Analysis of prokaryotic genomes revealed a wide distribution of genes encoding Ni and Co transporters as well as Ni- and Co-dependent proteins [see Additional files 1 and 2]. Table 1 shows the general distribution of both utilization traits in the three domains of life. This analysis was carried out by detecting known metalloproteins, metal transporters and cofactor biosynthesis pathways, and where possible, calls were based on multiple evidences. It should be noted, however, that these approaches may occasionally be insufficient to assign a function with complete confidence. For example, it cannot be excluded that some genes said to be associated with Ni or Co utilization may prove to have a different metal specificity or may not be functional. Therefore, our analysis is consistent with the current knowledge of Ni and Co pathways.

Among bacteria, 319 Ni-utilizing and 410 Co-utilizing organisms (59.1% and 75.9% of sequenced bacterial species, respectively) were identified, including 287 organisms (53.1%) that utilized both metals. In contrast, 98 organisms (18.1%) had neither Ni/Co transporters nor corresponding metalloenzymes and appeared to lack the ability to use either of the two trace elements. Only half of Co-utilizing organisms (209 out of 410) possessed the B₁₂ biosynthetic pathway. The other half likely acquires external B₁₂ via the vitamin uptake systems. Investigation of the occurrence of homologs of the BtuFCD transport system in these B₁₂-uptaking organisms showed that more than 90% of them had BtuFCD homologs, implying that essentially all of these organisms may use a BtuFCD system for B₁₂ uptake [see Additional file 1]. The remaining 10% B₁₂-uptaking organisms, such as Nitrosomonas europaea and Xanthomonas axonopodis, appeared to lack BtuFCD transporters, suggesting the presence of additional B₁₂ transport systems in these organisms. A small number of organisms which had either Ni-dependent proteins (but lacked both Ni transporters and transporters with unassigned function) or Ni transporters (but lacked known Ni-dependent proteins) were found among bacteria (62 and 10 organisms, respectively, Table 1). A similar situation was also observed in 13 B₁₂-synthesizing species that lacked both Co transporters and transporters with unassigned function. Therefore, our data suggest that dual-function Ni/Co transporters (i.e., some predicted Ni-specific transporters may also be involved in Co uptake), additional Ni- and Co-specific transporters, multifunctional metal transporters (e.g., magnesium/nickel/cobalt transport system) and/or novel metalloproteins may be present in a small number of analyzed organisms. Alternatively, metal acquisition might occur nonspecifically in some of these organisms using cation influx systems.

Except for phyla represented by few sequenced organisms (<3), Ni and Co utilization traits were detected in nearly all bacterial phyla (Fig. 1). Neither Ni- nor Co-utilizing organisms were found among the Chlamydiae and Alphaproteobacteria/Rickettsiales. Essentially all organisms in the two phyla are obligate intracellular parasites and have small genome size (<1.5 Mbp). In addition, most organisms in the Firmicutes/Mollicutes (88.2%) and Spirochaetes (62.5%), which are extracellular parasites with small genomes, also lost the ability to use both metals. Thus, it appears that parasitic lifestyle may result in the loss of utilization of both metals. Co utilization appeared to be more widely distributed than that of Ni. It is present in 90% Ni-utilizing organisms and in some phyla, such as the Spirochaetes and Thermotogae, which lack Ni utilization. However, the fact that Ni utilization is found in all sequenced Epsilonproteobacteria, which rarely use Co, suggests a mostly independent relationship between the two metal utilization traits. Nevertheless, significant overlap between the two traits observed in bacteria suggests that they may be related in some way, for example, common or similar transporter systems may be involved.

Similar but even wider Ni/Co utilization was observed in sequenced archaea (Fig. 2 and [Additional file 2]). 45 and 39 archaeal species were found to utilize Co and Ni, respectively. A total of 38 organisms use both metals, including all 18 sequenced methanogenic archaea. Approximately 75% of Co-utilizing archaea possessed the B₁₂ biosynthetic pathway (Table 1). Overall, it appears that utilization of both Ni and Co represent ancient traits which have been and remain common to most prokaryotes.

