The Complete Multipartite Genome Sequence of Cupriavidus necator JMP134, a Versatile Pollutant Degrader
Cupriavidus necator JMP134 is a Gram-negative beta-proteobacterium able to grow on a variety of aromatic and chloroaromatic compounds as its sole carbon and energy source. Its genome consists of four replicons (two chromosomes and two plasmids) containing a total of 6631 protein coding genes. Comparative analysis identified 1910 core genes common to the four genomes compared (C. necator JMP134, C. necator H16, C. metallidurans CH34, R. solanacearum GMI1000). Although secondary chromosomes found in the Cupriavidus, Ralstonia, and Burkholderia lineages are all derived from plasmids, analyses of the plasmid partition proteins located on those chromosomes indicate that different plasmids gave rise to the secondary chromosomes in each lineage. The C. necator JMP134 genome contains 300 genes putatively involved in the catabolism of aromatic compounds and encodes most of the central ring-cleavage pathways. This strain also shows additional metabolic capabilities towards alicyclic compounds and the potential for catabolism of almost all proteinogenic amino acids. This remarkable catabolic potential seems to be sustained by a high degree of genetic redundancy, most probably enabling this catabolically versatile bacterium with different levels of metabolic responses and alternative regulation necessary to cope with a challenging environment. From the comparison of Cupriavidus genomes, it is possible to state that a broad metabolic capability is a general trait for Cupriavidus genus, however certain specialization towards a nutritional niche (xenobiotics degradation, chemolithoautotrophy or symbiotic nitrogen fixation) seems to be shaped mostly by the acquisition of "specialized" plasmids. The availability of the complete genome sequence for C. necator JMP134 provides the groundwork for further elucidation of the mechanisms and regulation of chloroaromatic compound biodegradation.
The Complete Multipartite Genome Sequence of
JMP134, a Versatile Pollutant
*, Danilo Pe
, Thomas Ledger
, Kostantinos Mavromatis
, Iain J.
, Natalia N. Ivanova
, Sean D. Hooper
, Alla Lapidus
, Susan Lucas
, Bernardo Gonza
Nikos C. Kyrpides
1 Department of Energy (DOE)-Joint Genome Institute, Walnut Creek, California, United States of America, 2 Departamento de Gene
tica Molecular y Microbiologı
Facultad de Ciencias Biolo
gicas, NM-EMBA, NM-PFG, and CASEB, P. Universidad Cato
lica de Chile, Santiago, Chile, 3 Facultad de Ingenierı
a y Ciencia, Universidad Adolfo
ez, Santiago, Chile
Cupriavidus necator JMP134 is a Gram-negative b-proteobacterium able to grow on a variety of aromatic and
chloroaromatic compounds as its sole carbon and energy source.
Its genome consists of four replicons (two chromosomes and two plasmids) containing a
total of 6631 protein coding genes. Comparative analysis identified 1910 core genes common to the four genomes compared
(C. necator JMP134, C. necator H16, C. metallidurans CH34, R. solanacearum GMI1000). Although secondary chromosomes
found in the Cupriavidus, Ralstonia,andBurkholderia lineages are all derived from plasmids, analyses of the plasmid partition
proteins located on those chromosomes indicate that different plasmids gave rise to the secondary chromosomes in each
lineage. The C. necator JMP134 genome contains 300 genes putatively involved in the catabolism of aromatic compounds and
encodes most of the central ring-cleavage pathways. This strain also shows additional metabolic capabilities towards alicyclic
compounds and the potential for catabolism of almost all proteinogenic amino acids. This remarkable catabolic potential
seems to be sustained by a high degree of genetic redundancy, most probably enabling this catabolically versatile bacterium
with different levels of metabolic responses and alternative regulation necessary to cope with a challenging environment.
From the comparison of Cupriavidus genomes, it is possible to state that a broad metabolic capability is a general trait for
Cupriavidus genus, however certain specialization towards a nutritional niche (xenobiotics degradation, chemolithoautotrophy
or symbiotic nitrogen fixation) seems to be shaped mostly by the acquisition of ‘‘specialized’’ plasmids.
The availability of the complete genome sequence for C. necator JMP134 provides the
groundwork for further elucidation of the mechanisms and regulation of chloroaromatic compound biodegradation.
Citation: Lykidis A, Pe
rez-Pantoja D, Ledger T, Mavromatis K, Anderson IJ, et al. (2010) The Complete Multipartite Genome Sequence of Cupriavidus necator
JMP134, a Versatile Pollutant Degrader. PLoS ONE 5(3): e9729. doi:10.1371/journal.pone.0009729
Editor: Niyaz Ahmed, University of Hyderabad, India
Received October 23, 2009; Accepted February 17, 2010; Published March 22, 2010
Copyright: ß 2010 Lykidis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The work presented in this article was performed under the auspices of the US Department of Energy’s Office of Science, Biological and Environmental
Research Program and by the University of California, Lawrence Livermore National Laboratory under contract No. W-7405-Eng-48, Lawrence Berkeley National
Laboratory under contract No. DE-AC02-05CH11231 and Los Alamos National Laboratory under contract No. DE-AC02-06NA25396. The funders had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript. Additional support from the FONDECYT grant 1070343; the
Millennium Nuclei grants P/04-007-F and P/06-009-F; the research program FONDAP 1501-0001 and the grant PBCT RED-12 is acknowledged. D.P.P. is a CONICYT-
DAAD PhD fellow.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Cupriavidus necator JMP134 (formerly Ralstonia eutropha JMP134) is
a Gram-negative b-proteobacterium able to degrade a variety of
chloroaromatic compounds and chemically-related pollutants. It
was originally isolated based on its ability to use 2,4 dichlor-
ophenoxyacetic acid (2,4-D) as a sole carbon and energy source
. In addition to 2,4-D, this strain can also grow on a variety of
aromatic substrates, such as 4-chloro-2-methylphenoxyacetate
(MCPA), 3-chlorobenzoic acid (3-CB) , 2,4,6-trichlorophenol
, and 4-fluorobenzoate . The genes necessary for 2,4-D
utilization have been identified. They are located in two clusters
on plasmid pPJ4: tfd
[5,6,7,8]. The sequence and analysis
of plasmid pJP4 was reported and a congruent model for bacterial
adaptation to chloroaromatic pollutants was proposed .
According to this model, catabolic gene clusters assemble in a
modular manner into broad-host-range plasmid backbones by
means of repeated chromosomal capture events.
Cupriavidus and related Burkholderia genomes are typically
multipartite, composed of two large replicons (chromosomes)
accompanied by classical plasmids. Previous work with Burkholderia
xenovorans LB400 revealed a differential gene distribution with core
functions preferentially encoded by the larger chromosome and
secondary functions by the smaller . It has been proposed that
PLoS ONE | www.plosone.org 1 March 2010 | Volume 5 | Issue 3 | e9729
the secondary chromosomes in many bacteria originated from
ancestral plasmids which, in turn, had been the recipient of genes
transferred earlier from ancestral primary chromosomes . The
existence of multiple Cupriavidus and Burkholderia genomes provides
the opportunity for comparative studies that will lead to a better
understanding of the evolutionary mechanisms for the formation
of multipartite genomes and the relation with biodegradation
Materials and Methods
Genome sequencing and assembly
The complete genome of C. necator JMP134 was sequenced at
the Joint Genome Institute using a combination of 3 kb and
fosmid (40 kb) libraries. Library construction, sequencing, finish-
ing, and automated annotation steps were performed as described
at the JGI web page (http://www.jgi.doe.gov/sequencing/index.
html). Gene prediction was performed using CRITICA  and
Glimmer  followed by manual inspection of the automatically
predicted gene models. Predicted coding sequences (CDSs) were
manually analyzed and evaluated using an Integrated Microbial
Genomes (IMG) annotation pipeline (http://img.jgi.doe.gov) .
CLUSTALW was used for sequence alignments ; phylogenetic
trees were built using Phylip.
Functional annotation and comparative analysis of C. necator with
related organisms was performed using a set of tools available in
IMG. Unique and orthologous C. necator genes were identified using
BLASTp (reciprocal best BLASTp hits with cutoff scores of E ,10
and 60% identity). Signal peptide cleavage sites were identified using
SignalP 3.0  and transmembrane proteins were predicted using
TMHMM , both with their default settings. Synteny plots were
made using Promer, a subroutine of Mummer .
GenBank accession numbers
The sequences of the four genomic replicons described here
have been deposited in GenBank (accession numbers CP000090-
CP000093), and the project information to the GenomesOnline
Database (Gc00292) .
