Phenotypic difference between ∆(Srl–reca)306 and ∆recA::Km elucidated by next-generation sequencing combined with a long-PCR system

Abstract

Many significant gene mutations in E. coli have contributed to the development of genetics. Among these, a commonly used recA mutation, ∆(srl– recA)306 has been sequenced by a next-generation sequencer combined with a long PCR. An original report described that ∆(srl–recA)306 cells were deleted from srlR to recA genes in their genome. The next-generation sequencer enables more accurate details to be determined. We ask whether both surrounding genes from hypF to norV for srlR and alaS for recA is there first. The long PCR was carried out with primers, norR and alaS, and amplified DNA fragments differed in length from wild to ∆(srl–recA)306 cells, suggesting that an entire ∆(srl–recA)306 mutation was included. Sequences of those DNA fragments indicated that 9147 bp, from srlR to recA including 10 genes, were replaced by a Tn10 DNA sequence. Junction points at both srlR-Tn10 and Tn10-recA were determined precisely. The results indicate that the first 97% of recA gene sequences were lost with a downstream recX gene remaining intact. The phenotypic difference between ∆(srl–recA)306 and ∆recA::Km is discussed.