Two‐step induction of cdsA promoters leads to upregulation of the glycolipid MPIase at cold temperature

Abstract

Glycolipid MPIase, essential for membrane protein integration into the cytoplasmic membrane of Escherichia coli, is upregulated at cold temperatures. This upregulation is rapid and sustainable. CdsA, a CDP‐diacylglycerol synthase, is a rate‐limiting enzyme for MPIase biosynthesis. Upregulation of CdsA is responsible for the increase in the MPIase level at low temperature. Investigation of cdsA regulatory regions revealed at least two cold‐inducible promoters, a cold‐shock promoter that functions transiently and immediately in the cold, and one that is sustainable in the cold. The stability of the cdsA transcript was comparable with that of tufA, which is not cold‐inducible. Thus, cdsA is induced through two‐step cold‐induction to maintain MPIase at a high level rapidly and sustainably in the cold.

CdsA, a CDP‐diacylglycerol synthase involved in phospholipid and glycolipid MPIase biosynthesis, possesses multiple initiation codons

“…As a positive control, the cdsA ORF starting with “TTG,” as assigned in the database as an initiation codon, was cloned under the control of the tac promoter and the SD sequence in pUSI2 (pTac‐CdsA(TTG)) (Sawasato, Sato, et al, 2019). To evaluate the ability to complement the cdsA knockout, we used two cdsA knockout strains, KS23 (Sawasato, Sato, et al, 2019) and YS23 (Sawasato, Sekiya, & Nishiyama, 2019), both of which confer the cdsA :: cat allele and a complementing plasmid, pAra‐CdsA. To critically examine the effects of mutants, we employed these cdsA knockouts, which also confer the Δ ynbB mutation, since YnbB is a CdsA paralogue dedicated to MPIase biosynthesis (Sato et al, 2019).…”

“…Such regulation of CdsA expression seems to be conserved among Gram‐negative bacteria, since the sequence at the 5′ region of cdsA is highly conserved (Table S1). Nonetheless, CdsA is upregulated at low temperature through an increase in the level of mRNA (Sawasato, Sekiya, & Nishiyama, 2019; Sawasato, Suzuki, & Nishiyama, 2019).…”

“…4.1 | Bacterial strains and plasmids E. coli strains EK413 (F À Δ[argF-lac]U168 rpsL150 relA1 thi deoC7 ptsF25 flbB5301) (Nishiyama et al, 1996) and its derivatives with cdsA knockout KS23 (EK413 ΔcdsA::cat ΔynbB) (Sawasato, Sato, et al, 2019) or YS23 (EK413 ΔcdsA::cat ΔynbB ΔpcnB:: Tn10) (Sawasato, Sekiya, & Nishiyama, 2019) were used. KS23 and YS23 harbor plasmid pAra-CdsA, from which CdsA is expressed upon arabinose addition (Sawasato, Sato, et al, 2019).…”

“…Such regulation of CdsA expression seems to be conserved among Gram-negative bacteria, since the sequence at the 5 0 region of cdsA is highly conserved (Table S1). Nonetheless, CdsA is upregulated at low temperature through an increase in the level of mRNA (Sawasato, Sekiya, & Nishiyama, 2019;. Originally, the initiation codon was suggested to be codon 37 (ATG) (Icho et al, 1985), but it was not the true initiation codon, since CdsA Δ1-36 could not complement the cdsA knockouts.…”

Expression of Cds4/5 of Arabidopsis chloroplasts in E. coli reveals the membrane topology of the C‐terminal region of CDP‐diacylglycerol synthases

“…The first step of MPIase biosynthesis is catalyzed by CdsA (Sawasato, Sato, et al., 2019). A part of CDP‐DAG is converted to GlcNAc‐PP‐DAG (compound I) on CdsA, through incorporation of GlcNAc‐1‐phosphate, CMP being released (Kamemoto et al., 2020; Sawasato, Sato, et al., 2019; Sawasato et al., 2019). Depletion of CdsA resulted not only in PA accumulation but also in MPIase depletion.…”