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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Mycobacterium spp., rod-shaped cells belonging to the phylum Actinomycetes, lack the Min- and Noc/Slm systems responsible for preventing the placement of division sites at the poles or over the nucleoids to ensure septal assembly at mid-cell. We show that the position for establishment of the FtsZ-ring in exponentially growing Mycobacterium marinum and Mycobacterium smegmatis cells is nearly random, and that the cells often divide non-medially, producing two unequal but viable daughters. Septal sites and cellular growth disclosed by staining with the membrane-specific dye FM4-64 and fluorescent antibiotic vancomycin (FL-Vanco), respectively, showed that many division sites were off-centre, often over the nucleoids, and that apical cell growth was frequently unequal at the two poles. DNA transfer through the division septum was detected, and translocation activity was supported by the presence of a putative mycobacterial DNA translocase (MSMEG2690) at the majority of the division sites. Time-lapse imaging of single live cells through several generations confirmed both acentric division site placement and unequal polar growth in mycobacteria. Our evidence suggests that post-septal DNA transport and unequal polar growth may compensate for the non-medial division site placement in Mycobacterium spp.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Mycobacterium spp. are acid-fast, rod-shaped, Gram-positive and high-GC-content microorganisms categorized as Actinomycetes, a phylum that includes pathogenic as well as non-pathogenic bacteria. In fact, some of the oldest and most successful pathogens known to man, Mycobacterium tuberculosis and Mycobacterium leprae (the aetiological agents of tuberculosis and leprosy respectively), belong to this group of bacteria (Sasaki et al., 2001; Russell, 2007). Members of the genus Mycobacterium inhabit various environmental niches such as water, soil, animals and humans. They are able to adapt to a variety of lifestyles, viz., form biofilms and invade macrophages where they can enter into a passive state for prolonged periods (Wayne, 1994; Carter et al., 2003; Chacon et al., 2004; Monack et al., 2004; Ramakrishnan, 2004; Vaerewijck et al., 2005). The molecular mechanisms that allow this switch to an alternative lifestyle are not yet clear. Adaptation to different lifestyles requires a precise cell cycle control responsive to environmental stimuli and an ability to slow down or even temporarily cease growth, chromosome duplication and cell division. A detailed understanding of mycobacterial growth, division and their control mechanisms might therefore provide valuable insights as to how the complex lifestyles of these organisms are regulated. To this end, we have followed the complete life cycle of a relatively fast-growing model mycobacterial strain, Mycobacterium marinum, as its cell population underwent changes in morphology and DNA content from fresh inocula through vigorous vegetative growth to late stationary phase. This led to the counter-intuitive conclusion that Mycobacterium spp. can form spore particles under certain, yet to be characterized, conditions (Ghosh et al., 2009). This finding of course was challenged (Traag et al., 2010); but additional evidence supporting sporulation in different Mycobacterium spp. has also emerged (see Singh et al., 2010; Lamont et al., 2012). In the present work, we examined the mechanisms of growth and division of mycobacterial cells from exponentially growing cultures for some insight into the complex life cycles of these organisms.

Rod-shaped bacteria are known to grow along their longitudinal axis up to a critical size after which they divide into two identical progeny by binary fission. The placement of the division site is under strict spatiotemporal control such that it is localized at the mid-cell region between the segregating/segregated nucleoids (Goehring and Beckwith, 2005; Harry et al., 2006). The earliest observable event in cell division is the polymerization of the tubulin analogue FtsZ into a ring at the mid-cell region; this Z-ring then acts as the nucleating site for the components of division machinery, also known as the divisome (Bi and Lutkenhaus, 1991; Harry, 2001; Lutkenhaus, 2007). In both Gram-negative and Gram-positive model systems, viz., Escherichia coli and Bacillus subtilis, respectively, the choice of mid-cell as division site is determined by a combination of negative control mechanisms such as the Min system and nucleoid occlusion. The MinCDE (or MinCDJDivIVA) system prevents Z–ring formation and subsequent divisome assembly near the cell poles. The nucleoid occlusion factors SlmA (or Noc) act by preventing the assembly of FtsZ as a functional ring over the nucleoids. This leaves the post-segregation, nucleoid-free mid-cell region as the only zone available for divisome assembly and septation (de Boer et al., 1989; Mulder and Woldringh, 1989; Wu and Errington, 2004; Bernhardt and de Boer, 2005; Margolin, 2005; Lutkenhaus, 2007; Adams and Errington, 2009).

