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Journal of Bacteriology, August 2006, p. 5958-5965, Vol. 188, No. 16
0021-9193/06/$08.00+0     doi:10.1128/JB.00524-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Relationship among Several Key Cell Cycle Events in the Developmental Cyanobacterium Anabaena sp. Strain PCC 7120

Samer Sakr,¹ Melilotus Thyssen,² Michel Denis,² and Cheng-Cai Zhang^1*

Laboratoire de Chimie Bactérienne, CNRS-UPR 9043, Institut de Biologie Structurale et Microbiologie, 31 Chemin Joseph Aiguier, 13402 Marseille cedex 20, France,¹ Laboratoire de Microbiologie, Géochimie et Ecologie Marines, CNRS-UMR 6117, Université de la Méditerranée, Centre d'Océanologie de Marseille, 163 avenue de Luminy, Case 901, 13288 Marseille cedex 09, France²

Received 12 April 2006/ Accepted 31 May 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
When grown in the absence of a source of combined nitrogen, the filamentous cyanobacterium Anabaena sp. strain PCC 7120 develops, within 24 h, a differentiated cell type called a heterocyst that is specifically involved in the fixation of N₂. Cell division is required for heterocyst development, suggesting that the cell cycle could control this developmental process. In this study, we investigated several key events of the cell cycle, such as cell growth, DNA synthesis, and cell division, and explored their relationships to heterocyst development. The results of analyses by flow cytometry indicated that the DNA content increased as the cell size expanded during cell growth. The DNA content of heterocysts corresponded to the subpopulation of vegetative cells that had a big cell size, presumably those at the late stages of cell growth. Consistent with these results, most proheterocysts exhibited two nucleoids, which were resolved into a single nucleoid in most mature heterocysts. The ring structure of FtsZ, a protein required for the initiation of bacterial cell division, was present predominantly in big cells and rarely in small cells. When cell division was inhibited and consequently cells became elongated, little change in DNA content was found by measurement using flow cytometry, suggesting that inhibition of cell division may block further synthesis of DNA. The overexpression of minC, which encodes an inhibitor of FtsZ polymerization, led to the inhibition of cell division, but cells expanded in spherical form to become giant cells; structures with several cells attached together in the form of a cloverleaf could be seen frequently. These results may indicate that the relative amounts of FtsZ and MinC affect not only cell division but also the placement of the cell division planes and the cell morphology. MinC overexpression blocked heterocyst differentiation, consistent with the requirement of cell division in the control of heterocyst development.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The filamentous cyanobacterium Anabaena sp. strain PCC 7120 is able to use a source of combined nitrogen, such as nitrate or ammonium, and to fix N₂ through a process catalyzed by the nitrogenase complex (24, 42). Nitrogenase is confined within a particular cell type called a heterocyst, which is differentiated from a vegetative cell in its response to the deprivation of combined nitrogen in the growth medium. Heterocysts are distributed in a semiregular pattern along each filament and represent about 5 to 10% of all cells. The differentiation process of heterocysts is regulated by multiple signals (47). Recent studies indicate that the level of 2-oxoglutarate is a signal of nitrogen deprivation and that the experimental addition and accumulation of a nonmetabolizable analogue of 2-oxoglutarate are enough to trigger heterocyst development, even in the presence of ammonium (22). The level of free calcium ions is also modulated during heterocyst development, and a low level of calcium ions prevents heterocyst formation (36, 48).

