Critical Reviews in Microbiology, 31:101–135, 2005 Copyright c Taylor & Francis Inc. ISSN: 1040-841X print / 1549-7828 online DOI: 10.1080/10408410590922393

Protein Signatures Distinctive of Alpha Proteobacteria and Its Subgroups and a Model for α-Proteobacterial Evolution

Radhey S. Gupta Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada

on the distribution patterns of these signatures, it is now possi- Alpha (α)proteobacteria comprise a large and metabolically ble to logically deduce a model for the branching order among diverse group. No biochemical or molecular feature is presently α-proteobacteria, which is as follows: Rickettsiales → Rhodo- known that can distinguish these bacteria from other groups. The spirillales-Sphingomonadales → Rhodobacterales-Caulobacterales evolutionary relationships among this group, which includes nu- → Rhizobiales (Rhizobiaceaea-Brucellaceae-Phyllobacteriaceae, merous pathogens and agriculturally important microbes, are also and Bradyrhizobiaceae). The deduced branching order is also con- not understood. Shared conserved inserts and deletions (i.e., indels sistent with the topologies in the 16 rRNA and other phylogenetic or signatures) in molecular sequences provide a powerful means trees. Signature sequences in a number of other proteins provide ev- for identification of different groups in clear terms, and for evo- idence that α-proteobacteria is a late branching taxa within Bacte- lutionary studies (see www.bacterialphylogeny.com). This review ria, which branched after the δ,-subdivisions but prior to the β,γ- describes, for the first time, a large number of conserved indels in proteobacteria. The shared presence of many of these signatures in broadly distributed proteins that are distinctive and unifying char- the mitochondrial (eukaryotic) homologs also provides evidence of acteristics of either all α-proteobacteria, or many of its constituent the α-proteobacterial ancestry of mitochondria. subgroups (i.e., orders, families, etc.). These signatures were iden- tified by systematic analyses of proteins found in the Rickettsia Keywords Bacterial Phylogeny; Alpha Proteobacteria Trees; Pro- prowazekii (RP) genome. Conserved indels that are unique to α- tein Signatures; Rickettsiales; Rhodobacterales; Branch- proteobacteria are present in the following proteins: Cytochrome ing Order; Mitochondrial Origin; Rickettsia prowazekii; c oxidase assembly protein Ctag, PurC, DnaB, ATP synthase α- Rhizobiales subunit, exonuclease VII, prolipoprotein phosphatidylglycerol transferase, RP-400, FtsK, puruvate phosphate dikinase, cyto- For personal use only. chrome b, MutY,and homoserine dehydrogenase. The signatures in succinyl-CoA synthetase, cytochrome oxidase I, alanyl-tRNA syn- INTRODUCTION thetase, and MutS proteins are found in all α-proteobacteria, ex- The alpha (α) proteobacteria comprise an important group cept the Rickettsiales, indicating that this group has diverged prior to the introduction of these signatures. A number of proteins con- within Bacteria, which has contributed seminally to many as- tain conserved indels that are specific for Rickettsiales (XerD inte- pects of the history of life (Margulis 1970; Kersters et al. 2003). grase and leucine aminopeptidase), Rickettsiaceae (Mfd, ribosomal It is now established that mitochondria, which enable eukary- protein L19, FtsZ, Sigma 70 and exonuclease VII), or Anaplasmat- otic cells to produce energy via oxidative phosphorylation, are aceae (Tgt and RP-314), and they distinguish these groups from the result of endosymbitotic capture of an α-proteobacteria by all others. Signatures in DnaA, RP-057, and DNA ligase A are commonly shared by various Rhizobiales, Rhodobacterales, and the primitive eukaryotic cell (Margulis 1970; Falah & Gupta Caulobacter, suggesting that these groups shared a common an- 1994; Viale & Arakaki 1994; Andersson et al. 1998; Gray et al. Critical Reviews in Microbiology Downloaded from informahealthcare.com by Cornell University cestor exclusive of other α-proteobacteria. A specific relationship 1999; Karlin & Brocchieri 2000; Emelyanov 2001a; Esser et al. between Rhodobacterales and Caulobacter is indicated by a large 2004). There is also strong evidence indicating that the ances- insert in the Asn-Gln amidotransferase. The Rhizobiales group tral eukaryotic cell itself may have originated via a fusion, or of species are distinguished from others by a large insert in the α Trp-tRNA synthetase. Signature sequences in a number of other long-term symbiotic association, event between one or more - proteins (viz. oxoglutarate dehydogenase, succinyl-CoA synthase, proteobacteria and an archaebacteria (or Archaea) (Gupta et al. LytB, DNA gyrase A, LepA, and Ser-tRNA synthetase) serve to 1994; Lake & Rivera 1994; Gupta & Golding 1996; Margulis distinguish the Rhizobiaceae, Brucellaceae, and Phyllobacteriaceae 1996; Gupta 1998; Martin & Muller 1998; Ribeiro & Golding families from Bradyrhizobiaceae and Methylobacteriaceae. Based 1998; Andersson et al. 1998; Karlin et al. 1999; Lang et al. 1999; Kurland & Andersson 2000; Emelyanov 2001a, 2003b). The symbiosis between α-proteobacteria (viz. Rhizobiaceae species) and plant root nodules plays a central role in the fixation of at- Received 20 December 2004; accepted 8 December 2005. Address correspondence to Radhey S. Gupta, Department of Bio- mospheric nitrogen by plants (Sadowsky & Graham 2000; Van chemistry and Biomedical Sciences, McMaster University, Hamilton, Sluys et al. 2002; Kersters et al. 2003; Sawada et al. 2003). Ad- Ontario, Canada L8N 3Z5. E-mail: [email protected] ditionally, many α-proteobacterial species (viz. Rickettsiales, 101 102 R. S. GUPTA

Brucella, Bartonella) are adapted to intracellular life style and ettsia conorii, Ri. prowazekii, Ri. typhi, and Wolbachia sp. are major human and animal pathogens (Moreno & Moriyon (Drosophila endosymbiont)) have become available (Anders- 2001; Kersters et al. 2003; Yu & Walker 2003). son et al. 1998; Kaneko et al. 2000, 2002; Nierman et al. 2001; The α-proteobacteria exhibit enormous diversity in terms of Wood et al. 2001; Ogata et al. 2001; Galibert et al. 2001; their morphological and metabolic characteristics and they in- DelVecchio et al. 2002; Paulsen et al. 2002; Larimer et al. 2004; clude numerous phototrophs, chemolithotrophs and chemoorgan- McLeod et al. 2004). These provide valuable resources for iden- otrophs (Stackebrandt et al. 1988; De Ley 1992; Kersters et al. tifying novel molecular features that are likely distinctive char- 2003). This group also harbors all known aerobic photoheterotro- acteristics of α-proteobacteria and its various subgroups, and hic bacteria, which contain bacteriochlorophyll a,but are unable which may prove helpful in clarifying the evolutionary rela- to grow photosynthetically under anaerobic conditions (Yurkov tionships among them. This article, describes for the first time, & Beatty 1998). These bacteria are abundant in the upper layers a large number of conserved indels in widely distributed pro- of oceans (Kolber et al. 2001). The α-proteobacterial species are teins that are either uniquely shared by all α-proteobacteria, or presently recognized on the basis of their branching pattern in which are shared by only particular subgroups (i.e., families or the 16S rRNA trees, where they form a distinct clade within the orders) of this Class. These signatures provide novel and defini- proteobacterial phylum (Woese et al. 1984; Stackebrandt et al. tive molecular means for distinguishing α-proteobacteria and 1988; Olsen et al. 1994; Gupta 2000; Kersters et al. 2003). This many of its subgroups from all other bacteria. The distribution group has been given the rank of a Class or subdivision within of these signatures in different α-proteobacteria also enables one the Proteobacteria phylum (Stackebrandt et al. 1988; Murray to logically deduce the relative branching orders and interrela- et al. 1990; De Ley 1992; Stackebrandt 2000; Ludwig & Klenk tionships among different α-proteobacteria subgroups. Phylo- 2001; Garrity & Holt 2001; Kersters et al. 2003). Other than genetic studies have also been carried out based on 16S rRNA their distinct branching in the 16S rRNA or other phylogenetic and a number of proteins sequences. Based on this informa- trees (De Ley 1992; Viale et al. 1994; Eisen 1995; Gupta et al. tion, a detailed model for the evolutionary relationships among 1997; Gupta 2000; Stepkowski et al. 2003; Emelyanov 2003a; α-proteobacteria has been developed. Battistuzzi et al. 2004), there is no reliable phenotypic or molec- ular characteristic known at present that is uniquely shared by PHYLOGENETIC TREE FOR ALPHA PROTEOBACTERIA different α-proteobacteria which distinguish them from all other BASED ON 16S rRNA SEQUENCES bacteria (Kersters et al. 2003). On the basis of 16S rRNA trees the Although α-Proteobacteria comprise a major group within α-proteobacteria have been divided into seven main subgroups Bacteria (Garrity & Holt 2001) with >5200 sequences in the or orders (viz. Caulobacterales, Rhizobiales, Rhodobacterales, Ribosomal Database Project II (Maidak et al. 2001), there is no Rhodospirillales, Rickettsiales, Sphingomondales, and Parvu- detailed review or article that discusses the evolutionary relation-