Mycobacterium spp. are Gram-positive, but they differ from other Gram-positive rod-shaped bacteria such as B. subtilis in thickness of the cell wall as well as the presence of an outer surface consisting of mycolic acid which confers upon them some of the characteristics of Gram-negative bacteria (Thanky et al., 2007; Hett and Rubin, 2008). The absence of analogues or orthologues for mreB, minCDE and nucleoid occlusion systems (noc/slmA) in the annotated mycobacterial genomes make the issue of division site selection even more intriguing. Recent investigations into cell division mechanisms of Mycobacterium smegmatis and M. tuberculosis have revealed apical growth at polar peptidoglycan layers and division by snapping (Chauhan et al., 2006; Thanky et al., 2007; Kang et al., 2008; Singh et al., 2010). In this study, we examined the process of growth and division site selection in M. marinum and M. smegmatis with fluorescence microscopy and followed individual cells for several generations by time-lapse microscopy. Our results show that division site placement in Mycobacterium spp. occurred over a large area from mid-cell to the poles. There was no evidence for nucleoid occlusion as the septal sites frequently overlapped the nucleoid(s). However, there was no detectable production of DNA-less mini-cells or cells with guillotined partial chromosomes, as might be expected when division occurs at non-medial sites. Such deleterious consequences appeared to be prevented by chromosome translocation across asymmetrically placed septa and by unequal polar growth in subsequent generations. In this context, Aldridge et al. (2012) showed that M. smegmatis and M. tuberculosis grew asymmetrically in a unipolar fashion, giving rise within a few generations to heterogeneity in size, consistent with our findings.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Random placement of division sites in microbial cells

To identify the distinguishing features of growth and cell division in Mycobacterium spp., if any, we first decided to visualize the division site placement in E. coli, which is known to divide precisely at mid-cell to produce daughter cells of identical length (Yu and Margolin, 1999). Figure 1A and B demonstrates the precision of mid-cell division site placement in this rod-shaped, Gram-negative bacterium. The micrograph (Fig. 1A) shows dividing E. coli cells with their Z-rings made visible by in-frame fusion of FtsZ with the green fluorescence protein (GFP). The Z-ring positions, measured from the nearest poles of 120 dividing cells, were plotted as fractions of the cell lengths normalized to 1.0 against the actual cell lengths (Fig. 1B); all Z-rings are positioned in a tight group localized at the cell centre consistent with the findings of Yu and Margolin (1999). Similar narrow, mid-cell distribution of Z-rings has also been demonstrated in dividing cells of the model Gram-positive bacterium B. subtilis (Migocki et al., 2004).

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Figure 1. Division sites placements in E. coli and in Mycobacterium spp.

A. Fluorescence micrographs of dividing E. coli cells carrying FtsZ–GFP fusions and stained with DAPI (blue) and FM4-64 (red).

B. Distribution of FtsZ-ring positions in 120 cells plotted along their axial length. The distance of the FtsZ-rings was measured from the nearest pole in fractions of cell length normalized to 1.0; the y-axis gives the size of individual cells in microns (μm).

C. Micrographs of M. smegmatis cells with FtsZ–mCherry fusions.

D. Distribution of FtsZ-ring positions of 100 dividing M. smegmatis cells plotted as in (B).

E. Micrographs of M. marinum cells from an exponentially growing culture stained with the membrane-specific stain FM4-64 (see Experimental procedures). All cells show off-centre division sites. The middle cell shows completed division with two unequal daughter cells. The inset shows the enlarged image of an asymmetrically placed septal membrane.

F. Positions of septal membranes in dividing M. marinum (Mm) and M. smegmatis (Msm) cells plotted as in (B); 200 cells were counted in each case.

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Next, we studied the positioning of the Z-ring in an M. smegmatis strain containing a plasmid carrying the FtsZ–mCherry fusion construct (see Table 1). The dividing merodiploid cells in the micrograph show the positioning of Z-rings at mid-cell, as well as in pole-proximal regions (Fig. 1C). This is in sharp contrast to those in E. coli (cf. Fig. 1A). A plot of the Z-ring positions for 100 dividing M. smegmatis cells (as in Fig. 1B) showed Z-rings over the entire length of the cell, from centre to poles, with some clustering at 20% and 50% of the cell length (Fig. 1D). In M. marinum cells too, Z-ring positions were not restricted to the cell centre (see Supplement Fig. S1), whether examined in live cells with fluorescent FtsZ [Fig. S1(i)] or in fixed cells immunostained with an anti-FtsZ antibody [see Fig. S1(ii)].

Table 1. Strains and plasmids used in the current study
StrainsRelevant genotypeSource
  1. a

    Uppsala University Hospital, Uppsala, Sweden.

  2. b
  3. c

    Inst for Cell and Molecular Biosciences, Newcastle University, Newcastle, UK.

  4. d

    School of Public Health, University of California at Berkeley, USA.

BS532E. coli MG1655 with FtsZ–GFP plasmid pEG12Current study
BSm103M. marinum NCTC 2275 (ATCC 927)Dr Björn Herrmanna
BSm106M. smegmatis mc2 155 ATCC 700084LGC Promochem
DSM43239M. phleiDSMZb
BSm131BSm106attB::attP<PG13–GFP>Current study
BSm156BSm106attB::attP<MSMEG2690–GFP>Current study
RG1MmM. marinum CCUG 20998/pRG1ZmCurrent study
RG1MsmBSm 106/pRG1ZmCurrent study
B. subtilis Bacillus subtilis subsp. subtilis (BGSC #1A1; 168)Bacillus Genetic Stock Center
PlasmidsRelevant gene(s) 
pEG12E. coli FtsZ–GFP plasmidDr Elisa Gallic
pG13G13 promoter driven mGFPBarker et al. (1999)
pPGSYGFP containing integration vector with attPDr Lee W. Rileyd
pBS101pPGSY with G13 promoter inserted at the SapI/PacI sitesCurrent study
pBS104pPGSY with MSMEG2690, with 500 bp upstream sequence, inserted at EcoRI/PacI sitesCurrent study
pBS401pIGn (Casali et al., 2006) derivative containing TetRO promoter from pMind (Blokpoel et al., 2005) at the KpnI/SpeI sites.Current study
pRG1ZmpBS401<PTetROFtsZ–mCherry>Current study

