The temperature-sensitive dnaA46 and dnaA508 mutants exhibit synthetic lethality with priC303::kan even at 30 °C (Hinds and Sandler, 2004), suggesting that PriC can rescue blocks in DnaA-dependent replication initiation. However, the mechanism underlying PriC-promoted rescue of blocks in replication initiation remains a mystery. Based on previous reports demonstrating PriC binding to short oligo-ssDNA, SSB, and DnaB and its role in DnaB loading at stalled replication forks (Wessel et al., 2013; Wessel et al., 2016), we considered the possibility that PriC permits bypass of DnaA-dependent stable DUE unwinding and/or DnaB loading at the oriC ssDUE. To genetically test these ideas, we analyzed the impact of PriC on the growth of a temperature-sensitive dnaC2 mutant, which is competent for ongoing replisome progression at 37–42°C, but defective in DnaB loading specifically at the oriC ssDUE (Withers and Bernander, 1998).

In spotting assays performed using serial dilutions of the cell cultures and LB agar plates, the cell growth of dnaA46 cells was slightly inhibited at 37℃ (Figure 2A). Consistent with the previous report, when crossed with the ∆priC allele, the cell growth of dnaA46 cells but not WT dnaA cells was severely inhibited at 37℃ (Figure 2A). Unlike the dnaA46 priC303::kan cells, the dnaA46 ∆priC cells grew at 30 °C without prominent inhibition (Figure 2A). This difference may stem from the different strain backgrounds. Notably, the cell growth of dnaC2 mutant at 35℃ was moderately inhibited, whereas it was severely inhibited in dnaC2 ∆priC double mutant (Figure 2A). Also, the cell growth of dnaC2 cells and dnaC2 ∆priC cells was similar at 30 °C. The observed requirement of PriC for cell growth of dnaC2 cells at 35℃ supports the idea that PriC assists in the DnaC-dependent DnaB loading step at oriC.

Introduction of a priC-encoded plasmid (pBRpriC), but not an empty vector (pBR322), restored the cell growth of dnaA46 ∆priC double mutant at 37℃ (Figure 2—figure supplement 1A, B) and that of dnaC2 ∆priC double mutant at 35℃. Also, introduction of pBRpriC to dnaA46 and dnaC2 mutants with intact priC (priC⁺) might only slightly enhance the cell growth at these temperatures (Figure 2—figure supplement 1A, B), suggesting that the intrinsic level of PriC is functionally nearly sufficient.

To investigate replication initiation from oriC, we used flow cytometry of synchronized cells bearing dnaA46 or dnaC2 with or without ∆priC. In these experiments, cells were incubated at 42 °C or 37 °C for 80–90 min to synchronize the replication cycle at the pre-initiation step, after which incubation was continued at 30 °C for 5 min to allow initiation and then 4 hr in the presence of rifampicin and cephalexin to inhibit both replication initiation and cell division and allow replication run-out of the whole chromosome. The resultant number of chromosomes per cell, measured by flow cytometry, is known to correspond to the number of oriC copies per cell at the time of drug addition, an indicator of initiation activity.

A majority of the synchronized dnaA46 and dnaC2 cells had only a single oriC copy per cell after incubation at 42 °C or 37 °C, regardless of the presence of ∆priC (Figure 2B and C). In cells with intact priC (priC⁺), after release of initiation at 30 °C for 5 min, most cells had two oriC copies. However, in dnaA46 ∆priC cells, only about half of the cells had two oriC copies after the release (Figure 2B), indicating moderate inhibition of initiation. In dnaC2 ∆priC cells, the number of two-oriC cells was slightly lower than that of dnaC2 cells (Figure 2C). These observations are consistent with the cell growth ability (Figure 2A) and with our hypothesis that PriC contributes to the rescue of blocked replication initiation by assisting DnaB loading onto the oriC ssDNA.

