BACKGROUND
the epsilon  proteobacteria comprise one of the five classes within the phylum proteobacteria  <cit> .
these bacteria inhabit a wide variety of ecological niches ranging from gastrointestinal tracts of animals to water reservoirs, sewage, oil-field community and deep-sea hydrothermal vents  <cit> .
recent studies show that ε-proteobacteria comprise a significant proportion of the microbial population in deep-sea hydrothermal vents where, because of their ability to carry out different types of metabolism using a variety of alternate electron donors  and acceptors , they play important role in carbon, nitrogen and sulfur cycles  <cit> .
a great deal of interest in these bacteria stems from the fact that many of these species are host-associated  and comprise important human and animal pathogens  <cit> .
of these bacteria, helicobacter pylori is the causative agent for gastric and peptic ulcers  <cit>  and infections with this and the related species h. hepaticus are important predisposing factors in gastric cancers in humans and liver cancers in rodents  <cit> .
campylobacter jejuni and c. coli are the most common causes of food-born illnesses such as diarrhea worldwide  <cit> .
c. jejuni infection can also lead to the neuromuscular disease guillain-barre syndrome  <cit> , which causes weakness and paralysis of muscles.
in contrast to the pathogenic nature of helicobacter and campylobacter, wolinella succinogenes is a commensal in the gastrointestinal tract of cattle and it is not known to cause any illness in either animals or humans  <cit> .
in addition to the host-associated species, many free-living members which include chemolithotrophic and autotrophic bacteria  also form part of the ε-proteobacterial group  <cit> .
the ε-proteobacteria are presently distinguished from other bacteria based their branching in the 16s rrna trees  <cit> .
although most of these bacteria assume a spiral shape sometime during their life cycle  <cit>  and they can also utilize a variety of electron donors and acceptors , these characteristics are not unique to this group  <cit> .
presently, there is no molecular or biochemical characteristic known that is unique to this group of bacteria.
within ε-proteobacteria, two main orders, campylobacterales and nautiliales, are presently recognized  <cit>  the campylobacterales is made up of three families, campylobacteraceae, helicobacteraceae and hydrogenimonaceae, whereas the nautiliales order is comprised of three genera   <cit> .
except for the 16s rrna, very little sequence information is available for species belonging to the hydrogenimonaceae family and the nautiliales order.
in the past few years, genomic sequences of several ε-proteobacterial species from the campylobacterales order have become available.
the completely sequenced genomes include those from: helicobacter pylori  <dig>  <cit> , h. pylori j <dig> <cit> , h. hepaticus atcc  <dig>  <cit> , campylobacter jejuni nctc  <dig>  <cit> , c. jejuni rm <dig>  <cit> ,wolinella succinogenes dsm  <dig>  <cit>  and thiomicrospira denitrificans atcc  <dig>  <cit> .
in addition, genomes of several campylobacter species  and c. jejuni subsp.
jejuni strains  are now at assembly stage <cit>  and sequence information from them is available in the ncbi database.
the availability of these sequences has opened new windows for discovering novel molecular characteristics that are unique to these bacteria and can be used for their diagnostics as well as for biochemical and functional studies.
earlier comparative genomic studies on ε-proteobacteria have examined a number of aspects of their gene/protein contents  <cit> .
of these, the studies by eppinger et al.
<cit>  and fouts et al.
<cit>  are particularly detailed.
in these works, genes/proteins that are unique to individual genomes were identified as well as genes that are commonly shared by, but not uniquely present, in a number of these bacteria .
pair-wise comparison of the gene contents of these bacteria, functional classification of their genomic inventory, synteny and co-linearity of genes in various genomes, and examples of gene losses as well as recombination were also reported  <cit> .
additionally, coenye and vandamme  <cit>  have carried out studies to identify genes that have been laterally transferred between ε-proteobacteria and other bacteria.
however, thus far no comparative study has examined or identified genes/proteins that are uniquely found in ε-proteobacteria at different taxonomic levels.
such genes and proteins, because of their specificity, provide novel means for diagnostics and taxonomic studies  <cit>  and for discovering important physiological characteristics that are unique to these bacteria.
in our recent work, we have used comparative genomics to identify a large number of signature proteins that are specific for either alpha proteobacteria  <cit> , chlamydiae  <cit>  or actinobacteria  <cit> .
in the present work, we have carried out systematic blast searches on all open reading frames  in the genome of wolinella succinogenes dsm  <dig> and campylobacter jejuni rm <dig> to identify whole genes/proteins  that are unique to ε-proteobacteria.
these studies have led to identification of  <dig> genes/proteins that are uniquely present in various sequenced ε-proteobacteria , as well as many other proteins that are limited to certain subgroups within the campylobacterales order.
additionally, we also describe a number of conserved indels in widely distributed proteins that are specific for either all-available ε-proteobacteria or for certain subgroups among them.
the identified signature proteins and indels comprise rare genetic changes that have been introduced at various stages during the evolution of campylobacterales  and their species distribution patterns are supported by the branching order of these species in phylogenetic trees.