It has been proposed that the cell cycle could be involved in the process of heterocyst development (1, 33). Heterocysts are terminally differentiated cells, unable to divide again. The expression of FtsZ, a key component in the initiation of bacterial cell division, is downregulated in heterocysts (21, 38). The hetC mutant is unable to stop the division of proheterocysts, resulting in chains of small proheterocysts which still express the ftsZ gene, in contrast to proheterocysts of the wild-type filaments (38, 44). Although it is expected that DNA replication no longer occurs in heterocysts, DnaE, one component of the DNA polymerase, is still present in heterocysts (39). This finding may underlie the requirement for the DNA replication machinery in processes such as DNA recombination and DNA repair, which may still be necessary in heterocysts. Consistent with the complex relationship between the cell cycle and heterocyst development, the inhibition of cell division leads to the absence of heterocyst differentiation (33). When Anabaena sp. strain PCC 7120 cells are treated with the antibiotic aztreonam, targeted at FtsI or the expression of sulA, which encodes an inhibitor of the GTPase activity of FtsZ, cells on filaments appear in elongated forms as a result of the inhibition of cell division. When the inhibition of cell division is relieved, cell division occurs first, before heterocyst differentiation resumes normally (33). In addition to FtsZ, which recruits more than 10 proteins to assemble in the septum and drives the process of bacterial cell division, three proteins, MinC, MinD, and MinE, constitute a regulatory system for the proper placement of the FtsZ ring at the midcell position (13, 31, 40). In Escherichia coli, the constantly oscillating behavior of these proteins along the axis of the cell length ensures that only the cell division site at the midcell position is available for FtsZ to form a ring structure (29). MinC interacts with FtsZ and inhibits the polymerization of FtsZ, while another cell division inhibitor, SulA, inhibits the GTPase activity of FtsZ (18, 26, 37).

Most cyanobacteria contain multiple copies of the chromosome. Anabaena sp. strain PCC 7120 may contain 10 to 20 copies of the same chromosome (17). The DNA content in spores or heterocysts in comparison to that in vegetative cells in cyanobacteria has been investigated, but the results were controversial (34). Using fluorescent dyes that target DNA and measuring the fluorescence intensities, the authors of one report found that heterocysts contained slightly less DNA than vegetative cells, regardless of the age of heterocysts (34). The cycle of DNA replication seems to differ according to the cyanobacterial strain investigated. For the freshwater unicellular strain Synechococcus sp. strain PCC 6301 (previously known as Anacystis nidulans), it has been proposed that the initiation of DNA replication is asynchronous, in contrast to the situation for E. coli or the marine cyanobacterium Synechococcus sp. strain WH-8101 (3, 5). In Synechococcus sp. strain WH-8101, two gaps of DNA synthesis during the cell cycle have been found, and the progression of the cell cycle is strongly influenced by light. It appears that the current model of the cell cycle based on E. coli is not suitable for cyanobacteria (3, 5). In E. coli, DNA replication occurs during a particular phase, the C period, during the cell cycle (7). In this organism, cells grown with a long generation time inherit a single copy of the chromosome, and cells with a short generation time may inherit more than one chromosome equivalent; even when several chromosome equivalents are present, a new round of DNA replication is initiated synchronously (7).

In this study, we have investigated several parameters of the cell cycle, such as DNA synthesis and cell division in Anabaena sp. strain PCC 7120, and compared the DNA contents of heterocysts and vegetative cells. Our results suggest that proteins involved in cell division may also regulate cell size and morphology in this cyanobacterium.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Strains and culture conditions. The culture conditions of Anabaena sp. strain PCC 7120 in BG11 medium with nitrate or BG11₀ medium without a source of combined nitrogen (30) were previously described (33). For strains containing the petE promoter, we used BG11 or BG11₀ medium without copper sulfate and took precautions to remove trace amounts of copper from glassware (6, 33), and 0.2 or 0.7 µM copper was added for the induction of gene expression. Conjugation was carried out as described previously (12, 41).

Heterocyst preparation, microscopy, and DAPI staining. Heterocysts were prepared as previously described (15, 21). A Nikon DXM 1200 digital camera mounted on a Nikon Eclipse E800 microscope was used to capture images. For DAPI (4'-6-diamidino-2-phenylindole) staining, cultures were incubated for 30 min in the presence of lysozyme (3 µg · ml^–1), rinsed twice in phosphate-buffered saline (0.85% NaCl, 10 mM Na₂HPO₄ · 2H₂O, pH 7.2), fixed for 1 hour at 4°C with 4% paraformaldehyde, and again rinsed twice in phosphate-buffered saline. Staining was performed by the addition of DAPI (2 µg · ml^–1) for 10 min.

