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Journal of Bacteriology, September 2008, p. 5870-5878, Vol. 190, No. 17
0021-9193/08/$08.00+0     doi:10.1128/JB.00479-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Excess SeqA Leads to Replication Arrest and a Cell Division Defect in Vibrio cholerae

Djenann Saint-Dic,¹ Jason Kehrl,¹ Brian Frushour,¹ and Lyn Sue Kahng^1,2*

Section of Digestive Diseases and Nutrition, University of Illinois at Chicago,¹ Jesse Brown VA Medical Center, University of Illinois at Chicago, Chicago, Illinois 60612²

Received 8 April 2008/ Accepted 30 June 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Although most bacteria contain a single circular chromosome, some have complex genomes, and all Vibrio species studied so far contain both a large and a small chromosome. In recent years, the divided genome of Vibrio cholerae has proven to be an interesting model system with both parallels to and novel features compared with the genome of Escherichia coli. While factors influencing the replication and segregation of both chromosomes have begun to be elucidated, much remains to be learned about the maintenance of this genome and of complex bacterial genomes generally. An important aspect of replicating any genome is the correct timing of initiation, without which organisms risk aneuploidy. During DNA replication in E. coli, newly replicated origins cannot immediately reinitiate because they undergo sequestration by the SeqA protein, which binds hemimethylated origin DNA. This DNA is already methylated by Dam on the template strand and later becomes fully methylated; aberrant amounts of Dam or the deletion of seqA leads to asynchronous replication. In our study, hemimethylated DNA was detected at both origins of V. cholerae, suggesting that these origins are also subject to sequestration. The overproduction of SeqA led to a loss of viability, the condensation of DNA, and a filamentous morphology. Cells with abnormal DNA content arose in the population, and replication was inhibited as determined by a reduced ratio of origin to terminus DNA in SeqA-overexpressing cells. Thus, excessive SeqA negatively affects replication in V. cholerae and prevents correct progression to downstream cell cycle events such as segregation and cell division.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
DNA replication in bacteria is a highly complex process, requiring controlled initiation in a manner allowing progeny cells to inherit a normal genetic complement. Multiple factors influencing this process have been described for model bacteria with single chromosomes, such as Escherichia coli. A variety of bacteria from different subgroups have been found to have multipartite genomes, but it is unclear how correct replicon stoichiometry is maintained or may be modulated. All Vibrio species characterized thus far have divided genomes consisting of one large and one small chromosome, and in the last few years, Vibrio cholerae has become a primary model system for studying the replication of a complex bacterial genome.

Although the timing of the replication of the V. cholerae chromosomes remains under investigation, a number of factors are known to be important for replication itself. DnaA and Dam methylation are both required for the replication of V. cholerae origin minichromosomes in an E. coli host (12). The novel factors RctA and RctB, an RNA and a protein, are involved in the replication of the small V. cholerae chromosome (12, 41). In the initial characterization of the V. cholerae replication origins, SeqA was also implicated as a required factor for the replication of its large chromosome origin in the E. coli host. Although the SeqA gene can be deleted in E. coli, a null mutant of V. cholerae could not be generated (12). Thus, although the E. coli and V. cholerae SeqA homologs are fairly similar (54% identical and 69% similar), they may have different functions in the two organisms.

SeqA is best characterized in E. coli, in which it plays a critical role in synchronizing DNA replication and ensuring that replication occurs once per cell cycle and in which it also affects chromosome structure. In E. coli, several factors affect the proper timing of DNA replication. While the titration of the initiator protein DnaA and the regulation of its activity are critical to this process, DNA methylation and sequestration are also extremely important (5). Newly synthesized DNA strands are unmethylated at Dam recognition (GATC) sites and modified by the Dam enzyme shortly thereafter (7, 22). The binding of SeqA to hemimethylated DNA in oriC (containing the methylated old strand and the unmethylated new strand) sequesters the DNA and prevents this rapid methylation. The interplay between SeqA and Dam methylation influences origin refiring such that a seqA null mutant and strains expressing too little or excess Dam methyltransferase display abnormal initiation timing and aberrant DNA content in flow cytometry analyses (4, 6). It is unclear whether this mechanism may also be operative in V. cholerae.

