JOURNAL OF BACTERIOLOGY, Jan. 2006, p. 317–327
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 188, No. 1
Comparative and Functional Genomic Analysis of Prokaryotic Nickel
and Cobalt Uptake Transporters: Evidence for a Novel Group of
ATP-Binding Cassette Transporters†
Dmitry A. Rodionov,1,2* Peter Hebbeln,2Mikhail S. Gelfand,1and Thomas Eitinger2
Institute for Information Transmission Problems RAS, Moscow, Russia,1and Institut fu ¨r Biologie/Mikrobiologie,
Humboldt-Universita ¨t zu Berlin, Berlin, Germany2
Received 27 August 2005/Accepted 17 October 2005
The transition metals nickel and cobalt, essential components of many enzymes, are taken up by specific
transport systems of several different types. We integrated in silico and in vivo methods for the analysis of
various protein families containing both nickel and cobalt transport systems in prokaryotes. For functional
annotation of genes, we used two comparative genomic approaches: identification of regulatory signals and
analysis of the genomic positions of genes encoding candidate nickel/cobalt transporters. The nickel-responsive
repressor NikR regulates many nickel uptake systems, though the NikR-binding signal is divergent in various
taxonomic groups of bacteria and archaea. B12riboswitches regulate most of the candidate cobalt transporters
in bacteria. The nickel/cobalt transporter genes are often colocalized with genes for nickel-dependent or
coenzyme B12biosynthesis enzymes. Nickel/cobalt transporters of different families, including the previously
known NiCoT, UreH, and HupE/UreJ families of secondary systems and the NikABCDE ABC-type transport-
ers, showed a mosaic distribution in prokaryotic genomes. In silico analyses identified CbiMNQO and
NikMNQO as the most widespread groups of microbial transporters for cobalt and nickel ions. These unusual
uptake systems contain an ABC protein (CbiO or NikO) but lack an extracytoplasmic solute-binding protein.
Experimental analysis confirmed metal transport activity for three members of this family and demonstrated
significant activity for a basic module (CbiMN) of the Salmonella enterica serovar Typhimurium transporter.
The transition metals nickel and cobalt are essential cofac-
tors for a number of prokaryotic enzymes involved in a variety
of metabolic processes (36, 41). Among the known nickel-
dependent enzymes are urease (8), [NiFe] hydrogenase, car-
bon monoxide dehydrogenase (Ni-CODH) (35), acetyl-coen-
zyme A decarbonylase/synthase (21), superoxide dismutase
SodN (22), methyl-coenzyme M reductase (20), and glyoxylase
I (50). In contrast to the diverse roles of nickel in microbial
metabolism, cobalt is mainly found in the corrin ring of coen-
zyme B12, a cofactor involved in methyl group transfer and in
rearrangement reactions (36). Since in natural environments,
soluble Ni2?and Co2?are usually present only in trace
amounts, the synthesis of the respective metalloenzymes re-
quires high-affinity uptake of metal ions. Until recently, two
major types of microbial high-affinity nickel and cobalt trans-
porters were known: ATP-binding cassette (ABC) systems and
secondary permeases of the NiCoT family (reviewed in refer-
The NikABCDE system of Escherichia coli belongs to the
nickel/peptide/opine ABC transporter family and is composed
of the periplasmic binding protein NikA, two integral mem-
brane components (NikB and -C), and two ATPases (NikD
and -E) (43). The molecular basis of selective high-affinity
binding of Ni2?remains elusive, although crystal structures of
E. coli NikA have been determined by two approaches (10, 31).
The two studies uncovered that NikA does not coordinate
Ni2?directly but argue for the requirement of a metallophore.
Though distantly related ABC transporter systems from patho-
genic Yersinia pseudotuberculosis and Brucella suis are also im-
plicated in the high-affinity nickel uptake (33, 49), many other
representatives of this ABC transporter family are involved in
uptake of other compounds, i.e., dipeptides and oligopeptides
Nickel/cobalt permeases of the NiCoT family are widely
distributed in bacteria and are also present in some archaea
and fungi. The substrate preferences of many representatives
have been analyzed in detail (17, 18, 30). The NiCoT family
includes at least one nickel-specific permease and many pro-
teins with mixed metal ion specificities that have a preference
for either nickel or cobalt ions.
Two other families of putative secondary metal transporters,
HupE/UreJ and UreH, are distantly related to NiCoTs, and
certain members of these families have recently been shown to
mediate nickel transport (24). HupE/UreJ proteins are wide-
spread among bacteria and often encoded within [NiFe] hy-
drogenase (HupE) and urease (UreJ) gene clusters. Subgroups
of UreH proteins are found in marine cyanobacteria and in
plants. The cyanobacterial variants are encoded adjacent to
[Ni] superoxide dismutase genes predicting a role in nickel
Several hypothetical transporters (CbiMNQO) with some
similarity to ABC transporters have been annotated as cobalt
uptake systems based on their genomic colocalization with B12
biosynthesis genes or on the presence of the regulatory B12
elements in their upstream regions (46, 47). In two studies,
cbiM, cbiQ, and cbiO genes were identified adjacent to bacte-
* Corresponding author. Present address: The Burnham Institute,
10901 N. Torrey Pines Rd., La Jolla, CA 92037. Phone: (858) 646-3100,
ext. 3082. Fax: (858) 713-9949. E-mail: firstname.lastname@example.org.
† Supplemental material for this article may be found at http://jb
rial urease genes (7, 9) and were shown to be important for the
urease activity in cells grown under nickel limitation.
