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BMC Microbiology

  • Research article
  • Open Access

Phylogenomics and signature proteins for the alpha Proteobacteria and its main groups

BMC Microbiology20077:106

https://doi.org/10.1186/1471-2180-7-106

©  Gupta and Mok; licensee BioMed Central Ltd. 2007

Received: 20 July 2007

Accepted: 28 November 2007

Published: 28 November 2007

Abstract

Background

Alpha proteobacteria are one of the largest and most extensively studied groups within bacteria. However, for these bacteria as a whole and for all of its major subgroups (viz. Rhizobiales, Rhodobacterales, Rhodospirillales, Rickettsiales, Sphingomonadales and Caulobacterales), very few or no distinctive molecular or biochemical characteristics are known.

Results

We have carried out comprehensive phylogenomic analyses by means of Blastp and PSI-Blast searches on the open reading frames in the genomes of several α-proteobacteria (viz. Bradyrhizobium japonicum, Brucella suis, Caulobacter crescentus, Gluconobacter oxydans, Mesorhizobium loti, Nitrobacter winogradskyi, Novosphingobium aromaticivorans, Rhodobacter sphaeroides 2.4.1, Silicibacter sp. TM1040, Rhodospirillum rubrum and Wolbachia (Drosophila) endosymbiont). These studies have identified several proteins that are distinctive characteristics of all α-proteobacteria, as well as numerous proteins that are unique repertoires of all of its main orders (viz. Rhizobiales, Rhodobacterales, Rhodospirillales, Rickettsiales, Sphingomonadales and Caulobacterales) and many families (viz. Rickettsiaceae, Anaplasmataceae, Rhodospirillaceae, Acetobacteraceae, Bradyrhiozobiaceae, Brucellaceae and Bartonellaceae). Many other proteins that are present at different phylogenetic depths in α-proteobacteria provide important information regarding their evolution. The evolutionary relationships among α-proteobacteria as deduced from these studies are in excellent agreement with their branching pattern in the phylogenetic trees and character compatibility cliques based on concatenated sequences for many conserved proteins. These studies provide evidence that the major groups within α-proteobacteria have diverged in the following order: (Rickettsiales(Rhodospirillales (Sphingomonadales (Rhodobacterales (Caulobacterales-Parvularculales (Rhizobiales)))))). We also describe two conserved inserts in DNA Gyrase B and RNA polymerase beta subunit that are distinctive characteristics of the Sphingomonadales and Rhodosprilllales species, respectively. The results presented here also provide support for the grouping of Hyphomonadacea e and Parvularcula species with the Caulobacterales and the placement of Stappia aggregata with the Rhizobiaceae group.

Conclusion

The α-proteobacteria-specific proteins and indels described here provide novel and powerful means for the taxonomic, biochemical and molecular biological studies on these bacteria. Their functional studies should prove helpful in identifying novel biochemical and physiological characteristics that are unique to these bacteria.

Keywords

NeptuniumPhylogenomic AnalysisBrucella SpeciesCompatibility AnalysisAlpha Proteobacteria

Background

The α-proteobacteria form one of the largest groups within bacteria that includes numerous phototrophs, chemolithotrophs, chemoorganotrophs and aerobic photoheterotrophs [1]. They are abundant constituents of various terrestrial and marine environments [2]. Pelagibacter oblique, which is the smallest known free-living bacteria, is believed to be the most numerous bacteria on this planet (about 1028 cells) comprising about 25% of all microbial cells in the oceans [2]. The intimate association that many α-proteobacteria exhibit with the eukaryotic organisms is of central importance from agricultural and medical perspectives [3, 4]. Symbiotic association of the Rhizobiaceae family members with the plant root nodules is responsible for most of the atmospheric nitrogen fixation by plants [4–6]. Many other α-proteobacteria such as Rickettsiales, Brucella and Bartonella have adopted intracellular life styles and they constitute important human and animal pathogens [3, 7–9]. Additionally, the α-proteobacteria have also played a seminal role in the origin of the eukaryotic cell [10, 11].

In the current taxonomic scheme based on 16S rRNA, α-proteobacteria are recognized as a Class within the phylum Proteobacteria [1, 12, 13]. They are subdivided into 7 main subgroups or orders (viz. Caulobacterales, Rhizobiales, Rhodobacterales, Rhodospirillales, Rickettsiales, Sphingomonadales and Parvularculales) [12]. The α-proteobacteria and their main subgroups are presently distinguished from each other and from other bacteria primarily on the basis of their branching in phylogenetic trees [14, 15]. However, we have previously described several conserved inserts and deletions (indels), as well as whole proteins, that are specific for these bacteria [16, 17].

In the past 3–4 years, the numbers of α-proteobacterial genomes that have been sequenced has increased markedly. In addition to > 60 complete genomes (Table 1), sequence information is available for a large number of other species. These genomes cover all of the main groups within α-proteobacteria (Table 1) and provide an enormously valuable resource for identifying molecular characteristics that are unique to them. This information can be used to identify unique sets of genes or proteins that are distinctive characteristics of various higher taxonomic groups (e.g. families, orders, etc.) within α-proteobacteria. Such genes/proteins provide valuable tools for taxonomic, biochemical and molecular biological studies [17–26]. With this goal in mind, in the present work, we have performed comprehensive phylogenomic analyses of α-proteobacterial genomes to identify proteins/ORFs that are distinctive characteristics of the various higher taxonomic groups within α-proteobacteria. The phylogenomic distribution of these proteins is also compared with the branching patterns of these species in phylogenetic trees and in the character compatibility cliques to develop a reliable picture of α-proteobacterial evolution.
Table 1

Genome sizes, protein numbers and GC contents of sequenced alpha-proteobacteria

Species Name

Order

Family

Genome Size (Mb)

GC content (%)

Protein Number

Reference

Bartonella bacilliformis KC583

Rhizobiales

Bartonellaceae

1.4

38.2

1283

TIGR

Bartonella henselae str. Houston-1

Rhizobiales

Bartonellaceae

1.93

38.2

1488

[7]

Bartonella quintana str. Toulouse *

Rhizobiales

Bartonellaceae

1.58

38.8

1142

[7]

Bradyrhizobium japonicum USDA 110*

Rhizobiales

Bradyrhizobiaceae

9.1

64.1

8317

[5]

Bradyrhizobium japonicum BT Ail

Rhizobiales

Bradyrhizobiaceae

8.53

64.8

7622

DOE-JGI

Bradyrhizobium sp. ORS278

Rhizobiales

Bradyrhizobiaceae

7.5

65.5

6717

Genoscope

Nitrobacter hamburgensis X14

Rhizobiales

Bradyrhizobiaceae

5.01

61.6

4326

DOE-JGI

Nitrobacter winogradskyi Nb-255*

Rhizobiales

Bradyrhizobiaceae

3.4

2.5

3122

[42]

Rhodopseudomonas palustris BisA53

Rhizobiales

Bradyrhizobiaceae

5.51

64.4

4878

DOE-JGI

Rhodopseudomonas palustris BisB18

Rhizobiales

Bradyrhizobiaceae

5.51

65.0

4886

DOE-JGI

Rhodopseudomonas palustris BisB5

Rhizobiales

Bradyrhizobiaceae

4.89

64.8

4397

DOE-JGI

Rhodopseudomonas palustris CGA009

Rhizobiales

Bradyrhizobiaceae

5.47

65.0

4820

[41]

Rhodopseudomonas palustris HaA2

Rhizobiales

Bradyrhizobiaceae

5.33

66.0

4683

DOE-JGI

Brucella abortus biovar 1 str. 9–941

Rhizobiales

Brucellaceae

3.29

57.2

3085

[69]

Brucella melitensis 16M

Rhizobiales

Brucellaceae

3.29

57.2

3198

[38]

Brucella melitensis biovar Abortus 2308

Rhizobiales

Brucellaceae

3.28

57.2

3034

[39]

