BACKGROUND
the short-chain dehydrogenase/reductase  superfamily consists of nad-dependent oxidoreductases that are distinct from the medium-chain dehydrogenase/reductase  and aldo-keto reductase  superfamilies  <cit> .
our knowledge of these superfamilies initially emerged from observations made concerning alcohol dehydrogenases of drosophila and mammalian liver, which were seen to be clearly different  <cit> .
insect and bacterial alcohol and polyol dehydrogenases initially received less attention, since these enzymes were found to be different, and were only considered to be of prokaryotic and lower eukaryotic origin  <cit> .
however, the discovery of similarities between these latter enzymes and human or mammalian prostaglandin, hydroxyl-steroid and other dehydrogenases, changed that view dramatically  <cit> .
in addition, in recent years, interest in sdr enzymes has increased, since they are useful in biotechnological and analytical processes.
about 25% of all dehydrogenases belong to the sdr superfamily  <cit> .
common to all types of oxidoreductases is the occurrence of a rossmann-fold dinucleotide cofactor-binding motif, which has been found to be one of the most common protein folds  <cit> .
among sdr, no high sequence identity between different members is observed , but all of them display a highly similar 3d structure, typically folding into a simple one-domain architecture with distinct conserved motifs, including the cofactor binding site at the n-terminal, structure stabilizing motifs, the active center, catalysis-enhancing sites and the substrate binding site, located in the highly variable c-terminal region  <cit> .
such a degree of 3d structure conservation indicates that ancestral dehydrogenases existed within each sdr family, and after multiple events, these ancestral dehydrogenases gave rise to the present system of subfamilies and classes found within each family  <cit> .
given the early origin of sdr, the subsequent divergence has had time to become quite pronounced.
hundreds of sdr enzyme activities and their corresponding families have been detected.
based on the similar coenzyme-binding structure, their active-site relationship and repetitive patterns, five sdr superfamily types have been discerned from different data banks, named as “classical”, “extended”, “intermediate”, “divergent” and “complex” sdr enzymes  <cit> .
this divergence also includes different enzymatic activities, most of them dehydrogenases or reductases, but also lyases and some isomerases.
the active-site tyr residue, assisted by adjacent lys, asn and ser residues, has been found to fit to the basic reaction mechanism in most cases, but also to reflect acid–base catalysis and proton transfers  <cit> .
thus, sdr proteins not only have a very distant origin, including a viral representation  <cit> , presumably from a time when virally-mediated lateral gene transfer commonly occurred  <cit> , but also show a wide range of activities, involving half of all enzyme activity types.
few gene/protein superfamilies exhibit this great divergence.
recently, a sustainable and expandable nomenclature sdr database has been proposed, based on hidden markov models   <cit> .
this database has identified  <dig> sdr families, encompassing about  <dig>  members  <cit> .
among them, the sdr196c family  groups bacterial sorbitol dehydrogenases , which are of industrial interest for the specific determination of sorbitol , a natural acyclic polyol found in food, and in pharmaceutical and cosmetic preparations  <cit> .
in this study, we provide a comprehensive insight into the distribution, diversity, evolution and classification of the sdr196c superfamily in bacteria.
the phylogenetic analysis revealed different lineages related to some sequence differences in the conserved sdr motifs and in the characteristic sdh blocks, allowing, for the first time, the classification of this sdr family  into  <dig> different lineages and three sub lineages.
this could permit a more efficient data curation, and a new nomenclature for the classification of incoming sequences into the sdr196c family.
RESULTS
distribution of sdh gene
the sdr database  is a sustainable and expandable nomenclature database  <cit> , which includes  <dig> bacterial sorbitol dehydrogenases  within the sdr196c family.
some identical sequences have been included two or more times in the database, representing different strains.
in order to simplify the study, only one strain of these species was included in the analysis.
the sdh gene was found in  <dig> bacterial species, all of them gram-negative belonging to alpha -, beta - and gamma -proteobacteria , with both low and high gc representatives .
this distribution of sdh agrees with the prevalence of sdr enzymes, which are present in all forms of life  <cit>  and, according to recent data from random genome screenings of microorganisms and viruses from sea water, are also among the most abundant genes in nature  <cit> .
α-proteobacteria, with six families, is the largest group with a total of  <dig> species, including members of the acetobacteriace, rhodospirillaceae, rhodobacteriaceae, brucellaceae, phyllobacteriaceae and rhizobiaceae families .
two of these species, ochrobactrum anthropi and o. intermedium  are human pathogens that cause septicemia  <cit> .
the acetobacteriaceae family is represented by two members, acidiphilium cryptum and gluconoacetobacter hansenii, which are common in vinegar and used as iron contamination indicators  <cit> .
members of the rhodospirilaceae, phyllobacteriaceae and rhizobiaceae families are usually nitrogen-fixing microorganisms found in soil and aquatic habitats, with the exception of agrobacterium tumefaciensa.
radiobacter and a. vitis from the rhizobiaceae family, which are well known plant pathogens, causing tumors.
rhodobacteriaceae is the most numerous family, with  <dig> members, including sea water microorganisms, photosynthetic bacteria  and two extremophiles .