In contrast to prokaryotes, only 51 Ni-utilizing and 49 B₁₂-utilizing organisms were identified in eukaryotes (31.9% and 30.6% of sequenced eukaryotic genomes, respectively). Among them, 9 organisms (belonging to the Stramenopiles, Viridiplantae/Chlorophyta and Metazoa/Coelomata/Others) use both trace elements (Fig. 3 and [Additional file 3]). On the other hand, almost half of analyzed eukaryotic organisms appeared to lack the ability to use either Ni or B₁₂, including insects (Metazoa/Coelomata/Arthropoda), saccharomycotina and most unicellular parasites. The fact that no organism contained orphan Ni transporter and that more than 96% of Ni-utilizing eukaryotes possessed both known Ni transporters and urease (the only known Ni-dependent enzyme in eukaryotes) strongly suggested excellent correspondence between the occurrence of the Ni uptake system and Ni-dependent proteins in eukaryotes. Although the mechanism of B₁₂ uptake is unclear in eukaryotes excluding mammals, we could examine B₁₂ utilization by analyzing the occurrence of B₁₂-dependent enzymes. It is interesting that most Ni-utilizing eukaryotes were fungi (including the Ascomycota/Pezizomycotina, Ascomycota/Schizosaccharomycetes and Basidiomycota subdivisions) and plants, and that most B₁₂-utilizing organisms were animals (except insects) which lack the ability to use Ni (Fig. 3). The data suggest that the majority of lower eukaryotes lost the Co (or more precisely, B₁₂) utilization trait whereas higher eukaryotes lost the Ni utilization trait. Although less likely, an alternative hypothesis is that the Co utilization trait was independently acquired by some ancient eukaryotes, for example, the ancestor of all animals, and then lost by certain groups such as arthropoda.

We analyzed all well-characterized Ni/Co transport systems in prokaryotes 26314047. Members of these transporter families in sequenced genomes were identified by homology searches and the function of each protein was predicted based on genome context (see Methods). Orthologs of these transporters showed a mosaic distribution in bacteria. A summary of the distribution of these Ni/Co transporters in bacteria is shown in Table 2. Considering that many transporters do not have clear substrate preference (either Ni or Co or both), our analyses focused on predicted Ni- or Co-specific transporters. Although some transporters with unassigned function were clustered with multiple predicted Ni- or Co-specific transporters in phylogenetic trees, we considered them as being of unclear function.