We further performed flow cytometry analysis to assess the role of PriC in replication initiation of the WT dnaA/dnaC cells. To estimate the number of oriC copies per unit cell mass (ori/mass) as a proxy for initiation activity (Sakiyama et al., 2017; Sakiyama et al., 2022), exponentially growing cells were subjected to run-out replication in the presence of rifampicin and cefalexin (Figure 2—figure supplement 2). When cells were grown in LB medium at 25 °C, 30 °C, or 42 °C, the ori/mass values for ∆priC mutant were comparable to those of priC⁺. Similar results were observed in cells grown in M9 minimal medium at 30 °C. However, when grown in M9 minimal medium at 37 °C, ∆priC mutant cells exhibited slightly reduced ori/mass values. Also, it is noteworthy that in all conditions we tested, the fraction of cells with non-2ⁿ oriC copies was slightly higher in ∆priC cells compared to priC⁺. The asynchronous initiations would be caused by partial inhibition of initiation of multiple origins in ∆priC cells, as representatively seen for the case of ∆priC cells growing in M9 minimal medium at 37 °C where four-oriC cells reduced and three-oriC cells increased, compared to priC⁺ cells. A similar case might be shown for ∆priC cells growing in LB medium at 25 °C in that eight-oriC cells reduced and asynchronous initiations (A.I. %) as well as four-oriC cells increased. As such, inhibition of replication initiation would occur intrinsically at low frequency in the WT dnaA/dnaC background. PriC function would be effective to suppress the inhibition, thereby ensuring replication initiation of multiple origins.

DnaA contains two regions for interactions with DnaB during DnaB loading at oriC ssDNA: one is a high-affinity region in domain I containing Phe46, and the other is a low-affinity region in domain III containing His136. To analyze the mechanism of PriC-promoted DnaB loading at oriC, we introduced a series of pING1 vector-based plasmids encoding WT DnaA (pKA234) or the DnaA F46A H136A double mutant (pFH) into dnaA46 cells with or without priC. Growth of dnaA46 cells containing pING1 was inhibited at 42℃, but the leaky expression of dnaA from pKA234 enabled the growth of these cells, as previously reported (Sakiyama et al., 2018; Figure 3). Introduction of pFH moderately supported the cell growth of dnaA46 cells at 42℃. By contrast, dnaA46 ∆priC cells carrying pFH exhibited little growth at 42℃ (Figure 3).

Western blotting analysis supported that the expression levels of WT DnaA and DnaA F46A H136A proteins in dnaA46 cells bearing pKA234 or pFH were comparable, regardless of the presence or absence of priC⁺ (Figure 3—figure supplement 1). As DnaA46 is unstable and thermolabile (Shimuta et al., 2004), these cells were grown at 30 °C, and were incubated at 42 °C for 90 min before Western blotting analysis. The DnaA level in dnaA46 cells bearing the vector pING1 plasmid was minimum.

These results suggest that PriC is crucial for the growth of cells defective in specific DnaA-DnaB interactions, possibly because it can bypass the DnaA-DnaB interaction step required for DnaB loading onto oriC ssDNA.

DiaA, a homotetrameric protein, binds to multiple DnaA molecules and stimulates DnaA multimer assembly at oriC (Keyamura et al., 2007). The Phe46 residue in DnaA domain I is part of the DiaA-binding site and DnaB binding site. Oversupply of DiaA inhibits timely initiation of replication in cells growing in tryptone medium at 30 °C, probably because of binding competition between DiaA and DnaB for DnaA domain I (Ishida et al., 2004; Keyamura et al., 2009). If PriC can bypass the DnaA-DnaB interaction requirement for DnaB loading onto oriC, PriC should suppress inhibition of initiation by DiaA oversupply. To test this possibility, we introduced pBR322 or its derivative encoding diaA (pNA135) into cells with or without priC (Figure 4). priC⁺ carrying pBR322 or pNA135 similarly grew on LB agar plates containing ampicillin between 25°C and 42°C (Figure 4A, Figure 4—figure supplement 1). However, cell growth of ΔpriC cells carrying pNA135, but not pBR322, was moderately inhibited at 25 °C (Figure 4A), supporting our conjecture that PriC can bypass the requirement for the DnaA-DnaB interaction.

Rep helicase stimulates PriC-mediated rescue of arrested replication forks containing nascent strands (Heller and Marians, 2005b; Heller and Marians, 2005a; Sandler, 2000). To test the contribution of Rep helicase to PriC-promoted rescue of blocked replication initiation, we introduced pBR322 or pNA135 into ∆rep cells. Unlike ∆priC cells, ∆rep cells carrying pNA135 grew similarly to ∆rep cells carrying pBR322 (Figure 4A), suggesting no involvement of Rep in the PriC-promoted rescue of blocked replication initiation. This is consistent with the features of unwound oriC, which has a fork structure similar to that of the replication fork but without nascent strands, and the specific role of Rep in rescuing abandoned replication forks with nascent strands in the PriC-dependent pathway. Rep is specifically required for the unwinding of the nascent leading strand of abandoned replication forks, which is needed to expand the ssDNA region required for entry of PriC (Heller and Marians, 2005b; Heller and Marians, 2005a).

