Molecular Microbiology (2013) 87(5), 9981012
12146
ParA of Mycobacterium smegmatis co-ordinates chromosome
segregation with the cell cycle and interacts with the polar
growth determinant DivIVA
Katarzyna Ginda,1 Martyna Bezulska,2
Magorzata Zikiewicz,3 Jarosaw Dziadek,3
Jolanta Zakrzewska-Czerwinska1,2 and
Dagmara Jakimowicz1,2*
1
Faculty of Biotechnology, University of Wrocaw,
Wrocaw, Poland.
2
Ludwik Hirszfeld Institute of Immunology and
Experimental Therapy, Polish Academy of Sciences,
Wrocaw, Poland.
3
Medical Biology Institute, Polish Academy of Sciences,
Lodowa 106, 93-232 dz, Poland.
Summary
Mycobacteria are among the clinically most important
pathogens, but still not much is known about the
mechanisms of their cell cycle control. Previous
studies suggested that the genes encoding ParA and
ParB (ATPase and DNA binding protein, respectively,
required for active chromosome segregation) may
be essential in Mycobacterium tuberculosis. Further
research has demonstrated that a Mycobacterium
smegmatis parB deletion mutant was viable but
exhibited a chromosome segregation defect. Here,
we address the question if ParA is required for the
growth of M. smegmatis, and which cell cycle proc-
esses it affects. Our data show that parA may be
deleted, but its deletion leads to growth inhibition and
severe disturbances of chromosome segregation and
septum positioning. Similar defects are also caused
by ParA overproduction. EGFPParA localizes as
pole-associated complexes connected with a patch
of fluorescence accompanying two ParB complexes.
Observed aberrations in the number and positioning
of ParB complexes in the parA deletion mutant indi-
cate that ParA is required for the proper localization
of the ParB complexes. Furthermore, it is shown
that ParA colocalizes and interacts with the polar
growth determinant Wag31 (DivIVA homologue). Our
Accepted 24 December, 2012. *For correspondence. E-mail
dagmara.jakimowicz@uni.wroc.pl; Tel. (+48) 713752926; Fax (+48)
713752608.
2013 Blackwell Publishing Ltd
doi:10.1111/mmi.
First published online 28 January 2013
results demonstrate that mycobacterial ParA medi-
ates chromosome segregation and co-ordinates it
with cell division and elongation.
Introduction
Due to their enormous human health impact, bacteria
belonging to the genus Mycobacterium, particularly Myco-
bacterium tuberculosis, have mainly been studied in the
contexts of pathogenicity and infection. Critical for the
treatment of M. tuberculosis infection is the ability of these
bacteria to exist in a dormant, non-replicating state in which
they remain metabolically active and are able to return to
their normal cell cycle. Although this state is closely con-
nected with persistence of M. tuberculosis and lowered
susceptibility of these bacteria to drugs, particularly those
targeting cell division and cell wall synthesis, the mecha-
nisms controlling the cell cycle during dormancy have not
yet been fully explored (Wayne and Sohaskey, 2001). Until
now, studies on mycobacterial cell cycle processes have
largely focused on the main proteins involved in chromo-
some replication, cell division and cell elongation, i.e.
DnaA (Yamamoto et al., 2002; Zawilak et al., 2004;
Zawilak-Pawlik et al., 2005; Madiraju et al., 2006; Kumar
et al., 2009), FtsZ (Dziadek et al., 2002; 2003; Rajagopa-
lan et al., 2005a,b; Dziedzic et al., 2010) and the DivIVA
homologue, Wag31 (Nguyen et al., 2007; Kang et al.,
2008) respectively. Unlike in Bacillus subtilis, where DivIVA
interacts with MinC and MinD to control septum formation,
it is mainly responsible for controlling cell wall synthesis at
the cell poles in Mycobacterium (Nguyen et al., 2007; Kang
et al., 2008). Sequence analysis of the M. tuberculosis
(Hett and Rubin, 2008) and Mycobacterium smegmatis
genomes revealed a lack of genes encoding some cell
division regulators, such as FtsA and the Min system
proteins. Studies of the co-ordination of key steps of the
Mycobacterium cell cycle (i.e. chromosome replication and
segregation, cell division and elongation) should shed light
on persistence of tuberculosis infection.
Up to now the question of Mycobacterium chromosome
segregation has been addressed by the studies of segre-
gation proteins ParB (Jakimowicz et al., 2007a; Chaudhuri
and Dean, 2011) and ParA (Nisa et al., 2010), which are

components of the active chromosome segregation
machinery found in a number of bacterial species
(e.g. B. subtilis, Caulobacter crescentus, Vibrio cholerae,
Pseudomonas spp., Corynebacterium glutamicum, and
Streptomyces coelicolor). In all studied bacteria, ParB
homologues bind to a cluster of DNA sequences called the
parS sites, which range in number from 3 in V. cholerae
and C. glutamicum to about 20 in S. coelicolor and are
usually localized near oriC (Bartosik and Jagura-Burdzy,
2005). Upon binding to parS sites, ParB oligomerizes to
form large nucleoprotein complexes called segrosomes.
Segrosomes organize the oriC proximal region of newly
replicated chromosomes and facilitate their proper posi-
tioning in the future daughter cells, either at the cell centre
(in B. subtilis) or close to the pole(s) (in C. crescentus,
C. glutamicum and V. cholerae chromosome I) (Leonard
et al., 2005; Chaudhuri and Dean, 2011; Chaudhuri et al.,
2011). This process is followed by segregation and con-
densation of other regions of the chromosome (Glaser
et al., 1997; Fogel and Waldor, 2006; Donovan et al., 2010;
Shebelut et al., 2010).
The formation and positioning of ParB complexes
depends on ParA homologues. These are Walker
ATPases, whose activity is enhanced in the presence of
ParB. ParAs are able to dimerize and polymerize into
filaments in a process that is ATP binding-dependent and
requires the presence of DNA in some cases (e.g. Thermus
thermophilus) but not others (e.g. S. coelicolor) (Leonard
and Grimwade, 2005; Ditkowski et al., 2010). Most ParA
homologues, such as those in C. crescentus (Ptacin
et al., 2010), V. cholerae (Fogel and Waldor, 2006) and
C. glutamicum (Donovan et al., 2010), show dynamic
localization and extend from one pole towards the middle
of the cell; however, the B. subtilis Soj protein (ParA homo-
logue) seems to be associated with septa and with the
chromosomal origin regions (Murray and Errington, 2008).
ParA filaments undergo ParB-dependent polymerization/
depolymerization, and are believed to provide motive force
for segrosomes within bacterial cells. A growing body of
evidence suggests that ParA proteins link chromosome
segregation with other cellular processes, including the
initiation of chromosome replication in B. subtilis (Murray
and Errington, 2008; Scholefield et al., 2012) and V. chol-
erae (Kadoya et al., 2011), cell division in C. crescentus
and S. coelicolor (Mohl et al., 2001; Jakimowicz et al.,
2007b), polar growth in S. coelicolor (B. Ditkowski, submit-
ted) and motility in Pseudomonas aeruginosa (Lasocki
et al., 2007).
Even though the parA and parB genes were shown to be
essential only in C. crescentus, in most studied bacteria
elimination of the ParB protein results in the formation of
anucleate cells after division, at frequencies ranging from
1% in B. subtilis (Ireton et al., 1994) to 43% in a C. glutami-
cum parB deletion strain grown on minimal medium
2013 Blackwell Publishing Ltd, Molecular Microbiology, 87, 9981012
The role of ParA in Mycobacterium smegmatis 999
(Donovan et al., 2010). A transposon mutagenesis study
performed by Sassetti et al. (2003) indicated that the
parAB genes could not be deleted from M. tuberculosis. In
M. smegmatis, which is a relatively fast growing model
organism often used for studies on mycobacterial cell
biology, parB was found to be non-essential, but its deletion
resulted in chromosome segregation defects and gener-
ated 10% anucleate cells (Jakimowicz et al., 2007a). In
M. smegmatis, ParB was shown to bind three parS sites
localized around the oriC region in vivo and in vitro, and this
binding was enhanced in the presence of ParA (Jakimow-
icz et al., 2007a). Lowering the level of ParA was shown to
decrease the growth rate of M. smegmatis (Nisa et al.,
2010), but the function of ParA in the cell cycle of Myco-
bacterium has not yet been studied more extensively.
Here, the relative scarcity of knowledge of the mycobac-
terial cell cycle, particularly in terms of chromosome
dynamics and cell division, prompted us to address the role
of ParA in chromosome segregation and its co-ordination
with other cell cycle processes in M. smegmatis.
Results
In M. smegmatis, parA is non-essential but required for
normal growth
The work of Sassetti et al. (2003) suggested that ParA
could not be eliminated from M. tuberculosis H37Rv while
our previous studies indicated that Mycobacterium ParA
protein, as a component of the partitioning system, may
play an important role in the assembly of ParBDNA
complexes (Jakimowicz et al., 2007a). This notion and the
complex functions of ParA homologues in other bacteria
led us to examine the role of ParA during the cell cycle of
Mycobacterium. To analyse if parA (gene msmeg 6939) is
essential for the viability of M. smegmatis, a two-step
recombination protocol (Parish and Stoker, 2000) was
used to generate an unmarked deletion of the parA gene
in the M. smegmatis chromosome. The deletion was
constructed successfully and the obtained DparA strain
(KG22; Table 1) was verified by PCR, Southern blotting
(data not shown) and Western blotting (Fig. S1). RT-PCR
transcript analysis (data not shown) and Western blotting
confirmed that the level of ParB was only marginally
affected in the parA deletion strain (Fig. S1). Although parA
was found to be non-essential for the viability of M. smeg-
matis, growth rate analysis of the DparA strain in rich liquid
medium 7H9 + OADC (see Experimental procedures)
revealed that the lack of ParA substantially inhibited cell
growth (Fig. 1A and B). To verify if the observed growth
delay was solely dependent on parA deletion, a comple-
mentation strain was constructed. Since parA is believed to
be transcribed from its own promoter (Casart et al., 2008),
the parA gene and its promoter region (332 bp upstream of
1000 K. Ginda et al.