the β-proteobacteria group includes  <dig> members of the burkholderiaceae family and  <dig> of the commamonadaceae family .
these two families are composed of soil and free-living microorganisms, which are usually nitrogen-fixing  or symbionts .
the burkholderiaceae family also includes a commensal of the earthworm nephridia , a plant pathogen  and human pathogens from genera burkholderia, which cause opportunistic infections in diseases, such as cystic fibrosis  <cit> .
the γ-proteobacteria group, which includes only  <dig> species from three different bacterial families, is the smallest group among the sdr196c family.
aquatic bacteria from the halophilic family halomonadaceae  and from the marine family oceanospirillaceae  form part of this group.
the third family of this group  includes a plant commensal , a plant pathogen  which cause olive knot  <cit> , a saprophyte , and a soil bacterium .
taken together, these results indicate that the sdh gene is widely distributed in nature, and is an important enzyme in the polyol metabolism of some bacteria, to use sorbitol as an alternative source of carbon and energy  <cit> .
genetic organization of sdh gene
the three main bacterial groups that contain the sdh gene, α-, β- and γ-proteobacteria, encode this gene in different polyol clusters with a different gene order .
in general terms, the sdh gene in the polyol operon is usually surrounded by a transporter , an sdr protein  and a sugar related kinase, such as ribitol kinase.
however, the companion genes and the order in the cluster, vary between bacterial families, and, to a lesser extent, within families .
only one overall organization of sdh genes has been described for the polyol operon of rhodobacter sphaeroides si <dig>  where smos gene encodes for an sdh, smok for an abc transporter, and mtlk for a mannitol dehydrogenase  <cit> .
within the α-proteobacteria, there are twelve variants of the sdh cluster, each family having its own gene order.
the rhodobacteriaceae family shows three variants of this polyol operon, in which a cluster of four genes related with abc transporter are on one side of the sdh gene, and a dehydrogenase  plus an extra gene  on the other side .
the rhizobiaceae family has more diversity in its polyol cluster, which displays  <dig> different variants , but still shows the pattern of at least three abc genes on one side of the sdh gene, except for agrobacterium tumefaciens and rhizobium etli, in which two abc genes are replaced by two sugar kinase genes  .
on the other side of the sdh gene, a dehydrogenase  is also present, except for agrobacterium radiobacter, which presents an arac regulator, followed by metal-accepting chemotaxis sensory transducer macst .
this microorganism also has a lysr gene, indicating a tight regulation of the sdh related genes in order to use this sugar and to control its metabolism under adverse conditions.
this control is also seen in a. tumefaciens and r. etli , with the presence of laci.
the rest of the α-proteobacteria families  have the common pattern of at least three abc proteins on one side, but are more diverse on the other side, having not only kinases  but also two singular enzymes in sdh clusters, which are related with phosphogluconate  , or two consecutive dehydrogenases .
the lysr gene is also present, except in the rhodospirillaceae family.
among the β-proteobacteria group, the commamonadaceae family has its own order, but with the presence of an intercalating mdh gene between sdh and abc transporter genes .
the latter order is also similar to that of verminephrobacter eiseniae, a member of the burkholderiaceae family , which is in fact, quite different from the common pattern displayed for the rest of the burkholderiaceae members .
the latter clusters show a ferric uptake regulator gene and its corresponding cation abc transporter genes on one side, and sorbitol/mannitol abc transport genes followed by had gene on the sdh gene side , and sometimes interrupted by two sugar related genes .
finally, the γ-proteobacteria group has no specific pattern, and it is easy to differentiate the halomonadaceae family , with an haloacid dehalogenase gene intercalating the sdh and three abc transporter proteins, from the pseudomonadaceae family , in which only one  abc transporter gene is present, together with an arac gene.
this latter family also lacks the second sdr gene, indicating that the sdh gene is not close to other the sugar-utilizing genes as it is in all of the above described families.
signatures of horizontal gene transfer
basically, there are two main methods to identify putative horizontal gene transfer events, phylogenetic methods and surrogate methods based on nucleotide composition.
also, the presence of transposases and/or integrases within a region may suggest another mode of transfer.
however, no such enzymes genes were found in the proximity of any of the polyol clusters described above.
on the other hand, the differences between the average gc content of whole genome  and the gc content of the sdh genes  , showed only one unknown possible horizontal gene transfer event  in gluconoacetobacter hansenii with a gc difference of − <dig>  .
in addition, loktanella vestfoldensis, agrobacterium radiobacter, chromohalobacter salexigensis, pseudomonas savastanoi and oceanicola batsensis have a high, but not significant, gc difference , which suggest the possibility of horizontal gene transfer for sdh in these species, too.
phylogenetic analysis of sdh gene
in order to examine further the evolutionary history of the sdh gene, a multiple sequence alignment  was carried out with guidance  <cit> , using the msa algorithm prank  <cit> , which gave an overall quality assessment exceeding  <dig>   .
the phylogenetic analysis and the topology obtained were compared with that found for the species tree based on 16s rrna sequences aligned with the above algorithm .
phylogenetic analyses of sdh amino acid sequences resulted in a well-resolved tree, which was quite similar, regardless of the method used .
