Mar. Drugs 2014, 12(3), 1495-1511; doi:10.3390/md12031495
Article

Construction and Characterization of an Escherichia coli Mutant Producing Kdo2-Lipid A

1,2email, 1,2email, 1email, 1email and 1,3,* email
Received: 8 December 2013; in revised form: 6 February 2014 / Accepted: 13 February 2014 / Published: 13 March 2014
(This article belongs to the Special Issue Marine Lipopolysaccharides)
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract: 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo)2-lipid A is the conserved structure domain of lipopolysaccharide found in most Gram-negative bacteria, and it is believed to stimulate the innate immune system through the TLR4/MD2 complex. Therefore, Kdo2-lipid A is an important stimulator for studying the mechanism of the innate immune system and for developing bacterial vaccine adjuvants. Kdo2-lipid A has not been chemically synthesized to date and could only be isolated from an Escherichia coli mutant strain, WBB06. WBB06 cells grow slowly and have to grow in the presence of tetracycline. In this study, a novel E. coli mutant strain, WJW00, that could synthesize Kdo2-lipid A was constructed by deleting the rfaD gene from the genome of E. coli W3110. The rfaD gene encodes ADP-l-glycero-d-manno-heptose-6-epimerase RfaD. Based on the analysis by SDS-PAGE, thin layer chromatography (TLC) and electrospray ionization mass spectrometry (ESI/MS), WJW00 could produce similar levels of Kdo2-lipid A to WBB06. WJW00 cells grow much better than WBB06 cells and do not need to add any antibiotics during growth. Compared with the wild-type strain, W3110, WJW00 showed increased hydrophobicity, higher cell permeability, greater autoaggregation and decreased biofilm-forming ability. Therefore, WJW00 could be a more suitable strain than WBB06 for producing Kdo2-lipid A and a good base strain for developing lipid A adjuvants.
Keywords: Kdo2-lipid A; lipopolysaccharide; rfaD; Escherichia coli; ESI/MS; membrane permeability; autoaggregation; biofilm
PDF Full-text Download PDF Full-Text [1136 KB, uploaded 13 March 2014 10:30 CET]

Export to BibTeX |
EndNote


MDPI and ACS Style

Wang, J.; Ma, W.; Wang, Z.; Li, Y.; Wang, X. Construction and Characterization of an Escherichia coli Mutant Producing Kdo2-Lipid A. Mar. Drugs 2014, 12, 1495-1511.

AMA Style

Wang J, Ma W, Wang Z, Li Y, Wang X. Construction and Characterization of an Escherichia coli Mutant Producing Kdo2-Lipid A. Marine Drugs. 2014; 12(3):1495-1511.

Chicago/Turabian Style

Wang, Jianli; Ma, Wenjian; Wang, Zhou; Li, Ye; Wang, Xiaoyuan. 2014. "Construction and Characterization of an Escherichia coli Mutant Producing Kdo2-Lipid A." Mar. Drugs 12, no. 3: 1495-1511.


Mar. Drugs EISSN 1660-3397 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert