BACKGROUND
kdo2-lipid a is a complex glycolipid consisting of glucosamine, 3-oh fatty acids, and unusual sugar 3-deoxy-d-manno-octulonsonic acid  .
kdo2-lipid a is the principle and essential component of the outer leaflet of the outer cell wall of gram-negative bacteria  <cit> .
it is the membrane anchor for a wide and variable range of polysaccharide repeating units extending beyond the cell wall.
together with kdo2-lipid a the polysaccharide repeating units constitute lipopolysaccharide , a class of molecules believed to occur solely in gram-negative bacteria and cyanobacteria and essential for their viability  <cit> .
lipid a is a potent immunoreactive factor responsible for triggering a macrophage mediated and frequently overwhelming immune response resulting in endotoxic shock, a potentially lethal condition.
indeed, as far as is known, all vertebrates and an assortment of invertebrates exhibit a robust and highly specific anti-lps response.
for decades lipid a/lps has been essentially synonymous with gram-negative organisms, yet, despite the relative diversity of the gram-negative bacteria, the origin and evolutionary pathway of this important molecule is virtually unknown.
presently the complex, nine-enzyme biosynthetic process of kdo2-lipid a is best known from escherichia coli .
the genetic sequences for the enzymes have been derived from the e. coli genome .
this pathway and the concomitant genes in e. coli probably represent the most highly evolved kdo2-lipid a biosynthetic pathway given the highly adapted association of e. coli with vertebrate enteric habitats, which depends heavily on the structure of lps.
thus, the central question becomes: can the evolutionary radiation of lps be described by understanding the comparative genomics of kdo2-lipid a biosynthetic pathway?
traditional classification of bacteria: gram-positive vs. gram-negative, is based on the gram-staining procedure  <cit> .
gram-positive bacteria retain the crystal-violet stain, whereas gram-negative bacteria are decolorized with alcohol or acetone and stained red with safranin or basic fuchsin.
different responses to the staining are caused by the chemical properties of the cell walls.
gram-positive bacteria have an impermeable cell wall that is made of thick peptidoglycan and is not affected by decolorization.
gram-negative bacteria, on the other hand, have a thin peptidoglycan layer and an outer membrane containing lps, which can be disrupted by decolorization.
phylogenetic analysis of biomolecular sequences is now considered to be more reliable for bacterial classification.
molecular phylogenies do not support the traditional simple grouping of eubacteria: gram-negative vs. gram-positive .
the most important and often used phylogenetic marker for bacteria is 16s ribosomal rna  due to their highly conserved nature of sequences.
the second edition of bergey's manual of systematic bacteriology  <cit> , the gold standard of bacterial classification, is mostly based on 16s rrna phylogenies.
gram-positive bacteria are now grouped into two paraphyletic groups: the phylum firmicutes  and the phylum actinobacteria .
gram-negative bacteria are composed of more than  <dig> of highly diverged phyla.
firmicutes, a gram-positive phylum, forms a sister cluster with a gram-negative phylum cyanobacteria.
the phylum proteobacteria, the largest gram-negative bacteria group that includes e. coli and many pathogens, is the most derived group of bacteria.
understanding the distribution and diversity of the kdo2-lipid a biosynthetic pathway among bacteria is important for a multitude of reasons.
notwithstanding its limitation, gram-staining is still widely used in clinical practice.
it is often the first diagnostic test, which is crucial for the initial diagnostic and treatments.
kdo2-lipid a is the highly immunoreactive endotoxic center of lps.
the endotoxicity of lps is dependent on and mediated by the kdo2-lipid a component.
furthermore, the kdo2-lipid a pathway is being considered as a target for new antibiotic development .
kdo2-lipid a is required for growth of e. coli and most other gram-negative bacteria.
inhibitors of the kdo2-lipid a biosynthesis, therefore, can become good antibiotics against these bacteria.
in order to elucidate how the kdo2-lipid a biosynthetic pathway has evolved in the bacterial kingdom, we examined the distribution of the nine enzymes involved in this pathway across  <dig> bacterial genomes.
with kdo2-lipid a as the final product, the entire pathway was expected to be highly conserved among gram-negative bacteria.
on the other hand, gram-positive bacteria would lack some or all of the enzymes required for the kdo2-lipid a biosynthesis.
on the contrary, we identified a widely varied level of conservation in this pathway among gram-negative bacteria.
we showed that the currently known, considered to be "canonical", nine-enzyme pathway, which has been characterized mainly in e. coli and related bacteria, does not represent nor should be considered as ancestral to all gram-negative bacteria.
rather, the nine-enzyme pathway represents the product of genomic plasticity, evolved in highly-derived proteobacteria, especially in those closely related to e. coli.
