Download Evolution of gram-negative bacteria: a view from lipid A biosynthesis

Evolution of gram-negative bacteria: a view from lipid A biosynthesis
enzymes
Stephen O. Opiyo1, Rosevelt L. Pardy1, Hideaki Moriyama1, and Etsuko N. Moriyama1,2
1
School of Biological Sciences, University of Nebraska-Lincoln
Center for Plant Science Innovation, University of Nebraska-Lincoln
2
Lipid-A, a complex glycolipid, is the highly immunoreactive endotoxic center of lipopolysaccharide
(LPS). It anchors the LPS into the outer membrane of most gram-negative bacteria. Lipid A can be
recognized by animal cells, triggers some defense-related responses, and causes gram-negative sepsis.
The lipid-A biosynthesis pathway consists of nine enzymatic steps. The enzymes involved are: LpxA,
LpxC, LpxD, LpxH, LpxB, LpxK, WaaA, LpxL, and LpxM. Traditionally bacteria are classified into two
major groups as gram-positive and gram-negative based on Gram-staining procedure. Gram-negative
bacteria have LPS-containing outer membranes; hence all these bacteria are expected to possess all genes
encoding lipid-A biosynthesis-pathway enzymes. These genes, on the other hand, are likely to be missing
from gram-positive bacteria. In order to elucidate how the lipid-A biosynthesis pathway has evolved in
the bacterial kingdom, we have examined the distribution of the lipid-A biosynthesis enzymes across 62
bacteria genomes (from 16 phyla including five species of gram-positive bacteria as control). Similarity
search was performed using BLASTP and TBLASTN, and also profile hidden Markov models. We
started our search using the nine enzyme sequences obtained from the Escherichia coli K12 genome as
queries against the other 61 bacterial genomes. We repeated reciprocal searches against all genomes using
each of newly identified protein sequences.
As we expected, none of the five gram-positive bacteria had genes encoding the lipid A biosynthesis
enzymes. On the other hand, we found surprisingly a wide range of presence/absence patterns with these
genes among 57 gram-negative bacteria we studied. Only one group of gammaproteobacteria (we call it
as the group I) had all nine genes required for lipid A biosynthesis. E. coli K12, and Vibrio cholera are
the representatives from this group. The other group of gammaproteobacteria (the group II; e.g.,
Pseudomonas syringae ) as well as betaproteobacteria (e.g., Bordetella parapertussis, but Shewanella sp.
MR-4 as one exception) had all except lpxM gene. All other gram-negative bacteria are missing both of
lpxH and lpxM genes. This implies that the lipid-A biosynthesis pathway consisting of the nine enzymes
is not canonical, but rather a specialized, derived form found only in E. coli K12 and closely related group
I gammaproteobacteria. The LpxM protein, found only in this group, shared a high similarity with the
LpxL protein, and appeared to be a product of duplication. We also found two surprising exceptions
among proteobacteria. Nitrosomonas europaea (betabacteria) had only one (lpxK) of the nine, and two
Walbachia species (W. pipientis and W. strain TRS of Brugia malayi) had none of the nine genes. Seven
other gram-negative bacteria lacked all of the nine genes although they are classified as gram-negative.
Based on the complete lack of the genes required for lipid-A biosynthesis, these gram-negative bacteria
could not be distinguished from gram-positive bacteria. Based on phylogenetic analysis using
concatenated proteins of lipid A enzymes, Dictyoglomi and cyanobacteria were the outmost groups
among the gram-negative bacteria. Interestingly, all these bacteria had only four of the nine genes; lpxA,
lpxC, lpxD, and lpxB. These genes encode the first four enzymes of the lipid-A biosynthesis up to the
point of producing the lipid A disaccharide. It indicates that these bacteria may have a different type of
Lipid A, which is probably the ancestral status. Another interesting observation was that although lpxH
gene is considered to be essential in lipid-A biosynthesis, it is missing from the majority of the bacteria
we examined. lpxH gene, which encodes pyrophosphatase, appeared to have arisen from a duplication of
lpxH2 gene after beta/gammaproteobacteria were diverged from other proteobacteria. lpxH2 gene encodes
phosphatase , and is not involved with lipid-A biosynthesis. Thus this new gene creation was followed by
a functional change from pyrophosphatase of LpxH to phosphatase of LpxH2. It is still not clear what
enzyme replaces the LpxH activity in bacteria with no lpxH gene found. This study clearly showed that
the currently known nine-enzyme pathway for lipid A biosynthesis, which has been mainly studied in E.
coli K12 and related bacteria, should not be considered as a representative nor ancestral to all bacteria.