Department of Bacteriology, Swedish Institute for Infectious Disease Control, 171 82, Solna, Sweden1
Department of Cell and Molecular Biology, Biomedical Center, Uppsala University, Box 596, S-751 24, Uppsala, Sweden2
Author for correspondence: Kurt Nordström. Tel: +46 18 471 45 26. Fax: +46 18 53 03 96. e-mail: Kurt.Nordstroem{at}icm.uu.se
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ABSTRACT |
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Keywords: cell size, min mutants, minicells
a Thomas
kerlund and Björn Gullbrand contributed equally to this work.
b Present address: Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, CA 94720, USA.
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INTRODUCTION |
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The Min system consists of three proteins, MinC, MinD and MinE (encoded by the genes minC, minD and minE, respectively, forming the minB operon) and directs cell division to the mid-cell by a mechanism that is independent of the nucleoid. Overexpression of MinC and/or MinD results in a block of cell division, whereas overexpression of MinE leads to a phenotype similar to that of a strain lacking all three min genes, including the formation of polar septa and a broad cell size distribution (de Boer et al., 1989 ). These observations suggest that MinC and MinD acts to block cell division in a non-specific manner and that MinE is required to relieve this inhibition at the mid-cell. MinC has since been shown to interact directly with FtsZ and thereby inhibit the formation of stable FtsZ rings (Hu et al., 1999
). MinD is a peripheral membrane protein with ATPase activity that binds MinC and recruits it to the cell membrane (de Boer et al., 1991
; Hu & Lutkenhaus, 2000
). In the absence of MinD, a 25-fold increase in the level of MinC is required to inhibit cell division, suggesting that MinD activates MinC by increasing its concentration at the cell membrane (de Boer et al., 1992
). Remarkably, all three Min proteins have been shown by GFP fusions to oscillate between the two cell halves (Hu & Lutkenhaus, 1999
; Raskin & de Boer, 1997
, 1999a
, b
; Hale et al., 2001
). MinCD forms a membrane-associated cloud, extending inwards from one cell pole at a time, and MinE forms a ring-like structure, probably associated with the innermost edge of the MinCD cloud. MinE has been shown to stimulate the ATPase activity of MinD under certain conditions in vitro (Hu & Lutkenhaus, 2001
). As it is probably only the ATP-bound form of MinD that associates with the membrane, MinE might cause the dissociation of MinD from the membrane by stimulating the ATPase activity of MinD (de Boer et al., 1991
; Hu & Lutkenhaus, 2001
). The oscillation means that the time a certain site along the membrane is occupied by MinC increases with its distance from the cell centre. This might explain how the Min system inhibits cell division at sites away from the mid-cell.
Strains lacking a functional Min system have disturbed nucleoid segregation (Jaffé et al., 1988 ; Mulder et al., 1990
;
kerlund et al., 1992
) and there are several possible explanations for this disturbance. For example, polar divisions might interfere with the partition system. Alternatively, a delayed localization of FtsZ to the cell centre, caused by FtsZ polymerization at the cell poles, might delay the recruitment of FtsK to the cell centre, which is required for the resolution of chromosome dimers (Boyle et al., 2000
; Recchia et al., 1999
). Results obtained here argue against these explanations, leaving open the possibility that the Min proteins might directly affect nucleoid segregation (
kerlund et al., 1992
). We found that nucleoid distribution in a strain deleted for all min genes (EC1005
minB) was more disturbed during growth in minimal medium than in rich medium, and the more disturbed nucleoid segregation was associated with a higher frequency of polar septa. Overexpression of MinE impaired nucleoid segregation in cells devoid of polar septa. Overexpression of MinD, on the other hand, had no effect on nucleoid distribution, despite blockage of cell division.
