YabA of Bacillus subtilis controls DnaA-mediated replication initiation but not the transcriptional response to replication stress

Effects of YabA on DNA replication require the DnaA-dependent oriC

Null mutations in yabA cause an increase in replication initiation (Noirot-Gros et al., 2002; Hayashi et al., 2005; Noirot-Gros et al., 2006; Cho et al., 2008). We found that this increase was not observed in cells initiating replication from a heterologous origin inserted into the chromosome. We compared the effects of deletion and overexpression of yabA on replication in cells initiating from either oriC or the heterologous origin oriN. Initiation from oriN requires its cognate initiator protein RepN and is independent of DnaA (Hassan et al., 1997). We monitored replication relative to cell growth by measuring the DNA to protein ratio (DNA/protein) of cells in culture (Kadoya et al., 2002).

In cells growing in minimal medium and initiating replication from oriC, a yabA null mutation caused an increase in the DNA to protein ratio of nearly 2-fold relative to that of yabA⁺ cells (Fig. 1A). Conversely, overexpression of yabA from a heterologous promoter caused a decrease in the DNA to protein ratio (Fig. 1A). The effect in the yabA deletion mutant was more severe in minimal medium than in rich medium (data not shown). These results are consistent with previous findings that YabA is a negative regulator of replication (Noirot-Gros et al., 2002; Hayashi et al., 2005; Noirot-Gros et al., 2006; Cho et al., 2008).

In contrast to the effects of yabA on replication from oriC, there was little or no effect when replication initiated from the DnaA-independent oriN. We constructed oriC-mutant strains by integrating the heterologous origin of replication, oriN, and its specific replication initiator, repN (Hassan et al., 1997; Kadoya et al., 2002; Goranov et al., 2005; Berkmen & Grossman, 2007), close to the location of the endogenous origin, and deleting part of oriC (oriC⁻). Replication from oriN does not require DnaA, but appears to require all other known replication initiation factors that are needed at oriC (Hassan et al., 1997). Neither deletion nor overexpression of yabA had any detectable effect on the DNA to protein ratios in oriN⁺ oriC⁻ strains (Fig.1B).

Subcellular localization of YabA

The use of strains capable of initiating replication from oriN makes dnaA dispensable for replication and viability (Hassan et al., 1997; Kadoya et al., 2002; Goranov et al., 2005; Berkmen & Grossman, 2007) and allowed us to determine if dnaA is required for formation of foci of YabA. Previous cell biological analyses indicate that GFP-YabA forms foci that correspond to the subcellular positions of the replisome (Hayashi et al., 2005; Noirot-Gros et al., 2006). Analyses of YabA mutants defective in interacting with either DnaA or DnaN (β-clamp) indicate that YabA needs to interact with both DnaA and the β-clamp in order to form foci (Noirot-Gros et al., 2006). Mutations in dnaA that cause altered interactions between DnaA and YabA were also found to prevent formation of foci of GFP-YabA (Cho et al., 2008), consistent with the prior findings. However, loss of interaction between YabA and DnaA is not the only effect of these mutations. The mutant cells also over-initiate replication (Noirot-Gros et al., 2006; Cho et al., 2008) and there could be other effects on YabA and DnaA as well. Therefore, it is possible that the YabA:DnaA interaction may not actually be required for focus formation by GFP-YabA and that the effects of the mutations on foci of GFP-YabA are indirect.

To test directly if DnaA and oriC are required for formation of GFP-YabA foci, we monitored the formation of GFP-YabA foci in oriC mutant cells initiating replication from oriN. We disrupted dnaA in the oriN⁺ oriC⁻cells by integrating a plasmid into dnaA and placing dnaN, the gene downstream, under control of the IPTG inducible promoter Pspac(hy). GFP-YabA still formed foci in the dnaA null mutant strain (Fig. 2A). Approximately 84% of the mutant cells (91 of 108 analyzed) had at least one focus of GFP-YabA. Similarly, in dnaA⁺ cells initiating from oriC, approximately 92% of cells (96 of 104 analyzed) had a detectable focus of GFP-YabA. From these results, we conclude that DnaA is not required for formation of GFP-YabA foci.