Z-rings are the earliest detectable structures that provide the templates for assembly of the multi-protein divisome complex, which in turn brings about septation and cytokinesis. In Fig. 1E we show the positions of septal membranes at matured division sites for several M. marinum cells using the membrane-specific fluorescent dye FM4-64. The three cells shown are at different stages of growth and division – the ones on the left and right show slightly asymmetric division sites; the left one is bent at the septum. The cell in the middle has already undergone an asymmetric division resulting in unequal-sized progeny cells. The inset shows an enlarged view of a dividing cell with a non-medial septum. FM4-64 staining at the division site was more intense than in the rest of the cell suggesting the presence of double layers of membrane at the septum prior to division (Fig. 2). The distribution of actual division sites in populations of dividing M. marinum and M. smegmatis cells (approximately 200 each) are shown in Fig. 1F. In these two plots, measurements were made as in Fig. 1B and D; both M. marinum and M. smegmatis exhibit division sites distributed over broad areas covering half (M. marinum), or more (M. smegmatis) of the cell length. For both M. marinum and M. smegmatis, the cells with septa were always larger in size (by 25–35%) than those without (see Supplement Fig. S2).

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Figure 2. Apical growth of mycobacterial cells. Fluorescence micrographs of FL-Vanco stained cells of M. marinum (Mm) and M. smegmatis (Msm), chosen at random from exponentially growing cultures. We have arranged them approximately in order of increasing size (top to bottom) to show the different patterns of growth during the life cycle of the bacteria. The fluorescent sites represent areas of nascent peptidoglycan incorporation stopped due to binding of the antibiotic FL-Vanco. The size bar = 5 μm.

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The locations of the septal membranes (labelled with FM4-64) or walls (labelled with FL-Vanco, a fluorescent complex of the antibiotic vancomycin (Vanco); see below and Experimental procedures) appeared less random and more clustered near the mid-cell region than expected from the location of the Z-rings (cf. Fig. 1D and Fig. 1F-Mm). Furthermore, shorter cells showed a relatively higher degree of asymmetry in septal position than longer cells (see Fig. 1F-Msm) suggesting that cells with asymmetric division sites were frequently younger. The higher than expected frequency of division sites at the mid-cell location with respect to the more random Z-ring placement suggests that there was unequal polar growth after Z-ring formation. Newly formed cells of Mycobacterium spp. would be expected to show greater heterogeneity in size distribution than model rod-shaped cells that divide at medially placed septa. Greater size heterogeneity in exponential phase compared with stationary phase has been reported earlier for cells of Mycobacterium spp. (Thanky et al., 2007). Phenotypic size heterogeneity within a clonal population of Mycobacterium spp. has also recently been confirmed (Aldridge et al., 2012).

Uni- and bi-polar apical growth in Mycobacterium spp

Figure 2 shows cellular growth in M. marinum (Mm) and M. smegmatis (Msm) from exponentially growing cultures treated with FL-Vanco (see above and Experimental procedures). Unlike FM4-64, which stains all the membranous areas (see Fig. 1E), FL-Vanco labelling is restricted to the area(s) of new cell wall synthesis where nascent peptidoglycan subunits are being incorporated. Thus the FL-Vanco dye reveals the locations of ongoing cell wall growth just before the lethal effect of the antibiotic. Cells with different patterns of growth are displayed; from non-dividing cells to cells with almost completed division. It should be pointed out that an increase in cell size does not necessarily imply an older age of the cell because cells can be unequal in size at birth and can grow at different rates (see below). Both uni- and bipolar growth, as visualized with polar fluorescence, were common as were dividing cells with nascent cell walls formed at the septal membrane. In cells showing bipolar growth, fluorescence at both ends was equally intense, indicating symmetric growth at both poles; bipolar growth was more frequent in smaller, non-dividing cells [Mm: (i, ii); Msm: (i)]. Older dividing cells, with the septal membrane already formed, showed little peptidoglycan incorporation either at the poles [Mm: (iii)] or only at one of the poles [Mm: (iv); Msm – (ii–v)]. Cell division by snapping was evident from the fluorescent V formed by nascent cell walls at the new poles [Mm: (v, vi); Msm: (vi, vii)]; the daughter cells could be equal in size [Mm: (v); Msm: (vi)] or unequal [Mm: (vi); Msm: (vii)]. A surface scan of a cell with bipolar growth confirmed the predominant addition of nascent peptidoglycan at the poles (see Supplement Fig. S3A). Some weak fluorescence along the sidewalls was also noticeable suggesting possible low level of peptidoglycan incorporation in a helical pattern (see the cartoon representation of FL-Vanco incorporation in a growing cell accompanying Fig. S3A). In contrast, exponentially growing, non-dividing B. subtilis cells showed only lateral incorporation of nascent peptidoglycan in the mid-cell region (see Supplement Fig. S3B and the cartoon inset). Very few stationary-phase cells stained with the FL-Vanco, suggesting that cell wall synthesis had ceased. However, both uni- and bipolar labelling could be seen among the rare small spheroidal cells (see Supplement Fig. S3C). Double labelling with FM4-64 and FL-Vanco showed colocalization at all cross-wall membrane sites, mid-cell or otherwise, suggesting that these were the true division sites (data not shown). Thus, either of the labels could be used to locate the septal position in dividing cells. We used FM4-64 to locate mature division sites in subsequent experiments; DAPI was used to localize the nucleoids.