Flow cytometry. To generate single cells of Anabaena sp. strain PCC 7120, cultures grown in BG11 medium were sonicated using a probe three times over 20 seconds and centrifuged at 3,000 rpm for 3 min to collect cells (the pellet) and cell debris (the supernatant). Cell fractions and supernatants were incubated for 15 min in the presence of PicoGreen (Molecular Probes; 1/100 dilution) and analyzed on a flow cytometer (Cytoron Absolute; Ortho Diagnostic Systems) equipped with an air-cooled 488-nm argon laser. The concentration of PicoGreen and the incubation time used in these experiments were determined following a titration assay using different concentrations of PicoGreen and different incubation times. Standard beads of uniform size (3.6-µm diameter) and fluorescence were added to each sample. Forward light scatter (FWS) was used as an indication of cell size, and DNA content was expressed as green fluorescence (FL1; 515 to 530 nm) after the sample was stained with PicoGreen (35). Data were collected and analyzed with Immunocount software (Ortho Diagnostic Systems) and Winlist software (Verity Software House). Removal of the background in the cell fraction cytograms was based on analysis of the supernatant. Additional statistical analyses were run with Microsoft Excel software.

Constructs of plasmids. The plasmid pRLFTS bearing the ftsZ coding region disrupted by the insertion of the element conferring resistance to spectinomycin and streptomycin was described previously (46). For the overexpression of minC (alr3455), the coding region of minC was amplified by PCR using the following pair of primers: MinC-f (5'-GGAATTCCATATGACTTCTGATTCTGCCATC-3') and MinC-r (5'-GGGGTACCGGATCCTTATGGTGTCTGATTGATCTT-3'). The DNA fragment was cloned into the NdeI and KpnI sites of the vector pBS-PetE (with the petE promoter region in the plasmid pBluescript [see reference 33]). The minC coding region together with the petE promoter was cloned into the BamHI site of the shuttle vector pRL25c, and the final construct was named pSS11b. For the translational fusion of ftsZ with gfp (a gene encoding the green fluorescent protein [GFP]) under the control of the promoter of ftsZ itself, the promoter region of ftsZ together with the coding region of ftsZ was amplified by PCR using the two following primers: FtsZ-f (5'-ATAAGAATGCGGCCGCCTTAATAGTTGACTCC-3' [the NotI site is underlined]) and FtsZ-r (5'-AAAACTGCAGATTTTTGGGTGGTCGCCG-3' [the PstI site is underlined]). The PCR fragment was cloned into the NotI and PstI sites of pBluescript. The gfp coding region was amplified as described previously (33) and cloned in frame with ftsZ using PstI and EcoRI. The whole insert was then moved into the shuttle vector pRL25c using NotI and EcoRI. The final construct was called pSS20b.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Correlation between cell length and DNA synthesis. To measure the DNA content and the size of individual cells by flow cytometry, it is necessary to break filaments of Anabaena sp. strain PCC 7120 into single cells. For this purpose, a culture at the exponential phase grown in the presence of nitrate, which suppresses heterocyst differentiation, was sonicated (Fig. 1). Microscopic observation of the cell fraction after sonication indicated that single cells, doublets, and small chains (three to four cells) represented 83% ± 4%, 15% ± 1.5%, and 2% ± 1.5%, respectively, of the cell fraction. Most of the doublets are possibly dividing daughter cells, judging from the constricting septa. Samples were centrifuged, and the pellet was first analyzed by flow cytometry. Cells were incubated in the presence of PicoGreen, which stained double-stranded DNA and generated green fluorescence for DNA quantification by flow cytometry (Fig. 2). Cell size and DNA content were measured based on FWS and FL1, respectively (35). As shown in Fig. 2A, the analysis of the data obtained was difficult due to the presence of particles or components that are relatively small. Since sonication also causes many cells to lyse, giving rise to cell debris and free DNA, which may interfere with the measurement, we also analyzed the supernatant after sonication and centrifugation. As shown in Fig. 2B, the data obtained from such a sample overlapped the data obtained using the pellet, as shown in Fig. 2A. We therefore considered the data shown in Fig. 2B to reflect cell debris and free DNA that were still present in the cell pellet and that were difficult to eliminate by washing and centrifugation (data not shown). For subsequent analysis, we used data obtained from the supernatant to subtract data corresponding to the cell debris, and the remaining data, which more likely reflected those from intact cells, are shown in Fig. 2C. These results indicate that FL1 increased linearly with FWS, indicating a strong correlation (R = 0.74; P < 0.001) between DNA content and cell size (Fig. 2D). These results suggest that DNA synthesis occurs during the whole cycle of vegetative cell growth in Anabaena sp. strain PCC 7120.

