# The Complete Genome Sequence of Cupriavidus metallidurans Strain CH34, a Master Survivalist in Harsh and Anthropogenic Environments

Paul J Janssen, Rob van Houdt, Hugo Moors, Pieter Monsieurs, Nicolas Morin, Arlette Michaux, Mohammed A Benotmane, Natalie Leys, Tatiana Vallaeys, Alla Lapidus, et al.

► To cite this version:

Paul J Janssen, Rob van Houdt, Hugo Moors, Pieter Monsieurs, Nicolas Morin, et al. The Complete Genome Sequence of Cupriavidus metallidurans Strain CH34, a Master Survivalist in Harsh and Anthropogenic Environments. PLoS ONE, 2010, 5 (5), pp.e10433. 10.1371/journal.pone.0010433. hal-04580603

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Submitted on 20 May 2024

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# The Complete Genome Sequence of Cupriavidus metallidurans Strain CH34, a Master Survivalist in Harsh and Anthropogenic Environments

Paul J Janssen, Rob van Houdt, Hugo Moors, Pieter Monsieurs, Nicolas Morin, Arlette Michaux, Mohammed A Benotmane, Natalie Leys, Tatiana Vallaeys, Alla Lapidus, et al.

► To cite this version:

Paul J Janssen, Rob van Houdt, Hugo Moors, Pieter Monsieurs, Nicolas Morin, et al. The Complete Genome Sequence of Cupriavidus metallidurans Strain CH34, a Master Survivalist in Harsh and Anthropogenic Environments. PLoS ONE, 2010, 5 (5), pp.e10433. 10.1371/journal.pone.0010433. hal-04580603

HAL Id: hal-04580603

https://hal.science/hal-04580603v1

Submitted on 20 May 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>
# The Complete Genome Sequence of *Cupriavidus metallidurans* Strain CH34, a Master Survivalist in Harsh and Anthropogenic Environments

Paul J. Janssen¹*, Rob Van Houdt¹, Hugo Moors¹, Pieter Monsieurs¹, Nicolas Morin¹, Arlette Michaux¹, Mohammed A. Benotmane¹, Natalie Leys¹, Tatiana Vallaeya s², Alla Lapidus³, Sébastien Monchy⁴, Claudine Médigue⁵, Safiyh Taghavi⁶, Sean McCorkle⁶, John Dunn⁶, Daniël van der Lelie⁶, Max Mergeay¹

¹ Molecular and Cellular Biology, Belgian Nuclear Research Center SCKCEN, Mol, Belgium, ² Laboratoire Ecosystèmes Lagunaires (ECOLAG), Centre National de la Recherche Scientifique - UMR 5119, Université Montpellier II, Montpellier, France, ³ Joint Genome Institute, Lawrence Berkeley National Laboratory, Walnut Creek, California, United States of America, ⁴ Laboratoire Microorganismes: Génome et Environnement (LMGE), Centre National de la Recherche Scientifique - UMR 6023, Université Blaise Pascal, Aubière, France, ⁵ Laboratoire d'Analyses Bioinformatiques pour la Génomique et le Métabolisme (LABGeM), Centre National de la Recherche Scientifique - UMR8030 and CEA/DSV/IG/Genoscope, Evry, France, ⁶ Biology Department, Brookhaven National Laboratory, Upton, New York, United States of America