RESULTS
these studies were undertaken to identify molecular characteristics that are uniquely shared by either all sequenced ε-proteobacteria species, or their subgroups, but which generally are not found in any other organism.
three different kinds of molecular signatures that are specific for ε-proteobacteria are described in the present work.
the first of these consists of whole proteins or open reading frames  that are uniquely found in ε-proteobacteria.
the other two characteristics are comprised of rare genetic changes  consisting of either conserved inserts or deletions  in widely distributed proteins that are specific for the ε-proteobacterial homologs as well as a gene-fusion event within this group of bacteria.
a brief description of these molecular signatures and their evolutionary significances are discussed below.
whole proteins or orfs that are unique for the epsilon-proteobacteria  and helicobacteraceae family
the ε-proteobacteria-specific proteins were identified as described in the methods section.
generally, a protein was considered to be epsilon-proteobacteria specific if all significant alignments  in a psi-blast search with the query protein were from ε-proteobacteria species.
in a few cases, where the e values of  <dig> or  <dig> hits from other species also exhibited borderline significance, but there was a large increase in e value from the last ε-proteobacteria hit in the search to these other proteins, such proteins were also regarded as ε-proteobacteria-specific.
in table  <dig>  i list some characteristics of  <dig> proteins that could be regarded as specific for most sequenced ε-proteobacteria based on these criteria.
forty-one of these  <dig> proteins were present in all sequenced ε-proteobacteria genomes and for them all significant alignments/hits were from this group.
however, in three instances  the e value for one ε-proteobacteria was just above the default threshold value  for significance.
for three other proteins, ws <dig>  ws <dig> and ws <dig>  1– <dig> hits from other bacteria exhibited borderline significance, but there was a large jump in e values from the last ε-proteobacteria hit to these other proteins , indicating that these proteins are also ε-proteobacteria-specific.
eight other proteins in this table  are missing in 1– <dig> ε-proteobacteria species, which could be due to selective gene loss  <cit> .
of these  <dig> proteins, ws <dig>  ws <dig> and ws <dig> are present in almost all sequenced ε-proteobacteria except t. denitrificans.
the phylogenetic position of t. denitrificans within ε-proteobacteria is presently not clear .
hence, absence of these proteins in t. denitrificans could be explained by either earlier divergence of this species in comparison to other sequenced ε-proteobacteria, or due to gene loss.
for the protein ws <dig> listed in table  <dig>  in addition to various ε-proteobacteria, homologs with very low e values  were also found in two δ-proteobacteria belonging to the desulfovibrio genus.
in phylogenetic trees based on 16s rrna  <cit> , various proteins  <cit> , and in analyses based on conserved indels  <cit> , δ-proteobacteria generally branch in close proximity to the ε-proteobacteria.
hence, the shared presence of the ws <dig> homologs in desulfovibrio genus and ε-proteobacteria may reflect either a deep phylogenetic relationship that exist between these two groups  <cit> , or it could result from lateral gene transfer  <cit> .
based on the available data we are unable to distinguish between these possibilities.
however, it is interesting to note that a  <dig> aa insert in a conserved region of the reca protein, which was previously indicated to be specific for ε-proteobacteria  <cit> , and is present in all available ε-proteobacteria homologs, is also commonly present in desulfovibrio and lawsonia species  .
several of the proteins listed in table  <dig>  exhibit a high degree of sequence conservation across various ε-proteobacteria species.
a partial nucleotide sequence alignment for the ws <dig> coding sequence for various ε-proteobacterial species is shown in figure  <dig>  a large number of positions in the alignments are completely conserved in various campylobacterales species and there are several long stretches  showing a high degree of sequence conservation.
the pcr primers and other molecular probes based on these conserved regions could provide novel and specific means for identification of both new, as well existing campylobacterales species and possibly different ε-proteobacteria.
the comparative analysis of w. succinogenes genome has also identified  <dig> proteins that are uniquely found in wolinella and helicobacter species .
of these  <dig> proteins, the first  <dig> are present in all  <dig> of the sequenced species/strains from these genera, whereas the last  <dig> proteins are only found in w. succinogenes and h. hepaticus but missing in the two h. pylori strains.
all of these proteins are of unknown function.
the wolinella and helicobacter genera are part of the helicobacteraceae family and these uniquely shared proteins provide potential molecular markers for this family.
our analysis also reveals that  <dig> proteins in the genome of w. succinogenes dsm  <dig> show no significant similarity to any other protein in the databases .
barr et al.
<cit>  have previously indicated a much higher number  of such proteins.
however, since their analysis, genomes of several ε-proteobacteria as well as numerous other organisms have become available  <cit> .
because of this, and our employment of more stringent criteria for identification of group-specific proteins, the number of such proteins is considerably smaller than indicated originally  <cit> .
sixteen of these proteins are present in seven clusters  in the w. succinogenes dsm  <dig> genome.
proteins specific for the campylobacter genus
we have also performed blast searches on various proteins found in the genome of c. jejuni rm <dig> to identify proteins that are unique to the campylobacter species.
fouts et al.