Fig. 1. Influence of ParA protein levels on the growth rate, cell morphology and chromosome segregation of M. smegmatis.
A and B. (A) Growth curves determined by optical density measurements and (B) representative CFU counts for particular culture time points
(optical density) of the wild type, parA deletion (KG22), parA complementation (KG23) (left panel), ParA overproduction (KG11) and control
strain (KG08) (right panel) grown in 7H9/OADC medium. Experiments were performed in triplicate; bars indicate standard errors. For the
parA-overexpressing KG11 strain and the control strain KG08, acetamide (0.1%) was added to growing cultures. Insets show colony
morphology of the wild type and the parA deletion strain (KG22) observed after 11 days of incubation on 7H10 + OADC plates at 37C; scale
bar: 1 cm.
C. Statistical analysis of cell length in the wild type, the parA deletion (KG22) and the parA-overexpressing (KG11; compared with the control
strain KG08, both grown in the presence of 0.1% acetamide) strain. In each case, cell length was measured for about 2000 cells.
D. Images of cells stained with DAPI (DNA), in Nomarski contrast, and overlaid images, as indicated. Anucleate cells are indicated by white
stars. Scale bar: 5 mm.

Table 1. Strains used in this study.

Strain Relevant genotype Source

E. coli
DH5a supE44DlacU169(f80lacZDM15)hsdR17 recA1 endA1 gyrA96 thi-1 relA1 Laboratory stock
BL21(DE3) F-, ompT, gal, dcm, lon, hsdSB(rB- mB-), l(DE3) Laboratory stock
BTH101 F-, cya-99, araD139, galE15, galK16, rpsL1 (Str r), hsdR2, mcrA1, mcrB1 Karimova et al. (2000)
M. smegmatis
WT M. smegmatis mc2 155 Laboratory stock
DparB M. smegmatis mc2 155 DparB Jakimowicz et al. (2007a)
KG02 M. smegmatis mc2 155 egfpparA This study
KG03 M. smegmatis mc2 155 DparAB This study
KG11 M. smegmatis mc2 155 attBL5:: pMV306pamiparA This study
KG13 M. smegmatis mc2 155 egfpparA, DparB This study
KG16 M. smegmatis mc2 155 parBmcherry This study
KG22 M. smegmatis mc2 155 DparA This study
KG23 M. smegmatis mc2 155 DparA attBL5:: pMV306pnatparA This study
KG29 M. smegmatis mc2 155 egfpparA, parBmcherry This study
KG37 M. smegmatis mc2 155 attBL5:: pMV306pnatdivIVAmcherry This study
KG40 M. smegmatis mc2 155 DparA, parBmcherry This study
KG56 M. smegmatis mc2 155 DparA attBL5:: pMV306pnategfpparAS This study

the start codon) were cloned into vector pMV306. In the
complementation strain, KG23 (Table 1), the obtained
plasmid pMV306pnatparA was integrated into the phage L5
attachment site of the parA deletion strain (KG22). The
growth curve of complementation strain KG23 did not differ
significantly from that of the wild-type strain (Fig. 1A),
proving that the growth defect observed in KG22 cells
resulted solely from deletion of parA.
Elimination of ParA affects chromosome segregation
and cell length
To study the influence of ParA on chromosome segrega-
tion, the parA deletion strain (KG22) was examined micro-
scopically. DNA staining (DAPI) of cells collected in the
exponential growth phase showed a larger fraction of
arisen from the insertion of an additional parS site within
the parA gene delivered in trans at the phage L5 attach-
ment site, which is far from the oriC region.
Interestingly, microscopic analysis of the parA deletion
strain revealed an increased variation in cell length. There
was a substantial fraction of shortened, usually anucleate
cells and a large number of elongated cells compared to
the wild type [of ~ 2000 cells analysed for each strain,
33% of DparA (KG22) and 16% of wild-type cells were
longer than 6 mm; Fig. 1C].
Table 2. Frequencies of anucleate cells in various M. smegmatis
strains observed under the microscope (DAPI staining).
% of anucleate cells
(~ 2000 cells counted)

anucleate cells for DparA (30.5% of 2162 cells analysed) M. smegmatis mc2 155 0.2%
compared to the wild type (0.2% of 2013 cells analysed) DparA (KG22) 30.5%
DparB 11.3%
(Table 2, Fig. 1D). In the complementation strain (KG23), DparAB (KG03) 1.1%
less than 4% of cells were anucleate (2118 cell analysed; DparA/pnatparA (KG23) 3.8%
Table 2, Fig. 1D), confirming that the severe segregation pamiparA (KG11) 9.2%
pMV306 (KG08) 0.3%
defect in KG22 was due to elimination of ParA. The slight
missegregation in the complementation strain may have

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2013 Blackwell Publishing Ltd, Molecular Microbiology, 87, 9981012

1002 K. Ginda et al.

These results indicated that, compared to the elimina-

tion of ParB (10% anucleate cells; Jakimowicz et al.,
2007a), elimination of ParA has more severe effects on
chromosome segregation in M. smegmatis. Moreover,
they revealed that ParA elimination also influenced cell
length of M. smegmatis.