For personal use only. larucales) (Maidak et al. 2001; Garrity & Holt 2001; Kersters ships among this group (i.e. indicating the relationships among et al. 2003). However, the branching order and interrelation- different subgroups and orders within this Class) (Kersters et al. ships among these subgroups are presently not resolved and no 2003). Most of the articles on α-Proteobacteria are aimed at clar- distinctive features that can distinguish these groups from each ifying the phylogenetic placement of particular species at either other are known (Kersters et al. 2003). genus or family levels (Dumler et al. 2001; Gaunt et al. 2001; In our recent work, we have been utilizing a new approach Young et al. 2001; Taillardat-Bisch et al. 2003; van Berkum based on identification of conserved indels (also referred to as et al. 2003; Broughton 2003; Stepkowski et al. 2003; Sawada signatures) in proteins sequences that is proving very useful in et al. 2003). The second edition of Bergey’s Manual (Ludwig & identifying different groups within Bacteria in clear molecu- Klenk 2001) and the third edition of Prokaryotes (Kersters et al. lar terms and clarifying evolutionary relationships among them 2003) present condensed phylogenetic trees for the α-Proteo-

Critical Reviews in Microbiology Downloaded from informahealthcare.com by Cornell University (see www.bacterialphylogeny.com) (Gupta 1998, 2003, 2004; bacteria (or Proteobacteria) as a whole to indicate presumed Griffiths & Gupta 2002, 2004a; Gupta & Griffiths 2002; Gupta relationships among different subgroups comprising this sub- et al. 2003). We have previously described many protein sig- division. However, most of these trees do not show any boot- natures that are distinctive characteristics of the proteobacte- strap scores or even individual species (Ludwig & Klenk 2001; rial phylum and which also provided information regarding its Kersters et al. 2003), making it difficult to get a clear sense of the branching position relative to other bacterial groups (Gupta 1998, reliability of the observed (or indicated) relationships. Hence, 2000; Griffiths & Gupta 2004b). This review focuses on ex- as an initial step toward understanding the evolutionary rela- amining the evolutionary relationships among α-proteobacteria tionships among α-Proteobacteria, a phylogenetic tree based on using the signature sequence as well as traditional phyloge- 16S rRNA sequences was constructed from 65 α-proteobacterial netic approaches. In recent years, complete genomes of sev- species, covering its major subgroups. The resulting neighbor- eral α-proteobacteria (viz. Bartonella henselae, Bart. quintana, joining bootstrapped consensus tree is presented in Figure 1. Bradyrhizobium japonicum, Brucella melitensis, Bru. suis, The tree shown was rooted using the 16S rRNA sequences from Caulobacter crescentus, Mesorhizobium loti, Sinorhizobium loti, epsilon proteobacteria, which show deeper branching than the Rhodopseudomonas palustris, Agrobacterium tumefaciens, Rick- α-subdivision in the rRNA as well as various other trees (Olsen PHYLOGENY AND SIGNATURES DISTINCTIVE OF α-PROTEOBACTERIA 103 For personal use only. Critical Reviews in Microbiology Downloaded from informahealthcare.com by Cornell University

FIG. 1. A neighbor-joining bootstrap consensus tree for α-proteobacteria based on 16S rRNA sequences. The tree was bootstrapped 100 times and bootstrap scores which were >60 are indicated on the nodes. The tree was rooted using H. pylori.However, the tree topologies was not altered on rooting with other deep branching bacteria (e.g., Aq. aeolicus). The groups of species corresponding to some of the main subgroups within α-proteobacteria are marked. ∗indicates anomalous branching in the tree. 104 R. S. GUPTA

et al. 1994; Viale et al. 1994; Eisen 1995; Gupta 1998). The A. Signature Sequences That are Common to All bootstrap scores for all nodes, which were >60 (out of 100) are α-Proteobacteria indicated on the tree. Signature sequences in the following proteins are uniquely In the resulting tree a number of different clades are either shared by different α-proteobacteria. Cytochrome c oxidase clearly (>90% bootstrap score) or reasonably well resolved. (CoxI) is an integral component of the respiratory chain in mi- These included the clades corresponding to group of species tochondria and various aerobic bacteria, and it serves as the which are recognized as major orders within the α-Proteobacteria terminal electron acceptor (Stryer 1995; Andersson et al. 1998; (Rhizobiales, Rhodospirillales, Caulobacterales, Sphingomon- Emelyanov 2003a). This membrane-associated complex requires adales, Rhodobacterales, and Rickettsiales) (Ludwig & Klenk the association of several protein subunits and the formation of 2001; Garrity & Holt 2001; Kersters et al. 2003). Within Rhi- many different metal centers. One of the proteins involved in zobiales, the Bradyrhizobiaceae family of species was clearly its assembly is Ctag (Cox11), which is required for the for- separated from some of the other families within this order mation of CuB and magnesium centers of Cox I (Hiser et al. (viz. Rhizobiaceae, Brucellaceae, and Phyllobacteriaceae) 2000). In the Ctag protein,a5aainsert in a conserved region is (Wang et al. 1998; Sadowsky & Graham 2000; Dumler et al. present in all α-proteobacteria, but not found in any other bacte- 2001; van Berkum et al. 2003; Stepkowski et al. 2003). Within ria (Figure 2). Within bacteria the homologs of this protein are α-Proteobacteria, the deepest branching was observed for the mainly restricted to α, β, and γ -subdivisions of proteobacteria. Rickettsiales group of species. Within the Rickettsiales, the Rick- Although a protein which carries out a similar function (also ettsia, and Orientia genera, which form part of the Rickttsi- known as Ctag) is present in gram-positive bacteria, it does not aceae family, were clearly resolved from the Anaplasmataceae show any sequence similarity to the proteobacterial homologs family comprised of Ehrlichia, Wolbachia, Anaplasma, and Ne- (Bengtsson et al. 2004). The observed insert in the Ctag protein orickettsia species (Dumler et al. 2001; Yu & Walker 2003). In is also present in various eukaryotic homologs, supporting their contrast to these well-resolved clades or relationships, various derivation from α-proteobacteria. nodes indicating the interrelationships among different orders Another conserved insert that is specific for α-proteobacteria had lower bootstrap scores (<60%), indicating that interrela- is present in the enzyme 5-phosphoribosyl-5-aminoimidazole- tionships among them were not resolved. The relationships ob- 4-N-succinocarboxamide (SAICAR or PurC) synthetase, which served here are very similar to those reported in earlier studies carries out the seventh step in the de novo purine biosynthetic (Olsen et al. 1994; Sadowsky & Graham 2000; Dumler et al. pathway (Hui & Morrison 1993; Stryer 1995). The enzyme is en- 2001; Yu et al. 2001; Kersters et al. 2003; Yu & Walker 2003; coded by the purC gene and it is broadly distributed in bacteria. Stepkowski et al. 2003). The tree shown here will serve as a A3aa insert in this protein is present in various α-proteobacteria useful reference for determining the evolutionary significance (Figure 3), but not in any other bacteria, indicating that it is a For personal use only. of various signature sequences. distinctive characteristic of the group. The eukaryotic homologs of the SAICAR synthetase do not contain this insert, but their SIGNATURE SEQUENCES DISTINCTIVE OF overall similarity in this region is limited (not shown). In ad- α-PROTEOBACTERIA dition to α-proteobacteria, a 2 aa insert is also present in this To identify conserved indels that might be distinctive of α- position in Magnetococcus sp. MC-1, suggesting that it may be Proteobacteria, or a particular groups of species within this distantly related to this group. The phylogenetic assignment of Class, multiple sequence alignments of all proteins that are found Magnetococcus sp. MC-1 is presently uncertain (Garrity & Holt in the genome of Ri. prowazekii (Andersson et al. 1998) were cre- 2001). ated using the CLUSTAL X program (Jeanmougin et al. 1998). Two other signatures that are specific for α-proteobacteria are These alignments were visually inspected for any conserved in- present in the replicative DNA helicase (DnaB) and the α subunit

Critical Reviews in Microbiology Downloaded from informahealthcare.com by Cornell University dels that were mainly restricted to the α-proteobacterial species. of ATP synthase complex. DnaB helicase is a multifunctional The indels that we focussed on were generally of defined size enzyme involved in the DNA replication process (Soni et al. and they were present in the same position in a given protein. 2003). It interacts with a number of proteins involved in DNA The indels of interest were also required to be flanked on both replication and exhibits multiple enzymatic activities including sides by conserved regions to ensure that the sequence align- helicase, ATP hydrolysis and DNA binding. An insert of between ment in the region was reliable and that the indel under con- 8 and 14 aa is present in a conserved region of DnaB, which is sideration was not resulting from any alignment artefact (Gupta unique to various α-proteobacteria (Figure 4). Most of the Rhizo- 1998, 2000; Rokas & Holland 2000; Gupta & Griffiths 2002). biales as well as Rhodobacter and Caulobacter species are found The indels that appeared unique to other groups of bacteria, or to contain the 14 aa insert, whereas a smaller insert is present in which were present in only a single α-proteobacterial species, various Rickettsiales and certain other α-proteobacteria. In Mag. were not further investigated. This has led to identification of magnetotacticum, three different homologs of DnaB are found many conserved indels that are specific for α-proteobacteria or and they all contained the 14 aa insert. Based upon the fact that its subgroups. A brief description of these signatures as well as this insert is present in the same position in all α-proteobacteria, of the proteins in which they are found is given below. it is likely that it was introduced only once in a common ancestor PHYLOGENY AND SIGNATURES DISTINCTIVE OF α-PROTEOBACTERIA 105 For personal use only.