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FIG. 1. Anabaena sp. strain PCC 7120 cells before (A) and after (B) sonication.

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FIG. 2. Analysis of DNA content and cell size by using flow cytometry. The FWS and the green fluorescence (FL1) represent the relative size and DNA content of cells, in arbitrary units, respectively. (A) Cytogram obtained with the pellet fraction after sonication and centrifugation. (B) Cytogram obtained with the supernatant fraction after sonication and centrifugation (see Materials and Methods). (C) Cytogram as described for panel A, but the data corresponding to those of panel B were subtracted. Beads with diameters of 3.6 µm (gray) were used as the control. (D) Relationship between FWS and FL1 illustrating a link between cell size and DNA content.

DNA content in heterocysts and vegetative cells. We compared the relative DNA content of the whole cell population to that of purified heterocysts by using flow cytometry. The sum of the fluorescence intensities was divided by the number of particles counted by the flow cytometer, and this result was considered to reflect the relative amount of DNA in each cell on average. It was found that the relative DNA content in heterocysts on average was higher than that of the whole cell population (Fig. 3A). The average DNA content (indicated by FL1) of the heterocyst preparation was 81.4 ± 26.1 arbitrary units (AU), similar to that of the large cells, whereas it was 54.2 ± 23.6 AU for the average of the whole cell population. Eighty-five percent of the vegetative cells have a DNA content similar to the average content of heterocyst cells. These vegetative cells corresponded to the subpopulation of cells which were the largest in size; they presented a FWS value of 145.8 ± 1.4 AU, compared to 69.45 ± 1.46 AU for the entire population of vegetative cells on average.

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FIG. 3. DNA in heterocysts and vegetative cells analyzed by flow cytometry (A) and optical microscopy after DAPI staining (B and C). In panel A, cells before induction and purified heterocysts were analyzed by flow cytometry, and their FL1 values representing DNA content were compared. In panels B and C, filaments were induced for heterocyst differentiation, and 13 h after induction, DAPI staining was performed and filaments were visualized with a 60x objective under 340-nm fluorescence excitation (B) or bright-field microscopy (C). Arrows indicate proheterocysts.

The results of analysis by flow cytometry were consistent with those obtained by DAPI staining (Fig. 3B and C). Filaments were stained with DAPI at 13 and 24 h after nitrogen reduction, when proheterocysts and mature heterocysts, respectively, appeared. Two condensed nucleoids in each cell were observed for 66% of the proheterocysts (Fig. 3B) and for 71% of the mature heterocysts (data not shown). Forty-eight hours after the induction of heterocyst differentiation, DAPI staining revealed only one nucleoid in each of the heterocysts. These results suggest that proheterocysts have already finished DNA replication and that the chromosomes were segregated. At 48 h after the induction of heterocyst differentiation, since only one nucleoid was found in each these heterocysts, either a loss of one nucleoid occurred or the nucleoids had merged. The DNA contents in heterocysts 24 h or 48 h after the induction, as measured by flow cytometry, did not display significant differences. These results argue against an important loss of DNA in mature heterocysts and are consistent with those previously published (34).

Effect of inhibition of cell division on DNA content. To further understand the relationship between cell division and DNA synthesis, we compared the relative amounts of DNA content after the inhibition of cell division in Anabaena sp. strain PCC 7120.

We have shown previously that in Anabaena sp. strain PCC 7120, the expression of the E. coli gene sulA, encoding an inhibitor of the GTPase activity of FtsZ, blocked cell division and gave rise to elongated cells (33). The DNA content in cells overexpressing sulA for 48 or 96 h was analyzed by flow cytometry and compared to that of cells without induction of sulA. The mean value of cell fluorescence, as determined by flow cytometry, was used as an indication of the average DNA content per cell. While cells continued to elongate as a result of the inhibition of cell division, the relative fluorescence intensity per cell, reflecting the DNA content, remained stable (data not shown). Similar results were also obtained with filaments treated with aztreonam to inhibit cell division (data not shown) (33). These results suggest that once cell division was blocked, DNA synthesis was also arrested.

