AdpA interacts specifically with the oriC region in vivo and in vitro. (a) Schematic of the S. coelicolor oriC region. The positions of AdpA and DnaA protein-binding sites (boxes) are presented by red triangles and black/grey pentagons (spatial arrangement and numbering of DnaA boxes adapted from Zawilak-Pawlik et al. ), respectively. In the rectangle next to legend, AdpA consensus sequence  (bold and upper one) and A1–3 boxes were aligned; underlined nucleotides indicate mismatches. Radiolabelled P2 (poriC−Br3) primer was used together with P1 (poriC−Bf1) primer to amplify a 283-bp fragment of the oriC region for DNase I footprinting and sequencing reactions (see (c)). (b) EMSA. A 32P-labelled, 981-bp oriC fragment (PCR-amplified with poriC−Bf1 and poriC−Br4 primers, table 2) was incubated in Marians' buffer in the presence of a non-specific competitor with increasing amounts of AdpA protein, and the nucleoprotein complexes were analysed on a 4 per cent polyacrylamide gel. (c) DNase I footprinting of wild-type and ΔA1A2 oriC fragments. A 32P-labelled, 283-bp DNA fragment (see (a)) was incubated in Marians' buffer with increasing amounts of AdpA protein and then subjected to DNase I digestion. Lanes: A, C, G and T represent sequencing reactions for the WT and ΔA1A2 oriC fragments, respectively. Sequences above the gel schematically represent mutations introduced within oriC region (blue, nucleotides to be mutated; underlined, introduced nucleotides). Radiolabelled P2 (poriC−Br3) primer was used to perform sequencing reactions. (d) In vivo identification of the AdpA–oriC complex using immunoprecipitation. Anti-AdpA polyclonal antibodies  were used to immunoprecipitate AdpA–DNA complexes cross-linked with formaldehyde. PCR was carried out with the primers, poriC−Bf1 and poriC−Br3 (see (a) and electronic supplementary material, table S2), flanking the putative AdpA-binding sites. Lanes: S, sample (immunoprecipitated DNA); plus symbols (+), input (not immunoprecipitated) DNA (positive control); strains: 1, strain containing the adpA gene under the control of the inducible promoter, M851 ptipAadpA; 2, adpA deletion mutant M851+pIJ6902-hyg.
AdpA and DnaA proteins compete for oriC-binding in an ATP-dependent manner. (a) Binding of AdpA reduces accessibility of the oriC to DnaA. SPR analysis: comparison of AdpA and DnaA protein interactions with wild-type and ΔA1A2 oriC fragments. Sensograms were obtained by binding AdpA (i), DnaA (ii), or both proteins (co-injection, (iii) and (iv)) to biotinylated wild-type and ΔA1A2 oriC fragments (283-bp, amplified with biotinylated pb−Scoric and poriC−Br3 primers) immobilized on a streptavidin-coated chip in the BIAcore apparatus. Proteins were injected in HBS200 buffer supplemented with 3mM ATP (for DnaA) or without this nucleotide (for AdpA). (b) Presence of ATP reduces the affinity of AdpA for the oriC region. (i)–(ii) SPR: comparison of AdpA and DnaA protein interactions with the oriC fragment in the presence and absence of ATP. Sensograms were obtained by incubating AdpA (i) and DnaA (ii) proteins with biotinylated wild-type oriC fragment (283-bp, amplified with biotinylated pb−Scoric and poriC−Br3 primers) immobilized on a streptavidin-coated chip of the BIAcore apparatus. The concentration of ATP was 3 mM. (iii) Cross-linking of AdpA–oriC complexes formed in the presence or absence of ATP. A 283-bp DNA fragment (100 ng) was incubated with AdpA protein (100 nM) in the absence or presence of increasing amounts of ATP, and then nucleoprotein complexes were cross-linked with glutaraldehyde (final concentration: 0.5 mM). After electrophoresis (5% polyacrylamide), the gel was stained with ethidium bromide and analysed.
Mutation of AdpA-binding sites within the oriC region results in earlier maturation of aerial hyphae and more frequent replication. (a) Growth of wild-type and ΔA1A2 mutant strains of S. coelicolor on R2 and R2YE media. (b) Quantitative PCR analysis of frequency of initiation in wild-type and ΔA1A2 mutant strains of S. coelicolor on R2 medium. Real-time qPCR analyses were performed as described in §5 (see electronic supplementary material to quantify the ratio of oriC to argG, which reflects the frequency of initiation). Grey bars, M145; black bars, ΔA1A2.