RESULTS
distribution of kdo2-lipid a biosynthetic enzymes across bacterial genomes
gram-negative bacteria, by definition, should have lps-containing outer membranes; hence all these bacteria are expected to possess all genes encoding kdo2-lipid a biosynthetic enzymes.
these genes, on the other hand, are likely to be missing from gram-positive bacteria unless they are used in alternative functions in these bacteria.
as we expected, none of the seven gram-positive bacteria we examined had the genes encoding these enzymes.
on the other hand, we found surprisingly a wide range of presence/absence patterns with these genes among  <dig> gram-negative bacteria we studied .
only one group of gammaproteobacteria including escherichia, vibrio, and shewanella species had all nine genes required for kdo2-lipid a biosynthesis.
we call them group ii gammaproteobacteria .
remaining gammaproteobacteria  as well as betaproteobacteria  had all genes except lpxm.
all other gram-negative bacteria are missing lpxh as well as lpxm genes.
this implies that the kdo2-lipid a biosynthetic pathway consisting of the nine enzymes is not ancestral, but rather a specialized, derived form found only in e. coli and closely related group ii gammaproteobacteria.
the lpxm protein, found only in this group, shares a sequence similarity of 47% with the lpxl protein, and appeared to be a product of gene duplication.
we also found two exceptions among proteobacteria.
nitrosomonas europaea  had only one  and two species of walbachia  we examined had none of the nine genes.
seven other bacteria also lacked all of the nine genes although they are classified as gram-negative .
gene duplication and functional specialization characterize the evolution of the kdo2-lipid a biosynthetic pathway
we identified multiple gene-duplication events during the evolution of the kdo2-lipid a biosynthetic pathway.
lpxa and lpxd are duplicated genes and share 45% sequence similarity at the protein level.
lpxa and lpxd proteins have trimer structures with different quaternary assembly and active sites  <cit> .
all bacteria we examined either possess or lack both enzymes.
therefore, generation of this pair of enzymes must have been an early event in gram-negative bacteria.
further duplications of either of the genes have happened in some lineages independently  .
lpxh gene, which encodes pyrophosphatase  <cit> , appeared to have arisen from a duplication of lpxh <dig> gene within the proteobacteria lineage but before beta- and gammaproteobacteria divergence .
furthermore, lpxl gene seems to be prone to duplicate.
we identified several such duplication events including one that generated lpxm in the group ii gammaproteobacteria .
lpxh/lpxh <dig> duplication within proteobacteria
lpxh and lpxh <dig> proteins share 42% sequence similarity  <cit> .
lpxh <dig> candidates were found mainly in the phylum proteobacteria, but also in some other gram-negative bacteria .
the duplication event that created the lpxh gene happened within the phylum proteobacteria before the divergence of beta- and gammaproteobacteria .
interestingly, after the duplication event, lpxh <dig> gene was lost from the group ii gammaproteobacteria except for shewanella sp.
mr- <dig> .
it implies that lpxh <dig> gene is dispensable when lpxh gene exists.
since some of the gram-negative bacteria that have seven enzymes in the pathway have only lpxh <dig> gene or neither lpxh nor lpxh <dig> genes, it is tempting to speculate that functions of lpxh and lpxh <dig> proteins are interchangeable or can be replaced by other non-specific phosphatases .
babinski et al.
<cit>  showed that lpxh gene from pseudomonas aeruginosa compensated for the loss of e. coli k <dig> lpxh, but lpxh <dig> of p. aeruginosa did not.
for those proteobacteria that have both of lpxh and lpxh <dig> proteins, the function of each protein may have been optimized for their specific roles.
such specialized proteins may not be as flexible as the ancestral form.
lpxh and lpxh <dig> proteins belong to the metallophosphoesterase superfamily .
the lpxh and lpxh <dig> protein groups form two distinct clusters among similar bacterial proteins identified .
all these proteins contain a five-block motif: d-xn-gd-xn-gnh-xn-h-xn-ghxh, a signature of the metallophosphoesterase superfamily.
mutational and structural analyses showed that these residues are involved in metal-ion binding and catalysis  <cit> .
the five-block residues are completely conserved among all proteins we examined including lpxh and lpxh <dig>  except for the third block, gnh, where the histidine  is substituted to arginine  only in lpxh .
after the lpxh2/h ancestral protein was generated by a duplication of a gene encoding a protein similar to calcineurin phosphohydrolase , another duplication produced the two protein families, lpxh <dig> and lpxh .
although the function of lpxh <dig> has been unknown, comparisons of structural models of lpxh and lpxh <dig> proteins indicate that the five-block regions form similar cavities in these proteins .
the histidine-to-arginine substitution in the third block in lpxh may have increased the lpxh activity significantly in beta- and gammaproteobacteria.
recently, metzger and raetz  <cit>  identified a gene located between lpxa and lpxb in caulobacter crescentus .
this gene, named lpxi, which does not share similarity with lpxh/lpxh <dig> genes, was found to catalyze udp- <dig> -diacylglucosamine hydrolysis by a mechanism different from lpxh.
it also rescued lpxh-deficient e. coli.
lpxi orthologues were found in many, but not all of, bacteria that lack lpxh  <cit>  .
metzger and raetz  <cit>  reported that lpxi was found even from gram-positive bacteria .
it suggests that replacement of lpxh gene function appears to be not very difficult, and multiple gene recruitments may have happened during the evolution of kdo2-lipid a biosynthetic pathway.
multiple lpxl duplications within gram-negative bacteria
functional specialization in lpxl and its paralogues
although it is not clear if types- <dig> and - <dig> lpxl function differently in all beta/gammabacteria, in some bacteria duplicated lpxl copies have apparently evolved to have slightly different functions.
type- <dig> lpxl  of bordetella pertutssis adds secondary 2-hydroxy laurate at the position  <dig> of kdo2-lipid a, and type- <dig> lpxl  adds myristate at the position 2' of kdo2-lipid a  <cit> .
francisella novicida has species-specific duplicated copies of type- <dig> lpxl, lpxl <dig> and lpxl <dig>  and they function as kdo-dependent and -independent acyltransferases, respectively.
