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Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Konoe-cho, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan
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 Top Abstract IntroductionResultsDiscussionConclusionsExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionConclusionsExperimental proceduresReferences |
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The SeqA protein is a negative modulator of replication initiation, and preferentially binds to hemimethylated GATC sequences (Lu et al. 1994). SeqA protein thus binds to newly synthesized hemimethylated DNA segments and forms clusters in the growing cells (for a review, see Hiraga 2000). Fluorescent foci of SeqA represent the localization of replication forks (Hiraga et al. 1998, 2000; Onogi et al. 1999; Brendler et al. 2000; Molina & Skarstad 2004). A single SeqA focus separates into two when chromosomal replication proceeds to one-fourth (Onogi et al. 1999; Hiraga et al. 2000; Yamazoe et al. 2005). Subcellular localization of the foci of β-subunit (DnaN/sliding clump) of DNA Pol III holoenzyme is similar to SeqA (Onogi et al. 2002). These results support the translocating replisome model: a pair of replisomes acting on clockwise and anticlockwise replication forks first localizes in the same place and then the pair separates and each replisome migrate in opposite directions to the 1/4 and 3/4 cellular positions during ongoing replication in the culture undergoing non-multifork replication (Hiraga 2000; Hiraga et al. 2000).
The MukB protein forms a large complex with MukF and MukE, which is essential for proper positioning and partitioning of chromosomes into both daughter cells (Niki et al. 1991, 1992; Yamanaka et al. 1996; Yamazoe et al. 1999; for a review, see Hiraga 2000). MukB condenses DNA in vivo (Onogi et al. 2000; Sawitzke & Austin 2000; Weitao et al. 2000; Adachi & Hiraga 2003) and in vitro (Chen et al. personal communication), and belongs to the Structural Maintenance of Chromosomes superfamily (Melby et al. 1998). Fluorescence microscopy revealed that MukB–GFP is localized as fluorescent foci in the living cell and that the foci localized to subcellular positions in ordered manners (Ohsumi et al. 2001).
Previously, cohesion between sister chromosomes was observed by FISH in mutant cells that exhibit temperature-sensitive initiation of chromosomal replication. The mutant cells were synchronized for replication initiation by repeated temperature shifts (Hiraga et al. 2000; Sunako et al. 2001). Bipolar migration of replication forks during ongoing replication was also found in similar synchronized cultures (Hiraga et al. 2000; Sunako et al. 2001; Yamazoe et al. 2005).
In this work, we demonstrate the cohesion between sister chromosomes and the translocation of replication forks using cultures growing exponentially and asynchronously in various media to avoid possible artificial physiological influences caused by repeated temperature shifts performed for synchronization. We first analyzed the length of cell phases of the cell cycle by flow cytometry and by immunofluorescence microscopy of nascent DNA pulse-labeled with 5-bromo-2'-deoxyuridine (BrdU). Then we analyzed subcellular localization of oriC and the terminal region (ter) of replication in living cells of E. coli by fluorescence microscopy. In parallel, we also analyzed the number of oriC copies per cell by the run-off replication method using flow cytometry. The results strongly indicate existence of cohesion of sister chromosomes between sister oriC or ter copies. In addition, analysis of the fluorescent foci of MukB–GFP indicated that the number of MukB foci is always larger than the number of BrdU foci. Analysis of nascent DNA pulse-labeled with BrdU suggests that paired replication forks were first closely associated with each other at a place, and then separated from each other during ongoing replication in poor medium. Together with the analysis of cell length, nucleoid and cell membrane morphology, subcellular localization of FtsZ (bacterial tubulin homologue acts for cell division) and SeqA, we discuss the order of these chromosomal events in the E. coli cell cycle.
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First, we analyzed newly synthesized nascent DNA pulse-labeled with BrdU in IL05 cells growing in various media at 30 °C in order to calculate the lengths of the B, C and D periods. The B period is the period between cell birth and initiation of chromosomal replication (Helmstetter 1996). The C period is the chromosomal replication period between initiation and termination (Cooper & Helmstetter 1968; Helmstetter & Cooper 1968). The D period is the period between termination of chromosomal replication and cell division (Cooper & Helmstetter 1968; Helmstetter & Cooper 1968). These periods can overlap each other in rich media. We carefully cultivated exponential cultures, which were in a phase of balanced growth. Doubling times of IL05 were 150, 75 and 35 min in media C, A and L, respectively. Fluorescent BrdU foci were clearly observed as shown in Fig. 1A. The number of BrdU foci increased together with the cell length (Fig. 1C and Supplementary Table S1-A). As can be seen in Fig. 1C, cells without BrdU focus seem to separate into two groups in the cell length, which might correspond to cells of B and D periods, respectively. The proportion of the cells in D period was calculated by subtract the proportion of one oriC copy (in B period) in flow cytometry data from the proportion of cells without BrdU focus (in B + D period), indicating that there were non-replicating cells in B and D periods in medium C (Supplementary Table S1-A).