We further analyzed replication initiation in ∆priC cells carrying pNA135 at 30 °C by flow cytometry (Figure 4B). Under these conditions, priC⁺ bearing pBR322 mainly had eight or four oriC copies per cell with eight-oriC cells predominating (Figure 4B). The number of oriC copies per cell was similar to that in priC⁺-bearing pNA135, with only slight increase in asynchronous initiation, as shown by the slight increase in the number of cells with non-2ⁿ oriC copies, which is basically consistent with our previous data (Ishida et al., 2004). However, in ∆priC cells bearing pNA135, severe inhibition of initiation occurred, as shown by the substantial decrease in the number of eight-oriC cells and the large increase in the number of cells with only one to three oriC copies (Figure 4B). This result supports the proposed function of PriC at oriC mentioned above.

To test whether PriC rescues replication initiation inhibited by deletion of the diaA gene, we conducted similar flow cytometry analysis of ∆priC, ∆diaA, and ∆priC ∆diaA cells (Figure 4C). ∆diaA cells showed severe asynchrony of replication initiation, as shown by the increase in the number of five-to-seven-oriC cells, which supports the stimulatory role of DiaA in DnaA-oriC complex formation (Figure 4C). However, ∆priC ∆diaA cells did not show more asynchronization of replication initiation than ∆diaA cells, as shown by the similar asynchrony index (A.I.). These results suggest that PriC does not support or bypass DnaA-oriC complex formation.

Right DnaA-subcomplex plays a stimulatory role in DnaB helicase loading (Sakiyama et al., 2022; Stepankiw et al., 2009). The loading of two DnaB hexamers at oriC ssDNA regions requires multiple steps that depend on the distinct functions of two DnaA subcomplexes (Figure 1): the Left-DnaA subcomplex stably unwinds M-R regions of the DUE and the Right-DnaA subcomplex expands the unwound region to the AT-L region, supporting efficient DnaB helicase loading (Sakiyama et al., 2017; Sakiyama et al., 2022; Yoshida et al., 2023). The expanded ssDNA regions in this open oriC complex efficiently promote the loading of one DnaB hexamer onto the lower (A-rich) strand M-R region and a second DnaB hexamer onto the upper (T-rich) strand of the DUE (Fang et al., 1999; Sakiyama et al., 2022). In the absence of the Right-DnaA subcomplex, the AT region of the AT-L region assists in DnaB loading (Sakiyama et al., 2022).

To determine whether PriC rescues defects in DnaB loading when the oriC sequence is altered to prevent Right-DnaA subcomplex formation, we first assessed the cell growth of cells lacking the Middle- and Right-DOR of oriC (Left-oriC) (Figure 5A). The Left-oriC mutant formed colonies at 37 °C but showed severe growth defects at 25°C and 30°C (Figure 5B). At 37 °C, thermal energy might be sufficient to expand DUE unwinding into the AT region and allow DnaB loading in the absence of the Right-DnaA subcomplex (Sakiyama et al., 2022), but not at 25 °C or 30 °C. By contrast, the cell growth of the Left-oriC ∆priC double mutant was markedly compromised at 37 °C and moderately reduced at 25°C and 30°C (Figure 5B). These findings support the idea that PriC functions to rescue defective processes in DnaB loading caused by deletions of the oriC sequence that prevent DnaA subcomplex formation.

Next, similar experiments were performed using a mutant in which the R4 DnaA box within Right-DOR was substituted with a sequence (R4Tma) defective in binding of E. coli DnaA (Figure 5A and C; Noguchi et al., 2015; Sakiyama et al., 2022). We reasoned that formation of the Right-DnaA subcomplex would be inhibited by the introduction of R4Tma and PriC would be required for robust initiation in this context. As the Left-oriC strain, the priC⁺ R4 Tma strain showed normal cell growth. By contrast, cell growth of the R4Tma ΔpriC double mutant was impaired moderately at 25 °C and slightly at 30 °C (Figure 5C), indicating that PriC can rescue the blocked replication initiation caused by the absence of the R4 box. The reduced cell growth of R4Tma ∆priC strain was alleviated at higher temperatures, similar to the phenotype of the Left-oriC mutant.