Brucella suis 1330*

Rhizobiales

Brucellaceae

3.32

57.3

2123

[40]

Brucella ovis ATCC 25840

Rhizobiales

Brucellaceae

3.3

57.2

2892

TIGR

Mesorhizobium loti MAFF303099*

Rhizobiales

Phyllobacteriaceae

7.6

62.5

7372

[6]

Mesorhizobium sp. BNC1

Rhizobiales

Phyllobacteriaceae

4.94

61.1

4543

DOE-JGI

Agrobacterium tumefaciens str. C58

Rhizobiales

Rhizobiaceae

5.67

59.0

4661

[37]

Rhizobium etli CFN 42

Rhizobiales

Rhizobiaceae

6.53

61.0

5963

[35]

Rhizobium leguminosarum bv. viciae 3841

Rhizobiales

Rhizobiaceae

7.75

61.0

7263

[81]

Sinorhizobium meliloti 1021

Rhizobiales

Rhizobiaceae

6.69

62.2

6205

[36]

Caulobacter crescentus CB15*

Caulobacterales

Caulobacteraceae

4.02

67.2

3737

[51]

Hyphomonas neptunium ATCC 15444

Caulobacterales+

Hyphomonadaceae

3.71

61.9

3505

[46]

Maricaulis maris MCS10

Caulobacterales+

Hyphomonadaceae

3.37

62.7

3063

DOE-JGI

Jannaschia sp. CCS1

Rhodobacterales

Rhodobacteraceae

4.4

62.2

4212

DOE-JGI

Paracoccus denitrificans PD1222

Rhodobacterales

Rhodobacteraceae

5.25

66.8

5077

DOE-JGI

Rhodobacter sphaeroides 2.4.1*

Rhodobacterales

Rhodobacteraceae

4.45

68.8

4242

DOE-JGI

Rhodobacter sphaeroides ATCC 17025

Rhodobacterales

Rhodobacteraceae

4.54

68.2

4333

DOE-JGI

Rhodobacter sphaeroides ATCC 17029

Rhodobacterales

Rhodobacteraceae

4.42

69.0

4132

DOE-JGI

Roseobacter denitrificans OCh 114

Rhodobacterales

Rhodobacteraceae

4.3

58.9

4129

[44]

Silicibacter pomeroyi DSS-3

Rhodobacterales

Rhodobacteraceae

4.6

64.1

4252

[45]

Silicibacter sp. TM1040*

Rhodobacterales

Rhodobacteraceae

4.15

60.1

3864

DOE-JGI

Gluconobacter oxydans 621H*

Rhodospirillales

Acetobacteraceae

2.92

60.8

2664

[56]

Granulibacter bethesdensis CGDNIH1

Rhodospirillales

Acetobacteraceae

2.7

59.1

2437

Rocky Mountain Lab

Acidiphilium cryptum JF-5

Rhodospirillales

Acetobacteraceae

3.97

67.1

3564

DOE-JGI

Magnetospirillum magneticum AMB-1

Rhodospirillales

Rhodospirillaceae

4.97

65.1

4559

[57]

Rhodospirillum rubrum ATCC 11170*

Rhodospirillales

Rhodospirillaceae

4.41

65.4

3841

DOE-JGI

Candidatus Pelagibacter ubique HTCC1062

Rickettsiales

-

1.3

29.7

1354

[2]

Anaplasma marginale str. St. Maries

Rickettsiales

Anaplasmataceae

1.2

49.8

949

[70]

Anaplasma phagocytophilum HZ

Rickettsiales

Anaplasmataceae

1.47

41.6

1264

[71]

Ehrlichia canis str. Jake

Rickettsiales

Anaplasmataceae

1.32

29.0

925

DOE-JGI

Ehrlichia chaffeensis str. Arkansas

Rickettsiales

Anaplasmataceae

1.18

30.1

1105

[71]

Ehrlichia ruminantium str. Gardel

Rickettsiales

Anaplasmataceae

1.5

27.5

950

[72]

Ehrlichia ruminantium str. Welgevonden

Rickettsiales

Anaplasmataceae

1.52

27.5

888

[73]

Neorickettsia sennetsu str. Miyayama

Rickettsiales

Anaplasmataceae

0.86

41.1

932

[71]

Rickettsia bellii RML369-C

Rickettsiales

Rickettsiaceae

1.52

31.6

1429

[74]

Rickettsia conorii str. Malish 7

Rickettsiales

Rickettsiaceae

1.27

32.4

1374

[75]

Rickettsia felis URRWXCal2

Rickettsiales

Rickettsiaceae

1.59

32.5

1512

[76]

Rickettsia prowazekii str. Madrid E

Rickettsiales

Rickettsiaceae

1.11

29.0

835

[77]

Rickettsia typhi str. Wilmington

Rickettsiales

Rickettsiaceae

1.11

28.9

838

[78]

Wolbachia endosymbiont (Drosophila)*

Rickettsiales

Rickettsiaceae

1.27

35.2

1195

[79]

Wolbachia endosymbiont (Brugia malayi)

Rickettsiales

Rickettsiaceae

1.08

34.2

805

[80]

Erythrobacter litoralis HTCC2594

Sphingomonadales

Erythrobacteraceae

3.05

63.1

3011

GBM Foundation

Novosphingobium aromaticivorans DSM 12444*

Sphingomonadales

Sphingomona-daceae

3.56

65.2

3937

DOE-JGI

Sphingopyxis alaskensis RB2256

Sphingomonadales

Sphingomona-daceae

3.37

65.5

3195

DOE-JGI

Sphingomonas wittichii RW1

Sphingomonadales

Sphingomona-daceae

5.93

67.9

5345

DOE-JGI

Zymomonas mobilis subsp. mobilis ZM4

Sphingomonadales

Sphingomona-daceae

2.06

46.3

1998

[53]

* indicates species that were used in blast searches

+ Although these species are classified as Rhodobacterales, results presented here and elsewhere [29,47] suggest their placement in the Caulobacterales.

DOE-JGI, Department of Energy, Joint Genome Institute; GBM, The Gordon and Betty Moore Foundation Marine Microbiology Initiative; TIGR, The Institute for Genome Research.

Results and discussion

Phylogeny of alpha proteobacteria

For comparing and interpreting the results of phylogenomic analysis, it was necessary at first to examine the evolutionary relationships among α-proteobacteria in phylogenetic trees. Phylogenetic analyses for α-proteobacteria was carried out based on concatenated sequences for 12 highly conserved proteins (see Methods). The relationships among these species were examined by both traditional phylogenetic methods (viz. neighbour-joining (NJ), maximum parsimony (MP) and maximum-likelihood (ML)) and by the character compatibility approach [27]. Figure 1 presents a neighbour-joining distance tree for α-proteobacteria, showing the bootstrap scores for various nodes using the NJ, MP and ML methods. In this tree, all of the main groups or orders within α-proteobacteria (viz. Caulobacterales, Rhizobiales, Rhodobacterales, Rhodospirillales and Sphingomonadales), except the Rickettsiales, formed well-resolved clades by different methods. For the Parvularculales, sequence information was available from a single species and it branched with the Caulobacterales. The two main families of the Rickettsiales i.e. Rickettsiaceae and Anaplasmataceae did not form a monophyletic clade, although they constituted the deepest branching lineages within α-proteobacteria. Except for the NJ method, the relative branching of different main groups within α-proteobacteria was not resolved by other methods. The branching orders of different groups as seen here is similar to that observed previously in the rRNA trees [1, 15, 16, 28]. Recently, after this work was completed, Williams et al. [29] have reported similar results based on phylogenetic analysis of a different large set of protein sequences.
Figure 1
Figure 1

A neighbour-joining distance tree based on concatenated sequences for 12 conserved proteins. The numbers on the nodes indicate bootstrap scores (out of 100) observed in the neighbour-joining (NJ), maximum parsimony (MP) and maximum-likelihood (ML) analyses (NJ/MP/ML). The species marked with * are presently not part of the Caulobacterales order, but the results of phylogenetic and phylogenomic studies presented here suggest their placement in this group.

