Persisters are a small population of slowly growing or nongrowing bacteria that are phenotypically resistant to antibiotics, but the mechanisms involved are not well understood. The aim of this study is to determine new mechanisms underlying antibiotic-tolerant persisters. The Escherichia coli deletion mutant library was screened to identify mutants that had a defect in persister survival after exposure to ampicillin for 24 h or 5 days. The identified mutants and the parent strain were subjected to minimum inhibitory concentration (MIC) and minimum bactericidal tests and antibiotic or stress conditions in exposure assays. sucB and ubiF mutants deficient in energy production were identified from the mutant screens to have defective persister survival as demonstrated by higher susceptibility to various antibiotics, including ampicillin, norfloxacin, tetracycline and gentamicin, and different stresses such as oxidative stress, acid pH and weak acid compared with the parent strain. In addition, both sucB and ubiF had a twofold lower MIC than the parent strain. The above sucB and ubiF mutant phenotypes could be complemented by their respective functional genes. Defective energy production through mutations in sucB and ubiF affects persister survival and could serve as new drug targets for persister bacteria.