While SeqA may be required for V. cholerae replication, its possible negative influence on this process has not been explored. During previous cytologic studies, we found that wild-type V. cholerae may contain more than two replication origins per chromosome within a single cell under rapid-growth conditions (31). Our flow cytometry studies described here demonstrate that the DNA content increases with the bacterial growth rate, suggesting parallels to E. coli DNA replication. Because of the clustered Dam methylation sites present in both V. cholerae replication origins, we investigated the methylation state of origin DNA. Under steady-state conditions, Southern blots detected hemimethylated DNA in both chromosome origins, indicating that these origins are sequestered from rapid Dam methylation. We demonstrated that SeqA is in fact essential for viability and thus investigated the phenotype of SeqA overexpression. Inducible seqA overexpression led to a severe filamentous phenotype and DNA condensation, although the cell mass continued to increase. We also observed a loss of recoverable CFU that preceded the most marked morphological abnormalities. Flow cytometry analyses of SeqA-overexpressing bacteria demonstrated the appearance of aberrant peaks in a small percentage of cells, correlating with DNA content differing from integral genome units. Finally, the normal ratio of origin to terminus DNA on both chromosomes decreased following seqA induction, consistent with the inhibition of replication initiation. Thus, the two replication origins of V. cholerae are sequestered, and the overproduction of SeqA perturbs both replication and cell division.

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Bacterial strains, plasmids, and media. Bacterial strains and plasmids are listed in Table 1. E. coli and V. cholerae were grown at 37°C in Luria-Bertani (LB) broth (1% [wt/vol] tryptone, 0.5% [wt/vol] yeast extract, 1% [wt/vol] sodium chloride) (32) or in AB minimal medium (8) supplemented with 0.2% glycerol (V. cholerae) or 0.2% glycerol plus 0.01% thiamine and 0.2% Casamino Acids (E. coli). As needed, carbenicillin at 50 µg/ml, kanamycin at 30 µg/ml, or tetracycline at 1.2 µg/ml was used. The induction of the arabinose promoter was through the addition of 0.02 or 0.2% arabinose. Cells were studied by microscopy or flow cytometry at an optical density at 600 nm (OD₆₀₀) of between 0.4 and 0.8.

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TABLE 1. Strains and plasmids used in this study

Essentiality of seqA. The upstream and downstream regions of seqA were amplified and used to create a construct in which the coding region of seqA was replaced with a kanamycin resistance cassette (GenBlock; Pharmacia). This construct was then transferred into the suicide vector pWM91 and introduced into V. cholerae N16961 by conjugation (24, 31). pWM91 provides resistance to carbenicillin and also carries the Bacillus subtilis sacB gene. The levansucrase encoded by sacB generates a toxic metabolite when bacteria are grown on sucrose, allowing counterselection against the presence of the plasmid. Merodiploid strains were grown without antibiotic selection and plated onto sucrose to force allelic exchange.

Complementing and inducible seqA constructs. The V. cholerae seqA gene was amplified by PCR for the production of two constructs: one including the upstream putative promoter region (250 bp) and one including only the coding region and translational start site information. The products were cloned using the pCR-BLUNT-TOPO kit for sequencing (Invitrogen) and then checked by sequencing. The complementing construct was cloned into the broad-host-range RK2-derived vector pMR20 in the opposite direction from the lac promoter by using HindIII and XhoI enzymes, and the vector was transferred into V. cholerae by conjugation. The promoterless seqA was cloned into the pBAD18 plasmid, under the control of the arabinose-inducible promoter, by using SacI and XbaI enzymes. The P_BAD-seqA construct was then cloned into the broad-host-range RK2-derived vector pMR10 in the opposite orientation from the lac promoter by using PstI and EcoRV enzymes, and the plasmid was transferred into V. cholerae by conjugation. The construct was also recovered from V. cholerae and resequenced to confirm that there was no mutation; similar phenotypic effects were observed in independently constructed strains and in experiments using a separate construct synthesized with different primers (data not shown).