FIG. 2. Partial sequence alignment for Cytochrome c oxidase assembly protein (Ctag) showinga5aainsert (boxed), which is specific for α-proteobacteria. Dashes in this sequence alignment as well as all others indicate identity with the amino acid on the top line. The position of this sequence in the proteinismarked

Critical Reviews in Microbiology Downloaded from informahealthcare.com by Cornell University on the top. The accession numbers of various sequences are shown in the second column. Only representative sequences from different bacteria are shown. The identified insert is also present in various eukaryotic (mitochondrial) homologs indicating their derivation from an α-proteobacterial ancestor. The abbreviations used in the species names are listed at the end of this review.

of the group and that subsequent genetic changes led to the ob- is commonly present in all α-proteobacteria, but which is not served variation in its length. The DnaB homologs are not found found in any other proteobacterial species (Figure 5). Besides, in eukaryotic species. α-proteobacteria, inserts of variable lengths are also present in The synthesis of ATP in different organisms is carried out this position in various Actinobacteria and Bacteriodetes (not by F1F0ATP synthase, a multisubunit complex located in the shown). In phylogenetic tree based on ATP synthase (α), these cytoplasmic membrane of bacteria or inner membrane of mito- latter groups do not show any affinity for each other or to the α- chondria (Stryer 1995; Leyva et al. 2003). The F1 portion of this proteobacteria (Gupta 2004), indicating that these inserts have complex is a heteromer made up of five subunits, α, β, γ , δ and likely been introduced independently. The observed insert in  with the stoichiometry α3β3γδ. The α subunit of ATP syn- ATP synthase α is also present in various eukaryotic homologs thase contains an 8 aa insert in a highly conserved region that providing evidence of their α-proteobacterial ancestry. 106 R. S. GUPTA For personal use only.

FIG. 3. Partial sequence alignment of PurC (SAICAR synthetase) showinga3aainsert that is specific for various α-proteobacteria.

The enzyme exonuclease VII degrades single-stranded DNA supported by the phylogenetic analysis. The evolutionary stages bidirectionally and processively (Chase et al. 1986). In the large where different identified signatures have likely been introduced subunit of exonuclease VII, encoded by the xseA gene, a number in this gene/protein are marked on the tree (Figure 7). of useful signatures for α-proteobacteria are present. These sig- Another insert that is specific for α-proteobacteria is present natures includea3aainsert anda1aadeletion that are present in the enzyme prolipoprotein-phosphatidylglycerol (PLPG) trans- in all known α-proteobacterial homologs (Figure 6A), but not ferase, which carries out the first committed step in the pathway Critical Reviews in Microbiology Downloaded from informahealthcare.com by Cornell University found in any other groups of bacteria. In the same position where leading to synthesis of lipid modified proteins (Figure 6B) (Qi the 3 aa insert is found, an additional 3 aa insert is present in et al. 1995). The indicated 3 aa insert in PLPG- transferase is all α-proteobacteria except the Rickettsiales. Elsewhere in this unique to α-proteobacteria and not found in other bacteria. The protein, a 1 aa deletion (at position 141 in the Ri. prowazekii se- homologs of exonuclease VII and PLPG-transferase were not quence) that is unique to various Rickettsia species is also found detected in eukaryotes. (not shown). In a phylogenetic tree based on exonuclease VII se- Two other proteins where α-proteobacteria-specific inserts quences (Figure 7), all of the α-proteobacterial homologs formed are found are, RP-400 and puryvuate phosphate dikinase (PPDK). a well-defined clade, which was strongly supported by bootstrap The first of these is a protein of unknown function present in the scores. Similar to the rRNA tree, the Rickettsiales species formed Ri. prowazekii genome (RP-400), which is distantly related to the earliest branching group within the α-proteobacteria, and murein transglycosylases. This protein contains a 4–6 aa insert both the Rickettsiales clade, as well as a clade comprising of the in a conserved region that is a distinctive characteristic of var- remainder of the α-proteobacteria were clearly resolved in this ious α-proteobacteria, except Zymomonas mobilis (Figure 8). tree. The inferences from signature sequences are thus strongly The absence of this insert in the latter species could result from PHYLOGENY AND SIGNATURES DISTINCTIVE OF α-PROTEOBACTERIA 107 For personal use only. Critical Reviews in Microbiology Downloaded from informahealthcare.com by Cornell University

FIG. 4. Partial sequence alignment of replicative DNA helicase, DnaB, showing an 8–14 aa insert that is specific for various α-proteobacteria.

either selective loss, or exchange of this gene from some other found in various other α-proteobacteria (Figure 9). Interestingly, species lacking the insert. PPDK is a key enzyme in photosyn- an insert of 10 aa is also present in the same position in various thesis, which catalyzes the reversible conversion of phospho- δ-proteobacteria, suggesting a distant relationship of this group enolpyruvate to pyruvate (Ku et al. 1996). This enzyme is not to the α-proteobacteria. Because the insert sequence in vari- found in mammalian cells but it is broadly distributed in bacteria ous species appears to be related, it is possible that this insert and plants. A conserved insert in PPDK provides an informative was originally introduced in a common ancestor of the α- and δ- signature for α-proteobacteria. Rickettsiales species contain a proteobacteria. The varying lengths of the inserts in Rickettsiales 5aainsert in this position, whereas a larger insert of 12 aa is and other α-proteobacteria could then result from subsequent 108 R. S. GUPTA For personal use only. Critical Reviews in Microbiology Downloaded from informahealthcare.com by Cornell University

FIG. 5. Sequence alignment of ATP synthase α subunit showing an 8 aa insert in a highly conserved region that is present in various α-proteobacterial homologs, but not found in any other proteobacteria. The shared presence of this insert in various eukaryotic homologs provides evidence of their α-proteobacterial ancestry.

genetic changes in the branches leading to these groups. Alter- divergent and it is possible that this may have been acquired by natively, the inserts of different lengths could have been inde- means of lateral gene transfer (LGT) from other bacteria. pendently introduced in these groups and the observed sequence Two different proteins, FtsK and Cytochrome b (PetB) con- similarity may be a consequence of their related function. It is tain deletions which are mainly limited to the α-proteobacterial of interest that in contrast to other α-proteobacteria, which con- species. In the FtsK protein, which plays a central role in cell tain only a single PPDK homolog, two homologs of this protein division and chromosome segregation in bacteria (Capiaux et al. are found in Bradyrhizobium (Brad.) japonicum. Of these, only 2002; Espeli et al. 2003), two 1 aa deletions are present in con- one contained the insert. The homolog lacking the insert is quite served regions that are largely distinctive of α-proteobacteria PHYLOGENY AND SIGNATURES DISTINCTIVE OF α-PROTEOBACTERIA 109 For personal use only. Critical Reviews in Microbiology Downloaded from informahealthcare.com by Cornell University

FIG. 6. Signature sequence in exonucleaseVII (A) and prolipoprotein-phosphatidylglycerol (PLPG) transferase (B) proteins that are distinctive of α- proteobacterial species. Exonuclease VII contains a 3 aa insert and a 1 aa deletion that is unique to all α-proteobacteria. The presence of an additional 3 aa insert distinguishes Rickettsiales species from other α-proteobacteria. The enzyme PLPG-transferase also contains a 3 aa insert that is specific for α-proteobacteria. 110 R. S. GUPTA For personal use only. Critical Reviews in Microbiology Downloaded from informahealthcare.com by Cornell University FIG. 7. A neighbor-joining bootstrap consensus tree based on exonucleaseVII sequences. Bootstrap scores >50% are indicated on various nodes. All inserts and deletions were excluded from the sequence alignment used for phylogenetic analysis. The α-proteobacteria formed a well-defined clade in this tree, however, their branching position relative to other groups was not resolved. The Rickettsiales order formed the deepest branch within α-proteobacteria and they were also clearly resolved from other α-proteobacteria. The arrows mark the suggested positions where the identified signatures were introduced in this protein.

(Figure 10). One of these deletions is a distinctive characteris- position corresponding to aa 513–520 in Ri. prowazekii protein). tic of all α-proteobacteria and not found in any other bacteria. Since the region where this insert is found exhibits variability The other deletion, in addition to the α-proteobacteria, is in other bacteria, this signature is not shown. The FtsK protein also commonly present in the two Desulfovibrio species has also been previously shown to contain an 8–9 aa insert in a (δ-proteobacteria), suggesting a distant relationship of this group different region of the protein that is a distinctive characteristic to α-proteobacteria, as also seen with the PPDK protein (Figure of various Bacteriodetes and Chlorobium species (Gupta 2004). 9). In addition to these deletions, the FtsK protein also con- The FtsK homologs are not found in most eukaryotic organisms. tains a 5–6 aa insert that is unique to various α-proteobacteria However, a homolog of this protein is present in Plasmodium in comparison to the other groups of proteobacteria (present in yoelii (Genebank accession number 23485217). The origin and PHYLOGENY AND SIGNATURES DISTINCTIVE OF α-PROTEOBACTERIA 111