<cit> , who sequenced the genomes of several campylobacter species/strains had reported comparative studies on them.
their work identified several proteins that were specific for the c. jejuni rm <dig> and c. jejuni nctc  <dig> strains , but they did not look for proteins that were uniquely shared by either all or different campylobacter species.
our analyses have identified  <dig> proteins  that are uniquely present in all of the sequenced campylobacter species viz.
c. fetus, c. lari, c. upsaliensis, c. coli and c. jejuni .
three additional proteins listed in table  <dig>  cje <dig>  cje <dig> and cje <dig> are missing in only one of the campylobacter species, which is likely due to gene loss.
eighteen other proteins  are present in all of the campylobacter species, except c. fetus.
among the sequenced campylobacter species, c. fetus exhibits deepest branching in various phylogenetic trees .
hence, the absence of these proteins in c. fetus could be explained by their introduction in a common ancestor of the other campylobacter species after branching of c. fetus.
ten other proteins  are commonly present in c. upsaliensis, c. coli and c. jejuni only indicating a closer relationship among these species.
the genes for these proteins were likely introduced or evolved in a common ancestor of these three species.
likewise,  <dig> other proteins listed in table  <dig>  which are only found in c. coli and c. jejuni  points to a specific relationship between these species to the exclusion of all others.
most of these proteins are of unknown function.
however, in a few cases, where any similarity to conserved domain present in other proteins has been identified by blast searches, such information is noted in various tables.
these analyses have also identified a large number of proteins that are specific for the c. jejuni species .
the first  <dig> proteins listed in this table are present in all sequenced c. jejuni strains , whereas the remainder are missing or have been lost from a few of the strains.
conserved indels and other rare genetic changes specific for epsilon proteobacteria
conserved indels in protein sequences provide another useful kind of molecular signatures for taxonomic and diagnostic studies.
in our recent work, conserved indels that are distinctive characteristics of many different groups of bacteria  have been identified  <cit> .
to identify conserved indels that may be specific for ε-proteobacteria, the sequence alignments of various proteins constructed in earlier work were examined.
these studies have led to identification of  <dig> conserved indels that are specific for this group.
the characteristics of these indels and of the proteins in which they are found are briefly described below.
in figure  <dig>  i present sequence information for two conserved indels that are uniquely present in various sequenced ε-proteobacterial homologs, but which are not found in the corresponding proteins from any other organism.
the first of these indels is a  <dig> aa insert in the b protein of the uvr abc system , which plays a key role in the nucleotide excision repair process  <cit> .
the second indel consists of a  <dig> aa deletion in the enzyme phenylalanyl-trna synthetase , which is required for protein synthesis.
both these proteins are widely distributed in bacteria and sequence information for only representative species from other bacteria is presented.
the indels in both these proteins are flanked by highly conserved regions and the unique presence of these indels in all available ε-proteobacteria homologs strongly indicate that they are distinctive molecular characteristics of these bacteria.
two additional conserved indels that are specific for only certain ε-proteobacteria are shown in figure  <dig>  the top panel in this figure shows a  <dig> aa insert in the ftsh protease that is uniquely present in all sequenced ε-proteobacteria, except t. denitrificans.
the absence of this indel in various other bacteria as well t. denitrificans indicates that this indel is an insert that was introduced in a common ancestor of helicobacter, campylobacter and wolinella, after the branching of t. denitrificans.
the lower panel in fig.
<dig> shows a highly conserved insert in the β '-subunit of rna polymerase  that is uniquely present in various campylobacter species, except c. fetus.
rpoc homologs are present in all sequenced genomes and the identified insert is not found in any other ε-proteobacteria or other organism.
this insert was likely introduced in a common ancestor of the campylobacter after branching of c. fetus.
in addition to these conserved indels, zakaharova et al.
<cit>  have identified a rare genetic event that causes fusion of two different genes within certain groups of ε-proteobacteria.
the two largest and highly conserved subunits of rna polymerase  are encoded by two distinct genes in various bacteria  <cit> .
however, a rare genetic event has led to the fusion of these genes in helicobacter and wolinella species, such that rpob and rpoc are now made as a single large polypeptide  .
in contrast, in campylobacter and t. denitrificans, similar to other bacteria, separate genes encode for these proteins.
this rare genetic event provides evidence of a specific relationship between helicobacter and wolinella species, which are part of the helicobacteraceae family.
evolutionary significance of the signature proteins and conserved indels
it is important to understand at what point during the evolution of ε-proteobacteria, the above-described molecular characteristics evolved or were introduced.
to determine their evolutionary significance, phylogenetic trees were constructed for the sequenced ε-proteobacteria species based on 16s rrna and a concatenated dataset of sequences for  <dig> highly conserved proteins -tu, ef-g, gyrase a, gyrase b and alanyl-trna synthetase).
in the 16s rrna tree, the ε-proteobacterial species under consideration formed two clades .