The evolutionary relationships among α-proteobacteria were also examined using the character compatibility approach [27]. This method removes all homoplasic and fast-evolving characters from the dataset [27, 30, 31] and it has proven useful in obtaining correct topology in cases which have proven difficult to resolve by other means [31–33]. These analyses were carried out on a smaller set of 27 species containing all main groups of α-proteobacteria and two ε-proteobacteria. The concatenated sequence alignment for the 12 proteins contained 896 positions that were useful for these studies (i.e. those sites where only two amino acids were found with each present in at least two species). The compatibility analysis of these sites resulted in 12 largest cliques each containing 350 mutually compatible characters. The two main relationships observed in these cliques are shown in Fig. 2. The other cliques differed from those shown only in the relative branching positions of various Rhizobiaceae species, which varied from each other by a single character and are shown as unresolved in Fig. 2. In both these cliques, the species from all main orders within α-proteobacteria were clearly distinguished by multiple unique characters. Further, in contrast to the phylogenetic trees where Rickettsiaceae and Anaplasmataceae did not form a distinct clade (Fig. 1), their monophyletic grouping was strongly supported by 9 unique shared characters (Fig. 2). The Rickettsiales and Rhodospirillales formed the deepest branching lineages in these cliques and other groups within α-proteobacteria branched in the following order: (Sphingomonadales (Rhodobacterales (Caulobacterales (Rhizobiales)))). This branching order was supported by multiple unique characters at each node giving confidence in the results.
Figure 2
Figure 2

Character compatibility cliques showing the two largest cliques of mutually compatible characters based on the two states sites in the concatenated sequence alignment for 12 conserved proteins. The cliques consisted of 350 mutually compatible characters. The numbers of characters that distinguished different clades are indicated on the nodes. Rooting was done using the sequences for Helicobacter pylori and Campylobacter jejuni. *, as in Figure 1.

The two main cliques obtained in these analyses differed from each other in terms of the branching position of the species belonging to the order Rhodospirillales. In one clique (Fig. 2A), the Rhodospirillales branched with the Rickettsiales, whereas in the other this group of species was found to branch after the Rickettsiales and it formed outgroup of the other α-proteobacteria (Fig. 2B). However, only a single character supported the former relationship indicating that it was not reliable. The two families within the Rhodospirillales order (Rhodospirillaceae and Acetobacteraceae), although they branched close to each other, no unique character common to them was identified, indicating that they are highly divergent. The exact branching position of the Xanthobacter was also not resolved in these cliques. In some cliques, it appeared as an outgroup of the Bradyrhizobiaceae (as shown by the dotted lines in Fig. 2), whereas in others it was placed in the middle of the Rhizobiaceae and Bradyrhizobiaceae families (as seen for the Aurantiomonas).

Phylogenomic analyses of alpha proteobacteria

Table 1 lists some characteristics of various α-proteobacterial genomes that have been sequenced. The genomes vary in size from less than 1 Mb for Neorickettsia sennetsu to more than 9.0 Mb for Bradyrhizobium japonicum. To identify proteins that are distinguishing features of various higher taxonomic groups within α-proteobacteria, systematic Blastp searches were performed on each ORF in the genomes of B. japonicum USDA 110, Brucella suis 1330, Caulobacter crescentus CB15, Gluconobacter oxydans 621H, Mesorhizobium loti MAFF303099, Nitrobacter winogradskyi Nb-255, Novosphingobium aromaticivorans DSM 12444, Rhodobacter sphaeroides 2.4.1, Silicibacter sp. TM1040, Rhodospirillum rubrum ATCC 11170 and Wolbachia (Drosophila melanogaster) endosymbiont (see Methods section). The genomes chosen covered all main taxonomic groups within the sequenced α-proteobacteria. These analyses have identified large numbers of proteins that are uniquely found in particular groups of α-proteobacteria. A brief description of these results is given below.

Proteins that are distinguishing features of all (or most) alpha proteobacteria

We previously described 6 proteins (viz. CC1365, CC1725, CC1887, CC2102, CC3292 and CC3319) that appeared distinctive characteristics of α-proteobacteria [17]. The α-proteobacterial specificity of these proteins in earlier work was only assessed by means of Blastp searches and it was not confirmed by PSI-Blast, as in the present work (see Methods). Further, since the earlier work, the number of sequenced α-proteobacteria and other genomes has more than doubled. Hence, it was important to confirm the α-proteobacteria specificity of these proteins. Our results reveal that four of these proteins viz. CC1365, CC2102, CC3292 and CC3319, are indeed specific for the α-proteobacteria as a whole, whereas for the remaining two proteins homologs showing significant similarities are also found in other bacteria. Of these four proteins, CC3292 is present in all sequenced α-proteobacteria including Candidatus Pelagibacter ubique, which is the smallest known free-living bacterium [2]. The protein CC2102 is only missing in P. ubique, while the other two proteins are only missing in 1–2 rickettsiae species (CC1365) and P. ubique (CC3319). These proteins, which are uniquely present in virtually all α-proteobacteria, provide distinguishing markers for this Class of bacteria (Fig. 3).
Figure 3
Figure 3

Summary of the phylogenomic analyses showing the species distribution of various α-proteobacteria-specific proteins and the suggested evolutionary stages where the genes for these proteins have likely evolved. The genes IDs for some proteins described in earlier work are indicated [17]. The information for all other proteins can be found in the indicated Tables or Additional files. A large numbers of conserved indels that are specific for different groups or clades within α-proteobacteria shown here have also been identified in our earlier work [16] (not shown here). The branching order of α-proteobacteria relative to other bacteria has been established in earlier work [32,58].

In earlier work, 9 proteins were identified that were present in nearly all α-proteobacteria, except the Rickettsiales [17]. These latter species are all intracellular bacteria that have lost many genes that are not required under these conditions [3, 34]. The Blastp and PSI-Blast reexamination of these proteins have confirmed that 7 of these proteins (CC0100, CC0520, CC1211, CC1886, CC2245, CC3010 and CC3470) exhibit the indicated specificity. Except for CC0520 and CC3470, the other five proteins are also found in P. ubique, providing evidence for its placement within α-proteobacteria [2]. These results also provide evidence that P. ubique is not specifically related to the Rickettsiales. The phylogenomic distributions of these genes/proteins can be explained by either their evolution after the divergence of the Rickettsiales (Fig. 3), or by gene loss from this lineage.

Proteins that are distinguishing features of the Rhizobiales species

The Rhizobiales species comprise more than 1/3rd of the sequenced α-proteobacterial genomes (see Table 1). This order includes a wide assortment of species many of which interact with the eukaryotic organisms to produce diverse effects. This group includes various rhizobia (Rhizobium, Mesorhizobium, Sinorhizobium) and Bradyrhizobia species that induce root nodules in plants and live symbiotically within them to enable nitrogen fixation [4–6, 35, 36]. Another Rhizobiaceae species, A. tumefaciens, induces Crown gall disease (tumors) in plants [37]. Bartonella and Brucella species are intracellular pathogens responsible for a number of diseases in human and animals including trench fever and brucellosis [7, 38–40]. Other members of this order exhibit enormous versatility in terms of their metabolic capabilities and life styles [1, 41, 42]. Earlier studies identified six proteins that appeared distinctive characteristics of the Rhizobiales species [17]. Reexamination of these proteins by the more stringent criteria used in the present work confirmed that three of these proteins (BQ00720, BQ07670 and BQ12030) are indeed specific for the Rhizobiales and they are present in virtually all sequenced species from this large order. In addition to the Rhizobiales, these proteins are also present in Stappia aggregata, which is presently grouped with the Rhodobacterales [12], but was originally known as a strain of Agrobacterium [43].

New blast searches on the genomes of B. japonicum, B. suis, M. loti and N. winogradskyi have identified large number of other proteins that are specific for different subgroups within the Rhizobiales order. Seven proteins listed in Table 2A are uniquely present in most of the sequenced species belonging to the Rhizobiaceae (Rhizobium, Sinorhizobium, Agrobacterium), Brucellaceae, Phyllobacteriaceae (Mesorhizobium) and Aurantimonadaceae families. The absence of many of these proteins in Bartonella species is probably due to gene loss [7, 34]. These groups form a well-defined clade with high bootstrap scores in the NJ, MP and ML trees (see Fig. 1) and the genes for them likely evolved in a common ancestor of these genera/families (Fig. 3). Another 9 proteins (Table 2B) are uniquely present in most of the above species except Aurantimonas, which forms outgroup of the Rhizobiaceae, Brucellaceae, Bartonellaceae and Phyllobacteriaceae families (Figs. 1 and 2) [29]. Thus, the genes for these proteins have evolved after the branching of Aurantimonadaceae (Fig. 3). For six of the proteins in Tables 2A and 2B (marked with *), homologs with low E values are also found in S. aggregata, providing further evidence for its relatedness to the Rhizobiaceae family. Nine other proteins in Table 2C are found in most Rhizobiaceae species and M. loti, but they are missing in Bartonella and Brucella species. Although these proteins suggest a closer relationship between the Rhizobiaceae and Phyllobacteriaceae families, it is more likely that their genes have been lost from the Bartonella and Brucella species [34], which are intracellular bacteria. Additionally, some proteins were only found in either Mesorhizobium and Rhizobium, or Mesorhizobium and Sinorhizobium (Table 2D). These analyses have also identified 43 proteins that are uniquely present in all four sequenced Brucella species and many other proteins that are present in either three or two of the sequenced Brucella species (see Additional file 1).
Table 2

Proteins specific for the Rhizobiaceae and related species

Gene ID

Accession Number

Function

Gene ID

Accession Number

Function

A. Proteins unique to Aurantimonas, Mesorhizobium, Sinorhizobium, Rhizobium, Agrobacterium, Bartonella and Brucella

mll00621

NP_101943

Hypothetical

mlr07891

NP_102519

Hypothetical

mll40681

NP_105027

Hypothetical

mlr3016

NP_104217

Omp10

mll77911,4

NP_108034

Hypothetical

msl65261

NP_107016

Hypothetical

mlr0777 1

NP_102510

Hypothetical

   

B. Proteins unique to Mesorhizobium, Sinorhizobium, Rhizobium, Agrobacterium, Bartonella and Brucella (Missing in Aurantimonas )

mll0122 1,4

NP_101988

Hypothetical

mll5001 1,4

NP_105743

Hypothetical

mll1268 2

NP_102895

Hypothetical

mll8359 1,4

NP_108472

Hypothetical

mll2847 2,3

NP_104087

Hypothetical

mlr1823 1

NP_103319

Hypothetical

mll2898 4

NP_104130

Hypothetical

mlr0094 1,4

NP_101965

MhpC (COG0596)

mll4298 1,2

NP_105201

Hypothetical

   

C. Proteins unique to Mesorhizobium and Rhizobiaceae species

mll0080

NP_101954

Hypothetical

mll67034

NP_107159

Hypothetical

mll0867

NP_102577

Hypothetical

mlr1904

NP_103376

Hypothetical

mll9619

NP_109472

Hypothetical

mlr3274

NP_104418

Hypothetical

mlr5174 4

NP_105883

Hypothetical

mlr4951

NP_105704

NodF

mll6303

NP_106835

Hypothetical

   

D. Proteins specific to Mesorhizobium and either Rhizobium or Sinorhizobium

mll0459

NP_102252

Hypothetical

mll2007

NP_103455

Hypothetical

mll1779

NP_103286

Hypothetical

mlr1999

NP_103450

Hypothetical

mll6195

NP_106741

Hypothetical

mlr2029

NP_103476

Hypothetical

mll8758

NP_106740

Hypothetical

mlr6601

NP_107075

Hypothetical

mlr3037

NP_104236

Transcriptional regulator

   

1 Missing in Bartonella

2 Missing in Agrobacterium

3 Missing in Rhizobium.

4 Also found in Stappia aggregata

The analyses of proteins in the genomes of B. japonicum and N. winogradskyi have identified 12 proteins that are uniquely present in either all (or most) of the sequenced Bradyrhizobiaceae species as well as X. autotrophicus (Table 3A). The species from these two families form a strongly supported clade in the phylogenetic tree (Fig. 1)[29]. Sixty-two additional proteins in Table 3B are uniquely present in various species belonging to the Bradyrhizobiaceae family (i.e. Bradyrhizobium, Nitrobacter, Rhodopseudomonas). Many other proteins (see Additional file 2) are only found in two of the three Bradyrhizobiaceae genera and their distributions can result from gene losses, lateral gene transfers (LGTs), or other mechanisms.
Table 3