Analysis of the assembly of the FtsZ ring in the cell population. We have shown previously, using fusion to a GFP reporter, that FtsZ of Anabaena sp. strain PCC 7120 was able to form a ring-like structure at the midcell position, similar to that observed for E. coli (33). The ftsZ-gfp fusion used in that analysis was under the control of the heterologous promoter from the petE gene and carried on a plasmid called pSS10c. The expression of ftsZ-gfp was, thus, independent of the transcriptional control normally found in cells. In the present study, we made a construct in which the ftsZ-gfp fusion was under the control of the promoter of ftsZ itself. The plasmid bearing such a fusion, pSS20b, was transferred by conjugation into Anabaena sp. strain PCC 7120. As shown in Fig. 4A, only some cells bearing pSS20b had a ring-like structure, in contrast to the observation made using pSS10c, which led to the presence of a fluorescent ring-like structure in almost all cells. We have analyzed the cell size distribution of the whole population and correlated the cell size to the presence of the FtsZ-GFP ring at the midcell position. The cell lengths displayed a Gaussian type of distribution characteristic of a cell population in the exponential growth phase (Fig. 4B). The subpopulation of cells that clustered around a length of 2 or 2.5 µm did not exhibit a visible FtsZ ring structure (Fig. 4C). A small proportion (16%) of those cells that clustered around a length of 3 µm had an FtsZ ring, 52% of the cells that clustered around a length of 3.7 µm had an FtsZ ring, and all cells that clustered around a length of 5 µm, corresponding to the cell subpopulation with the longest cell length, had an FtsZ ring at the midcell position. These results indicated that the assembly of FtsZ into a ring structure was subjected to regulation during the cell growth phase, a situation similar to that observed for E. coli (32).

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FIG. 4. Analysis of the formation of the FtsZ ring in a strain bearing plasmid pSS20b containing a translational fusion of ftsZ and gfp under the control of the promoter region of ftsZ. (A) Fluorescent image of filaments. Ring-like structures are indicated by arrowheads. (B) Analysis of cell size distribution of the whole cell population showing the percentage of cells represented by each subpopulation. (C) Correlation between the presence of the FtsZ ring and cell size showing the percentage of cells with an FtsZ ring in each subpopulation.

Using antibodies against FtsZ (21) or a transcriptional fusion of gfp and the promoter region of ftsZ (38), it has been shown that ftsZ was developmentally regulated during heterocyst formation. In order to determine if FtsZ is regulated at a posttranscriptional level, we used the plasmid pSS10c bearing a translational fusion, FtsZ-GFP, driven by the heterologous promoter petE (33). In such a construct, the transcription of the fusion is uncoupled from the normal transcriptional regulation of ftsZ. The petE gene is transcribed in both vegetative cells and heterocysts, as shown by use of the gfp reporter (43); the petE transcript could be detected at similar levels in vegetative cells and heterocysts by semiquantitative reverse transcription-PCR (data not shown). Studies using microarrays based on DNA segments indicated that the cluster of four genes, including petE, showed a level of expression that was twofold lower than that in vegetative cells (11). It is not known how much, if any, of such a decrease could be attributed to petE expression.

Proheterocysts could be recognized by the staining of their polysaccharide layers with Alcian blue, about 12 h after the induction. As shown in Fig. 5, in the presence of the plasmid pSS10c, the FtsZ-GFP fusion formed a ring-like structure in most of the proheterocysts (96%). However, only a few of the proheterocysts (3%) still retained a FtsZ-GFP ring at the midcell position 19 h after the induction or had only a weakly recognizable ring structure (Fig. 5). In mature heterocysts formed 24 h or 48 h after induction, FtsZ-GFP was rarely found as a ring-like structure at the midcell position. These results suggest that the expression of ftsZ or the function of FtsZ is negatively regulated at a posttranscriptional level during the period between 12 h and 19 h after the induction of heterocyst differentiation.

