BACKGROUND
motile archaeal species swim by means of rotating flagellum structures driven by a proton gradient force  <cit> , as in bacteria  <cit> .
interestingly, although they are both responsible for swimming, archaeal and bacterial flagella are not homologous, either in terms of overall structure, components and assembly .
the bacterial flagellum is a complex rotary structure made up of as much as  <dig> proteins and composed of three major parts -the basal body, the hook, and the filament.
rotation is provided by an atpase exploiting a proton gradient force, and can be switched by specific proteins in response to attractants or repellents in the environment through the chemotaxis system.
the filament is a hollow structure about  <dig> nm in diameter and is composed of a single type of protein called flagellin.
bacterial flagellins are assembled by a complex type iii secretion system located in the basal body and are added to the distal tip of the flagellum after passing through the hollow cavity  <cit> .
much less is known about the archaeal flagellum.
it has been extensively studied in terms of components, assembly, and mutation experiments, in halobacteria and methanococcales .
the archaeal flagellum is a structure thinner than its bacterial counterpart, where at least a filament and a hook are evident  <cit> .
the archaeal flagellum has been shown to have a unique symmetry in halobacterium salinarium.
in fact, it has  <dig>  subunits/turn of a  <dig>  nm pitch left-handed helix compared to  <dig>  subunits/turn of a  <dig>  nm pitch right-handed helix for plain bacterial flagellum filaments  <cit> .
the archaeal filament can be made up of different types of homologous flagellin proteins .
the filament is ~ <dig> nm in diameter and is not hollow, resembling more to bacterial type iv pili in this respect  <cit> .
a few other characteristics of archaeal flagella make them more alike bacterial pili than flagella: as bacterial pilins, archaeal flagellins  are made as preproteins with short signal peptides that are processed by a recently identified archaeal-specific signal peptidase   <cit>  that shows weak sequence similarity with the bacterial pili leader peptidase pild,  are likely added at the base of the filament as in bacterial pili, and  undergo glycosylation as post-translational modification  <cit>  .
moreover, one component of archaeal flagella  is homologous to bacterial pilt, an atpase involved in bacterial pilin export  and pilus retraction during twitching motility  <cit> .
however, none of the remaining archaeal flagellum components are homologous to those of bacterial pili  <cit> .
moreover, bacterial pili are not rotating structures, and no specific anchoring structures have ever been observed, indicating substantial differences between these two cellular structures.
a number of putative flagellum accessory genes lie close to flagellin genes in archaeal genomes   <cit> .
their putative role in flagellum structure and assembly was tentatively deduced based on their sequences, cellular location, and mutation experiments  <cit> .
flac, flad, flah and flai are associated with the membrane fraction in methanococcus voltae and may thus be peripheral components of the archaeal flagellum  <cit> .
flah, flai and flaj may be important for the assembly of archaeal flagella and possibly form a secretion complex  <cit> .
flah harbors a domain similar to that found in bacterial reca-like atpases, and flah mutants are nonmotile and nonflagellated  <cit> .
flaj contains many transmembrane domains, while flai probably encodes an atpase that may be important for flagellins export, similarly to the role of its bacterial homologue, the pilin export atpase pilt, and/or for providing the force for rotation.
no experimental data are presently available for flag and flaf, although flag may be a component of the anchoring system between the hook and the filament  <cit> .
it may be possible that some of the multiple flagellin proteins have different roles in flagellum substructures other than the filament  <cit> .
finally, additional components of the archaeal flagellum may be encoded by genes that have not yet been identified.
the uniqueness of the archaeal flagellum in terms of components, structure, and assembly indicates that archaeal and bacterial flagella have distinct origins .
interestingly, homologues of most bacterial chemotaxis genes are found in archaeal genomes  <cit> , suggesting that archaeal and bacterial chemotaxis systems are evolutionary related.
however, their interaction with the flagellum system in archaea remains largely unknown .
in this work, we sought to contribute to the research on archaeal flagella and archaeal motility in general performing an accurate phylogenomic study  of the archaeal motility apparatus in terms of taxonomic distribution of the genes coding for its components, their genomic context, and their phylogeny.
this allowed us to sketch a fairly detailed image on the origin and evolution of this macromolecular structure.
RESULTS
taxonomic distribution and genomic context
the taxonomic distribution of the genes coding for archaeal flagellum components is congruent with that presented in a recent review <cit>  and is generally consistent with species descriptions  <cit> .
gene homologues for all components of the archaeal flagellum are found in the complete genomes of archaea that are described as motile  <cit>  .
conversely, no homologues of genes coding archaeal flagellum proteins are found in the complete genomes of archaea that are described as non motile  <cit>  .
although the representative of the methanosarcina genus  are described as non-motile  <cit> , at least a full complement of homologues of the genes coding for archaeal flagellum components is present in the complete genomes of these species  <cit>  .
conversely, no homologues of the genes coding for archaeal flagellum components are found in the complete genome of pyrobaculum aerophilum  <cit>  .
this is surprising since the genus pyrobaculum is described as "motile due to flagellation" in the bergey's manual and a picture is included of a "platinum shadowed cell showing flagella of p. aerophilum"  <cit> .
similarly, no homologues of the genes coding for archaeal flagellum components are present in the complete genome of methanopyrus kandleri  <cit>  , which is described as motile in the bergey's manual  <cit> .
in all archaeal genomes harboring flagellum components, the corresponding genes are always organized into one or two very well conserved clusters  <cit>  .