Results and Discussion
General genome features
The genome of C. necator JMP134 consists of four DNA
molecules: two circular chromosomes and two plasmids (Table 1
and Figure 1). The four replicons combined contain 6,631 protein
coding sequences (CDSs), of which 4,898 (73.8%) could be
assigned a putative function. There are 87 RNA genes including
66 tRNAs and six rRNA loci, each arranged in the order of 5S-
23S-16S. Also identified were 83 pseudogenes. Analysis of the
distribution of genes representing major functional categories
reveals that chromosome 1 encodes most of the key functions
required for transcription, translation, and DNA replication, while
chromosome 2 encodes functions involved in energy production
and conversion, secondary metabolism, and amino acid transport
Various comparisons were made between the genome of C.
necator JMP134 and four other closely-related b-proteobacteria that
also possess multipartite genomes (Table 1). Synteny plots
comparing C. necator JMP134 with other closely related Cupriavi-
dus/Ralstonia genomes (C. necator H16; C. metallidurans CH34; and
Ralstonia solanacearum GM1000) reveal extensive conservation of
chromosome 1 but a lack of synteny in chromosome 2 (Figure 2).
The origin and evolutionary history of chromosome 2 probably
includes multiple occurrences of gene duplication and lateral gene
transfer (see below). Notably, in all four species chromosome 2
contains three copies of the rRNA locus, thus indicating past
recombination between chromosomes 1 and 2.
These four genomes were also compared by determining the
numbers of genes encoded by each that are unique to one
organism and the number that are shared by two, three, or all four
strains (Figure 3). Protein identity was defined conservatively using
reciprocal best BLASTp hits with a cutoff of 60% identity of the
amino acid sequence. By that criterion, 1910 genes are found in all
four strains (1713 on chromosome 1, 197 on chromosome 2).
Approximately 28.7% of the CDSs in the genome of C. necator
JMP134 (1904 out of 6,631) were not found in any of the other
three genomes. These 1904 unique genes are distributed among all
four replicons: 552 on chromosome 1, 841 on chromosome 2, 432
in the megaplasmid, and 80 in plasmid pPJ4. Of the 552 unique
genes on chromosome 1: 43 (8%) have no orthologs or paralogs in
the current version of IMG; 87 (15%) have a best BLASTp hit
within C. necator JMP134 indicating that they arose from gene
duplication; 422 (76%) have a best BLASTp hit to other organisms
within the database (Figure 4). The majority of those organisms
are other b-proteobacteria, particularly Burkholderiaceae, with a
minor percentage also from the Alcaligenaceae and the Comamona-
daceae b-proteobacterial families. A sizable minority of them
(,30%) are found in other phylogenetically diverse soil bacteria.
Of the 841 unique genes on chromosome 2 of C. necator, 47 (6%)
have no orthologs or paralogs, 181 (22%) have a best BLASTp hit
Table 1. Comparative genome statistics of five b-proteobacteria.
Chromosome 1 Chromosome 2 Plasmid 1 Plasmid 2
C. necator JMP134 3.80 3537 3 54 2.72 2449 3 11 0.63 555 - 1 0.08 90 - -
C. eutropha H16 4.05 3723 3 49 2.91 2571 2 5 0.45 424 - 3 - - - -
3.92 3684 2 54 2.58 2341 2 8 0.23 241 - - 0.02 164 - -
3.71 3521 3 53 2.09 1686 1 - - - - - - - -
B. xenovorans LB4004.89 4615 3 57 3.36 3054 3 8 1.47 1390 - - - - - -
Genome of C. necator JMP134
PLoS ONE | www.plosone.org 2 March 2010 | Volume 5 | Issue 3 | e9729
within the C. necator JMP134 genome, and 612 (73%) have a best
BLASTp hit to other genomes (Figure 4). These data indicate that
the evolution of these two chromosomes has involved substantial
gene duplication and extensive lateral gene transfer events
(preferentially with related organisms, i.e., b-proteobacteria).
To analyze the functional content of these unique genes we
examined their distribution towards particular COGs (Figure S1).
Excluding COGs R and S (categorized as General features and
Hypothetical Functions, respectively), the data indicate that the
majority of the unique genes belong to COG K. COG K refers to
transcription and the majority of these unique genes are
transcriptional regulators. Although the distribution of unique
genes to various COG categories differs among the four
organisms, a significant number of unique genes belong to signal
transduction pathways (COG T, mainly histidine kinases and
response regulators), energy production and conversion (COG C,
mainly dehydrogenases, oxidases and hydroxylases), amino acid
transport and metabolism (COG E, mainly transporters), and lipid
metabolism (COG I, mainly acyl-CoA synthetases and dehydro-
genases, enoyl-CoA hydratases).
Similarly, C. eutropha H16 has 2000 genes that are not present in
any of the other three strains: 784 on chromosome 1, 956 on
chromosome 2, and 258 in its megaplasmid pHG1. Interestingly,
orthologs for 122 genes found in megaplasmid pHG1 are present
on the chromosomes of the other two Cupriavidus strains: 35 in C.
necator JMP134 and 82 in C. metallidurans CH34.
Of the 2,449 genes identified on chromosome 2 of C. necator
JMP134, 460 (18.8%) have orthologs on chromosome 1 of either
C. eutropha H16, C. metallidurans CH34, or R. solanacearum, but only
45 of them have orthologs in more than one genome.
The prevailing hypothesis for the origin of the secondary
chromosome in the multipartite genomes of Cupriavidus and
Burkholderia posits that it evolved from ancestral plasmids. We
sought to determine whether these putative ancestral plasmids
were the same in the Cupriavidus/Ralstonia, and Burkholderia lineages.
Since chromosome 2 encodes homologs of ParA and ParB
Figure 1. Circular representations of the four replicons of the
genome. 1A: chromosome 1; 1B: chromosome 2; 1C: megaplasmid;
1D: plasmid pPJ4. Circle 1 (from outside to inside): COG assignments for CDSs on the plus strand. Circle 2: COG assignments for CDSs on the minus
strand. Circle 3: RNA genes (green = tRNAs; red = rRNAs; black = other RNAs). Circle 4 (for chromosome 1 and 2, only): genes not found in C.
eutropha H16, C. metallidurans CH34, or R. solanacearum GMI1000. Circle 5: % G+C. Circle 6: GC skew (G-C/G+C). Colors indicate the following: dark
gray, hypothetical proteins; light gray, conserved hypothetical and unknown function; brown, general function prediction; red, replication and repair;
green, energy metabolism; blue, carbon and carbohydrate metabolism; cyan, lipid metabolism; magenta, transcription; yellow, translation; orange,
amino acid metabolism; pink, metabolism of cofactors and vitamins; light red, purine and pyrimidine metabolism; lavender, signal transduction; and
blue sky, cellular processes.
Genome of C. necator JMP134
PLoS ONE | www.plosone.org 3 March 2010 | Volume 5 | Issue 3 | e9729
(proteins involved in the active partitioning of low-copy-number
plasmids), we investigated the similarity and phylogenetic
relationships of the ParA and ParB proteins encoded by
chromosome 2 in 19 b-proteobacteria from those three genera
(Figure 5). Figure 5A shows the similarity of the C. necator ParB and
DnaA (present in chromosome 1) to the corresponding proteins of
the other lineages. Although the identity of the DnaA proteins is
preserved to around 70%, the identity of the ParB proteins is
significantly lower among Cupriavidus/Ralstonia and Burkholderia
species (,28%). Phylogenetic analysis (Figure 5B) also indicates
that ParB proteins from the Cupriavidus and Ralstonia lineages form
distinct groups. Taken together, these data suggest that two
distinct plasmids (one for Cupriavidus/Ralstonia and one for
Burkholderia) may have been the origin of the secondary
chromosomes present in the genera Cupriavidus/Ralstonia, and
Catabolism of aromatic compounds
We have reconstructed the metabolic pathways for aromatic
compound degradation in C. necator JMP134, com paring the
catabolic abilities found in silico with the range of compounds that
support growth of this strain . C. necator is able to use 60
aromatic comp ounds as a sole carbon and energy source.
Aromatic degradation pathways have been classified to central
and peripheral. Peripheral pathways transform a large variety of
aromatic compounds into a few key intermediates (such as
gentisate, catechol, benzoyl-CoA etc) which are subsequently
degraded via the central pa thways. All of the central ring-
cleavage pathways for aromatic compounds known in Proteo-
bacteria, with the exception of the homoprotocatechuate
pathway, are fou nd in this strain: the b-ketoadipate pathway,
with its catechol, chlorocatechol and pr otocatechuate ortho ring-
cleavage branc hes (cat, tfd and pca genes, r espectively); the 4-
methylcatechol ortho ring-cleavage pathway (mml genes); the
gentisate ring-cleavage pathway (mhb genes ); the phenylacetyl-
CoA ring-cleavage pathway (paa genes); the homogentisate ring-
cleavage pathway (hmg genes); the 2,3-dihydroxyphenylpropio-
nate meta ring-cleavage pathway (mhp genes); the catechol meta
ring-cleavage pathway (phl genes); the chlorohydroxyquinol ortho
ring-cleavage pathway (tcp genes); the aminohydroquinone ring-
cleavage pathway (mnp genes); and the 2- aminobenzoyl-CoA ring-
cleavage pathway (abm genes).