Furthermore, we assessed replication initiation in R4Tma cells at 30 °C using flow cytometry (Figure 5D). R4Tma cells grew slightly slower than WT oriC cells and showed clear inhibition of initiation; in contrast to the WT oriC strain, the R4Tma mutant cell population contained more four-oriC cells and fewer eight-oriC cells, and showed severe asynchronous initiation. Notably, these negative effects of the R4Tma mutation were amplified by deletion of priC, i.e., the number of one to three-oriC cells increased and the ori/mass ratio was further reduced.

We next analyzed the role of PriC in cells lacking the AT-L region of oriC (subATL oriC) (Figure 6A). L-DUE and the flanking AT region assist in DnaB helicase loading by stimulating oriC unwinding, which is essential in a strain defective in Right-DnaA subcomplex formation (Sakiyama et al., 2022). SubATL oriC cells and WT oriC cells grew similarly on LB agar plates between 25°C and 37°C, regardless of the presence of ∆priC (Figure 6B). However, flow cytometry analysis revealed that deletion of priC specifically inhibited initiation in subATL oriC cells (Figure 6C); the number of eight-oriC cells was lower while the number of four-to-seven oriC cells was higher in subATL oriC ∆priC cells than in priC⁺ oriC cells. Taken together, these results are consistent with the idea that PriC restores initiation and DnaB loading at such truncated oriC sequences.

To analyze the mechanism of PriC rescue of blocked initiation at oriC, we assessed PriC activity in an in vitro reconstituted system for DnaB loading and DnaB-promoted DNA unwinding using the supercoiled circular form (form I) of the oriC plasmid pBSoriC and purified proteins, namely DnaA, N-terminally histidine-tagged DnaB (His-DnaB), DnaC, IHF, SSB, and gyrase. In this system, DnaA and IHF unwind the DUE and stimulate His-DnaB loading to the ssDUE region with the aid of DnaC. The loaded His-DnaB expands the ssDNA regions in concert with the ssDNA-binding activities of SSB. Concomitantly, the positive supercoiling generated through DNA unwinding by His-DnaB is resolved by DNA gyrase, resulting in the formation of plasmid DNA topoisomers (form I*). Form I* can be separated from form I by agarose gel electrophoresis (Baker et al., 1986; Sakiyama et al., 2022).

First, we assessed the effect of PriC on WT DnaA. WT DnaA stimulated form I* formation in the absence of PriC (Figure 7A, lanes 1 and 5) and increasing concentrations of PriC moderately inhibited WT DnaA-mediated form I* formation (Figure 7A, lanes 5–8), probably because of competition between DnaA and PriC for binding to DnaB. Next, we focused on the DnaA F46A H136A double mutant. The DnaA F46A and DnaA H136A mutant proteins alone are reported not to support form I* formation (Keyamura et al., 2009; Sakiyama et al., 2018). Consistently, the DnaA F46A H136A double mutant protein was inactive in form I* formation (Figure 7A, lane 9). However, when PriC was added to the assay, DnaA F46A H136A promoted form I* formation to a level comparable to that of WT DnaA in the presence of PriC (Figure 7A, lanes 5–12 and 7B), indicating that PriC rescues defective DnaB loading by DnaA F46A H136A-oriC complexes. These findings are consistent with the idea that PriC can bypass the strict reliance on DnaA-DnaB interactions for DnaB loading at oriC.

To corroborate this idea, we also determined whether PriC rescues blocked initiation resulting from unstable DUE unwinding. oriC complexes containing DnaA V211A cannot bind to the ssDUE and consequently fails in stable DUE unwinding (Ozaki et al., 2008). The DnaA V211A mutant was virtually inactive for form I * formation irrespective of the presence or absence of PriC (Figure 7A, lanes 13–16, and 7B), indicating that PriC cannot efficiently rescue a defect of DUE unwinding.

We further evaluated the DnaB loading activity of PriC in the presence of DiaA using a similar in vitro reconstituted system. Previously, DiaA was shown to stimulate oriC DUE unwinding, but to inhibit form I* formation, because DiaA-DnaA binding competitively inhibits DnaB-DnaA binding; DiaA and DnaB share the same binding site in DnaA domain I (Figure 7C lanes 1, 3, 5, and 7 and 7D) (Keyamura et al., 2009). PriC moderately stimulated form I* formation specifically in the presence of both DnaA and DiaA (Figure 7C lanes 7 and 8, and 7D). These results further support the idea that PriC can bypass the specific requirement for the DnaA-DnaB interaction in DnaB loading at oriC. DiaA bound to oriC-DnaA complexes might be a physical obstacle reducing efficiency of DnaB loading by PriC.