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In medium C, although replisomes acting on bidirectional replication might be two in the cell, cells with three BrdU foci were observed, although proportion of this type of cells is small (Fig. 1C). This can be explained as follows. In the small number of cells, nascent sister DNA segments in one of replication forks might separate from each other and form two BrdU foci. Thus, a total of three BrdU foci existed in the cell.
In medium A, the average cell length of cells without BrdU focus was larger than the average of cells with one BrdU focus (Fig. 1C). Cells with two BrdU foci seem to separate into two groups. These results are important to investigate the cell cycle in the medium as described in the Discussion section.
All cells growing in medium L had BrdU foci, that is, all cells were replicating chromosomal DNA as expected (Fig. 1B and C). This indicates that this method of pulse labeling with BrdU is highly reliable to detect replication forks.
From the results of BrdU labeling and flow cytometry (Supplementary Table S1-A), we calculated the length of the B, C and D periods in the cells grown at 30 °C in medium C as 45, 75 and 30 min, respectively. The foci localized in ordered manners in the cell (Fig. 1D). In the cells with a single BrdU focus, the focus localized at mid-cell or at the 1/4 cellular position. In the cells with two BrdU foci, the foci localized at the 1/4 and 3/4 cellular positions (Fig. 1D). This is consistent with previous data (Adachi et al. 2005).
Nucleoid separation and septum formation in various media
We analyzed simultaneously IL05 cells in the same culture used for BrdU pulse labeling by staining the nucleoid with 4',6-diamidino-2-phenylindole (DAPI) or by staining the cell membrane with [N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl) pyridinium dibromide] (FM4–64) (Fig. 2A). The shapes of nucleoid were classified as follows: a single nucleoid with no constriction (d-1), a single nucleoid with a constriction (d-2), two nucleoids with no constriction (d-3) and two nucleoids each of which has a constriction (d-4). The d-4 type of cells was observed only in medium L. The types of cell membrane were classified as follows: no septum (f-1), a septum with no cell constriction (f-2) and a septum with cell constriction (f-3). The distributions of the cell length of each category are shown in Fig. 2B. According to the average cell lengths, the order of the events was the same as the order of the number of type as expected (Fig. 2B and Supplementary Table S1-C and D). These results are summarized in the Discussion section.
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We analyzed IL05 cells in the same sample used for DAPI and FM4-64 staining by immunofluorescence microscopy for SeqA and FtsZ (Fig. 2C and D). SeqA foci showed similar localization to BrdU foci in ordered manner (data not shown). This is consistent with previous data that SeqA protein binds preferentially to hemimethylated nascent DNA segments, resulting in co-localization or overlapping of SeqA foci and BrdU foci (Adachi et al. 2005). In medium C, cells without SeqA focus separated into two groups (Supplementary Table S1-B). The major group with small cell length might correspond to cells in the B period. The minor group with large cell length might correspond to cells in the D period. Double staining of SeqA and FtsZ by immunofluorescence method revealed that the former group of cells did not have Z ring and that the latter group had a strong fluorescent Z ring as expected.
In medium A, the proportion of cells with one SeqA focus was slightly smaller than that of cells with one BrdU focus. This indicates that a single SeqA focus separated into two prior to separation of a single BrdU focus (Fig. 6A), suggesting that hemimethylated nascent DNA segments were presumably smaller than BrdU labeled DNA segments in the 8-min labeling condition with BrdU and that BrdU labeled DNA segments remained in the same cellular position upon separation of a single SeqA focus into two.