Proteins that are specific for the Bradyrhizobiaceae group

Gene ID

Accession Number

Function

Gene ID

Accession Number

Function

A. Proteins Unique to Bradyrhizobiaceae Family and Xanthobacter

bll60141,2

NP_772654

Putative general secretion pathway protein M

Nwi_2179

YP_318785

Hypothetical

Nwi_1093

YP_317707

Hypothetical

Nwi_24321

YP_319038

Hypothetical

Nwi_1227

YP_317841

Hypothetical

Nwi_24761

YP_319081

Putative bacterioferritin

Nwi_17861

YP_318399

Hypothetical

Nwi_2572

YP_319177

Hypothetical

Nwi_1788

YP_318401

Hypothetical

Nwi_2623

YP_319228

Hypothetical

Nwi_21471,3

YP_318753

Hypothetical

Nwi_2707

YP_319312

Hypothetical

B. Proteins Unique to Bradyrhizobiaceae Family

bll58992

NP_772539

Hypothetical

Nwi_2021

YP_318632

Hypothetical

blr61061,2

NP_772746

Hypothetical

Nwi_2063

YP_318673

Hypothetical

Nwi_0278

YP_316897

Hypothetical

Nwi_2064

YP_318674

Hypothetical

Nwi_0503

YP_317122

Hypothetical

Nwi_2163

YP_318769

Hypothetical

Nwi_0528

YP_317147

Hypothetical

Nwi_2173

YP_318779

Hypothetical

Nwi_06051

YP_317224

Hypothetical

Nwi_21831

YP_318789

Hypothetical

Nwi_07101,d

YP_317328

Hypothetical

Nwi_22083

YP_318814

Hypothetical

Nwi_0925

YP_317539

Hypothetical

Nwi_2244

YP_318850

Hypothetical

Nwi_09661,d

YP_317580

Hypothetical

Nwi_2247

YP_318853

Hypothetical

Nwi_1084

YP_317698

Hypothetical

Nwi_2379

YP_318985

Hypothetical

Nwi_1092

YP_317706

Hypothetical

Nwi_23812

YP_318987

Hypothetical

Nwi_11073

YP_317721

Hypothetical

Nwi_24143

YP_319020

Hypothetical

Nwi_1108

YP_317722

Hypothetical

Nwi_2489

YP_319094

Hypothetical

Nwi_1336

YP_317949

Hypothetical

Nwi_249213

YP_319097

Hypothetical

Nwi_1139

YP_317753

Hypothetical

Nwi_2500

YP_319105

Hypothetical

Nwi_12473

YP_317861

Hypothetical

Nwi_25063

YP_319111

Hypothetical

Nwi_1270

YP_317883

Hypothetical

Nwi_25093

YP_319114

Hypothetical

Nwi_12753

YP_317888

Hypothetical

Nwi_2531

YP_319136

Hypothetical

Nwi_14541

YP_318067

Hypothetical

Nwi_2575

YP_319180

Hypothetical

Nwi_1498

YP_318111

Hypothetical

Nwi_2577

YP_319182

Hypothetical

Nwi_1512

YP_318125

Hypothetical

Nwi_258813

YP_319193

Hypothetical

Nwi_15813

YP_318194

Hypothetical

Nwi_2630

YP_319235

Hypothetical

Nwi_1582

YP_318195

Hypothetical

Nwi_2676

YP_319281

Hypothetical

Nwi_1586

YP_318199

Dihydrofolate reductase

Nwi_26773

YP_319282

Hypothetical

Nwi_16491,3

YP_318262

Hypothetical

Nwi_2769

YP_319374

Hypothetical

Nwi_1674

YP_318287

Hypothetical

Nwi_27891

YP_319394

Hypothetical

Nwi_1705

YP_318318

Hypothetical

Nwi_29843

YP_319586

Hypothetical

Nwi_1711

YP_318324

Hypothetical

Nwi_2959

YP_319561

Hypothetical

Nwi_1785

YP_318398

Hypothetical

Nwi_3035

YP_319637

Hypothetical

Nwi_1793

YP_318406

Hypothetical

Nwi_3140

YP_319739

Hypothetical

Nwi_18001,2

YP_318413

Hypothetical

Nwi_31411

YP_319740

Hypothetical

1 Missing in one or more strains of Rhodopseudomonas

2 Missing in one or more species of Nitrobacter

3 Missing in Bradyrhizobium japonicum USDA 110

Proteins that are distinguishing features of the Rhodobacterales species

The order Rhodobacterales is a heterogeneous lineage of bacteria that exhibit much diversity in terms of their metabolism and cell division cycles [1, 13]. This group includes many photosynthetic bacteria that are capable of CO2 as well as nitrogen fixation and also many chemoorganotrophs that can metabolize various sulfur-containing compounds [44, 45]. A number of budding, stalk forming and prosthecate bacteria also belong to this group [46]. In addition to many completely sequenced genomes (see Table 1), information for several other species belonging to this order (e.g. Sulfitibacter, Oceanicola, Loktanella, Jannaschia, Dinoroseobacter, Roseovarius and Sagittula) is available in the NCBI database. To identify proteins that are specific for the Rhodobacterales, phylogenomic analyses were carried out on various ORFs in the genomes of R. sphaeroides 2.4.1 and Silicibacter sp. TM1040.

These studies have identified 29 proteins that are present in all-available Rhodobacterales species (Table 4A), but these proteins as well as those listed in Table 4B and 4C are not found in H. neptunium, Oceanicaulis alexandrii, Maricaulis maris or Stappia aggregata. These latter species are presently grouped with the Rhodobacterales [12], however, the absence of various Rhodobacterales-specific proteins in them and phylogenetic studies indicate that the placement of these species within this order is incorrect and needs be revised. In phylogenetic trees based on concatenated sequences for many proteins, O. alexandrii and M. maris consistently branched with the Caulobacter rather than the well-defined clade of Rhodobacterales (Figs. 1 and 2) [29]. The studies by Badger et al. [47] also provide strong evidence for the grouping of H. neptunium with the Caulobacterales.
Table 4

Proteins that are specific for the Rhodobacterales

Gene ID

Accession Number

Function

Gene ID

Accession Number

Function

A. Proteins specific for Rhodobacterales ( Oceanicola, Loktanella, Paracoccus, Roseovarius, Roseobacter, Jannaschia, Silicibacte r, Sulfitobacter, Dinoroseobacter, Sagittula )

TM1040_00931

YP_612088

Hypothetical

TM1040_1842

YP_613837

Phasin, PhaP

TM1040_01842

YP_612179

Hypothetical

TM1040_19673

YP_613961

Putative CheA signal transduction

TM1040_02361,2

YP_612231

Hypothetical

TM1040_1988

YP_613982

Hypothetical

TM1040_0471

YP_612466

Putative rod shape-determining protein MreD

TM1040_22632

YP_614257

Hypothetical

TM1040_05862

YP_612581

Hypothetical

TM1040_2370

YP_614364

Hypothetical

TM1040_05872

YP_612582

Hypothetical

TM1040_24253

YP_614419

Hypothetical

TM1040_0697

YP_612692

Hypothetical

TM1040_2466

YP_614460

GCN5-related N-acetyltransferase COG045

TM1040_07502,4

YP_612745

Hypothetical

TM1040_24872

YP_614481

Hypothetical

TM1040_07521

YP_612747

Lipoprotein, putative

TM1040_25823

YP_614576

Hypothetical

TM1040_1063

YP_613058

Gene transfer agent

TM1040_2999

YP_614993

Hypothetical

TM1040_1064

YP_613059

Gene transfer agent

TM1040_30773

YP_611313

Hypothetical

TM1040_12473

YP_613242

Hypothetical

TM1040_3749

YP_611978

Hypothetical

TM1040_13502

YP_613345

Hypothetical

TM1040_3759

YP_611988

Lipoprotein, putative

TM1040_1406

YP_613401

Outer membrane chaperone Skp (OmpH)

TM1040_37643

YP_611993

Putative transmembrane protein

TM1040_1567

YP_613562

Hypothetical

   

B. Proteins unique to various Rhodobacterales but missing in Rhodobacter and Paracoccus

TM1040_15581

YP_613553

Hypothetical

TM1040_21574

YP_614151

Hypothetical

TM1040_17351

YP_613730

Hypothetical

TM1040_24431,4

YP_614437

Lipolytic enzyme, G-D-S-L

TM1040_18441,5,6

YP_613839

Hypothetical

TM1040_26807

YP_614674

Hypothetical

C. Proteins Unique to Silicibacter and Roseobacter

TM1040_10998

YP_613094

Hypothetical

TM1040_3189

YP_611425

 

TM1040_14238

YP_613418

Hypothetical

TM1040_32028

YP_611438

 

TM1040_1451

YP_613446

Hypothetical

TM1040_32088

YP_611444

 

TM1040_19868

YP_613980

Hypothetical

TM1040_32268

YP_611462

 

TM1040_2106

YP_614100

Hypothetical

TM1040_35298

YP_611763

 

TM1040_21398

YP_614133

Hypothetical

TM1040_36268

YP_611855

 

TM1040_30758

YP_611311

Hypothetical

   

1 Missing in Loktanella vestfoldensis SKA53

2 Missing in one or more Rhodobacter sphaeroides strains

3 Missing in Paracoccus denitrificans PD12222

4 Missing in Rhodobacterales bacterium HTCC2654

5 Missing in one or more species of Roseovarius

6 Missing in Oceanicola batsensis HTCC2597

7 Missing in one or more species of Roseobacter

8 Missing in Silicibacter pomeroyi DSS-3

Six additional proteins in Table 4B are present in most of the Rhodobacterales species, but they are missing in R. sphaeroides and P. denitrificans, which form a distinct clade that appears as the outgroup of other Rhodobacterales species (Fig. 1)[29]. Thus, the genes for these proteins have likely evolved after the branching of these two genera. Thirteen additional proteins, which are only found in Silicibacter and Roseobacter genera (Table 4C) support a close relationship among them, as seen in phylogenetic trees (Fig. 1).

Of the proteins that are specific for Rhodobacterales, two of them (YP_613058 and YP_613059 in Table 4A, corresponding to proteins ABK27256 and ABK27255 in R. capsulatus genome) were previously identified as part of a complex referred to as gene transfer agent [48], based on similarity to certain virus-like elements. Another protein in the same category (viz. ABK27253) is specific for the Rhodobacter genus. The significance of these results is presently unclear.