ParA overproduction has phenotypic effects similar to

those of parB deletion

Our earlier studies showed that both elimination and over-

expression of parB reduced the growth rate of M. smeg-
matis, but overexpression did not affect chromosome
segregation (Jakimowicz et al., 2007a). This could
suggest that proper maintenance of the ratio between
ParA and ParB is important for the cell cycle of M. smeg-
matis. Therefore, we investigated whether the overex-
pression of parA could affect growth and/or chromosome
segregation in M. smegmatis. An additional copy of the
parA gene under the control of the inducible pami promoter
in the pMV306 vector was integrated into the chromo-
some of the wild-type strain to yield the KG11 strain
(Table 1). The induction (with 0.1% acetamide) of parA
expression in KG11 elevated the protein level of ParA to
approximately 20-fold that seen in the wild-type strain, as
estimated by Western blotting (Fig. S1). parA overexpres-
sion led to growth inhibition (Fig. 1A) and chromosome
missegregation (9.3% anucleate cells; Table 2), which
was similar to the defects observed for the parB deletion
strain. Additionally, we observed an increased variation in
cell length (Fig. 1C); 46.3% of ParA-overproducing cells
were longer than 6 mm compared to only 24% of cells in
the control strain (KG08 harbouring insert-free pMV306
grown on acetamide, or the strain expressing mcherry Fig. 2. The influence of ParA on septum positioning in
from the pami promoter in pMV306; data not shown). Thus, M. smegmatis.
A. Examples of wild-type and DparA cells stained with
parA overexpression affects chromosome segregation in
Vancomycin-BODIPY (newly synthetized peptidoglycan) and
a manner similar to ParB elimination, and also influences FM4-64 (membrane). Scale bars: 5 mm. White stars indicate septa.
cell length. B. Septum positioning in relation to % of cell length in the parA
deletion strain compared to the wild type (top panel), and in the
parA-overexpressing strain compared to control strain KG08, both
grown in presence of 0.1% of acetamide (bottom panel). In each
Deletion and overexpression of parA affect cell division
case, the distance from the septum to the closest pole was
measured; 400 cells of each strain were analysed.
The high variation of cell length in M. smegmatis strains
lacking or overexpressing parA could be the result of
impaired cell elongation or division (e.g. changes in the Septum position measurements showed that the localiza-
positioning and/or timing of septation). Moreover, distur- tion of the septum was highly variable in the DparA strain,
bances in cell division in the parA mutant could account whereas septa were typically localized within 4060% of
for its growth inhibition and severe chromosome misseg- the cell length in the wild type (Fig. 2B).
regation phenotypes, which exceeded the segregation Septum position was also analysed in the parA-
defects caused by parB deletion. In order to test the overexpressing strain (KG11) and compared to the control
influence of ParA on septum formation, parA deletion strain (containing an insert-free integrative pMV306
(KG22) and wild-type cultures were harvested at the plasmid; KG08). Both strains were grown in the presence
same point of exponential growth phase (OD = 0.4) and of 0.1% acetamide, and samples were prepared for micro-
subjected to cell wall and membrane staining (with scopy using cultures from the exponential phase of
Vancomycin-BODIPY and FM4-64 respectively) (Fig. 2A). growth (OD = 0.4). Septum positioning was found to be

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 The role of ParA in Mycobacterium smegmatis 1003

disturbed in KG11 cells compared to KG08 cells (Fig. 2B),

albeit not as severely as in the parA deletion strain.
Thus, the modifications of ParA level appear to affect
also the positioning of the septum and possibly the timing
of its formation which may be the result of defective chro-
mosome organization.

In parA deletion mutants, the segregation phenotype is

suppressed by parB deletion, whereas the other
defects are not
To examine which of the defects observed in the parA
deletion strain are dependent on ParB, a double-deletion
strain was constructed. The DparAB strain, KG03
(Table 1), was constructed by two-step recombination and
verified by PCR, Southern blotting (data not shown) and
Western blotting (Fig. S1). The growth of the DparAB
strain was impaired, although the CFU (colony-forming
unit) count showed that this impairment was not as severe
as that of the parA deletion strain (Fig. 3A and B). Sur-
prisingly, microscopic analysis showed that the fraction of
anucleate cells in the DparAB strain was very low (1.2% of
2014 cells counted; Table 2). Thus, the chromosome seg-
regation defect in the DparAB strain was diminished in
comparison to the parA or parB single-deletion strain
(30% and 10% respectively). In the DparAB strain aber-
rations of cell length were also observed (Fig. 3C); 26% of
cells were longer than 6 mm. Cell length abnormalities
were only slightly reduced in comparison to the parA
deletion strain (33% cells were longer than 6 mm) and still
noticeable in comparison to the control strain (16%), indi-
cating that the effect of ParA on cell length may be inde-
pendent of ParB.
Thus, surprisingly, although the parA phenotype
exceeded the parB phenotype in terms of segregation
defects, parB deletion suppressed segregation defects
caused by the elimination of ParA, but did not significantly
Fig. 3. Growth rates and cell length of M. smegmatis mutant
alter the other defects, such as disturbed cell length. strains.
A and B. (A) Growth curves and (B) CFU counting at particular
EGFPParA forms pole-associated complexes with a culture time points of the wild-type, DparA (KG22), DparB and
DparAB (KG03) strains grown in 7H9 + OADC medium.
patch of fluorescence in between Experiments were performed in triplicate; bars indicate standard
errors.
To examine subcellular localization of ParA in M. smeg- C. Cell length analysis of the wild-type, DparA (KG22), DparB and
matis cells, strain KG02 was constructed, in which parA DparAB (KG03) strains; for each strain 2000 cells containing DNA
was replaced with an egfpparA fusion at the original were taken into account.
locus (Table 1). Western blotting demonstrated that the
expression level of the EGFPParA fusion protein was the egfpparA fusion gene). The fusion protein did not
similar to that of ParA in the wild-type strain (Fig. S1), disturb the growth or chromosome segregation of KG02
but it also showed that in addition to EGFPParA the cells (1.2% anucleate). Additionally, a new strain was con-
wild-type ParA protein was also present in KG02. The structed (KG56), in which the pMV306 plasmid containing
observed expression of a native parA gene may result egfp fused to the downstream start codon of parA,
from start codon misannotation (the alternative start expressing EGFPParAS, was integrated into the chromo-
codon for parA gene is located 102 bp downstream of the some of the parA deletion strain (Table 1). In the strain
annotated start codon, and may be the start of translation KG56 no wild-type ParA could be detected (Fig. S1) and
due to the presence of an intact ribosome binding site in EGFPParAS was fully functional as demonstrated by the

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1004 K. Ginda et al.

Fig. 4. Localization of EGFPParA in M. smegmatis cells. Cells were grown to the exponential phase in 7H9 + OADC medium at 37C.
A. Representative images of M. smegmatis cells expressing egfpparA. Left panel represents a schematic of EGFPParA localization and the
percentage of cells with each observed localization pattern. A minor fraction of cells (about 1%) showed multiple ParA complexes. Scale bars:
5 mm.
B and C. (B) Fluorescence intensity profiles along cells and (C) differences of foci intensities in cells with two polar EGFPParA complexes.
The intensity value of the fainter focus was shown as the percentage of that of the brighter focus (100% intensity); 1218 cells were studied.
D. Histogram showing the position of the middle complex in relation to the closest pole (in 235 cells, each with three EGFPParA foci).
E. Length of M. smegmatis cells with one, two or three EGFPParA foci. Inset shows the percentage of cells longer than 6 mm.
For A and E, a total of 5461 cells were analysed.

complementation of the parA deletion phenotype (1.2%
anucleate cells). Microscopic analyses performed on live
cells collected from liquid 7H9 + OADC cultures and
re-suspended in PBS revealed that the localization pat-
terns of EGFPParA and EGFPParAS were identical.
Both proteins were visible as foci at the cell pole(s), with
extended patches of diffuse fluorescence (Figs 4A, B and
S2). In the majority of the cells (76% of 5461 and 84% of
1020 cells analysed in strains KG02 and KG56 respec-
tively), intensive fluorescence was visible at both poles.
The other cells contained either only a single complex at
one pole or three foci. The additional ParA complex in the
middle was mostly visible in cells longer than 6 mm
(average cell length of 5 mm) (Fig. 4A and E). Measure-
ments of the fluorescence intensity of EGFPParA indi-
cated that cells with bipolar complexes frequently had
unequal fluorescence intensities at the two poles; only
27% of cells (1218 KG02 cells analysed; Fig. 4C) showed
identical intensities at both poles. To address the question
if the localization of ParA complexes is affected by the
elimination of ParB, strain KG13 was constructed to
express egfpparA in the parB deletion background
(Table 1). The level of EGFPParA in KG13 was similar to
that in KG02, and the distribution of ParA complexes was
not visibly affected (data not shown). However, still
images analysis does not fully address the question of
ParA dynamics which may be affected in the DparB strain.
In summary, our data demonstrate that ParA is associated
with the poles, while the presence of diffuse fluorescence
between the poles may indicate a dynamic pattern of
localization.
ParA affects the number and positioning of
ParB complexes
In most studied bacteria, ParA has been shown to affect
the assembly and/or positions of ParB complexes. Our
observation that the parA segregation phenotype was
suppressed by parB deletion suggested that formation of
ParB complexes in the absence of ParA severely disturbs
chromosome segregation in Mycobacterium. To investi-
gate this further, we compared the localizations of ParB in
the presence and absence of ParA. For this purpose, we
constructed strain KG29 (expressing egfpparA and
parBmcherry) and two strains expressing parBmcherry
from the original locus in the wild-type (KG16) or parA

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 The role of ParA in Mycobacterium smegmatis 1005

Fig. 5. Influence of ParA elimination on ParBmCherry localization in M. smegmatis cells.