Maintenance of GFP-YabA foci was dependent on ongoing replication. We monitored GFP-YabA foci in cells treated with HPUra, an inhibitor of DNA Polymerase III that blocks replication elongation (Brown, 1970; Neville & Brown, 1972). Within 15 min after addition of HPUra, foci of GFP-YabA were very faint or undetectable in >99% of cells in both the dnaA null mutant initiating from oriN (Fig. 2B; no foci in 141 cells analyzed) and dnaA⁺ cells initiating from oriC (1 cell with a focus, of 113 cells analyzed) (compare Figs. 2C and 2D). The loss of visible foci does not necessarily mean that GFP-YabA is not there, just that it is below the limit of detection, and significantly different from that is untreated cells.

Loss of GFP-YabA foci correlated with loss of DnaN-GFP foci. We monitored the effects of replication arrest on foci of the β-clamp (DnaN-GFP), the catalytic subunit of DNA polymerase (PolC-GFP), the HolB subunit of the clamp loader (HolB-GFP), and the clamp loader/τ-subunit (DnaX-GFP). Foci of PolC-GFP (Fig. 2E, F), DnaX-GFP (Fig. 2G, H), and HolB-GFP (data not shown) persisted for greater than 60 min after replication arrest. In each case >80% of cells (of >75 analyzed) had at least one focus of the GFP-tagged replisome subunit. In contrast, foci of the β–clamp (DnaN-GFP) disappeared from most of the cells within 15 min after addition of HPUra (Fig. 2I, J). Without treatment, 97% of cells (124 of 128 analyzed) had at least one focus of DnaN-GFP. After treatment with HPUra, only 53 of 185 cells (29%) had a visible focus, and the foci detected were not as bright as those in cells without HPUra. These results indicate that after replication arrest with HPUra, several of the replisome components remain assembled, but that the β–clamp is largely dissociated. Taken together, our results indicate that formation of foci of GFP-YabA is independent of dnaA and oriC, but correlates with foci of DnaN-GFP, and that YabA and DnaN are released from the replication complex following replication arrest.

Overproduction of β-clamp (DnaN) stimulates DNA replication

Since both DnaA and YabA regulate replication initiation and YabA interacts with both DnaA and the β-clamp, we tested whether alterations in expression of dnaN (β-clamp) might also modulate replication initiation. We found that the β-clamp stimulates replication initiation, likely by inhibiting the activity of YabA. In these experiments, we manipulated transcription of dnaN using a xylose-inducible PxylA-dnaN fusion. Induction of PxylA-dnaN, in the presence of a wild type copy of dnaN, caused an approximately 8-fold increase in the amount of dnaN mRNA as determined using DNA microarrays (data not shown). We were unable to measure differences in levels of β-clamp protein since antibodies were not available. Nonetheless, alterations in expression of dnaN caused changes in replication.

Increased transcription of dnaN caused an increase of approximately 60% in the DNA to protein ratio relative to that of wild type (Fig. 3A), indicating that an increase in β-clamp stimulated DNA synthesis. This effect was not general for overexpression of any replisome component as overexpression of dnaX (the clamp loader/tau–subunit of DNA polymerase holoenzyme) for 3–4 generations caused a decrease in the DNA to protein ratio (Fig. 3A).

We also found that decreased transcription of dnaN caused a decrease in replication initiation. By placing the only copy of dnaN under the regulation of PxylA and growing without inducer (xylose) for 3 generations, the level of dnaN mRNA was reduced to ~60% of normal. The reduced transcription of dnaN caused a decrease of 18 ± 3% in the DNA to protein ratio (Fig. 3A). Severe decreases in expression of B. subtilis dnaN can cause replication fork stalling and induction of the SOS response {(Ogura et al., 2001) and data not shown}. Under the conditions in which mRNA levels of dnaN were reduced to ~60% of normal and replication was decreased, there was no detectable increase in mRNA levels of SOS genes (data not shown), indicating that the decrease in replication is either due to a very mild decrease in replication elongation or a decrease in replication initiation.

Overproduction of β-clamp stimulates DnaA-dependent replication initiation from oriC

Since dnaN encodes the β-clamp of DNA polymerase holoenzyme and is necessary for replication elongation, it is possible that the increase in DNA synthesis from overexpression of dnaN is due to either an effect of β-clamp on replication elongation, or due to an ability of β-clamp to modulate replication initiation. We found that the increase in DNA synthesis due to overexpression of dnaN was due to an increase in replication initiation. We monitored DNA replication in asynchronous populations of exponentially growing cells using DNA microarrays, essentially as described (Khodursky et al., 2000; Simmons et al., 2004; Goranov et al., 2006; Wang et al., 2007a; Wang et al., 2007b). Overexpression of dnaN for 25 (data not shown) or 50 min (Fig. 3B) in an oriC⁺ strain caused an increase in the amount of origin region DNA compared to other chromosomal regions. This type of increase is typically seen in cells overinitiating DNA replication {e.g., (Simmons et al., 2004)}.

