BACKGROUND
chitin is a biological polymer consisting of carbohydrate molecules bonded together to form long chains of polysaccharides.
unlike starch and glycogen that are storage polysaccharides in plants and animals respectively, chitin is a structural polysaccharide organised as crystalline microfibrils and with enormous tensile strength.
it contributes to the rigidity and integrity of cells, tissues or body surfaces in a wide range of organisms, protecting and giving them shape, as seen with cellulose or pectin in plant and algal cell walls.
so far, the presence of this structural polysaccharide has been mainly demonstrated in the cell walls of mycota, the exoskeleton of hexapoda or crustacea, and in the radula or beak of mollusca  <cit> , where it plays a major role in development and growth.
in addition, the presence and subcellular location of chitin in invertebrate hemocytes suggests another role for this polysaccharide in the immune system of diverse animals  <cit> .
it was generally thought that there was no chitin in vertebrates but this polymer has been described in several ray-finned fishes   <cit>  and in some amphibians  <cit> .
the role of endogenous chitin in the biology of these vertebrates remains elusive.
chitin has also been sporadically found in structures from a diverse range of eukaryotic microorganisms, such as the cell wall of a few chlorophyta , the cyst wall or lorica, of ciliophora , the theca of choanoflagellida , and the test or cyst wall, of amoebozoa   <cit> .
it is also present in the large family of heterokonta protists, for example in the cell wall of oomycota, the spines of diatomae and the stalk of chrysophyta  <cit> .
chitin is an hexosamine polymer composed of beta--linked linear chains of more than  <dig>  n-acetylglucosamine residues that are highly cross-linked with hydrogen bonds.
in insects, chitin is deposited exclusively on the apical sides of epithelial cells, facing the external environment   <cit> .
in fungal cell walls, there is a common fibrillar core composed of branched beta--glucan to which chitin and other polysaccharides are covalently bound.
chitin accounts for 1-2% of the cell wall mass in yeasts and up to 30% in molds  <cit> .
elongation of the chitin polymer is catalyzed by a highly conserved enzyme called chitin synthase, chs .
the chs enzyme belongs to the gt <dig> family of processive polymerizing glycosyltransferases which includes the synthases for cellulose, callose, curdlan, mannan, hyaluronate and alginate polymers  <cit> .
in fungi, chitin biosynthesis requires a set of multiple chs isoenzymes that are encoded by a multigenic family.
although they share a common central catalytic domain, chs isoenzymes from fungi and other species  can differ greatly in their n- and c-termini parts.
all chs utilize udp-n-acetylglucosamine  from the cytoplasm as a substrate and catalyze multiple transfers of the activated sugar donor to the non-reducing end of the growing chain.
multiple transmembrane domains are found in every chs protein and these probably form a channel in the cell membrane through which linear chitin is extruded into the extracellular space as it is described for cellulose biosynthesis  <cit> .
nevertheless, the exact mechanism by which chitin is assembled into microfibrils and cross-linked with other components of the cell wall is still poorly understood.
the importance of chs genes in fungal biology has been extensively investigated by reverse genetics.
mutants have been constructed by disrupting or deleting particular chs genes in fungal species and no less than a hundred mutants have been made so far in more than twenty fungi .
the multiplicity of chs genes in fungal genomes has necessitated their classification for comparative functional genomics, and fungal chs isoenzymes have been classified into multiple divisions and classes according to protein similarities in their catalytic domain.
more than  <dig> phylogenetic analyses have been published and differences, in the names and in the number of classes have complicated the situation .
initial attempts to classify chs mainly involved the ascomycota sequences .
organising classes i, ii and iii into the division  <dig> and the classes iv and v into the division  <dig> was the result of working on a small number of fungal sequences .
then, several other classes  or a new division  <dig>  were added but nomenclature was unfortunately decided heterogeneously between the different study groups .
more recently, chs classification was extended to several basidiomycota species but by still following the same nomenclature used for ascomycota species  <cit>  .
finally, a class viii was recently proposed but it was for three completely distinct clades of putative chs, resulting in an unusable classification  <cit> .
moreover, all these different classifications were initially based upon chs sequences from ascomycota and the actual classifications are not well suited to other fungi .table  <dig> summarized history of chitin synthase protein classifications in fungi
in bold, the chitin synthase classification issued from our study
ainversion between classes iva and ivb and inversion between classes va and vb were corrected in ortiz-castellanos and ruiz-herrera  <cit>
bcv: chlorovirus-like chs class; esv: ectocarpus siliculosus-like chs class; recchs: recombined chitin synthase
in order to update and standardize chs classifications, we performed a comparative multi-species analysis across many sequenced and annotated genomes.
a databank of chs proteins was generated from a similarity search of chs pfam domains in the complete proteomes.
maximum likelihood  and bayesian phylogenetic trees were constructed to provide a global view of the whole chs family.
applying a rigorous method of phylogenetic analysis, we organised the fungal sequences and obtained a more robust classification.
we extended the study to chitin synthases from other species in order to elucidate chs gene evolution.
in particular, this genome-wide phylogenetic analysis confirms the occurrence of multiple gene losses, duplications and horizontal gene transfers within this family of glycosyltransferases.
surprisingly, this work provides, for the first time, evidence of a chitin synthase horizontal gene transfer from eukaryota to bacterial genomes.
RESULTS
in silico detection of putative chs in annotated and complete proteomes enabled us to identify more than eight hundreds of chs sequences in about  <dig> eukaryota species.
we also found chs sequences from a few viruses and bacteria, but no chs could be found in archeal species .
most fungal chitin synthases fall into two distinct divisions .fig.
<dig> distribution of chitin synthases among eukaryotes, bacteria and viruses.
eukaryotic species phylogeny was adapted from  <cit> , with modifications for hacrobia  <cit> , ciliophora  <cit> , coelacanthimorpha  <cit> , and amoebozoa, apusozoa, filasterea and choanoflagellida  <cit> .
groups in which the presence of chitin synthase protein was detected are shaded in grey and written in bold.
in each group, we include, in brackets, the number of species harboring chitin synthase proteins compared to the number of species analyzed.
these species are listed in additional file 3: table s <dig> and additional file 14: table s7
fig.
<dig> evolution of chitin synthases .
a ml phylogeny based on  <dig> amino acid alignment positions of  <dig> sequences was constructed with phyml using nodc proteins and hyaluronan synthases as outgroups.
bootstraps of interest ≥ <dig> are shown above the branches.
black circles with a + correspond to root nodes of the subtrees detailed in another figure.
horizontal gene transfers  are indicated on the branches leading to transferred sequences.
the bayesian phylogenetic approach gave similar results .
the abbreviations used in sequence names are listed in additional file 13: table s <dig>   acanthamoeba and ichtyosporea sequences were also described in this group  <cit> .
picochlorum sp.
sequence is also present in this group  <cit>
update of chs fungi classification
we observed that in dikarya fungi, ascomycota and basidiomycota, up to  <dig> chs-encoding genes were found in the same species .
in ascomycota yeasts, the number of chs is usually lower than in filamentous fungi and ranges from one  to seven   <cit> .
in hemiascomycota yeasts, the presence of seven chs in yarrowia lipolytica and only three in saccharomyces cerevisiae can only be explained by several losses in this clade .
in mucoromycotina, chytridiomycota and blastocladiomycota,  <dig> to  <dig> chs genes were found depending on the species suggesting an even larger expansion of the chs family in these early-branching fungal lineages .
finally, none or only one, chs gene was found in four microsporidia fungal genomes .
this characteristic seems consistent with the extreme reduction and compaction that was observed in these particular genomes  <cit> .