A. Example of ParBmCherry localization in egfpparA (KG29) and DparA (KG40) strains. White stars indicate ParBmCherry foci. Scale bars:
5 mm.
B. Occurrence of cells with a particular ParBmCherry localization. A total of 542 (egfpparA parBmcherry) and 204 (DparA parBmcherry)
cells were examined.
C. Positions of pairs of ParBmCherry foci (violet and yellow) in the wild-type (top panel) and DparA (bottom panel) cells with two
ParBmCherry complexes; 108 cells of each strain were analysed.

deletion (KG40) backgrounds (Table 1) and verified them
as described above. Fluorescent fusions of ParA and
ParB were functional, as judged by the low fraction of
anucleate or elongated cells for the modified strains (data
not shown). Microscopic analysis of the egfpparA parB
mcherry expressing strain (KG29) revealed that in the
majority of cells (80% of 542 cells analysed, Fig. 5A and
B), bipolar ParA fluorescence was accompanied by two
ParB complexes, which were positioned at 2025% and
7580% of the cell length (Fig. 5C). Only a minor fraction
(9% of cells) contained only one ParB complex close to or
slightly shifted from the cell centre or more than two ParB
complexes (3% of cells) (Fig. 5B). In 9% of cells, three
ParA complexes were accompanied by more than two
ParB foci. The third ParA complex was positioned in
the middle of these cells, which were elongated (longer
6 mm), suggesting that they were undergoing division. The
increased number of ParB foci and their positions in these
cells indicated that a new round of chromosome replica-
tion followed by segregation had begun, and ParB com-
plexes in one or both of the daughter cells had already
doubled. The pattern of ParB localization in cells of the
parBmcherry strain (KG16) was almost identical to that
in cells of the egfpparA parBmcherry strain (KG29),
indicating that the positioning of ParB complexes was not
affected by the expression of EGFPParA.
To study the influence of ParA on the localization of ParB,
KG40 (DparA parBmcherry; Table 1) cells were studied
microscopically. ParB complexes were less intense and
more varied in number in cells of the DparA parBmcherry
strain (KG40) compared to the parBmcherry control strain
(KG16) (Fig. 5A). Statistical analysis showed that 51.7% of
DparA parBmcherry cells (of 204 analysed) contained two
ParB complexes, and only 21.2% contained one ParB
complex, whereas 27.1% of cells contained three or
more ParB complexes (Fig. 5B). Whereas wild-type cells
showed a maximum of five ParB complexes, parA deletion
cells contained up to seven ParB foci. This difference was
not due to a change in the level of ParBmCherry, since the
levels of ParBmCherry were similar in the DparA parB

2013 Blackwell Publishing Ltd, Molecular Microbiology, 87, 9981012

1006 K. Ginda et al.

mcherry (KG40), egfpparA parBmcherry (KG29) and

parBmcherry (KG16) strains (Fig. S1). Additionally, the
distribution of ParB complexes in the parA deletion strain
differed from that observed in KG16 (parBmcherry) cells.
The ParB foci were not confined to positions at 20% and
80% of the cell length (as in the wild type); instead, they
were rather randomly distributed along the long axis of the
cell (Fig. 5A and C). This pattern was not observed in the
control strains, where the ParB foci were always close to
the cell axis. Thus, the number and positioning of ParB
complexes was found to be highly disturbed in cells lacking
ParA.

Mycobacterium smegmatis ParA colocalizes with the

polar growth determinant Wag31 (DivIVA)
The polar localization of ParA resembled that described
previously for the growth determinant Wag31 (a DivIVA
homologue, called further DivIVA) in M. smegmatis
(Nguyen et al., 2007; Kang et al., 2008), suggesting
that the proteins may colocalize. To address this ques-
tion, we constructed strain KG37, in which vector
pMV306pnatdivIVAmcherry (expressing the divIVA
mcherry gene under the control of the divIVA promoter)
was integrated into the chromosome in the KG02 (egfp
parA) background (Table 1). Microscopic analysis of the
divIVAmcherry egfpparA strain (KG37) revealed that
ParA complexes usually overlapped with the DivIVA foci.
Interestingly, a large fraction of KG37 cells showed bulging
poles and branching. This could be due to the overexpres- Fig. 6. Colocalization of EGFPParA and DivIVA (Wag31).
A. Representative images of M. smegmatis cells expressing
sion of divIVA via the additional copy of the divIVA gene egfpparA and divIVAmcherry. Scale bar: 5 mm.
(Nguyen et al., 2007). In about half of the cells (48%, 514 B. Occurrence of cells with a particular DivIVA and ParA
cells counted) the fluorescence signals of DivIVAmCherry localization. A total of 514 cells were examined.
and EGFPParA were equally distributed between the
poles. At the bulged poles of asymmetric cells (52% of
obtained for constructs expressing C-terminally truncated
cells), however, DivIVA complexes were enlarged and
(lacking the C-terminal coiled-coil domain IV) and N-
usually accompanied by more intensive ParA fluorescence
terminally truncated (lacking the N-terminal coiled coil)
(Fig. 6). These data suggest that ParA not only colocalizes
DivIVA, as well as for a version of the protein lacking the
with DivIVA, but that its localization may be influenced by
variable region between the coiled-coil domains (Fig. 7A).
DivIVA.
From these results, we conclude that fragments of both
coiled-coil regions of DivIVA are involved in the interaction
ParA interacts with DivIVA
with ParA.
Our microscopic studies demonstrated that ParA and In order to confirm the ParADivIVA interaction identified
DivIVA colocalize in M. smegmatis cells, leading us to in the BTH studies, pull-down assays were applied. GST
speculate that they could interact. To verify this notion, a DivIVA was purified from lysates of Escherichia coli BL21
bacterial two-hybrid (BTH) system was applied (Karimova (DE3) cells containing pGEX6P-2divIVA, and then mixed
et al., 2000). Both N-terminal and C-terminal fusions of with purified ParA protein (10 mg of protein per extract from
M. smegmatis ParA and DivIVA with the T18 and T25 100 ml of culture). To exclude contamination of ParA with
domains of adenylate cyclase (CyaA) were studied. A GSTParA and unspecific binding of ParA to the chroma-
positive signal was observed only when ParA was tography resin, a control experiment was performed in
N-terminally fused. To pinpoint the interaction interface in which ParA was added to lysates from E. coli BL21 (DE3)
DivIVA, an extended BTH analysis was performed using cells containing the empty pGEX6P-2 vector expressing
pUT18C and pKT25 constructs containing fragments of the GST protein. In an additional control experiment, no
the divIVA gene (Fig. 7A and B). Positive signals were ParA protein was added to E. coli BL21 (DE3) pGEX6P-