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Dynamics of MukB–GFP foci during the cell cycle
We also analyzed MQ325, which is a mukB–gfp derivative of IL05, growing in various media at 30 °C for the number of MukB–GFP foci and oriC copies per cell (Fig. 3). Doubling times were 155, 60 and 35 min at 30 °C in media C, A and L, respectively. The average cell length and the average numbers of oriC copies were slightly larger than that of the parental IL05 strain. The majority of cells had one to four MukB–GFP foci in all the media examined. The number of the foci increased together with the cell length (Supplementary Table S1, exp. 3 and Fig. 3C). The average cell length of MQ325 was larger than that of the parental IL05 (Supplementary Table S1). The number of MukB–GFP foci did not correlate with the number of oriC copies in the three media (Fig. 3B). One focus localized at mid-cell, two foci localized at the 1/4 and 3/4 cellular positions, and three or four foci were also localized in ordered manner (Fig. 3D), being consistent with previous data (Ohsumi et al. 2001).
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Next, to examine the chromosome dynamics, especially cohesion between sister chromosomes, we used strain IL05 (pLAU53), in which the tetO cassette locates near the oriC region and the lacO cassette locates in the terminal region (Lau et al. 2003). This strain harbors the ampicillin resistant plasmid pLAU53, which carries a TetR–EYFP fusion (TetR and Enhanced Yellow Fluorescent Protein) gene and LacI–ECFP fusion (LacI and Enhanced Cyan Fluorescent Protein) gene. Both fusion proteins can be induced by the addition of L-(+)-arabinose to the cultures of the strain. IL05 (pLAU53) cells were incubated with L-(+)-arabinose, anhydrotetracycline and isopropyl-β-D-fucoside (IPTG) in various media containing ampicillin. The addition of both the TetR inhibitor anhydrotetracyclene and the LacI inhibitor IPTG can avoid physiological impairment caused by over-expression of TetR–EYFP and LacI–ECFP (Lau et al. 2003). The living cells were analyzed by foci of TetR–EYFP (named "oriC foci") and LacI–ECFP (named "ter foci") using fluorescence microscopy (Fig. 4A). In parallel, samples of the same cultures were analyzed by the number of oriC copies per cell using the run-off replication method to compare it with the number of oriC foci per cell.
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Additionally, we analyzed cohesion of oriC copies by FISH in YK1100 cells growing in M9 glucose media with or without casamino acids at 37 °C and obtained results similar to IL05 (pLAU53) cells growing in media A or C, respectively (data not shown).
Cohesion between sister chromosomes in a mukB null mutant
To examine whether MukB protein is involved in the cohesion between sister chromosomes, we analyzed IL05 (pLAU53) and mukB null derivative MQ142 grown at 22 °C. Doubling times of IL05 at 22 °C were 270, 180 and 90 min in media C, A and L, respectively. Doubling times of MQ142 were 300, 180 and 100 min in media C, A and L, respectively. In IL05, the cohesion of oriC copies was observed in media A and L, however, not in medium C (Table 1). In MQ142, the average numbers of oriC foci were significantly smaller than that of oriC copies in media C and A (Table 1). However, the difference between the number of oriC copies and oriC foci was small in medium L. This unexpected result in medium L is discussed later.
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When oriC foci were analyzed by FISH, results of YK1100 (the wild-type mukB gene) and AZ5372 (mukB null mutant) cells growing in M9 glucose media at 22 °C were similar to the above results of IL05 (pLAU53) and MQ142 cells growing in medium C, respectively (data not shown).
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Discussion |
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TopAbstractIntroductionResultsDiscussionConclusionsExperimental proceduresReferences |
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We summarize the present results of the chromosomal events and cell cycle in three media at 30 °C as schemes shown in Fig. 6A. The average cell lengths of the cell types suggest the order of the events. The pattern of the histogram of the cell length showed that different cell types were not frequently observed in the same cell age, and the majority of the cells might undergo a single cell cycle, because the differences of the cell length between short cells and the long cells were no more than twofold (Figs 1C, 2B, 3C and 4C, D, and Supplementary Table S1-A to F). Thus, the initial cell age of each chromosomal event was calculated from the proportion of each cell type (Powell 1956). A large number of cells having a non-replicating chromosome existed in the balanced growth culture in medium C, and the cells lacking BrdU focus are classified into two groups according to their cell length (Fig. 1C). In medium C, chromosomal replication might initiate at the 0.3 cell age and terminate at the 0.8 cell age, 75 min in total. Two models have been proposed for the initiation of chromosomal replication in E. coli cells growing slowly in poor media. Model 1 suggests that chromosomal replication is initiated at the same time of cell birth in poor media (Cooper & Helmstetter 1968; Helmstetter & Cooper 1968). On the other hand, model 2 suggests that there is a pre-replication period, termed the B period between cell birth and initiation of chromosomal replication (Helmstetter 1996). The present results in medium C strongly supports model 2 (Fig. 6B).