overall, the sdh genes in the three proteobacteria groups studied did not form three distinct lineages  as it does, in the 16s rrna tree .
indeed, the sdh tree could be subdivided into six main lineages  , lineage  <dig> being the most divergent, encompassing α- and γ-proteobacteria from five different families .
also within this lineage, three sublineages  were found, all with a common origin.
lineages  <dig>  and  <dig>  were formed by species of the rhodobacteriaceae family, except hoeflea phototrophobica, which is member of the phyllobacteriaceae family.
lineage  <dig>  was the most divergent group, with members of α- and γ-proteobacteria from five different families: phyllobacteriaceae, pseudomonaceae, rhodobacteriaceae, rhodospirillaceae and rhizobiaceae .
this lineage  <dig> is also grouped the most sdh from the rhodobacteriaceae family, except r. bacterium and thalassiobium sp.
interestingly, the three members of genus rhodobacter did not group as closely as might be expected, r. capsulatus being a member of lineage  <dig>  and r. sphaeroides and rhodobacter sp.
members of lineage  <dig> .
this indicates a common ancestor, with a divergence in the time of sdh gene acquisition.
similarly, pseudomonaceae members were grouped in lineage  <dig>  except for pseudomonas sp., which belongs to lineage  <dig>  together with all members of burkholderiaceae family .
the distribution of the sdh from different pseudomonas species in divergent branches of the tree  indicates that in these species, the sdh genes were acquired many times and from different sources.
lineage  <dig> contained most of the members of β-proteobacteria family, except for the commamonadaceae representatives, which branches away in lineage  <dig>  in addition, lineage  <dig> branches firmly away from the rest of the lineages, suggesting that the origin of sdh in lineage  <dig> is unique, and that there are two different origins for β-proteobacteria sdh.
lineage  <dig> is also a divergent group which includes, apart from commamonadaceae sdh proteins, members of α- and γ-proteobacteria from the rhodobacteriaceae and oceanospirillaceae families.
the presence of members of the rhodobacteriaceae family  clearly separated from lineage  <dig>  indicated the possibility of a horizontal gene transfer event, although this is not supported by the gc difference, with values of − <dig>  and  <dig> , respectively, or by the presence of transposases/integrases within its polyol cluster.
lineages  <dig>   <dig> and  <dig> are well separated groups, of different but close origin.
members of halomonadaceae , acetobacteriaceae , brucellaceae and rhizobiaceae  families are included in these lineages.
agrobacterium tumefaciens and rhizobium etli did not group with the rest of their family in lineage  <dig>  indicating a divergent origin of sdh in this family.
interestingly, γ-proteobacteria had at least one member in four of the six lineages described , which suggests a divergent origin of the sdh gene among γ-proteobacteria.
this widespread and variable origin of sdh detailed here is related to the distribution and evolution of sdr, which were mentioned above, occurs in all kingdoms of life  <cit> .
however, this variability is not observed in the structure of these enzymes in all six lineages , which all share a common rossmann-fold motif for dinucleotide cofactor binding, and a substrate binding site in the highly variable c-terminal region  <cit> .
this variability in the distribution and the homogeneity in the structure, together with the recombinatorial formation of the catalytic subunit from building blocks, suggest that sdr, and consequently sdh, emerged early from α/β elements to a form a rossman-fold domain in the universal cellular ancestor prior to darwinian evolution in cells of all kingdoms of life  <cit> .
sequence comparison of sdh lineages
with the aim of further exploring the evolution of sdh proteins,  <dig> characteristic sdh blocks  were described using the sequence alignment of the six different lineages and the  <dig> sublineages of lineage  <dig>  overall, differences in the blocks between the six lineages were well defined, and it was possible to establish different consensus sequences for each lineage with guidance scores above  <dig>  , which supports the classification obtained from the phylogenetic analysis.
lineages  <dig> and  <dig> were the most variable of the lineages , as might be expected according to the phylogenetic analysis , although it was still possible to establish consensus sequences.
the eleven sdh conserved blocks were also observed in the three sublineages of lineage  <dig> with guidance scores above  <dig>  .
sequence alignment of lineage  <dig> showed highly conserved blocks  among its members.
this high degree of sequence similarity was in agreement with that described in the phylogenetic analysis, since lineage  <dig> was basically composed of members of the burkholderiaceae family, except for pseudomonas sp.
the specific block sequence for this lineage is indicated in figure  <dig>  and, interestingly, the sequence of block i , which is involved in nad+ binding  <cit>  and the sequence of block x , which is related to nad stabilization and tetramer formation, can be considered as fingerprints for this lineage, since these sequences were only present in this lineage.
lineage  <dig>  as described above, was highly divergent, as also shown in the sequence alignment , where only the blocks corresponding to the characteristic fingerprints for sdh  and some parts of the c-ter are conserved.
block xi and its sequence nvms could be considered as its fingerprint.
lineage  <dig>  which comprises only two members, showed a high sequence identity .
its long and conserved block iii, whose function is to stabilize the central β-sheet  <cit> , could be considered as its fingerprint, ending in the conserved sequence dmapvlev .
the most notable feature of lineage  <dig> alignment is the absence of block i in g. hansenii, which is involved in cofactor recognition .