one clade consisted of various campylobacter species whereas the other clade included helicobacter, wolinella and t. denitrificans.
in the latter clade, t. denitrificans formed a deep branching outgroup of the helicobacter and wolinella species, but a specific association of t. denitrificans to these species was not supported by the bootstrap score of the node    <cit> .
in contrast to the rrna tree, in the tree based on concatenated protein sequences, all of the internal nodes were reliably resolved.
in this tree, t. denitrificans formed a deep branching lineage showing no specific relationship to either the helicobacter/wolinella clade or to the campylobacter species .
a similar deep branching of t. denitrificans in comparison to other sequenced ε-proteobacteria is observed in phylogenetic trees based on hsp <dig>  rpoc, gyrase a, gyrase b and ef-tu protein sequences .
using the above trees as reference points, the evolutionary stages where different ε-proteobacteria-specific genes/proteins or other molecular signatures likely evolved is depicted in fig.
5c.
the genes for the first  <dig> proteins listed in table  <dig> as well as the conserved indels in phers and exinuclease b protein, which are unique to almost all sequenced ε-proteobacteria, were likely introduced in a common ancestor of the campylobacterales or ε-proteobacteria.
the genes for the last three proteins listed in table  <dig>  that are absent in t. denitrificans but present in all  other ε-proteobacteria were likely introduced in a common ancestor of the helicobacter, wolinella and campylobacter after the divergence of t. denitrificans.
the insert in the ftsh protease was also likely introduced at this stage.
the proteins listed in table  <dig> were introduced in a common ancestor of the wolinella and helicobacter genera, and it is expected that some of them will constitute distinctive characteristics of the helicobacteraceae family.
the rare genetic event leading to the fusion of rpob and rpoc genes also occurred at a similar stage.
the proteins listed in tables  <dig> to  <dig> that are unique to either all sequenced campylobacter species or various species within this genus, were introduced at different stages in the evolution of this group .
the observed species distribution patterns of these proteins strongly support the branching pattern of campylobacter species in the phylogenetic trees .
the inference from these proteins and the phylogenetic trees that c. fetus is one of the deepest branching species within the campylobacter genus is also strongly supported by the large insert in rpoc , which is present in all campylobacter species except c. fetus.
CONCLUSIONS
the comparative genomics of ε-proteobacteria reported here have led to identification of a large number of molecular signatures  that are distinctive characteristics of these bacteria.
our analyses indicate that these characteristics have been introduced at various stages in the evolution of ε-proteobacteria, but once introduced, they were generally stably retained in various descendents of these lineages with minimal gene loss or lateral gene transfer to other bacteria.
sequence information for these proteins or molecular signatures is presently available only from the campylobacterales species and no information is available from the nautiliales order, which comprise the other main group within ε-proteobacteria.
however, the genomes of several ε-proteobacteria  covering all of its main groups are currently in progress .
based upon our work on signature sequences for other groups of bacteria  <cit> , we expect that many of the signatures identified in the present work  will also be found in different ε-proteobacteria, whereas several other will prove to be specific for only the campylobacterales order.
the primary sequences of many of these genes/proteins are highly conserved and they provide novel diagnostic tools for these bacteria by means of pcr amplification and fluorescence in situ hybridization methods.
monoclonal and polyclonal antibodies based upon these proteins provide another means for their detection.
additionally, these campylobacterales or ε-proteobacteria specific proteins also provide potential targets for developing therapeutics and vaccines that are specific for these bacteria.
the identified signature proteins and rgcs also provide novel and definitive molecular means for circumscribing a number of taxonomic groups within campylobacterales  and for identifying species belonging to these groups.
the cellular functions of most of the ε-proteobacteria-specific proteins are not known.
although a number of these proteins exhibit weak sequence similarity to conserved domains in other proteins, their actual functions may be quite different, and determining them constitute an important task for the future.
likewise, it is also of much interest to understand the functional significance of the conserved indels in various proteins  that are specific for different taxonomic groups/clades of ε-proteobacteria.
since these indels, which are located in highly conserved regions, are retained by all  members of these clades it is highly likely that they are functionally important  for these bacteria.
thus, it is of much importance to understand how the functions of these proteins are modified by these indels and the physiological significance of these modifications for these bacteria.
further studies on these ε-proteobacteria specific proteins and indels thus may lead to the discovery of novel biochemical and physiological characteristics that are uniquely shared by these bacteria.