the large number of detected chs, and their diversity, provided the opportunity to establish a more robust fungal classification for this protein family.
in our definition, a class means a set of chs sequences, from different ascomycota, basidiomycota and/or mucoromycotina species, which forms a well-supported group in the trees constructed with two phylogenetic approaches .
for now, we consider the genome quality and number from chytridiomycota and blastocladiomycota species insufficient to accurately classify these sequences.
ascomycota yeast chs sequences were excluded from most phylogenetic analyses in order to reduce the risk of long-branch attraction  as substantial artefact was described in the phylogeny of saccharomyces species  <cit> .
lba could have contributed to erroneously cluster together long branches, irrespective of the true relationships of sequences  <cit> .
such exclusion is not a problem as classification of chs from yeasts is easy .
fungal division  <dig> : five new classes a to e in addition to classes i, ii and iii
fungal division  <dig> consists mainly of protein sequences with an n-terminal chitin_synth_1n domain , a conserved catalytic site and  <dig> predicted transmembrane domains in the c-terminal region .
in this division, different fungal lineage-specific classes were found with only one, corresponding to class iii, which was common to ascomycota and basidimycota .
previous attempts to group the classes a to e mainly with ascomycota classes i or ii resulted in different classifications as these groups were not well supported by the phylogenies .
a possible explanation for the observed phylogeny is that several duplications arose for this division at an early stage in fungal evolution and, thereafter, different gene losses might have occurred in the different lineages.
more than forty fungal mutants impaired in a division  <dig> chs gene were generated in ascomycota species and a few in basidiomycota species .
some differences in chs activity in vitro, chitin content and conidiation were observed for chs mutants impaired in ascomycota classes i or ii.
these classes probably have redundant roles as the single corresponding mutants were not affected in their growth and morphology.
on the contrary, class iii chs seems to play a crucial role in the hyphal tip growth as several class iii mutants were strongly affected in their morphology, with reduced growth resulting in small colonies and abnormal highly-branched hyphae .
this class was lost early on during saccharomycotina yeast evolution, as only yarrowia lipolytica possess a class iii chs gene .
conversely, this class was expanded in some pezizomycotina species as already mentioned for fusarium sp.
and aspergillus sp.
<cit> .
some duplicated copies are fast evolving sequences, which contrasts with the strong sequence conservation observed in this class, and they might have acquired different roles .fig.
<dig> structural features of the chitin synthase proteins.
the core of chs proteins is always composed of conserved motifs named a-h and playing a role in the active site of the enzyme .
the domains related to chitin synthase used for their detection, chitin_synth_ <dig>  and chitin_synth_ <dig> , correspond to the a-c and a-h regions respectively.
other domains from the pfam library, 1n , aminotransferase , myosin head , c <dig>  and d  were often detected in some chs clades and are indicated by dashed boxes.
the length variation of the myosin head domain in the clade v is represented by a dashed line in the dashed box of this domain.
n-terminal and c-terminal regions of chs are variable and transmembrane segments detected in almost all the sequences of one clade are shown with black solid squares.
the less frequent additional segments are shown with white solid squares.
predicted outside or cytoplasmic localizations of eukaryota chs protein segments are indicated thanks to a dotted line .
cytoplasmic localization of the conserved motifs a-h in homologous gt <dig> glycosyltransferases was confirmed with lacz, phoa and/or gfp reporter fusions .
according to these studies, some predicted transmembrane domains are, in fact, putative membrane domains that do not cross the membrane .
the distant motif h might interact with the other conserved motifs as its cytoplasmic localization was confirmed
fig.
<dig> evolution of fungal chs belonging to division  <dig>  a ml phylogeny, based on  <dig> amino acid alignment positions of  <dig> sequences, was constructed with phyml.
the root was placed according to the phylogeny in the additional file 16: file s <dig>  the bayesian phylogenetic approach gave similar results
fungal division  <dig> : two superclasses iv and v
fungal division  <dig> is clearly divided into two monophyletic superclasses iv and v .
these are composed of protein sequences which contain, in addition to the chitin synthase catalytic site, one or two predicted transmembrane domains in the n-ter and c-ter regions plus a cytochrome-b5-like domain  in the n-ter region, which has a proposed role as a binding site for lipid ligands  <cit>  .
sequences of the superclass v often have two additional domains: a myosin motor domain  fused to their n-terminus extremity, involved in intracellular trafficking of chs and site specificity of chitin secretion  <cit>  , and a dek-c domain , of unknown function, at their c-terminus.
each superclass was divided into several classes, most of which are conserved between ascomycota, basidiomycota and mucoromycotina .
the class ivb was lost in ascomycota and the ancestor of most hemiascomycota yeasts lost all division  <dig> classes except the class iva .
the class ivc was only found in mucoromycotina, where an expansion of classes iva, va and vb was also observed.
proteins from superclasses iv and v were also detected in the proteomes of chytridiomycota and blastocladiomycota whereas microsporidia proteomes only contained one member of the superclass iv.
despite the fact that the superclass iv was found in the largest number of fungal species, the corresponding mutants do not usually exhibit any apparent phenotypic change compared to the wild-type strain .
by contrast, mutants of class va or vb are usually strongly affected in their morphology , in the response to cell wall stresses and in virulence  <cit>  .fig.
<dig> evolution of fungal chs belonging to division  <dig>  a ml phylogeny, based on  <dig> amino acid alignment positions of  <dig> sequences, was constructed with phyml.
the root was placed according to the phylogeny in the additional file 18: file s <dig>  the bayesian phylogenetic approach gave similar results
fungal division 2: two virus-like classes
in fungal division  <dig>  in addition to the two superclasses iv and v, two others classes were found .
these fungal classes, that we have called esv  and cv , were found in proteomes of some ascomycota , some basidiomycota , a chytridiomycota  and some phycodnaviridae giant algal-viruses .
the cv class was recently proposed as a new class viii  <cit>  but we do not recommend this denomination as class viii is used for other distinct groups of putative chs  <cit>  .
independent horizontal transfers of these chs genes with their neighboring genes, in fungal genomes from chloroviruses and phaeoviruses respectively, have already been suggested  <cit> .
in addition to the strong similarity between cv and esv viral and fungal sequences, the corresponding genes are clustered on genomes with an udp-n-acetylglucosamine 6-dehydrogenase gene .
this two-gene cluster, conserved in some fungal species and in viruses of distantly related algae, suggests that these two genes were probably transmitted together and that this gene pair might work in concert : ungd family enzymes provide precursors for glycosyltransferase enzymes  <cit> .
moreover, the fungal and viral cv genes are also colocated with a polysaccharide deacetylase.
these genetic material exchanges between fungi and algal-viruses are perplexing.
however, as exemplified by the  <dig> million year old lichen-fossils, interactions betwen fungi and algae have long existed  <cit> .
furthermore, gene transfers from the ancestor of dothideo/sordariomycetes to the ancestor of the terrestrial alga trebouxia decolorans have also been proposed  <cit> .
there is currently no evidence for the functionality of the fungal proteins encoded by the genes from the esv class.
however, the cv class genes of glomerella graminicola and gibberella zeae were found to be differentially expressed during plant infection  <cit>  and a deletion mutant in a cv class chs gene was recently described in fusarium graminearum   <cit> .
disruption of this gene resulted in a reduced accumulation of chitin, decreased chs activity, sensitivity to sds and reduced pathogenicity.
it has been suggested that the fgchs <dig> gene is required for cell wall development in f. graminearum.
the chs genes from the cv and esv classes were duplicated in some ascomycota lineages and they were lost in others  <cit>  .
they also show higher evolutionary rates than other fungal chs .
the virus containing a chs gene from the esv class is, more precisely, a lysogenic phaeovirus.