taking this into account, its assignation as an sdh or even as an sdr might be erroneous, although the remaining conserved domains of both sdr and sdh, including the catalytic tetrad n-y-s-k, responsible of the oxidoreductase activity  <cit> , are present.
thus, the absence of block i is an error which arises from wrongly determining the starting point of sequence.
lineage  <dig> was also highly conserved , and showed the eleven blocks of sdh with high identity.
specific fingerprints of this lineage are located in block iv , and at the end of block ix, which in the other lineages usually ends with grmg, while in lineage  <dig> it ends in fatp .
the heterogeneity of lineage  <dig> makes it difficult to establish a fingerprint, except for the consensus sequence of block v .
however, when the three sublineages were aligned independently, a high degree of conservation was observed .
lineage  <dig>  was the most divergent, showing identity only in the eleven conserved blocks .
although consensus sequences are shown for every block, only a specific sequence for this sublineage was observed at the beginning of block viii .
similarly, in lineage  <dig> , specific fingerprints could be found in block iii , and at the end of block viii .
lineage  <dig>  had its own fingerprint at the end of block iii .
in addition, some lineages also presented highly conserved sequences between conserved blocks.
thus, lineages  <dig>   <dig> and  <dig> showed such a sequence between motifs i and ii , and another between block iv and v in lineages  <dig>   <dig>   <dig> and  <dig> .
these latter residues are part of the large α <dig>  which is part of the sorbitol binding domain in sdh.
such a degree of conservation and the 3d proximity to blocks iv and v, which are part of the active site, suggests a role for these interblock sequences in making up the correct structure of the active site or the substrate binding site.
to expand the above analysis, a study of sdh family functional divergence was carried out to detect amino acid sites that have varying evolutionary conservation among member genes, using diverge  software  <cit> .
the analysis grouped the amino acids residues responsible for altered functional constraints into two categories:  conserved in the first lineage, but variable in the second lineage;  conserved in the second lineage, but variable in the first lineage.
a site-specific profile based on probability  was used to identify critical amino acids  <cit> , with a qk >  <dig>  .
among the six lineages, only i, ii, v and vi were relevant .
in fact, when lineages i-ii were compared, only  <dig> amino acids  were conserved in category ii.
lineages i-v showed only one amino acid  in category i, and two  in category ii.
the divergence was clearly more pronounced between lineage i and vi, with four amino acids  in category i and  <dig>  in category ii.
this divergence pattern was also observed between lineages ii and vi, with four amino acids  in category i and  <dig>  in category ii.
finally, the divergence between lineage v and vi was reduced to only one amino acid  in category i.
to visualize these divergence sites, a 3d representation was carried out for each lineage  and for all sites together using rhodobacter sphaeroides sequence and its crystal structure   <cit>  .
at first sight, it was clear that divergent amino acids are basically outside the main conserved blocks, clearly indicating that the drift at these sites  is well tolerated by the structure with no loss of activity.
the changes are outside block i , block iii , block vii  and blocks viii and ix .
however, as shown in figure 6b, the main changes are located in and around β <dig>  which contains one of the amino acids of the catalytic tetrad , which is not affected.
thus, it could be concluded that the sequence alignment of the different lineages obtained according to the phylogenetic and functional divergence analysis showed specific fingerprints for each lineage, whose conserved sequences could be very useful for the future classification of sdh enzymes.
CONCLUSIONS
sdr196c family encompasses short chain sdh of prokaryotic origin.
the distribution of this family is limited to gram-negative bacteria, grouping members of the α-, β- and γ-proteobacteria.
this distribution is in agreement with the widespread nature of sdr, and indicates that sorbitol metabolism is of importance among these bacteria as an alternative source of carbon and energy.
this variability is also observed in the genetic organization of polyol operon among the different species, and in the phylogenetic analysis, which clearly point to different origins for sdh in bacteria, although the 3d structure among groups is highly conserved, with the typical rossmann fold motif.
such a degree of divergence in the origin but similarity in the structure suggests that sdhs are of extremely old, and emerged early in the evolution, giving rise to the six different lineages and the three sublineages observed in the phylogenetic analysis.
this phylogenetic classification is supported by the sequence differences found in the conserved blocks of sdh, allowing for the first time, the classification of the sdr196c family into different subgroups, and introducing the possibility of expanding the actual classification of sdr enzymes by two extra numbers, the lineage and sublineage, separated by a point.
as an example, r. capsulatus sdh could be classified as sdr196c <dig> .