The increase in replication initiation caused by overproduction of the β-clamp was dependent on replication initiation from oriC. We tested whether overexpression of dnaN affects replication initiation in a strain initiating solely from the heterologous origin oriN. We found that overexpression of dnaN (β-clamp) in oriN⁺ oriC⁻ mutant strains had no significant effect on the DNA to protein ratio (Fig. 3A). In addition, overexpression of dnaN for 25 (data not shown) or 50 min (Fig. 3C) in the oriN⁺ oriC⁻ mutant strains had no detectable effect on replication initiation as monitored using DNA microarrays to profile DNA content. These results indicate that the regulatory effects of β-clamp on replication depend on initiation from oriC, and that overexpression of β-clamp, to the levels tested here, stimulates replication initiation. There did not appear to be any significant effects on replication elongation as those are expected to be independent of the origin of replication.

Increased replication initiation caused by overproduction of the β-clamp depends on yabA

YabA and β-clamp directly interact (Noirot-Gros et al., 2002; Noirot-Gros et al., 2006), both affect replication initiation {(Noirot-Gros et al., 2002) and above}, and both require DnaA-dependent initiation of replication from oriC to manifest their effects (see above). Based on these properties, we wondered if the effects of overproduction of the β-clamp were dependent on YabA, or if the effects of a yabA null mutation and overproduction of β-clamp were additive. We constructed a strain that contains a deletion of yabA and overexpresses β-clamp (PxylA-dnaN). Each single allele causes overreplication as determined by DNA to protein ratios (Fig. 3A). Overexpression of dnaN in the yabA null mutant did not increase the DNA to protein ratio above that in the yabA null mutant alone (Fig. 3A), indicating that these effects were not additive and that yabA is required for overproduction of β-clamp to stimulate replication initiation. These results also indicate that YabA and the β-clamp are likely affecting the same rate-limiting step in replication initiation.

To be sure that we could detect an increase in replication initiation above that caused by the yabA null mutation alone, we tested for effects of overproduction of another replication protein. We found that the potential of the cells to initiate replication was not saturated in the yabA null mutant. There was an increase in the DNA to protein ratio in the yabA null mutant when the dnaBS371P (a.k.a., dnaB75) allele was expressed ectopically from Pspank(hy) (Fig. 3A). dnaBS371P causes a serine to proline change at amino acid 371 in the essential replication initiation protein DnaB. dnaBS371P causes aberrant replication (Rokop et al., 2004), bypasses the need for priA in replication restart (Bruand et al., 2001), and suppresses temperature sensitive mutations in the replication initiation gene dnaD(Rokop et al., 2004; Bruand et al., 2005; Rokop & Grossman, 2009). Expression of Pspank(hy)-dnaBS371P in cells growing in minimal medium caused a significant increase in the DNA to protein ratio in both yabA⁺ and yabA null mutant cells (Fig. 3A). These results indicate that the increase in replication caused by overexpression of dnaBS371P does not depend on yabA and that cells have the potential to increase replication above the level in the yabA mutant. The increase in replication appears to be additive, consistent with the notion that yabA and dnaBS371P affect different rate-limiting steps.

Gene expression in a yabA null mutant

YabA is an attractive candidate for regulating the activity of DnaA in response to replication status. YabA affects replication initiation from the DnaA-dependent oriC, it interacts with both DnaA and DnaN, and its association with the replisome correlates with that of DnaN. Expression of genes known or thought to be controlled by DnaA changes in response to replication stress (Goranov et al., 2005; Breier & Grossman, 2009). DnaA binds to the promoter regions of many of the proposed target genes in vivo (Goranov et al., 2005; Ishikawa et al., 2007; Breier & Grossman, 2009) and binding to some of these regions increases when replication is inhibited (Breier & Grossman, 2009). YabA could be required to couple the activity of DnaA to replication elongation, perhaps by sequestering DnaA at active replication forks as previously proposed (Noirot-Gros et al., 2006; Soufo et al., 2008). If this is the case, then the DnaA-mediated transcriptional response to replication arrest should be severely compromised in a yabA null mutant. Furthermore, expression of DnaA-regulated genes might be different in the absence of yabA, even during ongoing replication.