## Abstract

Many bacteria in the environment have adapted to the presence of toxic heavy metals. Over the last 30 years, this heavy metal tolerance was the subject of extensive research. The bacterium *Cupriavidus metallidurans* strain CH34, originally isolated by us in 1976 from a metal processing factory, is considered a major model organism in this field because it withstands milli-molar range concentrations of over 20 different heavy metal ions. This tolerance is mostly achieved by rapid ion efflux but also by metal-complexation and -reduction. We present here the full genome sequence of strain CH34 and the manual annotation of all its genes. The genome of *C. metallidurans* CH34 is composed of two large circular chromosomes CHR1 and CHR2 of, respectively, 3,928,089 bp and 2,580,084 bp, and two megaplasmids pMOL28 and pMOL30 of, respectively, 171,459 bp and 233,720 bp in size. At least 25 loci for heavy-metal resistance (HMR) are distributed over the four replicons. Approximately 67% of the 6,717 coding sequences (CDSs) present in the CH34 genome could be assigned a putative function, and 9.1% (611 genes) appear to be unique to this strain. One out of five proteins is associated with either transport or transcription while the relay of environmental stimuli is governed by more than 600 signal transduction systems. The CH34 genome is most similar to the genomes of other *Cupriavidus* strains by correspondence between the respective CHR1 replicons but also displays similarity to the genomes of more distantly related species as a result of gene transfer and through the presence of large genomic islands. The presence of at least 57 IS elements and 19 transposons and the ability to take in and express foreign genes indicates a very dynamic and complex genome shaped by evolutionary forces. The genome data show that *C. metallidurans* CH34 is particularly well equipped to live in extreme conditions and anthropogenic environments that are rich in metals.

**Citation:** Janssen PJ, Van Houdt R, Moors H, Monsieurs P, Morin N, et al. (2010) The Complete Genome Sequence of *Cupriavidus metallidurans* Strain CH34, a Master Survivalist in Harsh and Anthropogenic Environments. PLoS ONE 5(5): e10433. doi:10.1371/journal.pone.0010433

**Editor:** Niyaz Ahmed, University of Hyderabad, India

**Received:** February 1, 2010; **Accepted:** March 29, 2010; **Published:** May 5, 2010

**Copyright:** © 2010 Janssen 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 was partially supported by internal funds of SCK/CEN, by the European Space Agency through the MESSAGE contracts (PRODEX agreements No. 90037 and 90094), and by Laboratory Directed Research and Development funds at the Brookhaven and Lawrence Berkeley National Laboratories. JD, SMcC, ST and DvdL acknowledge support by the United States Department of Energy, Office of Science, Biological and Environmental Research (BER), project number KP1102010 under contract DE-AC02-98CH10886, and by Laboratory Directed Research and Development funds (LDRD09-005) at the Brookhaven National Laboratory under contract with the U. S. Department of Energy. SM and NM acknowledge the support via a SCK/CEN PhD grant (AWM-programme). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

**Competing Interests:** The authors have declared that no competing interests exist.

* E-mail: pjanssen@sckcen.be

## Introduction

The abundant metal-containing minerals in our environment have shaped the world we live in. While metals such as calcium or sodium are essential micronutrients sustaining life, other ('heavy') metals, such as cobalt, copper, nickel, and zinc are vital cofactors for metallo-proteins and enzymes. At excessive concentrations, these metals may be toxic. Nonessential heavy metals, such as mercury, lead, and cadmium are considered toxic at any concentration: hence they are termed 'toxic metals'.

Perhaps the largest natural environmental sources of heavy metals are volcanic emissions, forest fires, deep-sea vents, and geysers [1]. Because of geobiochemical transformations and

natural routes of accumulation, metal-rich sites are fairly common across the planet. Such sites became colonised by long-lasting microbial consortia that overcame metal toxicity by adapting to metal stress and developing the underlying cellular mechanisms to overcome metal toxicity (such as complexation, efflux, or reduction of metal ions). However, consequent upon industrialisation, the concentrations of heavy and toxic metal in soils further increased worldwide over the past few hundred years [2]. A major contribution to this acceleration is the large-scale burning of fossil fuels that releases a wide range of toxic metals into the environment. Anthropogenic sources of metal contamination also include mining and smelting sites, metal-manufacturing plants, painting- and coating-industries, and tanneries. Other human

<figure><img src="image_2.png" /></figure>
# The Complete Genome Sequence of *Cupriavidus metallidurans* Strain CH34, a Master Survivalist in Harsh and Anthropogenic Environments