lpxl <dig> of f. novicida can add laurate at the position 2' of lipid iva without kdo addition by waaa, which probably accounts for a large amount of 'free lipid a'   <cit> .
acyltransferases lpxl and lpxm share 47% sequence similarity.
in e. coli, lpxl and lpxm add laurate at the position 2' and myristate at the position 3' of kdo2-lipid a, respectively  <cit> , and have conserved catalytic dyad motifs hx4d and hx4e, respectively  <cit> .
lpxm is not required for growth of e. coli k <dig>  <cit> .
while e. coli k <dig> mutants for lpxl and lpxm genes can grow on minimum medium and at all temperatures, they do not grow on rich media at temperatures above 32°c  <cit> .
in haemorphilus influenzae, lpxl and lpxm are both myristoyl transferases  <cit> .
therefore, four types of related enzymes appear to have evolved after duplications: "generalist" lpxl enzyme that catalyzes the transfer of both laurate and myristate, more "specialized" lpxl enzyme that acts only either on laurate or myristate, and lpxm newly "specialized" as myristoyl transferase.
lpxm is especially optimized for group ii gammaproteobacteria, many of which live in warm body temperatures of the hosts.
cold-temperature adaptation with lpxp
another lpxl duplicate, lpxp, is found in e. coli and other closely related enterobacteria.
while e. coli and s. enterica have all three paralogous genes , y. pestis has only two derived copies .
lpxp encodes the palmitoleoyl transferase, which is induced upon cold shock   <cit> .
in e. coli, palmitoleate is incorporated to kdo2-lipid iva by lpxp at the position where normally lpxl incorporates laurate   <cit> .
palmitoleoyl residue changes the properties  of the outer membrane and makes bacteria adaptable to low growth temperatures.
while survival outside of an animal host is necessary in bacteria such as e. coli, h. influenzae, for example, is transmitted from animal to animal without being exposed in the colder environment.
these gram-negative bacteria do not have lpxp gene.
another example of lipid-a associated adaptation is found in y. pestis.
y. pestis changes its host from flea to mouse, cold to warm temperature environment.
due to the absence of the lpxl gene, y. pestis produces only tetra-acylated kdo2-lipid iva  but not hexa-acylated kdo2-lipid a at 37°c  contributing bacteria's poor toll-like receptor  <dig>  stimulating activity  <cit> .
with lpxp activated, however, at 21°c  hexa-acylated kdo2-lipid a with palmitoleate is synthesized in y. pestis.
four-enzyme pathway in cyanobacteria and dictyoglomi: the primordial form?
cyanobacteria  and dictyoglomi  have only four of the nine genes: lpxa, lpxc, lpxd, and lpxb .
these genes encode the first four enzymes of kdo2-lipid a biosynthesis up to the point of producing the lipid a disaccharide , implying that this may be the primordial form of lipid a. lps from cyanobacteria is simpler than that of lps of enteric bacteria and lacks kdo, heptose, and phosphate  <cit> .
it is reported that lps from e. coli is more toxic than lps from cyanobacteria  <cit> .
kdo2-lipid a biosynthetic pathway gene clusters
in prokaryotic genomes, many functionally related gene sets exist as gene clusters .
as shown in additional file  <dig>  genes encoding some or all of the first four enzymes  are often found in a gene cluster.
dictyoglomi and cyanobacteria have only these four genes and all of them exist in a conserved gene cluster, lpxd-lpxc-fabz-lpxa-lpxb.
note that the majority of these clusters include fabz, which encodes -hydroxymyristoyl acyl carrier protein  dehydratase  <cit> .
-hydroxymyristoyl-acp serves as an important biosynthetic branch point.
it is transferred to udp-glcnac to initiate lipid a biosynthesis .
or it is elongated by fabz and other enzymes of fatty acid synthesis to palmitate, which is a major component of the membrane glycerophospholipids.
therefore, this gene cluster is important for regulating the proportion of lps and phospolipids in bacterial membranes.
in some bacteria , the lpxc-fabz part of the cluster has been fused and exists as a single gene .
although lpxh is not found as part of these gene clusters, as mentioned before, its functional equivalent lpxi is found as part of the cluster  in alphaproteobacteria and some other bacteria .
another often found gene cluster is waaa-lpxk.
again these genes encode enzymes that catalyze the two consecutive steps  indicating the importance of their functional association in kdo2-lipid a biosynthesis.
bacterial phylogeny based on kdo2-lipid a biosynthetic enzymes
there have been a number of studies to reconstruct bacterial phylogenies using various molecular data .
our phylogenies based on kdo2-lipid a biosynthesis enzymes, analyzed concatenated  or individually , are largely consistent with previous studies.
the monophyletic cluster including both gamma- and betaproteobacteria is strongly supported by high bootstrap values in both lpx protein and 16s rrna phylogenies  as well as by the shared events with lpxh2/lpxh and lpxl duplications.
group ii gammaproteobacteria, which includes the orders enterobacteriales , pasteurellales , vibrionales , and alteromonadales , also form a highly-supported cluster  and all share the newly emerged lpxm gene.
this group ii gammaproteobacteria clustering is also supported by the phylogenetic study by gao et al.