FIG. 8. Partial sequence alignment of RP-400 protein showing a 4–6 aa insert that is specific for various α-proteobacteria, except Z. mobilis. For personal use only. possible significance of this gene/protein is presently unclear. branching clade within α-proteobacteria (see Figures 1 and 7) A1aa deletion that is specific for various α-proteobacteria is (Dumler et al. 2001; Gaunt et al. 2001; Yu et al. 2001; Kersters also present in the Cytochrome b (Cyt b; PetB) protein (Fig- et al. 2003; Yu & Walker 2003; Stepkowski et al. 2003). We ure 11), which is a subunit of the cytochrome reductase, which have identified several signatures that are present in various α- is an integral part of the electron transport chain (Daldal et al. proteobacteria, except the Rickettsiales. These signatures are 1987; Stryer 1995; Emelyanov 2003a). This indel is not present described below. in other bacteria including that from Aquifex aeolicus, indicat- The enzyme succinyl CoA-synthetase, which is part of the ing that it is a deletion in α-proteobacteria, rather than an insert citric acid cycle, carries out cleavage of the thioester bond in in other bacteria. Cyt b is one of the 13 proteins that is still succinyl-CoA in a coupled reaction to generate succinate and encoded by mitochondrial DNA (Lang et al. 1999). Sequence producing GTP (Bridger et al. 1987; Stryer 1995). It is the Critical Reviews in Microbiology Downloaded from informahealthcare.com by Cornell University information for Cyt b is available from a large number (>500) only step in the citric acid cycle that directly leads to the for- of mitochondrial genomes and phylogenetic studies based on mation of a high-energy phosphate bond. The beta subunit of this protein provides evidence for the origin of mitochondria this protein contains a conserved insert of 10 aa, that is com- from within the Rickettsiaceae (Sicheritz-Ponten et al. 1998; monly present in all other α-proteobacteria, except the Rick- Emelyanov 2003a). Similar to the α-proteobacteria, Cyt b from ettsiales (Figure 12). Surprisingly, this insert is also present all eukaryotic mitochondrial homologs was found to lack this 1 in Ral. metallidurans (a β-proteobacterium), but not in any aa indel, providing evidence of their specific relationship to the other β-proteobacteria, including the closely related species Ral. α-proteobacteria. solanacearum. This suggests that the Succ-CoA synthetase gene in Ral. metallidurans has likely originated by non-specific means such as LGT. A smaller unrelated insert in this region, which is B. Signature Sequences Distinguishing Rickettsiales from presumably of independent origin, is also present in Cytophaga Other α-Proteobacteria and Rhodopirellula species (not shown). It is of interest that a In phylogenetic trees based on 16S rRNA, as well as many 7–8 aa insert is also present in this position in various eukary- protein sequences, the Rickettsiales are found to form the deepest otic homologs. It is unclear at present, whether this latter insert 112 R. S. GUPTA For personal use only.

FIG. 9. Excerpt from sequence alignment for pyruvate phosphate dikinase (PPDK) protein showing a signature for α-proteobacteria. The Rickettsiales species contain a 5 aa long insert, where all other α-proteobacteria have a 12 aa insert in the same position. Two different homologs of PPDK are found in Brad. japonicum, only one of which is found to contain the insert. A smaller conserved insert of 10 aa is also present in this position in various δ-proteobacteria suggesting that they may be specifically, but distantly, related to the α-proteobacteria.

has originated from an α-proteobacterial ancestor or it is of in- Another signature showing a similar distribution pattern has dependent origin. If these inserts are of common origin, then been identified in cytochrome oxidase polypeptide I (Cox I). this would suggest that the eukaryotic homologs of Succ-CoA- In this case,a5aainsert in a conserved region is commonly

Critical Reviews in Microbiology Downloaded from informahealthcare.com by Cornell University synthetase have originated from an α-proteobacterial ancestor present in various α-proteobacterial species except the Rick- other than the Rickettsiales. This observation will be at variance ettsiales (Figure 13). It should be noted that α-proteobacteria with other evidence pointing to a closer relationship of mito- contain two different related proteins. One of these, which har- chondria to the Rickettsiales species (Viale & Arakaki 1994; bors this insert seems to correspond to Cox I, whereas the other Gupta 1995; Andersson et al. 1998; Sicheritz-Ponten et al. 1998; homologs lacking the insert are mainly those from Cytochrome Gray et al. 1999; Lang et al. 1999; Emelyanov 2001a, 2001b, o ubiquinol oxidase (Davidson & Daldal 1987). However, all 2003a). Emelyanov (2001a, 2001b) has observed a closer rela- Rickettsiales species contain only a single homolog of this pro- tionship of mitochondrial homologs to certain rickettsial species tein, corresponding to Cox I. The observed insert in both Succ- (e.g. Holospora obtusa, Caedibactera caryophila), for which CoA-synthetase and Cox I were thus likely introduced in a com- sequence information for this protein is lacking at present. It mon ancestor of the remainder of the α-proteobacteria after the is possible that Succ-CoA synthetase from these species may branching of Rickettsiales. Similar to the Cyt b, the Cox I in eu- contain this insert. Presently, the possibility that the insert in karyotic cells is also encoded by mitochondrial DNA (Andersson eukaryotic homologs was independently introduced also cannot et al. 1998; Gray et al. 1999) and sequence information for be excluded. this protein is available from a large number of mitochondrial PHYLOGENY AND SIGNATURES DISTINCTIVE OF α-PROTEOBACTERIA 113 For personal use only.

FIG. 10. Partial sequence alignments of FtsK protein showing two different signatures (1 aa deletions) that are informative characteristics of α-proteobacteria. The deletion on the left is unique to various α-proteobacteria, whereas the one on the right is also commonly shared by two Desulfovibrio species (δ-proteobacteria) suggesting their relatedness to the α-proteobacteria.

genomes. The eukaryotic homologs of Cox I do not contain for these signatures is that they were introduced in an ancestral

Critical Reviews in Microbiology Downloaded from informahealthcare.com by Cornell University the identified insert (results not shown) indicating their possible α-proteobacterial lineage, after the branching of Rickettsiales derivation from Rickettsiales (Emelyanov 2003a). (and also possibly Mag. magnetotacticum). The observed vari- Two other proteins were found to contain inserts of variable ations in the lengths of these inserts have presumably resulted lengths in highly conserved regions in various α-proteobacterial from subsequent genetic changes. species, with the exception of Rickettsiales (Figure 14). In We have also identified a number of α-proteobacteria-specific alanyl-tRNA synthetase (AlaRS), which is ubiquitously found signatures in proteins for which no homologs are found in all organisms, an insert of between 5–11 aa is present in in the Rickettsiales.Inthe MutY protein, which is an A-G a highly conserved region in various α-proteobacteria, except specific DNA glycosylase involved in DNA repair (Parker & the Rickettsiales (and also Mag. magnetotacticum) (Figure 14A). Eshleman 2003; Martins-Pinheiro et al. 2004), a 4–9 aa in- Another signature showing a similar distribution pattern is found sert in a conserved region is present in various α-proteobacteria in the MutS protein, which is involved in the DNA mismatch re- (Figure 15A). An insert of similar length is also present in most pair (Sixma 2001; Martins-Pinheiro et al. 2004). In this case, a eukaryotic homologs (with the exception of Anopheles gambiae) conserved insert of 2–5 aa is present in various α-proteobacteria indicating their possible derivation from α-proteobacteria. An- (Figure 14B), but not in Rickettsiales. The simplest explanation other signature showing similar species distribution is present in 114 R. S. GUPTA For personal use only. Critical Reviews in Microbiology Downloaded from informahealthcare.com by Cornell University

FIG. 11. Partial sequence alignment for Cyt b protein showing a 1 aa deletion that is specific for various α-proteobacteria. This deletion is also present in all mitochondrial homologs (Cyt b is encoded by mitochondrial DNA) providing strong evidence of their α-proteobacterial ancestry.

the protein homoserine dehydrogenase (Figure 15B). This indel observed inserts in these genes could have been introduced in a consists ofa1aainsert in a conserved region that is present common ancestor of the α-proteobacteria, either before or after in various α-proteobacteria, but not any other proteobacteria. the loss of these genes in Rickettsiales. The homologs of both these proteins were not detected in the Several proteins contain conserved inserts that are either unique Rickettsiales species and their absence is very likely due to selec- for the Rickettsiales or for the two main families, Rickettsi- tive loss of these genes in a common ancestor of the Rickettsiales aceae and Anaplasmataceae, comprising this order (Dumler (Martins-Pinheiro et al. 2004), presumably due to the intracel- et al. 2001; Yu& Walker 2003). The Rickettsiales-specific signa- lular life-style of these organisms (Boussau et al. 2004). The tures are present in the proteins XerD and leucine aminopeptidase PHYLOGENY AND SIGNATURES DISTINCTIVE OF α-PROTEOBACTERIA 115 For personal use only.