Flow cytometry. Overnight cultures were diluted 200-fold in the appropriate medium and harvested at log phase (usually at an OD₆₀₀ of 0.4). As appropriate, 0.02 or 0.2% L-arabinose or glucose was added, and samples were harvested at subsequent time points. Samples were processed for flow cytometry as described in reference 11 but using fixation with 1% paraformaldehyde plus 0.05% glutaraldehyde. Prior to flow cytometry, the fixed cells were pelleted and resuspended in 1 ml of a buffer containing 1x Tris-EDTA (pH 7.2), 50 mM potassium citrate, 0.1% (vol/vol) Triton X-100, 0.1 mg of RNase A (DNase free)/ml, and SYBR green (10x). Resuspended cells were incubated at 37°C for 2.5 h and protected from light, and samples were analyzed using a FACSCalibur instrument (Becton Dickinson) and the CellQuest Pro program. The flow cytometer acquisition settings were adjusted as needed for E. coli and V. cholerae to better visualize peaks of different sizes, as larger peaks (e.g., corresponding to a DNA content of eight genome units [8N]) would be off the scale if this adjustment were not done. The bacterium Caulobacter crescentus, which contains only one or two chromosomes and has a genome size of 4.02 Mb (23, 25, 44), was used as a standard during acquisitions to gauge the DNA content of test samples.

Southern blotting. Southern blotting was performed using standard methodologies (32). For analyzing the methylation state of the DNA, genomic DNA was digested with methylation-sensitive restriction enzymes overlapping Dam recognition sequences. Test sites were located at bases 233 (MboII), 15534 (MboII), and 415302 (TacI) for chromosome I and 1973 (TacI), 5921 (TacI), 11915 (TacI), 17527 (MboII), and 162365 (HphI) for chromosome II. The band intensities were quantitated, and the results were used to calculate the percentage of hemimethylation as described previously (17). Equal amounts of sample DNA were digested and loaded; however, each sample could be assessed independently, as both cut and uncut bands were present in a lane. For determining origin/terminus DNA ratios, the genomic DNA was digested with PstI and HindIII (chromosome I) or with SacI (chromosome II). These enzymes were chosen to yield distinct band sizes when hybridized with our probes. Southern blot probes were synthesized by PCR using the oligonucleotides listed in Table 2.

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TABLE 2. Oligonucleotides used in this study

The digested DNA was run on 1% agarose gels and then transferred onto Hybond N+ membranes (Amersham Biosciences). The membranes were hybridized in Church and Gilbert solution (7% sodium dodecyl sulfate [wt/vol]-1 mM EDTA in 0.5 M Na phosphate buffer, pH 7.2 [0.5 M Na₂HPO₄ buffered with 0.5 M NaH₂PO₄]) at 65°C. Probes were radiolabeled with ³²P by using the Amersham Rediprime II random prime labeling system (GE Healthcare) and quantitated using a Packard 1900TR liquid scintillation counter. Blots were exposed to a phosphor screen (Amersham Biosciences) and developed using a Storm 860 PhosphorImager (Molecular Dynamics).

Western blotting. Western blotting was performed using standard methodologies (32). Samples were normalized using culture ODs for the loading of equivalent bacterial cell masses into the lanes of the gel. Blotting was performed with a rabbit anti-E. coli SeqA antiserum at a 1:5,000 dilution (1), horseradish peroxidase-conjugated donkey anti-rabbit secondary antibody at a 1:10,000 dilution (Jackson ImmunoResearch), and a Pierce SuperSignal West Pico kit. Anti-SeqA antiserum was a generous gift of Kirsten Skarstad.

Microscopy. Fluorescence in situ hybridization (FISH) and microscopy were performed with fixed cells as described previously (31). Agarose pads for the examination of live cells were prepared using cultures grown as described above. At the desired time points, 2 µg of DAPI (4',6-diamidino-2-phenylindole)/ml was added to a small sample, 10- to 20-µl aliquots were applied to the surface of solidified 1% agarose on slides, the slides were covered with coverslips, and the cells were immediately visualized using a Leica DM4000B microscope with a 100x lens objective, a cooled charge-coupled device camera (Q Imaging), and Slidebook 4.0 software (Intelligent Imaging Innovations).