2013 Blackwell Publishing Ltd, Molecular Microbiology, 87, 9981012


2divIVA cell extracts. Western blotting performed using
anti-ParA antibodies detected the protein solely in fractions
containing resin-bound GSTDivIVA (Fig. 7C).
In summary, the results of the pull-down assay con-
firmed the ability of M. smegmatis ParA to interact with
DivIVA.
Discussion
In all bacteria studied to date, the ParA proteins primarily
function in active chromosome segregation. However,
these proteins are also engaged in other cellular proc-
esses, including the regulation of chromosome replication
(Murray and Errington, 2008; Scholefield et al., 2012) and,
indirectly, cell division (Easter and Gober, 2002). It seems
likely, therefore, that these proteins may co-ordinate chro-
mosome segregation with specific cell cycle processes.
Here, we investigated this possibility by studying the func-
tions of ParA in Mycobacterium.
Sassetti et al. (2003) suggested that the parA gene may
be essential in M. tuberculosis, while Nisa et al. showed
that lowering the level of ParA inhibited the growth of
M. smegmatis (Nisa et al., 2010). Our present results show
that the M. smegmatis parA deletion strain is viable but
shows an altered morphology and slower growth, which
confirms the importance of ParA for proper cell cycle
progression.
In M. smegmatis, we found that elimination of ParA leads
to chromosome segregation defects; this is consistent with
findings in the other bacteria studied to date, but the
2013 Blackwell Publishing Ltd, Molecular Microbiology, 87, 9981012
The role of ParA in Mycobacterium smegmatis 1007
Fig. 7. Examination of ParADivIVA
(Wag31) interactions in vivo and in vitro.
A. Bacterial two-hybrid analysis of the
interaction of ParA with DivIVA and its
truncated derivatives (Dd abbreviation for
DivIVA domain). Symbols: ***, ** and *
indicate strong, moderate and weak
interactions respectively.
B. Diagram of DivIVA structure showing
the conserved N-terminus, coiled-coil
structure and M. tuberculosis
phosphorylation site. Selected domains of
DivIVA (Dd-Roman numerals) were
cloned into bacterial two-hybrid system
vectors.
C. GST pull-down assay. GSTDivIVA or
GST were purified using GSH-Sepharose
from E. coli BL21 (DE3) lysates
containing ParA (except for negative
control, panels 2 and 4 from the left side).
Proteins bound to GSH-Sepharose were
analysed by Western blotting using an
anti-ParA antibody. Lanes: 1 proteins
not bound to resin during incubation; 2
and 3 elution fractions.
fraction of anucleate cells is remarkably high in M. smeg-
matis cells lacking ParA (30%). Our observation that ParB
complexes are mislocalized in the parA deletion strain
suggests that the segregation defect is a direct conse-
quence of disturbances in the numbers and positions of
ParB complexes. In wild-type cells, ParB forms one or two
distinct complexes positioned at 20% and 80% of the cell
length. Random positioning of ParB foci within parA-
deleted cells suggests that ParA ensures the attachment
and/or movement of these foci from the centre to the poles.
This function has previously been attributed to ParAs in
other bacteria, such as V. cholerae (Fogel and Waldor,
2006) and C. crescentus (Ptacin et al., 2010). Our obser-
vation that the elimination of ParA leads to more pro-
nounced segregation defects than those seen with deletion
of parB may indicate that either chromosome segregation
is more severely perturbed by mislocalized (rather than
absent) ParB complexes, or other function(s) of ParA
contribute to the observed disorders. Similarly, differences
between the segregation phenotypes of the parA and parB
deletion strains have been observed in S. coelicolor (26%
of DparA spores were anucleate versus 17% of DparB
spores), C. glutamicum (16% of DparA cells were anucle-
ate versus 11% of DparB cells) (Jakimowicz et al., 2007b;
Donovan et al., 2010) and Pseudomonas (Lewis et al.,
2002).
Overproduction of ParA leads to the formation of about
10% of anucleate cells, which is similar to the phenotype
seen for parB deletion strains. Interestingly, the segrega-
tion phenotype is suppressed in parAB deletion mutant,
1008 K. Ginda et al.
supporting the notion that the presence of mislocalized
segrosomes is particularly deleterious. The lack of a seg-
regation phenotype in the parAB deletion strain could also
be the result of an elevated copy number of the chromo-
some, which might ensure random segregation sufficient
to provide necessary genetic material to daughter cells.
The increased number of chromosomes was detected
using the FROS method, in both the parA and the parB
deletion strains (I. Santi, pers. comm.). This and the
increased number of ParB complexes in the M. smegma-
tis parA deletion strain may be the result of missegrega-
tion. It may also suggest that ParA may be involved in
regulating DNA replication, as has been shown for ParA
proteins in B. subtilis and V. cholerae (Murray and Err-
ington, 2008; Kadoya et al., 2011). The mechanism of
such a regulation could be analogous to that observed in
B. subtilis, in which a direct interaction between ParA and
the replication initiator protein, DnaA, has been reported
(Murray and Errington, 2008; Scholefield et al., 2012).
Although no interaction between DnaA and ParA has yet
been detected in mycobacteria, the involvement of the
ParA protein in the regulation of replication cannot be
excluded. In addition to observing an increased number of
ParB foci in the parA deletion strain, we also found that
their fluorescence intensities were lower. An earlier study
(Jakimowicz et al., 2007a) showed that ParA facilitates
formation of ParB complexes in vitro. Thus, it is likely that
ParB complexes are not properly assembled in the parA
deletion strain. Another possible explanation for the weak
fluorescence intensity could be a lowered availability of
ParB molecules for complex formation.
The other phenotypic defect observed for the parA dele-
tion strain was aberrant septum placement. An influence of
ParA on septum positioning has previously been observed
in other bacteria, such as S. coelicolor (Jakimowicz et al.,
2007b). Aberrant septation may be a direct consequence
of chromosome mislocalization and/or missegregation,
and the disturbance of ParB complex positioning in the
parA deletion background is likely to be connected with
altered chromosome organization and/or replication. The
presence of a mechanism for co-ordinating septum place-
ment with chromosome positioning could explain this
aspect of parA deletion phenotype. In addition, the
observed parA overexpression phenotype is consistent
with the notion of a direct link between septum placement
and segregation, since both septum mispositioning
and segregation defects were moderate in parA-
overexpressing cells. Until now, however, there has not
been any evidence of proteins playing roles equivalent to
the Min system or nucleoid occlusion in Mycobacterium
(Hett and Rubin, 2008). Since mycobacteria contain other
ParA-like proteins, it is also possible that a ParA paralogue
which interacts with ParB to regulate cell division (as is the
case in C. glutamicum and C. crescentus) becomes mis-
2013 Blackwell Publishing Ltd, Molecular Microbiology, 87, 9981012
placed if chromosome segregation fails to proceed prop-
erly in the absence of ParA. This would be reminiscent of
the function of C. crescentus MipZ which is also a MinD
homologue and a ParB interacting protein. MipZ is postu-
lated to be a checkpoint that co-ordinates assembly of the
divisome by control of FtsZ polymerization with the onset of
DNA replication (Kiekebusch et al., 2012). Another possi-
ble, although less likely, explanation for irregular septation
is that ParA may exert direct control on septum positioning.
The ParA-mediated direct control of septum positioning
could lead to the formation of both short (often anucleate)
and elongated cells, contributing to the severe segregation
phenotype observed for the parA deletion strain. The
alteration of cell length observed in parA mutant strains
(both deletion and overexpression) may also result from a
disturbance in the co-ordination between elongation and
the timing of cell division. An influence of ParA on the cell
length has also been observed in other bacteria, including
C. glutamicum (Donovan et al., 2010) and P. putida
(Godfrin-Estevenon et al., 2002), and deletion of parA in
S. coelicolor yielded spores of uneven length (Jakimowicz
et al., 2007b).
In most M. smegmatis cells, we observed that EGFP
ParA localizes at the poles with the visible patch of fluo-
rescence in between, while longer cells (probably dividing
cells) often have an additional complex in the middle of
the cell. The localization of ParA suggests that it is asso-
ciated with the septum during division. A similar septal
localization of ParA was previously observed in B. subtilis
(Murray and Errington, 2008). The patch of diffuse fluo-
rescence observed between the EGFPParA complexes
may indicate that ParA has dynamic properties in
M. smegmatis cells. Many other ParA homologues, such
as those in V. cholerae, C. glutamicum and C. crescentus,
exhibit dynamic localization patterns, forming filamentous
structures that extend from the pole in a manner depend-
ent on their ATPase activity and interaction with ParB
(Fogel and Waldor, 2006; Donovan et al., 2010; Ptacin
and Shapiro, 2010). The dynamic properties of M. smeg-
matis ParA are subject of further analyses.
The polar localization of EGFPParA prompted us to
speculate that it may interact with a homologue of the pole
determinant DivIVA (called Wag31) in M. smegmatis, and
our studies confirmed that these proteins colocalize and
interact. A previous study showed that DivIVA homologues
in actinomycetes are required for polar peptidoglycan
sythesis and cell elongation, and overproduction of DivIVA
results in overgrowth of one of the cell poles, while its
depletion leads to the formation of rounded M. smegmatis
cells (Scherr and Nguyen, 2009). An increased amount of
ParA was associated with the enlarged DivIVA complexes
(possibly due to some disfunction of the DivIVAmCherry
fusion protein), suggesting that localization of ParA
depends on DivIVA.