<cit>  based on  <dig> protein data as well as the existence of the unique indel within the rna polymerase β-subunit .
using the dictyoglomi  as the outgroup , phylogenies based on both of the kdo2-lipid a biosynthetic enzymes and the 16s rrna  showed that most bacteria that have no or only four enzymes  are located at basal relative to those that have seven or more of the enzymes.
exceptions include brachyspira hyodysenteriae  and thermodesulfovibrio yellowstonii .
fusobacterium nucleatum  was also located outside of cyanobacteria cluster in kdo2-lipid a enzyme phylogeney .
note, however, that none of their phylogenetic positions was supported with high bootstrap values in 16s rrna phylogeny .
gram-negative bacteria with no kdo2-lipid a biosynthetic enzyme
some gram-negative bacteria  as well as gram-positive bacteria  have none of the nine enzymes.
as mentioned above, since both dictyoglomi  and cyanobacteria  have the first four enzymes of the pathway, having the four-enzyme pathway appears to be the ancestral form.
it implies that these enzymes must have been lost later in some groups of bacteria.
many of the gram-negative bacteria with no kdo2-lipid a biosynthetic enzyme have specialized life-styles: endosymbiomes , obligate chemolithoautotroph , or hyperthermophiles .
wolbachia is either a parasite in arthropod hosts or a mutualist in nematode hosts.
it belongs to the family anaplasmataceae of the class alphaproteobacteria.
bacteria in this family include ehrlichia chaffeensis, anaplasma phagocytophilum, and neorickettsia sennetsu; all are obligatory intracellular and infect mononuclear cells and granulocytes.
they have been found to lack kdo2-lipid a biosynthetic genes  <cit> .
therefore, the loss of these genes must have happened in the ancestral lineage after the divergence from other alphaproteobacteria group .
lipid a is directly recognized by hosts to trigger innate-immune responses  <cit> .
it is likely that the endosymbiotic bacteria have adapted to their symbiosis conditions by losing these enzymes and not producing lipid a to avoid the induction of defense response in the hosts.
b. burgdorferi is also an endosymbiotic bacterium that lives in ticks and transmits lyme disease.
t. denticola is the cause of periodontal disease.
they belong to the family spirochaetaceae .
while there have been conflicting reports whether or not these bacteria have lps  <cit> , genomic analyses showed that these bacteria as well as t. pallidum lack kdo2-lipid a biosynthetic enzymes  <cit>  .
the genes encoding these enzymes have been identified from two other spirochaetes families , which diverged earlier than the family spirochaetaceae .
therefore, the gene loss event is specific to the family spirochaetaceae.
n. europaea is a obligate chemolithoautotroph that derives all its energy and reductant for growth from the oxidation of ammonia to nitrite  <cit> .
only lpxk was detected from this betaproteobacteria species.
from the closely related nitrosospira multiformis genome, only lpxa gene instead has been identified  <cit> .
these ammonia-oxidizing bacteria have the smallest genomes  among the betaproteobacteria  <cit> .
such genome reduction is consistent with their limited lifestyles as we also described earlier for obligate endosymbionts and pathogens.
large numbers of insertion sequence elements and pseudogenes have been also found in these genomes, implying that the genomes are still undergoing reductive evolution.
the origin of lpx genes found in plants
although kdo2-lipid a biosynthesis is generally considered to be a prokaryote-specific function, at least six of the nine lpx genes have been identified in arabidopsis thaliana, oryza sativa, and other plant genomes .
furthermore, armstrong et al.
<cit>  showed that green algae and chloroplasts of garden pea were stained with affinity reagents for lipid a, indicating that these plants synthesize lipid a-like molecules.
since cyanobacteria possess only the first four genes, all or some of the lpx genes in plants must have been transferred from bacteria other than cyanobacteria.
despite the presence of lipid a genes in plants, attempts to isolate canonical lipid a from plants using standard methods have been unsuccessful ; no structural data for putative lipid a in higher plants has been reported.
however, as mentioned earlier, using a novel lipid a preparation technique, snyder et al.
<cit>  recently provided chemical composition and structural data for a simple lipid a from strains of a marine cyanobacteria, synechoccoccus.
the composition and structure of this lipid a differs significantly from that of canonical lipid a from enteric bacteria in ways hypothesized to be adaptive to marine synechoccoccus.
although not only the first four genes exist in plants, lipid a in plants likely has a structure different from canonical lipid a. further investigation is required to elucidate the origin and functions of eukaryotic lpx genes.
CONCLUSIONS
bacterial genomes are extremely plastic.
although the kdo2-lipid a biosynthesis is one of the most fundamental and most conserved pathways among gram-negative bacteria, this study showed that gene duplications as well as partial or complete losses of the genes encoding these enzymes have happened multiple times independently during bacterial evolution.
each group of bacteria took advantage of such evolutionary events to optimize the pathway and adapted to their specialized life style.
the most optimized form of the pathway is found in the proteobacteria lineage, especially among gammaproteobacteria.
the nine-enzyme pathway currently known for the kdo2-lipid a biosynthesis, which is mainly studied in e. coli and related bacteria, is the most optimized, derived form of this pathway.

 kdo2-lipid a is a complex glycolipid consisting of glucosamine, 3-oh fatty acids, and unusual sugar 3-deoxy-d-manno-octulonsonic acid. kdo2-lipid a is the principle and essential component of the outer leaflet of the outer cell wall of gram-negative bacteria. it is the membrane anchor for a wide and variable range of polysaccharide repeating units extending beyond the cell wall. together with kdo2-lipid a the polysaccharide repeating units constitute lipopolysaccharide, a class of molecules believed to occur solely in gram-negative bacteria and cyanobacteria and essential for their viability. lipid a is a potent immunoreactive factor responsible for triggering a macrophage mediated and frequently overwhelming immune response resulting in endotoxic shock, a potentially lethal condition. indeed, as far as is known, all vertebrates and an assortment of invertebrates exhibit a robust and highly specific anti-lps response. for decades lipid a/lps has been essentially synonymous with gram-negative organisms, yet, despite the relative diversity of the gram-negative bacteria, the origin and evolutionary pathway of this important molecule is virtually unknown. presently the complex, nine-enzyme biosynthetic process of kdo2-lipid a is best known from escherichia coli. the genetic sequences for the enzymes have been derived from the e. coli genome. this pathway and the concomitant genes in e. coli probably represent the most highly evolved kdo2-lipid a biosynthetic pathway given the highly adapted association of e. coli with vertebrate enteric habitats, which depends heavily on the structure of lps. thus, the central question becomes: can the evolutionary radiation of lps be described by understanding the comparative genomics of kdo2-lipid a biosynthetic pathway? traditional classification of bacteria: gram-positive vs. gram-negative, is based on the gram-staining procedure. gram-positive bacteria retain the crystal-violet stain, whereas gram-negative bacteria are decolorized with alcohol or acetone and stained red with safranin or basic fuchsin. different responses to the staining are caused by the chemical properties of the cell walls. gram-positive bacteria have an impermeable cell wall that is made of thick peptidoglycan and is not affected by decolorization. gram-negative bacteria, on the other hand, have a thin peptidoglycan layer and an outer membrane containing lps, which can be disrupted by decolorization. phylogenetic analysis of biomolecular sequences is now considered to be more reliable for bacterial classification. molecular phylogenies do not support the traditional simple grouping of eubacteria: gram-negative vs. gram-positive. the most important and often used phylogenetic marker for bacteria is 16s ribosomal rna due to their highly conserved nature of sequences. the second edition of bergey's manual of systematic bacteriology, the gold standard of bacterial classification, is mostly based on 16s rrna phylogenies. gram-positive bacteria are now grouped into two paraphyletic groups: the phylum firmicutes and the phylum actinobacteria. gram-negative bacteria are composed of more than of highly diverged phyla. firmicutes, a gram-positive phylum, forms a sister cluster with a gram-negative phylum cyanobacteria. the phylum proteobacteria, the largest gram-negative bacteria group that includes e. coli and many pathogens, is the most derived group of bacteria. understanding the distribution and diversity of the kdo2-lipid a biosynthetic pathway among bacteria is important for a multitude of reasons. notwithstanding its limitation, gram-staining is still widely used in clinical practice. it is often the first diagnostic test, which is crucial for the initial diagnostic and treatments. kdo2-lipid a is the highly immunoreactive endotoxic center of lps. the endotoxicity of lps is dependent on and mediated by the kdo2-lipid a component. furthermore, the kdo2-lipid a pathway is being considered as a target for new antibiotic development. kdo2-lipid a is required for growth of e. coli and most other gram-negative bacteria. inhibitors of the kdo2-lipid a biosynthesis, therefore, can become good antibiotics against these bacteria. in order to elucidate how the kdo2-lipid a biosynthetic pathway has evolved in the bacterial kingdom, we examined the distribution of the nine enzymes involved in this pathway across bacterial genomes. with kdo2-lipid a as the final product, the entire pathway was expected to be highly conserved among gram-negative bacteria. on the other hand, gram-positive bacteria would lack some or all of the enzymes required for the kdo2-lipid a biosynthesis. on the contrary, we identified a widely varied level of conservation in this pathway among gram-negative bacteria. we showed that the currently known, considered to be "canonical", nine-enzyme pathway, which has been characterized mainly in e. coli and related bacteria, does not represent nor should be considered as ancestral to all gram-negative bacteria. rather, the nine-enzyme pathway represents the product of genomic plasticity, evolved in highly-derived proteobacteria, especially in those closely related to e. coli.  distribution of kdo2-lipid a biosynthetic enzymes across bacterial genomes gram-negative bacteria, by definition, should have lps-containing outer membranes; hence all these bacteria are expected to possess all genes encoding kdo2-lipid a biosynthetic enzymes. these genes, on the other hand, are likely to be missing from gram-positive bacteria unless they are used in alternative functions in these bacteria. as we expected, none of the seven gram-positive bacteria we examined had the genes encoding these enzymes. on the other hand, we found surprisingly a wide range of presence/absence patterns with these genes among gram-negative bacteria we studied. only one group of gammaproteobacteria including escherichia, vibrio, and shewanella species had all nine genes required for kdo2-lipid a biosynthesis. we call them group ii gammaproteobacteria. remaining gammaproteobacteria as well as betaproteobacteria had all genes except lpxm. all other gram-negative bacteria are missing lpxh as well as lpxm genes. this implies that the kdo2-lipid a biosynthetic