FIG. 12. Partial sequence alignment of Succ-CoA synthase showing a 10 aa insert that is present in various α-proteobacteria, except the Rickettsiales. This insert is not found in other bacteria, except Ral. metallidurans, which has likely acquired it by non-specific means. A smaller insert is also present in this position in various eukaryotic homologs. Critical Reviews in Microbiology Downloaded from informahealthcare.com by Cornell University (Figure 16). XerD protein (Figure 16B) is a part of the XerCD (Figure 17B) anda1aainsert in the FtsZ protein (Figure 17C). integrase/recombinase that is involved in the cell division pro- Two additional Rickettsia-specific signatures consisting of a 1 cess and decatenation of DNA duplexes (Ip et al. 2003).A7aa aa insert in the major sigma factor-70 (at position 141 in the R. insert is present in a conserved region of this protein which is prowazekii sequence) anda1aadeletion in exouclease VII (at uniquely shared by all Rickettsiales and not found in any other position 137 in the Ri. prowazekii homolog) were also identi- bacteria (Figure 16A). Another 2 aa insert that is specific for fied, but they are not shown here. The identified signatures in Rickettsiales is present in leucine aminopeptidase (Figure 16A), these proteins are present only in various Rickettsiaceae species which is an exopeptidase that selectively releases N-terminal and not found in other Rickettsiales (viz. Ehrlichia, Wolbachia, amino acids from peptides and proteins (Gonzales & Robert- Anaplasma)orother groups of bacteria. Within eukaryotes, a Baudouy 1996). The signatures that are specific for Rickettsia homolog of the transcription repair-coupling factor is only de- include a 4 aa insert in a highly conserved region of the tran- tected in Arabidoposis thaliana and it lacks the identified insert scription repair coupling factor (Mfd) (Martins-Pinheiro et al. (results not shown). The homologs of ribosomal protein L19 are 2004) (Figure 17A), a 10 aa insert in ribosomal protein L19 found in various plants and algae but not in any of the animal 116 R. S. GUPTA

FIG. 13. Partial sequence alignment of Cox I showing a 5 aa insert that is present in various α-proteobacteria, except Rickettsiales. The other α-proteobacteria also contains a second more distantly related homolog that lacks this insert.

species. Of these, an 8 aa insert in the same position is present et al. 2003; Emelyanov 2003a). These signatures were likely in- For personal use only. only in the homolog from Cyanophora paradox (not shown). The troduced in a common ancestor of the Anaplasmataceae family, significance and possible origin of this insert is not clear. Simi- which now includes all Ehrlichia, Anaplasma, Cowdria, Wol- lar to the ribosomal protein L19, FtsZ homologs are also found bachia, and Neorickettsia species (Dumler et al. 2001; Yu & only in plants but not in animals. These homologs also lacked the Walker 2003). insert that is present in Rickettsiaceae. The plant homologs of these proteins likely correspond to those of the plastids, which because of their cyanobacterial ancestry (Gray 1989; Morden C. Signature Sequences for Other Subgroups of et al. 1992; Margulis 1993; Gupta et al. 2003) are expected to α-Proteobacteria and Providing Information Regarding be lacking Rickettsia-specific signatures. Their Interrelationships We have also identified two large inserts that are commonly Signature sequences in a number of other proteins are use- Critical Reviews in Microbiology Downloaded from informahealthcare.com by Cornell University shared by the Ehrlichia, Wolbachia, and Anaplasma species but ful in distinguishing other subgroups of α-proteobacteria and not found in any of the Rickettsia species or other bacteria. These they also provide information clarifying the interrelationships signatures include a 15 aa insert in the HlyD family of secretory among them. In the DnaA protein involved in chromosomal protein (Figure 18A) and a 10–11 aa insert in the tRNA guanine replication (Messer 2002),a5aainsert is present in various Rhi- transglycosylase (Tgt) protein (Figure 18B), involved in the syn- zobiales and Caulobacter/Rhodobacter species (Figure 19A). thesis of hypermodified nucleoside queousine (Reuter & Ficner However, this insert is not found in any of the Rickettsiales,as 1995). The eukaryotic homologs of Tgt do not contain this insert well most α-proteobacterial species belonging to the orders Sph- providing evidence against their origin from Anaplasmatacaea ingomonadales and Rhodospirillales. The species Mag. mag- family of species (results not shown). The homologs of HlyD are netotacticum contains two different homologs of this protein, not found in eukaryotes. These signatures point to a close rela- only one of which is found to contain the insert. Another insert tionship between Ehrlichia, Wolbachia, and Anaplasma species, showing a similar distribution pattern is present in the protein which is also seen in phylogenetic trees based on many other se- RP057, which is a homolog of the glucose-inhibited division quences (Dumler et al. 2001; Gaunt et al. 2001; Yu et al. 2001; protein B (Romanowski et al. 2002). This protein contains a 3 aa Taillardat-Bisch et al. 2003; Yu & Walker 2003; Stepkowski insert that is common to the same subgroups of α-proteobacteria PHYLOGENY AND SIGNATURES DISTINCTIVE OF α-PROTEOBACTERIA 117 For personal use only. Critical Reviews in Microbiology Downloaded from informahealthcare.com by Cornell University

FIG. 14. Signature sequences in alanyl-tRNA synthetase (AlaRS) and MutS proteins that are informative for the α-proteobacteria. In AlaRS (upper panel) an insert of variable length in a highly conserved region is present in various α-proteobacteria, except the Rickettsiales and Mag. magentotacticum. The DNA mismatch repair protein MutS (lower panel) also contains a 3–5 aa insert in various α-proteobacteria, except Rickettsiales. The inserts lengths in this case also serve to differentiate Rhodospirillales and Sphingomonadales species from the Rickettsiales, Rhodobacterales, and Caulobacterales. 118 R. S. GUPTA For personal use only. Critical Reviews in Microbiology Downloaded from informahealthcare.com by Cornell University

FIG. 15. Partial sequence alignments of MutY (upper panel) and homoserine dehydrogenase (lower panel) proteins showing inserts (boxed) in conserved regions that are specific for α-proteobacteria. The homologs of both these proteins are not found in the Rickettsiales. For MutY, an insert of approximately similar length is also present in various eukaryotic homologs, with the exception of Anopheles gambiae.

as the insert in the DnaA protein, but which is not found in α-proteobacteria. Based on the distribution patterns of these the Rickettsiales or Rhodospirillales/Sphingomonadales species signatures, these inserts were likely introduced in a common (Figure 19B). The variable length inserts are also present in ancestor of the Rhizobiales and Caulobacter/Rhodobacter this position in other bacteria (not shown). However, within after the branching of Rickettsiales and Rhodospirillales/ proteobacteria this insert is limited to the above subgroups of Sphingomonadales orders (Figure 19C). PHYLOGENY AND SIGNATURES DISTINCTIVE OF α-PROTEOBACTERIA 119 For personal use only. Critical Reviews in Microbiology Downloaded from informahealthcare.com by Cornell University

FIG. 16. Signature sequences in XerD integrase (upper panel) and leucine aminopeptidase (lower panel) that are distinctive of the Rickettsiales order and not found in other α-proteobacteria or other bacteria. 120 R. S. GUPTA For personal use only. Critical Reviews in Microbiology Downloaded from informahealthcare.com by Cornell University

FIG. 17. Signature sequences in transcription repair coupling factor Mfd (A), Ribosomal protein L19 (B), and FtsZ (C) proteins that are distinctive of Rickettsia species and not found in other α-proteobacteria including Anaplasmataceae family (e.g., Wolbachia, Ehrlichia, Anaplasma)ofspecies. Two additional signatures showing similar distribution are found in the sigma factor-70 and exonuclease VII proteins. PHYLOGENY AND SIGNATURES DISTINCTIVE OF α-PROTEOBACTERIA 121 For personal use only. Critical Reviews in Microbiology Downloaded from informahealthcare.com by Cornell University

FIG. 18. Signature sequences in RP-314 (A) and tRNA guanine transglycosylase (Tgt) (B) proteins that are distinctive of the Anaplasmataceae family of species and not found in Rickettsia or various other bacteria.

The protein DNA ligase (NAD dependent; Lig A) contains a ingomonadales (Z. mobilis, Novo. armoaticivorans). The ab- 12 aa insert in a highly conserved region that is commonly shared sence of this insert in the Mesorhizobium sp. BNC1,issome- by various Rhizobiales as well as Rhodobacterales species what surprising, but it could result from non-specific mecha- (Figure 20A), but which is not found in C. crescentus, Rho- nisms. This signature suggests that Rhizobiales species may dospirillales (Rhodo. rubrum, Mag. magnetotacticum), and Sph- be more closely related to Rhodobacterales in comparison to 122 R. S. GUPTA For personal use only. Critical Reviews in Microbiology Downloaded from informahealthcare.com by Cornell University

FIG. 19. Partial sequence alignments of DnaA (panel A) and RP-057 (panel B) proteins showing inserts in conserved regions (boxed) that are only present in various Rhizobiales, Rhodobacterales, and Caulobacter,but not found in other α-proteobacteria or bacteria. These inserts were likely introduced in a common ancestor of the above groups after the branching of Rickettsiales, Rhodospirillales, and Sphingomonadales as indicated in panel C.

Caulobacter and other α-proteobacteria. However, another promi- a distinct clade in the tree, but they consisted of only certain nent insert (11 aa) in a highly conserved region of the protein Rhizobiaceace and Caulobacter species (Martins-Pinheiro et al. aspargine-glutamine amidotransferase points to a specific re- 2004). To fully understand the evolutionary significance of these lationship between Rhodobacterales and Caulobacter species signatures, it would be necessary to obtain sequence information (Figure 20B), to the exclusion of all other α-proteobacteria. for these proteins from additional Caulobacterales. Martins-Pinheiro et al. (2004) have reported phylogenetic anal- We have also identified many conserved inserts that are ysis based on LigA sequences. The α-proteobacteria formed specific for species belonging to the Rhiziobiales order. The PHYLOGENY AND SIGNATURES DISTINCTIVE OF α-PROTEOBACTERIA 123 For personal use only.

FIG. 20. Signature sequences in DNA ligase A and (upper panel) and Asn-Gln amidotransferase (lower panel) that are informative for α-proteobacteria. The signature in DNA ligase is commonly shared by various Rhizobiales as well as C. crescentus species, while that in the Asn-Gln amidotransferase is uniquely shared by Rhodobacterales and Caulobacter, indicating a specific relationship between these subgroups.