 the epsilon proteobacteria comprise one of the five classes within the phylum proteobacteria. these bacteria inhabit a wide variety of ecological niches ranging from gastrointestinal tracts of animals to water reservoirs, sewage, oil-field community and deep-sea hydrothermal vents. recent studies show that ε-proteobacteria comprise a significant proportion of the microbial population in deep-sea hydrothermal vents where, because of their ability to carry out different types of metabolism using a variety of alternate electron donors and acceptors, they play important role in carbon, nitrogen and sulfur cycles. a great deal of interest in these bacteria stems from the fact that many of these species are host-associated and comprise important human and animal pathogens. of these bacteria, helicobacter pylori is the causative agent for gastric and peptic ulcers and infections with this and the related species h. hepaticus are important predisposing factors in gastric cancers in humans and liver cancers in rodents. campylobacter jejuni and c. coli are the most common causes of food-born illnesses such as diarrhea worldwide. c. jejuni infection can also lead to the neuromuscular disease guillain-barre syndrome, which causes weakness and paralysis of muscles. in contrast to the pathogenic nature of helicobacter and campylobacter, wolinella succinogenes is a commensal in the gastrointestinal tract of cattle and it is not known to cause any illness in either animals or humans. in addition to the host-associated species, many free-living members which include chemolithotrophic and autotrophic bacteria also form part of the ε-proteobacterial group. the ε-proteobacteria are presently distinguished from other bacteria based their branching in the 16s rrna trees. although most of these bacteria assume a spiral shape sometime during their life cycle and they can also utilize a variety of electron donors and acceptors, these characteristics are not unique to this group. presently, there is no molecular or biochemical characteristic known that is unique to this group of bacteria. within ε-proteobacteria, two main orders, campylobacterales and nautiliales, are presently recognized the campylobacterales is made up of three families, campylobacteraceae, helicobacteraceae and hydrogenimonaceae, whereas the nautiliales order is comprised of three genera. except for the 16s rrna, very little sequence information is available for species belonging to the hydrogenimonaceae family and the nautiliales order. in the past few years, genomic sequences of several ε-proteobacterial species from the campylobacterales order have become available. the completely sequenced genomes include those from: helicobacter pylori, h. pylori j, h. hepaticus atcc, campylobacter jejuni nctc, c. jejuni rm,wolinella succinogenes dsm and thiomicrospira denitrificans atcc. in addition, genomes of several campylobacter species and c. jejuni subsp. jejuni strains are now at assembly stage and sequence information from them is available in the ncbi database. the availability of these sequences has opened new windows for discovering novel molecular characteristics that are unique to these bacteria and can be used for their diagnostics as well as for biochemical and functional studies. earlier comparative genomic studies on ε-proteobacteria have examined a number of aspects of their gene/protein contents. of these, the studies by eppinger et al. and fouts et al. are particularly detailed. in these works, genes/proteins that are unique to individual genomes were identified as well as genes that are commonly shared by, but not uniquely present, in a number of these bacteria. pair-wise comparison of the gene contents of these bacteria, functional classification of their genomic inventory, synteny and co-linearity of genes in various genomes, and examples of gene losses as well as recombination were also reported. additionally, coenye and vandamme have carried out studies to identify genes that have been laterally transferred between ε-proteobacteria and other bacteria. however, thus far no comparative study has examined or identified genes/proteins that are uniquely found in ε-proteobacteria at different taxonomic levels. such genes and proteins, because of their specificity, provide novel means for diagnostics and taxonomic studies and for discovering important physiological characteristics that are unique to these bacteria. in our recent work, we have used comparative genomics to identify a large number of signature proteins that are specific for either alpha proteobacteria, chlamydiae or actinobacteria. in the present work, we have carried out systematic blast searches on all open reading frames in the genome of wolinella succinogenes dsm and campylobacter jejuni rm to identify whole genes/proteins that are unique to ε-proteobacteria. these studies have led to identification of genes/proteins that are uniquely present in various sequenced ε-proteobacteria, as well as many other proteins that are limited to certain subgroups within the campylobacterales order. additionally, we also describe a number of conserved indels in widely distributed proteins that are specific for either all-available ε-proteobacteria or for certain subgroups among them. the identified signature proteins and indels comprise rare genetic changes that have been introduced at various stages during the evolution of campylobacterales and their species distribution patterns are supported by the branching order of these species in phylogenetic trees.  these studies were undertaken to identify molecular characteristics that are uniquely shared by either all sequenced ε-proteobacteria species, or their subgroups, but which generally are not found in any other organism. three different kinds of molecular signatures that are specific for ε-proteobacteria are described in the present work. the first of these consists of whole proteins or open reading frames that are uniquely found in ε-proteobacteria. the other two characteristics are comprised of rare genetic changes consisting of either conserved inserts or deletions in widely distributed proteins that are specific for the ε-proteobacterial homologs as well as a gene-fusion event within this group of bacteria. a brief description of these molecular signatures and their evolutionary significances are discussed below. whole proteins or orfs that are unique for the epsilon-proteobacteria and helicobacteraceae family the ε-proteobacteria-specific proteins were identified as described in the methods section. generally, a protein was considered to be epsilon-proteobacteria specific if all significant alignments