Non-dividing persisters, bacteria that can survive in the presence of antibiotics by pausing their metabolic activity, are among the many causes of the refractory nature of bacterial infections. Here we constructed a recombinant Escherichia con strain that enables to distinguish non-dividing from dividing cell based on Z-ring during cell division. Then, non dividing cells and dividing cells were successfully separated using a fluorescence activated cell sorter. The sorted non dividing cells showed significantly higher tolerance toward ofloxacin than dividing cells, which indicates that per sisters were concentrated with the methodology. Transcriptional analysis revealed that genes involved in guanosine tetraphosphate synthesis are upregulated in persisters, which represses transcription and DNA replication and leads to ofloxacin tolerance. Lactate dehydrogenase and several ATP-binding cassette transporters were upregulated in persisters to adapt to anaerobic metabolism. In addition, nitrite and dimethyl sulfoxide (DMSO) may be used as reducible substrates for alternative energy generation pathways. Our methodology revealed a unique transcriptional profile of E. coli persisters. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.