Cbi/NikMNQO transporter is the most widespread transport system for Ni and Co uptake in bacteria, which is consistent with previous observations 47. These modular transporters belong to a novel class of ATP-dependent transporters (named energy-coupling factor or ECF transporters) that use membrane proteins to capture substrate 48. Comparison of subunits of Cbi/NikMNQO systems in different organisms revealed that M, Q and O are universal components and are present in almost all predicted transport systems. No significant similarity was detected between NikN and CbiN, although they have similar topology (two transmembrane domains, 47). It is known that two additional components, NikK and NikL, are involved in Ni uptake in the absence of NikN, which form the NikKMLQO system [see Additional file 4]. Phylogenetic analyses of all these components are shown [see Additional files 5, 6, 7, 8, 9, 10, 11]. In general, except for CbiO/NikO, all components showed separate Ni- and/or Co-related branches although the function of some members of these components was unclear. Almost all CbiN proteins contained the same domain (COG1930, CbiN) and had similar sequences (e-value < 0.1 based on BL2SEQ pairwise alignment). In contrast, more sequence diversity was observed for NikN, NikK and NikL proteins. Sometimes, multiple distant homologs were present in the same organism (e.g., Desulfotalea psychrophila and Desulfovibrio vulgaris contained two distantly related sequences of both NikK and NikL). Here, we divided NikN, NikK and NikL into different groups based on sequence similarity and phylogenetic analyses. Three types of NikN (named N1–N3), two of NikL (L1, L2) and three of NikK (K1–K3) were identified in bacteria. Distribution of different types of these components is shown [see Additional file 12]. Approximately 90% NikL1 co-occurred with NikK1 (the other 10% co-occurred with NikK2 or NikK3), whereas NikL2 only co-occurred with NikK2 or NikK3. Interestingly, in five proteobacteria (most are alpha- and gammaproteobacteria), such as Rhodopseudomonas palustris and Shewanella sediminis, operons for NikK1ML1QO orthologs were found to be adjacent to B₁₂ biosynthesis genes or were preceded by B₁₂-dependent riboswitch elements 49, implying that they are involved in Co uptake in these organisms. Phylogenies of all components showed a relatively small branch for these evolutionarily distant organisms [see Additional file 5 and Additional files 8, 9, 10, 11] although each component belonged to a large Ni-related group. These observations suggest that the Co uptake function recently evolved for NikK1ML1QO system in these organisms. However, it is not clear whether they are still involved in Ni uptake. Orphan NikK and/or NikL orthologs were also observed in several organisms which lack NikMQO but contain Ni-dependent proteins, or even lack Ni utilization (see Additional files 1, 10 and 11]. We checked their gene neighborhoods and could not find proteins directly implicating their function. Thus, they may be involved in Ni-independent pathways. In several organisms where no NikQ could be detected, a hypothetical transporter component (5 transmembrane domains, similar topology as NikQ but no sequence similarity) was always found encoded next to nikO. Orthologs of this hypothetical transmembrane protein were only detected in six sequenced organisms and most of them were predicted to be involved in Ni uptake [see Additional file 13], suggesting that novel Ni-related transporter component evolved in organisms lacking NikQ. In addition, different NikMs in NikMNQO or NikKMLQO system clustered in separate branches [see Additional file 5], indicating that the evolutionary process of NikM correlates with the usage of N or K+L components. However, no correlation was observed for NikM based on different subtypes of NikN, NikK and NikL components. Similarly, phylogeny of the core transporter components Q and O did not show significant similarity to that of M, N, K or L component. It should be noted that no organism that contained both NikMNQO and NikKMLQO was detected, indicating a complementary or mutually exclusive relationship between these two systems.

Two other transporter families, HupE/UreJ and NiCoT, were also found to be frequently used in bacteria (Table 2). The HupE/UreJ transporter family is widely utilized in the Cyanobacteria and various proteobacterial subdivisions except for the Deltaproteobacteria and Epsilonproteobacteria. Phylogenetic analysis of all collected members of this transporter family showed two separate branches of predicted Ni- and Co-specific subgroups although there were still several members with unassigned function in each branch [see Additional file 14]. The NiCoT family was detected in diverse taxonomic groups of bacteria. Compared to HupE/UreJ, NiCoT showed much more complex functional diversity and predicted Ni- and Co-specific transporters were scattered in various branches of the phylogenetic tree [see Additional file 15].

ABC transporter systems are typically major and the most active transporters of organic compounds and metals, such as zinc, manganese, amino acids and peptides. In our study, only a fraction of organisms were predicted to possess the NikABCDE system, including distant Ni ABC-type transporters identified in Yersinia species, YntABCDE 29. Besides genomic context, we attempted to utilize residues which may be involved in Ni-binding (see Methods for details) to distinguish NikABCDE from homologous peptide import systems. Multiple alignment of NikA sequences and other homologs showed that most of the residues proposed to be involved in Ni-NikA interaction are conserved in predicted NikA proteins but absent in other homologs [see Additional file 16]. Except for members of the NikABCDE family in Clostridium tetani and Desulfitobacterium hafniense, which were previously predicted to be preceded by a B₁₂-dependent riboswitch element 47, all NikA orthologs appeared to be Ni-specific [see Additional file 17]. Although YntA (the periplasmic Ni-binding component in the YntABCDE system) is evolutionarily distant from NikA, and it is still unclear how YntA binds Ni, gene neighborhoods could be used to identify this distant Ni ABC-type transporter family.

In addition, only 20 organisms possessed orthologs of the UreH transporter. This family was previously predicted to be Ni-specific because these genes were always located adjacent to the genes for Ni-dependent enzymes, such as urease, Ni-Fe hydrogenase and SodN 2647. There have been no reports that showed that UreH may also be involved in Co uptake. Here, we found that a member of the UreH family is adjacent to several B₁₂ biosynthesis genes (such as CbiD and CobB), in a gammaproteobacterium, Moritella sp. PE36, suggesting that UreH is involved in Co uptake in this organism [see Additional file 18].

Besides the above well-characterized Ni/Co transporter families, several recently predicted Co transporters, including CbtAB, CbtC-CbtG and CbtX 3140, were detected in 87 species, mostly in the Proteobacteria and Actinobacteria (Table 2). Essentially all of these organisms possessed the B₁₂ biosynthetic pathway and many lacked known Co transporters.

In E. coli, the nickel repressor gene nikR is positioned immediately next to its target, the nikABCDE operon. NikR-dependent regulation was also predicted for other Ni transporters, such as NikMNQO and Ni-specific NiCoT, and Ni-dependent enzymes such as Ni-Fe hydrogenase 47. In this study, NikR was found in less than half of the organisms containing NikABCDE, suggesting the presence of NikR-independent regulation of the NikABCDE system [see Additional files 1 and 19]). Here, the occurrence of NikR was used to supplement the searches for Ni-related transporters.

Only three Ni/Co transporter families were detected in archaea: Nik/CbiMNQO, NikABCDE, and NiCoT (Table 3). As in bacteria, Nik/CbiMNQO was the most widespread transporter system. Compared to variations in the bacterial NikMNQO and NikKMLQO systems, only NikMN1QO and NikMN2QO were detected in archaea. In contrast, the distribution of the other two transporters was not very pronounced and most NiCoT transporters did not show clear function. In the case of other predicted Co transporters, only CbtX was detected, in 7 archaeal species.

Among bacterial Ni-dependent enzymes, urease (catalyzes the hydrolysis of urea to carbon dioxide and ammonia) and Ni-Fe hydrogenase (catalyzes hydrogen evolution and uptake; it includes Ni-Fe hydrogenase I (COG0374, HyaB), Ni-Fe hydrogenase III (COG3261, HycE) and F420-reducing hydrogenase (COG3259, FrhA)) were the two most widespread families (Table 4). In the analyzed dataset, 185 organisms (58.0% of Ni-utilizing bacteria) possessed urease and 168 (52.7%) Ni-Fe hydrogenase. Occurrence of other Ni-dependent proteins was limited and mosaic (Table 4). For example, CODH/ACS, a key enzyme in the Wood-Ljungdahl pathway of anaerobic CO(2) fixation 50, was identified only in 11 organisms belonging to the Firmicutes/Clostridia, Chloroflexi and Deltaproteobacteria, whereas SodN was detected in 21 organisms in the Actinobacteria, Bacteroidetes, Cyanobacteria and some Gammaproteobacteria. As mentioned above (Table 1), 10 organisms containing Ni-specific transporters (mostly NikABCDE) lacked known Ni-dependent proteins. We examined the genes adjacent to the predicted transporter genes in these organisms, but did not find good candidates for Ni-dependent proteins. It is possible that these organisms possess additional Ni users which are not strictly Ni-dependent such as GlxI. We found that all these organisms containing orphan Ni transporters also contain GlxI proteins, although it is unclear which of these proteins bind Ni. Although incorrect functional assignment of some transporters (e.g., a predicted Ni-specific transporter may be involved in Co or peptide import) cannot be excluded, misassignment of function should be not significant.