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V. cholerae increases its DNA content and contains increased numbers of origins during periods of rapid growth. During our previous studies of replication origin positioning in V. cholerae, we performed FISH analyses of fixed cells (31). Our studies differed from the initial studies by other groups in that we used a fixed-cell model and more rapid growth in LB medium (13, 31, 37). In a small percentage (<10%) of cells examined under these conditions, we detected three and sometimes four fluorescent foci in nonseptate cells by using individual probes for each origin (Fig. 1A). To more closely analyze the DNA content of V. cholerae at different growth rates, we used flow cytometry to analyze both E. coli AB1157 and V. cholerae N16961, using C. crescentus as a control as described in Materials and Methods. Bacteria were cultured in AB minimal medium or LB medium supplemented with glucose. In early log phase, bacteria were treated with rifampin and cephalexin to allow the runout of replication prior to the harvesting and fixing of the cells for flow cytometry. Consistent with data from other labs (11, 28, 33, 36), E. coli AB1157 contained mainly one or two chromosomes per cell at a low growth rate (80-min doubling time) and two, four, or eight chromosomes per cell at a high growth rate (25-min doubling time). Histograms for V. cholerae in minimal medium (94-min doubling time) corresponded to one or two genome equivalents, but those for V. cholerae in LB medium (25-min doubling time) corresponded to two or four genome equivalents (Fig. 1B to E). Flow cytometry analysis of the dam mutant E. coli strain SCS110 was performed as a control for our ability to detect aberrant numbers of chromosomes (Fig. 1F). The pattern for the bulk DNA content without antibiotic treatment contained a broad peak between the two discrete peaks that resulted after antibiotic treatment (data not shown). Taken together, the combined flow cytometry and microscopy data suggested that V. cholerae contains increased numbers of origins within a cell when growing rapidly. This finding bears similarity to data for E. coli but differs from previous observations from flow cytometry analyses of rapidly growing alphaproteobacteria with multipartite genomes (17, 30, 45).

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FIG. 1. Increased DNA content in V. cholerae under conditions of rapid growth in LB medium. (A) FISH analysis showing cells with more than two foci of origin I (left) and origin II (right). Scale bars, 1 µm. (B and C) Runout flow cytometry analysis of E. coli AB1157 grown in AB minimal medium (doubling time, 80 min) (B) or LB medium-glucose (doubling time, 25 min) (C). The DNA content increases along the x axis; numbers denote numbers of chromosomes corresponding to DNA content peaks. (D and E) Runout flow cytometry analysis of V. cholerae N16961 in AB minimal medium (doubling time, 94 min) (D) or LB medium-glucose (doubling time, 25 min) (E). Axes are as described above; labels of peaks correspond to DNA content in genome equivalents, where "1N" indicates one large and one small chromosome. The x axis scales in the histograms for each individual bacterium are the same; however, they differ between the two bacteria to demonstrate the larger DNA content peaks for E. coli. (F) Flow cytometry analysis of dam mutant E. coli SCS110.

Hemimethylated DNA can be detected in the origins of both V. cholerae chromosomes, consistent with DNA sequestration. Dam methylation sites are overrepresented in both chromosome origins of V. cholerae (12, 16), and we hypothesized that sequestration might be important in the proper control of initiation. To obtain evidence of origin sequestration in V. cholerae, we performed restriction digestion and Southern blotting to assay the degree of Dam methylation of DNA at different locations on the chromosome. As shown in Fig. 2A, these assays were performed by digesting genomic DNA at restriction sites that partially overlap Dam methylation sites. When fully methylated, DNA cannot be digested. However, after the passage of a replication fork, one of the hemimethylated daughter strands can be cut. The ability of DNA at a particular test site to be cleaved indicates that under steady-state growth conditions, hemimethylated DNA is present. Under steady-state conditions, cleaved DNA can be found in small amounts in the origin of E. coli but not elsewhere on the chromosome due to rapid Dam methylation, leading to the initial description of DNA sequestration (7).

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FIG. 2. Southern blotting assays to assess the methylation state of V. cholerae genomic DNA. (A) Diagram of methylation assay. Fully methylated DNA is uncut by a restriction enzyme whose recognition site overlaps the Dam site. Following replication, each hemimethylated daughter molecule will have one methylated and one unmethylated strand. One of the two daughters can then be cleaved by the test enzyme, although the other cannot. (B) Southern blots at the indicated test sites. Multiple replicates were performed, and two independent samples are shown. U, uncut DNA band; C, cut DNA band. (C) Graph of the percentages of hemimethylated DNA at test sites used in Fig. 2B; the approximate distance from the origin is used to allow comparison. The means of results for at least three independent replicates are shown with the standard deviations.