dna from this virus, including the esv chs gene, is integrated into the genome of the pluricellular brown alga ectocarpus siliculosus  <cit> .
this integrated viral esv chs gene is transcriptionally silent in the algae and is probably not functional  <cit> .
viruses with at least one chs gene from the cv class include some chloroviruses infecting the unicellular green alga chlorella with a lytic infection style .
some chloroviruses form chitin on the surface of infected cells which might protect virus-infected algae from uptake by other organisms  <cit> .
indeed, the heterologous expression of the chlorovirus cvk <dig> chs gene was performed in e. coli leading to the production of fibers by the bacterium  <cit> .
outside fungal divisions : a class of chs probably emerging from a recombination event
in our analysis, a complete chs class, previously called vi, vii  or sometimes division  <dig>  <cit> , was treated separately.
we suspect a recombination event of being at the origin of this class .
associated with a reduced taxonomic distribution in fungi, including some ascomycota groups  and a mucoromycotina species , this class was also recently detected in a chromalveolate , plasmodiophora brassicae, an obligate biotrophic pathogen of crucifers  <cit> .
in this study, we suggest that the corresponding protein sequences have characteristics associated with a rearrangement in their ancestor: a duplication of the qxxxy motif  and a phylogenetic signal which seems to differ in the n- and c-termini sequence fragments located on either side of this duplication .
indeed, while the c-terminal region appears to be similar to that in other chs , the n-terminal region is closer to hyaluronan synthase proteins in the phylogenetic tree  and shares the same organization of transmembrane domains .
this observation could be explained by an ancient recombination between the ancestor’s sequences of two glycosytransferase family  <dig> proteins.
an alternative explanation, that we can not completely exclude, is that the ancestor of this class underwent a period of an accelerated rate of evolution which blurred the phylogenetic signal.
in both cases , we excluded them from phylogenetic analyses as they might have provoked artefactual groups due to long-branch attractions.
hgts are probably at the origin of this chs class in one mucoromycotina , one chytridiomycota  and one chromalveolata  .
in ascomycota groups, where this class is present, the corresponding genes are probably functional as they are strictly fixed in one well-conserved copy .
however, their chitin synthase activity has not yet been proved so we recommend using the term recchs  for these sequences.
few fungal deletion mutants were obtained for this class and their phenotypes are divergent.
recchs orthologs have probably evolved with different roles in these fungi during growth and development  <cit> .
origin of chs in eukaryotes
the phylogeny was obtained with a protein sequences sample representative of the entire set of chitin synthases detected in this study .
at least three chitin synthase genes were present in the ancestor genome of opistokonta: one ancestral chs for the metazoa division, one for the division  <dig> and one for the division  <dig> .
a previous study suggested four ancestral chs genes in the last opisthokonta common ancestor   <cit>  but we propose that two of them belong to division  <dig> and they may have diverged from a common ancestral sequence in loca .
the chitin synthase genes probably appeared earlier, given the basal positions in the metazoa division of one sequence of the amoebozoa entamoeba histolytica and the chs of the apusomonad thecamonas trahens.
the distribution of taxa possessing a chs gene in metazoa indicates that chs genes were lost several times independently in this phylum .
on the other hand, family expansion occurred in some species, such as in the gastropod lottia gigantea  <cit>  and the amphioxus branchiostoma floridae  <cit> .
it is noteworthy that a second chs sequence from entamoeba histolytica did not significantly group with any of the chitin synthase divisions, which raises the question of its origin.
however, heterologous expression of this protein in the yeast saccharomyces cerevisiae confirmed its chs activity  <cit> .
the presence of chitin synthases belonging to different divisions in diverse chromalveolates, such as ciliates, diatoms, oomycetes and other protists, might be the result of a deeper evolutionary origin of chs in the last common eukaryotic ancestor  <cit>  .
however, an alternative plausible model for the origin of chromalveolate chs could imply several independent horizontal gene transfers at different times during the chromalveolate evolutionary history  <cit> , as we suggested for the chs of the rhizaria plasmodiophora brassicae .
diatom chs genes from thalassiosira pseudonana  and phaeodactylum tricornutum , were probably acquired by an hgt from opistokonta .
other hgt must have occurred to explain the actual taxonomic distribution of chs genes and their sequence diversity.
if the division  <dig> chs of the green algae  chlorella  <cit>   and picochlorum  <cit>  were vertically inherited from the plantae ancestor, it would imply that these genes were independently lost in many lineages of the plantae.
as mentioned for the cv and esv classes of division  <dig> , chs gene exchanges might have occurred between algae-related viruses, including chloroviruses and fungi.
however, the chlorella chs belongs to division  <dig> and seems to resemble oomycete and ciliate chs more than fungal ones .
therefore, a possible source of the transfer could be a ciliate living in symbiosis with a green alga, such as paramecium bursaria.
hence, the evolutionary history of chitin synthase genes suggests different independent horizontal gene transfers among diverse eukaryotic microorganisms .
while viruses are known to be gene transfer agents, membrane vesicles, which are not impaired by receptor recognition in the way that viruses are, could be large spectrum transducing agents .
bacterial chitin synthase genes
the analysis of bacterial proteomes gave unexpected results as it revealed a dozen of bacteria possessing at least one division  <dig> chs whereas no bacterial chs had been previously described .
these bacteria correspond mainly to gammaproteobacteria  but there is also an alphaproteobacteria .
finally, one chs gene was detected in the genome of the cyanobacteria tolypothrix campylonemoides  but it was not included as it is split by a transposase insertion and is probably not functional.
the well supported monophyletic group, formed by the corresponding chs proteins, suggests a unique transfer of a eukaryotic division  <dig> chs gene to a bacterial genome and multiple hgt might have occurred then between bacterial species.
several cases of the lateral transfer of genes, protein domain-encoding fragments or repeat elements have been described, from animals to bacteria  and from plantae to bacteria .
however, the detected eukaryote to bacteria transfers are fewer than the bacteria to eukaryote ones  <cit>  for the following possible reasons:  the barrier formed by spliceosomal introns in eukaryotic genes  <cit> ;  the smaller population size of eukaryotes which, therefore, offers a reduced pool of potential donors  <cit> ; and  the small number of eukaryotic genes that might present a selective advantage for bacteria and which could, thus, persist in their genome after being transferred  <cit> .
interestingly, the family  <dig> of carbohydrate-binding modules , which are short chitin-binding modules predominantly found in animal and fungal genomes, were also detected in seven bacterial genomes as putative hgt  <cit> .
however, the bacterial species involved were different from those harboring chitin synthase genes.
most of the bacterial chs genes were co-localized with a set of co-oriented genes, in a classical bacterial operon organization  including a sugar epimerase and hypothetical protein encoding-genes.
this unusual pattern of gene order conservation between distantly related bacteria suggest that the corresponding genes were transferred and maintained together, possibly because they participate in a common function in these bacteria.
noticeably, two of the hypothetical proteins possess bacterial domains of unknown functions duf <dig> and duf <dig>  that are predicted to be part of the same operon in operondb  <cit> .
enterobacteriales species  clearly acquired these duf- and chs-encoding genes during the same transfer because close enterobacteriales relatives without chs have neither the duf1501- nor the duf1800-encoding genes .
the alphaproteobacteria agrobacterium vitis has two pairs of duf1501-duf <dig> encoding genes organized in tandem.
phylogenetic trees revealed that the pair of proteins encoded by the genes co-localized with the chs are more closely related to their homologs in gammaproteobacteria than to their homologs in alphaproteobacteria .
hence, these duf-encoding genes were also transferred with the chs in a. vitis.
some transfers seem to be recent as the corresponding chs belongs to a genomic region with a different g + c content, compared with the neighboring genomic context .
this is the case for one of the two dickeya_dadantii_ech <dig> chs genes  and the chs of cedecea neteri and pseudomonas cichorii, which are localized in regions with a lower g + c content.
the secondary metabolism gene cluster, containing one chs in teredinibacter turnereae, also appeared with a different g + c composition higher than the neighboring genomic context.
some bacterial chitin synthase genes have evolved additional features that differentiate them from eukaryotic division  <dig> chs .
first, they have a short n-terminal region and they lack the eukaryota chitin_synt_1n domain  .
secondly, an accretion of an aminotransferase domain probably occurred in the ancestor of pectobacterium and brenneria .fig.