in a psi-blast search with the query protein were from ε-proteobacteria species. in a few cases, where the e values of or hits from other species also exhibited borderline significance, but there was a large increase in e value from the last ε-proteobacteria hit in the search to these other proteins, such proteins were also regarded as ε-proteobacteria-specific. in table i list some characteristics of proteins that could be regarded as specific for most sequenced ε-proteobacteria based on these criteria. forty-one of these proteins were present in all sequenced ε-proteobacteria genomes and for them all significant alignments/hits were from this group. however, in three instances the e value for one ε-proteobacteria was just above the default threshold value for significance. for three other proteins, ws ws and ws 1– hits from other bacteria exhibited borderline significance, but there was a large jump in e values from the last ε-proteobacteria hit to these other proteins, indicating that these proteins are also ε-proteobacteria-specific. eight other proteins in this table are missing in 1– ε-proteobacteria species, which could be due to selective gene loss. of these proteins, ws ws and ws are present in almost all sequenced ε-proteobacteria except t. denitrificans. the phylogenetic position of t. denitrificans within ε-proteobacteria is presently not clear. hence, absence of these proteins in t. denitrificans could be explained by either earlier divergence of this species in comparison to other sequenced ε-proteobacteria, or due to gene loss. for the protein ws listed in table in addition to various ε-proteobacteria, homologs with very low e values were also found in two δ-proteobacteria belonging to the desulfovibrio genus. in phylogenetic trees based on 16s rrna, various proteins, and in analyses based on conserved indels, δ-proteobacteria generally branch in close proximity to the ε-proteobacteria. hence, the shared presence of the ws homologs in desulfovibrio genus and ε-proteobacteria may reflect either a deep phylogenetic relationship that exist between these two groups, or it could result from lateral gene transfer. based on the available data we are unable to distinguish between these possibilities. however, it is interesting to note that a aa insert in a conserved region of the reca protein, which was previously indicated to be specific for ε-proteobacteria, and is present in all available ε-proteobacteria homologs, is also commonly present in desulfovibrio and lawsonia species. several of the proteins listed in table exhibit a high degree of sequence conservation across various ε-proteobacteria species. a partial nucleotide sequence alignment for the ws coding sequence for various ε-proteobacterial species is shown in figure a large number of positions in the alignments are completely conserved in various campylobacterales species and there are several long stretches showing a high degree of sequence conservation. the pcr primers and other molecular probes based on these conserved regions could provide novel and specific means for identification of both new, as well existing campylobacterales species and possibly different ε-proteobacteria. the comparative analysis of w. succinogenes genome has also identified proteins that are uniquely found in wolinella and helicobacter species. of these proteins, the first are present in all of the sequenced species/strains from these genera, whereas the last proteins are only found in w. succinogenes and h. hepaticus but missing in the two h. pylori strains. all of these proteins are of unknown function. the wolinella and helicobacter genera are part of the helicobacteraceae family and these uniquely shared proteins provide potential molecular markers for this family. our analysis also reveals that proteins in the genome of w. succinogenes dsm show no significant similarity to any other protein in the databases. barr et al. have previously indicated a much higher number of such proteins. however, since their analysis, genomes of several ε-proteobacteria as well as numerous other organisms have become available. because of this, and our employment of more stringent criteria for identification of group-specific proteins, the number of such proteins is considerably smaller than indicated originally. sixteen of these proteins are present in seven clusters in the w. succinogenes dsm genome. proteins specific for the campylobacter genus we have also performed blast searches on various proteins found in the genome of c. jejuni rm to identify proteins that are unique to the campylobacter species. fouts et al., who sequenced the genomes of several campylobacter species/strains had reported comparative studies on them. their work identified several proteins that were specific for the c. jejuni rm and c. jejuni nctc strains, but they did not look for proteins that were uniquely shared by either all or different campylobacter species. our analyses have identified proteins that are uniquely present in all of the sequenced campylobacter species viz. c. fetus, c. lari, c. upsaliensis, c. coli and c. jejuni. three additional proteins listed in table cje cje and cje are missing in only one of the campylobacter species, which is likely due to gene loss. eighteen other proteins are present in all of the campylobacter species, except c. fetus. among the sequenced campylobacter species, c. fetus exhibits deepest branching in various phylogenetic trees. hence, the absence of these proteins in c. fetus could be explained by their introduction in a common ancestor of the other campylobacter species after branching of c. fetus. ten other proteins are commonly present in c. upsaliensis, c. coli and c. jejuni only indicating a closer relationship among these species. the genes for these proteins were likely introduced or evolved in a common ancestor of these three species. likewise, other proteins listed in table which are only found in c. coli and c. jejuni points to a specific relationship between these species to the exclusion of all others. most of these proteins are of unknown function. however, in a few cases, where any similarity to conserved domain present in other proteins has been identified by blast searches, such information is noted in various tables. these analyses have also identified a large number of proteins that are specific for the c. jejuni species. the first proteins listed in this table are present in all sequenced c. jejuni strains, whereas the remainder are missing or have been lost from a few of the strains. conserved indels and other rare genetic changes specific for epsilon proteobacteria conserved indels