the only exception is the gene coding for the preflagellin peptidase flak, which is located close to the fla cluster in methanococcus jannaschii only  <cit> .
flak homologues are nevertheless always present, at least in single copy at different locations in the other archaeal genomes, to the notable exception of aeropyrum pernix, thermoplasma acidophilum and thermoplasma volcanium.
we verified that these species do not harbor any homologue of pild -the bacterial prepilin peptidase- that they may have recruited by horizontal gene transfer, and how they cope with the absence of this enzymatic activity  remains puzzling.
a careful observation of gene order within each cluster revealed two types of organizations, that we will hereafter call fla <dig> and fla <dig> .
fla <dig> clusters are characterized by the presence of flac, plus one or few copies of flad , or by a fusion of flac and flad , whereas fla <dig> clusters lack these genes .
nevertheless, psi-blast searches revealed that the genes  lying between flab and flag in fla <dig> clusters from methanomicrobia  and archaeoglobales may be a very distant homologue of flad, as already suggested  <cit> .
a second characteristic differentiates fla <dig> and fla <dig> clusters: they show an inverted order of flag and flaf.
while the gene order in fla <dig> clusters is flab-flac-flad-flaf-flag-flah-flai-flaj, fla <dig> clusters  display the order flab-flag-flaf-flah-flai-flaj .
interestingly, all the archaeal genomes contain only a single type of fla clusters , except m. burtonii that is the only species that harbors both type i and ii fla clusters.
fla <dig> clusters are present only in euryarchaeota: thermococcales , methanococcales , thermoplasmatales , halobacteriales , and methanomicrobia ; whereas fla <dig> clusters are present in crenarchaeota: desulfurococcales  and sulfolobales  and in some euryarchaeota: methanomicrobia  and the archaeoglobale archaeoglobus fulgidus .
in halobacteriales and sulfolobales the clusters also include non-flagellum genes .
in particular, a gene coding for a homologue of a bacterial chemotaxis component  is present in halobacterium sp., and three genes coding for components of the chemotaxis system are present in h. marismortui  and in n. pharaonis .
in this archaeon, the operon is disrupted, the second half lying at ~ <dig> orfs downstream .
interestingly,m.
mazei and m. acetivorans each harbor two copies of the fla <dig> cluster , that differ in the number of flagellin gene copies , and in the presence of two different hypothetical genes  lying in between the genes coding for flab and flag .
according to these characteristics, the a. fulgidus, the m. hungatei and one of the m. burtonii gene clusters resemble more to the fla2a cluster, while that from m. barkeri resembles more to the fla2b cluster .
multiple copies of flagellin genes  are found in most archaeal genomes, especially in thermococcales, whereas thermoplasmatales and sulfolobales harbor single gene copies , confirming earlier studies on the composition of the flagella from t. volcanium and s. shibatae  <cit> .
in m. hungatei, the flagellin genes lie in another region of the chromosome ), suggesting a disruption of the original cluster.
this is also the case of the fla <dig> cluster of m. burtonii, which presents no nearby flagellin genes .
in s. solfataricus a transposase disrupts the gene coding for flag .
however, both the n- and c-ter sequences of flag are still very similar to flag homologues found in s. solfataricus close relatives , suggesting that the disruption of flag is recent.
indeed, the sequenced genome of s. solfataricus presents indeed a high number of insertion elements that may have recently invaded this strain  <cit> .
interestingly, cells of this strain appear non-flagellated under the electron microscope  even if the disruption of the flag gene does not affect the transcription of the downstream operon genes  <cit> .
this suggests that flag  is an essential flagellum component.
finally, additional copies of flagellum components lie in a few instances outside of the clusters  .
phylogenetic analysis
phylogenetic analyses were performed on six amino acid sequence datasets corresponding to flaa/b, flad/e, flag, flah, flai, and flaj.
phylogenetic analysis of flac, flaf and flak could not be performed due to a too restricted phylogenetic distribution of flac, and the poor sequence conservation of flaf and flak.
flag, flah, flai, flaj
among all archaeal flagellum components, flah, flai and flaj are the most conserved at the sequence level and always lie close to each other in all the analyzed genomes  strengthening their likely fundamental role in flagellum assembly and function .
flai has a number of bacterial homologues belonging to type iv and type ii secretion systems, including the typeiv pili component pilt  <cit> .
moreover, flai has additional archaeal homologues that are also probably part of yet to describe secretion machineries  <cit> .
in a phylogeny including all these homologues flai sequences form a monophyletic group and are most closely related to their archaeal counterparts .
flah shares a reca-like atpase domain with distant archaeal and bacterial homologues that are not involved in motility structures.
psi-blast searches revealed  that flaj harbours a few distant archaeal homologues annotated as involved in type ii secretion and  weak similarities with the bacterial pilus assembly protein tadc and tadb.
flaj shares the domain gspii f with tadc and tadb.
however, this may not be significant, given that the domain was defined on the basis of an alignment that included both archaeal and bacterial sequences.
the similarity between the flaj sequences and tadb and tadc sequences is very weak  and is mainly the result of the sharing of small hydrophobic amino acids.
to our point of view this sequence similarity is too weak to definitively conclude that these sequences are homologues although this has been claimed  <cit> .
after removal of ambiguously aligned positions,  <dig>   <dig>   <dig> and  <dig> amino acids could be kept for phylogenetic analysis of flag, flah, flai, and flaj, respectively.
the resulting trees are strikingly congruent .