pathway consisting of the nine enzymes is not ancestral, but rather a specialized, derived form found only in e. coli and closely related group ii gammaproteobacteria. the lpxm protein, found only in this group, shares a sequence similarity of 47% with the lpxl protein, and appeared to be a product of gene duplication. we also found two exceptions among proteobacteria. nitrosomonas europaea had only one and two species of walbachia we examined had none of the nine genes. seven other bacteria also lacked all of the nine genes although they are classified as gram-negative. gene duplication and functional specialization characterize the evolution of the kdo2-lipid a biosynthetic pathway we identified multiple gene-duplication events during the evolution of the kdo2-lipid a biosynthetic pathway. lpxa and lpxd are duplicated genes and share 45% sequence similarity at the protein level. lpxa and lpxd proteins have trimer structures with different quaternary assembly and active sites. all bacteria we examined either possess or lack both enzymes. therefore, generation of this pair of enzymes must have been an early event in gram-negative bacteria. further duplications of either of the genes have happened in some lineages independently. lpxh gene, which encodes pyrophosphatase, appeared to have arisen from a duplication of lpxh gene within the proteobacteria lineage but before beta- and gammaproteobacteria divergence. furthermore, lpxl gene seems to be prone to duplicate. we identified several such duplication events including one that generated lpxm in the group ii gammaproteobacteria. lpxh/lpxh duplication within proteobacteria lpxh and lpxh proteins share 42% sequence similarity. lpxh candidates were found mainly in the phylum proteobacteria, but also in some other gram-negative bacteria. the duplication event that created the lpxh gene happened within the phylum proteobacteria before the divergence of beta- and gammaproteobacteria. interestingly, after the duplication event, lpxh gene was lost from the group ii gammaproteobacteria except for shewanella sp. mr-. it implies that lpxh gene is dispensable when lpxh gene exists. since some of the gram-negative bacteria that have seven enzymes in the pathway have only lpxh gene or neither lpxh nor lpxh genes, it is tempting to speculate that functions of lpxh and lpxh proteins are interchangeable or can be replaced by other non-specific phosphatases. babinski et al. showed that lpxh gene from pseudomonas aeruginosa compensated for the loss of e. coli k lpxh, but lpxh of p. aeruginosa did not. for those proteobacteria that have both of lpxh and lpxh proteins, the function of each protein may have been optimized for their specific roles. such specialized proteins may not be as flexible as the ancestral form. lpxh and lpxh proteins belong to the metallophosphoesterase superfamily. the lpxh and lpxh protein groups form two distinct clusters among similar bacterial proteins identified. all these proteins contain a five-block motif: d-xn-gd-xn-gnh-xn-h-xn-ghxh, a signature of the metallophosphoesterase superfamily. mutational and structural analyses showed that these residues are involved in metal-ion binding and catalysis. the five-block residues are completely conserved among all proteins we examined including lpxh and lpxh except for the third block, gnh, where the histidine is substituted to arginine only in lpxh. after the lpxh2/h ancestral protein was generated by a duplication of a gene encoding a protein similar to calcineurin phosphohydrolase, another duplication produced the two protein families, lpxh and lpxh. although the function of lpxh has been unknown, comparisons of structural models of lpxh and lpxh proteins indicate that the five-block regions form similar cavities in these proteins. the histidine-to-arginine substitution in the third block in lpxh may have increased the lpxh activity significantly in beta- and gammaproteobacteria. recently, metzger and raetz identified a gene located between lpxa and lpxb in caulobacter crescentus. this gene, named lpxi, which does not share similarity with lpxh/lpxh genes, was found to catalyze udp- -diacylglucosamine hydrolysis by a mechanism different from lpxh. it also rescued lpxh-deficient e. coli. lpxi orthologues were found in many, but not all of, bacteria that lack lpxh. metzger and raetz reported that lpxi was found even from gram-positive bacteria. it suggests that replacement of lpxh gene function appears to be not very difficult, and multiple gene recruitments may have happened during the evolution of kdo2-lipid a biosynthetic pathway. multiple lpxl duplications within gram-negative bacteria functional specialization in lpxl and its paralogues although it is not clear if types- and - lpxl function differently in all beta/gammabacteria, in some bacteria duplicated lpxl copies have apparently evolved to have slightly different functions. type- lpxl of bordetella pertutssis adds secondary 2-hydroxy laurate at the position of kdo2-lipid a, and type- lpxl adds myristate at the position 2' of kdo2-lipid a. francisella novicida has species-specific duplicated copies of type- lpxl, lpxl and lpxl and they function as kdo-dependent and -independent acyltransferases, respectively. lpxl of f. novicida can add laurate at the position 2' of lipid iva without kdo addition by waaa, which probably accounts for a large amount of'free lipid a'. acyltransferases lpxl and lpxm share 47% sequence similarity. in e. coli, lpxl and lpxm add laurate at the position 2' and myristate at the position 3' of kdo2-lipid a, respectively, and have conserved catalytic dyad motifs hx4d and hx4e, respectively. lpxm is not required for growth of e. coli k. while e. coli k mutants for lpxl and lpxm genes can grow on minimum medium and at all temperatures, they do not grow on rich media at temperatures above 32°c. in haemorphilus influenzae, lpxl and lpxm are both myristoyl transferases. therefore, four types of related enzymes appear to have evolved after duplications: "generalist" lpxl enzyme that catalyzes the transfer of both laurate and myristate, more "specialized" lpxl enzyme that acts only either on laurate or myristate, and lpxm newly "specialized" as