Despite the importance of Ni2?and Co2?for bacterial me-
tabolism, their uptake must be tightly regulated to avoid toxic
effects. In E. coli, nickel overload is avoided via the repressor
NikR, which binds to the promoter region of the nikABCDE
operon when nickel is present (11, 19). NikR has both strong
(in the pM range) and weak (nM) Ni-binding sites, allowing
sensing of nickel at concentrations corresponding to the range
from 1 to 100 molecules per cell (6, 12). Recently determined
crystal structures of NikR from E. coli and Pyrococcus horiko-
shii reveal a plausible mechanism of the Ni-dependent pro-
moter recognition (13, 48). Known cobalt-transporting NiCoTs
are controlled on the level of translation initiation by B12
riboswitch elements (46). These RNA regulatory elements en-
coded in the leader regions of bacterial B12biosynthesis genes
are able to selectively bind coenzyme B12and repress expres-
sion of target genes (42).
The mechanism of nickel and cobalt uptake in many bacteria
and most archaea is not known, although, for instance in
methanogenes, Ni- and Co-containing enzymes are essential
for energy metabolism and anabolism. Comparative analysis of
binding sites for transcriptional regulators is a powerful ap-
proach to the gene annotation. Here, we analyzed prokaryotic
genomes for the presence of candidate NikR-binding sites and
B12riboswitches. We combined these data with additional
comparative genomics techniques to gain comprehensive in-
sight into the mechanism of nickel and cobalt uptake. This
analysis demonstrated that variants of the CbiMNQO-type
transporters are the most widespread uptake system for the
two metals. We propose the designations “Cbi” and “Nik” for
systems related to cobalt and nickel homeostasis, respectively.
Heterologous expression of the respective gene cassettes from
Salmonella enterica serovar Typhimurium and Rhodobacter
capsulatus in E. coli and metal accumulation assays confirmed
the substrate preferences of these transporters, as initially pre-
dicted by genomic analyses. The CbiMNQO systems of both
organisms are transporters that have a strong preference for
cobalt ions, whereas the Nik(MN)QO system of R. capsulatus,
in which the M and N components are fused and form a single
protein, is a high-affinity nickel transporter.
MATERIALS AND METHODS
Identification of nickel and cobalt transporter genes and their genomic loca-
tions. Complete and partial bacterial genomes were downloaded from GenBank
(5), the Institute for Genomic Research (http://www.tigr.org/), and the DOE
Joint Genome Institute (http://jgi.doe.gov/). Homologs of previously described
nickel and cobalt transporters (see the introduction) were identified in microbial
genomes using the Smith-Waterman algorithm implemented in the GenomeEx-
plorer software (40). Orthologous proteins were defined as bidirectional best hits
(51). Distant homologs were identified using PSI-BLAST and confirmed by
construction of phylogenetic trees (2). Furthermore, we analyzed positional gene
clustering of identified candidate nickel/cobalt transporters with known Ni- and
Co-containing enzymes using the SEED tool for comparative analysis and an-
notation of multiple microbial genomes (44) (http://theseed.uchicago.edu/FIG
/index.cgi; see the “Transport of Nickel and Cobalt” subsystem). To do that, we
first identified homologs of known Ni-dependent enzymes in microbial genomes
and also noted the presence or absence of the B12biosynthesis pathways. The
obtained distribution of nickel/cobalt transporters and Ni/Co-dependent en-
zymes in microbial genomes and their positional clusters are described in Table
S1 in the supplemental material. Genes encoding proteins from the HupE/UreJ
family were called ureJ if they were located within urease gene clusters (also
indicated by “U” in parentheses) and hupE otherwise. Likewise, for genes en-
coding members of the UreH/SodT family, ureH was the default name and sodT
was used only for those genes that clustered with sodN (encoding [Ni] superoxide
dismutase). Finally, the CbiMNQO-related genes were named using either the
cbi or nik prefix, depending on the predicted specificity. Candidate nickel- and
B12-responsive regulatory elements are also described in Table S1 in the sup-
plemental material (see below).
Identification of NikR-binding sites and B12regulatory elements. For identi-
fication of candidate NikR-binding sites, we started from sets of upstream re-
gions of potentially coregulated genes that are homologs of known nickel trans-
porters. A 28-bp inverted repeat, GTATGA-(16 bp)-TCATAC, is known to serve
as the NikR-binding site upstream of the nikA gene in E. coli (11). An iterative
signal detection procedure implemented in the program SIGNALX (27) was
used for construction of common NikR-binding signals in sets of upstream gene
fragments. Each NikR-encoding genome was scanned with the constructed pro-
file using the GenomeExplorer software, and additional genes with candidate
regulatory sites in the upstream regions were selected. Sequence logos for de-
rived regulatory signals were drawn using the WebLogo package v. 2.6 (15)
(http://weblogo.berkeley.edu/). For group I NikRs, we identified similar sites
upstream of orthologous nikA genes in eight proteobacterial genomes. A recog-
nition profile was constructed based on these eight NikR-binding sites. The
profile is highly selective: there are only one to three candidate sites with a score
of ?5.00 per genome. The procedure for identification of the signal for group II
NikRs in ?-proteobacteria was described previously (45). A recognition profile of
group III NikRs in ε-proteobacteria was constructed using the sites from up-
stream regions of putative nickel transporters from Wolinella succinogenes and
two Helicobacter species. It was less selective than other NikR profiles, since it
selected up to 10 other candidate sites with comparable scores per genome.
However, this analysis is supported by an experiment in which one NikR-binding
site has been shown to be involved in the negative regulation of the divergently
transcribed nikR and exbBD-tonB genes in Helicobacter pylori 26695 (14). To
identify a common signal of group IV NikRs, we collected the upstream regions
of the nikMNQO operons from Pyrococcus furiousus, Thermococcus kodakaren-
sis, and Thermoanaerobacter tengcongensis. To detect the candidate NikR signal
in methanogenic archaea (group VI), we collected upstream regions of the
nikMNQO, nikABCDE, and nikR operons and used the same procedure. In some
archaeal genomes, we failed to identify candidate NikR-binding sites. Surpris-
ingly, two other Pyrococcus species (P. abyssi and P. horikoshii) lack homologs of
known nickel transporters, and the search for similar NikR sites could not
identify candidate NikR targets in their genomes. Though the structure of Py-
rococcus horikoshii NikR protein has been determined (13), its in vivo target
genes are not known.
The RNA-PATTERN program and the B12-element RNA pattern were used
to search for additional B12riboswitch regulatory elements in bacterial genomes
as previously described (46).
Other bioinformatics programs. The phylogenetic trees were constructed by
the maximum likelihood method implemented in PHYLIP (26) using multiple
sequence alignments of protein sequences produced by CLUSTALX (52). Gene
identifiers from GenBank are used throughout in the phylogenetic trees. The
SIGNALP 3.0 (www.cbs.dtu.dk/services/SignalP/) and TMPRED (www.ch
.embnet.org/software/TMPRED_form.html) servers were used to predict signal
peptides and transmembrane helices, respectively (4).
Plasmid constructions. The cbi and nik genes were amplified by PCR using
genomic DNA from Salmonella enterica serovar Typhimurium lysotype 2 strain
SGSC1412 (kindly provided by Steffen Porwollik, Sidney Kimmel Cancer Center,
San Diego, CA) and Rhodobacter capsulatus strain SB1003 (kindly provided by
Gabriele Klug, Universita ¨t Gie?en, Gie?en, Germany) as the templates, Plati-
num Pfx DNA polymerase (Invitrogen), and a “touchdown” protocol. For con-
struction of plasmid pStcbiMNQO (containing cbiMNQO of S. enterica serovar
Typhimurium under control of a lac promoter and a ribosomal binding site),
primers were used that introduced a PciI site at the 5? end of cbiM and a BamHI
site at the 3? end of cbiO. The amplicon was treated with PciI and BamHI,
purified, inserted between the NcoI and BglII sites of plasmid pCH675-RP (30),
and introduced into E. coli XL1-Blue. Deletions within the cloned StcbiMNQO
operon were constructed by inverse PCR using pStcbiMNQO as the template,
reverse primers that inserted a BglII site immediately downstream of the last
codon of cbiQ, cbiN, and cbiM, respectively, and a forward primer that generated
a BglII site and was directed to the sequence adjacent to the 3? end of cbiO
containing the stop codon. The products were digested with BglII, religated to
give plasmids pStcbiMNQ, pStcbiMN, and pStcbiM, and transformed into E. coli
XL1-Blue. Deletions were verified by PCR techniques. pRccbiMNQO was con-
structed in two steps. First, the gene cluster was amplified with primers intro-
ducing NcoI and BglII sites to the 5? and 3? ends, respectively, and the NcoI/
BglII-treated amplicon was introduced into pCH675-RP to give pRccbiMNQO
318RODIONOV ET AL.J. BACTERIOL.
(MHis2Asp). The His-to-Asp exchange arose from the generation of the NcoI site.
Since position 2 in all mature CbiM and NikM proteins is occupied by a His
residue (see Fig. S1a in the supplemental material), the Asp codon was replaced
by a His codon in the second step. For this purpose, a PCR product was
generated with a forward primer that introduced a HindIII site at the 5? end,
overlapped the start codon of cbiM, and replaced the GAT (Asp) codon with
a CAT (His) codon. The amplicon was treated with HindIII and BglII and
used to replace the respective fragment of pRccbiMNQO(MHis2Asp) to give
pRccbiMNQO. The exchange was verified by restriction analysis with NsiI be-
cause the G-to-C nucleotide replacement resulted in a recognition site (ATG-
CAT) for this enzyme. Likewise, pRcnik(MN)QO—the parentheses indicate that
M and N are fused to a single open reading frame in the R. capsulatus nik(M-
N)QO cluster—was constructed in two steps. First, nik(MN)QO was amplified
with primers that generated AflIII and BamHI sites at the 5? and 3? ends,
respectively, and the digested product was inserted between the NcoI and BglII
sites of pCH675-RP to give pRcnik(MN)QO(MHis2Tyr). pRcnik(MN)QO with a
His-2 codon was generated after a second round of PCR.
Metal uptake assays.63Ni2?and57Co2?uptake of recombinant E. coli XL1-
Blue strains was analyzed as previously described (17, 18, 25, 30, 54). Cells were
grown in LB medium supplemented with the radiolabeled ions at concentrations
between 100 and 500 nM, ampicillin (100 ?g/ml), and isopropyl-?-D-thiogalac-
topyranoside (IPTG; 1 mM). Cells were harvested in the early stationary phase,
washed in 50 mM Tris-hydrochloride, pH 7.5, and concentrated. Radioactivity of
aliquots was quantitated in a Canberra-Packard Tri-Carb 2900 TR liquid scin-
tillation counter. Metal accumulation is expressed as pmol ? (mg protein)?1.
Each data point in Fig. 5 and bar in Fig. 6 represents the mean value of double
assays using independent cultures grown in the same lot of medium.
Genomic analysis of nickel/cobalt transporters in microbial
genomes. Initially, orthologs of known nickel and cobalt trans-
porter genes in available prokaryotic genomes were identified
by similarity search (see Table S1 in the supplemental material;
also see the supplemental material for genomic identification
numbers). We searched the genomic databases for the pres-
ence of secondary transporters of the NiCoT, HupE/UreJ,
and UreH families and the ABC transport systems of the
NikABCDE and CbiMNQO families. All five families of the
nickel/cobalt transporters showed a mosaic distribution along
bacterial lineages (see Table S1 in the supplemental material).