Further studies of DivIVAParA interaction suggested
that the C-terminal domain of ParA may be responsible for
binding to DivIVA (the C-terminal fusions of ParA did not
interact in BTH experiments), while the interaction inter-
face in DivIVA is probably formed by the coiled-coil
regions flanking the variable third domain, which itself is
not required for interaction. Interestingly, the coiled-coil
domain II is longer in Mycobacterium DivIVA compared
to the other DivIVA homologues, and is followed by a
long third domain that contains a phosphorylation site in
M. tuberculosis (Nguyen et al., 2007). Phosphorylation
of M. tuberculosis DivIVA by PknA/B serinethreonine
kinase(s) is growth phase-dependent and may affect the
function of DivIVA, as mutations of the phosphorylation
site trigger alterations in cell shape (Nguyen et al., 2007).
This observation opens the interesting possibility that
phosphorylation may affect the affinity of M. smegmatis
DivIVA towards ParA by changing the conformation of the
interaction interface. The interaction of the segregation
machinery with pole determinants has been recently sug-
gested to exist in other actinomycetes. In C. glutamicum,
for example, ParB is recruited to the cell poles via an
interaction with tip-anchored DivIVA (Donovan et al.,
2012), while in S. coelicolor, ParA interacts with another
tip-associated protein, Scy (B. Ditkowski, submitted). The
tip anchorage of ParA is not specific to actinomycetes; it
has also been identified in C. crescentus, mediated
through association with the polar protein TipN (Ptacin
et al., 2010). Interestingly, the pole-associated DivIVA of
B. subtilis interacts with MinD, which belongs to the same
family as ParA (Marston et al., 1998). The identified inter-
action between ParA and DivIVA in M. smegmatis may
therefore contribute to the complex phenotype of parA
deletion strain, especially the observed alterations in cell
length. This notion critically contributes to the growing
body of evidence that bacterial ParA homologues are
associated with cell division and/or cell elongation.
In summary, deletion of parA in M. smegmatis yields a
complex phenotype that may be associated with the mul-
tifunctional segregation machinery and is accompanied
by disturbed cell division/elongation control. A presumed
similar function of ParA in co-ordinating the cell cycle in
M. tuberculosis and the fact that it is postulated to be
essential together suggest that ParA and (more precisely)
the ParADivIVA interaction may be potential drug targets
for the treatment of tuberculosis.
Experimental procedures
DNA manipulation, bacterial strains and
growth conditions
DNA manipulations were carried out using standard protocols
(Sambrook et al., 1989). Enzymes were supplied by Fermen-
tas, and oligonucleotides were obtained from Genomed. All
2013 Blackwell Publishing Ltd, Molecular Microbiology, 87, 9981012
The role of ParA in Mycobacterium smegmatis 1009
PCR-derived clones were confirmed by DNA sequencing.
E. coli BL21 (DE3) was used as the host for overproduction of
fusion proteins. E. coli strains were grown in LuriaBertani
(LB) medium at 37C. M. smegmatis mc2 155 and its deriva-
tives were grown in Middlebrook 7H9 medium and on 7H10
agar plates supplemented with OADC (oleic acid, albumin,
glucose, sodium chloride; Difco), in NB broth [8.0 g l-1 nutrient
broth (Difco), 10.0 g l-1 glucose and 0.2% Tween 80, pH 6.0
6.2] or on NB agar plates. For selection, kanamycin
(25 mg ml-1) was used as needed. Acetamide (final concen-
tration 0.1%) was used to induce ParA overproduction in
KG11 cells (M. smegmatis mc2 pMV306pamiparA).
Construction of mutant strains in the M. smegmatis
mc2 155 background
To perform unmarked deletions and insertions in the
M. smegmatis mc2 chromosome, targeted gene replacement
was performed according to the protocol of Parish and
Stoker (2000). The constructs, which were based on vectors
p2Nil (KanR) and pGOAL17 (sacB, lacZ) (Parish and Stoker,
2000), were prepared according to the strategy described in
Supporting information and integrated into the M. smegmatis
chromosome by homologous recombination. For transfor-
mation of electrocompetent M. smegmatis cells, the plasmid
DNA was treated with NaOH/EDTA (0.2 mM/0.2 mM). Trans-
formants were plated on NB plates and selected for
kanamycin resistance. The KanR SCO (single-crossover
recombinant) colonies were blue and sensitive to sucrose
(2%). The SCO colonies were further plated on NB without
selection. Cells were then resuspended in liquid medium
and serial dilutions were plated onto NB plates supple-
mented with sucrose and X-Gal. The selected double-
crossover (DCO) mutants were white, KanS and resistant to
sucrose.
PCR and Southern hybridization were used to distinguish
between wild-type and DCO mutant cells. The desired dele-
tions and/or fusion proteins were confirmed by Western blot-
ting analyses.
For construction of M. smegmatis complementation or
meroploid strains, derivatives of the pJAM2 shuttle vector
(Triccas et al., 1998) or the mycobacteriophage L5-based
integration-proficient vector pMV306 (Murry et al., 2005)
were used (see Supporting information for details on their
construction). Vectors were introduced into M. smegmatis-
competent cells by electroporation, and transformants were
selected using kanamycin.
In all strains, the presence of plasmid DNA was con-
firmed by PCR analysis of DNA isolated from the mutant
strains, as described previously (Dziadek et al., 2003). The
protein levels in cell extracts were analysed by SDS-PAGE
(100 mg of cell extracts were loaded on the gel) followed by
Western blotting performed using polyclonal anti-ParA or
anti-ParB according to standard procedures (Towbin et al.,
1979). The presence of fluorescent fusion proteins was veri-
fied by scanning the fluorescence in the SDS-PAGE gel as