<dig> phylogeny and genomic context of bacterial chitin synthases.
a phylogenetic relationships of bacterial chitin synthases.
the ml tree is shown with numbers above the branches, indicating support in bootstrap analyses .
the tree was rooted with division  <dig> fungal chs from a. fumigatus and u. maydis.
the bayesian phylogenetic approach gave the exact same topology .
b gene organization of each variable region containing chitin synthase.
the limits of regions were obtained by comparison with ortholog regions in proximal species without the chs gene.
each represented region is aligned with the corresponding chs in the phylogenetic tree.
chs in arrows is the chitin synthase domain and at is the aminotransferase domain.
<dig> and 1501: proteins with a domain of unknown function, duf <dig>  and duf <dig>  respectively; se: sugar epimerase; a: transcriptional regulator with a sugar-binding domain; b:  <dig> -dihydroxyhept-2-ene- <dig> -dioic acid aldolase; c: osmc family protein; d: duf465; e: lipid a biosynthesis lauroyl  acyltransferase; f: cold-shock protein; g: duf2511; h: efflux protein; i: gluconate-5-dehydrogenase; j: taurine dioxygenase; k: sulfotransferase nodh; l: aminotransferase; m: hydroxylase; and n: arac family transcriptional regulator.
the dotted rectangles correspond to regions detected as a prophage, a secondary metabolism cluster or a genomic island.
empty arrows and those with stars represent hypothetical proteins.
among them, groups of arrows with the same number of stars are orthologs
in a large variety of bacteria, type  <dig> glycosyltransferases  homologous to chitin synthases have been described.
these transmembrane enzymes are localized at the inner membrane of gram negative bacteria and synthesize different exopolysaccharides  into the periplasm.
these eps are made of cellulose, curdlan, alginate or hyaluronic acid  <cit> .
in pseudomonas aeruginosa, secretion of alginate through the peptidoglycan and the outer membrane is ensured by an envelope-spanning multiprotein complex including channel proteins  <cit> .
we found that some bacterial chs genes are colocated with porin or efflux protein-encoding genes .
it is possible that these bacterial chs genes, and their neighbouring transporter encoding-genes, are involved in the production and secretion of an eps made of chitin.
eps are matrix components of bacterial biofilms which are known to play a major role in pathogenic or symbiotic interactions between bacteria and animals or plants  <cit> .
it is noticeable that, among bacteria in which a chs gene was found, pseudomonas cichorii and agrobacterium vitis are plant-pathogens and secrete an eps made of alginate and curdlan, respectively  <cit> .
indeed, a. vitis is able to build biofilms on abiotic as well as on plant root surfaces, probably due to its eps  <cit> .
other bacterial species displaying a chs gene are also plant pathogens: a brenneria spp., three pectobacterium species and two dickeya dadantii isolates.
the later two bacterial isolates, d. dadantii ech <dig> and d. dadantii ech <dig>  should be reclassified as two different species, dickeya zeae ech <dig> and dickeya paradisiaca ech <dig>  respectively  <cit> .
it may be that the bacterial chitin synthase plays a role in the parasitic interaction between these bacteria and their plant hosts.
chs activities in fungi or metazoa are usually described as producing long crystalline chains of chitin  but it is possible that a bacterial chs secretes small soluble chitosaccharides, or eventually chitooligosaccharides , instead.
indeed, the phytopathogenic oomycete aphanomyces euteiches does not secrete chitin fibers although two chs genes are present in this species  <cit> .
however, the encoded chs activities of a. euteiches are thought to be active because a small soluble and noncrystalline glucan-chitosaccharide was detected in its cell wall  <cit> .
interestingly, one of the two chs genes in d. dadantii ech <dig> and the chs gene of pseudomonas cichorii are co-located and have the same orientation as a putative chitoporin-encoding gene .
in the marine bacterium vibrio harveyi, chitoporin is a transporter of cos through the outer membrane  <cit> .
cos are known to play a key role as signal molecules in multiple plant-microbe interactions.
in parasitic interactions between plants and fungi, cos are released from the digestion of the fungal cell wall by secreted plant chitinases.
the resulting cos are then recognized by plant receptors as microbial associated molecular patterns  and they induce plant immunity  <cit> .
in symbiotic interactions, modified cos, called lipochitooligosaccharides , which include myc and nod factors, modulate plant host immunity.
myc factors are produced and secreted by arbuscular mycorrhizal fungi but the enzymes responsible for their biosynthesis have not yet been identified  <cit> .
nod factors are synthesized by nodc, a bacterial gt <dig> homologous to chs, and they induce nodule formation during a symbiotic interaction between rhizobiacea  and leguminous host plants  <cit> .
whether bacterial chs genes are able to synthesize cos implicated in plant interactions is still uncertain.
several bacterial chs genes are fused with a putative aminotransferase domain and they are colocated with a putative sugar epimerase-encoding gene .
these two additional functions could be associated with modifications of chitin or cos. a gene of unknown functions, and composed of the duf <dig> domain, was also found colocated with several bacterial chs.
the encoded duf1501-containing protein carries a twin-arginine motif which would imply that it is exported by the twin-arginine translocation  pathway .
the bacterial tat system allows folded proteins to be moved across membranes without significant ion leakage  <cit> .
the duf <dig> domain has also been described as forming part of a conserved machinery in compartimentalized species from the planctomycetes, verrucomicrobia and chlamydiae  super-phylum  <cit> .
these informations about duf <dig> proteins are hardly connectable with the biological role of known chs.
however, our hypothesis is that duf <dig>  duf <dig> and chs proteins might have a functional link in bacteria as they share a common conserved operon organization.
it would be very interesting to study the function of bacterial chs and also of the proteins that seem associated to them.
finally, in the particular cases of teredinibacter turnerae and cellvibrio mixtus subsp.
mixtus, the chs genes are localized in the middle of secondary metabolism gene clusters  and they could be involved in the production of a bioactive molecule  <cit> .
CONCLUSIONS
bacterial chitin synthase genes constitute a new example of genes acquired by a bacterium via horizontal transfer from a eukaryotic donor.
the chitin synthase activity of the bacterial chs and the possible selective advantage for the corresponding bacteria, often implied in plant interactions, needs further investigation.
chs-encoding genes have also been transferred between eukaryotic microorganisms.
we recommend avoiding the use of this multigenic family to elucidate the phylogenetic relationships between the different eukaryotic species, especially since many duplications and losses are also observed in different lineages.
fungal chs are, unfortunately, not an exception and the difficulty of classifying these sequences has led to discordant classifications.
we took advantage of the current study to determine which can be robustly classified and then to construct the most consensual classification possible.
to facilitate the use of this new classification, any information that corresponds with the previously published versions is provided , together with the databank, in fasta format, of all the classified chs sequences .
a website also permits blast queries to the databank .
this facilitates a search for the class of a previously identified chs, even if the corresponding accession number has changed .
it is also an aid for the classification of chs from species closely related to those analyzed.