In addition, we analyzed the DUE unwinding activities of DnaA mutants using the in vitro reconstituted system. As previously reported, DnaA V211A is largely inactive in DUE unwinding, but DnaA F46A H136A unwinds the DUE at a level comparable to that of WT DnaA (Figure 7—figure supplement 1A and B). PriC did not stimulate DUE unwinding irrespective of the presence of WT DnaA (Figure 7—figure supplement 1C and D). These results demonstrate the functional specificity of PriC in rescuing failed DnaB loading at oriC ssDNA and are consistent with the PriC mechanism mentioned above.

Finally, to analyze the role of PriC in other types of replication initiation, we examine whether PriC contributes to the initiation of cSDR, which does not require dnaA or oriC and is activated in cells lacking rnhA or recG. Although the PriA-PriB-DnaT primosome complex has previously been reported to be involved in the initiation of cSDR (Heller and Marians, 2006), it is unclear whether PriC has any role in this type of initiation.

Unlike dnaA46 cells, dnaA46 rnhA::cat double mutant cells grew even at 40 °C (Figure 8A), indicating that these cells were engaged in cSDR, as previously reported (Hinds and Sandler, 2004). The cell growth of ∆priC dnaA46 rnhA::cat triple mutant was severely inhibited at 40 °C (Figure 8A), suggesting that PriC contributes to the growth of dnaA46 rnhA::cat mutant cells.

To determine whether PriC contributes to the initiation of dnaA46-oriC replication or cSDR, we calculated the relative copy numbers of oriC (84.6 min) and ter (32.4 min: corresponding to insQ) using real-time quantitative PCR (qPCR). The ypaB (50.5 min) located at the middle position between oriC and ter was used as a reference. To calculate the copy number of the chromosomal ter region, the preferential initiation site of cSDR (Brochu et al., 2018; Maduike et al., 2014), dnaA46 rnhA::cat cells growing exponentially at 30 °C were shifted to 40 °C for 90 min and the copy number before and after incubation at 40 °C was calculated. Consistent with the cell growth data (Figure 8A), the ter copy number ratio of the dnaA46 rnhA::cat double mutant increased after 40 °C incubation, whereas the copy number ratio of the dnaA46 mutant expressing WT rnhA remained the same (Figure 8B). The ter copy number ratio of the dnaA46 rnhA::cat ∆priC triple mutant strain was also increased, indicating that cSDR could occur even in the absence of PriC.

To monitor initiation of DnaA-oriC replication, we determined the oriC copy number ratio (Figure 8B). At the permissive temperature, despite having similar growth rates, the oriC copy number ratio of the dnaA46 ∆priC double mutant cells was lower than that of the dnaA46 single mutant, confirming the importance of PriC for replication initiation in dnaA46 cells, as shown in Figure 2. In the priC⁺dnaA46 rnhA::cat mutant cells, the oriC copy number ratio was reduced, which is explained by activation of cSDR and increase in the relative copy number of the reference ypaB locus (50.5 min chromosomal map position). Together, these results suggest the idea that PriC stimulates replication fork progression during cSDR.

To consolidate these findings, we performed the chromosome loci copy-number analysis using whole-genome sequencing. The dnaA46 cells and its derivative cells were grown exponentially at 30 °C, followed by incubation at 40 °C for 90 min prior to DNA extraction. In growing WT cells, the highest copy numbers are observed near the oriC-proximal region and the lowest are near the ter-proximal region, resulting in a coverage bias from oriC to ter (Brochu et al., 2018; Maduike et al., 2014). However, in dnaA46 cells incubated at 40 °C, the coverage bias was largely diminished due to the run-out replication (Figure 8—figure supplement 1A). Notably, dnaA46 rnhA::cat cells incubated at 40 °C exhibited discrete coverage peaks at the preferential initiation sites of cSDR near the ter (or insQ) region with a decreasing coverage bias to oriC. Similar profiles were observed in the ∆priC mutant background, supporting the idea that cSDR can occur even in the absence of PriC (Figure 8—figure supplement 1A). Moreover, in the dnaA46 rnhA::cat background, the absence of priC appeared to decrease the coverage level at genomic positions near the 1.3 MB and 4 MB map positions, which are slightly outside of ter and oriC, respectively (Figure 8—figure supplement 1B). This observation may reflect the role of priC in the replication fork progression.