In archaea, the occurrence of these enzymes was different (Table 5). Ni-Fe hydrogenase was the most widespread protein, whereas urease was the least utilized one. SodN was not detected in archaea. In addition, the archaea-specific Ni-binding enzyme, MCR, a protein that contains a noncovalently bound Ni tetrapyrrolic cofactor (coenzyme F430) and catalyzes the final step in the biological synthesis of methane in methanogenic archaea 51, was found in all sequenced methanogens. It has been reported that MCR homologs (bind a modified F430) in some not yet cultured methanotrophic archaea (ANME) are involved in the anaerobic oxidation of methane in marine sediments 52.

We also analyzed the B₁₂ biosynthetic pathway in prokaryotes. By identifying key genes involved in B₁₂ biosynthesis (see Methods), half of B₁₂-utilizing bacteria were predicted to synthesize B₁₂ and all of them contained at least one known B₁₂-dependent enzyme [see Additional file 20]. The other half of B₁₂-utilizing bacteria lacked the complete B₁₂ biosynthetic pathway and, therefore, must be using external B₁₂ via specific uptake systems, such as BtuFCD whose homologs were detected in over 90% of these organisms (see above). It was previously reported that about one-fourth of B₁₂-utilizing bacteria lack the ability to synthesize B₁₂ 31. Our analysis shows that as the number of sequenced prokaryotic genomes increases, many additional organisms lacking B₁₂ biosynthesis will be identified.

In order to study further the Co/B₁₂ utilization in prokaryotes, we examined the occurrence of all known B₁₂-dependent enzymes as means of assessing Co utilization in organisms [see Additional file 20]. Except for MGM, which was previously found in an unsequenced bacterium Eubacterium barkeri 53, all known B₁₂-dependent proteins were detected, the most common being MetH (372 organisms), B₁₂-dependent RNR II (227 organisms) and MCM (including ICM and MeaA, 212 organisms). Other proteins, including GM, 5,6-LAM, DDH, MtrA and CprA, were found only in 2 through 26 organisms.

Surprisingly, some B₁₂-utilizing organisms had an extremely large number of B₁₂-dependent proteins, e.g., 7 MCM members in Nocardioides sp. JS614, 7 CprAs and 15 different B₁₂-dependent methyltransferases in D. hafniense DCB-2, 19 CprAs in Dehalococcoides ethenogenes and 32 CprAs in Dehalococcoides sp. CBDB1 [see Additional file 1]. Our results are consistent with previous findings which implicated these homologous enzymes in various B₁₂-dependent metabolic processes 54.

We also identified 31 bacteria containing Co-binding NHases [see Additional file 20] based on the presence of Co-binding motif (CTLCSCY, 23). All of them are B₁₂-utilizing organisms and most only have single copies of NHase [see Additional file 1]. Besides, iron-containing NHases (containing CSLCSCT sequence motif, 23) were predicted in four organisms that belong to the Actinobacteria, Betaproteobacteria/Burkholderiaceae and Gammaproteobacteria/Others. Phylogenetic analysis showed that these iron-containing NHases form a separate subbranch, suggesting that they might be newly evolved from Co-binding NHases [see Additional file 21].

In archaea, three-fourths of the sequenced B₁₂-utilizing organisms (including all methanogens) synthesize B₁₂ (Table 6). However, more than half of bacterial B12-dependent protein families were absent in archaea, including MetH, 5,6-LAM, DDH, EAL and CprA. B₁₂-dependent RNR II was the most widespread B₁₂-binding enzyme being present in 33 archaeal species. In addition, a variety of B₁₂-dependent methyltransferases were found in archaea, most of which were present in methanogens. The Methanosarcina species possessed an exceptionally large number of B₁₂-dependent methyltransferases, including MtaABC, MtmABC, MtbABC, MttABC, MtsABC and MtrAB (e.g., totally 15 methyltransferases in M. acetivorans and 12 in M. mazei). The presence of multiple B₁₂-dependent methyltransferases involved in different pathways is clearly important for these organisms. No Co-binding NHase could be detected in archaea.