Absence of nucleoid occlusion

The acentric distribution of Z-ring positions in mycobacterial cells (Fig. 1) implied that the precision of mid-cell placement of division sites between the segregated daughter nucleoids no longer held; symmetric localization of some of the matured septa may possibly have been due to unequal growth at the two poles. We examined the relative positions of FM4-64 labelled septa relative to the DAPI-stained chromosome(s) in dividing M. smegmatis cells. Figure 3A shows a micrograph with a large number of M. smegmatis cells from an exponentially growing culture stained with DAPI and the membrane-specific dye FM4-64. The micrograph shows several cells undergoing division; we have indicated these with arrowheads pointing at the septa (cross-wall membranes). As shown above (Fig. 1E), the septa spread broadly from mid-cell to polar regions and were often found overlapping the nucleoid(s). M. marinum cells also showed similar frequency of non-medial septal positions (data not shown).

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Figure 3. Absence of nucleoid occlusion.

A. shows the fluorescence micrograph of random sample of growing and dividing cells from an exponential culture of M. smegmatis stained with the DNA- and the membrane-specific dyes DAPI (blue) and FM4-64 (red) respectively. The cross-wall septal membranes, indicating division sites, are marked by arrowheads. As in Fig. 1C–F, the division sites appear to be quite randomly placed and the daughter cells are often unequal in length. Size bar = 5 μm.

B. Individual cells with different relative positions of the nucleoid(s) and the septa are shown along with cartoon representations in the panels below. Size bar = 5 μm.

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We have enlarged the images of several dividing cells and shown them in individual panels along with cartoon sketches (Fig. 3B) to elaborate some of the septal positions relative to nucleoid distribution in the cells. These show dividing cells with a non-medial septum on one side of the nucleoid(s) or overlapping the nucleoid as well as at mid-cell between two segregated nucleoids (see the explanatory cartoons below each micrograph). Dividing cells (marked with white arrows; Fig. 3A) with three distinct scenarios of septum-nucleoid positions are enlarged and shown in the Supplement Fig. S4(i–iii). All these images reiterate our initial conclusion that placement of division sites in Mycobacterium spp. is not constrained by chromosome position or segregation. Nucleoid occlusion did not appear to have a major role in determining septal position.

Translocation of nucleoids across the septal membrane

The results above show that asymmetry in division site placement is quite common in Mycobacterium spp. and the septa are often formed over or on one side of the nucleoids instead of between them (Fig. 3A and B and supplement Fig. S4). Cytokinesis at such asymmetrically placed septa with cell wall invagination and separation of poles would bisect the nucleoid to produce cells with either a guillotined chromosome or no chromosome at all. Since the mycobacterial cell cultures do not accumulate DNA-less cells or cells with bisected, fragmented chromosomes to any noticeable degree (Singh and Dasgupta, unpubl. obs.), it seems that Mycobacterium spp. may have evolved mechanisms to cope with the random placement of Z-rings at division. One such mechanism might be directional transport of the chromosome across an asymmetric septum. We used thin section transmission electron microscopy (TEM) to capture images of dividing cells in the process of undertaking DNA transfer through the septal barrier (Fig. 4A). Symmetric and asymmetric division sites were found in roughly equal abundance among dividing cells. Both M. smegmatis (Msm) and M. marinum (Mm) exhibited characteristic mid-cell [Fig. 4A (I)] and non-medial [Fig. 4A (II–IV)] septa. It should be pointed out that the observed septal positions in the TEM images depended partly upon the angle of the microtome knife relative to the orientation of the cell and hence might not represent their true position on the longitudinal axis of the cell. However, we observed different stages of progression of chromosome movement between the two cell-halves, varying from complete nucleoid separation [Fig. 4A: (I–III) Msm; (I–II) Mm] to nucleoid in-transit to the smaller compartment of the cell [Fig. 4A: (IV) Msm; (III, IV) Mm]. Thus we conclude that chromosome transfer across septa does occur between compartments of dividing M. marinum and M. smegmatis cells. Cells of another Mycobacterium spp., Mycobacterium phlei, were also examined in dividing stage for DNA transfer across septa. Panel V (Fig. 4) shows the magnified TEM cross-section of a symmetrically dividing M. phlei cell with a clearly visible connection (conceivably consisting partly of DNA) between the segregating nucleoids across the septal membrane.

figure

Figure 4. Transport of nucleoid DNA across the septa in mycobacterial cells.