The random selection of division sites in min mutants (Gullbrand & Nordström, 2000 ; Teather et al., 1974
; Yu & Margolin, 1999
), together with a limited division potential (Donachie & Begg, 1996
; Gullbrand & Nordström, 2000
), has been predicted to result in a population of cells with any number of nucleoids (1, 2, 3...) (Donachie & Begg, 1996
; Jaffé et al., 1990
). However, Jaffé et al. (1990)
found that the clear majority of cells in a min mutant population contained 2n origins of replication (n=0,1,2...). Measurements performed here on nucleoid segregation, DNA content and cell size distribution suggest an explanation for this controversy.
In the present communication we present a novel method to study disturbances in nucleoid segregation and use it to compare nucleoid segregation in a minB mutant and its wild-type parent. We also present studies of the interdependence of minicell production and cell elongation when a Min- phenotype is induced.
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METHODS |
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Microcolony experiments (kerlund et al., 1992
).
For growth of microcolonies, 10 µl of an exponentially growing culture was put on a microscope slide covered with the same growth medium with the addition of 1% agar. The microscope slides were incubated in a moisture chamber at 37 °C for 2 h before inspection by phase-contrast microscopy in a Nikon Optiphot-2 microscope. Images were digitized using a cooled CCD-camera (Meridian) and the cell lengths were measured with Image Analysis version 1.80 software (Bergström Instrument). For induction of MinE, 1 mM IPTG was added to the cultures 10 min before cells were transferred to the agar surface, and 1mM IPTG was also included in the agar medium.
Microscopic examinations (kerlund et al., 1992
).
Samples from exponentially growing cultures were fixed in 70% ethanol, centrifuged and resuspended in 0·9% NaCl, and spread on microscope slides covered with a flat surface of 1% agar containing 0·9% NaCl. The cells were observed by phase-contrast in a Nikon Optiphot-2 microscope and the projected cell area (µm2) was measured using software from Bergström Instrument. Inter-nucleoid distances were measured using the following method. First, wild-type and min mutant cells within a size range corresponding to a wild-type mother cell were chosen for each growth medium (see legend to Fig. 1). Next, the fluorescence intensities of the nucleoids in DAPI-stained cells of this size group were outlined using a grey-scale profile plot tool provided in the image analysis software. To account for differences in fluorescence intensities between cells, the mean of the peak intensities of the nucleoids located at about 1/4 and 3/4 of the total cell length was set to 1. Cells with deep constrictions were omitted from the analysis.
Flow cytometry analysis.
For flow cytometry, 1 ml cell culture was centrifuged at 15000 g for 7 min, resuspended in 0·35 ml 0·9% NaCl and fixed by the addition of 1 ml 95% ethanol. Samples were stored at 4 °C. Before analysis, the cells were centrifuged at 4 °C, washed in 1 ml 10 mM Tris buffer (4 °C, pH 7·5, adjusted at room temperature) containing 10 mM MgCl2, centrifuged again and resuspended in an appropriate amount of the same buffer. The DNA was stained by mixing 65 µl of the cell suspension with 65 µl buffer containing ethidium bromide (40 µg ml-1) and mithramycin A (200 µg ml-1). Samples were analysed on a Bryte-HS flow cytometer (Bio-Rad), calibrated with fluorescent beads. Light scatter and fluorescence signals were divided into 256 channels each and the number of particles in each channel was monitored by WinBryte version 2.03 software (Bio-Rad). At least 75000 cells were measured in each sample. The number of chromosomes represented by the positions of the peaks shown in Fig. 5 were determined by comparisons to the peak positions obtained from cultures grown to stationary phase and which were known to contain cells with one chromosome (Gullbrand & Nordström, 2000 ).
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RESULTS |
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Nucleoid segregation is impaired by overexpression of MinE
Overexpression of MinE in a wild-type strain causes a Min- phenotype (de Boer et al., 1989 ). By inducing MinE expression from a MinE expression vector (pDB156), we could therefore investigate whether the characteristics of the Min- phenotype appeared in any specific order (formation of polar septa, a decreased frequency of non-polar septa and a disturbed nucleoid segregation).