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FIG. 5. FtsZ ring in proheterocysts and mature heterocysts in a strain bearing plasmid pSS10c containing a translational fusion of ftsZ and gfp under the control of the heterologous promoter petE. Filaments were visualized under bright-field or fluorescence microscopy at time points in hours after the induction of heterocyst differentiation. Proheterocysts formed 12 h or 19 h after induction were visualized by staining with Alcian blue, which is specific for heterocyst polysaccharide. Arrowheads indicate proheterocysts or heterocysts.

Overproduction of MinC in Anabaena sp. strain PCC 7120. The ftsZ gene is likely essential in cyanobacteria (23, 25, 46). In E. coli, the Min system, composed of MinC, MinD, and MinE encoded by an operon, is required for the proper placement of the FtsZ ring and to ensure that cell division occurs at the middle of the cell (8, 29, 31). In Anabaena sp. strain PCC 7120, minC (alr3455), minD (alr3456), and minE (alr3457) are found in the same gene cluster in the same orientation (19). In E. coli, the MinC protein inhibits the polymerization of FtsZ but has no effect on the GTPase activity of FtsZ (26). When MinC was overproduced, cell division was inhibited, leading to the formation of elongated cells (8). We cloned the minC gene of Anabaena sp. strain PCC 7120 in a shuttle vector under the control of the petE promoter inducible by copper (plasmid pSS11b). When first obtained and grown on plates after conjugation, the colonies contained mostly single cells with spherical shapes or two to six cells arranged in the form of a cloverleaf (Fig. 6A). Many cells were also lysed under such conditions. Since the amount of copper on the plate was difficult to control and the petE promoter could respond to even trace amounts of copper, these colonies were picked from plates, extensively washed with copper-free medium, and further cultured in liquid medium depleted of copper. As shown in Fig. 6B, cells recovered from such culture conditions demonstrated mostly the phenotypes of the wild-type strain, with long filaments and cells of normal shapes and sizes. A 0.7 µM concentration of copper was then added to such a culture, and cell morphology in the presence of copper was observed under a microscope at different time points (Fig. 6C to F). During the exponential growth phase, cells of the wild type were rather rod or oval shaped, and the average length and width of the cells were 3.4 µm and 2.6 µm, respectively. As shown in Fig. 4B, the subpopulation of the smallest cells averaged 2.5 µm in length by 2.4 µm in diameter, and those in the subpopulation of the largest cells averaged 5 µm in length by 3 µm in diameter. These results indicated that wild-type cells grew longitudinally along the axis of the filament. With the induction of P_petE-minC by copper, some cells started to become spherical (Fig. 6C and D), and 5 days after the induction, almost all cells had become spherical and had expanded in such a form (Fig. 6E). Further incubation under such conditions led to extensive cell lysis, but some cells were able to divide, resulting in several cells attached together in the form of a cloverleaf due to incomplete cell division, similar to cells cultured on plates (Fig. 6E). Therefore, when minC was overexpressed, cell division was inhibited but cells expanded in a round shape rather than a rod shape, as is normally found for the wild type or the strain overexpressing sulA (33). While ftsZ is likely essential (46), partially segregated mutants with both wild-type copies as well as inactivated copies of ftsZ presented phenotypes that were similar to those of the strain overexpressing minC, although the phenotypes were less pronounced (Fig. 6G).

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FIG. 6. (A to E) Phenotypes of a strain overexpressing minC under the control of the copper-regulated promoter petE. (A) Colonies grown on plates which may contain trace amounts of copper. (B) Filaments grown in a liquid medium without copper. (C to E) Filaments incubated with copper for 1, 3, and 5 days, respectively. (F) Examples of cells after 5 days of incubation in the presence of copper. (G) Phenotypes of a strain with partially inactivated ftsZ. The lower part of the panel shows four examples of cells forming a cloverleaf-like structure (G1 and G2), cells with irregular sizes on a filament (G3), and a tortured filament (G4). Bar, 5 µm.

After the induction of minC by copper for 2 days in the presence of nitrate as a nitrogen source, the filaments were transferred into a medium without nitrate. As shown in Fig. 7, no or very few heterocysts could be induced even after 2 days, whereas the wild-type filaments could form heterocysts 1 day after the induction. These results are consistent with the previous observation that inhibition of cell division suppresses heterocyst differentiation (33).