To determine if YabA is required for the regulation of DnaA in response to replication stress, we analyzed global gene expression in a yabA null mutant, both during growth and after replication arrest with HPUra. We compared the results to those in isogenic yabA⁺ cells. To eliminate potential pleiotropic effects due to overreplication in yabA null mutants, we did these experiments in strains initiating replication from oriN and containing an inactive oriC. yabA has no effect on replication in these conditions (Fig. 1B).

We found that the yabA null mutation had little or no effect on gene expression during exponential growth (Fig. 4A) nor in response to replication arrest (Fig. 4B). We plotted the relative mRNA levels for each gene in yabA⁺ cells versus those in the yabA null mutant (Fig. 4A, B). Genes known or thought to be regulated directly by DnaA are indicated with black + or − symbols, depending on whether expression increases (+) or decreases (−) in response to replication arrest. All other genes are indicated with gray dots. Most of the values from yabA⁺ cells are the same as or similar to those from the yabA null mutant, both during ongoing replication in exponential growth (Fig. 4A) and after replication arrest in cells treated with HPUra (Fig. 4B). These results indicate that during exponential growth and after replication arrest, there is little or no effect of loss of yabA on gene expression.

Using the data from Figures 4A and 4B, we compared the fold change in mRNA levels caused by replication arrest (treatment with HPUra) in yabA⁺ cells to that in the yabA null mutant (Fig. 4C). Expression of many genes increases in response to HPUra treatment and many of these are part of the SOS regulon and depend on recA (Goranov et al., 2005; Goranov et al., 2006). The changes in expression of these genes were similar in the yabA⁺ and the yabA null mutant cells (Fig 4C). In addition, most of the genes known or thought to be directly regulated by DnaA (+’s and −‘s in Fig. 4) also changed expression in response to HPUra, and those changes were similar in the yabA⁺ and yabA null mutant cells (Fig. 4C).

The magnitudes of the changes in gene expression in response to replication arrest in both yabA+ and yabA null mutant cells initiating replication from oriN were somewhat less than those previously reported for cells initiating replication from oriC (Goranov et al., 2005; Goranov et al., 2006). In fact, there were many differences in gene expression simply comparing cells initiating replication from oriC to those initiating from oriN in the absence of added replication stress (unpublished results). These differences indicate that the apparently constitutive and asynchronous replication initiation from oriN (Noirot-Gros et al., 2002; Hayashi et al., 2005) might cause a small amount of replication stress. Since the basal expression of some of the genes is already changed in the oriN cells, the magnitude of the effect of replication arrest is muted and less dramatic in these cells compared to cells initiating from oriC. Nevertheless, for many DnaA-regulated genes, there was a significant response to replication stress and there was little or no effect of yabA on this response.

YabA does not affect replication initiation from the DnaA-independent origin of replication oriN

Models proposing that YabA modulates the activity of DnaA in response to replications stress {e.g., (Soufo et al., 2008)} are quite reasonable. Unfortunately, analyses of the effects of yabA on the activity of DnaA are complicated by the fact that yabA affects replication initiation and alterations in replication initiation or elongation alter the activity of DnaA (Noirot-Gros et al., 2002; Goranov et al., 2005). Therefore, to test the effects of YabA on the activity of DnaA, it was helpful to eliminate the effects of YabA on replication while maintaining the ability to monitor the activity of DnaA as a transcription factor. This was accomplished by integrating the heterologous DnaA-independent origin of replication oriN into the chromosome and inactivating oriC.

We found that neither a null mutation in nor overexpression of yabA affected the activity of oriN. These findings indicate that the function of YabA in the negative control of replication initiation is specific to the DnaA-dependent oriC. Like the effects of a yabA null mutation, we found that overexpression of dnaN stimulates replication initiation from oriC, but not from oriN. We suspect that this stimulation by increased production of DnaN is likely due to titration of YabA away from oriC. Since the essential chromosomal replication initiation proteins DnaB DnaD and the replicative helicase DnaC and the clamp DnaN are required for replication from oriN (Hassan et al., 1997), these results indicate that YabA is not likely to affect the activity of these other proteins, at least in the absence of DnaA. These findings also made it feasible to determine the effects of yabA on the activity of DnaA under conditions in which yabA had no detectable effect on replication and to directly determine the effects of dnaA on the subcellular positioning of YabA.