In the microcolony experiments, populations of strain EC1005(pDB156) were grown exponentially in M9gluc medium. To induce MinE synthesis, IPTG (1 mM) was added and after further incubation for 10 min samples were diluted and plated on microscope slides containing the same medium with 1 mM IPTG. The plated cells were allowed to grow for 2 h on the plates before examination under the microscope. During this period, only a few cell doublings occurred (see footnote to Table 2).
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Nucleoid segregation is not affected by overexpression of MinD
Localization of FtsZ to the cell centre is required for recruitment of FtsK to the cell centre (Yu et al., 1998 ), and FtsK in turn is essential for the resolution of chromosome dimers (Boyle et al., 2000
; Recchia et al., 1999
). Since min mutants have a decreased frequency of non-polar divisions and thereby possibly a higher frequency of chromosome dimers, it is possible that the impaired nucleoid segregation observed in min mutants reflects a higher frequency of chromosome dimers. Here, we investigated nucleoid segregation after overexpression of MinD, which blocks cell division, probably at the stage of FtsZ polymerization (Hu et al., 1999
) and thus prevents the resolution of chromosome dimers. MinD overexpression was induced from the expression vector pDB164 (de Boer et al., 1989
) with 1 mM IPTG. As expected, the cell size increased and the frequency of constricting cells decreased (Fig. 4a
, b
). The distribution of Y values at 40 min after induction was nearly identical to that of the non-induced culture (Fig. 4c
). Thus, overexpression of MinD and inhibition of cell division, and thereby inhibition of resolution of chromosome dimers, had no affect on nucleoid segregation, as measured here.
Division pattern in min mutants
A random selection of division sites in combination with a limited division potential in a minicell-producing strain has been predicted to lead to the production of cells with any number of nucleoids (Donachie & Begg, 1996 ; Jaffé et al., 1990
). The finding of Jaffé et al. (1990)
that very few cells in a
minB strain contained a number of origins of replication not equal to 2n (n=0, 1, 2...) is therefore surprising. This result was found by flow cytometry during analysis of the number of chromosomes per cell after a replication runout, where each chromosome represented one origin of replication before the runout. Replication runout was achieved by inhibition of initiation of replication by addition of rifampicin and cell division was inhibited by addition of cephalexin. Using the same approach, we obtained a similar result for EC1005
minB grown in M9gluc; 89% of the cells contained 2n origins of replication per cell, as compared to 93% for EC1005 (Fig. 5
). Even higher proportions of cells containing 2n origins of replication were obtained for EC1005
minB and EC1005 grown in M9glycerol and LBgluc. Thus, the random selection of division sites in EC1005
minB only lead to a very small increase in the proportion of cells with a number of origins not equal to 2n.
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About 36% of the cells of EC1005minB contained eight or more origins of replication, which corresponded to a cell length of 4·8 µm or more. The mean cell length for cells of EC1005 was 2·8 µm and this would correspond to a cell length of 2·1 µm for newborn cells, on the assumption that the cell-size distribution of EC1005 was ideal for an exponentially growing population. If a non-polar cell-division event in EC1005 and EC1005
minB required the same increase in cell length, non-polar, non-central division events in EC1005
minB would not occur until a cell length of 4x2·1 µm=8·4 µm was reached. Since the nucleoid segregation was markedly delayed in EC1005
minB grown in M9gluc, this is probably an underestimation of the minimum cell length at which cell division could occur at the cell quarter positions. It is therefore likely that there was a substantial period of time after the cells had gained eight origins of replication, during which they could not divide at the cell quarter positions.