Through our analysis, a novel B₁₂-dependent protein family was predicted in prokaryotes. Orthologs of this protein were detected in 11 sequenced bacteria belonging to four evolutionarily distant phyla (Firmicutes/Clostridia, Firmicutes/Lactobacillales, Chloroflexi and Thermotogae). A distant homolog of the B₁₂-binding domain (COG5012, found in MetH and other methyltransferases) was detected in its N terminus (Fig. 4). Structure prediction using HHpred 55 suggested that the N-terminus may contain a TIM-barrel-like structure involved in B₁₂ binding (data not shown). Analysis of genome context of this putative B₁₂-dependent protein showed that it is always adjacent to NAD/NADP octopine/nopaline dehydrogenase (pfam02317), which acts on the CH-NH substrate bond using NAD(+) or NADP(+) as an acceptor. Additional enzyme candidates included D-alanine:D-alanine ligase and asparagine synthase (glutamine-hydrolyzing), which were located in the vicinity of the gene for the novel B₁₂-dependent protein in several organisms. Further experiments are needed to confirm their dependence on B₁₂.

Distribution of Ni transporters, urease and B₁₂-dependent enzymes in eukaryotes is shown in Table 7. Except for two marine animals, Aplysia californica (sea slug) and Strongylocentrotus purpuratus (sea urchin), which contain an orphan urease, all Ni-utilizing eukaryotes contained at least one known Ni transporter and urease. However, analysis of the distribution of Ni transporters in different eukaryotic phyla showed high diversity of these proteins. NiCoT was only present in fungi (except for yeasts), whereas UreH was detected in plants (Viridiplantae/Chlorophyta and Viridiplantae/Streptophyta) and stramenopiles, and TgMTP1 was only present in land plants (Viridiplantae/Streptophyta). All B₁₂-utilizing eukaryotes contained MetH. Except for the Alveolata/Perkinsea and Viridiplantae/Chlorophyta, all organisms also possessed MCM. RNR II was only found in Dictyostelium discoideum (Dictyosteliida) and three phytophthora species (Stramenopiles) and lost in fungi and animals.

Based on the results shown above, it is possible to infer a general model of Ni and Co utilization in the three domains of life. Considering that the common property of various Ni- or Co-dependent proteins is to catalyze important reactions in the global carbon, hydrogen and nitrogen cycles, it is not surprising that both trace elements are essential for the majority of organisms. However, some organisms and even complete phyla/clades may have evolved alternative mechanisms for such reactions and are characterized by the loss of both transport systems and metalloenzymes.

Out of the five known Ni/Co transport systems in prokaryotes, only NiCoT family spans all three domains of life. If a protein family has many representatives in all domains of life and they cluster within their domains, it is thought that the family was present in the last universal common ancestor, LUCA 5657. We speculate that NiCoT evolved in the common ancestor of bacteria, archaea and eukaryotes. In addition, in spite of low occurrence, the presence of UreH transporter in several phyla of both bacteria and eukaryotes indicates that this family either could have been present in the last universal common ancestor but then lost in archaea, or evolved in early bacteria and was then acquired by the ancestor of eukaryotes through evolution of mitochondria. Phylogenetic analysis of UreH proteins suggested that the LUCA origin is more likely because the eukaryotic branch attaches near the bacterial root [see Additional file 18]. The B₁₂ biosynthetic pathway may have evolved only in prokaryotes or has been lost in eukaryotes. In most prokaryotic phyla, organisms retained Ni and/or Co utilization traits. A complete loss of both Ni and Co utilization was only observed in two phyla, Chlamydiae and Alphaproteobacteria/Rickettsiales. We noticed that their sister phyla (such as the Rhizobiaceae and other Alphaproteobacteria for the Rickettsiales) commonly utilize both traits, suggesting that the loss of Ni and Co utilization happened independently in the two divisions. Considering that essentially all sequenced organisms in the two phyla were obligate intracellular parasites, it is possible that both metals are not necessary for these organisms. However, the possibility that they exploit Ni/Co-binding proteins of the host cannot be excluded.