notably, all major archaeal groups except methanomicrobia  are well defined and strongly supported statistically , suggesting that no recent horizontal transfer of flah, flag, flai and flaj genes occurred across these groups.
the sequences from methanomicrobia form a well supported cluster  that also includes sequences from a. fulgidus.
it is not possible for the time being to decide whether this is due to a hgt or a hidden paralogy.
importantly, the corresponding trees are also strongly consistent with gene cluster organization.
in fact, homologues from fla <dig> and fla <dig> clusters  form two distinct strongly groups .
in particular, in all four trees, the homologues from the fla <dig> clusters of m. hungatei, the four methanosarcinales and a. fulgidus appear close to crenarchaeota .
this is in contrast to their expected position as sister-group of halobacteriales within euryarchaeota .
interestingly, such expected position is shown by the m. burtonii sequences belonging to its fla <dig> cluster .
this suggests that the fla <dig> and fla <dig> clusters from m. burtonii have different origins .
independent species-specific duplications of flag appear to have occurred in halobacterium sp., n. pharaonis, and m. hungatei .
moreover, in the flah, flai and flaj phylogenies, the sequences from m. burtonii, m. hungatei, m. barkeri and a. fulgidus fla <dig> clusters group with the sequences belonging to the fla2b cluster from m. mazei and m. acetivorans , supporting a close relationship of these clusters, as suggested by their gene organization .
flad/e
as discussed above, homologues of flad/e genes are missing in all fla <dig> clusters from crenarchaea.
in fla <dig> clusters from methanomicrobia and a. fulgidus the two hypothetical proteins hyp <dig> and hyp <dig> could be distantly related to flad/e .
however they are too distant to be included in any phylogenetic analysis.
the small number of unambiguously aligned positions  that could be kept for phylogenetic analysis gives a poor resolution of the relationships between major euryarchaeal groups .
however, sequences from phyla form monophyletic groups generally well supported .
this indicates that, similarly to flag, flai, flaj, and flah, no recent hgt involving the flad/e gene occurred among these groups.
interestingly, a tandem duplication event appears to have occurred in an ancestor of methanococcales, with the two copies having been conserved within the cluster.
halobacteriales also harbor two copies of flad.
one of the two copies is fused with flac and always resides within the fla cluster, whereas the second copy resides within the fla cluster only in halobacterium sp.
this suggests that the fusion of flac and flad genes occurred before the divergence of the three halobacteriales, but after the duplication event and that one of the two copies was displaced after the duplication event in the ancestor of h. marismortui and n. pharaonis.
a similar duplication of flad followed by a fusion of one of the two copies of flad and flac also appears to have occurred in m. burtonii.
as in most halobacteriales one of the two copies resides outside the fla cluster  suggesting its displacement after the duplication event.
the poor resolution of the relationships between groups  does not permit to determine if a single fusion event of flac and flad occurred in ancestor of halobacteriales and methanomicrobia or if this event occurred two times independently in both lineages.
flab
given the use of only  <dig> unambiguously aligned positions for analysis, the flab tree presents a number of poorly resolved nodes .
nevertheless, the monophyly of a number of groups is recovered and supported by bv > 850‰, except for thermococcales, methanococcales and methanomicrobia.
as for flag, flai, flaj, and flah, the flab tree is again strongly consistent with gene cluster organization .
in particular, the flab from the fla <dig> clusters of m. hungatei, the four methanosarcinales and a. fulgidus appear close to crenarchaeota, while the isolated flab from m. burtonii appear close to halobacteria, and thus likely functions with the flagellum components of fla <dig> cluster .
interestingly, the multiple copies of flagellins group in a group-specific manner , suggesting that flagellin gene family expansion occurred mainly by gene duplication and multiple times independently in different species, and not by hgt.
discussion and
CONCLUSIONS
the archaeal flagellum is a complex cellular structure composed of multiple subunits and anchored to the membrane.
the striking conservation of the genomic context of the genes coding for these subunits indicates a likely highly coordinated expression and assembly mechanisms.
coupled to the high sequence conservation of the different subunits across archaeal species inhabiting very different habitats, this underlines the importance for structural maintenance of the archaeal flagellum.
however, we highlighted an important dimorphism of the genetic context organization, with two types of gene clusters harboring differences in both gene content and gene order .
in fact, most euryarchaea exhibit the fla <dig> cluster, whereas all the crenarchaea and some euryarchaea have the fla <dig> cluster .
the difference between the two clusters is strongly supported by phylogenetic analysis, and indicates that methanomicrobia and a. fulgidus flagellum components encoded by fla <dig> gene clusters are more closely related to their crenarchaeal homologues than to the homologues encoded by the fla <dig> gene cluster of their close relatives .
two different evolutionary scenarios can be proposed to explain our results.
in the first scenario , the last ancestor of archaea was flagellated and had the two types of clusters , resulting from the duplication of an ancestral cluster , and these were secondarily and differently lost during archaeal lineages evolution .
importantly, some of these losses would have also occurred recently in the euryarchaea .
finally, a duplication event of the whole fla <dig> cluster occurred in an ancestor of methanosarcina  leading to the fla2b cluster .
this first scenario involves  <dig> losses, and implies that the ancestor of archaea and the ancestor of each euryarchaeal group had two types of flagella.
moreover, it does not explain the position of a. fulgidus sequences within the methanomicrobia group and not as sister of this group, as generally indicated by molecular phylogeny of multiple markers .
a second scenario involves less losses  .
here, the ancestor of archaea was also flagellated, but had only a single type of fla cluster .