Many bacterial species possess transporters from only one fam-
ily, and few species have a redundant set of nickel/cobalt trans-
porters from different families. For example, the Salmonella
enterica serovar Typhimurium genome contains cbiMNQO,
nikABCDE, and the gene for an NiCoT, whereas Rhodopseu-
domonas palustris has hupE, ureH, and an NiCoT gene. Over-
all, we have not found any candidate nickel/cobalt transporter
gene in approximately one-third of 200 analyzed microbial
To predict the substrate preferences of identified nickel/
cobalt transporters, we analyzed the genomic localization of
their genes. In Table S1 in the supplemental material, we
marked in parentheses the cases when a nickel/cobalt trans-
porter gene is located adjacent to genes encoding Ni-depen-
dent enzymes or enzymes involved in the coenzyme B12bio-
synthesis. We observed 89 cases of such genomic linkages
(Table 1). At that, 32 transport systems are encoded by genes
associated with B12biosynthesis gene clusters, allowing one to
annotate them tentatively as cobalt transporters. Genes for 57
candidate nickel transport systems are located adjacent to
genes encoding Ni-dependent enzymes. In most of these cases,
the predicted transporter genes form clusters with either ure-
ase or [NiFe] hydrogenase genes.
NikR regulons in bacteria and archaea. Orthologs of the E.
coli repressor NikR were found in the genomes of 23 pro-
teobacteria and 14 archaea (see Table S1 in the supplemental
material). Genomes of methanogenic archaea contain up to
four copies of nikR and multiple copies of candidate nickel
transporters, suggesting that the maintenance of Ni homeosta-
sis is of great importance for these organisms. The phyloge-
netic tree of the NikR proteins has several major branches
(Fig. 1): two large groups including proteins of most pro-
teobacteria and archaea; three smaller groups, consisting of
the NikR factors of ε- and ?-proteobacteria and Thermococ-
cales; and a diverged branch of pseudo-NikR factors from
some proteobacteria. The Thermococcales branch includes the
NikR proteins from hyperthermophilic archaea (Pyrococcus
spp. and Thermococcus kodakarensis), and a single extremely
thermophilic bacterium, Thermoanaerobacter tengcongensis
(Fig. 1). In order to identify nickel-regulated transporters, we
analyzed the NikR-binding signal in all of these groups sepa-
rately (see Materials and Methods for details). Candidate
NikR-binding sites identified in the analyzed microbial ge-
nomes are listed in Table S2 in the supplemental material.
TABLE 1. Number of nickel and cobalt transporters predicted from genomic localization and regulatory characteristics
No. of transporters with characteristic
CbiMNQO/NikMNQONikABCDENiCoT HupE/UreJ UreH/SodT
Predicted nickel transporters (96) 2325 16 239
Regulation by Ni repressor NikR (42)16 20700
Genomic linkage with Ni-dependent enzymes
Ni-superoxide dismutase (4)
Predicted cobalt transporters (44) 284860
Regulation by B12riboswitch (40)
Genomic linkage with B12biosynthesis genes (32)
VOL. 188, 2006 NICKEL AND COBALT TRANSPORT IN PROKARYOTES319
Sequence logos for the obtained group-specific NikR signals
are shown in Fig. 1.
In ?-, ?-, and ?-proteobacteria possessing group I NikR
proteins, the signal is conserved on the sequence level and is
similar to the previously known NikR-binding site in E. coli.
However, in three genomes the space between two dyad-sym-
metric consensus sequences is 13 bp instead of usual 14 bp.
The NikR proteins from these three organisms have an Arg-
Thr-Ser pattern of surface residues of the N-terminal beta-
sheet involved in DNA recognition, whereas all the other
group I proteins have an Arg-Thr-Thr pattern (see “RTS” and
“RTT” boxes in Fig. 1). Although the identified binding signals
of the NikR proteins from four other groups differ significantly
from the consensus of the group I NikR signal, they retain the
same palindromic structure and the distance between the two
half-sites (Fig. 1). The observed degeneracy in the recognition
sequences could be explained by differences in DNA-contact-
ing surface residues of the NikR proteins (Fig. 1 and see Fig.
S7 in the supplemental material).
A diverged branch of pseudo-NikR proteins from five pro-
teobacteria seems not to be involved in the regulation of nickel
uptake systems. Instead, we have observed that these pseudo-
nikR genes are located within a conserved cluster for an alter-
native pathway of urea degradation: i.e., urea amidolyase con-
sisting of biotin carboxylase, urea carboxylase, and allophanate
hydrolase activities (29, 34). The predicted binding sites of
pseudo-NikR regulators are present upstream of these gene
clusters but are absent upstream of the nickel transport genes.
Interestingly, Dechloromonas aromotica has two diverged cop-
ies of NikR: group I NikR is predicted to regulate adjacent
nickel uptake genes, whereas the pseudo-NikR regulon con-
tains solely urea amidolyase genes. In confirmation of our
hypothesis, the amino acids H76, H87, H89, and C95, corre-
sponding to the high-affinity Ni-binding site in the E. coli NikR
structure, are not conserved in the pseudo-NikR proteins (see
Fig. S7 in the supplemental material).
Tentative identification of NikR-binding sites in prokaryotic
genomes possessing the nickel repressor genes allowed us to
assign nickel specificity to many previously uncharacterized
transporters. Overall, we found 28 bacterial and 14 archaeal
transport systems under predicted NikR regulation (Table
1). The majority of them are ABC-type transporters (19
NikABCDE and 16 NikMNQO systems), whereas only seven
NiCoT transporters were predicted to belong to NikR regulons.
FIG. 1. Conservation of the NikR regulon in bacteria and archaea. (A) Sequence logos for the predicted NikR-binding signals in various
taxonomic groups. (B) Maximum likelihood phylogenetic tree of the NikR proteins. All NikR-binding sites identified in the genomes that encode
NikR from a certain group (marked by background gray) were used for construction of the sequence logo for the corresponding group. Candidate
NikR-binding sites were not identified in only a few archaeal genomes encoding NikR (unmarked proteins on the phylogenetic tree). Three surface
residues of the N-terminal beta sheets involved in DNA recognition are shown in boxes.
