The Origin of 8-Amino-3,8-dideoxy-<small>d</small>-manno-octulosonic Acid (Kdo8N) in the Lipopolysaccharide of <i>Shewanella oneidensis</i>

다국어 초록 (Multilingual Abstract)

<P>Lipopolysaccharide (LPS; endotoxin) is an essential component of the outer monolayer of nearly all Gram-negative bacteria. LPS is composed of a hydrophobic anchor, known as lipid A, an inner core oligosaccharide, and a repeating O-antigen polysaccharide. In nearly all species, the first sugar bridging the hydrophobic lipid A and the polysaccharide domain is 3-deoxy-<SMALL>d</SMALL>-manno-octulosonic acid (Kdo), and thus it is critically important for LPS biosynthesis. Modifications to lipid A have been shown to be important for resistance to antimicrobial peptides as well as modulating recognition by the mammalian innate immune system. Therefore, lipid A derivatives have been used for development of vaccine strains and vaccine adjuvants. One derivative that has yet to be studied is 8-amino-3,8-dideoxy-<SMALL>d</SMALL>-manno-octulosonic acid (Kdo8N), which is found exclusively in marine bacteria of the genus <I>Shewanella</I>. Using bioinformatics, a candidate gene cluster for Kdo8N biosynthesis was identified in <I>Shewanella oneidensis</I>. Expression of these genes recombinantly in <I>Escherichia coli</I> resulted in lipid A containing Kdo8N, and <I>in vitro</I> assays confirmed their proposed enzymatic function. Both the <I>in vivo</I> and <I>in vitro</I> data were consistent with direct conversion of Kdo to Kdo8N prior to its incorporation into the Kdo8N-lipid A domain of LPS by a metal-dependent oxidase followed by a glutamate-dependent aminotransferase. To our knowledge, this oxidase is the first enzyme shown to oxidize an alcohol using a metal and molecular oxygen, not NAD(P)<SUP>+</SUP>. Creation of an <I>S. oneidensis</I> in-frame deletion strain showed increased sensitivity to the cationic antimicrobial peptide polymyxin as well as bile salts, suggesting a role in outer membrane integrity.</P>