Trp-tRNA synthetase (TrpRS) contains a large insert in a in a common ancestor of the Rhizobiales and subsequent modifi-

Critical Reviews in Microbiology Downloaded from informahealthcare.com by Cornell University highly conserved region which is uniquely shared by various cation has led to the observed length variation. The distinctness Rhizobiales species (Figure 21A), but not found in any of the of Bradyrhizobium and Rhodopseudomonas from other Rhizo- other α-proteobacteria or other groups of bacteria (results for biales is also supported by a signature (3 aa insert) in Seryl-tRNA other groups of bacteria not shown). The absence of this insert in synthetase (SerRS), which is uniquely present in these species various Rickettsiales, Rhodospirillales, Sphingomonadales, and (Figure 21B) and it serves to distinguish them from other Rhizo- Rhodobacterales as well as Caulobacter provides evidence that biales as well as other α-proteobacteria. A schematic diagram these groups have branched off prior to the introduction of this indicating the suggested positions where signatures described in insert (Figure 21A). The length of the insert in TrpRS also serves Figures 20 to 23 have been introduced is presented in Figure 21C. to distinguish the Rhizobiaceae, Brucellaceae, and Phyllobacte- We have also identified several signatures that are uniquely riaceae family of species from those belonging to Bradyrhizobi- present in the Rhizobiaceae, Brucellaceae, and Phyllobacteri- aceae and Methylobacteriaceae. The insert in the former group aceae families of species, but not found in other α-proteobacteria of species is 19 aa long, whereas the latter species contain only a including Bradyrhizobium and Rhodopseudomonas. These sig- 9–10 aa insert. Because the insert sequence in all of these species natures include a 7 aa insert in Oxoglutarate dehydrogenease is conserved, it is likely that the insert was introduced only once (Figure 22A),a5aainsert in Succ-CoA synthase (Figure 22B), 124 R. S. GUPTA For personal use only.

FIG. 21. Signature sequences in Trp-tRNA synthetase (upper panel) and Ser-tRNA synthetase (lower panel) that are informative for α-proteobacteria. The first of these signatures is specific for Rhizobiales. The insert length in this signature also distinguishes Bradyrhizobiaceae and Methylobacteriaceae species from other Rhizobiales. The insert in the Ser-tRNA synthetase is specific for the Bradyrhizobiaceae species and distinguishes this family from other Rhizobiales.

a3aainsert in LytB metalloproteinase (Figure 23A) anda2aa well as Ehrlichia and a few other proteobacteria, has also been insert in DNA gyrase A subunit (Figure 23B). A smaller insert described by Stepkowski et al. (2003). in oxoglutarate dehydrogenase is also present in Novosphingob- acteria,but since its sequence is unrelated, it is either of in- D. Signature Sequences Indicating the Phylogenetic Critical Reviews in Microbiology Downloaded from informahealthcare.com by Cornell University dependent origin or could have resulted from LGT. In addition Placement of α-Proteobacteria to these proteins, a 1–2 aa insert that is specific for Rhizobi- A number of signatures described in earlier work have indi- aceae is also found in a conserved region of the LepA protein cated that proteobacteria is a late branching phylum in compar- (Figure 23C). The evolutionary positions where these signa- ison to other main groups within Bacteria (Gupta 1998, 2000, tures have been introduced are indicated in Figure 21C. It is 2003; Gupta & Griffiths 2002; Griffiths & Gupta 2004b). These of interest that in contrast to other Rhizobiaceae species, which signatures includeda4aainsert in alanyl-tRNA synthetase, an contain only 1 aa inserts, Sinorhizobium meliloti and Agrobac- insert of >100 aa in RNA polymerase β (RpoB) subunit, a 10 terium tumefacienes are found to contain 2 aa inserts in the LepA aa insert in CTP synthase, a 2 aa insert in inorganic pyrophos- protein (Figure 23C). This observation points to a specific rela- phatase, anda2aainsert in Hsp70 protein. The identified sig- tionship between these two Rhizobiaceae species, as has been natures in these proteins were present in all proteobacterial ho- suggested based on other lines of evidences (Young et al. 2001). mologs, but they were absent from most other bacterial phyla A2aainsert in the DnaK protein, which is commonly shared (viz. Firmicutes, Actinobacteria, Thermotogae, Deinococcus- by species belonging to Rhizobium and Sinrhizobium genera, as Thermus, Cyanobacteria, Spirochetes). In a number of cases, PHYLOGENY AND SIGNATURES DISTINCTIVE OF α-PROTEOBACTERIA 125 For personal use only.

FIG. 22. Signature sequences in Oxoglutarate dehydrogenase (upper panel) and Succ-CoA synthase (lower panel) proteins that are commonly shared by only certain Rhizobiales families (e.g., Rhizobiaceae, Brucellaceae, and Phyllobacteriaceae), and not found in Bradyrhizobiaceae or other α-proteobacteria. Critical Reviews in Microbiology Downloaded from informahealthcare.com by Cornell University where the corresponding proteins were present in Archaea (viz. Figure 24 shows the excerpt from a sequence alignment for RpoB, Hsp70, AlaRS), the archael homologs also lacked the in- the transcription termination factor Rho, which is an RNA- dicated inserts, indicating that the absence of these indels con- binding protein that plays a central role in the RNA chain stitute the ancestral states and that these signatures were intro- termination (Opperman & Richardson 1994). This protein is duced after branching of the groups lacking these indels (Gupta present in all main groups of bacteria, except cyanobacteria & Griffiths 2002; Gupta 2003; Griffiths & Gupta 2004b). A num- (Gupta & Griffiths 2002; Gupta 2003), where RNA chain termi- ber of identified signatures (7 aa insert in SecA, 1 aa deletion in nation presumably occurs via a Rho-independent mechanism. the Lon protease) were uniquely shared by only the α, β, and A3aa insert is present in a highly conserved region of Rho, γ -proteobacteria, providing evidence of the later branching of which is a distinctive characteristic of all α, β, and γ -proteo- these subdivisions (Gupta 2000, 2001, 2003). Two additional bacteria. The length of this insert is 2–3 aa longer in various signatures that are helpful in understanding the phylogenetic Rickettsiales species, which suggests an additional insert in this placement of α-proteobacteria are described in the following group of bacteria. In contrast to the α, β, and γ -proteobacteria, section. this insert is not present in δ, ε-proteobacteria or any other 126 R. S. GUPTA For personal use only. Critical Reviews in Microbiology Downloaded from informahealthcare.com by Cornell University

FIG. 23. Signature sequences in LytB (A), DNA gyrase A (B) and LepA proteins that are distinctive characteristics of only certain Rhizobiales families (e.g., Rhizobiaceae, Brucellaceae, and Phyllobacteriaceae), but not found in Bradyrhizobiaceae or other α-proteobacteria. PHYLOGENY AND SIGNATURES DISTINCTIVE OF α-PROTEOBACTERIA 127 For personal use only. Critical Reviews in Microbiology Downloaded from informahealthcare.com by Cornell University

FIG. 24. Partial sequence alignment of Rho protein showing a conserved insert that is commonly shared by various α, β, and γ -proteobacteria, but not found in any other groups of bacteria including the δ,-proteobacteria and all other phyla of gram-positive and gram-negative bacteria. This insert was likely introduced in a common ancestor of the α, β, and γ -proteobacteria after the branching of other bacterial phyla (see Figure 26). Many other signatures showing similar distribution pattern and supporting the indicated branching position of α, β, and γ -proteobacteria have been described in earlier work.

groups of Gram-negative and Gram-positive bacteria. This is present in various β and γ -proteobacteria, but it is not signature provides evidence that the groups consisting of α, found in any α-proteobacteria or other groups of bacteria (Fig- β, and γ -proteobacteria have branched off late in compari- ure 25). The absence of this insert in various other bacteria as son to the other groups of bacteria. Another novel signature well as archael homologs provides evidence that it was intro- that is useful in understanding the branching position of α- duced in a common ancestor of the β and γ -proteobacteria af- proteobacteria is present in the ATP synthase alpha subunit. ter the divergence of other bacteria, including α-proteobacteria In this case, an 11 aa insert in a highly conserved region (Figure 26). 128 R. S. GUPTA For personal use only. Critical Reviews in Microbiology Downloaded from informahealthcare.com by Cornell University

FIG. 25. Partial sequence alignment of ATP synthase α-subunit showing a highly conserved insert that is commonly shared by various β and γ -proteobacteria, but not found in any other groups of bacteria including the α- and δ,-proteobacteria and all other phyla of Gram-positive and Gram-negative bacteria. This insert is also not present in archael or eukaryotic homologs indicating that it was introduced in a common ancestor of the β and γ -proteobacteria after the branching of all other groups including α-proteobacteria. Other signatures showing similar relationships have been described in earlier work (Gupta 1998, 2000, 2001, 2003). PHYLOGENY AND SIGNATURES DISTINCTIVE OF α-PROTEOBACTERIA 129 For personal use only. Critical Reviews in Microbiology Downloaded from informahealthcare.com by Cornell University

FIG. 26. Evolutionary relationships among α-proteobacteria based on signature sequences in different proteins. The branching position of α-proteobacteria relative to other groups of bacteria is based on signature sequences such as those shown in Figures 24 and 25. The evolutionary stages where these signatures have been introduced are indicated by thick arrows. Many other signatures that are helpful in resolving the branching order of other groups have been described in our earlier work (Gupta 1998, 2000, 2001, 2003, 2004; Gupta & Griffiths 2002; Griffiths & Gupta 2004b (see also www.bacterialphylogeny.com)). The evolutionary relationship among α-proteobacteria shown here was deduced based on the distribution patterns of different signatures described in this review. The long thin arrows mark the positions where the signature sequences in various proteins have likely been introduced. 130 R. S. GUPTA