myristoyl transferase. lpxm is especially optimized for group ii gammaproteobacteria, many of which live in warm body temperatures of the hosts. cold-temperature adaptation with lpxp another lpxl duplicate, lpxp, is found in e. coli and other closely related enterobacteria. while e. coli and s. enterica have all three paralogous genes, y. pestis has only two derived copies. lpxp encodes the palmitoleoyl transferase, which is induced upon cold shock. in e. coli, palmitoleate is incorporated to kdo2-lipid iva by lpxp at the position where normally lpxl incorporates laurate. palmitoleoyl residue changes the properties of the outer membrane and makes bacteria adaptable to low growth temperatures. while survival outside of an animal host is necessary in bacteria such as e. coli, h. influenzae, for example, is transmitted from animal to animal without being exposed in the colder environment. these gram-negative bacteria do not have lpxp gene. another example of lipid-a associated adaptation is found in y. pestis. y. pestis changes its host from flea to mouse, cold to warm temperature environment. due to the absence of the lpxl gene, y. pestis produces only tetra-acylated kdo2-lipid iva but not hexa-acylated kdo2-lipid a at 37°c contributing bacteria's poor toll-like receptor stimulating activity. with lpxp activated, however, at 21°c hexa-acylated kdo2-lipid a with palmitoleate is synthesized in y. pestis. four-enzyme pathway in cyanobacteria and dictyoglomi: the primordial form? cyanobacteria and dictyoglomi have only four of the nine genes: lpxa, lpxc, lpxd, and lpxb. these genes encode the first four enzymes of kdo2-lipid a biosynthesis up to the point of producing the lipid a disaccharide, implying that this may be the primordial form of lipid a. lps from cyanobacteria is simpler than that of lps of enteric bacteria and lacks kdo, heptose, and phosphate. it is reported that lps from e. coli is more toxic than lps from cyanobacteria. kdo2-lipid a biosynthetic pathway gene clusters in prokaryotic genomes, many functionally related gene sets exist as gene clusters. as shown in additional file genes encoding some or all of the first four enzymes are often found in a gene cluster. dictyoglomi and cyanobacteria have only these four genes and all of them exist in a conserved gene cluster, lpxd-lpxc-fabz-lpxa-lpxb. note that the majority of these clusters include fabz, which encodes -hydroxymyristoyl acyl carrier protein dehydratase. -hydroxymyristoyl-acp serves as an important biosynthetic branch point. it is transferred to udp-glcnac to initiate lipid a biosynthesis. or it is elongated by fabz and other enzymes of fatty acid synthesis to palmitate, which is a major component of the membrane glycerophospholipids. therefore, this gene cluster is important for regulating the proportion of lps and phospolipids in bacterial membranes. in some bacteria, the lpxc-fabz part of the cluster has been fused and exists as a single gene. although lpxh is not found as part of these gene clusters, as mentioned before, its functional equivalent lpxi is found as part of the cluster in alphaproteobacteria and some other bacteria. another often found gene cluster is waaa-lpxk. again these genes encode enzymes that catalyze the two consecutive steps indicating the importance of their functional association in kdo2-lipid a biosynthesis. bacterial phylogeny based on kdo2-lipid a biosynthetic enzymes there have been a number of studies to reconstruct bacterial phylogenies using various molecular data. our phylogenies based on kdo2-lipid a biosynthesis enzymes, analyzed concatenated or individually, are largely consistent with previous studies. the monophyletic cluster including both gamma- and betaproteobacteria is strongly supported by high bootstrap values in both lpx protein and 16s rrna phylogenies as well as by the shared events with lpxh2/lpxh and lpxl duplications. group ii gammaproteobacteria, which includes the orders enterobacteriales, pasteurellales, vibrionales, and alteromonadales, also form a highly-supported cluster and all share the newly emerged lpxm gene. this group ii gammaproteobacteria clustering is also supported by the phylogenetic study by gao et al. based on protein data as well as the existence of the unique indel within the rna polymerase β-subunit. using the dictyoglomi as the outgroup, phylogenies based on both of the kdo2-lipid a biosynthetic enzymes and the 16s rrna showed that most bacteria that have no or only four enzymes are located at basal relative to those that have seven or more of the enzymes. exceptions include brachyspira hyodysenteriae and thermodesulfovibrio yellowstonii. fusobacterium nucleatum was also located outside of cyanobacteria cluster in kdo2-lipid a enzyme phylogeney. note, however, that none of their phylogenetic positions was supported with high bootstrap values in 16s rrna phylogeny. gram-negative bacteria with no kdo2-lipid a biosynthetic enzyme some gram-negative bacteria as well as gram-positive bacteria have none of the nine enzymes. as mentioned above, since both dictyoglomi and cyanobacteria have the first four enzymes of the pathway, having the four-enzyme pathway appears to be the ancestral form. it implies that these enzymes must have been lost later in some groups of bacteria. many of the gram-negative bacteria with no kdo2-lipid a biosynthetic enzyme have specialized life-styles: endosymbiomes, obligate chemolithoautotroph, or hyperthermophiles. wolbachia is either a parasite in arthropod hosts or a mutualist in nematode hosts. it belongs to the family anaplasmataceae of the class alphaproteobacteria. bacteria in this family include ehrlichia chaffeensis, anaplasma phagocytophilum, and neorickettsia sennetsu; all are obligatory intracellular and infect mononuclear cells and granulocytes. they have been found to lack kdo2-lipid a biosynthetic genes. therefore, the loss of these genes must have happened in the ancestral lineage after the divergence from other alphaproteobacteria group. lipid a is directly recognized by hosts to trigger innate-immune responses. it is likely that the endosymbiotic bacteria have adapted to their symbiosis conditions by losing these enzymes and not producing lipid