Further analyses of the Ni- or Co-dependent metalloproteomes (i.e., sets of Ni- and Co(B₁₂)-dependent enzymes) in different phyla provided us with an opportunity to explore the evolution of these metalloproteomes (Fig. 5, 6, 7). Normalized occurrence of these metalloproteins is shown [see Additional files 22 and 23]. There is no correlation between the number of Ni- or Co-dependent enzymes and the genome/proteome size (data not shown). In most bacteria, the size of the Ni-dependent metalloproteome was 1–4 (Fig. 5). Most of these proteins were ureases or Ni-Fe hydrogenases. However, half of sequenced Deltaproteobacteria appeared to have a larger Ni-dependent metalloproteome (≥ 5), including deltaproteobacterium MLMS-1, which possessed the largest Ni-dependent metalloproteome (16 Ni-binding proteins, half of which were Ni-Fe hydrogenases). Similarly, compared to most Co-utilizing species which had 1–4 Co-dependent metalloenzymes, the majority of organisms in some phyla, such as the Chloroflexi (including two Dehalococcoides species which have the largest number of B₁₂-binding proteins in prokaryotes), Spirochaetales, Actinobacteria and Deltaproteobacteria, had larger Co-dependent metalloproteomes (≥ 5, Fig. 6). Therefore, the Deltaproteobacteria appear to be the only bacterial phylum which favors the use of both metals. In archaea, large Ni- or Co-dependent metalloproteomes were observed in methanogens (Fig. 7). Three Methanosarcina species in the Methanosarcinales phylum had the largest metalloproteomes for both Ni and Co.

A somewhat different trend was observed in eukaryotes. Few organisms utilized both Ni and Co (in the form of B₁₂). Ni utilization was limited to plants and lower eukaryotes, such as fungi and stramenopiles, but was absent in vertebrates. Except for the bacterial-type NiCoT and UreH Ni transporters, additional Ni uptake systems have evolved from certain eukaryotic proteins (such as TgMTP1 in land plants). It is possible that ancient eukaryotic phyla inherited the Ni utilization trait and urease from the universal ancestor of all eukaryotes, whereas certain organisms (especially vertebrates) appeared to have lost both of them. Interestingly, urease orthologs were detected in two marine animals (A. californica and S. purpuratus) although we could not find Ni transporters in these organisms. It is unclear whether these orphan ureases still use Ni as a cofactor. Another interesting case was observed in fungi. All sequenced saccharomycotina lacked both Ni transporter and Ni-dependent urease, suggesting that this trait was lost in this fungal subgroup. Co utilization was mainly observed in animals (except for insects) and we could not detect any known B₁₂-dependent proteins in most unicellular eukaryotes.

We examined fully sequenced genomes from the Entrez Genome website at NCBI. A list of fully sequenced prokaryotic and eukaryotic genomes can be found on the NCBI website 75. Only one strain was used for each species (e.g., E. coli O157:H7 EDL933 was used as a representative of E. coli). In total, 540 bacterial, 47 archaeal, and 160 eukaryotic genomes were analyzed (as of Jun. 2008).