 the short-chain dehydrogenase/reductase superfamily consists of nad-dependent oxidoreductases that are distinct from the medium-chain dehydrogenase/reductase and aldo-keto reductase superfamilies. our knowledge of these superfamilies initially emerged from observations made concerning alcohol dehydrogenases of drosophila and mammalian liver, which were seen to be clearly different. insect and bacterial alcohol and polyol dehydrogenases initially received less attention, since these enzymes were found to be different, and were only considered to be of prokaryotic and lower eukaryotic origin. however, the discovery of similarities between these latter enzymes and human or mammalian prostaglandin, hydroxyl-steroid and other dehydrogenases, changed that view dramatically. in addition, in recent years, interest in sdr enzymes has increased, since they are useful in biotechnological and analytical processes. about 25% of all dehydrogenases belong to the sdr superfamily. common to all types of oxidoreductases is the occurrence of a rossmann-fold dinucleotide cofactor-binding motif, which has been found to be one of the most common protein folds. among sdr, no high sequence identity between different members is observed, but all of them display a highly similar 3d structure, typically folding into a simple one-domain architecture with distinct conserved motifs, including the cofactor binding site at the n-terminal, structure stabilizing motifs, the active center, catalysis-enhancing sites and the substrate binding site, located in the highly variable c-terminal region. such a degree of 3d structure conservation indicates that ancestral dehydrogenases existed within each sdr family, and after multiple events, these ancestral dehydrogenases gave rise to the present system of subfamilies and classes found within each family. given the early origin of sdr, the subsequent divergence has had time to become quite pronounced. hundreds of sdr enzyme activities and their corresponding families have been detected. based on the similar coenzyme-binding structure, their active-site relationship and repetitive patterns, five sdr superfamily types have been discerned from different data banks, named as “classical”, “extended”, “intermediate”, “divergent” and “complex” sdr enzymes. this divergence also includes different enzymatic activities, most of them dehydrogenases or reductases, but also lyases and some isomerases. the active-site tyr residue, assisted by adjacent lys, asn and ser residues, has been found to fit to the basic reaction mechanism in most cases, but also to reflect acid–base catalysis and proton transfers. thus, sdr proteins not only have a very distant origin, including a viral representation, presumably from a time when virally-mediated lateral gene transfer commonly occurred, but also show a wide range of activities, involving half of all enzyme activity types. few gene/protein superfamilies exhibit this great divergence. recently, a sustainable and expandable nomenclature sdr database has been proposed, based on hidden markov models. this database has identified sdr families, encompassing about members. among them, the sdr196c family groups bacterial sorbitol dehydrogenases, which are of industrial interest for the specific determination of sorbitol, a natural acyclic polyol found in food, and in pharmaceutical and cosmetic preparations. in this study, we provide a comprehensive insight into the distribution, diversity, evolution and classification of the sdr196c superfamily in bacteria. the phylogenetic analysis revealed different lineages related to some sequence differences in the conserved sdr motifs and in the characteristic sdh blocks, allowing, for the first time, the classification of this sdr family into different lineages and three sub lineages. this could permit a more efficient data curation, and a new nomenclature for the classification of incoming sequences into the sdr196c family.  distribution of sdh gene the sdr database is a sustainable and expandable nomenclature database, which includes bacterial sorbitol dehydrogenases within the sdr196c family. some identical sequences have been included two or more times in the database, representing different strains. in order to simplify the study, only one strain of these species was included in the analysis. the sdh gene was found in bacterial species, all of them gram-negative belonging to alpha -, beta - and gamma -proteobacteria, with both low and high gc representatives. this distribution of sdh agrees with the prevalence of sdr enzymes, which are present in all forms of life and, according to recent data from random genome screenings of microorganisms and viruses from sea water, are also among the most abundant genes in nature. α-proteobacteria, with six families, is the largest group with a total of species, including members of the acetobacteriace, rhodospirillaceae, rhodobacteriaceae, brucellaceae, phyllobacteriaceae and rhizobiaceae families. two of these species, ochrobactrum anthropi and o. intermedium are human pathogens that cause septicemia. the acetobacteriaceae family is represented by two members, acidiphilium cryptum and gluconoacetobacter hansenii, which are common in vinegar and used as iron contamination indicators. members of the rhodospirilaceae, phyllobacteriaceae and rhizobiaceae families are usually nitrogen-fixing microorganisms found in soil and aquatic habitats, with the exception of agrobacterium tumefaciensa. radiobacter and a. vitis from the rhizobiaceae family, which are well known plant pathogens, causing tumors. rhodobacteriaceae is the most numerous family, with members, including sea water microorganisms, photosynthetic bacteria and two extremophiles. the β-proteobacteria group includes members of the burkholderiaceae family and of the commamonadaceae family. these two families are composed of soil and free-living microorganisms, which are usually nitrogen-fixing or symbionts. the burkholderiaceae family also includes a commensal of the earthworm nephridia, a plant pathogen and human pathogens from genera burkholderia, which cause opportunistic infections in diseases, such as cystic fibrosis. the γ-proteobacteria group, which includes only species from three different bacterial families, is the smallest group among the sdr196c family. aquatic bacteria from the halophilic family halomonadaceae and from the marine family oceanospirillaceae form part of this group. the third family of this group includes a plant commensal, a plant pathogen which cause olive knot, a saprophyte, and a soil bacterium. taken together, these results indicate that the sdh gene is widely