Rifampicin

Bacteria grown under the required growth conditions were pelleted and RNA was isolated using the SV total RNA purification kit (Promega) as described [34 (link)]. Total RNA (20 μg) was separated on MOPS agarose gels (1.2%), transferred by vacuum blotting for 1.5 h onto positively charged membranes (Whatman) in 10 x SSC buffer (1.5 M NaCl, 0.15 M sodium citrate, pH7) using a semi-dry blotting system and UV cross-linked. Prehybridization, hybridization to DIG-labelled probes and membrane washing were conducted using the DIG Luminescent Detection Kit (Roche, Germany) according to the manufacturer's instructions. The DIG-labelled PCR fragments used as probes were produced by PCR using the DIG-PCR nucleotide mix (Roche, Germany) as described [34 (link)] with the following primer pairs: for the lcrF transcript—I214/I303, for the csrB and csrC transcripts—555/556 and 583/I82, for the rne transcript—IV529/IV530 and the pnp transcript—IV527/IV528 (see S2 Table).
To determine stability of the lcrF transcript, RNA stability assays were performed. In order to stop the de novo mRNA synthesis 0.5 mg/ml rifampicin (Serva) was added. 0, 1, 2, 3, 5 and 7.5 min after rifampicin treatment, 10% v/v phenol was added and the samples were snap frozen in liquid nitrogen. RNA isolation and northern blot analysis were performed as described above.

Prior to flow cytometry analysis, exponentially growing cells (OD₄₅₀ = 0.1–0.3) were treated with rifampicin and cephalexin to inhibit initiation of RNA synthesis and cell division, respectively (Lobner‐Olesen et al., 1989), or chloramphenicol and cephalexin to inhibit protein synthesis and cell division, respectively (Skarstad et al., 1986). Drugs were used at the following final concentrations: rifampicin, 300 μg/ml (SERVA Electrophoresis GmbH); chloramphenicol, 200 μg/ml (Sigma‐Aldrich); cephalexin, 36 μg/ml (Sigma‐Aldrich). Incubation continued for a minimum of 4 hr at 37°C, unless otherwise specified. When replication initiation is inhibited, and ongoing rounds of replication allowed to finish, the number of fully replicated chromosomes per cell (i.e., genome equivalents) represents the number of origins per cell at the time of drug addition. In case of initiation synchrony, the integral number of chromosomes is two, four, or eight (i.e., 2ⁿ). In cells with asynchronous initiation additional peaks appear representing three, five, six, or seven cellular origins (i.e., ≠2ⁿ). Flow cytometry was performed as described previously (Løbner‐Olesen et al., 1989) using an Apogee A10 Instrument (Apogee, Inc.). For each sample, 40,000–100,000 cells were analyzed.