Figure 2. Synteny plots between
JMP134 (horizontal axis) and
GMI1000. Red = leading strand; blue = lagging strand.
Genome of C. necator JMP134
PLoS ONE | www.plosone.org 4 March 2010 | Volume 5 | Issue 3 | e9729
Figure 3. Distribution of shared and unique chromosomal genes in the genomes of three
. X-axis: the
number of genomes (1–4) where the gene is found. Y-axis: the percentage of genes in the genome that are found in 1, 2, 3, or all 4 of the compared
Figure 4. Phylogenetic distribution of the best BLASTp hits to the unique genes in
JMP134. Unique genes are those not
present in the three other strains compared.
Genome of C. necator JMP134
PLoS ONE | www.plosone.org 5 March 2010 | Volume 5 | Issue 3 | e9729
Figure 5. ParB protein similarity and phylogeny. A. Percent identity plots of C. necator ParB (Reut_B5344) and DnaA (Reut_A0001) proteins. B.
ParB phylogeny. Neighbor joining tree of 19 ParB proteins. Sequences are from the following species: RALTA, Cupriavidus taiwanensis; Reut_H16,
Cupriavidus necator H16; Rmet, Cupriavidus metallidurans CH34; Rpic, Ralstonia pickettii 12J; Rpic12D Ralstonia pickettii 12D; RSp, Ralstonia
Genome of C. necator JMP134
PLoS ONE | www.plosone.org 6 March 2010 | Volume 5 | Issue 3 | e9729
The approximately 300 genes predicted to be directly involved
in catabolism of aromatic compounds were found to be more or
less equally distributed between chromosomes 1 and 2. Gene
redundancy is predicted to play a significant role in the catabolic
potential of C. necator. Redundant functions were observed in the
catechol, protocatechuate, salicylate, and phenylacetyl-CoA path-
ways; in the degradative pathways for benzoate and chloroaro-
matic compounds; in some of the p-hydroxybenzoate and
(methyl)phenols peripheral reactions; in the presence of several
meta ring-cleavage enzymes; in other oxygenases, maleylacetate
reductases and regulatory proteins. In total, the genome of C.
necator encodes more than 70 oxygenases belonging to the main
oxygenase groups that function in the catabolism of aromatic
compounds. Is this extensive catabolic versatility shared by other
soil bacteria? Genome-wide studies performed on P. putida
KT2440 , B. xenovorans LB400 , Rhodococcus jostii RHA1
, and ‘‘A. aromaticum’’ sp. EbN1  show a significant degree
of catabolic versatility, based on the high number of aromatic
pathways encoded, suggesting that bacteria with such capabilities
may be more common in nature than previously supposed.
Transport of aromatic compounds
A search for transporter genes in the vicinity of genes encoding
aromatic degradative enzymes located ABC transporters from
several families, including the family 4 ABC transporters. This
group, originally identified as branched-chain amino acid
transporters, has more recently been found to also transport other
amino acids and urea (http://www.tcdb.org). One member of this
family is known to function in transport of aromatic compounds
. C. necator JMP134 contains several family 4 ABC transporters
that are predicted to transport aromatic compounds, most—but
not all—of which are shared with other Cupriavidus strains.
One family 4 transporter (Reut_A1329-1333) shared by the
three Cupriavidus strains is adjacent to genes involved in benzoate
degradation. This one is similar to that found in the box operon in
Azoarcus evansii , and also to an hba operon in R. palustris
GCA009 that encodes hydroxybenzoate degradation . Anoth-
er family 4 ABC transporter (Reut_B3779-3783) adjacent to a
ring-hydroxylating dioxygenase is found only in C. necator JMP134
and C. eutropha H16. In a family 4 ABC transporter found also in C.
metallidurans CH34 and R. solanacearum GMI1000, the binding
protein (Reut_B4017) is separated by several genes from the
permease and ATPase components (Reut_B4007-4010) which are,
in turn, adjacent to a gene encoding a 4-hydroxybenzoate 3-
monooxygenase. However, the transporters (Reut_B3779-3783,
and (Reut_B4007-4010, Reut_B4017) do not cluster with
sequences related to the degradation of aromatic compounds.
Two putative aromatic compound ABC transporters that are
unique to C. necator JMP134 are located on plasmids. One
(Reut_C6326-6330) is found on the megaplasmid where it is one
gene away from a putative 3-chlorobenzoate 3,4-ring-hydroxylat-
ing dioxygenase. The other (Reut_D6487-6490) is on plasmid
pPJ4 . However, this transporter has a high similarity to a
probable urea transporter in the C. necator JMP134 genome
(Reut_A0986- 0990) that is adjacent to urease encoding genes.
Some ABC transporter families that have not been previously
known to transport aromatic compounds are found in the vicinity
of aromatic degradative enzymes, including two from families 15/
16 (COG0715). One full transporter (Reut_B5799-5801) and one
binding protein (Reut_C6311) may be involved in aromatic
compound transport. A family 2 ABC transporter (Reut_B4133-
4136) may also function in aromatic compound transport as it is
directly adjacent to a dioxygenase putatively involved in ring
hydroxylation. The only closely related transporter found is in
Bradyrhizobium japonicum where it, also, is adjacent to genes of
C. necator JMP134 has only two members of the benzoate:
proton symporter family (TC 2.A.46): Reut_A2362 that is shared
with C. metallidurans CH34 and R. solanacearum GMI1000, and
Reut_B5351 that is unique to strain JMP134. Also found in C.
necator JMP134 are 13 members of a family of aromatic acid
transporters—family 15 of the major facilitator superfamily (MFS).
In addition, C. necator JMP134 has one MFS family 27 transporter
and one family 30 transporter, both likely to be involved in
aromatic compound uptake.
We investigated the possible presence of permease-type
aromatic transporters by searching for homologs to the following
proteins: BenK from Acinetobacter baylyi ADP-1 (the only benzoate
transporter with a biochemically confirmed function); VanK,
MucK, and PcaK from A. baylyi ADP-1 (transporters with other
biochemically confirmed transport functions); and four putative
transporter proteins (BenK from Pseudomonas putida PRS2000,
PcaK from Azoarcus
sp. EbN1, BenK from Rhodococcus sp. RHA1,
and a putative transporter from A. baylyi ADP-1. This search
identified 30 possible transporters with varying degrees of
similarity to described aromatic acid transporters of this type.
Additional metabolic features
In addition to the broad catabolic potential towards aromatic
compounds, strain JMP134 degrades various other pollutants such
as cyclohexanecarboxylate, tetrahydrofurfuryl alcohol and ace-
tone. The pathways utilized for the degradation of the above
compounds correspond to the ones described in other bacteria
(Table S1) [26,27,28,29,30,31,32].
Some interesting groups of enzymes without specific physiolog-
ical role are also encoded in the genome of this bacterium: (i)
Bacterial dehalogenases are important in the metabolism of
diverse halogenated compounds originated from natural and
anthropogenic sources [33,34], and some representatives of
different kinds of dehalogenases seem to be encoded in the
genome of strain JMP134. They include homologs of the
hydrolytic (S)-2-haloacid dehalogenase (Reut_A1952 and Reut_
B5662) and a reductive dehalogenase belonging to glutathione S-
transferase (GST) superfamily (Reut_C5979), probably involved in
dechlorination of 2-chloro-5-nitrophenol . Additionally, two
contiguous genes (Reut_A1486 and Reut_A1487) both belonging to
the GST family, show high identity with ORF3 and ORF4 of the tft
cluster involved in metabolism of 2,4,5-trichlorophenoxyacetate by
Burkholderia cepacia AC1100 , suggesting a probably role as
dechlorinating enzymes in catabolism of chloroaromatic compounds.
(ii) Bacterial nitroreductases are flavoenzymes that catalyze the
NAD(P)H-dependent reduction of the nitro groups on nitroaromatic
and nitroheterocyclic compounds. These enzymes have raised a great
interest due to their potential applications in bioremediation and
biocatalysis . At least four nitroreductases probably involved in
metabolism of nitroaromatic or nitroheterocyclic compounds are
solanacearum GMI1000; RRSL, Ralstonia solanacearum UW551; Bcep18194, Burkholderia sp. 383; Bcen, Burkholderia cenocepacia AU 1054; Bamb,
Burkholderia cepacia AMMD; Bcep1808, Burkholderia vietnamiensis G4; Bxe, Burkholderia xenovorans LB400; Bphyt, Burkholderia phytofirmans PsJN;
BokIE, Burkholderia oklahomensis EO147; BthaT, Burkholderia thailandensis TXDOH; BPSS, Burkholderia pseudomallei K96243; BURPS1106, Burkholderia
pseudomallei 1106a; BURPS668, Burkholderia pseudomallei 668. The corresponding tree built for ParA was similar (not shown).