distributed in nature, and is an important enzyme in the polyol metabolism of some bacteria, to use sorbitol as an alternative source of carbon and energy. genetic organization of sdh gene the three main bacterial groups that contain the sdh gene, α-, β- and γ-proteobacteria, encode this gene in different polyol clusters with a different gene order. in general terms, the sdh gene in the polyol operon is usually surrounded by a transporter, an sdr protein and a sugar related kinase, such as ribitol kinase. however, the companion genes and the order in the cluster, vary between bacterial families, and, to a lesser extent, within families. only one overall organization of sdh genes has been described for the polyol operon of rhodobacter sphaeroides si where smos gene encodes for an sdh, smok for an abc transporter, and mtlk for a mannitol dehydrogenase. within the α-proteobacteria, there are twelve variants of the sdh cluster, each family having its own gene order. the rhodobacteriaceae family shows three variants of this polyol operon, in which a cluster of four genes related with abc transporter are on one side of the sdh gene, and a dehydrogenase plus an extra gene on the other side. the rhizobiaceae family has more diversity in its polyol cluster, which displays different variants, but still shows the pattern of at least three abc genes on one side of the sdh gene, except for agrobacterium tumefaciens and rhizobium etli, in which two abc genes are replaced by two sugar kinase genes. on the other side of the sdh gene, a dehydrogenase is also present, except for agrobacterium radiobacter, which presents an arac regulator, followed by metal-accepting chemotaxis sensory transducer macst. this microorganism also has a lysr gene, indicating a tight regulation of the sdh related genes in order to use this sugar and to control its metabolism under adverse conditions. this control is also seen in a. tumefaciens and r. etli, with the presence of laci. the rest of the α-proteobacteria families have the common pattern of at least three abc proteins on one side, but are more diverse on the other side, having not only kinases but also two singular enzymes in sdh clusters, which are related with phosphogluconate, or two consecutive dehydrogenases. the lysr gene is also present, except in the rhodospirillaceae family. among the β-proteobacteria group, the commamonadaceae family has its own order, but with the presence of an intercalating mdh gene between sdh and abc transporter genes. the latter order is also similar to that of verminephrobacter eiseniae, a member of the burkholderiaceae family, which is in fact, quite different from the common pattern displayed for the rest of the burkholderiaceae members. the latter clusters show a ferric uptake regulator gene and its corresponding cation abc transporter genes on one side, and sorbitol/mannitol abc transport genes followed by had gene on the sdh gene side, and sometimes interrupted by two sugar related genes. finally, the γ-proteobacteria group has no specific pattern, and it is easy to differentiate the halomonadaceae family, with an haloacid dehalogenase gene intercalating the sdh and three abc transporter proteins, from the pseudomonadaceae family, in which only one abc transporter gene is present, together with an arac gene. this latter family also lacks the second sdr gene, indicating that the sdh gene is not close to other the sugar-utilizing genes as it is in all of the above described families. signatures of horizontal gene transfer basically, there are two main methods to identify putative horizontal gene transfer events, phylogenetic methods and surrogate methods based on nucleotide composition. also, the presence of transposases and/or integrases within a region may suggest another mode of transfer. however, no such enzymes genes were found in the proximity of any of the polyol clusters described above. on the other hand, the differences between the average gc content of whole genome and the gc content of the sdh genes, showed only one unknown possible horizontal gene transfer event in gluconoacetobacter hansenii with a gc difference of −. in addition, loktanella vestfoldensis, agrobacterium radiobacter, chromohalobacter salexigensis, pseudomonas savastanoi and oceanicola batsensis have a high, but not significant, gc difference, which suggest the possibility of horizontal gene transfer for sdh in these species, too. phylogenetic analysis of sdh gene in order to examine further the evolutionary history of the sdh gene, a multiple sequence alignment was carried out with guidance, using the msa algorithm prank, which gave an overall quality assessment exceeding. the phylogenetic analysis and the topology obtained were compared with that found for the species tree based on 16s rrna sequences aligned with the above algorithm. phylogenetic analyses of sdh amino acid sequences resulted in a well-resolved tree, which was quite similar, regardless of the method used. overall, the sdh genes in the three proteobacteria groups studied did not form three distinct lineages as it does, in the 16s rrna tree. indeed, the sdh tree could be subdivided into six main lineages, lineage being the most divergent, encompassing α- and γ-proteobacteria from five different families. also within this lineage, three sublineages were found, all with a common origin. lineages and were formed by species of the rhodobacteriaceae family, except hoeflea phototrophobica, which is member of the phyllobacteriaceae family. lineage was the most divergent group, with members of α- and γ-proteobacteria from five different families: phyllobacteriaceae, pseudomonaceae, rhodobacteriaceae, rhodospirillaceae and rhizobiaceae. this lineage is also grouped the most sdh from the rhodobacteriaceae family, except r. bacterium and thalassiobium sp. interestingly, the three members of genus rhodobacter did not group as closely as might be expected, r. capsulatus being a member of lineage and r. sphaeroides and rhodobacter sp. members of lineage. this indicates a common ancestor, with a divergence in the time of sdh gene acquisition. similarly, pseudomonaceae members were grouped in lineage except for pseudomonas sp., which belongs to lineage together with all members of burkholderiaceae family. the distribution of the sdh from different pseudomonas species in divergent branches of the tree indicates that in these species, the sdh genes were acquired many times and from different sources. lineage contained most of the members of β-proteobacteria family, except for the commamonadaceae representatives, which branches away in lineage in addition, lineage branches firmly away