320 RODIONOV ET AL. J. BACTERIOL.
B12regulons in bacteria. B12riboswitches are widespread
regulatory RNA elements modulating gene expression in bac-
teria in response to changing coenzyme B12concentrations (42,
46). The presence of a B12riboswitch upstream of a putative
nickel/cobalt transporter gene is a strong indication that the
transporter would prefer the cobalt ion. We analyzed regula-
tory regions of identified transporter genes for the presence of
B12riboswitches (see Table S1 in the supplemental material).
Among analyzed protein families, we tentatively assigned co-
balt preference to 40 transport systems that were preceded by
B12regulatory elements on the DNA level (Table 1). The
CbiMNQO transporters constitute the largest and most di-
verse family of transporters regulated by the B12riboswitches.
Novel outer membrane proteins for Ni and Co transport?
Based on genomic linkage to NiCoT genes and on the presence
of regulatory elements, we assign a function in nickel and
cobalt transport across the outer membrane to a group of
TonB-dependent receptors in certain gram-negative bacteria.
Candidate NikR sites were identified at RPA4757 in R. palustris
and its orthologs in Rubrivivax gelatinosus and Oligotropha car-
boxidovorans (see Table S2 in the supplemental material).
Products of these genes are weakly homologous to TonB-
dependent outer membrane receptors for coenzyme B12and
ferric siderophores but lack a typical TonB box on their N-
terminal end. Orthologs of RPA4757 from some proteobacte-
ria constitute a unique family of putative outer membrane
R. gelatinosus has two genes for this type of outer membrane
protein, both of which are preceded by the candidate NikR-
binding sites. Also it has an additional gene cluster preceded by
a NikR site, exbBD-tonB, which encodes energizing compo-
nents of outer membrane transporters. In Bradyrhizobium ja-
ponicum and Dechloromonas aromatica, the candidate NikR
site precedes a two-gene cluster encoding the NiCoT trans-
porter and the outer membrane receptor (Fig. 2). These find-
ings are in agreement with the hypothesis of TonB-dependent
nickel transport across the outer membrane. On the other
hand, in N. aromaticivorans and D. aromatica, homologs of
these receptors are preceded by B12riboswitch elements. Thus,
this family seems to comprise both nickel and cobalt transport-
Phylogenetic and functional analysis of nickel/cobalt trans-
porters. (i) CbiMQO/NikMQO family. Based on genomic lo-
calization, regulatory characteristics, and phylogenetic analy-
sis, we divided the CbiMNQO-like systems into putative nickel
and cobalt transporters. The attribution to one of these two
groups is indicated by using the nik and cbi prefixes. Compar-
ison of operon structures revealed several variations in bacte-
rial and archaeal genomes. M, Q, and O are universal compo-
nents, which are present in all predicted transport systems,
whereas the transmembrane proteins CbiN, NikN, and NikL
and the bitopic transmembrane or periplasmic protein NikK
are additional components (Fig. 3). The N and L components
are not similar to each other on the sequence level but are
predicted to have the same topology with two membrane-
spanning segments flanking an extracytoplasmic loop.
The CbiM/NikM proteins constitute a unique family of
transmembrane proteins with seven predicted transmembrane
segments. We constructed a maximum likelihood phylogenetic
tree based on multiple alignment of 86 bacterial and archaeal
FIG. 2. Genomic organization of bacterial genes encoding nickel- or coenzyme B12-regulated outer membrane receptors. Black circles and
triangles indicate candidate NikR-binding sites and B12riboswitch regulatory elements, respectively.
VOL. 188, 2006 NICKEL AND COBALT TRANSPORT IN PROKARYOTES 321
CbiM/NikM proteins (Fig. 4). In general, the tree is subdivided
into two large branches, which correspond to 36 predicted
cobalt transporters and 50 candidate nickel transporters. Some
of the strongly conserved positions in the CbiM/NikM proteins,
including two histidines and several negatively charged amino
acids, could be involved in metal ion recognition (Fig. 3; and
see Fig. S1a in the supplemental material). Furthermore, there
are several distinctive positions within the CbiM/NikM pro-
teins which are conserved within each subgroup but differ
between CbiM and NikM and thus could be involved in Ni2?
or Co2?selectivity (Fig. 3). For example, the CbiM proteins
have the signature motif Leu-Ala-His-Gly-Gly in the extracy-
toplasmic loop between transmembrane helices 4 and 5 and a
conserved Gln residue at the beginning of helix 7, whereas the
NikM proteins have the signature motif Phe-Ala-Asp-Gly-Gly
and a conserved His residue at the respective positions. An-
other distinctive feature of many CbiM proteins, an additional
N-terminal signal peptide with a conserved cleavage site, was
detected in 21 out of 36 proteins (see Fig. S1a in the supple-
mental material). This observation agrees with the predicted
orientation of the mature CbiM/NikM proteins, with their N-
terminal Met-His motif located outside (Fig. 3).
Two other universal components of the CbiMNQO/
NikMNQO family are a transmembrane protein (CbiQ/NikQ)
and a cytoplasmic ATP-binding protein (CbiO/NikO). CbiO/
NikO proteins have the linker peptide and the Walker A and
FIG. 3. Topology prediction for nickel and cobalt ABC transporters of the CbiMNQO/NikMNQO family. Homologous protein components of
different transport systems are colored in the same way. Membrane-bound components are shown by rectangles with predicted transmembrane
helices shown by cylinders. Putative ATPase components are shown by pink ovals. Periplasmic domains of NikL and NikK are shown by turquoise
and yellow ovals, respectively. The fifth transmembrane helix is not conserved in some CbiQ/NikQ proteins and is shown in dotted lines. NikM
and NikN components are fused to form a single protein in many cases. The illustrated physical interaction between O and Q components is
speculative. The red arrow indicates a putative cleavage site (AnAMH) by bacterial signal peptidase I in all CbiM proteins that contain
transmembrane helix 0.