We performed similar assays at sites at or near the origins of both V. cholerae chromosomes (Fig. 2B). Cultures were grown in LB broth at 37°C to early log phase, when genomic DNA was prepared. Southern blotting was performed using probes that did not overlap the test site, and bands were quantitatively analyzed using a PhosphorImager Storm system. In samples of DNA isolated from bacteria in the exponential phase of growth (OD of 0.4 to 0.8), hemimethylated DNA could be detected in both origins. On chromosome I, DNA within the origin was cleaved but not at a test site approximately 15 kb away from the origin. On chromosome II, DNA within the origin was cleaved. However, we also detected hemimethylated DNA approximately 10 and 15 kb away from the original test site, in areas with overrepresented Dam sites, but not in an intervening test site with relatively few surrounding Dam sites. DNA samples from test sites approximately 45° away from either origin had minimal levels of cleaved DNA (Fig. 2C). Thus, DNA from both replication origins of V. cholerae appears to undergo sequestration.

V. cholerae seqA is essential, and its overexpression causes severe morphological abnormalities and the loss of viability. To explore the functions of the V. cholerae seqA homolog, a null mutant would be desirable; however, others had reported previously that the gene could not be deleted (12). To confirm essentiality, a deletion construct in which the coding region of the gene was replaced by a kanamycin resistance cassette was created using the pWM91 suicide plasmid, which itself confers carbenicillin resistance. The plasmid was then integrated into the chromosome, resulting in merodiploid isolates with both kanamycin and carbenicillin resistance. After growth without antibiotics, counterselection on sucrose was performed. In the presence of no additional vector or an empty vector, isolates either retained or simultaneously lost both carbenicillin and kanamycin resistance, indicating that they were wild-type or still merodiploid strains with sacB inactivation. However, if the complementing construct was present, carbenicillin-sensitive, kanamycin-resistant colonies were readily obtained. This result signified that V. cholerae seqA can be disrupted on the chromosome only if an intact copy is provided in trans and, hence, that the gene is essential for viability.

As a null mutant could not be generated, we created an overexpression construct under the control of the P_BAD promoter, as described in Materials and Methods; this construct was introduced into V. cholerae N16961 by conjugal transfer. Cells were grown overnight without induction, subcultured, and allowed to enter early log phase before the addition of arabinose. A wild-type strain containing P_BAD-seqA grew normally without induction and was compared to a control strain carrying an empty vector. At 75 min postinduction, Western blotting for SeqA protein levels using an antibody to the E. coli SeqA homolog (1) showed that the levels of protein without induction or with the induction of the control vector only were comparable to those of the wild type but increased severalfold over baseline with the induction of the P_BAD-seqA construct (Fig. 3A). After induction, all cultures continued to increase in OD (Fig. 3B). The growth rates of all strains grown in arabinose were slightly lower than those of uninduced strains but did not differ between the control vector and test strains. In contrast, when we assessed viability, the number of viable CFU recovered from the P_BAD-seqA strain decreased significantly after the first 60 to 90 min of induction with 0.2% arabinose (Fig. 3C).

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FIG. 3. The overexpression of SeqA causes a loss of viability. (A) Western blot showing SeqA levels in wild-type (WT) bacteria and bacteria containing either a control vector or PBAD-seqA, with or without induction with various levels of arabinose (ara). Samples were acquired at 75 min postinduction. (B) ODs over time. Induced cultures contained 0.2% arabinose. (C) Numbers of recovered viable CFU over time. Induced cultures contained 0.2% arabinose.

Cells containing induced seqA developed severe morphological abnormalities by 3 h postinduction (Fig. 4). The DNA became condensed at the center of the cell, and a small number of cells that did not contain DNA were noted. In addition, cells became very filamentous without pinching (Fig. 4C). We performed microscopy with DAPI staining using live cells on agarose pads and found that the DNA condensation and morphological abnormalities were present to the same degrees without the use of fixation; live cells are shown in Fig. 4. The seqA-containing strain and the control vector strain appeared the same before induction (Fig. 4A and F), and the uninduced strain and control strain continued to appear normal (Fig. 4D to J). At early time points, when cells were less strikingly elongated, some condensation of DNA in the induced strain was already evident (Fig. 4B). Thus, the overexpression of seqA leads to a loss of viability, as well as DNA condensation and a cell division defect. In addition to our experiments with a control vector, we included experiments with a seqA in-frame deletion construct and found that equivalent arabinose induction also had no effect on cells (data not shown). The same phenotype described above but with a slower onset was also observed when induction was carried out with 0.02% arabinose, as well as when cells were grown more slowly in AB minimal medium or when the slower-growing O395 classical strain of V. cholerae was used (data not shown).