The plasmids used in this study are listed in key resource table. pKA234, pKW44-1, pNA135, pBSoriC, pET22b(+)-priC, and pTKM601 were described previously (Aramaki et al., 2015; Ishida et al., 2004; Kawakami et al., 2005; Keyamura et al., 2009; Kubota et al., 1997; Ozaki et al., 2008). For the construction of pFH, an alanine substitution was introduced into pH136A with specific mutagenic primer sets using the QuikChange site-directed mutagenesis protocol (Stratagene [Agilent], Agilent, La Jolla, CA, United States) as described previously (Keyamura et al., 2007; Sakiyama et al., 2018).

WT DnaA and its derivative proteins were overproduced in E. coli strain KA450 from pKA234, pKW44-1, or pFH and purified as described previously (Noguchi et al., 2015; Ozaki et al., 2008; Sakiyama et al., 2017).

PriC protein was prepared as previously reported with minor modifications (Aramaki et al., 2013). Briefly, PriC protein was overproduced in E. coli strain BL21-codonPlus-RIL by inducing its expression from pET22b(+)-priC using 0.2 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG). The resulting cells were suspended in chilled buffer A (50 mM HEPES-NaOH [pH7.0], 10% sucrose, 1 mM EDTA, 2 mM dithiothreitol (DTT), and 1 mM PMSF) and disrupted by sonication. Proteins in the soluble fraction were precipitated with 0.24 g/mL ammonium sulfate, resuspended in a separation buffer (50 mM imidazole [pH 7.0], 20% glycerol, 2 mM DTT, 1 mM EDTA, and 40 mM ammonium sulfate) and loaded onto a 1 mL HiTrap SP HP column (Cytiva, Uppsala, Sweden). Bound proteins were eluted with a linear gradient from 0 to 1 M sodium chloride.

All E. coli strains used in this study are listed in key resource table. KYA018, MIT125, MIT162, SA103, NY20, and NY21 were described previously (Kasho and Katayama, 2013; Noguchi et al., 2015; Noguchi and Katayama, 2016). Strains bearing mutant oriC (NY20L and NY20ATL) were constructed using the λRed site-directed recombination system as previously described (Noguchi et al., 2015; Sakiyama et al., 2017; Sakiyama et al., 2022). NY20-frt strain and oriC mutant strains (NY21-frt, NY20L-frt, and NY20ATL-frt) were constructed by eliminating kan from the NY20 strain and strains NY21, NY20L, and NY20ATL. To remove the kanamycin-resistant cassette (kan) from oriC, FLP recombinase encoded on pCP20 was used. Elimination of kan was verified by checking sensitivity to 50 µg/ml kanamycin in LB agar plates. ∆priC::frt-kan from JW0456-KC and ∆rep::frt-kan from JW5604-KC were introduced into MG1655 by P1 transduction, resulting in the construction of KRC002 and FF001, respectively. For construction of other ∆priC::frt-kan strains (KRC004, KRC005, NY20-priC, NY21-priC, NY20L-priC, NY20ATL-priC), P1 phage lysates prepared from KRC002 were used for transduction of strains MIT125, KYA018, NY20-frt, SYM21-frt, NY20L-frt, and NY20ATL-frt. Transductants were screened on LB agar plates containing 50 µg/mL kanamycin. ∆diaA::frt-kan or rnhA::cat derivatives (KRC006, MIT125c, and KRC004c) were constructed using a similar protocol except that P1 phage lysates from SA103 (∆diaA::frt-kan) or MIT162 (rnhA::cat) were used. KRC003 was generated by removal of kan from KRC002.

Buffer P contained 60 mM HEPES–KOH (pH 7.6), 0.1 mM zinc acetate, 8 mM magnesium acetate, 30% [v/v] glycerol, and 0.32 mg/mL bovine serum albumin (BSA). Buffer N contained 50 mM HEPES–KOH (pH 7.6), 2.5 mM magnesium acetate, 0.3 mM EDTA, 7 mM DTT, 0.007% [v/v] Triton X-100, and 20% [v/v] glycerol. Form I* buffer contained 20 mM Tris-HCl (pH 7.5), 125 mM potassium glutamate, 10 mM magnesium acetate, 8 mM DTT, and 0.5 mg/mL BSA.