after the divergence of crenarchaea and euryarchaea , evolution led to the fla <dig> and fla <dig> clusters.
a single hgt of the fla <dig> cluster would have then occurred from some ancestors of sulfolobales and desulfurococcales to an ancestor of methanomicrobia , and was followed by a hgt from some ancestors of methanosarcinales to a. fulgidus .
methanosarcina, m. hungatei and a. fulgidus would thus have lost their original fla <dig> gene cluster before or after their replacement by a fla <dig> gene cluster of crenarchaeal origin.
we favor a hgt from crenarchaea to methanomicrobia rather than the opposite, since in this case we would expect to find the m. burtonii fla <dig> genes more closely related to their paralogues belonging to the fla <dig> cluster than to their fla <dig> orthologues from halobacteriales.
both scenarios imply that the archaeal flagellum would have appeared prior to the last archaeal ancestor
apart these two likely cases of hgt of the entire fla <dig> gene cluster, we found no clear evidence for recent transfers of the genes coding for flagellum components among archaeal species.
indeed, the poor resolution of inter-phyla relationships in some trees is more likely due to lack of phylogenetic signal rather than horizontal gene transfer.
one explanation for the rarity of hgt is that it is possible the result of the high level of integration of flagellum components within the macromolecular structure.
importantly, this contradicts the generally assumed notion that hgt of "informational" genes  are less frequent than those involving the remaining ones  genes.
nevertheless, the acquisition of a whole flagellum component coding gene cluster from distant donors seems possible.
interestingly, even if two clusters coexist within a genome  neither gene recombination is observed between homologous genes belonging to the two clusters, nor losses, suggesting that the components of a cluster may interact preferentially due to coevolution, although they form similar cellular structures.
the presence in m. burtonii of two types of fla clusters  represents an interesting experimental model to study.
it would be in fact particular interesting to know the difference between the components encoded by the two fla clusters in terms of expression and assembly, and how two different flagellum systems coexist in this archaeon.
finally, it has been suggested that archaea are modified actinobacteria and that archaeal flagella are derived from bacterial flagella following an adaptation to acidic environments by the recruitment of an already acid-stable glycoprotein from the pilus machinery that would have replaced the original flagellin while leaving intact the basal rotary motor  <cit> .
we find this hypothesis unlikely for the fact that archaeal flagella do not resemble to bacterial flagella in major structure, assembly, and components, and not only concerning flagellin.
indeed, no even distant homologues to any component, including the basal rotary motor, of bacterial flagella can be recovered in archaeal genomes, including the related type iii secretion system components  <cit> .
moreover, flagella of acidophilic bacteria  show a typical bacterial structure , so they adapted to acidic conditions without radically modifying their motility structures and components  <cit> .
indeed, the uncomplete genome of the extreme acidophilic bacterium  acidobacterium capsulatum contains a clear homologue of bacterial flagellin, indicating that adaptation to acidic environments was possible without its replacement.
the lack of congruence between the description of archaeal species as motile or non-motile and the taxonomic distribution of homologues of flagellum component coding genes <cit>  is particularly striking and underlines the need to explore more in depth the motility systems in archaea.
the presence of two gene clusters in non motile methanosarcinales is particularly puzzling.
either these species can be flagellated under particular conditions that have not yet been tested, or the flagellum component homologues are involved in other functions than motility .
it will be extremely interesting to study the expression and localization of the flagellum components in methanosarcinales, in the light of the fact that the flagellum genes of methanosarcinales may have been recruited from the distantly related crenarchaeota, since this event may have been an important step in the evolution of this archaeal lineage.
moreover, virtually nothing is known about other types of motility than swimming in archaea  <cit> , while in bacteria these are starting being investigated  <cit> .
the fact that no homologues of flagellum components are encoded in the genomes of at least two archaeal species that are described as motile  is also puzzling.
although it is possible that the strains used for genome sequencing have lost the flagellum operon following lab cultivation .
for each dataset, additional searches using psi-blast were performed to search for divergent homologues   <cit> .
tblastn searches on the unfinished genomes of the halobacteriale haloferax volcanii were performed at tigr  <cit> .
multiple alignments were done with clustalw  <cit>  and muscle  <cit> .
for each dataset, the quality of the alignments obtained with clustalw and muscle, was evaluated with t-coffee   <cit> .
all the alignments were edited and manually improved using the ed program from the must package  <cit> .
regions where the homology between sites was doubtful were manually removed from the datasets for phylogenetic analyses.
phylogenetic analysis
maximum likelihood  phylogenetic trees were computed with phyml  <cit>  and the jjt model of amino acid substitution  using phyml.
in addition, bayesian analyses were performed using mrbayes  <cit>  with a mixed model of amino acid substitution.
as for ml tree reconstruction, a gamma correction with  <dig> discrete classes of sites was used and the alpha parameter and the proportion of invariable sites were estimated.
mrbayes was run with four chains for  <dig> million generations and trees were sampled every  <dig> generations.
to construct the consensus tree, the first  <dig> trees were discarded as "burnin".
genomic context analysis
the genomic localization of each archaeal flagellum component was manually investigated in all archaeal complete genomes available at the ncbi.
authors' contributions
sg conceived the study and supervised the analyses.
ed carried out the analyses.
cb and sg refined and completed the analyses and wrote the manuscript.
all authors read and approved the final manuscript.