Paul J. Janssen¹*, Rob Van Houdt¹, Hugo Moors¹, Pieter Monsieurs¹, Nicolas Morin¹, Arlette Michaux¹, Mohammed A. Benotmane¹, Natalie Leys¹, Tatiana Vallaеys², Alla Lapidus³, Sébastien Monchy⁴, Claudine Médigue⁵, Safiyh Taghavi⁶, Sean McCorkle⁶, John Dunn⁶, Daniël van der Lelie⁶, Max Mergeay¹

¹ Molecular and Cellular Biology, Belgian Nuclear Research Center SCKCEN, Mol, Belgium, ² Laboratoire Ecosystèmes Lagunaires (ECOLAG), Centre National de la Recherche Scientifique - UMR 5119, Université Montpellier II, Montpellier, France, ³ Joint Genome Institute, Lawrence Berkeley National Laboratory, Walnut Creek, California, United States of America, ⁴ Laboratoire Microorganismes: Génome et Environnement (LMGE), Centre National de la Recherche Scientifique - UMR 6023, Université Blaise Pascal, Aubière, France, ⁵ Laboratoire d'Analyses Bioinformatiques pour la Génomique et le Métabolisme (LABGeM), Centre National de la Recherche Scientifique - UMR8030 and CEA/DSV/IG/Genoscope, Evry, France, ⁶ Biology Department, Brookhaven National Laboratory, Upton, New York, United States of America

## Abstract

Many bacteria in the environment have adapted to the presence of toxic heavy metals. Over the last 30 years, this heavy metal tolerance was the subject of extensive research. The bacterium *Cupriavidus metallidurans* strain CH34, originally isolated by us in 1976 from a metal processing factory, is considered a major model organism in this field because it withstands milli-molar range concentrations of over 20 different heavy metal ions. This tolerance is mostly achieved by rapid ion efflux but also by metal-complexation and -reduction. We present here the full genome sequence of strain CH34 and the manual annotation of all its genes. The genome of *C. metallidurans* CH34 is composed of two large circular chromosomes CHR1 and CHR2 of, respectively, 3,928,089 bp and 2,580,084 bp, and two megaplasmids pMOL28 and pMOL30 of, respectively, 171,459 bp and 233,720 bp in size. At least 25 loci for heavy-metal resistance (HMR) are distributed over the four replicons. Approximately 67% of the 6,717 coding sequences (CDSs) present in the CH34 genome could be assigned a putative function, and 9.1% (611 genes) appear to be unique to this strain. One out of five proteins is associated with either transport or transcription while the relay of environmental stimuli is governed by more than 600 signal transduction systems. The CH34 genome is most similar to the genomes of other *Cupriavidus* strains by correspondence between the respective CHR1 replicons but also displays similarity to the genomes of more distantly related species as a result of gene transfer and through the presence of large genomic islands. The presence of at least 57 IS elements and 19 transposons and the ability to take in and express foreign genes indicates a very dynamic and complex genome shaped by evolutionary forces. The genome data show that *C. metallidurans* CH34 is particularly well equipped to live in extreme conditions and anthropogenic environments that are rich in metals.

**Citation:** Janssen PJ, Van Houdt R, Moors H, Monsieurs P, Morin N, et al. (2010) The Complete Genome Sequence of *Cupriavidus metallidurans* Strain CH34, a Master Survivalist in Harsh and Anthropogenic Environments. PLoS ONE 5(5): e10433. doi:10.1371/journal.pone.0010433

**Editor:** Niyaz Ahmed, University of Hyderabad, India

**Received:** February 1, 2010; **Accepted:** March 29, 2010; **Published:** May 5, 2010

**Copyright:** © 2010 Janssen 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 was partially supported by internal funds of SCKCEN, by the European Space Agency through the MESSAGE contracts (PRODEX agreements No. 90037 and 90094), and by Laboratory Directed Research and Development funds at the Brookhaven and Lawrence Berkeley National Laboratories. JD, SMCC, ST and DvdL acknowledge support by the United States Department of Energy, Office of Science, Biological and Environmental Research (BER), project number KP1102010 under contract DE-AC02-98CH10886, and by Laboratory Directed Research and Development funds (LDRD09-005) at the Brookhaven National Laboratory under contract with the U. S. Department of Energy. SM and NM acknowledge the support via a SCKCEN PhD grant (AWM-programme). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

**Competing Interests:** The authors have declared that no competing interests exist.

* E-mail: pjanssen@sckcen.be

## Introduction

The abundant metal-containing minerals in our environment have shaped the world we live in. While metals such as calcium or sodium are essential micronutrients sustaining life, other ('heavy') metals, such as cobalt, copper, nickel, and zinc are vital cofactors for metallo-proteins and enzymes. At excessive concentrations, these metals may be toxic. Nonessential heavy metals, such as mercury, lead, and cadmium are considered toxic at any concentration: hence they are termed 'toxic metals'.

Perhaps the largest natural environmental sources of heavy metals are volcanic emissions, forest fires, deep-sea vents, and geysers [1]. Because of geobiochemical transformations and

natural routes of accumulation, metal-rich sites are fairly common across the planet. Such sites became colonised by long-lasting microbial consortia that overcame metal toxicity by adapting to metal stress and developing the underlying cellular mechanisms to overcome metal toxicity (such as complexation, efflux, or reduction of metal ions). However, consequent upon industrialisation, the concentrations of heavy and toxic metal in soils further increased worldwide over the past few hundred years [2]. A major contribution to this acceleration is the large-scale burning of fossil fuels that releases a wide range of toxic metals into the environment. Anthropogenic sources of metal contamination also include mining and smelting sites, metal-manufacturing plants, painting- and coating-industries, and tanneries. Other human

<figure><img src="image_2.png" /></figure>
practices such as applying heavy metals in herbicides and pesticides or using sewage sludge as fertiliser recently raised concern as they have led to the presence of toxic metals in crops, and subsequently, into the foodchain. Typical hot spots of metal contamination are urbanised areas with large industrial activities surrounded by agricultural areas.

The facultative chemolithoautotrophic β-proteobacterium *Cupriavidus* (formerly *Wautersia*, *Ralstonia*, *Alcaligenes*) *metallidurans* was isolated in 1976 as a cadmium-resistant and hydrogen-oxidising pseudomonad from a decantation tank at a metal processing factory in Belgium [3]. This strain, later called CH34, was found to be highly resistant to Zn²⁺, Cd²⁺, and Co²⁺; the extrachromosomal genetic determinants conferring this resistance were transferable to related bacteria. Subsequent studies identified multiple loci for metal resistance, many of which were located on two large plasmids pMOL28 and pMOL30 [4–14].

Many environmental microbes evolved new or better strategies to survive metal-stressed conditions via the exchange of DNA i.e. by gene transfer. Novick and Roth [15] were among the earliest to report on plasmid-mediated heavy metal resistance when they discovered separate genetic loci on the plasmids of *Staphylococcus aureus* encoding resistance to arsenate, arsenite, antimony, lead, cadmium, zinc, bismuth, and mercury [15]. Since then, the transfer of genes involved in heavy metal resistance (HMR) was documented for many different bacterial species [6,16–18]. For *C. metallidurans* strain CH34, some important observations related to horizontal gene transfer and the acquisition of HMR can be made. First, upon its discovery, strain CH34 was described as a good acceptor of foreign genes in homologous and heterospecific matings that involved the broad host range IncP plasmid RP4::miniMu (aka pULB113) containing a very efficient transposon (prophage miniMu3A). This transposon promotes the generation of R-prime plasmids via an “in vivo” cloning process [19,20] later used to map the largest chromosome of *C. metallidurans* CH34 [21,22]. Second, strain CH34 is a good recipient of large plasmids that are involved in the HMR or hydrogenotrophy of other *Cupriavidus* strains [23]. And third, strain CH34 served as recipient and donor in soil microcosms to either assess (recombinant) gene dissemination promoted by broad host range plasmids [24–26] or to directly capture new broad host range plasmids [27–30] and plasmids carrying genes for degradation of xenobiotics [31].