Genome of C. necator JMP134
PLoS ONE | www.plosone.org 7 March 2010 | Volume 5 | Issue 3 | e9729
encoded in the genome of strain JMP134: Reut_B3607, Reut_
C6301, Reut_C5940 and Reut_C5984. The last three of them are
encoded by genes located in the megaplasmid and without close
homologs in the rest of Cupriavidus/Ralstonia strains, suggesting that
this replicon could be specialized in catabolism of nitroaromatic
compounds, besides 3-nitrophenol catabolism . (iii) Baeyer-
Villiger monooxygenases (BVMO) are a type of flavoproteins that
play a role in hydroxylation of either alicyclic, aliphatic, or aryl
ketones to form a corresponding ester, which can easily be
hydrolyzed. These enzymes attract a huge interest on industrial
applications since they are able to perform highly regio- and enantio-
selective oxygenations on several substrates. The strain JMP134 has
four genes putatively encoding BVMO (Reut_B5461, Reut_C6279,
Reut_B4935 and Reut_B5155) that are scattered across the genome
and are present in clusters with other genes coding for subsequent
metabolism downstream of the monooxygenase reaction (i.e.,
esterases, hydrolases and alcohol/aldehyde dehydrogenases) but this
fact does not shed enough light about their physiological substrates. A
few related homologs are also found in the rest of Cupriavidus genomes.
Degradation of amino acids
C. necator JMP134 is able to grow on all the proteinogenic amino
acids except glycine, methionine, arginine and lysine . This
pattern of amino acids utilization is identical for C. necator H16 and
slightly different for C. metallidurans CH34, which is unable to use
tryptophane and cysteine but grows on glycine and lysine . It
should be noted that glutamine and asparagine were not included
in this study .
The inability of strain JMP134 to grow on arginine is consistent
with the absence of genes coding for any of the four arginine catabolic
pathways described in bacteria: the arginine deiminase, the arginine
decarboxylase, the arginine dehydrogenase and the arginine
succinyltransferase pathway . These genes are also absent in
Cupriavidus/Ralstonia strains H16, CH34, LMG19424, GMI1000 and
12J. On the other hand, the absence of genes coding for the
cadaverine pathway, the aminovalerate pathway and the aminoadi-
pate pathway involved in degradation of lysine  is consistent with
the inability of this bacterium to grow on this amino acid. Similarly,
these genes are not found in the rest of Cupriavidus/Ralstonia strains,
but the presence of a putative ornithine/lysine/arginine decarbox-
ylase (Reut_A068 9, H16_A2930, Rmet_2754, RALTA_A2412,
RSc2365, Rpic_2578) in all the Cupriavidus/Ralstonia strains is
intriguing, since the ability to grow on these amino acids is not a
metabolic trait of these genera. An explanation for this apparent
inconsistency is that the role of this putative ornithine/lysine/arginine
decarboxylase in Cupriavidus/Ralstonia strains is exclusively in acid
resistance and not in catabolism since this kind of amino acids
decarboxylases are acid-induced and are part of an enzymatic system
in E. coli that contributes to making this organism acid-resistant .
The inability of use methionine as growth substrate by JMP134
and the rest of Cupriavidus/Ralstonia strains is consistent with the
absence of L-methionine c-lyase, a pyridoxal 59-phosphate-
dependent enzyme that catalyzes the direct conversion of L-
methionine into a-ketobutyrate, methanethiol, and ammonia .
The presence of a putative glycine cleavage enzyme system in C.
necator JMP134, encoded by the gcvTHP genes (Table S1),
catalyzing the oxidative cleavage of glycine to CO
transferring a one-carbon unit to tetrahydrofolate would be
contradictory with the inability of this strain to grow in glycine.
However, it should be noted that the metabolism of one-carbon
compounds in C. necator JMP134 is not enough to support growth
on these compounds as sole carbon source and they are only used
as an auxiliary energy source , in contrast with chemolithoau-
totroph strains as H16 and CH34 (See energy metabolism section).
Glutamine is also included among the amino acids that are not
supporting growth of C. necator JMP134, since a glutaminase
encoding-gene, enabling the transformation of glutamine to
glutamate, is not found in this strain, although is present in strains
CH34 and GMI1000. A gene encoding a bifunctional proline
dehydrogenase/pyrroline-5-carboxylate dehydrogenase, catalyz-
ing the four-electron oxidation of proline to glutamate, is found
in the genome of strain JMP134 (Table S1) and the rest of
Cupriavidus/Ralstonia strains allowing the utilization of proline by
these bacteria. According to this trait, a glutamate dehydrogenase-
encoding gene, converting glutamate to a-ketoglutarate and thus
directly feeding the tricarboxylic acids cycle is found in strain
JMP134 (Table S1) and the rest of Cupriavidus strains, but not in
strains 12J and GMI1000.
The presence in strain JMP134 of an L-asparaginase-encoding
gene, enabling the hydrolysis of L-asparagine to L-aspartate and
ammonia (Table S1), would suggest that this strain is able to use
this amino acid as sole carbon and energy source. This gene is also
encoded in the genomes of the rest of Cupriavidus strains but not in
strains 12J and GMI1000. The formed aspartate can be
metabolized through conversion to oxaloacetate by L-aspartate
oxidase (NadB), or to fumarate by aspartate-ammonia-lyase
(AspA) (Table 1). The presence of an L-aspartate oxidase-encoding
gene is common to the rest of Cupriavidus/Ralstonia strains, but the
aspartate-ammonia-lyase is a peculiarity of C. necator JMP134.
Alternatively, aspartate may be transformed to alanine by an
aspartate 1-decarboxylase, however a gene encoding this enzyme
was not found in C. necator JMP134, in contrast with strains H16,
LMG19424, 12J and GMI1000 that harbor an aspartate 1-
The genomic analysis of strain JMP134 suggests that L-alanine
can be degraded by two different pathways. L-alanine can be
directly degraded to pyruvate and ammonia by a NADH-
dependent L-alanine dehydrogenase or converted to D-alanine
by an alanine racemase and subsequently degraded to pyruvate
and ammonia via D-alanine dehydrogenase (Table S1) . The
D-alanine pathway seems to be shared by the rest of Cupriavidus/
Ralstonia strains, but the L-alanine dehydrogenase is only found in
strains H16 and JMP134.
Serine and threonine seem to be used as carbon source by strain
JMP134 due to the presence of the respective deaminases (Table
S1). Serine would be directly converted into pyruvate and
ammonia by the action of serine deaminase whose gene is also
found in the genomes of the rest of Cupriavidus/Ralstonia strains. On
the other hand, threonine would be deaminated to 2-oxobutano-
ate by threonine deaminase that also seems to be encoded in the
genomes of the rest of Cupriavidus/Ralstonia strains.
A complete bifurcated pathway for degradation of histidine is
found in the genome of strain JMP134 consistent with its ability to
grow using this amino acid as only carbon and energy source.
Histidine catabolism proceeds in four or five steps pathways
overlapping in the first three reactions to transform this amino acid
into N-formimino-Lglutamate . At this point, N-formimino-
Lglutamate can be converted to L-glutamate via single- or two-
step reactions. Both routes are encoded in the genome of C. necator
JMP134 (Table S1) and in the genomes of the rest of Cupriavidus
strains, but only the single-reaction route is encoded in the
genomes of strains 12J and GMI1000.
The catabolism of branched-chain amino acids (BCAAs) starts by
the action of an a-oxoglutarate-dependent aminotransferase which
catalyzes the hydrolysis of leucine, isoleucine and valine to a-
oxoisocaproate, a-oxo-c-methylvalerate, and a-oxoisovalerate, re-
spectively, followed by decarboxylation of these
a-oxoacids to their
corresponding branched chain acyl-CoA, in a reaction catalyzed by
Genome of C. necator JMP134
PLoS ONE | www.plosone.org 8 March 2010 | Volume 5 | Issue 3 | e9729
abranchedchaina-oxoacid dehydrogenase complex. Both, the
BCAA aminotransferase and the a-oxoacid dehydrogenase complex
seem to be encoded in the genome of strain JMP134 (Table S1).
The catabolism of branched-chain amino acids (BCAA) starts
with leucine dehydrogenase or a-oxoglutarate-dependent amino-
transferase which catalyzes the hydrolysis isoleucine and valine to to
the corresponding a-oxoacids (a-oxoisocaproate, a-oxo-c-methylva-
lerate and a-oxoisovalerate, respectively). Subsequently, the
branched-chain a-oxoacid dehydrogenase complex catalyzes the
decarboxylation to the corresponding acyl-coenzyme A (CoA)
derivatives . Both BCAA aminotransferase and leucine dehy-
drogenase seems to be encoded in the genome of strain JMP134, in
addition to the common branched-chain a-oxoacid dehydrogenase
complex (Table S1). The branched-chain aa aminotransferase seems
to be also encoded in the rest of Cupriavidus strains, but only strain
H16 additionally encodes leucine dehydrogenase.