A. Transmission electron micrograph (TEM) of thin sections of M. smegmatis (Msm) and M. marinum (Mm) cells at various stages of acentric division. Passage of nucleoid DNA through the septal membrane is visible in Msm (I and IV) and Mm (III and IV). (V) shows a symmetrically dividing M. phlei cell in the process of transporting DNA across the septal membrane.

B. Time-lapse images of an asymmetrically dividing M. smegmatis cell stained with DAPI (blue), FM4-64 (red) and FL-Vanco (green). A small amount of the DAPI fluorescence appears to move across the septal membrane in 1 h (cf. the black and white image in the central panel with that on its left – see the explanatory cartoon representation below). Size bar = 5 μm.

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We attempted to demonstrate chromosome translocation across the septal membrane in real time by time-lapse microscopy of a single cell. Figure 4B shows the successive fluorescence micrographs at 0 h, 1 h and 2 h of the same M. smegmatis cell, chosen for its pole-proximal septum. It was stained with FM4-64 (red), FL-Vanco (green) and DAPI (blue) and images were recorded at intervals of 1 h. At the start (0 h), the cell showed growth at both poles (green fluorescence) and colocalized FM4-64 and FL-Vanco fluorescence (red + green = yellow) at the pre-septal membrane with the nucleoid (DAPI fluorescent blue) in the larger compartment on one side of septum; no DAPI fluorescence could be detected in the smaller compartment on the other side of the septum. After 1 h, a little of the DAPI fluorescence could be seen in the smaller segment of the dividing cell (shown in black and white for better resolution in the middle panels and explained in cartoon drawings in the bottom panels). This suggests transport of a small part of the chromosome across the septal membrane. This partial transfer of DNA across the septal membrane is significant if one considers that growth of the cell wall is inhibited by FL-Vanco incorporation and that exposure to UV radiation is lethal for DAPI stained cells. Some chromosomal DNA, however, is still translocated across the septum in the short lifespan of the cell. Cessation of cell wall growth is indicated by loss of green fluorescence at cell poles and conversion of the septal fluorescence to red (FM4-64) from yellow (FM4-64 + FL-Vanco). The partial transfer of DNA across the division site within this period suggests that directional chromosome translocation across the division site is feasible.

Septal association of MSMEG2690, the putative DNA translocase of M. smegmatis

Chromosome transport across the septum by DNA translocases has been reported during final stages of partition in E. coli and during sporulation in B. subtilis (Begg et al., 1995; Errington et al., 2001; Sherratt et al., 2010). MSMEG2690 is annotated as the putative gene encoding DNA translocase in M. smegmatis but it remains to be functionally characterized. We made a C-terminal translational fusion of MSMEG2690 with the gene encoding GFP (green fluorescent protein); the fusion protein was then integrated into the M. smegmatis chromosome (see Supplement Fig. S5). The resulting merodiploid strain BSm156 did not show any change in growth rate or morphology compared with the control strain (data not shown). Fluorescence microscopy of BSm156 cells revealed the presence of MSMEG2690–GFP foci in dividing (Fig. 5A-i, ii) as well as non-dividing cells (Fig. 5A-iii). No such foci were visible in the control strain BSm131, expressing GFP alone; fluorescence was spread throughout the cells without any localized focus (see Supplement Fig. S5). The GFP foci in BSm156 overlapped the DAPI fluorescence with very few exceptions indicating the preferred association of the fusion protein with the nucleoid (Fig. 5A-i, ii, iii). The majority of the cells (over 90%; number of cells 191, Fig. 5B) had multiple GFP foci; almost all of these had foci near both the poles. Among the cells with visible septal membranes, about 80% of the GFP foci were associated with symmetric as well as asymmetric septa. In ≈ 20% of the cells, the septa (symmetric or asymmetric) were not associated with GFP foci. Figure 5B shows the relative proportion of cells with different positions of the MSMEG2690–GFP; each bar represents a specific intracellular localization pattern of MSMEG2690 as indicated by the cartoon(s) below the columns. The height of the column indicates frequency. In Fig. 5A and B it seems that all growing cells have the putative DNA translocase foci associated with the nucleoid as well as localized at poles in non-dividing cells. Among the cells with septa (65% of all cells), over 75% had the FtsK analogue MSMEG2690 associated with the septal membrane; 20% had FtsK foci at the poles but not at the septa, while approximately 10% did not have any FtsK foci at all. GFP foci also formed at internal sites; these probably indicate the positions of future septa. The foci associated with septa may be involved in translocating the chromosome as part of the segregation process. GFP foci might lose their association with septa when segregation is complete or in situations where translocation is not needed (i.e. when the septum forms after segregation). More work is needed to establish the significance of the presence of MSMEG2690 at the observed locations and its role in partition if any, in Mycobacterium spp. The observed intracellular locations of MSMEG2690 and its association with septa and nucleoids permits us however to speculate that MSMEG2690 might indeed be the true DNA translocase in M. smegmatis with an active role in DNA transfer across the septa.

figure

Figure 5. Localization of putative DNA translocase, MSMEG2690, in dividing and non-dividing M. smegmatis cells.