Today, following several taxonomic amendments, strain CH34 is known as the type strain of the species *C. metallidurans* [32–34]. This strain tolerates high concentrations of heavy metal ions, including but not necessarily limited to Cu⁺, Cu²⁺, Ni²⁺, Zn²⁺, Co²⁺, Cd²⁺, CrO₄²⁻, Pb²⁺, Ag⁺, Au⁺, Au³⁺, HAsO₄²⁻, AsO₂⁻, Hg²⁺, Cs⁺, Bi³⁺, Tl⁺, SeO₃²⁻, SeO₄²⁻, and Sr²⁺, with at least 24 gene clusters distributed over four replicons, i.e., two main chromosomes and two megaplasmids ([8,10,14,35–37] and unpublished results). *C. metallidurans* CH34 accomplishes metal detoxification by the concerted action of efflux systems including at least eight P-type ATPases and a wide variety of systems for RND (for Resistance, Nodulation, cell Division) and CDF (for Cation Diffusion Facilitation). It also contains transporters specialized for certain metal ions, whereby efflux may be followed by metal sequestration or complexation (this study; [7,8,38–40]). Many of these metal responses overlap and are multilayered ([10] and unpublished results). In addition, we have noted changes in gene expression, cell morphology and physiology for *C. metallidurans* CH34 cells in response to spaceflight (i.e. cells grown at the International Space Station during 10-day visits) and oxidative stress [41,42], as well as simulated microgravity and ionizing radiation (unpublished). The organism has been isolated from very harsh environments such as ultra-clean spacecraft assembly rooms [43] and spent-nuclear-fuel pools [44]. Not

surprisingly, genome data indicate that CH34 has at its disposal intricate regulatory networks and many signal transduction systems.

Members of the genus *Cupriavidus* are metabolically diverse and occur in a wide range of habitats. In this study, we compared the CH34 genome with the genomes of three other species: The H₂-oxidising *C. eutrophus* H16 [45], the chloroaromatic pollutant degrading *C. pinatubonensis* JMP134 (unpublished), and the nitrogen-fixing plant symbiont *C. taiwanensis* [46]. While each of these four species carry plasmids that *de facto* determine their lifestyle, orthology and synteny analyses showed they share many genetic characteristics defined by their bipartite chromosomal structure (denoted as CHR1 and CHR2 throughout this study).

The CH34 genome was also compared with the genomes of more distantly related bacteria belonging to the family *Burkholderiaceae*. The species included in this analysis were selected on the basis of high overall synteny between their genomes and the CH34 genome, exemplifying their phylogenetic relationship. One striking feature across the analyzed genomes is the overrepresentation of periplasmic receptors containing the Bug domain, a carboxylate-binding motif first noted as overrepresented in host-restricted *Bordetella* species [47]. The prevalence of these receptors in *Cupriavidus* and closely related free-living bacteria may point to a common adaptation strategy to polluted environments rich in metal-carboxylates or carboxylated compounds (i.e., sewage, activated sludge, effluents, and industrial wastewaters), and in rhizospheres affected by anthropogenic pollution or in which carboxylates exuded from plant roots have accumulated.

Here we describe the complete sequencing of *C. metallidurans* strain CH34, and discuss its gene functions derived from manual curation. New insights into its genome structure, its anthology of mobile genetic elements, the many metal-resistance mechanisms, its regulatory networks, vitamin biosynthesis, and siderophore binding and translocation are revealed. Respiration and autotrophy, the oxidation of hydrogen and sulfur, the synthesis of PHB polymeric granules, its metabolic capacities, capsular-protein production, its signal sensory and transduction systems, and its secretion and transport machinery were also considered.