described before (Jakimowicz et al., 2005). The level of
parA overexpression in KG11 was assessed by immunob-
lotting different amounts of KG11 and control cells, and
quantifying the results (ImageQuant software, Molecular
Dynamics).
1010 K. Ginda et al.
Growth curves
Mycobacterium smegmatis wild-type, KG22, KG23, KG11,
KG03 and DparB (Jakimowicz et al., 2007a) strains were
cultured in rich (7H9 Middlebrook supplemented with OADC)
medium. Starter cultures were grown overnight in rich
medium, and growth assays were conducted with an initial
OD600 of 0.05. For overproduction of ParA, 0.1% acetamide
was added; the M. smegmatis mc2 pMV306 strain served as a
wild-type control for acetamide induction. Cells were incu-
bated with shaking for 24 h, and samples were collected every
3 h for optical density analysis and CFU determination. Assays
were performed at least in triplicate.
Microscopy
Mycobacterium smegmatis cells (log-phase cultures grown in
7H9 Middlebrook supplemented with OADC) were washed
and resuspended in phosphate-buffered saline (PBS; 10 mM
sodium phosphate, pH 7.4, 150 mM NaCl, 15 mM KCl).
Nascent peptidoglycan synthesis labelling was performed as
previously described (Daniel and Errington, 2003). Briefly,
BODIPY FL-conjugated vancomycin (Invitrogen; final concen-
tration 2 mg ml-1) mixed with an equal amount of unlabeled
vancomycin was added to growing cultures, which were then
incubated with shaking for 3 h to allow binding of the labelled
antibiotic within the cell wall. Cells were viewed directly by
microscopy. For analysis of chromosome segregation and
elongation defects, the cells were additionally permeabilized
by exposure to toluene (2%) for 25 min, washed, resus-
pended in PBS and stained with DAPI (2 mg ml-1) for 30 min at
room temperature. The cells were then incubated for 5 min in
1.5 mg ml-1 FM4-64 for visualization of membranes, and
examined under a Zeiss Axio Imager Z1 equipped with an
X100 objective.
Purification of mycobacterial ParA and DivIVA proteins
To overproduce recombinant GSTParA and GSTDivIVA
fusion proteins in M. smegmatis, the pGEX-6P-2 vector was
used. The parA and divIVA genes were PCR amplified using
appropriate primers (pJAMAMsF and pJAMAMsR for parA;
DivIVMsF and DivIVMsR for divIVA) and chromosomal DNA
as the template. The resulting PCR products were cloned into
the pGEM-T Easy vector (Promega) and recloned into the
BamHI/NotI (parA gene) or BamHI/EcoRI (divIVA gene) sites
of pGEX-6P-2 (GE Healthcare). The glutathione S-transferase
fusion proteins were purified on GSH-Sepharose 4B columns
(GE Healthcare) and treated with the PreScission protease
(GE Healthcare), as previously described (Jakimowicz et al.,
2007a). The purified proteins were more than 95% homoge-
neous, as assessed by SDS-PAGE analysis.
Pull-down assays
A modified affinity chromatography procedure was used
for the pull-down assays. Briefly, 10 mg of ParA and GSH-
Sepharose resin was added to 100 ml of E. coli BL21 lysate
(suspended in 50 mM Tris, pH 8.0, 150 mM NaCl and 1 mM
ATP) containing overproduced GSTDivIVA. After incubation
for 2 h at 4C, the GSH-Sepharose beads were extensively
2013 Blackwell Publishing Ltd, Molecular Microbiology, 87, 9981012
washed with buffer containing 50 mM Tris, pH 8.0, and
150 mM NaCl. Bound protein complexes were eluted with
glutathione solution (20 mM glutathione, 100 mM Tris, pH 8.0,
and 100 mM NaCl) and analysed by SDS-PAGE and Western
blotting with anti-MsParA.
Bacterial two-hybrid (BTH) assays
The recombinant plasmids used in the BTH system (Kari-
mova et al., 2000) were constructed by PCR amplification of
the parA and divIVA genes using appropriate primers and
chromosomal DNA templates (Table S1). The amplified
genes were cloned into the appropriate sites of the pUT18C
and pKT25, or pUT18 and pKNT25 vectors. The resulting
recombinant plasmids expressed the proteins of interest
fused to the C- or N-terminus (respectively) of the T18 and
T25 fragments of adenylate cyclase. For BTH assays, recom-
binant or empty pKT25 and pUT18C plasmids were used in
various combinations to co-transform BTH101 cells. The
transformants were plated onto LB/X-Gal medium and incu-
bated at 30C for 48 h.
Acknowledgements
This work was supported by the Wroclaw Research Center
EIT+ under the project Biotechnologies and advanced medical
technologies BioMed (POIG 1.1. Project 3.1.) financed from
the European Regional Development Fund (Operational
Program Innovative Economy, 1.1.2).
We are very grateful to John McKinney and Isabella Santi
for critical comments on the manuscript.
References
Bartosik, A.A., and Jagura-Burdzy, G. (2005) Bacterial chro-
mosome segregation. Acta Biochim Pol 52: 134.
Casart, Y., Gamero, E., Rivera-Gutierrez, S., Gonzalez,
Y.M.J., and Salazar, L. (2008) par genes in Mycobacterium
bovis and Mycobacterium smegmatis are arranged in an
operon transcribed from SigGC promoters. BMC Microbiol
8: 51.
Chaudhuri, B.N., and Dean, R. (2011) The evidence of large-
scale DNA-induced compaction in the mycobacterial chro-
mosomal ParB. J Mol Biol 413: 901907.
Chaudhuri, B.N., Gupta, S., Urban, V.S., Chance, M.R.,
DMello, R., Smith, L., et al. (2011) A combined global and
local approach to elucidate spatial organization of the
mycobacterial ParBparS partition assembly. Biochemistry
50: 17991807.
Daniel, R.A., and Errington, J. (2003) Control of cell morpho-
genesis in bacteria: two distinct ways to make a rod-
shaped cell. Cell 113: 767776.
Ditkowski, B., Troc, P., Ginda, K., Donczew, M., Chater, K.F.,
Zakrzewska-Czerwinska, J., and Jakimowicz, D. (2010)
The actinobacterial signature protein ParJ (SCO1662)
regulates ParA polymerization and affects chromosome
segregation and cell division during Streptomyces sporu-
lation. Mol Microbiol 78: 14031415.
Donovan, C., Schwaiger, A., Kramer, R., and Bramkamp, M.
(2010) Subcellular localization and characterization of the