a to avoid the induction of defense response in the hosts. b. burgdorferi is also an endosymbiotic bacterium that lives in ticks and transmits lyme disease. t. denticola is the cause of periodontal disease. they belong to the family spirochaetaceae. while there have been conflicting reports whether or not these bacteria have lps, genomic analyses showed that these bacteria as well as t. pallidum lack kdo2-lipid a biosynthetic enzymes. the genes encoding these enzymes have been identified from two other spirochaetes families, which diverged earlier than the family spirochaetaceae. therefore, the gene loss event is specific to the family spirochaetaceae. n. europaea is a obligate chemolithoautotroph that derives all its energy and reductant for growth from the oxidation of ammonia to nitrite. only lpxk was detected from this betaproteobacteria species. from the closely related nitrosospira multiformis genome, only lpxa gene instead has been identified. these ammonia-oxidizing bacteria have the smallest genomes among the betaproteobacteria. such genome reduction is consistent with their limited lifestyles as we also described earlier for obligate endosymbionts and pathogens. large numbers of insertion sequence elements and pseudogenes have been also found in these genomes, implying that the genomes are still undergoing reductive evolution. the origin of lpx genes found in plants although kdo2-lipid a biosynthesis is generally considered to be a prokaryote-specific function, at least six of the nine lpx genes have been identified in arabidopsis thaliana, oryza sativa, and other plant genomes. furthermore, armstrong et al. showed that green algae and chloroplasts of garden pea were stained with affinity reagents for lipid a, indicating that these plants synthesize lipid a-like molecules. since cyanobacteria possess only the first four genes, all or some of the lpx genes in plants must have been transferred from bacteria other than cyanobacteria. despite the presence of lipid a genes in plants, attempts to isolate canonical lipid a from plants using standard methods have been unsuccessful; no structural data for putative lipid a in higher plants has been reported. however, as mentioned earlier, using a novel lipid a preparation technique, snyder et al. recently provided chemical composition and structural data for a simple lipid a from strains of a marine cyanobacteria, synechoccoccus. the composition and structure of this lipid a differs significantly from that of canonical lipid a from enteric bacteria in ways hypothesized to be adaptive to marine synechoccoccus. although not only the first four genes exist in plants, lipid a in plants likely has a structure different from canonical lipid a. further investigation is required to elucidate the origin and functions of eukaryotic lpx genes.  bacterial genomes are extremely plastic. although the kdo2-lipid a biosynthesis is one of the most fundamental and most conserved pathways among gram-negative bacteria, this study showed that gene duplications as well as partial or complete losses of the genes encoding these enzymes have happened multiple times independently during bacterial evolution. each group of bacteria took advantage of such evolutionary events to optimize the pathway and adapted to their specialized life style. the most optimized form of the pathway is found in the proteobacteria lineage, especially among gammaproteobacteria. the nine-enzyme pathway currently known for the kdo2-lipid a biosynthesis, which is mainly studied in e. coli and related bacteria, is the most optimized, derived form of this pathway. 
in order to elucidate the evolutionary pathway of kdo2-lipid a biosynthesis, we examined the distribution of genes encoding the nine enzymes across bacteria. it anchors the lps into the outer membrane of most gram-negative bacteria. we found that not all gram-negative bacteria have all nine enzymes. some gram-negative bacteria have no genes encoding these enzymes and others have genes only for the first four enzymes. the nine-enzyme pathway, which was established based on the studies mainly in escherichia coli k appears to be the most derived and optimized form. lipid a is the highly immunoreactive endotoxic center of lipopolysaccharide. the kdo2-lipid a biosynthetic pathway exemplifies extremely plastic evolution of bacterial genomes, especially those of proteobacteria, and how these mainly pathogenic bacteria have adapted to their environment. two of such events happened within the proteobacteria lineage, followed by functional specialization of the duplicated genes and pathway optimization in these bacteria. it is found only in e. coli and related proteobacteria.
 lipid a is the highly immunoreactive endotoxic center of lipopolysaccharide. it anchors the lps into the outer membrane of most gram-negative bacteria. lipid a can be recognized by animal cells, triggers defense-related responses, and causes gram-negative sepsis. the biosynthesis of kdo2-lipid a, the lps substructure, involves with nine enzymatic steps.  in order to elucidate the evolutionary pathway of kdo2-lipid a biosynthesis, we examined the distribution of genes encoding the nine enzymes across bacteria. we found that not all gram-negative bacteria have all nine enzymes. some gram-negative bacteria have no genes encoding these enzymes and others have genes only for the first four enzymes. among the nine enzymes, five appeared to have arisen from three independent gene duplication events. two of such events happened within the proteobacteria lineage, followed by functional specialization of the duplicated genes and pathway optimization in these bacteria.  the nine-enzyme pathway, which was established based on the studies mainly in escherichia coli k appears to be the most derived and optimized form. it is found only in e. coli and related proteobacteria. simpler and probably less efficient pathways are found in other bacterial groups, with kdo2-lipid a variants as the likely end products. the kdo2-lipid a biosynthetic pathway exemplifies extremely plastic evolution of bacterial genomes, especially those of proteobacteria, and how these mainly pathogenic bacteria have adapted to their environment. 