Finally, L-cysteine would be degraded by two alternative
pathways in C. necator JMP134 since a L-cysteine desulfhydrase
transforming L-cysteine to ammonia, hydrogen sulphide and
pyruvate, and a Fe
-dependent cysteine dioxygenase that
performs sulfoxidation to form cysteine sulfinic acid, are found
in the genome of this strain. Both enzymes seem to be conserved in
the genomes of the rest of Cupriavidus/Ralstonia strains.
The pathways for the degradation of aromatic amino acids –
tryptophan, phenylalanine and tyrosine– have been analyzed in
detail, recently .
Degradation of carbohydrates
C. necator JMP134 is very limited in sugar or sugar acids
degradation, since only fructose and gluconate can be metabolized
by this strain, in contrast with other Cupriavidus strains that are able
to use glucose, 2-ketogluconate and N-acetyl-glucosamine .
Fructose and gluconate can be initially catabolized by fructokinase
and gluconate kinase, respectively, using a Entner-Doudoroff
pathway, with 2-keto-3-desoxy-6-phosphogluconate (KDPG) al-
dolase as key enzyme. The genes encoding this pathway are
equally distributed in both chromosomes and several examples of
gene redundancy are found (glucose-6-phosphate isomerase,
glucose-6-phosphate 1-dehydrogenase, 6-phosphogluconolacto-
nase and phosphogluconate dehydratase) (Table S1). It should
be noted that similar genes encoding gluconate kinase are found in
the rest of Cupriavidus/Ralstonia strains, but a homolog to
fructokinase gene is only found in the genome of strain H16. In
addition, genes encoding a glucosaminate deaminase and 2-keto-
3-deoxygluconate kinase are found in the genome of strain
JMP134 and in the rest of Cupriavidus/Ralstonia strains, putatively
enabling the utilization of glucosaminate by these strains.
However, the utilization of this sugar by strain JMP134 has not
been evaluated .
Although glucose would be metabolized by strain JMP134, since
a glucokinase gene is found in its genome, the absence of an
uptake system for this hexose would explain why this strain does
not use this sugar as a carbon source. In addition, the absence of 2-
ketogluconate kinase and N-acetylglucosamine-6-phosphate dea-
cetylase encoding genes is consistent with the inability of strain
JMP134 to use these sugars as growth substrates. C. necator JMP134
has incomplete Embden-Meyerhoff-Parnas and oxidative pentose
phosphate pathways due to the absence of genes encoding the key
enzymes phosphofructokinase and 6-phosphogluconate dehydro-
Metabolism of polyhydroxyalkanoate (PHA)
The microbial polyesters as poly-(R)-3-hydroxybutyrate (PHB),
belonging to the family of polyhydroxyalkanoic acids (PHA),
occurred as insoluble inclusions in the cytoplasm and served as a
storage compound for carbon and energy when the cells are
cultivated under imbalanced growth conditions. The metabolism
of PHA has been extensively studied in C. necator H16, a model for
microbial polyoxoester production . Analysis of genome
sequence revealed that strain JMP134 possesses the key enzymes
in PHA biosynthesis (Table S1): a type I poly(3-hydroxybutyrate)
polymerase (Reut_A1347), two b-ketoacyl-CoA thiolases (Re-
ut_A1348; Reut_A1353) and four NADPH-dependent b-ketoacyl-
CoA reductases (Reut_A1349, Reut_B3865, Reut_C6018, Re-
ut_B4127) which, together, convert acetyl-CoA into PHB. In
addition to type I PHA synthase, strain JMP134 contains also a
type II PHA synthase (Reut_A2138). Type II PHA synthases
utilize thioesters of at least five carbon atoms whereas type I
enzymes utilize thioesters of three to five carbon atoms. It should
be noted that C. necator H16 lacks apparent type II PHA synthases.
Additionally, four phasin (PHA-granule associated protein)
encoding genes are found in the genome of strain JMP134.
Phasins are most probably involved providing, together with
phospholipids, a layer at the surface of the PHA granules .
Finally, the intracellular depolymerization of PHB in C. necator
H16 is performed by multiple PHB depolymerases and PHB
oligomer hydrolases . Similarly, the mobilization of PHB in
strain JMP134 seems to be performed by two putative PHB
oligomer hydrolases (Reut_A1981, Reut_A1272) and five PHB
depolymerases (Reut_A1049, Reut_A0762, Reut_B4702, Re-
ut_B3626, Reut_B5113). Genes similar to the ones involved in
PHB metabolism are found in all the rest of Cupriavidus/Ralstonia
strains, indicating that this trait is widespread in these genera. It
should be noted that PHB accumulation in C. necator JMP134 has
been verified previously .
Among the genes participating in nitrogen metabolism found on
chromosome 1 of C. necator JMP134 are Reut_A3432, a putative
ammonium monooxygenase (amoA), and an NAD glutamate
dehydrogenase (NAD-gdh; 1371497–1376338 bp) putatively in-
volved in ammonification. The NAD-gdh protein has 55% and
57% amino acid identity with the NAD-gdh protein reported in
Azoarcus sp. and Pseudomonas aeruginosa, respectively .
Denitrification is encoded by three gene clusters on chromo-
some 2. The nitrate reduction nap genes (Reut_B4761-4765) have
.80% amino acid identity with the corresponding genes in C.
eutropha H16 ; likewise, the nitrite reduction genes (Reut_
B5010-5018) have .75% amino acid identity ; the nor genes
catalyzing later steps in denitrification (Reut_B5055-5057) have
.80% amino acid identity [50,51]. Two nitrogen metabolism
regulators, narX and narL (1804512–1807189 bp), also have high
identity to their counterparts in C. eutropha H16.
Aerobic energy metabolism
Genome analysis of strain JMP134 revealed a robust energy
metabolism typical of most free-living heterotrophs dwelling in an
environment with fluctuating O
levels. The presence of an
extensive inventory of genes for respiratory chain components
including at least nine distinct terminal oxidases indicates that the
aerobic respiration chain adapts to varying concentrations of O
Genes required for formation of complexes I, II and III of
oxidative phosphorylation are present in large chromosome of
strain JMP134: (i) a typical proton-pumping NADH:quinone
oxidoreductase encoded by a large cluster of 14 genes
(Reut_A0961– Reut_A0974); (ii) a succinate dehydrogenase
belonging to the four-subunit type C subgroup  encoded by
four genes (Reut_A2322–Reut_A2325); and (iii) the cytochrome
Genome of C. necator JMP134
PLoS ONE | www.plosone.org 9 March 2010 | Volume 5 | Issue 3 | e9729
bc1 complex, coupling electron transfer from ubiquinol to
periplasmic cytochromes c with proton pumping, encoded by
three genes (Reut_A3091– Reut_A3093). All of these genes are
highly conserved and share similarities to the relatives of
In addition to use of proton-translocating NADH dehydroge-
nase of complex I in energy production, strain JMP134 may
employ two different type II NADH dehydrogenases (Reut_
A0874/Reut_B4838) to optimize the (NADH)/(NAD+) balance
under changing environmental conditions . It should be noted
that the second of these genes seems to be unique to strain
JMP134, in contrast with the first one that is highly conserved in
the rest of Cupriavidus/Ralstonia strains.
The respiratory chain of strain JMP134 can be fueled, besides
NADH dehydrogenases, by at least three formate dehydrogenases
allowing the use of formate as an auxiliary energy source by this
strain , but not as a growth substrate since the product of
formate oxidation, CO
is not fixed by strain JMP134 . A
-reducing, molybdenum-containing formate dehy-
drogenase, previously characterized in strain C. necator H16 , is
encoded by the five genes of the fds cluster located in large
chromosome and seems to be conserved in all Cupriavidus strains,
but not in Ralstonia genus (Table S1). Another soluble formate
dehydrogenase may be encoded by fdw genes on small chromo-
some. The FdwA and FdwB gene products would form a dimeric
tungsten-containing formate dehydrogenase that recycles NADH
at the expense of formate oxidation to CO
, as proposed for C.
necator H16 . This soluble formate dehydrogenase is also found
in C. taiwanensis LMG19424 (Table S1). An additional membrane-
bound formate dehydrogenase is putatively encoded by fdhA, fdhB
and fdhC genes, which would encode a catalytic subunit, an iron-
sulfur subunit, and a transmembrane cytochrome b subunit,
respectively, as proposed for C. necator H16 . In addition, an
accessory gene fdhD is found in this cluster located in large
chromosome (Table S1). This kind of formate dehydrogenase
seems to be encoded in the genomes of all the rest of Cupriavidus/
Ralstonia strains. The presence of a second membrane-bound
formate dehydrogenase encoded by fdo genes, as described in
strain H16 , is not found in strain JMP134.
