Kato, unpublished data), a large-scale chromosome deletion mutant

Kato, unpublished data), a large-scale chromosome deletion mutant, termed Δ15a, that lacked deletion unit 21 but harbored the lambda red gene was constructed. Using Δ15a, 13 deletion units were combined using

the ApR-415S Sm system to obtain the additional deletion mutants, Δ16aK–Δ28a. As deletion of the dps gene in the chromosome near deletion unit 15 lowered cell viability during Trichostatin A stationary phase in the presence of other deletions (J. Kato, unpublished data), the dps gene was reintroduced into Δ28a to obtain Δ29a. The dps gene encodes the DNA-binding protein Dps which nonspecifically binds to and forms a nucleoprotein complex on DNA. In this complex, DNA is protected from a variety of stresses (Calhoun & Kwon, 2011). Next, four prophages were deleted using the

FRT4 system to construct Δ30a–Δ33a. For Δ15a–Δ27a, a series of dps+ derivatives were constructed by inserting the dps+ ApR fragment. Deletion mutant Δ33a, which had the largest number of deletions, lacks 38.9% of the original E. coli genome (2.8 Mb) (Figs 3 and 4, Fig. S2). The genome of Bcl 2 inhibitor deletion mutant Δ33a was resequenced with Genome Analyzer GAIIx (Illumina, CA) and the deletions were confirmed. Menadione sensitivity of the large-scale chromosome deletion mutants at stationary phase was examined. Deletion mutants were grown aerobically or anaerobically to stationary phase and the cells were then incubated at 4 °C in the presence of menadione (solubilized in ethanol) or

ethanol only (control) for 24 h. Viable cells were counted after plating the diluted culture onto plates containing antibiotic medium in triplicate. When mutants were grown aerobically, Δ21a, Δ22a, and Δ23a were sensitive to menadione (Fig. Aspartate 5), and among the combined deletion mutants constructed, Δ23a and Δ24a were the most sensitive. The combined deletion mutants Δ25a and Δ26a were resistant to menadione. When mutants were grown anaerobically, the combined deletion mutants Δ17a and Δ19a were sensitive to menadione (Fig. 6), and among the combined deletion mutants constructed, Δ19a was the most sensitive. The combined deletion mutant Δ20a was resistant to menadione. All of the mutants constructed still possessed the genes for superoxide dismutase, catalase, and RpoS, but some genes involved in the response to oxidative stress were deleted. The deletion mutant Δ7 lacked the gor gene, which encodes glutathione oxidoreductase (Greer & Perham, 1986), and the deletion mutant Δ14a lacked the tpx gene which encodes a thiol peroxidase (Cha et al., 1995). In addition, the deletion mutant Δ15a lacked grxA, which encodes glutaredoxin 1, a redox coenzyme for glutathione-dependent ribonucleotide reductase (Miranda-Vizuete et al., 1996), and the deletion mutant Δ17a lacked dsrA which encodes the regulatory sRNA that enhances the translation of RpoS (Sledjeski et al., 1996).

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