Characterization of a gene cluster containing four genes relevant to biosynthesis of inner core of lipopolysaccharide in Cronobacter sakazakii

Many genes in the biosynthetic pathway of lipopolysaccharide in Cronobacter sakazakii have not been identified. In this study, we demonstrate that an operon containing four genes ESA_RS18945, ESA_RS18950, ESA_RS18955, and ESA_RS18960 is responsible for L-glycero-D-mannoheptose addition on the inner core of lipopolysaccharide in C. sakazakii. The proteins encoded by these four genes are homologous to E. coli WaaQ, WaaC, WaaF, and WaaD. Lipopolysaccharide from the deletion mutants of ESA_RS18945, ESA_RS18950, ESA_RS18955, and ESA_RS18960 (named as oRS18945, oRS18950, oRS18955 and oRS18960, respectively) were analyzed by SDS-PAGE. oRS18945 synthesized lipopolysaccharide with similar length to the wildtype BAA-894, whereas oRS18950, oRS18955, and oRS18960 synthesized much shorter lipopolysaccharide. This suggests that the enzyme encoded by ESA_RS18945 might function as E. coli WaaQ on the sidechain of lipopolysaccharide. When E. coli WaaC, WaaF, and WaaD were overexpressed in oRS18950, oRS18955, and oRS18960, respectively, the full length of lipopolysaccharide was recovered. Mass spectrometry analysis indicates that oRS18950 and oRS18960 only synthesized Kdo(2)-lipid A, confirming that enzymes encoded by ESA_RS18950 and ESA_RS18960 have similar functions to E. coli WaaC and WaaD, respectively. Hep-Kdo(2)-lipid A with a phosphoethanolamine was produced in oRS18955, suggesting that the enzyme encoded by ESA_RS18955 has similar function to E. coli WaaF.