Proteins that are distinctive characteristics of the Caulobacterales

The order Caulobacterales is comprised of a single family with only four genera [12, 49]. These chemoorganotrophic bacteria are commonly found in marine aerobic environments and they are distinguished by their ability to form stalked cells and unusual cell division cycle [49, 50]. The complete genome of only C. crescentus, which is the best-studied organism from this group, is presently available [51]. However, as discussed above, a number of other species which are presently classified as Rhodobacterales viz. O. alexandrii, M. maris, H. neptunium and also Parvularcula bermudensis, consistently branch with C. crescentus in different phylogenetic trees (Figs. 1 and 2) [29, 47]. Thus, phylogenomic analysis of the ORFs from C. crescentus genome was of much interest.

These analyses have identified 2 proteins (CC0486 and CC2480), which are uniquely present in this species as well as O. alexandrii, M. maris, H. neptunium and P. bermudensis (Table 5) One additional protein, CC2764 is present in all of these species except H. neptunium. The remaining eight proteins in Table 5 are only found in C. crescentus, O. alexandrii and M. maris, indicating that the latter two species are more closely related to C. crescentus in comparison to either H. neptunium or P. bermudensis. These results are strongly supported by the branching patterns of these species in phylogenetic trees (Figs. 1 and 2) [29, 47]. Previously, Badger et al. [46] have reported identification of 62 proteins that were only found in C. crescentus and H. neptunium. However, the blast threshold used in this study to infer the absence of these proteins in other species was very high i.e. 1e-10. By the criteria used in the present work (see Methods), none of these 62 proteins was found to be unique to these two species.
Table 5

Proteins specific for the Caulobacter and related species

Proteins Unique to Caulobacter, Oceanicaulis and Maricaulis(Some also found in Hyphomonas and Parvularcula)

Gene ID

Accession Number

Function

Gene ID

Accession Number

Function

CC04861

NP_419305

Hypothetical

CC1066

NP_419882

Hypothetical

CC24801

NP_421283

Hypothetical

CC1586

NP_420397

Hypothetical

CC27642

NP_421560

Hypothetical

CC2207

NP_421010

Hypothetical

CC3101

NP_421895

Hypothetical

CC2628

NP_421428

hfaA protein

CC0512

NP_419331

Hypothetical

CC2639

NP_421438

Hypothetical

CC1064

NP_419880

Hypothetical

   

All of the proteins in this Table are present in C. crescentus as well as in O. alexandrii and M. maris.

1 These proteins are also present in H. neptunium and P. bermudensis.

2 Also found in P. bermudensis.

Proteins that are distinguishing characteristics of the Sphingomonadales

The species belonging to the order Sphingomonadales are present in both terrestrial and aquatic environments [52]. A distinguishing characteristic of many species from this group is the presence of glycosphingolipids in their cell envelope rather than lipopolysaccharides [52, 53]. Several species from this group (e.g. N. aromaticivorans) can degrade a wide variety of aromatic hydrocarbons [52], whereas others such as Zymomonas mobilis, can highly efficiently ferment sugar to ethanol [53], making them of much interest and importance from biotechnological standpoints. This group also includes phototrophic organisms (e.g. Erythrobacter litoralis), which contain bacteriochlorophyll a and can derive significant fraction of their metabolic energy via anaerobic photosynthesis [54]. The complete genomes of 5 species from this order are now available (see Table 1). In addition, large numbers of sequences for Sphingomonas sp. SKA58, are also available in the NCBI database. Blast searches on the ORFs in the genome of N. aromaticivorans have identified 16 proteins (Table 6A) that are uniquely present in all 6 of the Sphingomonadales species for which information is available. Thirteen additional proteins (Table 6B) are present in all other Sphingomonadales species, except Z. mobilis, which is the deepest branching species within this group (Fig. 1). The genes for these proteins likely evolved after the branching of Z. mobilis. Many other proteins are present in only 3 or 4 of these species (viz. N. aromaticivorans, E. litoralis, S. alaskensis, S. wittichiii and Sphingomonas sp. SKA58) (see Additional file 3) and their phylogenomic distribution can result from a variety of mechanisms including shared ancestry, gene loss and LGTs among these species.
Table 6

Proteins that are specific for the Sphingomonadales group of species

Gene ID

Accession Number

Function

Gene ID

Accession Number

Function

A. Proteins Unique to Sphingomonadales Order (Novosphingobium, Erythrobacter, Sphingomonas, Sphingopyxis and Zymomonas)

Saro_0018

YP_495301

Hypothetical

Saro_1291

YP_496569

Hypothetical

Saro_00521

YP_495335

Hypothetical

Saro_1378

YP_496656

Hypothetical

Saro_0087

YP_495370

Hypothetical

Saro_19141

YP_497188

Hypothetical

Saro_0150

YP_495433

Hypothetical

Saro_2130

YP_497403

Hypothetical

Saro_0232

YP_495514

Hypothetical

Saro_2788

YP_498058

Hypothetical

Saro_0409

YP_495691

Hypothetical

Saro_2958

YP_498227

Hypothetical

Saro_1088

YP_496367

Hypothetical

Saro_3138

YP_498407

Hypothetical

Saro_1144

YP_496423

Hypothetical

Saro_3213

YP_498482

Hypothetical

B. Proteins Unique to Sphingomonadales but missing in Zymononas

Saro_0044

YP_495327

Hypothetical

Saro_17482

YP_497022

 

Saro_0154

YP_495437

Hypothetical

Saro_1785

YP_497059

 

Saro_04153

YP_495697

Hypothetical

Saro_1972

YP_497246

 

Saro_0458

YP_495740

Hypothetical

Saro_2036

YP_497309

 

Saro_1078

YP_496357

Hypothetical

Saro_2037

YP_497310

 

Saro_1126

YP_496405

Hypothetical

Saro_2333

YP_497604

 

Saro_1160

YP_496439

Hypothetical

Saro_2548

YP_497818

 

Saro_1163

YP_496442

Hypothetical

   

1 Missing in Sphingomonas wittchii

2 Blast scores for C. crescentus and H. neptunium are also significant

3 Significant blast scores for C. crescentus

We have also identified a 4 aa insert in a highly conserved region of Gyrase B that is mainly specific for the species from this order (Fig. 4). This indel is present in all available Sphingomonadales species, but it is not found in most other alpha proteobacteria or other bacteria (results not shown). Besides Sphingomonadales, a similar size indel is also present in three other species (viz. C. leidyia, R. blasticus, and Pesudorhodobacter incheonensis). Because other Rhodobacterales or Caulobacter species lack this insert, the presence of this indel in these three species could be either due to LGTs or possibly due to taxonomic misclassification of these species.
Figure 4
Figure 4

Partial sequence alignments of DNA Gyrase B showing a 4 aa insert that is mainly specific for the Sphingomonadales species. A 4–5 aa insert present in some other α-proteobacteria could be due to either LGTs or taxonomic anomalies. The dashes (-) denote identity with the amino acid on the top line. Sequence information for other groups of bacteria (which do not contain this insert) is not shown.