As shown in Fig. 6A, in medium A, chromosome replication initiated at the 0.6 cell age and terminated at the 0.3 cell age of the next generation, according to the data from flow cytometry and BrdU pulse-labeling. The C period was calculated as 55 min immediately before the cells divided, the cells had two replicating chromosomes and four oriC copies in medium A (Fig. 6B). In medium L, multifork replication might occur because doubling time (35 min) was shorter than C period (55–75 min) (Fig. 6B).
A constriction of the nucleoid appeared in the late period of chromosome replication (0.6-cell age) in medium C. In media A, a constriction of the nucleoid appeared nearly at the time of replication termination (0.3-cell age) (Fig. 6A).
Cohesion between sister chromosomes
In the present work, cohesion between oriC or ter copies were observed in IL05 (pLAU53) cells, except in medium C at 22 °C (Table 1 and Supplementary Table S1). In medium C at 30 °C, two completed sister chromosomes existed upon cell division, resulting in newborn cells with one completed chromosome (Fig. 6B). In this medium, sister oriC copies might be cohesive for 0.1-cell age (from the 0.3-cell age to the 0.4-cell age), and were released from cohesion in early replication period. In medium A, chromosomal replication was initiated in two completed chromosomes prior to cell division, within the D period (Cooper and Helmstetter 1968; Helmstetter and Cooper 1968), resulting in cells having two replicating sister chromosomes upon cell division (Fig. 6B). In this medium, cohesion period is from the 0.6 cell age to the 0.2 cell age of the next generation (Fig. 6A). In medium L, chromosomal replication was initiated in two replicating chromosomes during cell cycle, resulting in two multifork replicating chromosomes. The cells having eight oriC copies had only four oriC foci and two ter foci upon cell division (Fig. 6B). In medium L, cohesion period is from the 0.6 cell age to the 0.3 cell age of the next generation (Fig. 6A). As described above, cohesion of oriC copies continued for only 0.1 cell age in medium C at 30 °C. However, in richer media A and L, the relative length of cohesion period in one division cycle was longer than that of medium C. We thus conclude that the relative time of cohesion period of oriC in one division cycle is variable and depends on growth conditions.
As illustrated in Fig. 6A, a single ter focus presumably moved from a pole proximal border of the nucleoid toward mid-cell at the 0.7, 0.4 and 0.1 cell age in medium C, A and L, respectively (Fig. 4). We speculated the cohesion of ter based on the period between the timing of replication termination and the timing of separation of ter sister copies from each other. Relative time of the ter cohesion period in one division cycle depends on growth conditions (cf. medium C and medium A of Fig. 6A).
Cohesion of sister oriC or ter copies is probably required for proper partitioning of replicating chromosomes to avoid tangling between replicating sister chromosomes. Cohesion might be important in cells in rich media, especially in media performing multifork replication. On the other hand, the cohesion activity of sister oriC and ter copies might be low in the slow growth conditions such as medium C at 22 °C (Table 1). The discrepancy in the timing of the release from cohesion between the present work and Sunako et al. (2001) might be due to possible artifacts from repeated temperature shifts producing the synchronization in Sunako et al. (2001). Only one round of replication occurred in those synchronized cultures in medium L, in which the cells usually undergo multifork replication.
Cohesion between sister copies of oriC was also observed in cultures of mukB null mutant cells (MQ142) growing at 22 °C in media C and A, but not L as shown in Table 1. The results in media C and A suggests that MukB is not the sole factor for cohesion of oriC copies, though it might be important in fast growing cells as no cohesion was observed in MQ142 cells in medium L (Table 1). It was previously described that the cohesion between sister oriC copies was not observed in a mukB null mutant culture synchronized for replication initiation in medium L at 42 °C (Sunako et al. 2001). Since the mukB mutant could not form colonies in medium L at 42 °C, the cohesion might not be observed under such nonviable conditions. On the other hand, the cohesion between oriC sister copies was observed in dam null mutant cells (Sunako et al. 2001), which were defective for DNA adenine methyltransferase. Molecular mechanism of the cohesion of sister chromosomes is not yet clear.