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FIG. 4. The overexpression of SeqA causes DNA condensation and a cell division defect. Micrographs are of live cells grown in LB medium supplemented with 0.2% arabinose where indicated. Bacteria were incubated with DAPI at 2 µg/ml and immediately visualized on agarose pads at the indicated times postinduction. Images from phase-contrast analysis and DAPI staining (falsely colored in red for visibility) are superimposed. (A and F) Strains containing PBAD-seqA or a control vector at time zero (t = 0). (B to E and G to J) PBAD-seqA-containing strain (B to E) or control vector strain (G to J), either induced with 0.2% arabinose for the specified time or uninduced. Scale bars, 2 µm.

Overexpression of SeqA leads to abnormalities of DNA content. We investigated whether SeqA overexpression also led to abnormalities of DNA content by performing flow cytometry analyses of cells grown and induced in AB minimal medium. Overall DNA content profiles of SeqA-overexpressing cells were similar to those of uninduced cells, and although the cells had begun to lengthen, they did not contain significantly increased numbers of chromosomes. However, a small percentage of cells yielded peaks intermediate in size between those corresponding to integral numbers of whole genomes, i.e., 1N or 2N (Fig. 5, center panel). These peaks were not apparent in profiles of control cultures. Since the distance from the y axis was proportional to the DNA content, these peaks corresponded to cells containing a DNA mass equivalent to that of one complete genome plus an extra large or an extra small chromosome. There were also smaller peaks to the left of the 1N peak, corresponding to cells containing less than one complete genome, i.e., only the large chromosome and likely only the small chromosome, although evidence of the latter was partly obscured by the debris peak in the histogram. Thus, the overexpression of SeqA led to a small subpopulation of cells with a perturbed relation between cell division and DNA segregation.

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FIG. 5. Flow cytometry analysis of strains containing PBAD-seqA or a control vector and grown in AB minimal medium. (Top panel) PBAD-seqA-containing strain with no induction. (Center panel) PBAD-seqA-containing strain induced with 0.2% arabinose. (Bottom panel) Control vector-containing strain induced with 0.2% arabinose. DNA content increases along the x axis; labels denote genome equivalents, where "1N" indicates one large and one small chromosome. Arrows indicate aberrant peaks.

Overexpression of seqA leads to replication arrest. The DNA content did not increase in proportion to the increase in cell mass, and the loss of cell viability was not accounted for by the relatively small number of cells with altered DNA content as judged by flow cytometry. The development of the cell division phenotype appeared to be a later event. We thus hypothesized that DNA replication could be affected early during SeqA overexpression, in addition to the cell division defects that became apparent with the continued growth of induced cultures. To assess this possibility, we prepared genomic DNA samples at 60 to 75 min after induction in LB medium and performed Southern blotting to detect the origin and terminus regions of the two chromosomes.

We found that arabinose induction of seqA led to a decrease in the ratios of origin to terminus DNA on both chromosomes. For chromosome I, the origin/terminus DNA ratio in the strain overexpressing SeqA was reduced to 28% of the ratio in the control strain. Similarly, for chromosome II, the origin/terminus DNA ratio in an induced culture was 64% of the ratio in the control strain. The average ratio of origin to terminus DNA for chromosome I was 3.2 in wild-type, control, and uninduced cultures and decreased to 1 in cultures in which seqA was induced (Fig. 6A). For chromosome II, the average ratio of origin to terminus DNA was 1.6 in wild-type, control, and uninduced cultures and decreased to 1 after seqA induction (Fig. 6B). Since a reduced copy number ratio of origin to terminus DNA indicates that fewer copies of the origin are present, seqA overexpression leads to the inhibition of replication initiation, affecting both chromosomes in V. cholerae.