 motile archaeal species swim by means of rotating flagellum structures driven by a proton gradient force, as in bacteria. interestingly, although they are both responsible for swimming, archaeal and bacterial flagella are not homologous, either in terms of overall structure, components and assembly. the bacterial flagellum is a complex rotary structure made up of as much as proteins and composed of three major parts -the basal body, the hook, and the filament. rotation is provided by an atpase exploiting a proton gradient force, and can be switched by specific proteins in response to attractants or repellents in the environment through the chemotaxis system. the filament is a hollow structure about nm in diameter and is composed of a single type of protein called flagellin. bacterial flagellins are assembled by a complex type iii secretion system located in the basal body and are added to the distal tip of the flagellum after passing through the hollow cavity. much less is known about the archaeal flagellum. it has been extensively studied in terms of components, assembly, and mutation experiments, in halobacteria and methanococcales. the archaeal flagellum is a structure thinner than its bacterial counterpart, where at least a filament and a hook are evident. the archaeal flagellum has been shown to have a unique symmetry in halobacterium salinarium. in fact, it has subunits/turn of a nm pitch left-handed helix compared to subunits/turn of a nm pitch right-handed helix for plain bacterial flagellum filaments. the archaeal filament can be made up of different types of homologous flagellin proteins. the filament is ~ nm in diameter and is not hollow, resembling more to bacterial type iv pili in this respect. a few other characteristics of archaeal flagella make them more alike bacterial pili than flagella: as bacterial pilins, archaeal flagellins are made as preproteins with short signal peptides that are processed by a recently identified archaeal-specific signal peptidase that shows weak sequence similarity with the bacterial pili leader peptidase pild, are likely added at the base of the filament as in bacterial pili, and undergo glycosylation as post-translational modification. moreover, one component of archaeal flagella is homologous to bacterial pilt, an atpase involved in bacterial pilin export and pilus retraction during twitching motility. however, none of the remaining archaeal flagellum components are homologous to those of bacterial pili. moreover, bacterial pili are not rotating structures, and no specific anchoring structures have ever been observed, indicating substantial differences between these two cellular structures. a number of putative flagellum accessory genes lie close to flagellin genes in archaeal genomes. their putative role in flagellum structure and assembly was tentatively deduced based on their sequences, cellular location, and mutation experiments. flac, flad, flah and flai are associated with the membrane fraction in methanococcus voltae and may thus be peripheral components of the archaeal flagellum. flah, flai and flaj may be important for the assembly of archaeal flagella and possibly form a secretion complex. flah harbors a domain similar to that found in bacterial reca-like atpases, and flah mutants are nonmotile and nonflagellated. flaj contains many transmembrane domains, while flai probably encodes an atpase that may be important for flagellins export, similarly to the role of its bacterial homologue, the pilin export atpase pilt, and/or for providing the force for rotation. no experimental data are presently available for flag and flaf, although flag may be a component of the anchoring system between the hook and the filament. it may be possible that some of the multiple flagellin proteins have different roles in flagellum substructures other than the filament. finally, additional components of the archaeal flagellum may be encoded by genes that have not yet been identified. the uniqueness of the archaeal flagellum in terms of components, structure, and assembly indicates that archaeal and bacterial flagella have distinct origins. interestingly, homologues of most bacterial chemotaxis genes are found in archaeal genomes, suggesting that archaeal and bacterial chemotaxis systems are evolutionary related. however, their interaction with the flagellum system in archaea remains largely unknown. in this work, we sought to contribute to the research on archaeal flagella and archaeal motility in general performing an accurate phylogenomic study of the archaeal motility apparatus in terms of taxonomic distribution of the genes coding for its components, their genomic context, and their phylogeny. this allowed us to sketch a fairly detailed image on the origin and evolution of this macromolecular structure.  taxonomic distribution and genomic context the taxonomic distribution of the genes coding for archaeal flagellum components is congruent with that presented in a recent review and is generally consistent with species descriptions. gene homologues for all components of the archaeal flagellum are found in the complete genomes of archaea that are described as motile. conversely, no homologues of genes coding archaeal flagellum proteins are found in the complete genomes of archaea that are described as non motile. although the representative of the methanosarcina genus are described as non-motile, at least a full complement of homologues of the genes coding for archaeal flagellum components is present in the complete genomes of these species. conversely, no homologues of the genes coding for archaeal flagellum components are found in the complete genome of pyrobaculum aerophilum. this is surprising since the genus pyrobaculum is described as "motile due to flagellation" in the bergey's manual and a picture is included of a "platinum shadowed cell showing flagella of p. aerophilum". similarly, no homologues of the genes coding for archaeal flagellum components are present in the complete genome of methanopyrus kandleri, which is described as motile in the bergey's manual. in all archaeal genomes harboring flagellum components, the corresponding genes are always organized into one or two very well conserved clusters. the only exception is the gene coding for the preflagellin peptidase flak, which is located close to the fla cluster in methanococcus jannaschii only. flak homologues are nevertheless always present, at least in single copy at different locations in the other archaeal genomes, to the notable exception of aeropyrum pernix, thermoplasma acidophilum and thermoplasma volcanium. we verified that these species do not harbor any homologue of pild -the bacterial prepilin peptidase- that they may have recruited by horizontal gene transfer, and how they cope with the absence of this enzymatic activity remains puzzling. a careful observation of gene order within each cluster revealed two types of organizations, that we will