from the rest of the lineages, suggesting that the origin of sdh in lineage is unique, and that there are two different origins for β-proteobacteria sdh. lineage is also a divergent group which includes, apart from commamonadaceae sdh proteins, members of α- and γ-proteobacteria from the rhodobacteriaceae and oceanospirillaceae families. the presence of members of the rhodobacteriaceae family clearly separated from lineage indicated the possibility of a horizontal gene transfer event, although this is not supported by the gc difference, with values of − and, respectively, or by the presence of transposases/integrases within its polyol cluster. lineages and are well separated groups, of different but close origin. members of halomonadaceae, acetobacteriaceae, brucellaceae and rhizobiaceae families are included in these lineages. agrobacterium tumefaciens and rhizobium etli did not group with the rest of their family in lineage indicating a divergent origin of sdh in this family. interestingly, γ-proteobacteria had at least one member in four of the six lineages described, which suggests a divergent origin of the sdh gene among γ-proteobacteria. this widespread and variable origin of sdh detailed here is related to the distribution and evolution of sdr, which were mentioned above, occurs in all kingdoms of life. however, this variability is not observed in the structure of these enzymes in all six lineages, which all share a common rossmann-fold motif for dinucleotide cofactor binding, and a substrate binding site in the highly variable c-terminal region. this variability in the distribution and the homogeneity in the structure, together with the recombinatorial formation of the catalytic subunit from building blocks, suggest that sdr, and consequently sdh, emerged early from α/β elements to a form a rossman-fold domain in the universal cellular ancestor prior to darwinian evolution in cells of all kingdoms of life. sequence comparison of sdh lineages with the aim of further exploring the evolution of sdh proteins, characteristic sdh blocks were described using the sequence alignment of the six different lineages and the sublineages of lineage overall, differences in the blocks between the six lineages were well defined, and it was possible to establish different consensus sequences for each lineage with guidance scores above, which supports the classification obtained from the phylogenetic analysis. lineages and were the most variable of the lineages, as might be expected according to the phylogenetic analysis, although it was still possible to establish consensus sequences. the eleven sdh conserved blocks were also observed in the three sublineages of lineage with guidance scores above. sequence alignment of lineage showed highly conserved blocks among its members. this high degree of sequence similarity was in agreement with that described in the phylogenetic analysis, since lineage was basically composed of members of the burkholderiaceae family, except for pseudomonas sp. the specific block sequence for this lineage is indicated in figure and, interestingly, the sequence of block i, which is involved in nad+ binding and the sequence of block x, which is related to nad stabilization and tetramer formation, can be considered as fingerprints for this lineage, since these sequences were only present in this lineage. lineage as described above, was highly divergent, as also shown in the sequence alignment, where only the blocks corresponding to the characteristic fingerprints for sdh and some parts of the c-ter are conserved. block xi and its sequence nvms could be considered as its fingerprint. lineage which comprises only two members, showed a high sequence identity. its long and conserved block iii, whose function is to stabilize the central β-sheet, could be considered as its fingerprint, ending in the conserved sequence dmapvlev. the most notable feature of lineage alignment is the absence of block i in g. hansenii, which is involved in cofactor recognition. taking this into account, its assignation as an sdh or even as an sdr might be erroneous, although the remaining conserved domains of both sdr and sdh, including the catalytic tetrad n-y-s-k, responsible of the oxidoreductase activity, are present. thus, the absence of block i is an error which arises from wrongly determining the starting point of sequence. lineage was also highly conserved, and showed the eleven blocks of sdh with high identity. specific fingerprints of this lineage are located in block iv, and at the end of block ix, which in the other lineages usually ends with grmg, while in lineage it ends in fatp. the heterogeneity of lineage makes it difficult to establish a fingerprint, except for the consensus sequence of block v. however, when the three sublineages were aligned independently, a high degree of conservation was observed. lineage was the most divergent, showing identity only in the eleven conserved blocks. although consensus sequences are shown for every block, only a specific sequence for this sublineage was observed at the beginning of block viii. similarly, in lineage, specific fingerprints could be found in block iii, and at the end of block viii. lineage had its own fingerprint at the end of block iii. in addition, some lineages also presented highly conserved sequences between conserved blocks. thus, lineages and showed such a sequence between motifs i and ii, and another between block iv and v in lineages and. these latter residues are part of the large α which is part of the sorbitol binding domain in sdh. such a degree of conservation and the 3d proximity to blocks iv and v, which are part of the active site, suggests a role for these interblock sequences in making up the correct structure of the active site or the substrate binding site. to expand the above analysis, a study of sdh family functional divergence was carried out to detect amino acid sites that have varying evolutionary conservation among member genes, using diverge software. the analysis grouped the amino acids residues responsible for altered functional constraints into two categories: conserved in the first lineage, but variable in the second lineage; conserved in the second lineage, but variable in the first lineage. a site-specific profile based on probability was used to identify critical amino acids, with a qk > . among the six lineages, only i, ii, v and vi were relevant. in fact, when lineages i-ii were compared, only amino acids were conserved in category ii. lineages i-v showed only one amino acid in category i, and two in category ii. the divergence was clearly more pronounced between lineage i and