It was previously described that the cohesion between other chromosomal regions far from oriC and ter was observed in a culture synchronized for replication initiation in medium L at 42 °C (Sunako et al. 2001). Cohesion of sister chromosomal regions for substantial period was previously detected using FISH (Hiraga et al. 2000; Sunako et al. 2001; Bates & Kleckner 2005). In addition, cohesion was detected by the TetR–EYFP/tetO labeling system and the LacI–ECFP/lacO labeling system inserted into the E. coli chromosome in this work. In addition, we detected cohesion of oriC copies in asynchronous cultures of YK1100 growing in M9 glucose medium with or without casamino acids at 37 °C by FISH (data not shown).
In the TetR–EYFP/tetO and LacI–ECFP/lacO labeling system, over-expression of these proteins inhibits progression of replication forks in the absence of anhydrotetracycline and IPTG, which inhibits binding of TetR or LacI to tetO or lacO, respectively (Payne et al. 2006; data not shown). Thus we added anhydrotetracycline and IPTG to the culture to avoid possible replication fork blockage, resulting in restoration of viability (Lau et al. 2003; data not shown).
Positioning of replication forks
Paired replication forks acting on clockwise and anticlockwise chromosome replication are first localized closely to each other. However, these forks appear to migrate in bipolar fashion when replication proceeds to about a half and oriC DNA segments are fully methylated (Hiraga et al. 2000; Onogi et al. 2002; Yamazoe et al. 2005). In the present results of the BrdU-labeling experiment in medium C, one round of replication initiated and completed within one division cycle as shown in the result by the run-off replication method (Fig. 6A and Supplementary Table S1). Under this condition, a single BrdU focus localized at mid-cell or the 1/4 cellular position (Fig. 1D). Thus, paired replication forks that are acting on bidirectional replication of the chromosome are closely localized to each other in the early stage of replication. Subsequently, the replication forks seem to separate and migrate in opposite directions to the 1/4 and 3/4 cell positions during ongoing replication (Fig. 6A). Thus, replication forks are mobile in the cell during replication. The separation of replication forks might be important for the separation of tangling nascent DNA strands. The separation of paired replication forks was not obvious in richer media A and L, because of a large number of replication forks. In medium L, a portion of the cells had eight oriC copies per cell, indicating that these cells had 12 or 8 replication forks. However, only up to four BrdU foci were observed in the majority of the cells (Supplementary Table S1 and Fig. 6A). Thus, some replication forks appeared to closely associate in medium L as shown in Molina & Skarstad (2004).
Cells replicating the terminal DNA region were detected as a minor group of cells with a single BrdU focus in medium C (Fig. 1C). We did not illustrate this step in Fig. 6A, because this final replication step might be very short (less than 0.1-cell age).
As the abnormal positioning of oriC and ter foci, abnormal positioning of replication forks pulse-labeled with BrdU was previously observed in a mukB null mutant (Adachi et al. 2005). These results are consistent with the previous results that cellular positioning of the bulk chromosome labeled with DAPI is abnormal in mukB null mutants (Niki et al. 1991; Yamanaka et al. 1996).
Positioning of MukB protein
Danilova et al. (2007) described recently that ori1 (same as oriC in our manuscript) foci co-localized preferentially with MukB foci in poor medium. Our results in medium C indicate strongly that co-localization of oriC and MukB foci is not mandatory, because the number of oriC foci did not correlate by one to one with the number of MukB–GFP foci (Fig. 6A). The number of MukB–GFP foci was always larger than the number of BrdU foci (Fig. 6A). Furthermore, two MukB–GFP foci existed in the cell having a single unreplicated chromosome with one oriC copy (Fig. 6A). This is an important finding to investigate the mechanism and role of MukB clusters in organization of sister chromosomes.
It is not yet clear how and why the number of MukB foci increased together with the cell length. MukB foci might act for the re-organization of sister chromosomes to form two compact nucleoids. In mukB null mutants, oriC, ter foci and replication forks did not localize in the regular cellular positions as described above. The abnormal localization of oriC, ter foci and replication forks might be caused by the impairment in the re-organization of sister chromosomes in mukB null mutant cells. These results suggest that MukB clusters act on separation of replication forks, that is, for separation of clockwise and anticlockwise replicating chromosomal arms to cell quarter positions during replication. Furthermore, MukB clusters may subsequently act on re-organization of sister chromosomes to two compact nucleoids in the cell quarter positions each other.