Strain JMP134 apparently contains an unusually large number
of genes for terminal oxidases catalyzing the reduction of O
water using cytochrome c or quinol as electron donors: (i) one
operon coding for an aa3-type cytochrome oxidase, which
typically operates at high oxygen concentrations; (ii) one operon
coding for a cbb3-type cytochrome oxidase having high affinity for
oxygen, and qualifying to operates at extremely low pressures of
oxygen; (iii) one operon for a bb3-type cytochrome oxidase; (iv)
two operons coding for bd-type quinol oxidases; and (v) three
operons coding for bo3-type quinol oxidases (Table S1). All these
terminal oxidases-encoding operons are also found in strain H16
and its putative function has been analyzed, according to previous
physiological and biochemical studies . All the rest of
Cupriavidus/Ralstonia strains have the aa3-, cbb3- and bb3-type
cytochrome oxidases-encoding operons but a lower number of
quinol oxidases-encoding operons (Table S1). Finally, it should be
mentioned the presence of a putative caa3-type high-potential iron
sulfur protein (HiPIP) oxidase-encoding operon, exclusively found
in the genome of strain JMP134. The HiPIP is a small soluble
protein functioning as the electron carrier between the cytochrome
bc complex and the HiPIP terminal oxidase of the respiratory
chain described in the strict aerobe and thermohalophile
Rhodothermus marinus . However, no homologous gene encoding
a HiPIP similar to that described in R. marinus is found in the
genome of strain JMP134, revealing that the identity of the
putative electron donor for this terminal oxidase remains unknown
in this bacterium.
Altogether, the genomic analysis of energy metabolism in strain
JMP134 confirms that this bacterium is well adapted to life in
habitats subject to fluctuating carbon sources and physicochemical
conditions. The existence of putative ecoparalogs or isoenzymes
having different kinetic properties (e.g., terminal oxidases) or metal
cofactor content (e.g., formate dehydrogenases) allows this
bacterium to cope with rapidly changing O
environments with varying metal supply.
Although several quorum-sensing systems employing N-acyl-
homoserine lactones (AHLs) have been identified in members of
the closely related Burkholderia and Ralstonia genera [58,59], none
were detected in the C. necator JMP134 genome. On the other
hand, a complete phenotype conversion (Phc) regulatory system
was found to be encoded by chromosome 1. This system has been
studied primarily in the phytopathogen R. solanacearum GMI1000
where it forms the core of the complex network that regulates
virulence and pathogenicity genes . At the center of this Phc
system is PhcA, a LysR-type transcriptional regulator, and the
products of the phcBSRQ operon that control levels of active PhcA
in response to cell density. The unique signaling molecule
employed for quorum sensing is the volatile 3-hydroxy palmitic
acid methyl ester (3-OH PAME) . 3-OH PAME post-
transcriptionally modulates the activity of PhcA by acting as the
signal for an atypical two-component regulatory system. This
system consists of a membrane-bound sensor-kinase, PhcS, which
phosphorylates PhcR, an unusual response regulator with a C-
terminal kinase domain in place of a DNA-binding domain .
The amino acid identity between the C. necator JMP134 and the R.
solanacearum GMI1000 Phc gene products range from 56% to 75%.
The presence of a phcA ortholog in a Cupriavidus strain capable of
fully complementing R. solanacearum phcA mutants was previously
reported . That strain also appears to make a form of 3-OH
PAME and to contain orthologs of phcB and phcS . The
possible physiological functions regulated by the Phc system in C.
necator JMP134 pose intriguing questions that are, as yet,
Members of the genus Cupriavidus, as well as the closely related
Ralstonia and Burkholderia, include a few plant pathogens and
symbionts. There is substantial evidence suggesting that members
of these two genera are able to interact with plants and to establish
diverse commensal or even mutualistic associations with these
hosts [62,63,64]. Although this area has not been the focus of
research in C. necator JMP134, specifically, recent experimental
evidence suggests that this bacterium is able to proliferate in the
rhizosphere and even within internal tissues of A. thaliana (Zu´n˜iga,
A, Ledger, Th. and B. Gonza´lez, unpublished data). For most of
the plant bacteria associations described so far, the bacterial genes
typically involved include those encoding protein or nucleotide
transport from the microorganism to the host, as well as those
involved in the production of extracellular enzymes and the
elicitors of the plant hypersensitive response [65,66]. C. necator
JMP134 has several genes related to protein transport. On
chromosome 1 are found several genes related to type IV transport
systems (Reut_A0401-0404, Reut_A0784-0788, Reut_A0779,
Reut_A1436, Reut_A2960-2962, and Reut_A3131-3135). Reut_
A2970 encodes a protein translocase with 72% amino acid identity
to the SecA of Burkholderia multivorans ATCC 17616. Chromosome
Genome of C. necator JMP134
PLoS ONE | www.plosone.org 10 March 2010 | Volume 5 | Issue 3 | e9729
2 also harbors a number of genes encoding putative components of
a type IV secretion system (Reut_B5405-5416).
On chromosome 1 of C. necator JMP134 is found a large phage-
like gene cluster that spans ,43 kb and includes 55 CDSs
(Reut_A2365-2419). Most of these putative proteins have no
homologs in other sequenced genomes of members of the Ralstonia
or the Cupriavidus genera. However, homologs for many of these
proteins, with amino acid sequence identities .60%, are present
in various Burkholderia species, including B. vietnamiensis G4, B.
cenocepacia HI2424, B. dolosa AUO158, and B. multivorans ATCC
17616. The overall sequence identity and arrangement of the
CDSs clustered in this region suggest that this putative phage is
related to the characterized temperate Burkholderia podophage,
A few additional phage-like sequences are found scattered in
chromosomes 1 and 2. These include phage-type integrases
(Reut_A0577, Reut_A1625, Reut_A2191, and Reut_B5345), two
DNA polymerases with similarity to the DNA polymerase of phage
SPO1 (Reut_A1937 and Reut_B4396), and two hypothetical
phage proteins (Reut_A0552 and Reut_A2198). Since these
sequences are not accompanied by other phage-like genes and
are instead adjacent to transposon-related sequences, they likely
correspond to transposon fragments rather than phage remnants.
One possible exception: Reut_A2191 is accompanied by genes
encoding putative phage regulatory proteins (Reut_A2193 and
Reut_A2195) and thus might be descended from a prophage.
The megaplasmid contains a higher density of phage-type
integrase genes and transposon elements than that found on either
chromosome. There are five integrase sequences (Reut_C5954,
Reut_C5993, Reut_C6147, Reut_C6164 and Reut_C6343) all of
which are adjacent to transposons, thus suggesting that these
integrases are part of transposon elements. This conclusion is
further supported by the identification of one such sequence in
plasmid pJP4 next to the transposase of a Tn3 family transposon
Protein transport, adherence, motility
C. necator JMP134 has a complete sec general protein secretion
system, including homologs of secA (Reut_A2970), secY (Reut_
A3159), secE (Reut_A3195), secG (Reut_A0960), secD (Reut_
A2810), secF (Reut_A2811) and yajC (Reut_A2809), as well as a
signal peptidase (Reut_A2254). It also has all the components of
the sec-independent twin-arginine translocation (TAT) system for
protein translocation: tatC (Reut_A3098), tatA/E (Reut_A3100),
tatB (Reut_3099), and tatD-related components (Reut_A1437 and
Reut_A1078). The TAT system is distinguished by the ability to
translocate fully-folded proteins and is found also in C. eutropha
H16, C. metallidurans CH34, and R. solanacearum GMI1000.
Complete type II and type IV secretion systems are also present
in these four organisms. In contrast, of the four, only the plant
pathogen R. solanacearum GMI1000 possesses a type III secretion
A full set of che genes encoding chemotaxis functions forms a
putative operon on chromosome 2 adjacent to fla genes encoding
the flagellum and motor proteins. Additional copies of all except
two of the che genes (cheY and cheZ) are scattered on chromosome 1.
These genes are also located on chromosome 2 in C. eutropha H16
and C. metallidurans CH34.
Analysis of the complete genome of C. necator JMP134 adds
further insights into the evolution of multipartite genomes in b-
proteobacteria, and the presence of aromatic catabolism and other
metabolic functions. It has been proposed that multipartite
genomes arise through intragenomic gene transfer between
progenitor chromosomes and ancestral plasmids. Our analysis
supports that hypothesis and further indicates that distinct
plasmids served as the scaffolds for the assembly of secondary
chromosomes in the Cupriavidus , Ralstonia, and Burkholderia lineages.