A. Putative M. smegmatis DNA translocase, MSMEG2690, was fused with GFP and localized in cells with or without septa. The GFP fusion appears as fluorescent foci. Control cells with free GFP show no such foci (see Supplement Fig. S5A). Three cells with polar (i), mid-cell (ii) and no (iii) division sites are shown with FM4-64, MSMEG2690–GFP and DAPI fluorescence separately and in combination. The cartoon sketches below show the membrane outlines at the cell boundaries and the septa (black lines), the nucleoids (blue) and the translocase foci (green). Size bar = 5 μm.

B. Frequencies of translocase positions in dividing and non-dividing Msm cells from an exponentially growing culture. Cells with mid-cell, non-medial or no septa are marked with different surface patterns.

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Asymmetric growth at poles

We followed the growth and division of FM4-64-stained individual M. smegmatis cells for two to three generations by time-lapse microscopy. Figure 6 shows micrographs of two cells (i and ii) growing on glass slides coated with a thin layer of agarose in nutrient medium. Cells were photographed at 3 h intervals except for a 12 h (overnight) gap between the 3rd and 15th hours; both cells underwent one division during this period. Under these conditions (see Experimental procedures) the cells grew with a doubling time of about 5 h which is similar to that in broth maintained at 30°C, shaken at 90 r.p.m. Cell growth and position of septa during the 27 h period were plotted for each cell with its length as abscissa and time as ordinate. On these plots, the first septum, (represented by a black line) was assigned position 0 on the x-axis. The positions of the poles of the growing cells with reference to the first mid-point were plotted along the y-axis at each time point and connected by grey lines showing total growth of the cells. Subsequent septal appearance were marked and connected with grey lines. The M. smegmatis cell (i) underwent its first division sometime during the initial 12 h period, producing two equal-sized daughter cells, presumably as a result of mid-cell division. Both of these daughter cells had non-medial septa at 15 and 18 h producing two unequal progeny from each. A follow-up of progenies of the same cells showed that the larger cells underwent another round of division with mid-cell septum formation (at 24 h) yielding two equal sized daughter cells. In contrast, the smaller cells grew for one generation without undergoing division and thereby attained the same size as newborn cells of the next generation from their larger sisters. A nearly identical pattern of asymmetric and symmetric divisions was shown by both cells (i and ii in Fig. 6) as well as by several other cells (data not shown). Thus, at any instant of growth, a clonal population would have cells of heterogeneous sizes – unequal cells born from non-medial division resulting in subpopulations with different growth rates.

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Figure 6. Cycles of growth and division of individual mycobacterial cells. Two M. smegmatis cells (i and ii) labelled with FM4-64, embedded in an agarose layer on a microscope slide, were allowed to grow for 27 h with infusions of nutrient medium under a fluorescence microscope and photographed every 3 h (with a 12 h gap between 3 h and 15 h). During the 27 h, they completed 3 generations (with an approximate generation time of 4.0–5.4 h). The first division site is marked by the black line at position 0 on the x-axis (start-time unknown, somewhere within the 12 h gap between 3 h and 15 h), and the subsequent ones by grey; the trajectory of cell growth is plotted with extension of the poles in reference to the first division line. Both mid-cell and non-medial division sites are seen in following generations. Size bar = 5 μm.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We investigated cell division patterns in Mycobacterium spp. in order to understand how these important pathogens undergo binary division in the absence of key regulatory mechanisms for mid-cell septum placement, such as Min and Noc/SlmA systems. Intense investigations into cell division mechanisms and its control for several decades with elegant microscopy techniques made possible by unprecedented developments of fluorescent probes have revealed some astonishing details about divisome assembly and cytokinesis in model rod-shaped bacteria (see for reviews: Rothfield et al., 2005; Harry et al., 2006; Margolin, 2006; Bramkamp and Baarle, 2009; Wu and Errington, 2011). However, despite many recent studies on divisome assembly in Mycobacterium spp., the mechanism controlling its division site placement remains uncharacterized (Datta et al., 2002; 2006; Dziadek et al., 2003; Kang et al., 2005; Rajagopalan et al., 2005; Thanky et al., 2007; Kiran et al., 2009; Maloney et al., 2009; Oleferenko et al., 2009; Sureka et al., 2010; Plocinski et al., 2011). The components of the division machinery and their assembly at the Z-ring in Mycobacterium spp. are similar to those in model bacteria (Datta et al., 2002; 2006; Rajagopalan et al., 2005; Mukherji et al., 2009; Sureka et al., 2010). Nonetheless, several unusual features in the site selection and the division process for Mycobacterium spp. (Thanky et al., 2007; Hett and Rubin, 2008) renders direct extrapolation of the detailed knowledge from the model systems inappropriate. The mid-cell location of the septum, in Mycobacterium spp., had until recently been taken for granted and models for achieving binary fission were proposed (Plocinski et al., 2011). However, none of these intuitive assumptions has been verified experimentally. Direct measurements of Z-ring positions in a population of dividing cells showed them to be distributed over a broad area from mid-cell to near the poles (Fig. 1D) and the division sites were accordingly found to be more dispersed than those in E. coli or B. subtilis (Fig. 1F; and Aldridge et al., 2012). Similarity of the FtsZ-ring positions seen in live cells with a FtsZ–mCherry fusion and in fixed cells visualized by immunofluorescence ruled out the possibility that the randomness in the former might have resulted from abnormal levels of ftsZ expression in the merodiploid strain. Furthermore, the asymmetry of division sites seen in time-lapse images of live cells rules out the possibility that asymmetry in the division site arose from asymmetry in polar growth. During revision of this article, Joyce et al. (2012) proposed that septum placement in M. smegmatis occurs accurately in the mid-cell and that asymmetry in division is a consequence of unequal polar growth after division site placement. These authors based their conclusions from growth measurements by PBP1a incorporation, reflecting a late stage of septal wall formation. We have measured the relative positions of the division sites at different stages from the earliest Z-ring placement to the late stage of septal wall synthesis. We noticed a reduced asymmetry in the locations of septa labelled with FM4-64 and FL-Vanco relative to the more dispersed locations of the Z-rings at the earliest step of division site placement (see Fig. 1F). We attribute this to the unequal growth at the two poles after Z-ring formation; Joyce et al. (2012) measured the position of division sites only at this late stage of septal wall synthesis and for this reason found it more symmetric than shown in this study.