The positional analysis of various foci showed multiple BrdU or oriC foci suddenly appeared at certain cell lengths, indicating that the foci moved toward ordered positions all at once, not gradually (Figs 1D and 4E). Similar results have been reported for SeqA foci and MukB–GFP foci (Hiraga et al. 1998, 2000; Ohsumi et al. 2001).
The order of chromosomal events during the cell cycle in poor medium
We propose a model of chromosomal events under slow growth conditions, such as in medium C (see Fig. 6A and B). (i) A newborn cell has one non-replicating chromosome and one MukB focus. (ii) MukB focus separates into two and they migrate toward 1/4 and 3/4 cellular positions prior to replication initiation. (iii) Chromosomal replication is initiated and sister oriC copiers are cohesive to each other and localized in the middle of the cell. Paired replication forks acting on bidirectional replication are first localized closely to each other at the middle of the cell or the 1/4 cellular position. SeqA protein molecules are recruited to hemimethylated nascent DNA segments and form a focus at replication fork. (iv) The oriC copies are released from cohesion and migrates separately to the 1/4 and the 3/4 cellular positions. (v) The paired replication forks separate from each other and migrate to the 1/4 and 3/4 cellular positions during ongoing replication. At almost the same cell age, a nucleoid constriction and a strong fluorescent Z ring of FtsZ forms. (vi) The numbers of MukB foci and replication forks increase to three. (Vii) The ter site moves toward mid-cell from a pole proximal border of the nucleoid. Replication is terminated, sister ter copies are cohesive to each other and localized at mid-cell. At this stage, a septum begins to form at mid-cell. (viii) Two ter copies are released from cohesion, the nucleoid separates into two, and a constriction of the cell forms. (ix) The cell divides.
In stages (vii) and (viii), the site-specific XerCD resolvase separates a chromosome dimmer to two monomers at the dif sites, and topoisomerase IV decatanates a catena of sister chromosomes. The DNA translocater FtsK helps the action of XerCD and topoisomerase IV at the septation site (Espeli et al. 2003). Our model illustrated in Fig. 6 is consistent with the results described recently by Bates & Kleckner (2005). In comparison of the data between different media (Supplementary Table S1), no direct correlation between cell length and chromosomal events was observed, as Bates & Kleckner (2005) pointed out.
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Experimental procedures |
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TopAbstractIntroductionResultsDiscussionConclusionsExperimental proceduresReferences |
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The strain IL05, which was provided from Dr David J. Sherratt, has the gentamycin resistant tetO repeat cassette at the attTn7 site (84.2-min map position) localized near the replication origin oriC (3909 kb position) and the kanamycin resistant lacO repeat cassette localized near the dif site (1803 kb position) (Lau et al. 2003). Its genotype is thr-1 leu-6 thi-1 lacY1 galK2 ara-14 xyl-5 mtl-1 proA2 his-4 argE3 str-31 tsx-33 supE44. IL05 (pLAU53) strain harbors plasmid pLAU53 encoding TetR–EYFP and LacI–ECFP fusion proteins, of which expression is controlled by the promoter PBAD of the ara operon (Lau et al. 2003). The MQ142 strain is a derivative of IL05 (pLAU53) and has a mukB null mutation (mukB::miniTn10). Strain MQ325, which is a derivative of IL05, has a mukB–gfpUV4 fusion gene instead of the native mukB gene on the chromosome (Ohsumi et al. 2001). YK1100 is a trypotophan auxotrophic derivative (trpC9941) from the wild-type strain W3110. AZ5372 is a derivative of YK1100 and has a mukB null mutation (trpC9941
mukB::kan) (Yamanaka et al. 1996).
Media
Medium C was M9 medium containing 0.5% glycerol, 50 µg/mL of L-threonine, L-leucine, L-(–)-proline, L-arginine, L-histidine, L-tryptophan, and 5 µg/mL of vitamin B1. Medium A was M9 medium containing 0.5% of glycerol, 0.4% of Bacto Casamino acids (Difco, Detroit, MI), 50 µg/mL of L-tryptophan, and 5 µg/mL of vitamin B1. Medium L consists of 1% Bactotryptone (Difco), 0.5% Yeast extract (Difco), and 0.5% NaCl (pH7.4). Ampicillin (50 µg/mL), kanamycin (30 µg/mL) or gentamycin (10 µg/mL) were added when necessary.