The actual distributions of septa for the populations shown in Fig. 5 were estimated by microscopy and are shown in Fig. 6
. Although a majority of cells longer than 4·8 µm had central septa (56% of the non-polar septa in cells longer than 4·8 µm were found within 10% of the cell length from the cell centre), many cells shorter than 8·4 µm had non-polar, non-central septa. The presence of these septa might in part be explained by the fact that some of these cells had recently undergone cell division and before that had been longer than 8·4 µm. Another explanation might be that the cell centre was less exactly determined in the absence of the Min system. It seems, however, that the observed frequency of non-polar, non-central divisions would lead to a higher frequency of cells with six origins of replication, than what was obtained by flow cytometry (2·7%, Fig. 5
). This discrepancy might in part be due to a shorter interdivision time after divisions at, e.g., quarter positions. This seems reasonable since when division occurs at a cell quarter position, nucleoid segregation can be expected to be relatively far advanced also at the other quarter position and, if not permanently blocked, also at the cell centre.
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DISCUSSION |
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We used a novel method to quantify nucleoid segregation in the wild-type and a minB mutant. The comparison was made with cells in the size range of dividing wild-type cells. It should be stressed that the growth rate was the same for the wild-type and the
minB mutant. We found that nucleoid segregation was delayed in the
minB mutant in the three different media used. We further found that nucleoid segregation in EC1005
minB was more disturbed during growth in minimal medium than in rich medium. The more disturbed nucleoid segregation in minimal medium was associated with an increased frequency of polar septa and a broader cell-size distribution. Although the ability to form polar septa was caused by the absence of a functional Min system, we speculate that the increased frequency of polar septa seen in minimal medium was caused by the more disturbed nucleoid segregation. This might have been due to unsegregated nucleoids at the cell centre, forcing cell division to occur at the cell poles. As disturbances in nucleoid segregation were observed before polar septa after induction of MinE, it seems less likely that the more disturbed nucleoid segregation in EC1005
minB grown in minimal medium was caused by the increased frequency of polar septa. The larger deviation from the wild-type cell-size distribution of EC1005
minB in minimal medium might have been caused by both the more disturbed nucleoid segregation and the increased frequency of polar septa.
Although the effect of growth medium on nucleoid segregation in EC1005minB is striking, there seems to be no obvious explanation for this effect. Nucleoids in cells grown in a rich medium appear to be divided into distinct lobules, as visualized by DAPI staining. No such structures can be seen for cells grown in minimal medium, where the nucleoids seem to fill out the cells uniformly. It is possible that these differences in apparent nucleoid structure could somehow directly affect the results of our measurements and/or nucleoid segregation in the absence of a functional Min system.
Overexpression of MinD inhibited cell division, but had no detectable effect on nucleoid segregation. Since cell division is required for the resolution of chromosome dimers (Boyle et al., 2000 ; Recchia et al., 1999
; Yu et al., 1998
), it is probable that the disturbances in nucleoid segregation observed in min mutants are not caused by an impaired resolution of chromosome dimers. Also, the disturbances in nucleoid segregation did not require the formation of polar septa. It is thus possible that the disturbed nucleoid segregation is a direct effect of a non-functional Min system. If true, both the absence of all Min proteins, as well as overexpression of MinE, but not of MinD, would directly cause the segregation disturbances. Both the nucleoid and MinD are assumed to interact with the cell membrane (de Boer et al., 1991
; Nanninga, 1998
). It is thus possible that the absence of MinD or some disturbances in its localization would cause the segregation disturbances. From studies on Min+ strains expressing either gfp-minD or gfp-minDE, the oscillation frequency of MinD was suggested to be inversely related to the MinD:MinE ratio (Raskin & de Boer, 1999a
). However, the expression levels used in this previous study might have been much lower than what was used here, as judging from the different phenotypes obtained. We therefore cannot assume any similar relationship between the oscillatory movement of MinD and the MinD:MinE ratios obtained here.
As shown in Fig. 4(c), overexpression of MinD and inhibition of cell division, and thereby inhibition of resolution of chromosome dimers, had no effect on nucleoid segregation, as measured here. It is possible that the resolution of chromosome dimers occurs too late in the cell cycle to produce a detectable effect on nucleoid segregation in cells of the sizes measured here. Another factor contributing to the failure to detect chromosome dimers could be that dimers form in only 14% of all cells (Steiner & Kuempel, 1998
; Pérals et al., 2000
).