To analyze the distribution of Ni/Co transporters, we used several well-characterized Ni/Co transport proteins (e.g., NikABCDE from E. coli, YntABCDE and NiCoT from Y. pseudotuberculosis, CbiMNQO from S. typhimurium and HupE from Rhizobium leguminosarum) and previously predicted Co transporters 3140 as initial seed sequences to search for homologous sequences in different organisms via TBLASTN 76 with an e-value < 0.1. Additional homologs were further identified using iterative TBLASTN searches. In parallel, three cycles of PSI-BLAST with default parameters were used for the identification of distant homologs. Orthologous proteins were defined using the conserved domain (COG/Pfam/CDD) database and bidirectional best hits 77. Considering that NikABCDE transporters have significant similarity to the ABC-type dipeptide and oligopeptide import systems 27, we also utilized the residues that were proposed to bind Ni in E. coli NikA as major discriminators. Residues involved in Ni binding are not well characterized and conflicting results have been reported in the literature. Cherrier et al. suggested that NikA binds Ni chelated by a small organic molecule, such as butane-1,2,4-tricarboxylate (BTC), and that some residues, including Tyr402, Arg137, Arg97 and His416, form a binding site that is involved in the BTC-Ni-NikA interaction 78. On the other hand, Addy and coworkers showed that Ni may bind E. coli NikA without chelators and is bound to two histidine residues (His56 and His442, although not conserved in other NikA proteins) at a position distant from the previously characterized binding site 79. Here, the presence of the majority of these residues was used to help predict NikA proteins. In each transporter family, subgroups specific for Ni or Co were identified based on either previous reports or gene neighborhoods (i.e., if a transporter gene in a certain organism was located adjacent to genes encoding Ni-dependent enzymes, NikR or B₁₂ biosynthesis proteins, it was considered as a predicted Ni- or Co-specific transporter). Other members of detected transporter families were considered as proteins with unassigned function. It is difficult to selectively identify B₁₂ transporter BtuFCD among other highly similar transport systems (such as iron/heme or siderophore transporters), although previous approaches, based on B₁₂ element regulation, were utilized for the identification of BtuFCD in some bacteria 31. Therefore, in this study, we only examined the presence of BtuFCD (or BtuBFCD in gram-negative bacteria) homologs in sequenced organisms for the possibility that the potential B₁₂ uptake system is present when we could not detect B₁₂ biosynthesis pathway. Orthologs of NikR were identified using a similar approach. Occurrence of B₁₂ biosynthesis was verified by the presence of most of the key components involved in B₁₂ biosynthetic pathway: CobE, CobF, CobG, CobM, CobN, CobS, CobT, CobW, CbiD, CbiG, CbiK and CbiX 3180818283.

Members of known Ni-dependent protein families were also identified. In this study, Ni-dependent proteins refer to strictly Ni-binding proteins that utilize Ni as a cofactor. We excluded proteins, which may bind other metals in different organisms, such as GlxI for which the contributor to shifts in metal activation is not clear 4. Conservation of Ni-binding ligands was also analyzed for each Ni-dependent protein and those lacking most of the ligands were discarded. Similarly, in this study, we only considered B₁₂-dependent enzymes as Co-dependent proteins because of the unspecificity of metal utilization, and limited distribution and information on non-corrin Co-binding enzymes. In addition, many B₁₂-dependent proteins contain multiple domains, some of which are B₁₂-independent. Therefore, only B₁₂-binding domain-containing proteins (most contain a conserved DXHXXG motif within the B₁₂-binding region 12) were viewed as B₁₂-dependent users. A complete list of query proteins is shown [see Additional files 1, 2, 3]. The presence of Ni/Co utilization trait was then verified by the requirement for occurrence of at least one predicted Ni/Co-specific transporter, or B₁₂ biosynthesis trait, or at least one Ni/Co-dependent enzyme. Protein sequences for transporters and users collected in this study are provided [see Additional files 26 and 27].

A recently reconstructed phylogenetic tree was adopted to analyze the distribution of organisms that utilize Ni/Co in different taxonomies 84. This tree of life was based on concatenation of 31 orthologs (most are ribosomal proteins) occurring in 191 species with sequenced genomes. The use of a common protein set across all three domains of life enables an objective, quantitative analysis of the consistency of traditional taxonomic groupings. Multiple sequence alignments were performed using CLUSTALW 85 with default parameters and ambiguous alignments in highly variable regions were excluded. Phylogenetic trees were reconstructed by PHYLIP programs 86. Pairwise distance matrices were calculated by PROTDIST to estimate the expected amino acid replacements per position. Neighbor-joining trees were obtained with NEIGHBOR and the most parsimonious trees were determined with PROTPARS. To evaluate robustness of the trees, we performed maximum likelihood (ML) with PHYML 87 using default parameters and likelihood test. If inconsistent topologies were obtained, a third program MrBayes 88, a Bayesian estimation of phylogeny, was used. The final phylogenetic tree was then manually refined for visualization purposes.