Proteins and indels that are specific for the Rhodospirillales species

The order Rhodospirillales is comprised of diverse species including some photosynthetic and magnetotactic bacteria, some acidophiles as well as other bacteria commonly associated with flowers, fruits and fermented beverages that are involved in the partial oxidation of carbohydrates and alcohols [55]. The order is made up of two main families, Rhodospirillaceae and Acetobacteraceae [12, 55]. The complete genomes of four species, two from each family, Rhodospirillaceae (Magnetospirillum magnticum, R. rubrum) and Acetobacteraceae (Acidiphilium cryptum and G. oxydans), are available (Table 1) [56, 57]. Phylogenomic analyses of various ORFs in the genomes of G. oxydans and R. rubrum have led to identification of one proteins, GOX0963, which is uniquely found in all of these species (Table 7A). Three other proteins in this Table are present in at least 3 of the 4 species from this order. This table also lists 14 proteins each that are distinctive characteristics of either the Acetobacteraceae (Table 7B) or the Rhodospirillaceae (Table 7C) families, providing molecular markers for these families. We have also identified a 25 aa insert in a conserved region of the RNA polymerase beta subunit (RpoB) that is unique to various sequenced Rhodospirillales species, but not found in any other bacteria (Fig. 5).
Table 7

Proteins that are specific for the Rhodospirillales group

Gene ID

Accession Number

Function

Gene ID

Accession Number

Function

A. Proteins Unique to Rhodospirillales Order ( Gluconobacter, Magnetospirillum, Rhodospirillum and Acidiphilium )

GOX06331

AAW60410

Hypothetical

GOX0963

AAW60735

Hypothetical

GOX06952

AAW60472

Hypothetical

GOX12583

AAW61019

Hypothetical

B. Proteins Unique to Acetobacteraceae Family ( Gluconobacter and Acidiphilium )

GOX0143

AAW59936

Hypothetical

GOX1616

AAW61357

Hypothetical

GOX0343

AAW60126

ANK, ankyrin repeats

GOX2216

AAW61951

Hypothetical

GOX1212

AAW60973

Phage portal protein

GOX2275

AAW62008

Hypothetical

GOX1215

AAW60976

Putative phage protein

GOX2316

AAW62049

Hypothetical

GOX1222

AAW60983

Putative phage protein

GOX2452

AAW62183

Hypothetical

GOX1224

AAW60985

Putative phage protein

GOX2454

AAW62185

Hypothetical

GOX1233

AAW60994

Hypothetical

GOX2456

AAW62187

Hypothetical

C. Proteins Unique to Rhodospirillaceae Family ( Rhodospirillum and Magnetospirillum )

Rru_A0125

YP_425217

Putative diguanylate phosphodiesterase

Rru_A2592

YP_427676

Hypothetical

Rru_A0152

YP_425244

Hypothetical

Rru_A2828

YP_427912

Hypothetical

Rru_A0531

YP_425622

Hypothetical

Rru_A3562

YP_428643

Hypothetical

Rru_A1689

YP_426776

Hypothetical

Rru_A3636

YP_428717

Hypothetical

Rru_A1756

YP_426843

Hypothetical

Rru_A3662

YP_428743

Hypothetical

Rru_A2112

YP_427199

Hypothetical

Rru_A3739

YP_428820

Hypothetical

Rru_A2510

YP_427597

Predicted transcriptional regulator

Rru_A3800

YP_428881

Hypothetical

1 Missing in Rhodospirillum

2 Missing in Acidiphilium

3 Missing in Magnetospirillum

Figure 5
Figure 5

Partial sequence alignments of RNA polymerase β subunit (RpoB) showing a large insert (boxed) that is a distinctive characteristic of various Rhodospirillales species and not found in any other bacteria. There are two homologs of RpoB in Magentospirillum (Mag.) magneticum and only one of these contains the insert. The dashes (-) denote identity with the amino acid on the top line.

Proteins that are specific for the Rickettsiales

The Rickettsiales species are intracellular pathogens responsible for a number of diseases in humans and other animals [3, 34]. This order is comprised of three families, Rickettsiaceae, Anaplasmataceae and Holosporaceae. All of the sequenced genomes are from the first two families. Phylogenomic analysis of various ORFs from the genome of Wolbachia (D. melanogaster) endosymbiont has identified 3 proteins viz. WD0161, WD0715 and WD0771 (Table 8A) that are specific for the entire Rickettsiales order. Five other proteins in Table 8B (viz. WD0083, WD0157, WD0821, WD0827 and WD0863) are present in all sequenced Ehrlichia, Anaplasma, Wolbachia and Neorickettsia species (belonging to the Anaplasmataceae family), but they are not found in any of the Rickettsiaceae or other bacteria. Ten additional proteins (Table 8C) are uniquely present in the Ehrlichia, Anaplasma and Wolbachia species, but are absent in Neorickettsia. In view of the deep branching of Neorickettsia in comparison to these other genera (Fig. 1), the genes for these proteins have likely evolved after the branching of Neorickettsia. In earlier work, a number of proteins that appeared specific for the Rickettsiaceae family were also identified [17]. Additional Blastp and PSI-Blast searches on these proteins confirm that three of these proteins viz. RP030, RP187 are RP192, are indeed specific for the Rickettsiaceae family. Of these, RP030 is also found in Orientia tsutsugamushi.
Table 8

Protein that are specific for the Rickettsiales group of species

Gene ID

Accession Number

Function

Gene ID

Accession Number

Function

A. Proteins Unique to Rickettsiales ( Wolbachia, Ehrlichia, Anaplasma, Rickettsia and Neorickettsia )

WD0161

NP_965979

Hypothetical

WD07711

NP_966526

Hypothetical

WD0715

NP_966474

Hypothetical

   

B. Proteins Unique to Anaplasmataceae Family ( Wolbachia, Ehrlichia, Anaplasma and Neorickettsia )

WD0083

NP_965909

Hypothetical

WD0827

NP_966580

Hypothetical

WD0157

NP_965975

Hypothetical

WD0863

NP_966613

Hypothetical

WD0821

NP_966574

Hypothetical

   

C. Proteins Unique to Anaplasmataceae Family but missing in Neorickettsia

WD0148

NP_965966

Hypothetical

WD0772

NP_966527

Hypothetical

WD0412

NP_966202

Hypothetical

WD1025

NP_966750

Hypothetical

WD0467

NP_966253

Preprotein translocase, SecG

WD1056

NP_966779

Hypothetical

WD0757

NP_966513

Hypothetical

WD1220

NP_966932

Hypothetical

WD0764

NP_966520

Hypothetical

WD1230

NP_966942

Hypothetical

1 Missing in Neorickettsia and Orientia

Conclusion

In this work, we have used a combined phylogenetic and phylogenomic approach to examine the evolutionary relationships among α-proteobacteria. Our analyses have identified large numbers of genes/proteins that are uniquely found in α-proteobacteria at various phylogenetic depths (Fig. 3). These include several proteins that are distinctive characteristics of all α-proteobacteria, as well as many proteins that constitute the unique repertoires of either all of the main orders of α-proteobacteria (viz. Rhizobiales, Rhodobacterales, Rhodospirillales, Rickettsiales, Sphingomonadales and Caulobacterales) or its different families (viz. Rickettsiaceae, Anaplasmataceae, Rhodospirillaceae, Acetobacteraceae, Bradyrhizobiaceae, Brucellaceae and Bartonellaceae). In addition, numerous other α-proteobacteria-specific proteins are present at different phylogenetic depths and they provide important information regarding the evolution of these bacteria. This work also describes two novel conserved indels in important housekeeping genes (viz. Gyrase B and RpoB) that are distinctive characteristics of the Sphingomonadales and Rhodospirillales orders, respectively. These indels are in addition to many other α-proteobacteria-specific indels that have been described in earlier work [16, 58].

Based upon these α-proteobacteria-specific proteins and conserved indels, it is now possible to define nearly all of the higher taxonomic groups (i.e. most orders and many families) within α-proteobacteria in clear and definitive molecular terms based upon multiple characteristics (Fig. 3). The species distribution profiles of these α-proteobacteria-specific proteins and indels also provide important information regarding their branching order and interrelationships, which are highly concordant with each other (c.f. Fig. 26 in ref. [16] with Fig. 3 in this work). Importantly, the relationships that emerge from these phylogenomic analyses (Fig. 3) are in excellent agreement with the branching patterns of these species in different phylogenetic trees (Figs. 1 and 2) [29], giving high degree of confidence in the derived inferences. It should be noted that both in our work (Fig. 1) and that by Williams et al. [29], when analyses were performed using the traditional phylogenetic methods (viz. NJ, MP or ML analyses), the branching of Caulobacterales with respect to Rhizobiales and Rhodobacterales was not resolved. In contrast, using the character compatibility approach, the Caulobacterales and related species were found to consistently branch in between the Rhizobiales and the Rhodobacterales (Fig. 2). Previously, this approach has also proven useful in clarifying the phylogenetic placement of Salinibacter ruber, which was not resolved by other methods [33, 59].