322 RODIONOV ET AL.J. BACTERIOL.
B motifs commonly found in the ATPase components of clas-
sical ABC-type transport systems (16). The topological model
for CbiQ/NikQ proteins predicts the presence of four con-
served transmembrane helices, a periplasmic domain between
the third and fourth transmembrane helices, and an optional
C-terminal transmembrane helix (Fig. 3). A cytoplasmic do-
main of CbiQ/NikQ, although lacking the conserved EAA mo-
tif, is likely to correspond to the cytoplasmic loop in other
membrane-spanning domains of ABC systems, which has been
identified previously as a contact site of ATP-binding proteins
with membrane-spanning proteins in ABC transporters (16).
Additional similarity searches identified a huge number of
gene pairs weakly homologous to CbiQ/NikQ and CbiO/NikO
(data not shown). However, the genomic analysis showed that
all of these cbiQO-like genes do not seem to be implicated in
nickel or cobalt homeostasis.
Almost all predicted Cbi transport systems possess a sepa-
rate CbiN component and are encoded by conserved gene
cluster cbiMNQO (Fig. 4). As an exception, the CbiM and
CbiN components in Geobacter sulfurreducens are fused to-
gether, encoded by a single gene, designated cbi(MN). The
predicted nickel transport systems may be subdivided into two
subclasses, dependent on the presence of one of two different
additional components, NikN or NikL (Fig. 2). The NikMNQO
system is widely distributed in bacteria and archaea, whereas
NikMLQO shows a mosaic distribution in several groups of
proteobacteria and in cyanobacteria (Fig. 4). Almost all
NikMLQO systems have an NikK component. In 19 out of 33
NikMNQO cassettes, the NikM and NikN components are
fused together and encoded by a single gene, named nik(MN).
To analyze the functionality of these novel transport systems
in nickel and cobalt transport and to validate the predicted
substrate preferences, we selected CbiMNQO of S. enterica
serovar Typhimurium and R. capsulatus and Nik(MN)QO of R.
capsulatus for an initial experimental analysis. Plasmids encod-
ing these transport systems were constructed and introduced
into E. coli, a bacterium that intrinsically does not produce
high-affinity nickel and cobalt transporters under aerobic con-
ditions. The results of metal uptake assays with the recombi-
nants are presented in Fig. 5 and indicate clearly that the three
plasmids encode functional metal transporters. CbiMNQO of
S. enterica serovar Typhimurium and R. capsulatus transport
both nickel and cobalt ions, but in accordance with the bioin-
formatic data have a strong preference for cobalt. The differences
in transport capacity between cells containing StCbiMNQO
and RcCbiMNQO may be explained by differences in heterol-
FIG. 4. Maximum likelihood phylogenetic tree of the M components of Cbi/Nik transport systems. Genes predicted to be regulated by the
nickel repressor NikR and coenzyme B12riboswitch are in blue and yellow background colors, respectively. Genomic colocalizations of cbi/nik
genes with genes encoding Ni-dependent enzymes and B12biosynthesis proteins are indicated by blue and yellow dots, respectively. The color of
each of the lines indicates the subunit composition of the respective nickel/cobalt transport system. Dashed lines indicate transporters containing
fused M and N subunits.
VOL. 188, 2006NICKEL AND COBALT TRANSPORT IN PROKARYOTES 323
ogous expression efficiency and stability or by true differences
in the transport mechanism. Similar results, i.e., apparent dif-
ferences in transport velocity after heterologous production,
had been obtained previously during characterization of vari-
ous members of the NiCoT family (30). Rcnik(MN)QO, on the
other hand, encodes a high-affinity nickel transporter. The
cobalt-transport activity of nik(MN)QO-expressing cells was
within the range of the background. Taken together, these data
suggest that variants of MNQO genes in more than 50 prokary-
otic genomes encode nickel or cobalt transporters. All of these
operons encode an ABC protein (CbiO and NikO), but none
contains a gene for a typical extracellular solute-binding pro-
tein. Since binding proteins are essential components of pro-
karyotic uptake ABC transporters (16), it was tempting to
speculate that the mechanism of Cbi/NikMNQO is different
from that of standard ABC systems. Experimental analysis of
the S. enterica serovar Typhimurium CbiMNQO system con-
firmed this hypothesis. Results illustrated in Fig. 6 demonstrate
that the basic modules CbiMN and CbiMNQ have significant
Co uptake activity in the absence of the CbiO ABC protein,
although these activities are considerably lower than those of
the four-component (CbiMNQO) system. Deletion of cbiN
abolished activity, suggesting that CbiM and CbiN are the
minimal requirements for a functional transporter.
(ii) NiCoT family. Nickel/cobalt permeases of the NiCoT
family were found in diverse taxonomic groups of bacteria, as
well as in two archaeal genera and several species of fungi (23).
The metal ion preferences of six NiCoTs extensively studied in
metal accumulation assays correlate with the genomic localiza-
serovar Typhimurium (St) or Rhodobacter capsulatus (Rc) or nik(MN)QO from R. capsulatus. Control cells contained an empty pBluescript II KS?
vector. Cells were grown in the presence of either of the radiolabeled metal salts. The cellular metal content was determined by liquid scintillation
counting. The inset in the upper right panel illustrates RcCbiMNQO-mediated nickel-uptake activity at a refined scale.
