Restart of DNA replication in Gram-positive bacteria: functional characterisation of the Bacillus subtilis PriA initiator

Abstract

The PriA protein was identified in Escherichia coli as a factor involved in the replication of extrachromosomal elements such as bacteriophage phiX174 and plasmid pBR322. Recent data show that PriA plays an important role in chromosomal replication, by promoting reassembly of the replication machinery during reinitiation of inactivated forks. A gene encoding a product 32% identical to the E.coli PriA protein has been identified in Bacillus subtilis. To characterise this protein, designated PriA(Bs), we constructed priA(Bs) mutants. These mutants are poorly viable, filamentous and sensitive to rich medium and UV irradiation. Replication of pAMbeta1-type plasmids, which is initiated through the formation of a D-loop structure, and the activity of the primosome assembly site ssiA of plasmid pAMbeta1 are strongly affected in the mutants. The purified PriA(Bs) protein binds preferentially to the active strand of ssiA, even in the presence of B.subtilis SSB protein (SSB(Bs)). PriA(Bs) also binds stably and specifically to an artificial D-loop structure in vitro. These data show that PriA(Bs) recognises two specific substrates, ssiA and D-loops, and suggest that it triggers primosome assembly on them. PriA(Bs) also displays a single-stranded DNA-dependent ATPase activity, which is reduced in the presence of SSB(Bs), unless the ssiA sequence is present on the ssDNA substrate. Finally, PriA(Bs) is shown to be an active helicase. Altogether, these results demonstrate a clear functional identity between PriA(Ec) and PriA(Bs). However, PriA(Bs) does not complement an E.coli priA null mutant strain. This host specificity may be due to the divergence between the proteins composing the E.coli and B.subtilis PriA-dependent primosomes.

Figure 1

The B.subtilis PriA protein. (A) Schematic representation of the B.subtilis PriA protein. The double signature which characterises PriA_Ec and which is conserved in PriA_Bs is presented: (i) the seven conserved motifs (I–VI) of the 3′→5′ helicase subfamily into which PriA_Ec is classified (filled boxes); (ii) the two putative zinc finger domains (hatched box); the spacing between the conserved cysteines is indicated. (B) Purification of PriA_Bs. The PriA_Bs protein was purified in E.coli using the IMPACT system. Proteins were resolved by 8% SDS–PAGE and stained with Coomassie brilliant blue. Lane 1, molecular weight standards; lane 2, soluble proteins of E.coli cells induced to express the fusion PriA_Bs–intein–CBD; lane 3, flow-through of the chitin column; lane 4, elution from the chitin column after DTT cleavage. The purity obtained at this step has been estimated to be >95%. (C) Immunodetection of PriA_Bs in B.subtilis. Protein preparations were resolved by 8% SDS–PAGE, transferred to PVDF membrane and probed with purified anti-PriA_Bs antibodies. Lanes 1–4, the indicated amounts (in ng) of purified PriA_Bs; lanes 5–8, total proteins extracted from an equal amount of cells grown in the presence of IPTG from strain 168 and derivatives PPBJ65, PPBJ69 and CBB294 (a derivative of PPBJ120 carrying an extragenic mutation suppressing the lack of PriA_Bs, designated dnaB75) (33). The priA_Bs allele present in each strain is indicated. (D) Schematic representation of the priA_Bs chromosomal region in B.subtilis strains used in this study. priA_Bs and flanking ORFs are represented by boxes. yloI is an ORF of unknown function; def and fmt are homologues of the E.coli def and fmt genes. In the lower part a schematic map of the pMUTIN2 integrative vector carrying the IPTG-inducible promoter P_spac is presented. Vertical arrows below the priA_Bs ORF indicate the insertion sites of pMUTIN2 derivatives. The names of the resulting strains with their associated priA allele are indicated.

Figure 2

UV sensitivity of B.subtilis priA mutant strains. Strains were grown in minimal medium supplemented with Em in the presence or absence of IPTG (1 mM), as indicated. Appropriate dilutions were plated on the same medium and immediately irradiated with different doses of UV. After incubation for 2 days at 37°C, the fraction of surviving cells was determined and plotted against the UV dose. Each point is the mean value of two independent determinations.