The phylogenetic studies presented here reveal that a number of species belonging to the Hyphomonadaceae family (viz. M. maris, O. alexandrii and H. neptunium), for which sequence information is available and that are presently grouped with the Rhodobacterales, branch reliably with the Caulobacterales (Figs 1 and 2)[29, 47]. The same is also true for P. bermudensis, which is the only species from Parvularculales order, for which sequence information is available. The grouping of these species with the Caulobacterales is independently strongly supported by phylogenomic studies, where a number of proteins that are unique for C. crescentus were present in these species and at the same time many proteins that are distinctive characteristics of the Rhodobacterales were absent in them. These results make a strong case for the transfer of these Hyphomonadaceae species and also P. bermudensis to an expanded Caulobacterales order [29, 47]. Another taxonomic anomaly identified by the present study concerns the phylogenetic position of Stappia aggregata. This species was originally identified as an Agrobacterium-related species, but later transferred to the Rhodobacterales order [43]. However, the shared presence of many Rhizobiales -specific proteins by S. aggregata and the absence of various Rhodobacterales-specific proteins in it, strongly suggest that it should be regrouped with the Rhizobiaceae-Phyllobacteriacea species.

The overwhelming majority of the identified α-proteobacteria-specific proteins do not have a homolog showing significant similarity in any other bacteria. The group-specificities of these proteins indicate that their genes have evolved in a common ancestor of these particular groups or clades of α-proteobacteria (Fig. 3). The clade specificities of these proteins also provide evidence that following their evolution, their genes have been transmitted primarily in a vertical manner, and that non-specific mechanisms such as LGTs have not played a significant role in their species distribution. Similar inferences have been reached in earlier studies for proteins that are specific for other higher taxa of bacteria [20, 22–24, 33, 60]. Most of the α-proteobacteria-specific proteins identified in the present work are of unknown function. A number of these proteins are present in the genomes in clusters of two or three, suggesting that they may form functional units and could be involved in related functions [18, 26, 48, 61]. The retention of these α-proteobacteria-specific proteins and conserved indels by the indicated clades of α-proteobacteria over long evolutionary periods strongly suggests that they serve essential functions in these groups of bacteria. Hence, studies on their cellular functions may lead to the discovery of novel biochemical and physiological characteristics that are distinctive characteristics of either all α-proteobacteria or their particular subgroups. Lastly, the primary sequences of many of these genes/proteins are highly conserved and they provide novel means for the identification and characterization of these bacteria by PCR-based and immunological methods.

Methods

Identification of proteins that are specific for alpha proteobacteria

The Blastp searches were carried out on each ORF in the genomes of B. japonicum USDA 110, B. suis 1330, C. crescentus CB15, G. oxydans 621H, M. loti MAFF303099, N. winogradskyi Nb-255, N. aromaticivorans DSM 12444, R. sphaeroides 2.4.1, Silicibacter sp. TM1040, R. rubrum ATCC 11170 and Wolbachia (D. melanogaster) endosymbiont to identify proteins that are uniquely present in α-proteobacteria species at different phylogenetic depths. The blast searches were performed against all organisms (i.e. non-redundant (nr) database) using the default parameters, without the low complexity filter [62]. The proteins that were of interest were those where either all significant hits were from the indicated groups (or orders) of α-proteobacteria, or which involved a large increase in E values from the last hit belonging to a particular group to the first hit from any other group and the E values for the latter hits were > 1e -4, indicating weak similarity that could occur by chance. However, higher E values were often considered significant for smaller proteins as the magnitude of the E value depends upon the length of the query sequence [62]. All promising proteins were further analyzed using the position-specific iterated (PSI)-Blast program [62]. In this study, we have also retained a few proteins where 1 or 2 isolated species from other groups had acceptable E values, as they provide possible cases of LGTs. For all of the proteins that are specific for α-proteobacteria, their protein ID's, accession numbers and any information regarding cellular functions (such as COG number or presence of any conserved domain) were tabulated and are presented. In describing various proteins in the text, "bll, bsl, blr", "BQ", "BR or BRA" "CC," "GOX" "ml", "Nwi", "Saro", "RSP", "TM1040", "Rru" and "WD" indicate the identification numbers of the proteins in the genomes of B. japonicum, Bartonella quintana, B. suis, C. crescentus, G. oxydans, M. loti, N. winogradskyi Nb-255, N. aromaticivorans, R. sphaeroides 2.4.1, Silicibacter sp. TM1040, R. rubrum and Wolbachia (Dros.) endosymbiont, respectively.

Phylogenetic analysis

The amino acid sequences for 12 conserved proteins (viz. RNA polymerase β and β' subunits, alanyl-tRNA synthetase, phenyalanyl-tRNA synthetase, arginyl-tRNA synthetase, protein synthesis elongation factors EF-Tu and EF-G, RecA, Gyrase A, Gyrase B, Hsp60 and Hsp70) for different species were downloaded from the NCBI database and aligned using the CLUSTAL × program [63]. In addition to the sequences for 50 α-proteobacteria species, sequences for two deep-branching species viz. Helicobacter pylori and Campylobacter jejuni [58], were also included for rooting purposes. The sequence alignments for all 12 proteins were concatenated into a single large file and poorly aligned regions were removed using the Gblocks 0.91b program [64]. The final sequence alignment that was used for phylogenetic analyses contained 7652 aligned positions. A neighbour-joining tree based on this alignment was constructed based on Kimura's two parameter model distances using the TREECON program [65]. Maximum-likelihood and MP trees were computed using the WAG+F model plus a gamma distribution with four categories using the TREE-PUZZLE [66] and Mega 3.1 program [67], respectively. All trees were bootstrapped 100 times [68], unless otherwise indicated.

The character compatibility analysis was performed on a concatenated sequences for the above 12 proteins for 25 α-proteobacteria species representing all its main orders plus two outgroup species (i.e. H. pylori and C. jejuni) [32]. Using the program "DUALSITE" [32], all sites in the sequence alignments where only two amino acid states were found, with each state present in at least two species, were selected. All columns where any gap was present in any of the species were omitted. The useful two state sites were converted into a binary file of "0, 1" characters using the DUALSITE program and this file was used for compatibility analysis [32]. The compatibility analysis was carried out using the CLIQUE program from the PHYLIP (ver. 3.5c) program package [68] to identify the largest clique(s) of compatible characters. The cliques were drawn and the numbers of characters that distinguished different nodes were indicated.

Identification of conserved indels specific for α-proteobacteria subgroups

Multiple sequence alignments for various proteins constructed in this work were visually inspected to search for any indels in a conserved region that was unique to particular subgroups or orders of α-proteobacteria. The group-specificity of any indel was evaluated by carrying out blast searches on a short segment of the sequence (between 80–120 aa) containing the indel and flanking conserved regions against the non-redundant database. The sequence information for various α-proteobacteria was compiled into signature files that are presented. Sequence information for all other groups of bacteria, which lack these inserts, is not shown.

Declarations

Acknowledgements

We thank Venus Wong and Yan Li for providing computer support for blast analyses and Beile Gao for help with phylogenetic analysis. We thank the investigators at US DOE Joint Genome Institute, The Institute of Genomic Research, Gordon and Betty Moore Foundation Marine Biotechnology Initiative and Rocky Mountain Laboratory, NIH, for making genome data for a number of alpha proteobacteria available in public databases prior to publication, which was very helpful in these studies. This work was supported by a research grant from the Canadian Institute of Health Research.

Authors’ Affiliations

(1)
Department of Biochemistry and Biomedical Science, McMaster University, Hamilton, Canada

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