Understanding the bacterial envelope is critical because it provides protection against environmental factors. Cardiolipin (CL) is one of the major phospholipids in the Escherichia coli cell envelope. This study aims to investigate the biological functions of CL by comparing the gene essentiality between the E. coli K-12 BW25113 WT and cardiolipin deficient E. coli using Transposon Directed Insertion Sequencing (TraDIS). The BW25113 ΔclsABC triple mutant library contained 2,708,478 transposon insertions equating to an insertion every 17.6 base pairs. The genes rpoE, degS, and rseP became non-essential in the absence of CL. These genes are involved in the σE stress response. We found that the abundance of OmpC and OmpF decreased in E. coli BW25113 ΔclsABC. Besides, the growth defect in rpoE sequestered E. coli BW25113 was recovered when Sec activity was inhibited. It was concluded that CL deficiency decreases OMPs secretion through Sec pathway, resulting in a decrease in unfolded OMPs density in the periplasm, removing the condition where rpoE is essential.