Reversible lysine acetylation is involved in DNA replication initiation by regulating activities of initiator DnaA in Escherichia coli

The regulation of chromosomal replication is critical and the activation of DnaA by ATP binding is a key step in replication initiation. However, it remains unclear whether and how the process of ATP-binding to DnaA is regulated. Here, we show that DnaA can be acetylated, and its acetylation level varies with cell growth and correlates with DNA replication initiation frequencies in E. coli. Specifically, the conserved K178 in Walker A motif of DnaA can be acetylated and its acetylation level reaches the summit at the stationary phase, which prevents DnaA from binding to ATP or oriC and leads to inhibition of DNA replication initiation. The deacetylation process of DnaA is catalyzed by deacetylase CobB. The acetylation process of DnaA is mediated by acetyltransferase YfiQ, and nonenzymatically by acetyl-phosphate. These findings suggest that the reversible acetylation of DnaA ensures cells to respond promptly to environmental changes. Since Walker A motif is universally distributed across organisms, acetylation of Walker A motif may present a novel regulatory mechanism conserved from bacteria to eukaryotes.

for 4 h at 37°C. Cells were harvested by centrifugation, resuspended in Binding Buffer (20 mM Tris-HCl (pH 7.6), 500 mM NaCl and 20mM imidazole), and then disrupted using an Ultrasonic Processor (Sonics, Newtown, CT, USA). The disrupted suspension was centrifuged and the resulting supernatant was loaded onto a 1-ml Ni-NTA column (GE Healthcare, USA). The column was washed initially with Washing Buffer (20 mM Tris-HCl (pH 7.6), 500 mM NaCl and 40 mM imidazole) and the histidine-tagged protein was eluted with Elution Buffer (40 mM Tris-HCl (pH 7.5), 500 mM NaCl and 500 mM imidazole). Protein purity was estimated to be > 90% on the basis of SDS-PAGE.

Purification of DnaA and its derivative mutants
For purification of DnaA, pET22b-dnaA, pET22b-dnaA(K178Q) and pET22b-dnaA(K178R) were constructed and confirmed by sequencing. All the constructed plasmids were introduced individually into E. coli strain BL21, and the resultant strains were grown in LB medium containing 100 μg/ml ampicillin at 37°C. IPTG was added to a final concentration of 0.5 mM at the time point when the absorbance of the culture at 600 nm reached 0.6-0.8. The culture was continuously incubated for 3-4 h. The cells were harvested by centrifugation and cell pellet was suspended in ice-cold PBS buffer (0.1 M Na2HPO4, 0.15 M NaCl, pH 7.0) containing 1.0 mM pheylmethylsulfonyl fluoride (PMSF). Cells were disrupted by using the Cell Ultrasonic system (Sonics, Newtown, CT, USA) with short bursts of 10 s followed by intervals of 30 s for cooling until they become clear. Unbroken cells and cell debris were removed by centrifugation at 21,000 x g for 40 min at 4°C. The supernatant was supplemented with 20 mM imidazole and injected into the GE Healthcare His-tag resin beans and purified by the ÄKTA™ system.
His-tag resin beans were washed with 10 volume binding buffer A (20 mM sodium phosphate, 500 mM NaCl and 20 mM imidazole, pH 7.5), recombinant protein was eluted using 10 volume of buffer B (20 mM sodium phosphate, 500 mM NaCl and 500 mM imidazole, pH 7.5). The protein was checked by SDS-PAGE before shock-frozen storage at -80°C.

Purification of chromosomally encoded DnaA
BL21(His-DnaA), BW25113(His-DnaA) and their ∆cobB, ∆ yfiQ and ∆ackA knockout derivative strains were grown to an OD600 of 0.2, 1.0 or overnight at 37°C in several liters of LB medium and collected. Cells were broken by high press cracker in buffer (40mM Tris-HCl, pH 7.5, 500mM NaCl, 20mM imidazole) containing DNase I. The lysate was centrifuged at 20,000 rpm for 1 h at 4°C. The resulting supernatant was loaded onto a 1-ml Ni-NTA column (GE Healthcare, USA). The column was washed with 10 volumes of binding buffer and the bound proteins were eluted with washing buffer (40mM Tris-HCl, pH 7.5, 500 mM NaCl, 500mM imidazole as described 4 . Fractions containing DnaA were desalted using a 5ml HiTrap TM desalting column (GE Healthcare, USA).

Protein concentration
Protein concentration was determined using the Bradford protein assay protocol (Bio-Rad, Hercules, CA, USA) with bovine serum albumin (BSA) as standard per the manufacturer's instructions.

EMSA
EMSA (Electrophoretic mobility shift assay) to test the interaction between DnaA and oriC was performed as described previously 5 , with some modifications. The 469 bp of oriC-containing fragment was amplified using a pair of FAM-labeled primers, 5'-FAM-oriC F and 5'-FAM-oriC R.

Filter-binding assay
ATP-and ADP-binding activity of DnaA protein was determined by the filter-binding assay as described previously 6 . Briefly, the standard reaction(40μl) containing buffer G (Tricine-KOH

Identification of acetylated lysine residues by mass spectrometry
The purified chromosomally encoded DnaA was separated by 10% one-dimensional SDS-PAGE and the bands containing DnaA were excised. The excised bands were destained and dehydrated. For trypsin digestion, proteins were treated with 100 mM DTT at 56°C for 30 min and then treated with 100 mM NH4HCO3 at room temperature for 15 min. The freeze-dried samples were incubated with 100-200 ng trypsin at 37°C for 20 h. Peptides generated after proteolytic digestion of DnaA were separated by the EASY-nLC HPLC system (Thermo Scientific, USA) and analyzed by Q-Exactive mass spectrometer (Thermo Scientific, USA).
Mass spectrometric data were analyzed using the Mascot 2.2 software for database search.

Real-Time quantitative PCR
Cells at the early logarithmic (EL) phase, late logarithmic (LL) phase and stationary phase were harvested by centrifuging at 8,000 x g for 10 min at 4°C. Extracts of DNA (2 μg) were analyzed by ABI PRISM 7500 fast Sequence Detection System (Applied Biosystems). The primers include: oriC RT (F-; R-), ter RT (F-; R-) and 16S rDNA RT (F-; R-) ( Table S2). The PCR reactions were carried out under the following conditions: 95°C for 1 minute, 40 cycles at 95°C for 15 s, 60°C for 15 s, and 72°C for 30 s. SyberGreen real-time quantitative PCR dissociation curves showed that each primer set gave a single and specific product. The 16S rDNA was used as internal standards to adjust for different qualities and quantities of DNA. All data are expressed as mean ± SEM.

Supplementary Figures
Supplementary Figure S1 Purification