Identification of Three Genes Encoding for the Late Acyltransferases of Lipid A in Cronobacter sakazakii

Abstract

Lipid A, the hydrophobic anchor of lipopolysaccharide, is an essential component in the outer membrane of most Gram-negative bacteria. Food-borne pathogen Cronobacter sakazakii synthesizes two lipid A species, differing by the length of the secondary acyl chain. In this work, we identified three genes ESA02293, ESA02951 and ESA01386 encoding for the late acyltransferases of lipid A biosynthesis pathway in C. sakazakii. Based on the sequence alignment, proteins YP_001438378.1 encoded by ESA02293, YP_001439016.1 encoded by ESA02951, and YP_001437482.1 encoded by ESA01386 are homologous to E. coli LpxL, LpxP and LpxM, respectively. Functions of the three acyltransferases were confirmed by overexpressing the genes in E. coli, isolating lipid As and analyzing their structures using an ESI/MS. C. sakazakii LpxL and LpxM transfer a C14:0 secondary acyl chain to the 2′- and 3′-position of lipid A, respectively. C. sakazakii LpxP can transfer either a C16:1 or a C14:0 secondary acyl chains to the 2′-position of lipid A.

Structural derivation of lipid A fromCronobacter sakazakiiusing tandem mass spectrometry

“…When compared to a similarly prepared, but previously determined structure for the E. coli lipid A, the structures shared similar compositions on the glucosamine disaccharide backbone with four primary acyl chains at the 2, 2′, 3 and 3′ positions, but differed at the 2'b and 3'b acyl chain substituents. The biochemical nature of these differences related to the biosynthetic enzymes LpxL and LpxM, which are known to add acyl chain substituents at the 2'b and 3'b positions, remains to be determined …”

The Global Response of Cronobacter sakazakii Cells to Amino Acid Deficiency

“…C. sakazakii is much more resistant than other Enterobacteriaceae to environmental stresses ( Dancer et al, 2009 ), which might be associated with its pathogenesis ( Alvarez-Ordóñez et al, 2014 ). Biofilm formation ( Jung et al, 2013 ), exopolysaccharide production, and lipopolysaccharide structure modification ( Wang and Quinn, 2010 ; Zhang et al, 2010 ; Wiederschain, 2011 ; Cai et al, 2013 ; Wang et al, 2015 ; Liu et al, 2016 ) are various strategies for C. sakazakii to cope with the environmental stress.…”

Identification of two secondary acyltransferases of lipid A inPseudomonas putidaKT2442

“…Loss of the phosphate group of lipid A usually occurs during the extraction procedure (Cai et al . ). The m/z values of peaks at m/z 1638·21 and 1468·12 differ from that of form A and form B by 22‐unit, respectively, suggesting that these peaks were derived from the sodium adducts of the two major lipid A species form A and form B (Chai et al .…”