Subcellular location of YabA

Analyses of GFP-YabA fusions indicated that YabA formed foci, apparently associated with the replisome during ongoing replication (Hayashi et al., 2005; Noirot-Gros et al., 2006; Cho et al., 2008). Formation of the GFP-YabA foci appeared to depend on DnaA based on analysis of YabA mutants unable to interact with DnaA (Noirot-Gros et al., 2006) and DnaA mutants unable to interact with YabA (Cho et al., 2008). YabA was also found be required to “tether” DnaA to the replisome (Soufo et al., 2008).

We also found that GFP-YabA formed foci. However, focus formation was not dependent on DnaA, nor was it dependent on oriC. These findings are not consistent with the interpretation that DnaA is required for the association of YabA with the replisome (Noirot-Gros et al., 2006; Cho et al., 2008; Soufo et al., 2008). The previous analyses used point mutations in yabA or dnaA that alter interactions between the two gene products (Noirot-Gros et al., 2006; Cho et al., 2008; Soufo et al., 2008). These point mutations are known to affect replication initiation (Noirot-Gros et al., 2006; Cho et al., 2008), which likely causes small amounts of replication stress. The point mutations in yabA and dnaA could also have other effects on those gene products.

The use of the heterologous origin of replication, oriN, allowed us to compare directly the ability of GFP-YabA to form foci in cells with and without dnaA. We observed no difference, indicating that neither oriC nor dnaA is required for formation of foci of GFP-YabA. We also found that after inhibition of replication elongation (replication fork arrest), several replisome subunits were still present in foci, but that foci of DnaN (β-clamp) and YabA became largely undetectable. These findings are consistent with previous results indicating that formation of GFP-YabA foci requires interaction with DnaN (Noirot-Gros et al., 2006).

yabA is not required for the transcriptional response to replication stress

Using strains in which yabA does not affect replication, we found no evidence that it affects the activity of DnaA or the ability of cells to respond to replication stress. In cells deleted for oriC and initiating replication from the DnaA-independent origin oriN, there was no significant effect of yabA on expression of genes known or thought to be regulated by DnaA during exponential growth. These findings are consistent with conclusions in a previous report that transcription of dnaA and association of DnaA with several chromosomal targets did not change significantly in a yabA null mutant (Cho et al., 2008). In addition, we found that genes known or thought to be regulated by DnaA still respond to replication stress in the absence of yabA. These results indicate that yabA is not required for modulating the activity of DnaA in response to disruptions in replication and are inconsistent with the model that YabA functions to tether DnaA at the replication fork and release it during replication stress (Soufo et al., 2008). Taken together, the simplest model for YabA function is that YabA acts locally at oriC to inhibit replication initiation. It is also formally possible that YabA affects DnaA globally, and that this effect is only manifest at oriC and does not alter the ability of DnaA to act as a transcription factor. Although we can not rule it out, we think this possibility is unlikely.

Models for the function of YabA and its interactions with DnaA and DnaN

YabA does not affect replication initiation from the DnaA-independent oriN, indicating that its function is specific to some aspect of oriC and/or DnaA. Since YabA does not affect DnaA in a distributive manner, that is, it does not appear to affect the ability of DnaA to function as a transcription factor, we favor models in which YabA functions at oriC to inhibit replication initiation.

It seems likely that there are at least three aspects to the ability of YabA to inhibit replication initiation from oriC without affecting the global activity of DnaA. First, YabA, although in bulk appears to be with the replication elongation machinery, must get to oriC. Second, it somehow inhibits replication initiation from oriC. Third, that inhibition is somehow relieved, or not complete, so that replication can initiate at the appropriate time in the cell cycle. Assuming that the interactions between YabA and both DnaA and DnaN are important for YabA function, then these interactions could be related to any of the three aspects of the ability of YabA to inhibit replication initiation.

There are many possible models that accommodate these three aspects of YabA function. For example, YabA could get to the origin via its interaction with DnaN, and then function to locally inhibit the activity of DnaA or some aspect of DnaA function at oriC. Versions of these types of models have been proposed (Hayashi et al., 2005; Cho et al., 2008). Missense mutations in yabA that cause altered interaction with either DnaA or DnaN cause phenotypes similar to those of a yabA null mutation (Noirot-Gros et al., 2006). If these mutant phenotypes are not due to general defects in YabA and are indicative of loss of YabA function, and are not due to secondary consequences of altered replication initiation, then the phenotypes appear to be most consistent with a model in which DnaN brings YabA to the oriC region, and then YabA inhibits a function of DnaA at oriC. For example, YabA might prevent the proper oligomerization or assembly of DnaA on oriC or prevent DnaA-mediated melting of oriC.

