Whole proteins or ORFs that are unique for the epsilon-proteobacteria (Campylobacterales order) and Helicobacteraceae family

For the protein WS0230 listed in Table 1, in addition to various ε-proteobacteria, homologs with very low E values (e-90 range) were also found in two δ-proteobacteria belonging to the Desulfovibrio genus. In phylogenetic trees based on 16S rRNA [2, 41], various proteins [42, 43], and in analyses based on conserved indels [44], δ-proteobacteria generally branch in close proximity to the ε-proteobacteria. Hence, the shared presence of the WS0230 homologs in Desulfovibrio genus and ε-proteobacteria may reflect either a deep phylogenetic relationship that exist between these two groups [43–45], or it could result from lateral gene transfer [46]. Based on the available data we are unable to distinguish between these possibilities. However, it is interesting to note that a 1 aa insert in a conserved region of the RecA protein, which was previously indicated to be specific for ε-proteobacteria [44], and is present in all available ε-proteobacteria homologs, is also commonly present in Desulfovibrio and Lawsonia species (belonging to Desulfovibrionaceae family) (results not shown).

Table 1 also lists the available information regarding possible cellular functions of these proteins. Most of these proteins are of unknown functions. However, in a number of cases weak but significant similarity is observed to conserved domains found in other proteins in the databases [47], or to particular COG families [48]. The information of this kind, along with the genomic context of these ORFs, provide useful leads for exploring the cellular functions of these conserved hypothetical proteins [49–52]. Of the proteins that are found in all sequenced ε-proteobacteria, WS0266 and WS0802 were experimentally identified as plasminogen binding proteins [53]. It has been suggested that these proteins may enable these bacteria to coat their exterior surface with plasminogen and thus they could be involved in enhancing their virulence. The putative functions of several other proteins are indicated in Table 1 and they include a putative helicase (WS0086), a Cbb3 type cytochrome oxidase (WS0180), a protein related to the FixH family (WS0185) of Rhizobium, a protein WS0316 containing the RDD domain, two proteins (WS0476 and WS0480) which contain molybdopterin_binding (MopB) domain found in NADH oxidoreductase I. Also found were two proteins implicated in flagellar function (WS0490 and WS0575) [23], a protein (WS0520) with TonB domain and another protein (WS1874) containing a domain related to the DNA polymerase delta subunit, a protein (WS2146) showing some similarity to Sua5 domain involved in binding to double stranded DNA, and a protein WS0230 showing similarity to deacylase domain. In addition, several proteins are predicted to be either periplasmic or membrane proteins. It should be emphasized that most of these functional predictions or annotations are based on weak similarity to conserved domains (CD) as identified by the CD search program implemented with the BLAST program [47]. Although this information is very useful, the actual functions of most of these proteins, which exhibit very little similarity to other molecules in the database, remain to be determined. Among the proteins listed in Table 1 that are missing in some ε-proteobacteria, WS1211 is a homolog of the C. jejuni invasion antigen (CiaB), which is recognized as an important factor in its pathogenicity[14, 54]. Of the proteins listed in Table 1, 10 proteins (WS133-WS134, WS184-WS185, WS447-WS448, WS1039-WS1040 and WS1495-WS1496) are present in clusters of two in the genome, and they could be involved in related functions [51, 52].

Several of the proteins listed in Table 1 (e.g., WS0086 and WS2123) exhibit a high degree of sequence conservation across various ε-proteobacteria species. A partial nucleotide sequence alignment for the WS0086 coding sequence for various ε-proteobacterial species is shown in Figure 1. A large number of positions in the alignments are completely conserved in various Campylobacterales species and there are several long stretches (boxed) 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.

Our analysis also reveals that 99 proteins in the genome of W. succinogenes DSM 1740 show no significant similarity to any other protein in the databases [see Additional file 1]. Barr et al. [23] have previously indicated a much higher number (i.e. 490) of such proteins. However, since their analysis, genomes of several ε-proteobacteria as well as numerous other organisms have become available [28, 30, 31, 55]. 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 [23]. Sixteen of these proteins are present in seven clusters (WS0261-WS0262; WS0531-WS0532; WS1446-WS1447; WS1573-WS1674; WS1888-WS1889; WS2027-WS2028-WS2029; WS2032-WS2033-WS2034) in the W. succinogenes DSM 1740 genome.

Proteins specific for the Campylobacter genus

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 (e.g., Chlamydiae, Proteobacteria, alpha proteobacteria, Actinobacteria, Cyanobacteria, Deinococcus-Thermus, Aquificae, etc.) have been identified [44, 56–60]. 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 4 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 2, 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 3 aa insert in the B protein of the Uvr ABC system (Fig. 2A), which plays a key role in the nucleotide excision repair process [61]. The second indel consists of a 2 aa deletion in the enzyme phenylalanyl-tRNA synthetase (Fig. 2B), 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 3. The top panel in this Figure shows a 1 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. 3 shows a highly conserved insert in the β '-subunit of RNA polymerase (RpoC) 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. [62] 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 (RpoB and RpoC, each approximately 1400 aa) are encoded by two distinct genes in various bacteria [62]. 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 (≈ 2900 aa) (Fig. 4). In contrast, in Campylobacte r 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 9 highly conserved proteins (viz. RpoB, RpoC, Hsp70, Hsp60, elongation factor (EF)-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 (Fig. 5A). 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 (<50%) (Fig. 5A) [8, 12]. 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 (Fig. 5B). A similar deep branching of T. denitrificans in comparison to other sequenced ε-proteobacteria is observed in phylogenetic trees based on Hsp70, RpoC, Gyrase A, Gyrase B and EF-Tu protein sequences (results not shown).