Furthermore, both chromosomes in the Cupriavidus show evidence
of significant gene duplication and lateral gene transfer, with
foreign DNA preferentially incorporated into the secondary
chromosomes. The C. necator JMP134 genome contains nearly
300 genes potentially involved in the catabolism of aromatic
compounds and encodes almost all of the central ring-cleavage
pathways. Although all these genomes possess a significant number
of aromatic catabolism functions, including central and peripheral
pathways, the genome of strain JMP134 is by far the one that
provides more versatile degradative abilities. The availability of
the complete genome sequence for C. necator JMP134 provides the
groundwork for further elucidation of the mechanisms and
regulation of chloroaromatic compound biodegradation, and its
interplays with several other key metabolic processes analyzed
Table S1 Functional annotation of key metabolic genes of C.
Found at: doi:10.1371/journal.pone.0009729.s001 (0.27 MB
Figure S1 Functional distribution of unique genes. COG
categories are as follows: Information storage and processing: A,
RNA processing, modification; B, chromatin structure; J, transla-
tion, ribosomal structure/biogenesis; K, transcription; L, DNA
replication, recombination, repair. Cellular processes: D, cell
division, chromosome partitioning; M, cell envelope biogenesis
outer membrane; N, Cell motility and secretion; P, Inorganic ion
transport and metabolism; T, Signal transduction mechanisms.
Metabolism: C, Energy production and conversion; G, Carbohy-
drate transport and metabolism; E, Amino acid transport and
metabolism; F, Nucleotide transport and metabolism; H, Coen-
zyme metabolism; I, Lipid metabolism; Q, Secondary metabolites
biosynthesis, transport and catabolism; Poorly characterized: R,
General function prediction only; S, Function unknown.
Found at: doi:10.1371/journal.pone.0009729.s002 (2.77 MB TIF)
Conceived and designed the experiments: AL DPP TL BG NCK.
Performed the experiments: AL SL. Analyzed the data: AL DPP TL
KM IA NI SDH BG. Contributed reagents/materials/analysis tools: BG.
Wrote the paper: AL DPP TL IA BG NCK.
1. Don RH, Pemberton JM (1981) Properties of six pesticide degradation plasmids
isolated from Alcaligenes paradoxus and Alcaligenes eutrophus. J Bacteriol 145:
2. Ghosal D, You IS, Chatterjee DK, Chakrabarty AM (1985) Genes specifying
degradation of 3-chlorobenzoic acid in plasmids pAC27 and pJP4. Proc Natl
Acad Sci U S A 82: 1638–1642.
Genome of C. necator JMP134
PLoS ONE | www.plosone.org 11 March 2010 | Volume 5 | Issue 3 | e9729
3. Clement P, Matus V, Cardenas L, Gonzalez B (1995) Degradation of
trichlorophenols by Alcaligenes eutrophus JMP134. FEMS Microbiol Lett
4. Schlomann M, Schmidt E, Knackmuss HJ (1990) Different types of dienelactone
hydrolase in 4-fluorobenzoate-utilizing bacteria. J Bacteriol 172: 5112–5118.
5. Laemmli CM, Leveau JH, Zehnder AJ, van der Meer JR (2000) Characteriza-
tion of a second tfd gene cluster for chlorophenol and chlorocatechol metabolism
on plasmid pJP4 in Ralstonia eutropha JMP134(pJP4). J Bacteriol 182:
6. Perez-Pantoja D, Guzman L, Manzano M, Pieper DH, Gonzalez B (2000) Role
of tfdC(I)D(I)E(I)F(I) and tfdD(II)C(II)E(II)F(II) gene modules in catabolism of 3-
chlorobenzoate by Ralstonia eutropha JMP134(pJP4). Appl Environ Microbiol
7. Perez-Pantoja D, Ledger T, Pieper DH, Gonzalez B (2003) Efficient turnover of
chlorocatechols is essen tial for growth of Ralstonia eutropha JMP134(pJP4) in 3-
chlorobenzoic acid. J Bacteriol 185: 1534–1542.
8. Plumeier I, Perez-Pantoja D, Heim S, Gonzalez B, Pieper DH (2002)
Importance of different tfd genes for degradation of chloroaromatics by
Ralstonia eutroph a JMP134. J Bacteriol 184: 4054–4064.
9. Trefault N, De la Iglesia R, Molina AM, Manzano M, Ledger T, et al. (2004)
Genetic organization of the catabolic plasmid pJP4 from Ralstonia eutropha
JMP134 (pJP4) reveals mechanisms of adaptation to chloroaromatic pollutants
and evolution of specialized chloroaromatic degradation pathways. Environ
Microbiol 6: 655–668.
10. Chain PS, Denef VJ, Konstantinidis KT, Vergez LM, Agullo L, et al. (2006)
Burkholderia xenovorans LB400 harbors a multi-replicon, 9.73-Mbp genome
shaped for versatility. Proc Natl Acad Sci U S A 103: 15280–15287.
11. Slater SC, Goldman BS, Goodner B, Setubal JC, Farrand SK, et al. (2009)
Genome sequences of three agrobacterium biovars help elucidate the evolution
of multichromosome genomes in bacteria. J Bacteriol 191: 2501–2511.
12. Badger JH, Olsen GJ (1999) CRITICA: coding region identification tool
invoking comparative analysis. Mol Biol Evol 16: 512–524.
13. Delcher AL, Harmon D, Kasif S, White O, Salzberg SL (1999) Improved
microbial g ene identification with GLIMMER. Nucleic Acids Res 27:
14. Markowitz VM, Szeto E, Palaniappan K, Grechkin Y, Chu K, et al. (2008) The
integrated microbial genomes (IMG) system in 2007: data content and analysis
tool extensions. Nucleic Acids Res 36: D528–533.
15. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, et al. (2007)
Clustal W and Clustal X version 2.0. Bioinformatics 23: 2947–2948.
16. Bendtsen JD, Nielsen H, von Heijne G, Brunak S (2004) Improved prediction of
signal peptides: SignalP 3.0. J Mol Biol 340: 783–795.
17. Krogh A, Larsson B, von Heijne G, Sonnhammer EL (2001) Predicting
transmembrane protein topology with a hidden Markov model: application to
complete genomes. J Mol Biol 305: 567–580.
18. Liolios K, Mavromatis K, Tavernarakis N, Kyrpides NC (2008) The Genome s
On Line Database (GOLD) in 2007: status of genomic and metagenomic
projects and their associated metadata. Nucleic Acids Res 36: D475–479.
19. Perez-Pantoja D, De la Iglesia R, Pieper DH, Gonzalez B (2008) Metabolic
reconstruction of aromatic compounds degradation from the genome of the
amazing pollutant-degrading bacterium Cupriavidus necator JMP134. FEMS
Microbiol Rev 32: 736–794.
20. Nelson KE, Weinel C, Paulsen IT, Dodson RJ, Hilbert H, et al. (2002) Complete
genome sequence and comparative analysis of the metabolically versatile
Pseudomonas putida KT2440. Environ Microbiol 4: 799–808.
21. McLeod MP, Warren RL, Hsiao WW, Araki N, Myhre M, et al. (2006) The
complete genome of Rhodococcus sp. RHA1 provides insights into a catabolic
powerhouse. Proc Natl Acad Sci U S A 103: 15582–15587.
22. Rabus R, Kube M, Heider J, Beck A, Heitmann K, et al. (2005) The genome
sequence of an anaerobic aromatic-degrading denitrifying bacterium, strain
EbN1. Arch Microbiol 183: 27–36.
23. Arias-Barrau E, Sandoval A, Naharro G, Olivera ER, Luengo JM (2005) A two-
component hydroxylase involved in the assimilation of 3-hydroxyphenyl acetate
in Pseudomonas putida. J Biol Chem 280: 26435–26447.
24. Gescher J, Zaar A, Mohamed M, Schagger H, Fuchs G (2002) Genes coding for
a new pathway of aerobic benzoate metaboli sm in Azoarcus evansii. J Bacteriol
25. Egland PG, Harwood CS (2000) HbaR, a 4-hydroxybenzoate sensor and FNR-
CRP superfamily member, regulates anaerobic 4-hydroxybenzoate degradation
by Rhodopseudomonas palustris. J Bacteriol 182: 100–106.
26. Ensign SA, Small FJ, Allen JR, Sluis MK (1998) New roles for CO2 in the microbial
metabolism of aliphatic epoxides and ketones. Arch Microbiol 169: 179–187.
27. Sluis MK, Larsen RA, Krum JG, Anderson R, Metcalf WW, et al. (2002)
Biochemical, molecular, and genetic analyses of the acetone carboxylases from
Xanthobacter autotrophicus strain Py2 and Rhodobacter capsulatus strain B10.
J Bacteriol 184: 2969–2977.
28. Stigter ECA, van den Lugt JP, Somers WAC (1997) Enantioselective oxidation
of secondary alcohols by quinohaemoprotein alcohol dehydrogenase from
Comamonas testosteroni. J Mol Catal B Enzym 2: 291–297.