Chromosome condensation has recently been suggested to play an important role in nucleoid segregation, by preventing the entanglement of newly replicated DNA and perhaps also by providing a force pulling the two strands of DNA in opposite directions (Nordström & Dasgupta, 2001 ). The effects seen here on nucleoid segregation might thus be caused by an impaired chromosome condensation in cells exhibiting the Min- phenotype. Indeed, Mulder et al. (1990)
found a correlation between the level of Min proteins and DNA supercoiling. It is also possible that the degree of superhelicity directly affected the results of our measurements, by affecting nucleoid expansion.
Division potential has been assumed to accumulate as cells grow (Teather et al., 1974 ) and the major limiting factor for cell division might be FtsZ (Bi & Lutkenhaus, 1990
). The number of septa per unit cell length has been found to be similar in Min+ and Min- strains (Donachie & Begg, 1996
), and an increase in cell length corresponding to one newborn Min+ cell has therefore been assumed to be required for each cell division. Polar and non-polar division events are assumed to consume equal amounts of division potential and polar division events would thus lead to an increase in the mean cell length for the DNA-containing cells. [It should, however, be noted that this simple quantal behaviour of cell division in minB mutants was questioned by
kerlund et al. (1992)
, who reported data suggesting that polar and central divisions are not mutually exclusive.] As cells get longer, more potential division sites would appear and a random selection between these division sites would lead to a population of cells of various lengths and various numbers of origins of replication. Here, the increase in mean cell length for EC1005
minB grown in M9gluc, as compared to the corresponding wild-type, was even larger than that predicted for a minicell-producing strain (Donachie & Begg, 1996
). [Also, these results are not in accord with the quantal theory of Teather et al. (1974)
]. Still, most non-polar divisions were central and most cells contained 2n origins of replication. Analyses performed here showed that there might be a long period of time during which cells with eight origins of replication could divide polarly or centrally, but not non-polarly/non-centrally, which could at least partially explain the low frequency of cells with six origins of replication. The minimum cell length for non-polar, non-central divisions was estimated to be 8·4 µm from the cell-size distribution of EC1005. If the delayed nucleoid segregation in EC1005
minB delayed cell division, however, this minimum cell length might have been even larger in many cells. Although the cell-size distribution of EC1005
minB was broader than predicted for a minicell-producing strain, few cells (10%) were long enough to divide non-polarly/non-centrally, as estimated from the cell-size distribution of EC1005.
A random selection of division sites and an equal delay in nucleoid segregation at central and non-polar, non-central sites would mean that non-polar, non-central septa would start to appear at a cell length double the length at which central septa started to appear. Here, a few non-polar, non-central septa were found in cells that were shorter than double the length at which central septa started to appear (Fig. 6). At least some of these cells might recently have undergone cell division, which would at least partially explain the occurrence of non-polar, non-central septa in these relatively short cells.
The size range at which central septa occurred was much larger in EC1005minB than in EC1005 (Fig. 6
). The smaller size range in EC1005 could have been caused by the Min system inhibiting cell division at the cell centre in cells above a certain length. This would be predicted from the observation that MinCD oscillates around the cell quarter positions instead of around the cell centre in cells above a certain cell length (Hu & Lutkenhaus, 1999
; Raskin & de Boer, 1999a
), inhibiting cell division at the cell centre. Accordingly, Min+ cells above a certain length have been found to divide at cell quarter positions rather than at the cell centre (Bernander et al., 1995
; Botello & Nordström, 1998
).
In this paper, we have presented further quantitative evidence that the Min- phenotype is characterized by impaired nucleoid segregation.
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ACKNOWLEDGEMENTS |
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Received 18 February 2002;
revised 31 May 2002;
accepted 24 June 2002.
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