Alternatively, the interaction between YabA and DnaN could function to move YabA away from the oriC region and YabA could get to oriC through its interaction with DnaA. Once at oriC, YabA could regulate a step in replication initiation that is downstream of but not directly involving DnaA. YabA could also regulate some aspect of DnaA function that is required for the initiation of DNA replication, for example interaction between DnaA and DnaD (required to load the replicative helicase) (Ishigo-Oka et al., 2001; Cho et al., 2008), formation of DnaA multimers (Mott et al., 2008), or another aspect of DnaA function that has not been yet appreciated. Both of these models predict that the effects of YabA would be oriC specific, but differ in what aspect of initiation is affected by YabA. It is not yet known how YabA inhibits replication initiation, but it seems to be by a mechanism different from those described for the various factors that regulate replication initiation in E. coli and its relatives {e.g., (Kaguni, 2006)}.

Growth media and culture conditions

Cells were grown with vigorous shaking at 37°C (unless indicated otherwise) in S7 defined minimal medium with MOPS (morpholinepropanesulfonic acid) buffer at a concentration of 50 mM rather than 100 mM (Jaacks et al., 1989). The medium was supplemented with 1% glucose, 0.1% glutamate, and required amino acids. In experiments utilizing expression from the xylose-inducible promoter PxylA, (PxylA-dnaN and PxylA-gfp-yabA), glucose was replaced with 1% arabinose and expression from PxylA was induced with 0.5% xylose. In experiments utilizing expression from the IPTG-inducible promoters Pspac(hy) or Pspank(hy), expression was induced with 1mM IPTG. Strains containing single crossover constructs were routinely grown in the appropriate antibiotic to maintain selection for the integrated plasmid. Standard concentrations of antibiotics were used (Harwood & Cutting, 1990). Where indicated, replication elongation was blocked by addition of HPUra (stock in 50mM KOH) to a final concentration of 38 µg/ml. Control cultures were mock treated with KOH. Samples were typically harvested 60 min after treatment with HPUra.

Strains and alleles

B. subtilis strains are listed in Table 1 and specific alleles are described below. Genetic manipulations were performed using standard protocols (Harwood & Cutting, 1990).

(ypjG-hepT)122 is a deviation in sequences of the ~24 kb chromosomal region from ~ypjG (201.4°) to ~hepT (203.5°) and was described previously (Berkmen & Grossman, 2007). Briefly, the ypjG-hepT region contains the tryptophan biosynthesis genes (trpABFCDE), and the (ypjG-hepT) 122 variant likely encodes a heterologous tryptophan operon as strains containing it were transformed to tryptophan-prototropy.

ΔyabA::cat is a deletion-insertion that inactivates yabA by replacing it with cat. The allele was generated by the long-flanking homology PCR method (Wach, 1996). The deletion starts at the 1^st codon (TTG) and ends 50bp downstream of the translational stop, removing a total of 407bp. The deletion stops 13bp upstream of the next gene, yabB. The yabA ORF is substituted with the 994bp chloramphenicol resistance cassette from pGEMcat. The cassette contains the cat ORF, 322bp upstream to include the promoter, and 20bp downstream of the stop codon, and does not include the transcriptional terminators. mRNA levels of yabB and other downstream genes in the yabA::cat mutant AIG109 were indistinguishable from those in wild type (yabA⁺) cells as assessed by microarray analysis (data not shown).

amyE::{Pspank(hy)-yabA spc} is a fusion of yabA to the IPTG-inducible promoter Pspank(hy) at amyE and was used to overexpress yabA. AIG80 was constructed by cloning the entire ORF of yabA with its endogenous ribosome binding site into a plasmid containing the Pspank(hy) promoter (pDR66, a gift from David Rudner) thus generating plasmid pAIG10. The Pspank(hy)-yabA construct was integrated into the genome of JH642 through a double crossover at the amyE locus to generate strain AIG80.

spoIIIJ::{oriN repN kan} is an insertion of the plasmid origin of replication, oriN, along with the plasmid initiator gene repN, into the chromosome at spoIIIJ, near oriC (Goranov et al., 2005; Berkmen & Grossman, 2007). The presence of this replication origin allowed the inactivation of oriC.

