Modification of lipid A structure and activity by the introduction of palmitoyltransferase gene to the acyltransferase‐knockout mutant of Escherichia coli

Abstract

Lauroyltransferase gene (lpxL), Myristoyltransferase gene (lpxM) and palmitoyltransferase gene (crcA) of Escherichia coli BL21 were independently disrupted by the insertional mutations. The knockout mutant of two transferase genes (lpxL and crcA) produced lipid A with no lauric or palmitic acids and only a little amount of myristic acid. The mutant was susceptible to polymyxin B, but showed comparable growth with the wild-type strain at 30°C. The palmitoyltransferase gene from E. coli (crcA) or Salmonella (pagP) was amplified by PCR, cloned in pUC119, and transferred into the double-knockout mutant by transformation. The transformant contained palmitic acid in the lipid A, and recovered resistance to polymyxin B. Mass spectrometric analysis revealed that palmitic acid was linked to the hydroxyl group of 3-hydroxymyristic acid at C-2 position of proximal (reducing-end) glucosamine. LPS from the double-knockout mutant showed reduced IL-6-inducing activity to macrophage-like line cells compared to that of the wild-type strain, and the activity was only slightly restored by the introduction of palmitic acid to the lipid A. These results suggested that the introduction of one palmitic acid was enough to recover the integrity of the outer membrane, but not enough for the stimulation of macrophages.

Structural modification of Escherichia coli lipid A by myristoyltransferase gene from Klebsiella pneumoniae

“…The first acyltransferase gene is a homolog of the lauroyltransferase gene of E. coli , and the second one, lpxL2 , expresses an enzyme that can transfer C 14:0 to the same position as for C 12:0, which is usually present in E. coli or many other enterobacterial lipid A, and considered to be critical for the receptor recognition. The finding of this myristoyltransferase gene prompted us to use it for the modification of the structure of lauroyloxymyristic acid in E. coli lipid A using the acyltransferase‐knockout mutants already constructed in our study …”

“…Crude LPS was prepared from about 200 mg of dry cells by hot phenol–water method, and used for chemical analysis. Fatty acid composition of crude LPS was measured by gas chromatographic analysis after methylesterification as described previously . Crude LPS was further purified by RNase and proteinase K treatment for the assay of IL‐6 induction.…”

“…For the assay of IL‐6 inducing activity, U937 cells (human macrophage cell line) were used. The U937 cells were cultured with phorbol 12‐myristate 13‐acetate, and treated with enzyme‐treated LPS preparations as reported in the previous paper . IL‐6 in the supernatants was measured by a commercial ELISA system of BD Biosciences (Franklin Lakes, NJ).…”

“…Each bar represents mean ± standard error (SE) of triplicated data of ELISA. KGU0107, wild‐type strain; KGU0221, myristoyltransferase‐knockout mutant; KGU0485, transformant derived from KGU0377; KGU0496, transformant derived from KGU0221. LPS, lipopolysaccharide…”

“…The finding of this myristoyltransferase gene prompted us to use it for the modification of the structure of lauroyloxymyristic acid in E. coli lipid A using the acyltransferase-knockout mutants already constructed in our study. 6 Two mutant strains are used as host strains for transformation: KGU0377, which has disrupted lpxL (lauroyltransferase gene) and crcA (palmitoyltransferase gene), and produces the lipid A with only four molecules of 3-OH-C 14:0 , similar to that used by Li et al, 7,8 and KGU0221, which has disrupted lpxM (myristoyltransferase gene), and produces the lipid A with four molecules of 3-OH-C 14:0 and one molecule of C 12:0 . For the cloning of lpxL2, chromosomal DNA of K. pneumoniae NBRC 14940 T was used as the template DNA for polymerase chain reaction amplification, and DNA primers were designed using the reported nucleotide sequence data.…”