57Co2?(empty circles) and63Ni2?(filled circles) uptake of recombinant E. coli cells expressing cbiMNQO from Salmonella enterica
FIG. 6. Cobalt uptake of recombinant E. coli expressing S. enterica
serovar Typhimurium cbiMNQO (white bars), cbiMNQ (diagonally
hatched bars), cbiMN (horizontally lined bars), or cbiM (gray bars) or
containing an empty vector (black bars).
324 RODIONOV ET AL.J. BACTERIOL.
tion of the transporter genes, which are often located adjacent
to genes for Ni-dependent enzymes or coenzyme B12biosyn-
thesis (30). In this study, we obtained additional indications of
the metal ion preferences of NiCoTs. We found that seven
NiCoT genes are preceded by candidate NikR binding sites,
whereas eight genes are preceded by B12riboswitches (see Fig.
S2 in the supplemental material). A known or predicted pref-
erence for either of the two metals does not reflect the phylo-
genetic neighborhood of individual NiCoTs, suggesting that
this is a result of adaptation to specific Ni2?or Co2?require-
ments in various species.
(iii) HupE/UreJ family. Transporters of the HupE/UreJ
family are widely distributed in proteobacteria and cyanobac-
teria. They contain an N-terminal signal peptide with a con-
served cleavage site and six predicted transmembrane helices.
The first helix has a conserved signature including two His
residues and one Asp residue (24). In most proteobacteria, as
well as in Aquifex aeolicus and Deinococcus radiodurans, the
HupE/UreJ proteins are encoded within Ni-hydrogenase and
urease gene clusters (see Fig. S3 in the supplemental material)
and therefore named either HupE or UreJ, respectively (3, 32,
39). HupE from R. palustris and UreJ from R. eutropha have
been heterologously produced in E. coli and shown to be in-
volved in nickel transport (24). The hupE/ureJ genes in 6 out of
10 cyanobacteria are preceded by B12riboswitches and thus
predicted to be involved in cobalt uptake linked to the biosyn-
thesis of coenzyme B12(46) (see Table S1 and Fig. S3 in the
supplemental material). Indeed, a very recent study showed
that mutagenesis of hupE in Synechocystis sp. strain PCC 6803
results in Co2?deficiency (Jens Appel, Department of Botany,
University of Kiel, Germany, personal communication).
(iv) UreH/SodT family. The ureH gene within the urease
operon in Bacillus sp. strain TB-90 is required for full urease
activity under Ni2?limitation (38). Orthologs of ureH are
present in similar urease gene clusters in two Geobacillus spe-
cies. The ureH genes were also identified in the genomes of
some proteobacteria and other species (see Fig. S4 in the
supplemental material). In Microbulbifer degradans and Cyto-
phaga hutchinsonii, ureH is located adjacent to urease genes,
whereas in O. carboxidovorans it is the promoter-distal gene in
a putative [NiFe]-hydrogenase operon (see Table S1 in the
supplemental material). Additional ureH homologs have been
identified in marine cyanobacteria and many plants. The cya-
nobacterial homologs are located adjacent to the genes encod-
ing Ni-dependent superoxide dismutase (24). There is no evi-
dence for the existence of cobalt transporters within the UreH
(v) NikABCDE transporters of PepT family. The NikABCDE
system of E. coli belongs to a large family of ABC transporters,
named the nickel/peptide/opine transporter family PepT.
Based on genomic colocalization with genes encoding Ni-
dependent enzymes and the presence of Ni- and B12-respon-
sive regulatory elements, we identified a set of candidate nick-
el/cobalt transporters within the PepT family (see Table S1 in
the supplemental material). In the phylogenetic tree con-
structed for the substrate-binding components, 25 predicted
nickel transporters fall into several separate branches. In par-
ticular, it contains two most diverse bacterial branches, named
NikA1 and NikA2, and two additional clusters that include
homologs from methanogenic archaea preceded by candidate
NikR-binding sites (see Fig. S5 in the supplemental material).
Strikingly, the predicted nickel transporters from proteobacte-
ria show a mosaic distribution between the diverged NikA1
and NikA2 groups. For example, among the closely related
taxonomic group of enterobacteria, the NikA1 branch is rep-
resented by proteins from E. coli, Klebsiella pneumoniae, Er-
winia chrysanthemi, Citrobacter rodentium, and Proteus mirabi-
lis, whereas the NikA2 branch contains proteins from Yersinia
species, S. enterica serovar Typhimurium, and the second NikA
paralog from P. mirabilis. In contrast, the predicted B12-regu-
lated transporters from the NikABCDE family are not closely
related on the sequence level. Crystallographic analyses of E.
coli NikA (10, 31) failed to uncover the molecular basis of
selective Ni2?recognition. Comparative genomics allows pre-
dicting the physiological substrate of various members of the
peptide/opine/nickel ABC transporter family.
Transition metals nickel and cobalt are essential compo-
nents of many enzymes and must be transported into the cell in
appropriate amounts. In this study, we combined bioinformat-
ics and experimental approaches to describe nickel and cobalt
uptake transporters in prokaryotes. Genome context tech-
niques including the analysis of nickel- and coenzyme B12-
specific regulatory elements allowed us to annotate 96 nickel
and 44 cobalt transport systems belonging to five different
types. Secondary nickel/cobalt transporters of the NiCoT,
HupE/UreJ, and UreH families, as well as NikABCDE ABC-
type nickel transporters, have been analyzed in previous ex-
perimental work (3, 17, 24, 25, 30–32, 38, 41, 43). In contrast,
experimental studies of Cbi/NikMNQO-like transporters were
very limited until this work, and involvement of any of these
systems in nickel or cobalt uptake has not yet been shown
directly. The cbiMNQO gene cassette in S. enterica serovar
Typhimurium has been considered to encode a cobalt trans-
porter because of its localization within the B12biosynthesis
superoperon and since some B12biosynthetic mutations in this
region were corrected by the addition of excess Co2?(47). Two
“NikMQO”-like systems, Nik(MN)QO in Streptococcus saliva-
rius (originally named UreMQO) (9) and NikKLMQO in Ac-
tinobacillus pleuropneumoniae (originally named CbiKLMQO)
(7), have been considered to provide Ni for urease activation
under Ni-limiting conditions. Insertional inactivation of NikM
in S. salivarius completely abolishes the ability of the cells to
accumulate63Ni2?during growth (9). In the present study, we
analyzed three different transport systems of this type in metal
accumulation assays and proved their involvement in the co-
balt or nickel uptake. The cbiMNQO gene cassettes from S.