oriC-S inactivates oriC replication functions by deleting ~150 bp of the sequence downstream of dnaA, including many essential DnaA binding sites and most of the AT-rich region that is normally unwound during replication initiation (Hassan et al., 1997; Kadoya et al., 2002; Berkmen & Grossman, 2007).

dnaN::{PxylA-dnaN cat} is a fusion of the only full copy of dnaN to the xylose-inducible promoter PxylA. This was constructed by amplifying a region of the genome of MMB26 that included PxylA and the 5’ end of dnaN. The amplified fragment was cloned into plasmid pGEMcat resulting in plasmid pAIG28. pAIG28 was integrated in the genome of JH642 by a single crossover to generate strain AIG260.

amyE::{PxylA-dnaN cat} is a fusion of PxylA to dnaN (encoding β-clamp) at amyE (Goranov et al., 2005; Berkmen & Grossman, 2007) and was used to ectopically express dnaN.

amyE::{PxylA-gfp-yabA cat} expresses a fusion of GFP to the N-terminus of YabA under control of PxylA and integrated at amyE in the chromosome. The gfp-yabA construct was obtained by cloning the entire yabA ORF in frame with gfp in the pEA18 plasmid (Gueiros-Filho & Losick, 2002). The resulting plasmid (pAIG58) was integrated into the chromosome through double cross over at the amyE locus. {The GFP-YabA fusion protein was at least partly functional as it complemented phenotypic characteristics of a yabA null mutant (data not shown) (Noirot-Gros et al., 2006)}.

dnaA::{Pspac(hy)-dnaN cat} disrupts dnaA while inserting Pspac(hy) to drive expression of dnaN. A 450bp DNA fragment internal to dnaA was PCR amplified and cloned into the SphI/HindIII cloning site of pJQ43 (Quisel et al., 2001) downstream of the Pspac-hy promoter to yield plasmid pAIG37. Integration of pAIG37 into the chromosome by a single crossover disrupts dnaA and places dnaN under the regulation of Pspac-hy. dnaA is normally essential, but it can be deleted in strains capable of initiating chromosomal replication from a heterologous origin such as oriN (Hassan et al., 1997; Moriya et al., 1997; Kadoya et al., 2002; Berkmen & Grossman, 2007). dnaA was in fact disrupted as evidenced by loss of detectable protein by Western blots and alterations in gene expression consistent with loss of DnaA (data not shown).

amyE::{Pspank(hy)-dnaB371 spc} places the dnaBS371P allele, containing a point mutation in the essential replication initiation gene dnaB that causes a serine to proline change at amino acid 371, under control of Pspank(hy) at amyE. dnaB371 confers a dominant phenotype of constitutive recruitment of the initiation protein DnaD to the cell membrane and overinitiation of replication (Rokop et al., 2004).

DNA/protein ratio determination

The ratio of DNA to protein was determined as previously described (Kadoya et al., 2002; Lee & Grossman, 2006). Briefly, 25ml of exponentially growing cells were collected at an OD600 ≤ 0.6. DNA and protein were extracted and the concentrations were determined using the diphenylamine reaction (DNA) and the Lowry BioRad DC Protein Assay Kit, with appropriate standards. The ratios for all strains were normalized to wild type (wt = 1.0) grown on the same day and under the same conditions. The average of three biological replicates is presented with error bars representing standard deviation.

DNA microarrays

DNA microarrays were prepared either using PCR products from >99% of the annotated B. subtilis open reading frames, or 65-mer oligonucleotide library representing all of the annotated ORFs in the B. subtilis genome (Sigma-Genosys) spotted onto Corning GAPS slides essentially as described previously (Britton et al., 2002; Au et al., 2005; Auchtung et al., 2005; Goranov et al., 2005). Culture samples were added to an equal volume of ice cold methanol and processed as described previously (Goranov et al., 2006).

Microscopy

Microscopy was performed essentially as described (Lee et al., 2003). Briefly, cells were grown in defined minimal medium at 30°C. Samples were taken and the vital membrane dye FM4-64 (Molecular Probes) was added to 0.05 µg/ml. Cells were placed on slides containing pads of 1% agarose in minimal salts with 1mM MgSO₄. Images were captured with a Nikon E800 microscope equipped with a Hamamatsu digital camera. Improvision OpenLabs 2.0 software was used to process images.