hereafter call fla and fla. fla clusters are characterized by the presence of flac, plus one or few copies of flad, or by a fusion of flac and flad, whereas fla clusters lack these genes. nevertheless, psi-blast searches revealed that the genes lying between flab and flag in fla clusters from methanomicrobia and archaeoglobales may be a very distant homologue of flad, as already suggested. a second characteristic differentiates fla and fla clusters: they show an inverted order of flag and flaf. while the gene order in fla clusters is flab-flac-flad-flaf-flag-flah-flai-flaj, fla clusters display the order flab-flag-flaf-flah-flai-flaj. interestingly, all the archaeal genomes contain only a single type of fla clusters, except m. burtonii that is the only species that harbors both type i and ii fla clusters. fla clusters are present only in euryarchaeota: thermococcales, methanococcales, thermoplasmatales, halobacteriales, and methanomicrobia; whereas fla clusters are present in crenarchaeota: desulfurococcales and sulfolobales and in some euryarchaeota: methanomicrobia and the archaeoglobale archaeoglobus fulgidus. in halobacteriales and sulfolobales the clusters also include non-flagellum genes. in particular, a gene coding for a homologue of a bacterial chemotaxis component is present in halobacterium sp., and three genes coding for components of the chemotaxis system are present in h. marismortui and in n. pharaonis. in this archaeon, the operon is disrupted, the second half lying at ~ orfs downstream. interestingly,m. mazei and m. acetivorans each harbor two copies of the fla cluster, that differ in the number of flagellin gene copies, and in the presence of two different hypothetical genes lying in between the genes coding for flab and flag. according to these characteristics, the a. fulgidus, the m. hungatei and one of the m. burtonii gene clusters resemble more to the fla2a cluster, while that from m. barkeri resembles more to the fla2b cluster. multiple copies of flagellin genes are found in most archaeal genomes, especially in thermococcales, whereas thermoplasmatales and sulfolobales harbor single gene copies, confirming earlier studies on the composition of the flagella from t. volcanium and s. shibatae. in m. hungatei, the flagellin genes lie in another region of the chromosome ), suggesting a disruption of the original cluster. this is also the case of the fla cluster of m. burtonii, which presents no nearby flagellin genes. in s. solfataricus a transposase disrupts the gene coding for flag. however, both the n- and c-ter sequences of flag are still very similar to flag homologues found in s. solfataricus close relatives, suggesting that the disruption of flag is recent. indeed, the sequenced genome of s. solfataricus presents indeed a high number of insertion elements that may have recently invaded this strain. interestingly, cells of this strain appear non-flagellated under the electron microscope even if the disruption of the flag gene does not affect the transcription of the downstream operon genes. this suggests that flag is an essential flagellum component. finally, additional copies of flagellum components lie in a few instances outside of the clusters. phylogenetic analysis phylogenetic analyses were performed on six amino acid sequence datasets corresponding to flaa/b, flad/e, flag, flah, flai, and flaj. phylogenetic analysis of flac, flaf and flak could not be performed due to a too restricted phylogenetic distribution of flac, and the poor sequence conservation of flaf and flak. flag, flah, flai, flaj among all archaeal flagellum components, flah, flai and flaj are the most conserved at the sequence level and always lie close to each other in all the analyzed genomes strengthening their likely fundamental role in flagellum assembly and function. flai has a number of bacterial homologues belonging to type iv and type ii secretion systems, including the typeiv pili component pilt. moreover, flai has additional archaeal homologues that are also probably part of yet to describe secretion machineries. in a phylogeny including all these homologues flai sequences form a monophyletic group and are most closely related to their archaeal counterparts. flah shares a reca-like atpase domain with distant archaeal and bacterial homologues that are not involved in motility structures. psi-blast searches revealed that flaj harbours a few distant archaeal homologues annotated as involved in type ii secretion and weak similarities with the bacterial pilus assembly protein tadc and tadb. flaj shares the domain gspii f with tadc and tadb. however, this may not be significant, given that the domain was defined on the basis of an alignment that included both archaeal and bacterial sequences. the similarity between the flaj sequences and tadb and tadc sequences is very weak and is mainly the result of the sharing of small hydrophobic amino acids. to our point of view this sequence similarity is too weak to definitively conclude that these sequences are homologues although this has been claimed. after removal of ambiguously aligned positions, and amino acids could be kept for phylogenetic analysis of flag, flah, flai, and flaj, respectively. the resulting trees are strikingly congruent. notably, all major archaeal groups except methanomicrobia are well defined and strongly supported statistically, suggesting that no recent horizontal transfer of flah, flag, flai and flaj genes occurred across these groups. the sequences from methanomicrobia form a well supported cluster that also includes sequences from a. fulgidus. it is not possible for the time being to decide whether this is due to a hgt or a hidden paralogy. importantly, the corresponding trees are also strongly consistent with gene cluster organization. in fact, homologues from fla and fla clusters form two distinct strongly groups. in particular, in all four trees, the homologues from the fla clusters of m. hungatei, the four methanosarcinales and a. fulgidus appear close to crenarchaeota. this is in contrast to their expected position as sister-group of halobacteriales within euryarchaeota. interestingly, such expected position is shown by the m. burtonii sequences belonging to its fla cluster. this suggests that the fla and fla clusters from m. burtonii have different origins. independent species-specific duplications of flag appear to have occurred in halobacterium sp., n. pharaonis, and m. hungatei. moreover, in the flah, flai and flaj phylogenies, the sequences from m. burtonii, m. hungatei, m. barkeri and a. fulgidus fla clusters group with the sequences belonging to the fla2b cluster from m. mazei and m. acetivorans, supporting a close relationship of these clusters, as suggested by their gene organization. flad/e as discussed above, homologues of flad/e genes are missing in all fla clusters from crenarchaea. in fla clusters from methanomicrobia and a. fulgidus the two hypothetical proteins hyp