## Results and Discussion

### Genome Structure and General Features

The total genome of *C. metallidurans* CH34, of 6,913,352 bp, comprises four circular replicons: Chromosome 1 (CHR1) (3,928,089 bp); chromosome 2 (CHR2) (2,580,084 bp); and two megaplasmids pMOL28 (171,459 bp), and pMOL30 (233,720 bp). Since the two megaplasmids are detailed by Monchy *et al.* [10] and Mergeay *et al.* [7] this report mainly covers the sequencing and expert annotation of the two large replicons. Nonetheless, in a separate subsection below, we give a brief update on the megaplasmids. Table 1 lists the general features of all four replicons. There are four sets of 5S, 16S, and 23S rRNA genes and 12 structural RNA genes that include one RNAaseP RNA (*mpB*; Rmet_R0004), one tmRNA (*ssr4*; Rmet_R0007), one SRP RNA (*ffs*; Rmet_R0026), and nine riboswitches as predicted by Rfam (http://rfam.sanger.ac.uk/) (*Rmet_R0001*, *Rmet_R0012*, *Rmet_R0043*, *Rmet_R0047*, *Rmet_R0067*, *Rmet_R0068*, *Rmet_R0083*, *Rmet_R0084*, and *Rmet_R0085*). Sixty-two transfer RNA (tRNA) genes with specificities for all 20 amino acids were identified, most located on CHR1. Five tRNA genes are duplicated on CHR2 (two genes once, and three genes twice) (Table 1). Genome analysis using the MaGe system [48] predicts 6,717 coding sequences (CDS). Of those, 4,518 CDS were assigned a function using one of five evidence classes, while 1,274 and 611 CDS remained, respectively, ‘conserved hypothetical’ or ‘hypothetical’. Using the automated COGnitor module [49]

<figure><img src="image_3.png" /></figure>
practices such as applying heavy metals in herbicides and pesticides or using sewage sludge as fertiliser recently raised concern as they have led to the presence of toxic metals in crops, and subsequently, into the foodchain. Typical hot spots of metal contamination are urbanised areas with large industrial activities surrounded by agricultural areas.

The facultative chemolithoautotrophic β-proteobacterium *Cupriavidus* (formerly *Wautersia*, *Ralstonia*, *Alcaligenes*) *metallidurans* was isolated in 1976 as a cadmium-resistant and hydrogen-oxidising pseudomonad from a decantation tank at a metal processing factory in Belgium [3]. This strain, later called CH34, was found to be highly resistant to Zn²⁺, Cd²⁺, and Co²⁺; the extrachromosomal genetic determinants conferring this resistance were transferable to related bacteria. Subsequent studies identified multiple loci for metal resistance, many of which were located on two large plasmids pMOL28 and pMOL30 [4–14].

Many environmental microbes evolved new or better strategies to survive metal-stressed conditions via the exchange of DNA i.e. by gene transfer. Novick and Roth [15] were among the earliest to report on plasmid-mediated heavy metal resistance when they discovered separate genetic loci on the plasmids of *Staphylococcus aureus* encoding resistance to arsenate, arsenite, antimony, lead, cadmium, zinc, bismuth, and mercury [15]. Since then, the transfer of genes involved in heavy metal resistance (HMR) was documented for many different bacterial species [6,16–18]. For *C. metallidurans* strain CH34, some important observations related to horizontal gene transfer and the acquisition of HMR can be made. First, upon its discovery, strain CH34 was described as a good acceptor of foreign genes in homologous and heterospecific matings that involved the broad host range IncP plasmid RP4::miniMu (aka pULB113) containing a very efficient transposon (prophage miniMu3A). This transposon promotes the generation of R-prime plasmids via an “in vivo” cloning process [19,20] later used to map the largest chromosome of *C. metallidurans* CH34 [21,22]. Second, strain CH34 is a good recipient of large plasmids that are involved in the HMR or hydrogenotrophy of other *Cupriavidus* strains [23]. And third, strain CH34 served as recipient and donor in soil microcosms to either assess (recombinant) gene dissemination promoted by broad host range plasmids [24–26] or to directly capture new broad host range plasmids [27–30] and plasmids carrying genes for degradation of xenobiotics [31].