 chitin is a biological polymer consisting of carbohydrate molecules bonded together to form long chains of polysaccharides. unlike starch and glycogen that are storage polysaccharides in plants and animals respectively, chitin is a structural polysaccharide organised as crystalline microfibrils and with enormous tensile strength. it contributes to the rigidity and integrity of cells, tissues or body surfaces in a wide range of organisms, protecting and giving them shape, as seen with cellulose or pectin in plant and algal cell walls. so far, the presence of this structural polysaccharide has been mainly demonstrated in the cell walls of mycota, the exoskeleton of hexapoda or crustacea, and in the radula or beak of mollusca, where it plays a major role in development and growth. in addition, the presence and subcellular location of chitin in invertebrate hemocytes suggests another role for this polysaccharide in the immune system of diverse animals. it was generally thought that there was no chitin in vertebrates but this polymer has been described in several ray-finned fishes and in some amphibians. the role of endogenous chitin in the biology of these vertebrates remains elusive. chitin has also been sporadically found in structures from a diverse range of eukaryotic microorganisms, such as the cell wall of a few chlorophyta, the cyst wall or lorica, of ciliophora, the theca of choanoflagellida, and the test or cyst wall, of amoebozoa. it is also present in the large family of heterokonta protists, for example in the cell wall of oomycota, the spines of diatomae and the stalk of chrysophyta. chitin is an hexosamine polymer composed of beta--linked linear chains of more than  n-acetylglucosamine residues that are highly cross-linked with hydrogen bonds. in insects, chitin is deposited exclusively on the apical sides of epithelial cells, facing the external environment. in fungal cell walls, there is a common fibrillar core composed of branched beta--glucan to which chitin and other polysaccharides are covalently bound. chitin accounts for 1-2% of the cell wall mass in yeasts and up to 30% in molds. elongation of the chitin polymer is catalyzed by a highly conserved enzyme called chitin synthase, chs. the chs enzyme belongs to the gt family of processive polymerizing glycosyltransferases which includes the synthases for cellulose, callose, curdlan, mannan, hyaluronate and alginate polymers. in fungi, chitin biosynthesis requires a set of multiple chs isoenzymes that are encoded by a multigenic family. although they share a common central catalytic domain, chs isoenzymes from fungi and other species can differ greatly in their n- and c-termini parts. all chs utilize udp-n-acetylglucosamine from the cytoplasm as a substrate and catalyze multiple transfers of the activated sugar donor to the non-reducing end of the growing chain. multiple transmembrane domains are found in every chs protein and these probably form a channel in the cell membrane through which linear chitin is extruded into the extracellular space as it is described for cellulose biosynthesis. nevertheless, the exact mechanism by which chitin is assembled into microfibrils and cross-linked with other components of the cell wall is still poorly understood. the importance of chs genes in fungal biology has been extensively investigated by reverse genetics. mutants have been constructed by disrupting or deleting particular chs genes in fungal species and no less than a hundred mutants have been made so far in more than twenty fungi. the multiplicity of chs genes in fungal genomes has necessitated their classification for comparative functional genomics, and fungal chs isoenzymes have been classified into multiple divisions and classes according to protein similarities in their catalytic domain. more than phylogenetic analyses have been published and differences, in the names and in the number of classes have complicated the situation. initial attempts to classify chs mainly involved the ascomycota sequences. organising classes i, ii and iii into the division and the classes iv and v into the division was the result of working on a small number of fungal sequences. then, several other classes or a new division were added but nomenclature was unfortunately decided heterogeneously between the different study groups. more recently, chs classification was extended to several basidiomycota species but by still following the same nomenclature used for ascomycota species. finally, a class viii was recently proposed but it was for three completely distinct clades of putative chs, resulting in an unusable classification. moreover, all these different classifications were initially based upon chs sequences from ascomycota and the actual classifications are not well suited to other fungi.table summarized history of chitin synthase protein classifications in fungi in bold, the chitin synthase classification issued from our study ainversion between classes iva and ivb and inversion between classes va and vb were corrected in ortiz-castellanos and ruiz-herrera bcv: chlorovirus-like chs class; esv: ectocarpus siliculosus-like chs class; recchs: recombined chitin synthase in order to update and standardize chs classifications, we performed a comparative multi-species analysis across many sequenced and annotated genomes. a databank of chs proteins was generated from a similarity search of chs pfam domains in the complete proteomes. maximum likelihood and bayesian phylogenetic trees were constructed to provide a global view of the whole chs family. applying a rigorous method of phylogenetic analysis, we organised the fungal sequences and obtained a more robust classification. we extended the study to chitin synthases from other species in order to elucidate chs gene evolution. in particular, this genome-wide phylogenetic analysis confirms the occurrence of multiple gene losses, duplications and horizontal gene transfers within this family of glycosyltransferases. surprisingly, this work provides, for the first time, evidence of a chitin synthase horizontal gene transfer from eukaryota to bacterial genomes.  in silico detection of putative chs in annotated and complete proteomes enabled us to identify more than eight hundreds of chs sequences in about eukaryota species. we also found chs sequences from a few viruses and bacteria, but no chs could be found in archeal species. most fungal chitin synthases fall into two distinct divisions.fig. distribution of chitin synthases among eukaryotes, bacteria and viruses. eukaryotic species phylogeny was adapted from, with modifications for hacrobia, ciliophora, coelacanthimorpha, and amoebozoa, apusozoa, filasterea and choanoflagellida. groups in which the presence of chitin synthase protein was detected are shaded in grey and written in bold. in each group, we include, in brackets, the number of species harboring chitin synthase proteins compared to the number of species analyzed. these species are listed in additional file 3: table s and additional file 14: table s7 fig. evolution of chitin synthases. a ml phylogeny based on amino acid alignment positions of sequences was constructed with phyml using nodc proteins and hyaluronan synthases as outgroups. bootstraps of interest ≥ are shown above the branches. black circles with a + correspond to root nodes of the subtrees detailed in another figure. horizontal gene transfers are indicated on the branches leading to transferred sequences. the bayesian phylogenetic approach gave similar results. the abbreviations used in sequence names are listed in additional file 13: table s acanthamoeba and ichtyosporea sequences were also described in this group. picochlorum sp. sequence is also present in this group update of chs fungi classification we observed that in dikarya fungi, ascomycota and basidiomycota, up to chs-encoding genes were found in the same species. in ascomycota yeasts, the number of chs is usually lower than in filamentous fungi and ranges from one to seven. in hemiascomycota yeasts, the presence of seven chs in yarrowia lipolytica and only three in saccharomyces cerevisiae can only be explained by several losses in this clade. in mucoromycotina, chytridiomycota and blastocladiomycota, to chs genes were found depending on the species suggesting an even larger expansion of the chs family in these early-branching fungal lineages. finally, none or only one, chs gene was found in four microsporidia fungal genomes. this characteristic seems consistent with the extreme reduction and compaction that was observed in these particular genomes. the large number of detected chs, and their diversity, provided the opportunity to establish a more robust fungal classification for this protein family. in our definition, a class means a set of chs sequences, from different ascomycota, basidiomycota and/or mucoromycotina species, which forms a well-supported group in the trees constructed with two phylogenetic approaches. for now, we consider the genome quality and number from chytridiomycota and blastocladiomycota species insufficient to accurately classify these sequences. ascomycota yeast chs sequences were excluded from most phylogenetic analyses in order to reduce the risk of long-branch attraction as substantial artefact was described in the phylogeny of saccharomyces species. lba could have contributed to erroneously cluster together long branches, irrespective of the true relationships of sequences. such exclusion is not a problem as classification of chs from yeasts is easy. fungal division: five new classes a to e in addition to classes i, ii and iii fungal division consists mainly of protein sequences with an n-terminal chitin_synth_1n domain, a conserved catalytic site and predicted transmembrane domains in the c-terminal region. in this division, different fungal lineage-specific classes were found with only one, corresponding to class iii, which was common to ascomycota and basidimycota. previous attempts to group the classes a to e mainly with ascomycota classes i or ii resulted in different classifications as these groups were not well supported by the phylogenies. a possible explanation for the observed phylogeny is that several duplications arose for this division at an early stage in fungal evolution and, thereafter, different gene losses might have occurred in the different lineages. more than forty fungal mutants impaired in a division chs gene were generated in ascomycota species and a few in basidiomycota species. some differences in chs activity in vitro, chitin content and conidiation were observed for chs mutants impaired in ascomycota classes i or ii. these classes probably have redundant roles as the single corresponding mutants were not affected in their growth and morphology. on the contrary, class iii chs seems to play a crucial role in the hyphal tip growth as several class iii mutants were strongly affected in their morphology, with reduced growth resulting in small colonies and abnormal highly-branched hyphae. this class was lost early on during saccharomycotina yeast evolution, as only yarrowia lipolytica possess a class iii chs gene. conversely, this class was expanded in some pezizomycotina species as already mentioned for fusarium sp. and aspergillus sp.. some duplicated copies are fast evolving sequences, which contrasts with the strong sequence conservation observed in this class, and they might have acquired different roles.fig. structural features of the chitin synthase proteins. the core of chs proteins is always composed of conserved motifs named a-h and playing a role in the active site of the enzyme. the domains related to chitin synthase used for their detection, chitin_synth_ and chitin_synth_, correspond to the a-c and a-h regions respectively. other domains from the pfam library, 1n, aminotransferase, myosin head, c and d were often detected in some chs clades and are indicated by dashed boxes. the length variation of the myosin head domain in the clade v is represented by a dashed line in the dashed box of this domain. n-terminal and c-terminal regions of chs are variable and transmembrane segments detected in almost all the sequences of one clade are shown with black solid squares. the less frequent additional segments are shown with white solid squares. predicted outside or cytoplasmic localizations of eukaryota chs protein segments are indicated thanks to a dotted line. cytoplasmic localization of the conserved motifs a-h in homologous gt glycosyltransferases was confirmed with lacz, phoa and/or gfp reporter fusions. according to these studies, some predicted transmembrane domains are, in fact, putative membrane domains that do not cross the membrane. the distant motif h might interact with the other conserved motifs as its cytoplasmic localization was confirmed fig. evolution of fungal chs belonging to division a ml phylogeny, based on amino acid alignment positions of sequences, was constructed with phyml. the root was placed according to the phylogeny in the additional file 16: file s the bayesian phylogenetic approach gave similar results fungal division: two superclasses iv and v fungal division is clearly divided into two monophyletic superclasses iv and v. these are composed of protein sequences which contain, in addition to the chitin synthase catalytic site, one or two predicted transmembrane domains in the n-ter and c-ter regions plus a cytochrome-b5-like domain in the n-ter region, which has a proposed role as a binding site for lipid ligands. sequences of the superclass v often have two additional domains: a myosin motor domain fused to their n-terminus extremity, involved in intracellular trafficking of chs and site specificity of chitin secretion, and a dek-c domain, of unknown function, at their c-terminus. each superclass was divided into several classes, most of which are conserved between ascomycota, basidiomycota and mucoromycotina. the class ivb was lost in ascomycota and the ancestor of most hemiascomycota yeasts lost all division classes except the class iva. the class ivc was only found in mucoromycotina, where an expansion of classes iva, va and vb was also observed. proteins from superclasses iv and v were also detected in the proteomes of chytridiomycota and blastocladiomycota whereas microsporidia proteomes only contained one member of the superclass iv. despite the fact that the superclass iv was found in the largest number of fungal species, the corresponding mutants do not usually exhibit any apparent phenotypic change compared to the wild-type strain. by contrast, mutants of class va or vb are usually strongly affected in their morphology, in the response to cell wall stresses and in virulence.fig. evolution of fungal chs belonging to division a ml phylogeny, based on amino acid alignment positions of sequences, was constructed with phyml. the root was placed according to the phylogeny in the additional file 18: file s the bayesian phylogenetic approach gave similar results fungal division 2: two virus-like classes in fungal division in addition to the two superclasses iv and v, two others classes were found. these fungal classes, that we have called esv and cv, were found in proteomes of some ascomycota, some basidiomycota, a chytridiomycota and some phycodnaviridae giant algal-viruses. the cv class was recently proposed as a new class viii but we do not recommend this denomination as class viii is used for other distinct groups of putative chs. independent horizontal transfers of these chs genes with their neighboring genes, in fungal genomes from chloroviruses and phaeoviruses respectively, have already been suggested. in addition to the strong similarity between cv and esv viral and fungal sequences, the corresponding genes are clustered on genomes with an udp-n-acetylglucosamine 6-dehydrogenase gene. this two-gene cluster, conserved in some fungal species and in viruses of distantly related algae, suggests that these two genes were probably transmitted together and that this gene pair might work in concert: ungd family enzymes provide precursors for glycosyltransferase enzymes. moreover, the fungal and viral cv genes are also colocated with a polysaccharide deacetylase. these genetic material exchanges between fungi and algal-viruses are perplexing. however, as exemplified by the million year old lichen-fossils, interactions betwen fungi and algae have long existed. furthermore, gene transfers from the ancestor of dothideo/sordariomycetes to the ancestor of the terrestrial alga trebouxia decolorans have also been proposed. there is currently no evidence for the functionality of the fungal proteins encoded by the genes from the esv class. however, the cv class genes of glomerella graminicola and gibberella zeae were found to be differentially expressed during plant infection and a deletion mutant in a cv class chs gene was recently described in fusarium graminearum. disruption of this gene resulted in a reduced accumulation of chitin, decreased chs activity, sensitivity to sds and reduced pathogenicity. it has been suggested that the fgchs gene is required for cell wall development in f. graminearum. the chs genes from the cv and esv classes were duplicated in some ascomycota lineages and they were lost in others. they also show higher evolutionary rates than other fungal chs. the virus containing a chs gene from the esv class is, more precisely, a lysogenic phaeovirus. dna from this virus, including the esv chs gene, is integrated into the genome of the pluricellular brown alga ectocarpus siliculosus. this integrated viral esv chs gene is transcriptionally silent in the algae and is probably not functional. viruses with at least one chs gene from the cv class include some chloroviruses infecting the unicellular green alga chlorella with a lytic infection style. some chloroviruses form chitin on the surface of infected cells which might protect virus-infected algae from uptake by other organisms. indeed, the heterologous expression of the chlorovirus cvk chs gene was performed in e. coli leading to the production of fibers by the bacterium. outside fungal divisions: a class of chs probably emerging from a recombination event in our analysis, a complete chs class, previously called vi, vii or sometimes division, was treated separately. we suspect a recombination event of being at the origin of this class. associated with a reduced taxonomic distribution in fungi, including some ascomycota groups and a mucoromycotina species, this class was also recently detected in a chromalveolate, plasmodiophora brassicae, an obligate biotrophic pathogen of crucifers. in this study, we suggest that the corresponding protein sequences have characteristics associated with a rearrangement in their ancestor: a duplication of the qxxxy motif and a phylogenetic signal which seems to differ in the n- and c-termini sequence fragments located on either side of this duplication. indeed, while the c-terminal region appears to be similar to that in other chs, the n-terminal region is closer to hyaluronan synthase proteins in the phylogenetic tree and shares