enterica serovar Typhimurium and R. capsulatus conferred
high-affinity cobalt uptake on recombinant E. coli cells,
whereas expression of the R. capsulatus nik(MN)QO genes
selectively enhanced nickel uptake.
Cbi/NikMNQO transporters are present in diverse species
of bacteria and archaea, where they are more widely distrib-
uted than nickel/cobalt transporters of other types. Based on
genome context analyses, we were able to assign a function in
nickel or cobalt transport to the majority of systems from this
family. The subunit composition of these unusual ATP-binding
cassette transporters is not uniform, but a typical solute-bind-
VOL. 188, 2006NICKEL AND COBALT TRANSPORT IN PROKARYOTES325
ing protein is not obvious in any case (Fig. 3). The cobalt
transporters consist of two transmembrane components (CbiM
and CbiQ), a small membrane-bound component (CbiN) and
an ATP-binding protein (CbiO). Similar components consti-
tute the nickel transporters with some variability in the small
membrane-bound component, either NikN or NikL, which are
not similar to CbiN on the sequence level. One-fourth of the
nickel transporters are accompanied by an additional mem-
brane-bound (or periplasmic) subunit (NikK), which could be
involved in binding of Ni2?, although NikK proteins are not
homologous to any known solute-binding protein of ABC sys-
tems. The mechanism of metal ion translocation for the Cbi/
NikMNQO transport systems is not clear. The presence of an
ATPase subunit suggests that, like classical ABC transporters,
these systems are energized by ATP hydrolysis. On the other
hand, results of an initial analysis of the S. enterica serovar
Typhimurium CbiMNQO system suggest that the transmem-
brane protein CbiQ and the ABC protein CbiO are at least not
essential for function of CbiMN, which we consider the basic
moiety of the cobalt transporter. These observations may be of
general relevance. Several lines of evidence support the notion
that the Cbi/NikMNQO members are representatives of a
mechanistically novel type of membrane transporters. (i) None
of about 50 “MNQO” operons identified by comparative
genomics codes for a protein similar to known solute-binding
proteins which are essential for activity of prokaryotic uptake
ABC transporters. (ii) Homologs of cbiQ and cbiO are linked
to genes for membrane proteins of unknown function in many
prokaryotic genomes. In some cases, however, a function in
methionine and thiamine metabolism and in biotin transport
can be ascribed to these operons due to genomic colocalization
and the presence of regulatory elements in the upstream re-
gions (see Fig. S6 in the supplemental material). A very recent
analysis of the bioMNY operon of Rhizobium etli (28), encoding
a CbiO homolog (BioM), a CbiQ homolog (BioN), and the
putative core biotin transporter (BioY), is in agreement with a
function in biotin transport. (iii) S. enterica serovar Typhi-
murium CbiMN retains a residual activity in the absence of
CbiQO. These observations lead us to speculate that CbiQ and
CbiO homologs form energizing modules and specifically in-
teract with different membrane transporters that are inde-
pendent of a solute-binding protein. The combination of a
secondary active transporter with an ABC domain is not un-
precedented. LmrA, an ABC-type multidrug exporter in Lac-
tococcus lactis, contains an N-terminal transmembrane domain
and a C-terminal nucleotide-binding domain. Removing the
nucleotide-binding domain results in a protein that functions
as a proton/multidrug symporter (53). In vitro studies with
reconstituted systems are required to elucidate the mechanism
of transporters with CbiO- and CbiQ-like components. These
studies are beyond the scope of the present investigation but
are currently under way.
In this study, we attempted to reconstruct pathways of nickel
and cobalt homeostasis in microbial genomes by identification
of gene sets for candidate metal transporters and metal-depen-
dent enzymes. Ni-dependent enzymes are absent from many
bacterial and archaeal lineages, including opportunistic and
obligate pathogens like chlamydia, rickettsia, and spirochetes.
Overall, distributions of genes for transporters and utilizing
enzymes coincide to a large extent, demonstrating that nickel-
and cobalt-dependent pathways are completed by high-affinity
metal uptake systems in most microorganisms (see Table S1 in
the supplemental material). However, some bacterial genomes
without any homolog of known nickel/cobalt transporters, in-
cluding Bacillus subtilis, Sinorhizobium meliloti, Campylobacter
jejuni, Neisseria meningitidis, and Corynebacterium spp., encode
Ni-dependent enzymes. This may indicate that other, currently
unknown, nickel transporters are present in these bacteria.
We thank Andrei Mironov (Moscow) for providing software for
genome analysis and useful discussions and Erwin Schneider (Berlin)
for many helpful comments on ABC-type transport systems.
This study has been supported by an exchange grant within the
European Science Foundation Program on Integrated Approaches for
Functional Genomics (to D.R.) and by grants from the Howard
Hughes Medical Institute (55000309 to M.G.), the Russian Fund of
Basic Research (04-04-49361 to D.R.), the Russian Science Support
Fund (to M.G.), the Russian Academy of Sciences (Programs “Mo-
lecular and Cellular Biology” and “Origin and Evolution of the Bio-
sphere” to M.G.), and the Deutsche Forschungsgemeinschaft (to
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