell cycle-dependent transcriptional and proteolytic regula-
ACKNOWLEDGEMENTS
tion of FtsZ in Caulobacter. Genes Dev 12, 880–893.
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.
Lowe, J. & Amos, L. A. (1998). Crystal structure of the bacterial cell-
division protein FtsZ. Nature 391, 203–206.
Lutkenhaus, J. & Addinall, S. G. (1997). Bacterial cell division and
the Z ring. Annu Rev Biochem 66, 93–116.
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.
REFERENCES
Anilkumar, G., Chauhan, M. M. & Ajitkumar, P. (1998). Cloning and
Ma, X., Sun, Q., Wang, R., Singh, G., Jonietz, E. L. & Margolin, W.
(1997). Interactions between heterologous FtsA and FtsZ proteins at
expression of the gene coding for FtsH protease from Mycobacterium
tuberculosis H37Rv. Gene 214, 7–11.
the FtsZ ring. J Bacteriol 179, 6788–6797.
Anilkumar, G., Srinivasan, R., Anand, S. P. & Ajitkumar, P. (2001).
Madiraju, M. V., Qin, M. H. & Rajagopalan, M. (2000). Develop-
Bacterial cell division protein FtsZ is a specific substrate for the AAA
family protease FtsH. Microbiology 147, 516–517.
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.
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.
Bi, E. & Lutkenhaus, J. (1990). FtsZ regulates frequency of cell
division in Escherichia coli. J Bacteriol 172, 2765–2768.
Bramhill, D. & Thompson, C. M. (1994). GTP-dependent poly-
merization of Escherichia coli FtsZ protein to form tubules. Proc Natl
Acad Sci U S A 91, 5813–5817.
1602
ment of simple and efficient protocol for isolation of plasmids
from mycobacteria using zirconia beads. Lett Appl Microbiol 30,
38–41.
Margolin, W. (2000). Themes and variations in prokaryotic cell
division. FEMS Microbiol Rev 24, 531–548.
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.
Mukherjee, A. & Lutkenhaus, J. (1994). Guanine nucleotidedependent assembly of FtsZ into filaments. J Bacteriol 176,
2754–2758.
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.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 14 Jun 2017 14:46:19
Microbiology 149
Conditional expression of M. smegmatis ftsZ
Quardokus, E., Din, N. & Brun, Y. V. (1996). Cell cycle regulation
Wang, X., Huang, J., Mukherjee, A., Cao, C. & Lutkenhaus, J. (1997).
and cell type-specific localization of the FtsZ division initiation
protein in Caulobacter. Proc Natl Acad Sci U S A 93, 6314–6319.
Analysis of the interaction of FtsZ with itself, GTP, and FtsA.
J Bacteriol 179, 5551–5559.
Snapper, S. B., Melton, R. E., Mustafa, S., Kieser, T. & Jacobs, W. R., Jr
(1990). Isolation and characterization of efficient plasmid trans-
Wang, Y., Jones, B. D. & Brun, Y. V. (2001). A set of ftsZ mutants
formation mutants of Mycobacterium smegmatis. Mol Microbiol 4,
1911–1919.
blocked at different stages of cell division in Caulobacter. Mol
Microbiol 40, 347–360.
Ward, J. E., Jr & Lutkenhaus, J. (1985). Overproduction of FtsZ
Triccas, J. A., Parish, T., Britton, W. J. & Gicquel, B. (1998). An
induces minicell formation in E. coli. Cell 42, 941–949.
inducible expression system permitting the efficient purification of
a recombinant antigen from Mycobacterium smegmatis. FEMS
Microbiol Lett 167, 151–156.
Wayne, L. G. & Hayes, L. G. (1996). An in vitro model for sequential
http://mic.sgmjournals.org
study of shiftdown of Mycobacterium tuberculosis through two stages
of nonreplicating persistence. Infect Immun 64, 2062–2069.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
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1603