Unequal divisions in the rod-shaped bacteria M. tuberculosis and Corynebacterium glutamicum have also been seen by others (Daniel and Errington, 2003; Dahl, 2004; Letek et al., 2008). Inactivation of Min- and SlmA/Noc-activity in model rod-shaped bacteria, E. coli and B. subtilis, results in division site placement at the poles (min-) and over the nucleoid(s) (slmA-, noc-, under certain conditions) giving rise to phenotypes including DNA-less mini-cells, cell filamentation and frequent bisection of the nucleoids (Bernhardt and de Boer, 2005; Lutkenhaus, 2007; Wu and Errington, 2011). In contrast, Mycobacterium spp. continues to produce viable cells albeit of unequal sizes despite random placement of the FtsZ-ring. Evidently, Mycobacterium spp. has evolved mechanisms to cope with absence of these regulators. Here, we show that directional DNA translocation and unequal polar growth are two novel tools acquired by these rod-shaped bacteria to compensate for the absence of controls to guide the division septa to mid-cell region in co-ordination with nucleoid segregation. The post-septal translocation of the nucleoid into the smaller compartment of the cell before cytokinesis prevents production of DNA-less cells or cells with a guillotined chromosome (Fig. 4). We demonstrate that the FtsK orthologue, MSMEG2690, is predominantly associated with septa in both equal and unequal divisions. This suggests that DNA translocation might be an integral part of the vegetative cell division apparatus in Mycobacterium spp. Furthermore, our results also attest to the identification of MSMEG2690 as a DNA translocator. In the time-lapse study covering several growth and division cycles, the unequal daughter cells appeared to grow and divide at different rates (Fig. 6). We speculate that the bigger cell underwent division earlier than the smaller sister as it reached the critical size for initiation of DNA synthesis from oriC (initiation mass) earlier. The cytokinesis step was also delayed until the duplicated chromosomes were partitioned into the two unequal compartments created by asymmetric division. Thus, checkpoints for co-ordinating replication, segregation, division and cell growth in Mycobacterium spp. might be very different from those of the well-known model systems. Instead of guiding the division site to mid-cell between segregated nucleoids, the mycobacterial cell adjusts cellular growth and DNA translocation to compensate for the randomness of the division site placement.

Non-medial septation, resulting in asymmetric cell division, has been reported in exponentially growing cells of M. tuberculosis and M. smegmatis (Thanky et al., 2007), and upon overproduction of Par proteins in M. tuberculosis (Maloney et al., 2009). In agreement with Thanky et al. (2007), our data suggest that randomness of septum position in mycobacterial populations is a part of their normal cell-cycle physiology and not a result of defective divisome assembly; Par overproduction may merely accentuate it further (Maloney et al., 2009). Our time-lapse images of individual M. smegmatis cells measured growth by the increase in total length and could not distinguish between uni- or bipolar growth until the appearance of the division septum; unequal growth at the two poles relative to the septum became obvious only during the second and subsequent rounds of cell division (Fig. 6). Asymmetric elongation at poles has been directly measured at a single cell level, which showed unipolar growth with a preference for growth at the old pole (Aldridge et al., 2012). Our time-lapse experiment measuring total extension of individual cells could neither confirm nor contradict these results until the appearance of the first division site. However, the FL-Vanco labelling showed that mycobacterial cells undergo bipolar as well as unipolar growth (Fig. 2A).

In conclusion, the novel features of mycobacterial cell division that distinguish it from those of the model rod-shaped bacteria can be summarized as follows: (i) imprecise placement of the Z-ring, resulting in asymmetric septa, (ii) absence of nucleoid occlusion, i.e. septum placement independent of nucleoid position, (iii) active translocation of chromosomal DNA during segregation, (iv) unequal growth at the two poles, and (v) different rates of growth for unequal siblings. Altogether, these characteristics suggest very different strategies for binary division and its control in Mycobacterium spp. They may also contribute to its unique pathogenic and virulence properties, although confirmation of this awaits further investigations.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Strains and growth conditions

Strains and plasmids are listed in Table 1. Strains BSm103 (from Dr Björn Herrmann, Uppsala University Academy Hospital, Uppsala) and BSm106 (LGC Promochem) were the wild-type M. marinum and M. smegmatis strains respectively. Strain DSM43239 was the wild-type M. phlei obtained from DSMZ strain collection of Germany. Strains BSm131 and BSm156 were derivatives of Bsm106. These were constructed by integrating plasmids pBS101 and pBS104, respectively, at the phage integration site attB on the Bsm106 chromosome using the integration vector pPGSY bearing attP. RG1Mm and RG1Msm were made by transforming M. marinum and M. smegmatis, respectively, with plasmid pRG1Zm carrying a FtsZ–mCherry fusion (Shaner et al., 2004) under tetracycline control. See Supporting information for the construction of the plasmids and Table S1.