Figure 3

PriA_Bs is required for ssiA activity and pAMβ1-type plasmid replication. (A) Bacillus subtilis PPBJ65 (PriA_Bs⁺) and PPBJ69 (priAind) strains harbouring pC194-derived plasmid pADG6406-1 (ssiA⁺) or pADG6406-2 (ssiA^–) were grown overnight in LB supplemented with IPTG, Em and Cm, then diluted 100-fold in fresh medium without IPTG and cultivated for ∼5 h. Total DNA was extracted and analysed by Southern hybridisation using ³²P-labelled pC194 DNA as probe. (B) Bacillus subtilis PPBJ65 (PriA_Bs⁺) and PPBJ69 (priAind) strains harbouring pAMβ1-derived plasmid pVA798ΔRCR were grown to mid-log phase without IPTG and their total DNA was extracted and analysed by Southern hybridisation, using ³²P-labelled pVA798ΔRCR as probe. ssDNA and dsDNA, single-stranded and double-stranded DNA, respectively.

Figure 4

PriA_Bs binds to the active strand of ssiA in the presence of SSB_Bs. Protein–DNA complexes were generated at 30°C for 30 min and electrophoresed on a 4% non-denaturing polyacrylamide gel in TGE buffer at 4°C. A 174 nt long ssDNA fragment containing ssiA⁺ or ssiA^– sequence was 5′ radiolabelled and used as a DNA binding substrate. Final protein concentrations of each binding experiment (expressed in nM) are indicated above each lane. SSB_Bs and PriA_Bs refer to B.subtilis SSB and PriA proteins, respectively. (A) Binding of SSB_Bs. (B) Binding of PriA_BS. (C) Binding of PriA_Bs to ssiA strands covered by SSB_Bs. The star indicates the PriA_Bs supershift observed with ssiA⁺ substrate covered by SSB_Bs.

Figure 5

PriA_Bs binds preferentially to a D-loop structure. The numbering on the schematic representation of the D-loop and bubble substrates indicates the size (in nt) of the dsDNA and ssDNA part, the star indicates the position of the ³²P radiolabelling present at the 5′ end. Purified PriA_Bs protein at the indicated concentrations (expressed in nM) was added to the substrates, incubated at 30°C for 30 min and the protein–DNA complexes were resolved on a 5% non-denaturing polyacrylamide gel run at 4°C in TGE buffer (A and B) or in TAM buffer (C and D). (A and C) PriA_Bs binding to the D-loop. (B and D) PriA_Bs binding to the bubble.

Figure 6

PriA_Bs binds more stably to ssDNA than PriA_Ec. Protein–DNA complexes were electrophoresed on a 5% non-denaturing polyacrylamide gel containing 5% glycerol in 0.25× TBE buffer at 4°C. The 90 nt long Ost4 oligonucleotide was 5′ radiolabelled and used as a DNA binding substrate with the indicated concentrations of PriA_Bs (A) or PriA_Ec (B) (expressed in nM). Binding assays were performed in buffer containing either 50 (left) or 200 mM NaCl (right).

Figure 7

PriA_Bs is a ssDNA-dependent ATPase displaying helicase activity. (A) PriA_Bs (55 nM) was incubated at 37°C for 30 min in the presence of increasing amounts of DNA at a fixed concentration of ATP (45 µM). The calculated amount of ATP hydrolysed is plotted against DNA concentration. Supercoiled dsDNA and ssDNA were prepared from the pAPJ9 phagemid as described in Materials and Methods. Purified oligonucleotides used for construction of the D-loop substrate were used as linear ssDNA substrates (cf. Fig. 5). (B and C) ssDNA-dependent ATPase activity of PriA_Bs was measured at 37°C in the presence of constant amounts (1 µM nt) of three ssDNA substrates, in the absence (B) or presence (C) of SSB_Bs protein. In the experiment conducted with SSB_Bs, ssDNA substrates were incubated with this protein (0.5 monomer/nt) for 30 min at 37°C prior to addition of PriA_Bs. A representative kinetic experiment at 0.1 mM ATP is shown; similar experiments were carried out at different ATP concentrations in order to calculate the k_cat and K_m values of the enzyme for ATP. These values are reported in the adjacent table and are the average of two independent experiments. M13, M13- mp19 ssDNA; M13-ssiA⁺, M13-mp19 ssDNA carrying the ssiA⁺ sequence; M13-ssiA^–, M13-mp19 ssDNA carrying the ssiA^– sequence. (D) PriA_Bs displays helicase activity. (Left) Native gel analysis of PriA_Bs unwinding activity. Lanes 1 and 2 contain, respectively, the synthetic forked DNA substrate and the labelled ssDNA liberated following heating at 95°C for 5 min, which are represented schematically on the left. Lanes 3–7 contain reactions performed with increasing amounts of PriA_Bs (indicated in nM at the top of the gel). Lane 8 contains a reaction performed with the same amount of PriA_Bs as in lane 7, but without ATP in the reaction buffer. (Right) Quantitation of PriA_Bs unwinding activity. Helicase activity is expressed as the percentage of the liberated ssDNA strand quantified in each sample lane in the gel presented in (A) (see Materials and Methods).