vi, with four amino acids in category i and in category ii. this divergence pattern was also observed between lineages ii and vi, with four amino acids in category i and in category ii. finally, the divergence between lineage v and vi was reduced to only one amino acid in category i. to visualize these divergence sites, a 3d representation was carried out for each lineage and for all sites together using rhodobacter sphaeroides sequence and its crystal structure. at first sight, it was clear that divergent amino acids are basically outside the main conserved blocks, clearly indicating that the drift at these sites is well tolerated by the structure with no loss of activity. the changes are outside block i, block iii, block vii and blocks viii and ix. however, as shown in figure 6b, the main changes are located in and around β which contains one of the amino acids of the catalytic tetrad, which is not affected. thus, it could be concluded that the sequence alignment of the different lineages obtained according to the phylogenetic and functional divergence analysis showed specific fingerprints for each lineage, whose conserved sequences could be very useful for the future classification of sdh enzymes.  sdr196c family encompasses short chain sdh of prokaryotic origin. the distribution of this family is limited to gram-negative bacteria, grouping members of the α-, β- and γ-proteobacteria. this distribution is in agreement with the widespread nature of sdr, and indicates that sorbitol metabolism is of importance among these bacteria as an alternative source of carbon and energy. this variability is also observed in the genetic organization of polyol operon among the different species, and in the phylogenetic analysis, which clearly point to different origins for sdh in bacteria, although the 3d structure among groups is highly conserved, with the typical rossmann fold motif. such a degree of divergence in the origin but similarity in the structure suggests that sdhs are of extremely old, and emerged early in the evolution, giving rise to the six different lineages and the three sublineages observed in the phylogenetic analysis. this phylogenetic classification is supported by the sequence differences found in the conserved blocks of sdh, allowing for the first time, the classification of the sdr196c family into different subgroups, and introducing the possibility of expanding the actual classification of sdr enzymes by two extra numbers, the lineage and sublineage, separated by a point. as an example, r. capsulatus sdh could be classified as sdr196c. 
finally, this subdivision was further confirmed by the differences observed in the sequence of the conserved blocks described for sdr and some specific blocks of sdh, and by a functional divergence analysis, which made it possible to establish new consensus sequences and specific fingerprints for the lineages and sub lineages. the phylogenetic analysis pointed to six clearly defined lineages and three sub lineages, and great variability in the origin of this gene, despite its well conserved 3d structure. the sdr196c family groups bacterial sorbitol dehydrogenases, which are of great industrial interest. this study also opens up a new and more accurate classification of sdr196c family, introducing two numbers at the end of the family name, which indicate the lineage and the sublineage of each member, i.e, sdr196c. the distribution of the bacterial sdh species analyzed was limited to gram-negative bacteria. sdh distribution agrees with that observed for sdr, indicating the importance of the polyol metabolism, as an alternative source of carbon and energy. six different bacterial families were found, encompassing α-, β- and γ-proteobacteria. according to the obtained trees, six different lineages and three sublineages can be discerned. short chain dehydrogenases/reductases are nad-dependent oxidoreductases with a highly conserved 3d structure and of an early origin, which has allowed them to diverge into several families and enzymatic activities. the phylogenetic analysis also suggested two different origins for sdh in β-proteobacteria and four origins for γ-proteobacteria. a cluster analysis of sorbitol dehydrogenase revealed different types of gene organization, although with a common pattern in which the sdh gene is surrounded by sugar abc transporter proteins, another sdr, a kinase, and several gene regulators.
 short chain dehydrogenases/reductases are nad-dependent oxidoreductases with a highly conserved 3d structure and of an early origin, which has allowed them to diverge into several families and enzymatic activities. the sdr196c family groups bacterial sorbitol dehydrogenases, which are of great industrial interest. in this study, we examine the phylogenetic relationship between the members of this family, and based on the findings and some sequence conserved blocks, a new and a more accurate classification is proposed.  the distribution of the bacterial sdh species analyzed was limited to gram-negative bacteria. six different bacterial families were found, encompassing α-, β- and γ-proteobacteria. this broad distribution in terms of bacteria and niches agrees with that of sdr, which are found in all forms of life. a cluster analysis of sorbitol dehydrogenase revealed different types of gene organization, although with a common pattern in which the sdh gene is surrounded by sugar abc transporter proteins, another sdr, a kinase, and several gene regulators. according to the obtained trees, six different lineages and three sublineages can be discerned. the phylogenetic analysis also suggested two different origins for sdh in β-proteobacteria and four origins for γ-proteobacteria. finally, this subdivision was further confirmed by the differences observed in the sequence of the conserved blocks described for sdr and some specific blocks of sdh, and by a functional divergence analysis, which made it possible to establish new consensus sequences and specific fingerprints for the lineages and sub lineages.  sdh distribution agrees with that observed for sdr, indicating the importance of the polyol metabolism, as an alternative source of carbon and energy. the phylogenetic analysis pointed to six clearly defined lineages and three sub lineages, and great variability in the origin of this gene, despite its well conserved 3d structure. this suggests that sdh are very old and emerged early during the evolution. this study also opens up a new and more accurate classification of sdr196c family, introducing two numbers at the end of the family name, which indicate the lineage and the sublineage of each member, i.e, sdr196c. 