DUE unwinding assays were performed essentially as described with minor modifications (Sakiyama et al., 2022; Yoshida et al., 2023). Briefly, DnaA was incubated with ATP in buffer N on ice for 3 min to generate ATP-DnaA. The indicated amount of PriC and ATP-DnaA or its mutant derivatives was incubated for 9 min at 30℃ in 10 µL buffer P containing 5 mM ATP, 125 mM potassium glutamate, 1.6 nM pBSoriC, and 42 nM IHF, followed by further incubation with 1.5 units of P1 nuclease (Wako) for 5 min. The reaction was stopped by the addition of 1% SDS and 25 mM EDTA, and DNA was purified by phenol-chloroform extraction and ethanol precipitation. One-third of each purified DNA was digested with AlwNI (NEB), which yielded 2.6 kb and 1.0 kb fragments after DUE unwinding of pBSoriC. The resultant DNA fragments were analyzed by 1% agarose gel electrophoresis with 1x Tris-acetate-EDTA buffer for 30 min at 100 V, followed by ethidium bromide staining. Gel images were taken using the GelDoc GO imaging system (Bio-Rad Laboratories, Hercules, CA), and products derived from unwound plasmids were quantified using ImageJ software.

This assay was performed as previously described with minor modifications (Baker et al., 1986; Noguchi et al., 2015; Sakiyama et al., 2022). The indicated amount of ATP-DnaA was incubated for 15 min at 30℃ in 12.5 µL of Form I* buffer containing 3 mM ATP, 1.6 nM pBSoriC, 42 nM IHF, 400 nM His-DnaB, 400 nM DnaC, 76 nM GyrA, 100 nM His-GyrB, and 760 nM SSB. The reaction was stopped by the addition of 0.5% SDS, and DNA was purified by phenol-chloroform extraction. Samples were analyzed by 0.65% agarose gel electrophoresis with 0.5x Tris-borate-EDTA buffer for 15 hr at 23 V, followed by ethidium bromide staining.

Flow cytometry analysis was performed essentially as described (Kasho et al., 2014). Briefly, cells were grown at 30℃ in LB medium until the absorbance of the culture (A₆₀₀) reached 0.1. Portions of the cultures were diluted a thousand-fold into 5 ml LB medium and incubated at 30℃ until the absorbance of the culture (A₆₀₀) reached 0.1. The remaining portions were further incubated to determine the doubling time (Td) by measuring A₆₀₀ every 20 min. At A₆₀₀ 0.1, aliquots of the cultures were fixed in 70% ethanol to analyze cell mass using the Multisizer 3 Coulter counter (Beckman Coulter, Brea, CA). The remaining cultures were further incubated for 4 hr with 0.3 mg/mL rifampicin and 0.01 mg/mL cephalexin to allow run-out replication of chromosomal DNA. The resultant cells were fixed in 70% ethanol. After DNA staining with SYTOX Green (Thermo Fisher Scientific, Waltham, MA), cellular DNA contents were analyzed on a FACS Calibur flow cytometer (BD Bioscience, Franklin Lakes, NJ). The number of the origins/cell (ori/cell) was determined from the histograms of flow cytometry analysis. The number of ori/cell was divided by the mean cell mass determined by cell mass analysis, resulting in ori/mass ratios. The numbers of cells containing non-2ⁿ copy number of oriC were divided by the number of cells containing 2ⁿ copy number of oriC to calculate the asynchrony index (A.I.). For the analysis of cells bearing plasmids, LB medium was supplemented with 100 µg/mL ampicillin.

MIT125 (dnaA46 tnaA::Tn10) cells or its ∆priC derivative KRC004 were grown at 30℃, a permissive temperature, in LB medium until the absorbance of the culture (A₆₀₀) reached 0.04, followed by further incubation for 90 min at 42℃, a restrictive temperature. Aliquots of the cultures were fixed in 70% ethanol as ‘Synchronization’ samples. The remaining cultures were further incubated for 5 min to initiate DNA replication, followed by further incubation for 4 hr at 42℃ with 0.3 mg/mL rifampicin and 0.01 mg/mL cephalexin to allow run-out replication of chromosomal DNA. The resultant cells were fixed in 70% ethanol as ‘5 min release’ samples. After DNA staining with SYTOX Green (Life Technologies), cellular DNA contents were analyzed on a FACS Calibur flow cytometer (BD Bioscience). Ratios of the origins/cell (ori/cell) were determined from the histograms of flow cytometry analysis.

For synchronization of dnaC2 mutant cells (KYA018 and KRC005), cells were grown in LB medium at 30℃ until the A₆₀₀ reached 0.04, followed by further incubation for 80 min at 37℃, at the restrictive temperature. The resulting synchronized cultures were released for 5 min at 30℃ and incubated in the presence of 0.3 mg/mL rifampicin and 0.01 mg/mL cephalexin at 30℃ to allow run-out of chromosomal DNA replication.