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 49 proteins listed in Table 1 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 1 (viz. WS1211, WS1752 and WS2059) that are absent in T. denitrificans but present in all (or most) 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 2 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 3 to 7 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 (Fig. 5C). The observed species distribution patterns of these proteins strongly support the branching pattern of Campylobacter species in the phylogenetic trees (Figs. 5A and 5B). 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 (Fig. 3B), which is present in all Campylobacter species except C. fetus.

Identification of proteins that are specific for epsilon proteobacteria

To identify proteins that are specific for ε-proteobacteria, all proteins in the genomes of W. succinogenes DSM 1740 [23] were analyzed. This genome was chosen for a number of reasons. First, of the sequenced ε-proteobacteria genomes, W. succinogenes genome is among the largest (2.11 Mb) with 2043 ORFs [23]. Hence, one expects that minimal gene loss has occurred in this bacterium and that it should contain maximal number of genes that may be present in other ε-proteobacteria. Second, phylogenetic and comparative studies have indicated that W. succinogenes forms an outgroup to various Helicobacter species and thus lies in an intermediate position between members of the Helicobacteraceae and Campylobacteraceae families [6, 14]. Thus, BLAST searches on proteins from this genome should enable us to identify proteins that are unique to the Helicobacteraceae family as well as those shared with other taxonomic groups of ε-proteobacteria. To identify proteins that are specific for the Campylobacter species, the genome of C. jejuni RM1221 was analyzed. The BLASTp searches were initially performed on each individual protein or ORF in these genomes against all available sequences in the NCBI sequence database, to identify all related gene/protein in other organisms [63, 64]. These searches were performed using the default parameters as set by the BLAST program, except that the low complexity filter was turned off. The expected values (E-values) of different hits from these searches were inspected to identify putative ε-proteobacteria-specific proteins [38, 40]. The proteins that were of interest to us generally involved large increase in E-values from the last ε-proteobacteria hit in the blast search to the first hit from any other organism. Further, the E values of these latter hits were expected to be in a range higher than 10^-4, which indicates weak level of similarity that could occur by chance. However, higher E-values are sometimes acceptable for smaller proteins as the magnitude of the E-value depends upon the length of the query sequence [63]. All promising proteins identified by the above criteria were further analyzed using the position-specific iterated (PSI) BLAST program [63]. This program creates a position-specific scoring matrix from statistically significant alignments produced by the BLASTp program and then searches the database using this matrix. The PSI-BLAST program is more sensitive in identifying weak but biologically relevant sequence similarity as compared to the BLASTp program [63]. The output of the PSI-BLAST program divides the various hits into two categories, i.e. sequences producing significant alignment versus those where the E values are worse than the threshold (default value set at .005). For most of the proteins that are indicated to be specific for different subgroups within ε-proteobacteria, all significant alignments were from the indicated groups. In a few cases, where an isolated hit has an E value slightly below the threshold value (arbitrarily set), but there was a large jump in E value from the last ε-proteobacteria hit, such proteins were also regarded as specific for the indicated groups. All of the identified group-specific proteins were also examined for the presence of any conserved domain [47] and this information along with the genome identification number of the protein, its accession number, sequence length, etc. was tabulated. In the description of various proteins in the text, the "WS" and "CJE" parts of the descriptors indicate the identification numbers of the proteins in the genomes of W. succinogenes DMS 1740 and C. jejuni RM1221, respectively.

Identification of conserved indels that are specific for epsilon proteobacteria

Multiple sequence alignments for large number of proteins have been created in our earlier work [44, 56, 60]. To search for conserved indels that might be specific for ε-proteobacteria, these alignments were visually inspected to identify any indel that was uniquely present in ε-proteobacteria species, and which was flanked by conserved sequences. The indels that were not flanked by conserved regions were not considered. The specificity of these indels for ε-proteobacteria was evaluated by carrying out detailed BLAST searches on short sequence segments (usually between 60–100 aa) containing the indel and the flanking conserved regions. The purpose of these BLAST searches was to obtain sequence information from all available bacteria homologs to determine the presence of the identified indels in various species. The sequence information for these indels was compiled into signature files such as those presented in Figures 2 and 3.

Phylogenetic analysis

Phylogenetic trees for the sequenced ε-proteobacteria species were constructed based on 16S rRNA sequences as well as a number of conserved proteins (viz. RNA polymerase β subunit (RpoB), RNA polymerase β ' subunit (RpoC), DNA gyrase A subunit (GyrA), DNA gyrase B subunit (GyrB), Hsp70, Hsp60, alanyl tRNA synthetase (AlaRS), elongation factor-G (EF-G) and elongation factor-Tu (EF-Tu) proteins) The 16S rRNA and protein sequences were downloaded from the Ribosomal Database Project-II site [65] and NCBI databases, respectively and aligned using the CLUSTALx program [66]. A neighbor-joining bootstrapped trees based on rRNA sequences was constructed by the Juke's and Cantor [67] method. The sequences for various proteins were concatenated into a large dataset containing 7919 aligned positions (RpoB (1440), RpoC (1559), GyrA (880), GyrB (814), Hsp70 (661), Hsp60 (552), AlaRS (912), EF-G (698) and EF-Tu (403)) and a neighbor-joining bootstrap tree based on this was constructed by Kimura's methods [68]. All gaps in the sequences were omitted during phylogenetic analyses. The trees were constructed using the PHYLIP [69] and the TREECON programs [70] and they were rooted using the chlamydiae species which is a deep branching group in comparison to ε-proteobacteria [41–43, 45].