the same organization of transmembrane domains. this observation could be explained by an ancient recombination between the ancestor’s sequences of two glycosytransferase family proteins. an alternative explanation, that we can not completely exclude, is that the ancestor of this class underwent a period of an accelerated rate of evolution which blurred the phylogenetic signal. in both cases, we excluded them from phylogenetic analyses as they might have provoked artefactual groups due to long-branch attractions. hgts are probably at the origin of this chs class in one mucoromycotina, one chytridiomycota and one chromalveolata. in ascomycota groups, where this class is present, the corresponding genes are probably functional as they are strictly fixed in one well-conserved copy. however, their chitin synthase activity has not yet been proved so we recommend using the term recchs for these sequences. few fungal deletion mutants were obtained for this class and their phenotypes are divergent. recchs orthologs have probably evolved with different roles in these fungi during growth and development. origin of chs in eukaryotes the phylogeny was obtained with a protein sequences sample representative of the entire set of chitin synthases detected in this study. at least three chitin synthase genes were present in the ancestor genome of opistokonta: one ancestral chs for the metazoa division, one for the division and one for the division. a previous study suggested four ancestral chs genes in the last opisthokonta common ancestor but we propose that two of them belong to division and they may have diverged from a common ancestral sequence in loca. the chitin synthase genes probably appeared earlier, given the basal positions in the metazoa division of one sequence of the amoebozoa entamoeba histolytica and the chs of the apusomonad thecamonas trahens. the distribution of taxa possessing a chs gene in metazoa indicates that chs genes were lost several times independently in this phylum. on the other hand, family expansion occurred in some species, such as in the gastropod lottia gigantea and the amphioxus branchiostoma floridae. it is noteworthy that a second chs sequence from entamoeba histolytica did not significantly group with any of the chitin synthase divisions, which raises the question of its origin. however, heterologous expression of this protein in the yeast saccharomyces cerevisiae confirmed its chs activity. the presence of chitin synthases belonging to different divisions in diverse chromalveolates, such as ciliates, diatoms, oomycetes and other protists, might be the result of a deeper evolutionary origin of chs in the last common eukaryotic ancestor. however, an alternative plausible model for the origin of chromalveolate chs could imply several independent horizontal gene transfers at different times during the chromalveolate evolutionary history, as we suggested for the chs of the rhizaria plasmodiophora brassicae. diatom chs genes from thalassiosira pseudonana and phaeodactylum tricornutum, were probably acquired by an hgt from opistokonta. other hgt must have occurred to explain the actual taxonomic distribution of chs genes and their sequence diversity. if the division chs of the green algae chlorella and picochlorum were vertically inherited from the plantae ancestor, it would imply that these genes were independently lost in many lineages of the plantae. as mentioned for the cv and esv classes of division, chs gene exchanges might have occurred between algae-related viruses, including chloroviruses and fungi. however, the chlorella chs belongs to division and seems to resemble oomycete and ciliate chs more than fungal ones. therefore, a possible source of the transfer could be a ciliate living in symbiosis with a green alga, such as paramecium bursaria. hence, the evolutionary history of chitin synthase genes suggests different independent horizontal gene transfers among diverse eukaryotic microorganisms. while viruses are known to be gene transfer agents, membrane vesicles, which are not impaired by receptor recognition in the way that viruses are, could be large spectrum transducing agents. bacterial chitin synthase genes the analysis of bacterial proteomes gave unexpected results as it revealed a dozen of bacteria possessing at least one division chs whereas no bacterial chs had been previously described. these bacteria correspond mainly to gammaproteobacteria but there is also an alphaproteobacteria. finally, one chs gene was detected in the genome of the cyanobacteria tolypothrix campylonemoides but it was not included as it is split by a transposase insertion and is probably not functional. the well supported monophyletic group, formed by the corresponding chs proteins, suggests a unique transfer of a eukaryotic division chs gene to a bacterial genome and multiple hgt might have occurred then between bacterial species. several cases of the lateral transfer of genes, protein domain-encoding fragments or repeat elements have been described, from animals to bacteria and from plantae to bacteria. however, the detected eukaryote to bacteria transfers are fewer than the bacteria to eukaryote ones for the following possible reasons: the barrier formed by spliceosomal introns in eukaryotic genes; the smaller population size of eukaryotes which, therefore, offers a reduced pool of potential donors; and the small number of eukaryotic genes that might present a selective advantage for bacteria and which could, thus, persist in their genome after being transferred. interestingly, the family of carbohydrate-binding modules, which are short chitin-binding modules predominantly found in animal and fungal genomes, were also detected in seven bacterial genomes as putative hgt. however, the bacterial species involved were different from those harboring chitin synthase genes. most of the bacterial chs genes were co-localized with a set of co-oriented genes, in a classical bacterial operon organization including a sugar epimerase and hypothetical protein encoding-genes. this unusual pattern of gene order conservation between distantly related bacteria suggest that the corresponding genes were transferred and maintained together, possibly because they participate in a common function in these bacteria. noticeably, two of the hypothetical proteins possess bacterial domains of unknown functions duf and duf that are predicted to be part of the same operon in operondb. enterobacteriales species clearly acquired these duf- and chs-encoding genes during the same transfer because close enterobacteriales relatives without chs have neither the duf1501- nor the duf1800-encoding genes. the alphaproteobacteria agrobacterium vitis has two pairs of duf1501-duf encoding genes organized in tandem. phylogenetic trees revealed that the pair of proteins encoded by the genes co-localized with the chs are more closely related to their homologs in gammaproteobacteria than to their homologs in alphaproteobacteria. hence, these duf-encoding genes were also transferred with the chs in a. vitis. some transfers seem to be recent as the corresponding chs belongs to a genomic region with a different g + c content, compared with the neighboring genomic context. this is the case for one of the two dickeya_dadantii_ech chs genes and the chs of cedecea neteri and pseudomonas cichorii, which are localized in regions with a lower g + c content. the secondary metabolism gene cluster, containing one chs in teredinibacter turnereae, also appeared with a different g + c composition higher than the neighboring genomic context. some bacterial chitin synthase genes have evolved additional features that differentiate them from eukaryotic division chs. first, they have a short n-terminal region and they lack the eukaryota chitin_synt_1n domain. secondly, an accretion of an aminotransferase domain probably occurred in the ancestor of pectobacterium and brenneria.fig. phylogeny and genomic context of bacterial chitin synthases. a phylogenetic relationships of bacterial chitin synthases. the ml tree is shown with numbers above the branches, indicating support in bootstrap analyses. the tree was rooted with division fungal chs from a. fumigatus and u. maydis. the bayesian phylogenetic approach gave the exact same topology. b gene organization of each variable region containing chitin synthase. the limits of regions were obtained by comparison with ortholog regions in proximal species without the chs gene. each represented region is aligned with the corresponding chs in the phylogenetic tree. chs in arrows is the chitin synthase domain and at is the aminotransferase domain. and 1501: proteins with a domain of unknown function, duf and duf respectively; se: sugar epimerase; a: transcriptional regulator with a sugar-binding domain; b: -dihydroxyhept-2-ene- -dioic acid aldolase; c: osmc family protein; d: duf465; e: lipid a biosynthesis lauroyl acyltransferase; f: cold-shock protein; g: duf2511; h: efflux protein; i: gluconate-5-dehydrogenase; j: taurine dioxygenase; k: sulfotransferase nodh; l: aminotransferase; m: hydroxylase; and n: arac family transcriptional regulator. the dotted rectangles correspond to regions detected as a prophage, a secondary metabolism cluster or a genomic island. empty arrows and those with stars represent hypothetical proteins. among them, groups of arrows with the same number of stars are orthologs in a large variety of bacteria, type glycosyltransferases homologous to chitin synthases have been described. these transmembrane enzymes are