Cells of MIT125 (dnaA46 tnaA::Tn10) cells and MIT125c (dnaA46 tnaA::Tn10 rnhA::cat) or its ∆priC derivative KRC004 (dnaA46 tnaA::Tn10 ∆priC::frt-kan) and KRC004c (dnaA46 tnaA::Tn10 rnhA::cat ∆priC::frt-kan) were grown at 30℃ in LB medium until the absorbance of the culture (A₆₀₀) reached 0.04 and aliquots of the cultures were withdrawn. The remaining samples were further incubated for 90 min at 40℃ and aliquots of the cultures were withdrawn. These samples were boiled for 5 min at 95℃ and the genome DNA was extracted. The levels of oriC (84.6 min), ter (32.4 min), and ypaB (50.5 min) were quantified by real-time qPCR using TB Green Premix Ex TaqII (Tli RNaseH Plus) (TaKaRa, Shiga, Japan) and the following primers: ORI_1 and KWoriCRev for oriC, qoriK fw, and qoriK rev for ter, STM419, and STM420 for ypaB.

The analysis was performed based on a previous paper with modifications (Brochu et al., 2018). Cells of the dnaA46 mutant derivatives MIT125 (dnaA46 tnaA::Tn10), MIT125c (dnaA46 tnaA::Tn10 rnhA::cat), KRC004 (dnaA46 tnaA::Tn10 ∆priC::frt-kan) and KRC004c (dnaA46 tnaA::Tn10 rnhA::cat ∆priC::frt-kan) were grown exponentially at 30℃ in LB medium (50 mL). When the absorbance of the culture (A₆₀₀) reached 0.04, cells were incubated for 90 min at 40℃ and collected by centrifugation. Cell pellets were dissolved by incubation at 37℃ for 1 hr in TE buffer (0.6 mL) containing 0.5% SDS and 25 µg/ml proteinase K, and were mixed with 0.2 mL of buffer containing 4% cetyltrimethylammonium bromide, followed by further incubation for 10 min at 65℃, phenol-chloroform extraction and isopropanol precipitation. Precipitates were resuspended in Tris (pH7.5) buffer containing 0.1 mg/ml RNase A, followed by incubation for 1 hr at 37℃ and DNA purification by FastGene Gel/PCR Extraction Kit (Nippon Genetics, Tokyo, Japan). The quality control of genomic DNA, library preparation, and whole-genome sequencing were performed by Rhelixa, Inc using NEBNext Ultra II DNA Library Prep Kit and Illumina NovaSeq X Plus. The number of reads (150 bp × two pair-ended) was at least 6.7 M (3.3 M pairs) per sample. The sequence data were analyzed using Galaxy (https://usegalaxy.org) and the coverage plot was generated essentially as described (Ivanova et al., 2015). Briefly, paired-end reads were mapped using the MG1655 reference genome (NC_000913) and the BWA-MEM algorithm, followed by calculation of the number of reads per genome coverage. DNA-sequence data are deposited in the NCBI Sequence Read Archive (Accession number: PRJNA1222470).

The assay was performed as described previously with minor modifications (Kawakami et al., 2005; Sakiyama et al., 2017; Yoshida et al., 2023). Briefly, MIT125 (dnaA46 tnaA::Tn10) and KRC004 (dnaA46 tnaA::Tn10 ∆priC::frt-kan) cells bearing pING1, pKA234, or pFH were grown at 30 °C in LB medium including 100 µg/ml ampicillin until the absorbance of the culture (A₆₀₀) reached 0.05, followed by incubation at 42 °C for 90 min. Cells were harvested by centrifugation and dissolved in SDS sample buffer to adjust the calculated A₆₀₀ of 0.5. Proteins were separated using SDS-12% polyacrylamide gel electrophoresis and were transferred to polyvinylidene difluoride membranes (Millipore) using a semi-dry blotting method (Bio-Rad). After blocking at 30 °C for 30 min in 3% gelatin-TBS (3% gelatin, 20 mM Tris–HCl, 500 mM NaCl, pH 7.5), the membrane was incubated overnight at 4 °C in 1% gelatin-TTBS (20 mM Tris–HCl, 500 mM NaCl, 0.05% Tween-20, pH 7.5) containing anti-DnaA rabbit antiserum (1:3000). After washing, the membrane was incubated at 30 °C for 1 hr in 1% gelatin-TTBS containing goat anti-rabbit IgG antibody conjugated to alkaline phosphatase (Bio-Rad), followed by development using AP Conjugate substrate Kit (Bio-Rad).