Next, the new deletion unit LD3-5-2 was added to Δ17aK to construct Δ18aK. The KmR marker was removed by the addition of the deletion unit OCL37 without the KmR marker using the ‘415S Sm system’

to construct Δ19a (Kato & Hashimoto, 2008). Similarly, Δ20a–Δ28a were constructed using the ‘ApR-415S Sm system. The dps gene was added to Δ28a to construct Δ29a. The DNA fragment, in which the chromosomal regions flanking the regions of the deletion unit 15 were joined to the sides of the ApR-dps fragment, was introduced into Δ28a. The region of the first DNA fragment was replaced with the second DNA fragment, in which the TcR–FRT fragment was flanked by one of the chromosomal regions and Ap. The third DNA fragment, in which the chloramphenicol-resistance Selleck Alvelestat (CmR)–FRT

and the dps fragments were joined to the sides of the chromosomal selleck products region, was cloned into the plasmid pSG76A (ApR) (Posfai et al., 1997; Kato & Hashimoto, 2008). Using this plasmid, the TcR and ApR markers were removed to yield Δ29a. The prophage regions were deleted to construct Δ30a–Δ33a by the ApR-415S Sm system (see Results and discussion). The primers used to construct the deletion units are shown in Supporting Information, Fig. S1, and Tables S1 and S2. The deletion mutants were grown on antibiotic medium 3 plates and then colonies were transferred to 2 mL of antibiotic medium 3 for 24 h at 37 °C with shaking. For aerobic cultures, the precultures were diluted 1/100 into 3 mL of antibiotic medium 3 and incubated for 24 h at 37 °C with shaking. The stationary culture (0.5 mL) was added to a sampling tube, mixed with menadione solution (in ethanol) or ethanol, and incubated for 24 h at 4 °C with rotation. These cultures were diluted, plated on antibiotic medium 3 plates, and the colonies were counted after incubation for 1–4 days at 37 °C. For anaerobic cultures, the precultures were diluted 1/100 into 3 mL of antibiotic medium 3 and, after bubbling with N2, were incubated Morin Hydrate for 24 h at 37 °C with rotation. The

stationary culture (0.5 mL) was added to a sampling tube with an O-ring, mixed with menadione solution (in ethanol) or ethanol and, after flashing with N2, was incubated for 24 h at 4 °C with rotation. These cultures were diluted and plated on antibiotic medium 3 plates, and the colonies were counted after incubation for 1–4 days at 37 °C. The concentrations of menadione were 1.0 mM for Δ1–Δ15a and 0.1 mM for Δ14a–Δ33a (anaerobic culture), and 1.0 mM for Δ1–Δ26a and 0.5 mM for Δ25a–Δ33a (aerobic culture). In order to obtain final concentrations of 1.0, 0.5, and 0.1 mM, 10 μL of 50 mM, 5 μL of 50 mM, and 2 μL of 25 mM menadione in ethanol were added to 0.5 mL cultures, respectively.