and hyp could be distantly related to flad/e. however they are too distant to be included in any phylogenetic analysis. the small number of unambiguously aligned positions that could be kept for phylogenetic analysis gives a poor resolution of the relationships between major euryarchaeal groups. however, sequences from phyla form monophyletic groups generally well supported. this indicates that, similarly to flag, flai, flaj, and flah, no recent hgt involving the flad/e gene occurred among these groups. interestingly, a tandem duplication event appears to have occurred in an ancestor of methanococcales, with the two copies having been conserved within the cluster. halobacteriales also harbor two copies of flad. one of the two copies is fused with flac and always resides within the fla cluster, whereas the second copy resides within the fla cluster only in halobacterium sp. this suggests that the fusion of flac and flad genes occurred before the divergence of the three halobacteriales, but after the duplication event and that one of the two copies was displaced after the duplication event in the ancestor of h. marismortui and n. pharaonis. a similar duplication of flad followed by a fusion of one of the two copies of flad and flac also appears to have occurred in m. burtonii. as in most halobacteriales one of the two copies resides outside the fla cluster suggesting its displacement after the duplication event. the poor resolution of the relationships between groups does not permit to determine if a single fusion event of flac and flad occurred in ancestor of halobacteriales and methanomicrobia or if this event occurred two times independently in both lineages. flab given the use of only unambiguously aligned positions for analysis, the flab tree presents a number of poorly resolved nodes. nevertheless, the monophyly of a number of groups is recovered and supported by bv > 850‰, except for thermococcales, methanococcales and methanomicrobia. as for flag, flai, flaj, and flah, the flab tree is again strongly consistent with gene cluster organization. in particular, the flab from the fla clusters of m. hungatei, the four methanosarcinales and a. fulgidus appear close to crenarchaeota, while the isolated flab from m. burtonii appear close to halobacteria, and thus likely functions with the flagellum components of fla cluster. interestingly, the multiple copies of flagellins group in a group-specific manner, suggesting that flagellin gene family expansion occurred mainly by gene duplication and multiple times independently in different species, and not by hgt. discussion and  the archaeal flagellum is a complex cellular structure composed of multiple subunits and anchored to the membrane. the striking conservation of the genomic context of the genes coding for these subunits indicates a likely highly coordinated expression and assembly mechanisms. coupled to the high sequence conservation of the different subunits across archaeal species inhabiting very different habitats, this underlines the importance for structural maintenance of the archaeal flagellum. however, we highlighted an important dimorphism of the genetic context organization, with two types of gene clusters harboring differences in both gene content and gene order. in fact, most euryarchaea exhibit the fla cluster, whereas all the crenarchaea and some euryarchaea have the fla cluster. the difference between the two clusters is strongly supported by phylogenetic analysis, and indicates that methanomicrobia and a. fulgidus flagellum components encoded by fla gene clusters are more closely related to their crenarchaeal homologues than to the homologues encoded by the fla gene cluster of their close relatives. two different evolutionary scenarios can be proposed to explain our results. in the first scenario, the last ancestor of archaea was flagellated and had the two types of clusters, resulting from the duplication of an ancestral cluster, and these were secondarily and differently lost during archaeal lineages evolution. importantly, some of these losses would have also occurred recently in the euryarchaea. finally, a duplication event of the whole fla cluster occurred in an ancestor of methanosarcina leading to the fla2b cluster. this first scenario involves losses, and implies that the ancestor of archaea and the ancestor of each euryarchaeal group had two types of flagella. moreover, it does not explain the position of a. fulgidus sequences within the methanomicrobia group and not as sister of this group, as generally indicated by molecular phylogeny of multiple markers. a second scenario involves less losses. here, the ancestor of archaea was also flagellated, but had only a single type of fla cluster. after the divergence of crenarchaea and euryarchaea, evolution led to the fla and fla clusters. a single hgt of the fla cluster would have then occurred from some ancestors of sulfolobales and desulfurococcales to an ancestor of methanomicrobia, and was followed by a hgt from some ancestors of methanosarcinales to a. fulgidus. methanosarcina, m. hungatei and a. fulgidus would thus have lost their original fla gene cluster before or after their replacement by a fla gene cluster of crenarchaeal origin. we favor a hgt from crenarchaea to methanomicrobia rather than the opposite, since in this case we would expect to find the m. burtonii fla genes more closely related to their paralogues belonging to the fla cluster than to their fla orthologues from halobacteriales. both scenarios imply that the archaeal flagellum would have appeared prior to the last archaeal ancestor apart these two likely cases of hgt of the entire fla gene cluster, we found no clear evidence for recent transfers of the genes coding for flagellum components among archaeal species. indeed, the poor resolution of inter-phyla relationships in some trees is more likely due to lack of phylogenetic signal rather than horizontal gene transfer. one explanation for the rarity of hgt is that it is possible the result of the high level of integration of flagellum components within the macromolecular structure. importantly, this contradicts the generally assumed notion that hgt of "informational" genes are less frequent than those involving the remaining ones genes. nevertheless, the acquisition of a whole flagellum component coding gene cluster from distant donors seems possible. interestingly, even if two clusters coexist within a genome neither gene recombination is observed between homologous genes belonging to the two clusters, nor losses, suggesting that the components of a cluster may interact preferentially due to coevolution, although they form similar cellular structures. the presence in m. burtonii of two types of fla clusters represents an interesting experimental model to study. it would be in fact particular interesting to know the difference between the components encoded by the two fla clusters in terms of expression and