in protein sequences provide another useful kind of molecular signatures for taxonomic and diagnostic studies. in our recent work, conserved indels that are distinctive characteristics of many different groups of bacteria have been identified. to identify conserved indels that may be specific for ε-proteobacteria, the sequence alignments of various proteins constructed in earlier work were examined. these studies have led to identification of conserved indels that are specific for this group. the characteristics of these indels and of the proteins in which they are found are briefly described below. in figure i present sequence information for two conserved indels that are uniquely present in various sequenced ε-proteobacterial homologs, but which are not found in the corresponding proteins from any other organism. the first of these indels is a aa insert in the b protein of the uvr abc system, which plays a key role in the nucleotide excision repair process. the second indel consists of a aa deletion in the enzyme phenylalanyl-trna synthetase, which is required for protein synthesis. both these proteins are widely distributed in bacteria and sequence information for only representative species from other bacteria is presented. the indels in both these proteins are flanked by highly conserved regions and the unique presence of these indels in all available ε-proteobacteria homologs strongly indicate that they are distinctive molecular characteristics of these bacteria. two additional conserved indels that are specific for only certain ε-proteobacteria are shown in figure the top panel in this figure shows a aa insert in the ftsh protease that is uniquely present in all sequenced ε-proteobacteria, except t. denitrificans. the absence of this indel in various other bacteria as well t. denitrificans indicates that this indel is an insert that was introduced in a common ancestor of helicobacter, campylobacter and wolinella, after the branching of t. denitrificans. the lower panel in fig. shows a highly conserved insert in the β'-subunit of rna polymerase that is uniquely present in various campylobacter species, except c. fetus. rpoc homologs are present in all sequenced genomes and the identified insert is not found in any other ε-proteobacteria or other organism. this insert was likely introduced in a common ancestor of the campylobacter after branching of c. fetus. in addition to these conserved indels, zakaharova et al. have identified a rare genetic event that causes fusion of two different genes within certain groups of ε-proteobacteria. the two largest and highly conserved subunits of rna polymerase are encoded by two distinct genes in various bacteria. however, a rare genetic event has led to the fusion of these genes in helicobacter and wolinella species, such that rpob and rpoc are now made as a single large polypeptide. in contrast, in campylobacter and t. denitrificans, similar to other bacteria, separate genes encode for these proteins. this rare genetic event provides evidence of a specific relationship between helicobacter and wolinella species, which are part of the helicobacteraceae family. evolutionary significance of the signature proteins and conserved indels it is important to understand at what point during the evolution of ε-proteobacteria, the above-described molecular characteristics evolved or were introduced. to determine their evolutionary significance, phylogenetic trees were constructed for the sequenced ε-proteobacteria species based on 16s rrna and a concatenated dataset of sequences for highly conserved proteins -tu, ef-g, gyrase a, gyrase b and alanyl-trna synthetase). in the 16s rrna tree, the ε-proteobacterial species under consideration formed two clades. one clade consisted of various campylobacter species whereas the other clade included helicobacter, wolinella and t. denitrificans. in the latter clade, t. denitrificans formed a deep branching outgroup of the helicobacter and wolinella species, but a specific association of t. denitrificans to these species was not supported by the bootstrap score of the node. in contrast to the rrna tree, in the tree based on concatenated protein sequences, all of the internal nodes were reliably resolved. in this tree, t. denitrificans formed a deep branching lineage showing no specific relationship to either the helicobacter/wolinella clade or to the campylobacter species. a similar deep branching of t. denitrificans in comparison to other sequenced ε-proteobacteria is observed in phylogenetic trees based on hsp rpoc, gyrase a, gyrase b and ef-tu protein sequences. using the above trees as reference points, the evolutionary stages where different ε-proteobacteria-specific genes/proteins or other molecular signatures likely evolved is depicted in fig. 5c. the genes for the first proteins listed in table as well as the conserved indels in phers and exinuclease b protein, which are unique to almost all sequenced ε-proteobacteria, were likely introduced in a common ancestor of the campylobacterales or ε-proteobacteria. the genes for the last three proteins listed in table that are absent in t. denitrificans but present in all other ε-proteobacteria were likely introduced in a common ancestor of the helicobacter, wolinella and campylobacter after the divergence of t. denitrificans. the insert in the ftsh protease was also likely introduced at this stage. the proteins listed in table were introduced in a common ancestor of the wolinella and helicobacter genera, and it is expected that some of them will constitute distinctive characteristics of the helicobacteraceae family. the rare genetic event leading to the fusion of rpob and rpoc genes also occurred at a similar stage. the proteins listed in tables to that are unique to either all sequenced campylobacter species or various species within this genus, were introduced at different stages in the evolution of this group. the observed species distribution patterns of these proteins strongly support the branching pattern of campylobacter species in the phylogenetic trees. the inference from these proteins and the phylogenetic trees that c. fetus is one of the deepest branching species within the campylobacter genus is also strongly supported by the large insert in rpoc, which is present in all campylobacter species except c. fetus.  