All mycobacterial (M. marinum, M. smegmatis and M. phlei) strains were grown on 7H10 agar and in 7H9 broth supplemented with 0.5% glycerol, 10% oleic acid albumin dextrose complex (OADC). Additional supplements were 0.05% Tween80 for M. marinum strain BSm103, 25 μg ml−1 kanamycin for M. smegmatis strains BSm131 and BSm156. The FtsZ–mCherry was induced by tetracycline (0.5–1 μg ml−1) followed by 3–6 h growth in 7H9 + OADC medium containing 100 μg ml−1 hygromycin.

The E. coli strain with FtsZ–GFP, BS532, was made by transforming E. coli MG1655 with plasmid pEG12 (kindly supplied by Dr Elisa Galli, Newcastle University, UK). E. coli (Laboratory stock) and B. subtilis (from Bacillus Genetic Stock Center, Ohio, USA) were grown on LB agar or in LB broth with appropriate antibiotics where needed. Liquid cultures were grown with continuous shaking at 90 r.p.m. All incubations were done at 30°C.

Electroporation

Derivatives of the integration vector pPGSY were introduced into competent mycobacterial cells by electroporating 1 μg of plasmid DNA at 2.5 kV, 25 μF and 1000 Ω using a pre-chilled 0.2 cm gap cuvette. The electroporation mixture was removed immediately after electroporation, mixed with 2 ml of 7H9 medium supplemented with 10% OADC and grown overnight; positive transformants were selected on 7H10 plates containing appropriate antibiotics.

Fluorescence microscopy

Cells in exponential growth phase, following staining (see below), were spotted on microscope slides covered with thin layers of 1% agarose in 0.9% NaCl. A Zeiss microscope (Axioplan 2) with a CCD camera linked to a computerized image analysis system was used for the micrographs. Images were analysed using the program Axiovision 4.7. Surface intensity plots were drawn using Image J software. Filters used for GFP and mCherry fluorescence were HQ480/40 (480 ± 20 nm) and ET-Texas Red (550–630 nm) respectively.

FM4-64 staining

Exponentially growing cells were washed from their growth medium and resuspended in 200 μl of PBS containing 0.1 μg ml−1 FM4-64FX® [N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylammino) phenyl) hexatrienyl) pyridinium dibromide] from Molecular Probes/Invitrogen. Cells were incubated for 20–30 min before microscopic examination using the HC F36-504 filter.

For time-lapse studies, 0.1 μg ml−1 FM4-64 was added to the 1% agarose-layer 7H9 broth media supplemented with OADC. The agarose-pad was infused with fresh 7H9-OADC broth containing 0.1 μg ml−1 FM4-64, at regular intervals, by capillary action.

FL-Vancomycin staining

Cells growing in broth were harvested during exponential growth phase, washed and resuspended in PBS. BODIPY® FL Vancomycin (FL-Vanco) from Molecular Probes/Invitrogen was added to the resuspended cells to a final concentration of 1 μg ml−1 together with non-fluorescent Vancomycin (1 μg ml−1). Cells were incubated with FL-Vanco in PBS for 20–30 min and examined under the microscope using HQ480/40 filter.

DAPI staining

Live cells from exponentially growing cultures were incubated with 0.5 μg ml−1 DAPI (4′, 6-diamidino-2-phenylindole; SIGMA) and examined using a D360/40 filter set.

Transmission electron microscopy (TEM)

Cells from exponentially growing cultures of M. marinum, M. smegmatis and M. phlei were fixed in 2.5% glutaraldehyde. Fixed cells were processed for TEM as described elsewhere (Ghosh et al., 2009).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We dedicate this report to the memory of Dr Kurt Nordström. We thank SIDA/SAREC, the Swedish Research Council for Environment, Agricultural Sciences, and Spatial Planning (FORMAS), the Swedish Research Council (Medicine), the Wennergren Foundation and the Söderberg Foundation for providing financial support for this work. We are thankful to Dr Kurt Nordström for his discussions on cell division in bacteria. We thank Dr Lee W Riley, Dr Lucia P Barker and Dr Malini Rajagopalan for kindly providing strains, plasmids and antibodies; Dr W. Margolin for the plasmid with FtsK–GFP, and Dr Norbert Vischer for providing Image J software. Leif A. Kirsebom is on the board of directors of Bioimics AB. Leif Kirsebom and Santanu Dasgupta are the holders of Swedish patent application number PCT/SE2008/051486.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting information

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