assembly, and how two different flagellum systems coexist in this archaeon. finally, it has been suggested that archaea are modified actinobacteria and that archaeal flagella are derived from bacterial flagella following an adaptation to acidic environments by the recruitment of an already acid-stable glycoprotein from the pilus machinery that would have replaced the original flagellin while leaving intact the basal rotary motor. we find this hypothesis unlikely for the fact that archaeal flagella do not resemble to bacterial flagella in major structure, assembly, and components, and not only concerning flagellin. indeed, no even distant homologues to any component, including the basal rotary motor, of bacterial flagella can be recovered in archaeal genomes, including the related type iii secretion system components. moreover, flagella of acidophilic bacteria show a typical bacterial structure, so they adapted to acidic conditions without radically modifying their motility structures and components. indeed, the uncomplete genome of the extreme acidophilic bacterium acidobacterium capsulatum contains a clear homologue of bacterial flagellin, indicating that adaptation to acidic environments was possible without its replacement. the lack of congruence between the description of archaeal species as motile or non-motile and the taxonomic distribution of homologues of flagellum component coding genes is particularly striking and underlines the need to explore more in depth the motility systems in archaea. the presence of two gene clusters in non motile methanosarcinales is particularly puzzling. either these species can be flagellated under particular conditions that have not yet been tested, or the flagellum component homologues are involved in other functions than motility. it will be extremely interesting to study the expression and localization of the flagellum components in methanosarcinales, in the light of the fact that the flagellum genes of methanosarcinales may have been recruited from the distantly related crenarchaeota, since this event may have been an important step in the evolution of this archaeal lineage. moreover, virtually nothing is known about other types of motility than swimming in archaea, while in bacteria these are starting being investigated. the fact that no homologues of flagellum components are encoded in the genomes of at least two archaeal species that are described as motile is also puzzling. although it is possible that the strains used for genome sequencing have lost the flagellum operon following lab cultivation. for each dataset, additional searches using psi-blast were performed to search for divergent homologues. tblastn searches on the unfinished genomes of the halobacteriale haloferax volcanii were performed at tigr. multiple alignments were done with clustalw and muscle. for each dataset, the quality of the alignments obtained with clustalw and muscle, was evaluated with t-coffee. all the alignments were edited and manually improved using the ed program from the must package. regions where the homology between sites was doubtful were manually removed from the datasets for phylogenetic analyses. phylogenetic analysis maximum likelihood phylogenetic trees were computed with phyml and the jjt model of amino acid substitution using phyml. in addition, bayesian analyses were performed using mrbayes with a mixed model of amino acid substitution. as for ml tree reconstruction, a gamma correction with discrete classes of sites was used and the alpha parameter and the proportion of invariable sites were estimated. mrbayes was run with four chains for million generations and trees were sampled every generations. to construct the consensus tree, the first trees were discarded as "burnin". genomic context analysis the genomic localization of each archaeal flagellum component was manually investigated in all archaeal complete genomes available at the ncbi. authors' contributions sg conceived the study and supervised the analyses. ed carried out the analyses. cb and sg refined and completed the analyses and wrote the manuscript. all authors read and approved the final manuscript.
as bacteria, motile archaeal species swim by means of rotating flagellum structures driven by a proton gradient force. in all complete archaeal genomes, the genes coding for flagellum components are co-localized in one or two well-conserved genomic clusters showing two different types of organizations. interestingly, experimental data have shown that the archaeal flagellum is non-homologous to the bacterial flagellum either in terms of overall structure, components and assembly. these support a history of mainly vertical inheritance for the components of this unique motility system, and an interesting possible ancient horizontal gene transfer event of a whole flagellum-coding gene cluster between euryarchaeota and crenarchaeota. we report here an exhaustive phylogenomic analysis of the components of the archaeal flagellum. we propose an evolutionary scenario for the evolution of the components of the archaeal flagellum. moreover, we show that the components of the archaeal flagellar system have not been frequently transferred among archaeal species, indicating that gene fixation following hgt can also be rare for genes encoding components of large macromolecular complexes with a structural role.
 as bacteria, motile archaeal species swim by means of rotating flagellum structures driven by a proton gradient force. interestingly, experimental data have shown that the archaeal flagellum is non-homologous to the bacterial flagellum either in terms of overall structure, components and assembly. the growing number of complete archaeal genomes now permits to investigate the evolution of this unique motility system.  we report here an exhaustive phylogenomic analysis of the components of the archaeal flagellum. in all complete archaeal genomes, the genes coding for flagellum components are co-localized in one or two well-conserved genomic clusters showing two different types of organizations. despite their small size, these genes harbor a good phylogenetic signal that allows reconstruction of their evolutionary histories. these support a history of mainly vertical inheritance for the components of this unique motility system, and an interesting possible ancient horizontal gene transfer event of a whole flagellum-coding gene cluster between euryarchaeota and crenarchaeota.  our study is one of the few exhaustive phylogenomics analyses of a non-informational cell machinery from the third domain of life. we propose an evolutionary scenario for the evolution of the components of the archaeal flagellum. moreover, we show that the components of the archaeal flagellar system have not been frequently transferred among archaeal species, indicating that gene fixation following hgt can also be rare for genes encoding components of large macromolecular complexes with a structural role. 