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FIG. 7. The strain overexpressing minC does not differentiate heterocysts. (A) The wild-type strain with heterocysts formed 24 h after deprivation of nitrate. (B and C) For the strain bearing pSS11b, copper was added over 2 days to induce minC expression, followed by the transfer of cells to a medium with copper but without nitrate for 24 h (B) or 48 h (C).

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the life cycle of a bacterium, there are several key processes: cellular growth, chromosome replication and nucleoid segregation, and septum formation and cell constriction. These processes must be coordinated in space and time, and they can be viewed as parts of a single cycle in which each step is dependent on the previous one. Alternatively, each step could be independently controlled and carefully tuned to the other steps without actually constituting a true cycle (10, 28). When E. coli cells fail to divide after the addition of cephalexin, a cell division inhibitor, or when fts mutants are grown at the nonpermissive temperature (2, 10, 28), they continue to replicate or segregate nucleoids. These results suggest the existence of parallel processes of the cell cycle (10, 28). In this study, we studied these several key events and their relationships in the cell cycle of Anabaena sp. strain PCC 7120. Our results suggest that while some events such as the formation of the FtsZ ring during the growth phase are similar to those observed for E. coli, other phenomena are different. In E. coli, while the relative amount of FtsZ is constant, the formation of the FtsZ ring starts to be found in cells halfway through the cell cycle (32). Using a translational fusion of ftsZ to gfp, we found that cells of a small size, presumably those that have just divided, or young cells of the cell cycle rarely contain a detectable FtsZ ring, whereas the FtsZ ring was more frequently found in cells of a bigger size. The expression of ftsZ was downregulated in heterocysts, based on immunodetection using antibodies against FtsZ (21) or the transcriptional fusion of gfp and the ftsZ promoter region (38). When the FtsZ-GFP fusion was produced under the control of a heterologous promoter, namely that of petE inducible by copper, the transcription should have bypassed the normal transcriptional control of ftsZ. In this case, we continued to observe the disappearance of the FtsZ ring in mature heterocysts. These results strongly suggest that FtsZ activity is controlled in heterocysts at a posttranscriptional level. These results are comparable to those reported for Caulobacter crescentus, in which the expression of ftsZ is controlled at multiple levels during the cell cycle (20).

Using flow cytometry to measure DNA content, we found that DNA synthesis was strongly correlated to the increase of cell size according to a linear relationship. This result may suggest that DNA synthesis occurs during the whole phase of cell growth. Our data cannot distinguish between the synchronous and asynchronous initiation of DNA replication, although data may favor the latter possibility. If the initiation of all copies of the chromosome were synchronous, one would have to assume that the replication period spans the entire phase of cell growth. These results are comparable to those obtained for Bacillus subtilis (16), except that the DNA content in B. subtilis ceases to increase at the late stages of the cell cycle, while that in Anabaena sp. strain PCC 7120 increases during the whole process of the cell cycle. The relative amount of DNA remains constant when cell division is arrested by either the expression of the cell division inhibitor SulA or the addition of the antibiotic aztreonam, suggesting that DNA synthesis is also inhibited under such conditions. It is possible that with Anabaena, the cell cycle can be viewed as a single integrated process, with interdependency of each step. Similar results were also reported for the unicellular cyanobacterium Microcystis aeruginosa (45). In contrast, when E. coli cells fail to divide as a result of the addition of the cell division inhibitor cephalexin or when fts mutants are grown at the nonpermissive temperature, they continue to replicate or segregate nucleoids (10, 28).