CONCLUSIONS guish them from all other bacteria. The unique presence of these The α-proteobacteria are a morphologically and metaboli- signatures in various α-proteobacteria, which is a very diverse cally very diverse group of organisms, which are presently rec- group (Kersters et al. 2003), strongly suggests that these indels ognized as a distinct group solely on the basis of their branching should be functionally important for this group of organisms. pattern in the 16S rRNA tree (Woese et al. 1984; Stackebrandt Hence, studies examining their functional effects should be of et al. 1988; Murray et al. 1990; De Ley 1992; Ludwig & Klenk much interest. 2001; Kersters et al. 2003). No biochemical, molecular or other Signature sequences in other proteins are helpful in defining features are presently known, which are uniquely shared by many of the α-proteobacteria subgroups and in clarifying evolu- various α-proteobacteria and that can clearly distinguish this tionary relationships among them. A number of proteins, which group from all others. The evolutionary relationships within include, Succ-CoA synthetase, Cox I, AlaRS, and MutS, contain this group of bacteria are also presently not understood. This conserved inserts that are shared by all other α-proteobacteria, review describes many novel signatures consisting of conserved except the Rickettsiales. The homologs of these proteins from inserts and deletions in widely distributed proteins that provide other bacteria also lack these indels providing evidence that these definitive means for defining the α-proteobacteria and many of signatures were introduced in a common ancestor of other α- its subgroups, and for understanding evolutionary relationships proteobacteria after the divergence of Rickettsiales. The Rick- among them. Because of the rarity and highly specific nature of ettsiales order also consistently forms the deepest branching these genetic changes, the possibility of their arising indepen- lineage in 16S rRNA and various protein trees (Dumler et al. dently by either convergent or parallel evolution is low (Gupta 2001; Gaunt et al. 2001; Kersters et al. 2003; Yu & Walker 1998; Rokas & Holland 2000). The simplest and most parsi- 2003; Stepkowski et al. 2003). Signature sequences in a num- monious explanation for such rare genetic changes, when re- ber of proteins were found to be specific for either the Rick- stricted to a particular clade(s), is that they were introduced ettsiales order (viz. XerD integrase and leucine aminopeptidase) only once in common ancestors of the particular group(s) and or the two main families, Rickettsiaceae (viz. transcription repair then passed on to various descendants. The signature approach coupling factor, ribosomal protein L19, and FtsZ proteins) and has proven very useful in the past in clarifying a number of Anaplasmataceae (RP-314 and Tgt proteins). These signatures important evolutionary relationships, which could not be reli- were likely introduced in the common ancestors of these groups. ably resolved based on phylogenetic trees (Rivera & Lake 1992; These groups are also clearly distinguished in the phylogenetic Baldauf & Palmer 1993). Our earlier work has identified many trees based on 16S rRNA (Figure 1) (Dumler et al. 2001; Yu & signatures that are either specific for particular groups of bac- Walker 2003) and various proteins (Figure 7) (Stepkowski et al. teria (viz. chlamydiae, cyanobacteria, Bacteroidetes-Chlorobi- 2003; Emelyanov 2003a). Fibrobacter, Deinococcus-Thermus, Proteobacteria) (Gupta Signature sequences in a number of proteins (viz. chromoso- For personal use only. 2000, 2004; Griffiths & Gupta 2002, 2004a; Gupta et al. 2003), mal replication factor, RP-057 and DNA ligase) were commonly or which are commonly shared by certain bacterial phyla provid- shared by various Rhizobiales, Rhodobacterales, and in most ing information regarding their interrelationships (Gupta 1998, cases Caulobacterales (currently represented by only C. cres- 2003; Gupta & Griffiths 2002; Griffiths & Gupta 2004b). centus),but they were not present in Rickettsiales, Rhodospir- A summary of the different signatures that were described illales as well as Sphingomonadales species. These results pro- in this review and the overall picture of α-proteobacterial evo- vide evidence that the groups lacking these signatures diverged lution that emerges based upon them is presented in Figure 26. prior to the introduction of these signatures. A unique signature Most of the signatures described here were unique for either all has also been identified for the Rhizobiales order (viz. TrpRS), α-proteobacteria or certain of its subgroups, and except for a few and one which is commonly shared by Rhodobacterales and C. isolated instances, they were not found in other bacteria. These crescentus. The latter signature suggests a specific relationship

Critical Reviews in Microbiology Downloaded from informahealthcare.com by Cornell University finding provides evidence that the genes containing these signa- between the Rhodobacterales and Caulobacter groups. The re- tures have not been laterally transferred from α-proteobacteria lationships indicated by these signatures are also generally sup- to other bacteria, although LGT for certain other genes have ported by the phylogenetic trees based on 16S rRNA and various been previously reported (Wolf et al. 1999). A large number proteins (Gaunt et al. 2001; Kersters et al. 2003; Stepkowski et al. of these signatures, present in broadly distributed proteins (cy- 2003; Emelyanov 2003a). Signatures sequences in a number tochrome assembly protein Ctag, SAICAR synthetase, DnaB, of other proteins (viz. oxoglutarate dehydrogenase, Succ-CoA ATP synthase α,exonuclease VII, PLPG transferase, RP-400, synthase, DNA gyrase A, LepA, and LytB), are able to distin- puruvate phosphate dikinase, FtsK, and Cyt b) were distinctive guish the Rhizobiaceae, Brucellaceae, and Phyllobacteriaceae characteristics of all α-proteobacteria. Two additional proteins, families from the Bradyrhizobiaceae species. The distinctness MutY and homoserine dehydrogenase, also contain signatures of Bradyrhizobiaceae from other Rhizobiales is also clearly that were specific for α-proteobacteria. However, the homologs indicated by a signature sequence in seryl-tRNA synthetase of these proteins were not found in Rickettsiales. These signa- that is specific for this group. These signatures are also con- tures, for the first time, describe molecular characteristics that sistent with the observation that Bradyrhizobiaceae species are unify all α-proteobacteria, and provide means to clearly distin- only distantly related to other Rhizobiales (viz. Rhizobeaceae, PHYLOGENY AND SIGNATURES DISTINCTIVE OF α-PROTEOBACTERIA 131

Brucellaceae, and Phyllobacteriaceae) (Figure 1) (Sadowsky within Bacteria (Woese et al. 1985; Stackebrandt 2000; Ludwig & Graham 2000; Gaunt et al. 2001; van Berkum et al. 2003; & Klenk 2001; Gupta & Griffiths 2002; Gupta 2002). Based on Kersters et al. 2003; Stepkowski et al. 2003; Moulin et al. 2004). the observations that α-proteobacteria can now be clearly dis- A specific relationship between Sinorhizobium and Agrobac- tinguished from all other bacteria based upon a large number of terium species was also indicated by the signature sequence in molecular characteristics, and that this group also branches dis- the LepA protein. tinctly from all other groups of bacteria including the β, γ - and On the basis of 16S rRNA or various genes/proteins trees, it δ,-proteobacteria, it is suggested that α-proteobacteri should be has proven difficult to reliably determine the interrelationships recognized as a main group or phylum within Bacteria, rather among different α-proteobacterial subgroups (Ludwig & Klenk than as a subdivision or class of the Proteobacteria (Gupta 2000, 2001; Kersters et al. 2003). However, based upon the distribu- 2004; Gupta & Griffiths 2002). Signature sequences in a few tion patterns of various signatures, it is now possible to logically proteins (viz. PPDK and FtsK) indicate that α-proteobacteria deduce the branching order of the main α-proteobacterial sub- might have shared a distant ancestry with the δ-proteobacteria groups (Figure 26). The model for α-proteobacterial evolution, exclusive of other bacteria, but this relationship needs to be fur- which has been developed here is based upon a large number of ther investigated and confirmed. proteins, which are involved in different functions. This model The α-proteobacteria have also given rise to mitochondria is internally highly consistent and it is difficult to logically ex- (Margulis 1970; Gray & Doolittle 1982; Andersson et al. 1998; plain the observed distributions of these signatures by alternate Sicheritz-Ponten et al. 1998; Gray et al. 1999; Gupta 2000; means. The model developed here is also consistent with the rela- Emelyanov 2001a, 2003a, 2003b) and very likely played a cen- tionships, which are resolved in the 16S rRNA or other phyloge- tral role in the origin of the ancestral eukaryotic cell (Gupta netic trees (viz. deep branching and distinctness of Rickettsiales, & Singh 1994; Gupta & Golding 1996; Margulis 1996; Gupta a closer relationship between Rhizobiaceae, Brucellaceae, and 1998; Martin & Muller 1998; Lopez-Garcia & Moreira 1999; Phyllobacteriaceae as compared to Bradyrhizobiaceae;acloser Karlin et al. 1999; Lang et al. 1999; Emelyanov 2003b; Rivera relationship between Rhodobacterales and Caulobacterales; dis- & Lake 2004). Many of the α-proteobacteria specific signa- tinctness of Rickettsiaceae from Anaplasmataceae species; dis- tures identified in the present work are also present in the mito- tinctness of Rhizobiales order containing various root nodule chondrial/eukaryotic homologs, providing additional evidence bacteria, etc.) (Sadowsky & Graham 2000; Dumler et al. 2001; of their derivation from an α-proteobacterial ancestor. In a few Kersters et al. 2003; Yu & Walker 2003; Moulin et al. 2004). cases, the α-proteobacterial signatures are present in genes which Afew minor inconsistencies seen at present (e.g., phyloge- are encoded by the mitochondrial DNA (viz. Cox I and Cyt b). netic placement of Ca. crescentus) should be clarified when The shared presence of these signatures in the mitochondrial ho- sequence information from additional species becomes avail- mologs provides further strong evidence for the α-proteobacterial