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FIG. 6. SeqA overexpression inhibits DNA replication. Southern blots (left panels) and corresponding PhosphorImager quantitation (right panels) demonstrating origin/terminus DNA ratios in strains carrying a control vector or PBAD-seqA, with (+) or without (–) induction by 0.2% arabinose (ara). ori, band corresponding to origin DNA; ter, band corresponding to terminus DNA; WT, wild type. The graphs show means of results for at least three independent replicates, with standard deviations.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Bacterial DNA replication is a precisely controlled process requiring balanced positive and negative regulation in order to prevent premature reinitiation. The proper timing of initiation in E. coli depends on a number of factors, including regulatory inactivation of DnaA, Dam methylation, sequestration by SeqA, and titration of DnaA by the datA locus. It is not clear whether bacteria with multipartite genomes may regulate their numbers of replicating origins differently from those with a single chromosome, and initial runout replication studies of V. cholerae indicated that during growth in minimal medium it contained one or two genome equivalents per cell (11). The alphaproteobacterium C. crescentus has been characterized previously to contain only one or two replicating origins per cell while growing in rich medium (48). Our flow cytometric studies of Agrobacterium tumefaciens, another alphaproteobacterium which contains four replicons, were performed during growth in rich medium yet similarly indicated that each cell contained only one or two genomes (17). Finally, the bacterium Neisseria gonorrhoeae contains multiple copies of its genome per coccus yet does not significantly alter its DNA content when the growth rate is varied (39). In contrast to the findings from studies of those organisms, our cytologic and flow cytometry data and those of others now suggest that V. cholerae modulates its DNA content as a function of the growth rate, in a manner somewhat analogous to that found in E. coli but not other multichromosomal bacteria thus far (28, 36).

Recently, a more detailed analysis of replication in V. cholerae by Rasmussen et al. indicated that the runout method of flow cytometry is limited by differential sensitivities of the two replication origins to rifampin and that in a novel paradigm, replication in this organism may be timed to achieve termination synchrony rather than initiation synchrony (28). New data from Srivastava and Chattoraj show that their flow cytometry studies were also limited by differential effects of antibiotics, that chromosome II does not increase its copy number similarly to chromosome I during rapid growth, and that their data are supportive of a model of coordinate initiation (36). Our data have similarities to and differences from the findings of these studies: the results of our origin-terminus Southern blotting studies are in agreement with findings of both groups that the average number of origins within a cell is approximately twofold higher for chromosome I than for chromosome II under rapid-growth conditions. However, during our FISH analyses of fixed wild-type cells harvested during growth in rich medium, three and sometimes four origin foci could be detected, not only for the large but also the small chromosome origin, in wild-type cells that were not septate. In contrast, Srivastava and Chattoraj observed additional fluorescent foci for the second chromosome origin upon the overexpression of the chromosome II initiator protein RctB (or increasingly with antibiotic treatment) but only very rarely during normal growth (36). As our origin-positioning data for cells with one or two foci correlate well with data from previously published live-cell studies (13, 31, 37), the reason for this discrepancy is unclear. Also, our flow cytometry studies yielded a profile of bacteria in LB medium with 2N and 4N peaks, as opposed to 4N and 8N, corresponding more closely to pretreatment DNA content and the findings of our cytologic experiments. Replication timing in this organism thus remains an intriguing question.

We find that the sequestration of DNA occurs in both origins of V. cholerae, in regions where GATC sites are statistically overrepresented. While our choice of test sites was limited by the choice of restriction enzyme sites, our test site at position 5921 on chromosome II had fewer surrounding GATC sites than the others and was not significantly protected. In E. coli, closely clustered GATC sequences are required but also sufficient to mediate sequestration, as nonorigin DNA with clustered GATC sites can be sequestered (2, 3, 47). Thus, SeqA appears to recognize DNA similarly in both organisms. We were able to detect hemimethylated DNA in regions (i.e., chromosome II, bases 11915 and 17527) where GATC sites are numerous but farther apart than in the E. coli origin. This finding may reflect the ability of SeqA to bind DNA with a somewhat lower GATC site density than that in the E. coli origin, as has been observed previously in vitro for E. coli SeqA with the lambda p_R promoter, or it may reflect binding over a larger area of DNA due to nearby GATC clustering and SeqA oligomerization (14, 35).

