Construction and Characterization of an Escherichia coli Mutant Producing Kdo2-Lipid A
1
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China
2
Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China
3
Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi 214122, China
*
Author to whom correspondence should be addressed.
Mar. Drugs 2014, 12(3), 1495-1511; https://doi.org/10.3390/md12031495
Received: 8 December 2013
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Revised: 6 February 2014
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Accepted: 13 February 2014
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Published: 13 March 2014
(This article belongs to the Special Issue Marine Lipopolysaccharides)
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.
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Keywords:
Kdo2-lipid A; lipopolysaccharide; rfaD; Escherichia coli; ESI/MS; membrane permeability; autoaggregation; biofilm
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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. https://doi.org/10.3390/md12031495
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. https://doi.org/10.3390/md12031495
Chicago/Turabian StyleWang, Jianli, Wenjian Ma, Zhou Wang, Ye Li, and Xiaoyuan Wang. 2014. "Construction and Characterization of an Escherichia coli Mutant Producing Kdo2-Lipid A" Marine Drugs 12, no. 3: 1495-1511. https://doi.org/10.3390/md12031495
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