Cells were grown in AB minimal medium (Clark and Maaløe, 1967) supplemented with 1 μg/ml thiamine, 0.2% glucose and 0.5% casamino acids (glucose-CAA medium). When necessary, antibiotic selection was maintained at the following final concentrations: kanamycin, 50 μg/ml; chloramphenicol, 20 μg/ml; tetracycline, 10 μg/ml; ampicillin, 150 μg/ml. All cells were cultured at 37°C, except when otherwise indicated. Cell growth was monitored by measuring optical density at 450 nm (OD₄₅₀).

All strains used were derived from E. coli K-12 MG1655 (F⁻, λ⁻, rph-1; Guyer et al., 1981) and are listed in Table 1. The ΔhipBA::frt::kan::frt (Germain et al., 2013) and dnaA46 tnaA600::Tn10 (Kogoma and von Meyenburg, 1983) alleles were moved by P1-phage-mediated transduction (Miller, 1972). To construct the chromosomal seqA mutant strains (seqA_S36A and seqA_S36D, respectively), base substitutions were made in the codon for Ser36 (5′-TCC-3′ to 5′-GCC-3′; Ser(S) to Ala(A), and 5′-TCC-3′ to 5′-GAC-3′; Ser(S) to Asp(D), respectively) using splicing by overlap extension (SOEing) polymerase chain reaction (PCR) (Horton et al., 1989). All primers are listed in Table 2. For each seqA variant two initial PCR products of the MG1655 chromosome were generated. 1) The lower region of the seqA gene, spanning residues 27–181, was amplified using primers “SeqA_down_bw_XmaI” and either “SeqA_pos36_SA_fw” or “SeqA_pos36_SD_fw”. 2) The upper region of the seqA gene was amplified using primers “SeqA_up_fw_SacI” and “SeqA_intern_bw” that generates a fragment with an overlap of 21 bp with the seqA downstream PCR product. A secondary amplification was performed using equimolar ratios of the two PCR products as template, and the oligonucleotides, SeqA_down_bw_XmaI, and SeqA_up_fw_SacI, as primers. The resulting PCR fragments were digested with XmaI and SacI, and cloned into the same sites of the 3.9 kb suicide vector, pRUC1437, a derivative of pSW29T (Demarre et al., 2005), carrying the aph gene encoding kanaymicn resistance, and the sacB gene. The resulting plasmids were transformed into strain S17-1 (recA thi pro hsdR⁻M⁺ RP4-2 Tc::Mu-Km::Tn7 λ-pir lysogen Tp^R Sm^R; (Simon et al., 1983) before being transferred into ALO 2956 cells by conjugation. Selection of exconjugants carrying the chromosomally integrated recombinant suicide plasmids as well as subsequent sucrose-mediated selection for loss of the sacB gene (i.e., loss of suicide vector sequences; (Donnenberg and Kaper, 1991), leaving either a wild-type or a mutant variant of the seqA gene on the MG1655 lacIZYA::cat chromosome, was performed as described previously (Riber et al., 2009). Chromosomal seqA mutant strains were verified by DNA sequencing of PCR fragments amplified from the seqA region using DNA oligonucleotides, “SeqA_chr_fw” and “SeqA_chr_bw,” as primers.

All plasmids used are listed in Table 3. Plasmids pLR77 and pLR75 were constructed by PCR amplifying the seqA variant genes (including the native seqA ribosome binding site) from MG1655 lacIZYA::cat cells carrying either the seqA_S36A or seqA_S36D chromosomal genes (see above), respectively, using DNA oligonucleotides, SeqA_up_fw_EcoRI and SeqA_down_bw_HindIII, as primers. The resultant PCR fragments were digested with EcoRI and HindIII and inserted downstream the IPTG inducible lacP_A1−04/03 promoter (Lanzer and Bujard, 1988) of plasmid pFH2102 (von Freiesleben et al., 2000), cut with the same enzymes. The inserted seqA mutant genes were later verified by DNA sequencing.

Exponentially growing cells (OD₄₅₀ = 0.15–0.30) were treated with rifampicin (300 μg/ml; SERVA Electrophoresis GmbH) and cephalexin (36 μg/ml; Sigma-Aldrich) to inhibit initiation of DNA replication and cell division, respectively (Løbner-Olesen et al., 1989). Incubation continued for a minimum of 4 h at 37°C to allow completion of ongoing rounds of replication. Cells were fixed in 70% ethanol and stained with 90 μg/ml mithramycin (SERVA Electrophoresis GmbH) and 20 μg/ml ethidium bromide (Sigma-Aldrich) as described (Løbner-Olesen et al., 1989). Flow cytometry was performed as previously described (Løbner-Olesen et al., 1989) using an Apogee A10 instrument (Apogee, Inc.). For all samples a minimum of 50.000 cells were analyzed. Numbers of origins per cell and relative cell mass were determined as previously described (Løbner-Olesen et al., 1989).

Samples of 2 ml of exponentially growing cells (OD₄₅₀ = 0.3–0.4) were harvested. Proteins were separated by SDS-PAGE and SeqA protein detected by Western blot using rabbit antiserum raised against SeqA protein (Torheim et al., 2000) as previously described (Riber and Lobner-Olesen, 2005). The membrane was scanned using a 230 V GenoView imaging system equipped with a UV transilluminator (VWR). Quantification was done using the ImageJ software.

Multiple alignment analysis of SeqA amino acids sequences was performed in the MEGA version 7.0.26 software (Kumar et al., 2016) using the default settings of the integrated ClustalW algorithm (Larkin et al., 2007). Selected species including SeqA protein accession numbers were: Escherichia coli K-12 (accession: AAA19855.1), Vibrio cholerae (accession: AOY47782.1), Pasteurella multocida PM70 (accession: AAK02440.1), Haemophilus influenzae Rd (accession: NP_438362.1), Yersinia enterocolitica (accession: CNB62546.1), Serratia marcescens (accession: KFL03527.1), Actinobacillus pleuropneumoniae (accession: SQF64393.1), and Glaesserella parasuis (accession: STO80764.1).

In order to determine any putative role of SeqA phosphorylation, we generated two mutations at the chromosomal codon 36 of seqA. In one strain, the codon for Ser36 was replaced with that of an aspartic acid (seqA_S36D). The SeqA_S36D mimics the conformation of Ser36 phosphorylated SeqA (Arany et al., 2013). In a second strain the codon for Ser36 was replaced with that of an alanine (seqA_S36A). The resulting protein, SeqA_S36A, is phosphorylation impaired at position 36 (Arany et al., 2013).

We used the RaptorX web server (Källberg et al., 2012) to predict the tertiary structures (Peng and Xu, 2011; Ma et al., 2012) of the wild-type SeqA and the SeqA_S36A and SeqA_S36D proteins. This revealed a significant level of resemblance (Figure 1A). By pairwise and multiple structural alignments of the SeqA protein variants, TMScore values above 0.9 were obtained, illustrating a significantly increased likelihood (>90% of chance) that the proteins pairwise and all together share similar folds, RaptorX Structure Alignment Server; (Wang et al., 2011, 2013), with SeqA and SeqA_S36D being structurally most alike [TMScore (WT vs. S36D) = 0.96]. The structural differences caused by changing the Ser36 residue seem to affect only the flexible linker region between SeqA-N and SeqA-C (Figure 1A; Chung et al., 2009).

We used flow-cytometry to determine cell cycle parameters of wild-type and seqA mutants. The two seqA mutants grew with similar doubling times as wild-type cells in minimal medium supplemented with glucose and casamino acids, whereas cells deficient in SeqA grew with an ~30% increased doubling time relative to that of wild-type cells (Figure 1B). Following treatment with rifampicin and cephalexin, wild-type, seqA_S36A and seqA_S36D cells were similar and contained mainly 2, 4, or 8 fully replicated chromosomes, indicative of initiation synchrony (Skarstad et al., 1986). As the average cell mass and numbers of origins per cell were similar, so was the origin concentration between these three cell types. SeqA deficient cells showed an asynchronous initiation phenotype with an increased average number of origins, which illustrates a lost ability to negatively regulate replication initiation. The average cell mass was similar to that of wild-type cells resulting in an increased origin concentration (Figure 1B).