In E. coli, a severalfold SeqA overexpression level has been shown previously to prolong sequestration and lead to segregation defects, as well as affect cell division (1, 42). The phenotype we observed in V. cholerae was more drastic, as overexpression in E. coli is not lethal unless strains are defective in replication initiation or methylation (1, 22, 42). Nonetheless, our findings have parallels with the observations made for E. coli (1). Most significantly, the overexpression of SeqA in V. cholerae inhibits the initiation of replication. Replication inhibition may be due to the physical sequestration of origins, but for E. coli, there are also differing reports as to whether and how SeqA may interfere with DnaA (26, 38, 40, 42). SeqA overexpression may also inhibit DNA replication by leading to the sequestration of promoter regions for important genes or by acting as a transcription factor (7, 34, 35). In E. coli, the dnaA promoter is sequestered, and Dam methylation sites are found upstream of V. cholerae dnaA (7, 22). However, work from Egan and Waldor indicated that the complementary actions of SeqA and Dam methylation found in E. coli also have additional layers of complexity in V. cholerae: while Dam is required for the replication of the second chromosome origin, this requirement cannot be eliminated by the simultaneous elimination of seqA, indicating that Dam is necessary in an additional capacity other than that related to the sequestration process (12). We did not observe consistent changes in the methylation state of the DNA during our SeqA overexpression studies, suggesting that lethality may not be due solely to the blocking of Dam methylation (data not shown). Furthermore, although methylation sites are found in the vicinity of P_rctB, Dam modulates its autoregulation by RctB but does not significantly change its baseline activity (10, 27). Finally, there is a positive requirement for seqA in the replication of a V. cholerae origin I minichromosome, although its role remains unclear; the overproduction of DnaA and RctB has been found to selectively lead to the overinitiation of chromosomes I and II, respectively (9).

SeqA in E. coli has additional previously studied functions that may also contribute to the phenotypes we observed. For example, V. cholerae SeqA overexpression led to significant condensation of DNA; E. coli SeqA is known to have supercoiling activity and to interact with topoisomerase IV (19, 20, 43). Previous SeqA studies of overexpression in E. coli demonstrated delayed nucleoid segregation, as well as the formation of larger SeqA foci and the possible recruitment of larger areas of hemimethylated DNA into these SeqA structures (1). In E. coli, not only does SeqA affect chromosome structure, but its absence also leads to global transcriptional changes similar to those seen in bacteria overexpressing Dam (21, 43). The overexpression of SeqA in V. cholerae may thus similarly influence DNA structure and affect the transcription of certain genes.

We hypothesize that SeqA overexpression in V. cholerae leads to the inhibition of replication and consequent defects in segregation and cell division. Our observation of relatively small numbers of cells with aberrant DNA content or no DNA staining suggests that these cells arise from residual cell division in the setting of defective segregation. This phenotype is distinct from effects observed during prior studies of aberrant V. cholerae chromosome II replication and partitioning. Yamaichi et al. reported that the large-scale loss of chromosome II in a population due to parAB2 deletion led to cells with condensed and/or fragmented DNA but that, in contrast to our observations, the cells retained chromosome I and were significantly bloated (46). Venkova-Canova et al. found that extra copies of rctA in trans hindered V. cholerae growth and led to elongated cells lacking DNA altogether (41). It appears that in our experiments, DNA condensation was more prominent and complete DNA loss was less common than in the experiments in previous studies. Thus, SeqA appears to function in negatively influencing the replication of both chromosomes in V. cholerae, in addition to being essential for the viability of this bacterium. The significant morphological defect we observed attests to the link between DNA replication and the successful completion of downstream cell cycle processes, including DNA segregation and cell division, as well as a role for SeqA in these critical events.

 
We are grateful to Kirsten Skarstad for SeqA antiserum, to L. Kenney, V. Viswanathan, and lab members for helpful comments, and to the reviewers for their suggestions.

This work was supported by a grant from the Department of Veterans Affairs (Merit Review) and by University of Illinois funds.

 
* Corresponding author. Mailing address: Section of Digestive Diseases and Nutrition, Jesse Brown VA Medical Center, University of Illinois at Chicago, 840 S. Wood St., M/C 716, Chicago, IL 60612. Phone: (312) 996-6387. Fax: (312) 996-5103. E-mail: lynsue{at}uic.edu

Published ahead of print on 11 July 2008.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Journal of Bacteriology, September 2008, p. 5870-5878, Vol. 190, No. 17
0021-9193/08/$08.00+0     doi:10.1128/JB.00479-08
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