For personal use only. able. In this context, it is important to acknowledge that se- ancestry of mitochondria, as previously shown by phylogenetic quence information is available at present from only a limited analysis (Andersson et al. 1998; Sicheritz-Ponten et al. 1998; number of α-proteobacterial species. Although, these species Emelyanov 2003a). The current evidence suggests that within include representatives from different α-proteobacterial orders, α-proteobacteria, the Rickettsiales group of species are the clos- it is necessary to obtain sequence information for many other est relatives of mitochondria (Gupta 1995; Andersson et al. 1998; species from different genera and families to test and validate this Sicheritz-Ponten et al. 1998; Gray et al. 1999; Lang et al. 1999; model. Emelyanov 2001a, 2001b). However, this view is supported by Signature sequences in a number of proteins, a few of which only some of the identified signatures and further work is needed are described here, also provide evidence that α-proteobacteria to clarify this aspect. is a late diverging group within Bacteria (Gupta 1998, 2000, δ Critical Reviews in Microbiology Downloaded from informahealthcare.com by Cornell University 2003; Gupta & Griffiths 2002). Within proteobacteria, and -subdivisions are indicated to have branched prior to α-proteo- LIST OF ABBREVIATIONS bacteria, whereas β and γ -subdivisions are indicated as later AlaRS, alanyl-tRNA synthetase; CFBG, Chlamydia- branching groups (see also www.bacterialphylogeny.com). The Fibrobacter-Bacteroidetes-Green sulfur bacteria; Cyt., Cyto- branching of α-proteobacteria in this position is also supported chrome; Cox I, Cytochrome oxidase polypeptide I; LGT, lat- by the16S rRNA and various protein trees (Olsen et al. 1994; eral gene transfer; PLPG, Prolipoprotein-phosphatidylgycerol; Viale et al. 1994; Eisen 1995; Kersters et al. 2003). The α- PPDK, pyruvate phosphate dikinase; RP, Rickettsia prowazekii; proteobacteria, which is a very large group within Bacteria SerRS, serine-tRNA synthetase; Succ-CoA, Succinyl-CoA; Tgt, (>5000 entries in the RDP-II database) (Maidak et al. 2001), tRNA-guanine transglycosylase; TrpRS, tryptophanyl-tRNA are presently recognized as a Class within the Proteobacteria synthetase; Abbreviations in the species names are: A., Agrobac- phylum (Woese et al. 1984; Stackebrandt et al. 1988; Murray terium; Ana., Anaplasma; Aqu., Aquifex; Azo., Azotobacter; et al. 1990; Ludwig & Schleifer 1999; Boone et al. 2001; Azospir.,Azospirillum; Bac., Bacillus; Bact., Bacteroides; Bart., Kersters et al. 2003). However, presently there are no clearly de- Bartonella; Bdello., Bdeollovibrio; Bif., Bifidobacterium; Bor., fined criteria for the higher taxa (viz. Phylum, Class, Order, etc.) Borrelia; Bord., Bordetella; Brad. Bradyrhizobium; Bru., 132 R. S. GUPTA

Brucella; Buch., Buchnera; Burk., Burkholderia; Ca., Caulobac- Boone, D.R., Castenholz, R.W., and Garrity, G.M. 2001. Bergey’s Manual of ter; Camp., Campylobacter; Cb., Chlorobium; Cfx., Chlorof- Systematic Bacteriology. Springer, New York. lexus; Chl., Chlamydia; Chlam, Chlamydophila; Chromo., Boussau, B., Karlberg, E.O., Frank, A.C., Legault, B.A., and Andersson, S.G. 2004. Computational inference of scenarios for alpha-proteobacterial genome Chromo-bacterium; Clo., Clostridium; Cor., Cornyebacterium; evolution. Proc. Natl. Acad. Sci. USA 101, 9722–9727. Cox., Coxiella; Cyt., Cytophaga; Dei., Deinococcus; Dechloro., Bridger, W.A., Wolodko, W.T., Henning, W., Upton, C., Majumdar, R., and Dechloromonas; Des., Desulfovibrio; Desulf., Desulfito- Williams, S.P. 1987. The subunits of succinyl-coenzyme A synthetase— bacterium; Dros. endo., Drosophila endosymbiont; E., Esche- function and assembly. Biochem. Soc. Symp. 103–111. richia; Ent., Enterococcus; Fuso., Fusobacterium; Geo., Geo- Broughton, W. J. 2003. Roses by other names: Taxonomy of the Rhizobiaceae. J. Bacteriol. 185, 2975–2979. bacter; H., Haemophilus; Hel., Helicobacter; Lac., Lactococ- Capiaux, H., Lesterlin, C., Perals, K., Louarn, J.M., and Cornet, F. 2002. A dual cus; Lactobac., Lactobacillus; Lep., Leptospira; Lis., Listeria; role for the FtsK protein in Escherichia coli chromosome segregation. EMBO Leg., Legionella; Mag., Magnetococcus; Meso., Mesorhizobium; Rep. 3, 532–536. Methano., Methanobacterium; Methyl., Methylobacillus; Mi- Chase, J.W., Rabin, B.A., Murphy, J.B., Stone, K.L., and Williams, K.R. 1986. crobul., Microbulbifer; Myc., Mycobacterium; Myx., Myxococ- Escherichia coli exonuclease VII. Cloning and sequencing of the gene encod- ing the large subunit (xseA). J. Biol. Chem. 261, 14929–14935. cus; Nei., Neisseria; Nit., Nitrosomonas; Nitro., Nitrosospira; Daldal, F., Davidson, E., and Cheng, S. 1987. Isolation of the structural genes Novo., Novosphingobacterium; Olig., Oligotropha; Para., Para- for the Rieske Fe-S protein, cytochrome b and cytochrome c1 all components coccus; Pas., Pasteurella; Photobac., Photobacterium; Por., of the ubiquinol: Cytochrome c2 oxidoreductase complex of Rhodopseu- Porphyromonas; Pse., Pseudomonas; Ral., Ralstonia; Rhi., Rhi- domonas capsulata. J. Mol. Biol. 195, 1–12. zobium; Rho., Rhodobacter; Rhodo., Rhodospirillum; Rhodo- Davidson, E., and Daldal, F. 1987. Primary structure of the bc1 complex of Rhodopseudomonas capsulata. Nucleotide sequence of the pet operon encod- pseud., Rhodopseudomonas; Ri., Rickettsia; Shew., Shewanella; ing the Rieske cytochrome b, and cytochrome c1 apoproteins. J. Mol. Biol. Sino., Sinorhizobium; Sta., Staphylococcus; Str., Streptomyces; 195, 13–24. Strep., Streptococcus; Syn., Synechococcus; Sulfo., Sulfolobus; De Ley, J. 1992. The Proteobacteria: Ribosomal RNA cistron similarities T., Thermotoga; Thermoan., Thermoanaerobacter; Thermosyn., and bacterial taxonomy. In The Prokaryotes, eds. A. Balows, H.G. Tr¨uper, Thermosynechococcus; Tre., Treponema; Vib., Vibrio; Xan., M. Dworkin, W. Harder, and K.H. Schleifer, 2111–2140. Springer-Verlag, New York. Xanthomonas; Thiobac., Thiobacillus; Wol., Wolinella; Xyl., DelVecchio, V.G., Kapatral, V., Redkar, R.J., Patra, G., Mujer, C., Los, T., Xylella; Yer., Yersinia; Z., Zymomonas. Ivanova, N., Anderson, I., Bhattacharyya, A., Lykidis, A., Reznik, G., Jablonski, L., Larsen, N., D’Souza, M., Bernal, A., Mazur, M., Goltsman, E., Selkov, E., Elzer, P.H., Hagius, S., O’Callaghan, D., Letesson, J. J., Haselkorn, ACKNOWLEDGMENTS R., and Kyrpides, N. 2002. The genome sequence of the facultative intracel- The competent technical assistance of Pinay Kanth, Jeveon lular pathogen Brucella melitensis. Proc. Natl. Acad. Sci. USA 99, 443–448. Clements, Larissa Shamseer, and Adeel Mahmood in creating Dumler, J.S., Barbet, A.F., Bekker, C.P., Dasch, G.A., Palmer, G.H., Ray, S.C., Rikihisa, Y., and Rurangirwa, F.R. 2001. Reorganization of genera in the

For personal use only. sequence alignments of proteins from Rickettsia prowazekii and families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: Uni- other genomes is thankfully acknowledged. I am also thankful to fication of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia YanLifor developing certain computer programs that facilitated and Ehrlichia with Neorickettsia, descriptions of six new species combinations the creation of signature sequence files and for help in setting up and designation of Ehrlichia equi and ‘HGE agent’ as subjective synonyms Int. J. Syst. Evol. Microbiol. the bacterial signatures website (www.bacterialphylogeny.com). of Ehrlichia phagocytophila. 51, 2145–2165. Eisen, J.A. 1995. The RecA protein as a model molecule for molecular systematic Thanks are also due to Emma Griffiths and Pinay Kanth for studies of bacteria: Comparison of trees of RecAs and 16S rRNAs from the helpful comments on the manuscript. The work on signature same species. J. Mol. Evol. 41, 1105–1123. sequences described here was mostly completed by August Emelyanov, V.V. 2001a. Evolutionary relationship of Rickettsiae and mitochon- 2004. This work was supported by a research grant from the dria. FEBS Letters 501, 11–18. rickettsia National Science and Engineering Research Council of Canada Emelyanov, V.V. 2001b. Rickettsiaceae, -like endosymbionts, and the origin of mitochondria. Biosci. Rep. 21, 1–17. and the Canadian Institute of Health Research.

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