Because the SeqA_S36A and SeqA_S36D protein levels were comparable or slightly elevated relative to that of wild-type SeqA protein (Figure 1C), these data altogether suggest that phosphorylation of SeqA at position 36 has little influence on its activity in replication initiation control. This was further corroborated by analyzing cells deficient in the HipA kinase, i.e., with a knock-out of the hipA gene. Here we found that ΔhipBA::kan mutant cells displayed similar cell cycle parameters as wild-type cells (Figure 1D).

We proceeded to examine whether overexpression of wild-type and mutant SeqA proteins could reveal any difference in activity among the phospho-impaired (S36A), phospho-mimetic (S36D) and wild-type SeqA proteins.

We expressed the seqA, seqA_S36A, and seqA_S36D genes from the IPTG-inducible lacP_A1/04−03 promoter in SeqA deficient cells. Exponentially growing cells were induced with 1 mM IPTG at time 0 min (T = 0 min). Immunoblot analysis of cells sampled at 120 min following the addition of IPTG indicated that all SeqA proteins were expressed to comparable levels corresponding to an ~12- to 14-fold increase in SeqA level relative to wild-type cells (Figure 2A). Both wild-type and mutant SeqA proteins complemented ΔseqA cells to the same extent when produced from a plasmid. Cells containing mainly two or four origins, indicative of initiation synchrony, dominated the population already after 30 min induction of the seqA variant genes (Figure 2B). A larger increase in mutant SeqA proteins (T = 120 min) resulted in no significant asynchrony relative to wild-type (Figure 2B). This is in agreement with earlier data on SeqA overproduction (Fossum et al., 2003).

Changes in the duration of sequestration by increasing or decreasing the level of Dam methylase (von Freiesleben et al., 2000) or by increasing the SeqA level (Charbon et al., 2011) were previously found to have relatively modest effects on the cell cycle relative to complete loss of sequestration. We therefore proceeded to determine whether the SeqA mutant proteins affected the length of the sequestration period, defined as the minimal time between successive initiations (von Freiesleben et al., 2000).

We introduced the dnaA46 allele into seqA_S36A and seqA_S36D cells by P1-transduction. The resultant strains are initiation proficient at 30°C (permissive temperature), but not at 42°C (non-permissive temperature) due to a reversible defect in nucleotide binding (Carr and Kaguni, 1996). Wild-type, seqA_S36A and seqA_S36D cells carrying the dnaA46 allele were grown exponentially at 30°C. The average number of origins per cell for all three strains was close to 2 (Figures 3A–C) and the SeqA proteins were produced in similar amounts (Figure 3D). When cells were shifted to 42°C, initiations ceased whereas cells continued to grow and divide, resulting in most cells ending up having one fully replicated chromosome after 90 min (Figures 3A–C). Upon a shift back to 30°C, where the DnaA46 protein was reactivated, all cells initiated replication, i.e., doubled their origin content, within a short period of time. This round of initiation was followed by a period of ~20 min where all newly formed origins were inert to further initiation, after which replication initiation resumed (Figures 3A–C). This 20-min period represents the minimal time between successive initiations (von Freiesleben et al., 2000), and it did not differ between wild-type and seqA mutant cells (Figures 3A–C). SeqA deficient cells were previously shown to reinitiate frequently without this 20-min delay (von Freiesleben et al., 2000).

We aligned SeqA amino acid sequences from the Vibrio cholerae, Pasteurella multocida PM70, Haemophilus influenzae Rd, Yersinia enterocolitica, Serratia marcescens, Actinobacillus pleuropneumoniae and Glaesserella parasuis with that of E. coli K12. All of these bacteria are known to carry hipBA genes. We looked for conservation of Ser36 along with the two flanking amino acids Phe35 and Ala37 (Table 4). None of these amino acids were conserved among the species with Ser36 showing the least degree of conservation. On the other hand Thr18, Ile21, and Ala25 which are instrumental in oligomerization of SeqA (Guarné et al., 2005), were completely conserved. For Arg116, Thr117, Arg118, Asn150, and Asn152 that make contact with the GATC sequence in DNA (Fujikawa et al., 2004) we also observed a high degree of conservation between species (Table 4). This may indicate a limited role of Ser36 for SeqA function.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.