the comparative genomics of ε-proteobacteria reported here have led to identification of a large number of molecular signatures that are distinctive characteristics of these bacteria. our analyses indicate that these characteristics have been introduced at various stages in the evolution of ε-proteobacteria, but once introduced, they were generally stably retained in various descendents of these lineages with minimal gene loss or lateral gene transfer to other bacteria. sequence information for these proteins or molecular signatures is presently available only from the campylobacterales species and no information is available from the nautiliales order, which comprise the other main group within ε-proteobacteria. however, the genomes of several ε-proteobacteria covering all of its main groups are currently in progress. based upon our work on signature sequences for other groups of bacteria, we expect that many of the signatures identified in the present work will also be found in different ε-proteobacteria, whereas several other will prove to be specific for only the campylobacterales order. the primary sequences of many of these genes/proteins are highly conserved and they provide novel diagnostic tools for these bacteria by means of pcr amplification and fluorescence in situ hybridization methods. monoclonal and polyclonal antibodies based upon these proteins provide another means for their detection. additionally, these campylobacterales or ε-proteobacteria specific proteins also provide potential targets for developing therapeutics and vaccines that are specific for these bacteria. the identified signature proteins and rgcs also provide novel and definitive molecular means for circumscribing a number of taxonomic groups within campylobacterales and for identifying species belonging to these groups. the cellular functions of most of the ε-proteobacteria-specific proteins are not known. although a number of these proteins exhibit weak sequence similarity to conserved domains in other proteins, their actual functions may be quite different, and determining them constitute an important task for the future. likewise, it is also of much interest to understand the functional significance of the conserved indels in various proteins that are specific for different taxonomic groups/clades of ε-proteobacteria. since these indels, which are located in highly conserved regions, are retained by all members of these clades it is highly likely that they are functionally important for these bacteria. thus, it is of much importance to understand how the functions of these proteins are modified by these indels and the physiological significance of these modifications for these bacteria. further studies on these ε-proteobacteria specific proteins and indels thus may lead to the discovery of novel biochemical and physiological characteristics that are uniquely shared by these bacteria. 
functional studies on these proteins and conserved indels should reveal novel biochemical or physiological characteristics that are unique to these groups of epsilon proteobacteria. of these proteins, are uniquely found in nearly all sequenced epsilon-proteobacteria, h. hepaticus, c. jejuni, c. lari, c. coli, c. upsaliensis, c. fetus, w. succinogenes dsm and thiomicrospira denitrificans atcc 33889), are unique for the wolinella and helicobacter species and many others are specific for either some or all of the species within the campylobacter genus. the inter-relationships amongst campylobacterales as deduced from these molecular signatures are in accordance with the phylogenetic trees based on the 16s rrna and concatenated sequences for nine conserved proteins. comparative analyses of proteins in the genomes of wolinella succinogenes dsm and campylobacter jejuni rm against all available sequences have identified a large number of proteins that are unique to various epsilon proteobacteria, but whose homologs are not detected in other organisms. although sequence information for these signatures is presently limited to campylobacterales species, it is likely that many of them will also be found in other epsilon proteobacteria. the primary sequences of many of these proteins are highly conserved and provide novel resources for diagnostics and therapeutics. these molecular signatures provide novel tools for identifying and circumscribing species from the campylobacterales order and its subgroups in molecular terms. the epsilon proteobacteria, which include many important human pathogens, are presently recognized solely on the basis of their branching in rrna trees. we also report four conserved indels in widely distributed proteins that are specific for either all epsilon proteobacteria or different subgroups. in addition, a rare genetic event that caused fusion of the genes for the largest subunits of rna polymerase in wolinella and helicobacter is also described.
 the epsilon proteobacteria, which include many important human pathogens, are presently recognized solely on the basis of their branching in rrna trees. no unique molecular or biochemical characteristics specific for this group are known.  comparative analyses of proteins in the genomes of wolinella succinogenes dsm and campylobacter jejuni rm against all available sequences have identified a large number of proteins that are unique to various epsilon proteobacteria, but whose homologs are not detected in other organisms. of these proteins, are uniquely found in nearly all sequenced epsilon-proteobacteria, h. hepaticus, c. jejuni, c. lari, c. coli, c. upsaliensis, c. fetus, w. succinogenes dsm and thiomicrospira denitrificans atcc 33889), are unique for the wolinella and helicobacter species and many others are specific for either some or all of the species within the campylobacter genus. the primary sequences of many of these proteins are highly conserved and provide novel resources for diagnostics and therapeutics. we also report four conserved indels in widely distributed proteins that are specific for either all epsilon proteobacteria or different subgroups. in addition, a rare genetic event that caused fusion of the genes for the largest subunits of rna polymerase in wolinella and helicobacter is also described. the inter-relationships amongst campylobacterales as deduced from these molecular signatures are in accordance with the phylogenetic trees based on the 16s rrna and concatenated sequences for nine conserved proteins.  these molecular signatures provide novel tools for identifying and circumscribing species from the campylobacterales order and its subgroups in molecular terms. although sequence information for these signatures is presently limited to campylobacterales species, it is likely that many of them will also be found in other epsilon proteobacteria. functional studies on these proteins and conserved indels should reveal novel biochemical or physiological characteristics that are unique to these groups of epsilon proteobacteria. 