Our analysis indicated that heterocysts contained on average more DNA than vegetative cells but an amount comparable to that in the subpopulation of large vegetative cells. The implication of this finding is not clear at the moment. The DnaA protein, which is the initiator of bacterial chromosome replication (14), is downregulated in heterocysts (unpublished results), suggesting that heterocysts no longer replicate their DNA although DNA polymerase may be required for DNA repair or recombination in heterocysts (39). We do not yet know when DNA replication is arrested during heterocyst development. Judging from the data obtained with DAPI staining, two nucleoids were found in most proheterocysts 13 h after the induction. That suggests that DNA replication should have finished before this time point, with segregated nucleoids. Further initiation of DNA replication is unlikely because two nucleoids were detected in most mature heterocysts at 24 h after the induction, and significant changes were not detected in DNA content later on. As the FtsZ ring is eliminated from proheterocysts, cell division will not follow, and in this case, the segregated nucleoids may merge again in mature heterocysts.

FtsZ has a GTPase activity, and the binding and the hydrolysis of GTP are necessary for the dynamic assembly of FtsZ polymers (27, 31). Two proteins inhibit the activity of FtsZ in E. coli (4, 18, 26, 37). The first one is SulA, which is involved in the SOS response and inhibits either the binding or both the binding and the hydrolysis of GTP by FtsZ, depending on the concentrations of SulA used. The second protein is MinC, a component of the Min system, which prevents the formation of FtsZ rings at polar locations. MinC has no effect on the GTPase activity of FtsZ but inhibits its polymerization (26). When either the sulA gene of E. coli or the minC gene of Anabaena sp. strain PCC 7120 is overexpressed using the petE promoter in Anabaena sp. strain PCC 7120, cell division is inhibited in both cases, and no heterocyst can be formed. However, strong differences in cell shape are found between these two situations. When cell division is arrested, cells of the SulA-producing strain expanded in size in an elongated form and did not seem to alter the division plane (33), whereas cells of the strain overproducing MinC grew in size in a spherical form and divided, with possibly alternating division planes, leading to a cloverleaf formation of several attached cells (Fig. 6). By analogy with E. coli, in which MinC has no effect on the GTPase activity of FtsZ, these results may suggest that the GTPase activity of FtsZ is involved not only in cell division per se but also in the control of cell shape and the placement of the division plane in Anabaena sp. strain PCC 7120.

A good marker for following the progression of the cell cycle is still lacking in Anabaena sp. strain PCC 7120. Treatment with a dark/light cycle, a method successfully used to synchronize unicellular cyanobacteria, did not lead to a good synchronization in Anabaena sp. strain PCC 7120 (data not shown). In this case, we propose the use of cell size as a marker for cell cycle analysis in this organism. For a cell population at the exponential growth phase, cell sizes display a Gaussian type of distribution. In addition, DNA content increases linearly with cell length; thus, cells at different lengths have different amounts of DNA, reflecting their age during the cell cycle.

Cyanobacteria exhibit an extraordinary diversity in shape, morphology, and placement of the division plane, and these characters are used for cyanobacterial classification (30). Some strains are unicellular, whereas others are filamentous, with or without ramification. As shown in this study, by simply altering the ratio of FtsZ, the cell division protein, to MinC, a cell division inhibitor, we could transform Anabaena filaments into single, giant spherical cells. When such cells divide, they result in cells attached together in the form of a cloverleaf. Similarly, a single mutation could also transform a unicellular cyanobacterial strain into a filamentous one, as reported for Synechococcus sp. strain PCC 7942 (9). These results indicate that a small change in the amount or the activity of a single protein is enough to modify the morphology of a cyanobacterial strain.

 
We thank Annick Janicki for technical assistance and G. Gregori for help with flow cytometry.

We acknowledge support from the Lebanese National Council for Scientific Research (Samer Sakr, Ph.D. fellowship) and the PACA region (Melilotus Thyssen, Ph.D. fellowship). This work was financially supported by the CNRS, the "ATIP-Microbiologie" program, and the "Environnement et Santé" program (AFSSE).

 
* Corresponding author. Mailing address: Laboratoire de Chimie Bactérienne, CNRS-UPR 9043, Institut de Biologie Structurale et Microbiologie, 31 Chemin Joseph Aiguier, 13402 Marseille cedex 20, France. Phone: 33-4-91164096. Fax: 33-4-91718914. E-mail: cczhang{at}ibsm.cnrs-mrs.fr.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Bacteriology, August 2006, p. 5958-5965, Vol. 188, No. 16
0021-9193/06/$08.00+0     doi:10.1128/JB.00524-06
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