ParAB system from Corynebacterium glutamicum. J Bac-
teriol 192: 34413451.
Donovan, C., Sieger, B., Kramer, R., and Bramkamp, M.
(2012) A synthetic Escherichia coli system identifies a con-
served origin tethering factor in Actinobacteria. Mol Micro-
biol 84: 105116.
Dziadek, J., Madiraju, M.V., Rutherford, S.A., Atkinson, M.A.,
and Rajagopalan, M. (2002) Physiological consequences
associated with overproduction of Mycobacterium tubercu-
losis FtsZ in mycobacterial hosts. Microbiology 148: 961
971.
Dziadek, J., Rutherford, S.A., Madiraju, M.V., Atkinson, M.A.,
and Rajagopalan, M. (2003) Conditional expression of
Mycobacterium smegmatis ftsZ, an essential cell division
gene. Microbiology 149: 15931603.
Dziedzic, R., Kiran, M., Plocinski, P., Ziolkiewicz, M.,
Brzostek, A., Moomey, M., et al. (2010) Mycobacterium
tuberculosis ClpX interacts with FtsZ and interferes with
FtsZ assembly. PLoS ONE 5: e11058.
Easter, J., Jr, and Gober, J.W. (2002) ParB-stimulated nucle-
otide exchange regulates a switch in functionally distinct
ParA activities. Mol Cell 10: 427434.
Fogel, M.A., and Waldor, M.K. (2006) A dynamic, mitotic-like
mechanism for bacterial chromosome segregation. Genes
Dev 20: 32693282.
Glaser, P., Sharpe, M.E., Raether, B., Perego, M., Ohlsen, K.,
and Errington, J. (1997) Dynamic, mitotic-like behavior of a
bacterial protein required for accurate chromosome parti-
tioning. Genes Dev 11: 11601168.
Godfrin-Estevenon, A.M., Pasta, F., and Lane, D. (2002) The
parAB gene products of Pseudomonas putida exhibit par-
tition activity in both P. putida and Escherichia coli. Mol
Microbiol 43: 3949.
Hett, E.C., and Rubin, E.J. (2008) Bacterial growth and cell
division: a mycobacterial perspective. Microbiol Mol Biol
Rev 72: 126156.
Ireton, K., Gunther, N.W.T., and Grossman, A.D. (1994)
spo0J is required for normal chromosome segregation as
well as the initiation of sporulation in Bacillus subtilis. J
Bacteriol 176: 53205329.
Jakimowicz, D., Gust, B., Zakrzewska-Czerwinska, J., and
Chater, K.F. (2005) Developmental-stage-specific assem-
bly of ParB complexes in Streptomyces coelicolor hyphae.
J Bacteriol 187: 35723580.
Jakimowicz, D., Brzostek, A., Rumijowska-Galewicz, A.,
Zydek, P., Dolzblasz, A., Smulczyk-Krawczyszyn, A., et al.
(2007a) Characterization of the mycobacterial chromo-
some segregation protein ParB and identification of its
target in Mycobacterium smegmatis. Microbiology 153:
40504060.
Jakimowicz, D., Zydek, P., Kois, A., Zakrzewska-Czerwinska,
J., and Chater, K.F. (2007b) Alignment of multiple chromo-
somes along helical ParA scaffolding in sporulating Strep-
tomyces hyphae. Mol Microbiol 65: 625641.
Kadoya, R., Baek, J.H., Sarker, A., and Chattoraj, D.K. (2011)
Participation of chromosome segregation protein ParAI of
Vibrio cholerae in chromosome replication. J Bacteriol 193:
15041514.
Kang, C.M., Nyayapathy, S., Lee, J.Y., Suh, J.W., and
Husson, R.N. (2008) Wag31, a homologue of the cell divi-
sion protein DivIVA, regulates growth, morphology and
2013 Blackwell Publishing Ltd, Molecular Microbiology, 87, 9981012
The role of ParA in Mycobacterium smegmatis 1011
polar cell wall synthesis in mycobacteria. Microbiology 154:
725735.
Karimova, G., Ullmann, A., and Ladant, D. (2000) A bacterial
two-hybrid system that exploits a cAMP signaling cascade
in Escherichia coli. Methods Enzymol 328: 5973.
Kiekebusch, D., Michie, K.A., Essen, L.O., Lwe, J., and
Thanbichler, M. (2012) Localized dimerization and nucle-
oid binding drive gradient formation by the bacterial cell
division inhibitor MipZ. Mol Cell 46: 245259.
Kumar, S., Farhana, A., and Hasnain, S.E. (2009) In-vitro
helix opening of M. tuberculosis oriC by DnaA occurs at
precise location and is inhibited by IciA like protein. PLoS
ONE 4: e4139.
Lasocki, K., Bartosik, A.A., Mierzejewska, J., Thomas, C.M.,
and Jagura-Burdzy, G. (2007) Deletion of the parA (soj)
homologue in Pseudomonas aeruginosa causes ParB
instability and affects growth rate, chromosome segrega-
tion, and motility. J Bacteriol 189: 57625772.
Leonard, A.C., and Grimwade, J.E. (2005) Building a bacte-
rial orisome: emergence of new regulatory features for
replication origin unwinding. Mol Microbiol 55: 978985.
Leonard, T.A., Butler, P.J., and Lowe, J. (2005) Bacterial
chromosome segregation: structure and DNA binding of
the Soj dimer a conserved biological switch. EMBO J 24:
270282.
Lewis, R.A., Bignell, C.R., Zeng, W., Jones, A.C., and
Thomas, C.M. (2002) Chromosome loss from par mutants
of Pseudomonas putida depends on growth medium and
phase of growth. Microbiology 148: 537548.
Madiraju, M.V., Moomey, M., Neuenschwander, P.F.,
Muniruzzaman, S., Yamamoto, K., Grimwade, J.E., and
Rajagopalan, M. (2006) The intrinsic ATPase activity of
Mycobacterium tuberculosis DnaA promotes rapid oli-
gomerization of DnaA on oriC. Mol Microbiol 59: 1876
1890.
Marston, A.L., Thomaides, H.B., Edwards, D.H., Sharpe,
M.E., and Errington, J. (1998) Polar localization of the
MinD protein of Bacillus subtilis and its role in selection of
the mid-cell division site. Genes Dev 12: 34193430.
Mohl, D.A., Easter, J., Jr, and Gober, J.W. (2001) The chro-
mosome partitioning protein, ParB, is required for cytoki-
nesis in Caulobacter crescentus. Mol Microbiol 42: 741
755.
Murray, H., and Errington, J. (2008) Dynamic control of the
DNA replication initiation protein DnaA by Soj/ParA. Cell
135: 7484.
Murry, J., Sassetti, C.M., Moreira, J., Lane, J., and Rubin,
E.J. (2005) A new site-specific integration system for myco-
bacteria. Tuberculosis (Edinb) 85: 317323.
Nguyen, L., Scherr, N., Gatfield, J., Walburger, A., Pieters, J.,
and Thompson, C.J. (2007) Antigen 84, an effector of
pleiomorphism in Mycobacterium smegmatis. J Bacteriol
189: 78967910.
Nisa, S., Blokpoel, M.C., Robertson, B.D., Tyndall, J.D., Lun,
S., Bishai, W.R., and OToole, R. (2010) Targeting the
chromosome partitioning protein ParA in tuberculosis drug
discovery. J Antimicrob Chemother 65: 23472358.
Parish, T., and 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: 19691975.
1012 K. Ginda et al.
Ptacin, J.L., and Shapiro, L. (2010) Initiating bacterial mitosis:
understanding the mechanism of ParA-mediated chromo-
some segregation. Cell Cycle 9: 40334034.
Ptacin, J.L., Lee, S.F., Garner, E.C., Toro, E., Eckart, M.,
Comolli, L.R., et al. (2010) A spindle-like apparatus guides
bacterial chromosome segregation. Nat Cell Biol 12: 791
798.
Rajagopalan, M., Atkinson, M.A., Lofton, H., Chauhan, A.,
and Madiraju, M.V. (2005a) Mutations in the GTP-binding
and synergy loop domains of Mycobacterium tuberculosis
ftsZ compromise its function in vitro and in vivo. Biochem
Biophys Res Commun 331: 11711177.
Rajagopalan, M., Maloney, E., Dziadek, J., Poplawska, M.,
Lofton, H., Chauhan, A., and Madiraju, M.V. (2005b)
Genetic evidence that mycobacterial FtsZ and FtsW
proteins interact, and colocalize to the division site in
Mycobacterium smegmatis. FEMS Microbiol Lett 250:
917.
Sambrook, J., Ftritsch, E.F., and Maniatis, T. (1989) Molecu-
lar Cloning: A Laboratory Manual, 2nd edn. Cold Spring
Harbor, NY: Cold Spring Harbor Laboratory.
Sassetti, C.M., Boyd, D.H., and Rubin, E.J. (2003) Genes
required for mycobacterial growth defined by high density
mutagenesis. Mol Microbiol 48: 7784.
Scherr, N., and Nguyen, L. (2009) Mycobacterium versus
Streptomyces we are different, we are the same. Curr
Opin Microbiol 12: 699707.
Scholefield, G., Errington, J., and Murray, H. (2012) Soj/ParA
stalls DNA replication by inhibiting helix formation of the
initiator protein DnaA. EMBO J 31: 15421555.
Shebelut, C.W., Guberman, J.M., van Teeffelen, S.,
Yakhnina, A.A., and Gitai, Z. (2010) Caulobacter chromo-
2013 Blackwell Publishing Ltd, Molecular Microbiology, 87, 9981012
some segregation is an ordered multistep process. Proc
Natl Acad Sci USA 107: 1419414198.
Towbin, H., Staehelin, T., and Gordon, J. (1979) Electro-
phoretic transfer of proteins from polyacrylamide gels to
nitrocellulose sheets: procedure and some applications.
Proc Natl Acad Sci USA 76: 43504354.
Triccas, J.A., Parish, T., Britton, W.J., and Gicquel, B. (1998)
An inducible expression system permitting the efficient
purification of a recombinant antigen from Mycobacterium
smegmatis. FEMS Microbiol Lett 167: 151156.
Wayne, L.G., and Sohaskey, C.D. (2001) Nonreplicating per-
sistence of Mycobacterium tuberculosis. Annu Rev Micro-
biol 55: 139163.
Yamamoto, K., Muniruzzaman, S., Rajagopalan, M., and
Madiraju, M.V. (2002) Modulation of Mycobacterium tuber-
culosis DnaA protein-adenine-nucleotide interactions by
acidic phospholipids. Biochem J 363: 305311.
Zawilak, A., Kois, A., Konopa, G., Smulczyk-Krawczyszyn,
A., and Zakrzewska-Czerwinska, J. (2004) Mycobacterium
tuberculosis DnaA initiator protein: purification and DNA-
binding requirements. Biochem J 382: 247252.
Zawilak-Pawlik, A., Kois, A., Majka, J., Jakimowicz, D.,
Smulczyk-Krawczyszyn, A., Messer, W., and Zakrzewska-
Czerwinska, J. (2005) Architecture of bacterial replication
initiation complexes: orisomes from four unrelated bacte-
ria. Biochem J 389: 471481.
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