29. Stoorvogel J, Kraayveld DE, Van Sluis CA, Jongejan JA, De Vries S, et al.
(1996) Characterization of the gene encoding quinohaemoprotein ethanol
dehydrogenase of Comamonas testosteroni. Eur J Biochem 235: 690–698.
30. Taylor DG, Trudgill PW, Cripps RE, Harris PR (1980) The microbial
metabolism of acetone. J Gen Microbiol 118: 159–170.
31. Zarnt G, Schrader T, Andreesen JR (1997) Degradation of tetrahydrofurfuryl
alcohol by Ralstonia eutropha is initiated by an inducible pyrroloquinoline quinone-
dependent alcohol dehydrogenase. Appl Environ Microbiol 63: 4891–4898.
32. Zarnt G, Schrader T, Andreesen JR (2001) Catalytic and molecular properties of
the quinohemoprotein tetrahydrofurfuryl alcohol dehydrogenase from Ralstonia
eutropha strain Bo. J Bacteriol 183: 1954–1960.
33. Copley SD (2003) Aromatic Dehalogenases: Insights into Structures, Mechanisms,
and Evolutionary Origins.; (eds) MMHaIDB, editor: Springer US. pp 227–259.
34. Janssen DB, Oppentocht JE, Poelarends GJ (2003) Bacterial Growth on Halogenated
Aliphatic Hydrocarbons: Genetics and Biochemistry; US S, editor. pp 207–222.
35. Daubaras DL, Hershberger CD, Kitano K, Chakrabarty AM (1995) Sequence
analysis of a gene cluster involved in metabolism of 2,4,5-trichlorophenoxyacetic
acid by Burkholderia cepacia AC1100. Appl Environ Microbiol 61: 1279–1289.
36. Roldan MD, Perez-Reinado E, Castillo F, Moreno-Vivian C (2008) Reduction
of polynitroaromatic compounds: the bacterial nitroreductases. FEMS Microbiol
Rev 32: 474–500.
37. Jenni B, Realini M, Aragno M, Tamer AU
(1988) Taxonomy of non H2-
lithotrophic, oxalate-oxidizing bacteria related to Alcaligenes eutrophus. System
Appl Microbiol 10: 126–133.
38. Lu CD, Yang Z, Li W (2004) Transcriptom e analysis of the ArgR regulon in
Pseudomonas aeruginosa. J Bac teriol 186: 3855–3861.
39. Revelles O, Espinosa-Urgel M, Fuhrer T, Sauer U, Ramos JL (2005) Multiple
and interconnected pathways for L-lysine catabolism in Pseudomonas putida
KT2440. J Bacteriol 187: 7500–7510.
40. Castanie-Cornet MP, Penfound TA, Smith D, Elliott JF, Foster JW (1999)
Control of acid resistance in Escherichia coli. J Bacteriol 181: 3525–3535.
41. Inoue H, Inagaki K, Eriguchi SI, Tamura T, Esaki N, et al. (1997) Molecular
characterization of the mde operon involved in L-methionine catabolism of
Pseudomonas putida. J Bacteriol 179: 3956–3962.
42. Fernandez M, Zuniga M (2006) Amino acid catabolic pathways of lactic acid
bacteria. Crit Rev Microbiol 32: 155–183.
43. Itoh Y, Nishijyo T, Nakada Y (2007) Histidine Catabolism and Catabolite
Regulation (eds) JLRaAF, editor: Springer Netherlands. pp 371–395.
44. Massey LK, Sokatch JR, Conrad RS (1976) Branched-chain amino acid
catabolism in bacteria. Bacteriol Rev 40: 42–54.
45. Reinecke F, Steinbuchel A (2009) Ralstonia eutropha strain H16 as model
organism for PHA metabolism and for biotechn ological production of
technically interesting biopolymers. J Mol Microbiol Biotechnol 16: 91–108.
46. Muller S, Bley T, Babel W (1999) Adaptive responses of Ralstonia eutropha to
feast and famine conditions analysed by flow cytometry. J Biotechnol 75: 81–97.
47. Lu CD, Abdelal AT (2001) The gdhB gene of Pseudomonas aeruginosa encodes
an arginine-inducible NAD(+)-dependent glutamate dehydrogenase which is
subject to allosteric regulation. J Bacteriol 183: 490–499.
48. Siddiqui RA, Warnecke-Eberz U, Hengsberger A, Schneider B, Kostka S, et al.
(1993) Structure and function of a periplasmic nitrate reductase in Alcaligenes
eutrophus H16. J Bacteriol 175: 5867–5876.
49. Rees E, Siddiqui RA, Koster F, Schneider B, Friedrich B (1997) Structural gene
(nirS) for the cytochrome cd1 nitrite reductase of Alcaligenes eutrophus H16.
Appl Environ Microbiol 63: 800–802.
50. Cramm R, Siddiqui RA, Friedrich B (1997) Two isofunctional nitric oxide
reductases in Alcaligenes eutrophus H16. J Bacteriol 179: 6769–6777.
51. Pohlmann A, Cramm R, Schmelz K, Friedrich B (2000) A novel NO-responding
regulator controls the reduction of nitric oxide in Ralstonia eutropha. Mol
Microbiol 38: 626–638.
52. Lemos RS, Fernandes AS, Pereira MM, Gomes CM, Teixeira M (2002)
Quinol:fumarate oxidoreductases and succinate:quinone oxidoreductases: phy-
logenetic relationships, metal centres and membrane attachment. Biochim
Biophys Acta 1553: 158–170.
53. Melo AM, Bandeiras TM, Teixeira M (2004) New insights into type II
NAD(P)H:quinone oxidoreductases. Microbiol Mol Biol Rev 68: 603–616.
54. Muller RH, Babel W (1996) Measurement of Growth at Very Low Rates ((mu)
. = 0), an Approach To Study the Energy Requirement for the Survival of
Alcaligenes eutrophus JMP 134. Appl Environ Microbiol 62: 147–151.
55. Oh JI, Bowien B (1998) Structural analysis of the fds operon encoding the
NAD+-linked formate dehydrogenase of Ralstonia eutropha. J Biol Chem 273:
56. Cramm R (2009) Genomic view of energy metabolism in Ralstonia eutropha
H16. J Mol Microbiol Biotechnol 16: 38–52.
57. Santana M, Pereira MM, Elias NP, Soares CM, Teixeira M (2001) Gene cluster
of Rhodothermus marinus high-potential iron-sulfur Protein: oxygen oxidore-
ductase, a caa(3)-type oxidase belonging to the superfamily of heme-copper
oxidases. J Bacteriol 183: 687–699.
58. Eberl L (2006) Quorum sensing in the genus Burkholderia. Int J Med Microbiol
59. Flavier AB, Schell MA, Denny TP (1998) An RpoS (sigmaS) homologue
regulates acylhomoserine lactone-dependent autoinduction in Ralstonia solana-
cearum. Mol Microbiol 28: 475–486.
60. Schell MA (2000) Control Of Virulence And Pathogenicity Genes Of Ralstonia
Solanacearum By An Elaborate Sensory Network. Annu Rev Phytopathol 38:
61. Garg RP, Yindeeyoungyeon W, Gilis A, Denny TP, Van Der Lelie D, et al.
(2000) Evidence that Ralstonia eutropha (Alcaligenes eutrophus) contains a
functional homologue of the Ralstonia solanacearum Phc cell density sensing
system. Mol Microbiol 38: 359–367.
Genome of C. necator JMP134
PLoS ONE | www.plosone.org 12 March 2010 | Volume 5 | Issue 3 | e9729
62. Barrett CF, Parker MA (2006) Coexistence of Burkholderia, Cupriavidus, and
Rhizobium sp. nodule bacteria on two Mimosa spp. in Costa Rica. Appl Environ
Microbiol 72: 1198–1206.
63. Chen WM, de Faria SM, Straliotto R, Pitard RM, Simoes-Araujo JL, et al.
(2005) Proof that Burkholderia strains form effective symbioses with legumes: a
study of novel Mimosa-nodulating strains from South Americ a. Appl Environ
Microbiol 71: 7461–7471.
64. Mendes R, Pizzirani-Kleiner AA, Araujo WL, Raaijmakers JM (2007) Diversity
of cultivated endophytic bacteria from sugarcane: genetic and biochemical
characterization of Burkholderia cepacia complex isolates. Appl Environ
Microbiol 73: 7259–7267.
65. Lugtenberg BJ, Chin AWTF, Bloemberg GV (2002) Microbe-plant interactions:
principles and mechanisms. Antonie Van Leeuwenhoek 81: 373–383.
66. Van Sluys MA, Monteiro-Vitorello CB, Camargo LE, Menck CF, Da Silva AC,
et al. (2002) Comparative genomic analysis of plant-associated bacteria. Annu
Rev Phytopathol 40: 169–189.
Genome of C. necator JMP134
PLoS ONE | www.plosone.org 13 March 2010 | Volume 5 | Issue 3 | e9729