localized at the inner membrane of gram negative bacteria and synthesize different exopolysaccharides into the periplasm. these eps are made of cellulose, curdlan, alginate or hyaluronic acid. in pseudomonas aeruginosa, secretion of alginate through the peptidoglycan and the outer membrane is ensured by an envelope-spanning multiprotein complex including channel proteins. we found that some bacterial chs genes are colocated with porin or efflux protein-encoding genes. it is possible that these bacterial chs genes, and their neighbouring transporter encoding-genes, are involved in the production and secretion of an eps made of chitin. eps are matrix components of bacterial biofilms which are known to play a major role in pathogenic or symbiotic interactions between bacteria and animals or plants. it is noticeable that, among bacteria in which a chs gene was found, pseudomonas cichorii and agrobacterium vitis are plant-pathogens and secrete an eps made of alginate and curdlan, respectively. indeed, a. vitis is able to build biofilms on abiotic as well as on plant root surfaces, probably due to its eps. other bacterial species displaying a chs gene are also plant pathogens: a brenneria spp., three pectobacterium species and two dickeya dadantii isolates. the later two bacterial isolates, d. dadantii ech and d. dadantii ech should be reclassified as two different species, dickeya zeae ech and dickeya paradisiaca ech respectively. it may be that the bacterial chitin synthase plays a role in the parasitic interaction between these bacteria and their plant hosts. chs activities in fungi or metazoa are usually described as producing long crystalline chains of chitin but it is possible that a bacterial chs secretes small soluble chitosaccharides, or eventually chitooligosaccharides, instead. indeed, the phytopathogenic oomycete aphanomyces euteiches does not secrete chitin fibers although two chs genes are present in this species. however, the encoded chs activities of a. euteiches are thought to be active because a small soluble and noncrystalline glucan-chitosaccharide was detected in its cell wall. interestingly, one of the two chs genes in d. dadantii ech and the chs gene of pseudomonas cichorii are co-located and have the same orientation as a putative chitoporin-encoding gene. in the marine bacterium vibrio harveyi, chitoporin is a transporter of cos through the outer membrane. cos are known to play a key role as signal molecules in multiple plant-microbe interactions. in parasitic interactions between plants and fungi, cos are released from the digestion of the fungal cell wall by secreted plant chitinases. the resulting cos are then recognized by plant receptors as microbial associated molecular patterns and they induce plant immunity. in symbiotic interactions, modified cos, called lipochitooligosaccharides, which include myc and nod factors, modulate plant host immunity. myc factors are produced and secreted by arbuscular mycorrhizal fungi but the enzymes responsible for their biosynthesis have not yet been identified. nod factors are synthesized by nodc, a bacterial gt homologous to chs, and they induce nodule formation during a symbiotic interaction between rhizobiacea and leguminous host plants. whether bacterial chs genes are able to synthesize cos implicated in plant interactions is still uncertain. several bacterial chs genes are fused with a putative aminotransferase domain and they are colocated with a putative sugar epimerase-encoding gene. these two additional functions could be associated with modifications of chitin or cos. a gene of unknown functions, and composed of the duf domain, was also found colocated with several bacterial chs. the encoded duf1501-containing protein carries a twin-arginine motif which would imply that it is exported by the twin-arginine translocation pathway. the bacterial tat system allows folded proteins to be moved across membranes without significant ion leakage. the duf domain has also been described as forming part of a conserved machinery in compartimentalized species from the planctomycetes, verrucomicrobia and chlamydiae super-phylum. these informations about duf proteins are hardly connectable with the biological role of known chs. however, our hypothesis is that duf duf and chs proteins might have a functional link in bacteria as they share a common conserved operon organization. it would be very interesting to study the function of bacterial chs and also of the proteins that seem associated to them. finally, in the particular cases of teredinibacter turnerae and cellvibrio mixtus subsp. mixtus, the chs genes are localized in the middle of secondary metabolism gene clusters and they could be involved in the production of a bioactive molecule.  bacterial chitin synthase genes constitute a new example of genes acquired by a bacterium via horizontal transfer from a eukaryotic donor. the chitin synthase activity of the bacterial chs and the possible selective advantage for the corresponding bacteria, often implied in plant interactions, needs further investigation. chs-encoding genes have also been transferred between eukaryotic microorganisms. we recommend avoiding the use of this multigenic family to elucidate the phylogenetic relationships between the different eukaryotic species, especially since many duplications and losses are also observed in different lineages. fungal chs are, unfortunately, not an exception and the difficulty of classifying these sequences has led to discordant classifications. we took advantage of the current study to determine which can be robustly classified and then to construct the most consensual classification possible. to facilitate the use of this new classification, any information that corresponds with the previously published versions is provided, together with the databank, in fasta format, of all the classified chs sequences. a website also permits blast queries to the databank. this facilitates a search for the class of a previously identified chs, even if the corresponding accession number has changed. it is also an aid for the classification of chs from species closely related to those analyzed. 
however, it is likely that several horizontal gene transfers also occurred in eukaryotic microorganisms and, even more surprisingly, in bacteria. there are several contradictory classifications of chs isoenzymes and, as regards their evolutionary history, their origin and diversity is still a matter of debate. this allowed us to revise and unify the fungal chs classification and to study the evolutionary history of the chs multigenic family. concerning the evolutionary history of chs, this family has mainly evolved via duplications and losses. it also highlights the importance of hgt in the evolutionary history of chs and describes bacterial chs genes for the first time. phylogenetic analyses were performed with special care to avoid any pitfalls associated with the peculiarities of these sequences. this polysaccharide is produced by type glycosyltransferases, called chitin synthases. a genome-wide analysis resulted in the detection of more than eight hundred putative chitin synthases in proteomes associated with about genomes. many of the bacteria that have acquired a chitin synthase are plant pathogens. chitin synthasebacteriaevolutionclassificationhorizontal gene transferfungiissue-copyright-statement© the author 2016 this comprehensive multi-species analysis contributes to the classification of fungal chs, in particular by optimizing its robustness, consensuality and accessibility.
 chitin, the second most abundant biopolymer on earth after cellulose, is found in probably all fungi, many animals, several protists and a few algae, playing an essential role in the development of many of them. this polysaccharide is produced by type glycosyltransferases, called chitin synthases. there are several contradictory classifications of chs isoenzymes and, as regards their evolutionary history, their origin and diversity is still a matter of debate.  a genome-wide analysis resulted in the detection of more than eight hundred putative chitin synthases in proteomes associated with about genomes. phylogenetic analyses were performed with special care to avoid any pitfalls associated with the peculiarities of these sequences. this allowed us to revise and unify the fungal chs classification and to study the evolutionary history of the chs multigenic family. this update has the advantage of being user-friendly due to the development of a dedicated website, and it includes any correspondences with previously published classifications and mutants. concerning the evolutionary history of chs, this family has mainly evolved via duplications and losses. however, it is likely that several horizontal gene transfers also occurred in eukaryotic microorganisms and, even more surprisingly, in bacteria.  this comprehensive multi-species analysis contributes to the classification of fungal chs, in particular by optimizing its robustness, consensuality and accessibility. it also highlights the importance of hgt in the evolutionary history of chs and describes bacterial chs genes for the first time. many of the bacteria that have acquired a chitin synthase are plant pathogens. whether they are able to produce a chitin exopolysaccharide or secrete chitooligosaccharides requires further investigation. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords chitin synthasebacteriaevolutionclassificationhorizontal gene transferfungiissue-copyright-statement© the author 2016 