To induce TetR–EYFP and LacI–ECFP, L-(+)-arabinose (0.01% for media C and A, 1% for medium L), anhydrotetracycline (12 nM) and IPTG (0.5 mM) were added to cultures. Cultures were incubated at 30 ºC for 1 h or at 22 ºC for 3 h for the induction.
M9 glucose medium was M9 medium containing 0.5% of glucose and 50 µg/mL of L-tryptophan. 0.4% of Bacto Casamino acids (Difco) were added when necessary.
Flow cytometry of the run-off replication method
Cells were inoculated at 106 cells/mL in medium and incubated at 30 °C or 22 °C, and when the culture reached 1 x 108/mL, the exponentially growing culture in a phase of balanced growth was used for analysis by the run-off replication method and fluorescence microscopy. Measuring of the copy number of the oriC region was analyzed by the run-off replication method according to Skarstad et al. (1986) with minor modification. Aliquots of exponentially growing cells at 30 °C were incubated with rifampicin (300 µg/mL) plus cephalexin (10 µg/mL) for further 3–4 h at 30 °C or 6–8 h at 22 °C. Rifampicin inhibits initiation of chromosomal replication, but allows completion of on-going rounds of chromosomal replication. Cephalexin inhibits cell division. After the treatment, with the drugs, the cells were fixed with 10-fold of 80% methanol and kept at 4 °C. Before analysis a small sample of the fixed cells was collected by low-speed centrifugation and suspended to a small volume of water. A solution of 10 µM SYTOX Green Nucleic Acid Stain (Molecular Probes, Inc., Eugene, OR) in DMSO was added to the cell suspension at the final concentration of 1 µM and kept for 1 h in a dark place. Intensity of fluorescence per cell was measured by a flowcytometer, FACS Calibur (Becton Dickinson Biosciences, K.K., Tokyo, Japan).
Fluorescence microscopy in living cells
Living cells incubated with L-(+)-arabinose, anhydrotetracycline and IPTG (in IL05 (pLAU53)) or without these chemicals (in MQ325) were collected by centrifugation at a low speed and 10 µL of the cell suspension was fixed on an agarose-coated glass slide covered with a cover glass by strong press with a finger. Images of fluorescence signal were observed using a phase-contrast and fluorescence microscope, Olympus AX70 (Olympus Corp., Tokyo, Japan) connected to C5810-01 Hamamatsu color chilled 3CCD camera (Hamamatsu Photonics, K.K., Hamamatsu, Japan) via an adapter with amplification lenses. Images of phase-contrast of cells and fluorescence were overlapped with a computer. Agarose-coated glass slides were prepared as follows: 30 µL of 90 °C melted agarose H solution (1%) was spread on a glass slide warmed at 80 °C by a plastic tip and dried up on an 80 °C hot plate.
Immunofluorescence microscopy in fixed cells
Method of immunofluorescence microscopy was described previously (Hiraga et al. 1998; Adachi et al. 2005).
Fluorescence in situ hybridization in fixed cells
Method of FISH was performed according to Niki & Hiraga (1997) with minor modification. oriC DNA probe was prepared from Kohara's 
phage clone #559 (Niki & Hiraga 1998). Cy3-dCTP labeled probe was applied to Sephadex® G-50 column (GE Healthcare UK Ltd., Buckinghamshire, UK) to remove non-incorporated substrates, and denatured at 80 °C for 10 min. Lysozyme was inactivated by 100% methanol. After hybridization, the slide was washed in wash buffer (50% formamide, 2x SSC) at 42 °C for 15 min. The slide was washed with a series of SSC solutions (2x, 1x, 4x and then 2x, for 15 min each except 4x for 5 min) at room temperature. The slide was not washed with PBS and observed immediately after wash with SSC.
Observation of cells stained with DAPI or FM4–64
Cultures of strain IL05 grown exponentially at 30 °C were fixed with 10-fold volumes of 80% methanol, and stained with 5 µg/mL DAPI or 1.5 µM FM4–64 (Molecular Probes, Inc., Eugene, OR). The cells were fixed on agarose-coated glass slides as described above.
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Acknowledgements |
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Footnotes |
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aPresent address: Laboratory of Developmental Immunology, Graduate School of Frontier Biosciences, Graduate School of Medicine, Osaka University, 2–2 Yamada-oka, Suita, Osaka, 565-0871, Japan.
* Correspondence: E-mail: hiraga{at}rg.med.kyoto-u.ac.jp |
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Accepted: 15 November 2007
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