Today, following several taxonomic amendments, strain CH34 is known as the type strain of the species *C. metallidurans* [32–34]. This strain tolerates high concentrations of heavy metal ions, including but not necessarily limited to Cu⁺, Cu²⁺, Ni²⁺, Zn²⁺, Co²⁺, Cd²⁺, CrO₄²⁻, Pb²⁺, Ag⁺, Au⁺, Au³⁺, HAsO₄²⁻, AsO₂⁻, Hg²⁺, Cs⁺, Bi³⁺, Tl⁺, SeO₃²⁻, SeO₄²⁻, and Sr²⁺, with at least 24 gene clusters distributed over four replicons, i.e., two main chromosomes and two megaplasmids ([8,10,14,35–37] and unpublished results). *C. metallidurans* CH34 accomplishes metal detoxification by the concerted action of efflux systems including at least eight P-type ATPases and a wide variety of systems for RND (for Resistance, Nodulation, cell Division) and CDF (for Cation Diffusion Facilitation). It also contains transporters specialized for certain metal ions, whereby efflux may be followed by metal sequestration or complexation (this study; [7,8,38–40]). Many of these metal responses overlap and are multilayered ([10] and unpublished results). In addition, we have noted changes in gene expression, cell morphology and physiology for *C. metallidurans* CH34 cells in response to spaceflight (i.e. cells grown at the International Space Station during 10-day visits) and oxidative stress [41,42], as well as simulated microgravity and ionizing radiation (unpublished). The organism has been isolated from very harsh environments such as ultra-clean spacecraft assembly rooms [43] and spent-nuclear-fuel pools [44]. Not

surprisingly, genome data indicate that CH34 has at its disposal intricate regulatory networks and many signal transduction systems.

Members of the genus *Cupriavidus* are metabolically diverse and occur in a wide range of habitats. In this study, we compared the CH34 genome with the genomes of three other species: The H₂-oxidising *C. eutrophus* H16 [45], the chloroaromatic pollutant degrading *C. pinatubonensis* JMP134 (unpublished), and the nitrogen-fixing plant symbiont *C. taiwanensis* [46]. While each of these four species carry plasmids that *de facto* determine their lifestyle, orthology and synteny analyses showed they share many genetic characteristics defined by their bipartite chromosomal structure (denoted as CHR1 and CHR2 throughout this study).

The CH34 genome was also compared with the genomes of more distantly related bacteria belonging to the family *Burkholderiaceae*. The species included in this analysis were selected on the basis of high overall synteny between their genomes and the CH34 genome, exemplifying their phylogenetic relationship. One striking feature across the analyzed genomes is the overrepresentation of periplasmic receptors containing the Bug domain, a carboxylate-binding motif first noted as overrepresented in host-restricted *Bordetella* species [47]. The prevalence of these receptors in *Cupriavidus* and closely related free-living bacteria may point to a common adaptation strategy to polluted environments rich in metal-carboxylates or carboxylated compounds (i.e., sewage, activated sludge, effluents, and industrial wastewaters), and in rhizospheres affected by anthropogenic pollution or in which carboxylates exuded from plant roots have accumulated.

Here we describe the complete sequencing of *C. metallidurans* strain CH34, and discuss its gene functions derived from manual curation. New insights into its genome structure, its anthology of mobile genetic elements, the many metal-resistance mechanisms, its regulatory networks, vitamin biosynthesis, and siderophore binding and translocation are revealed. Respiration and autotrophy, the oxidation of hydrogen and sulfur, the synthesis of PHB polymeric granules, its metabolic capacities, capsular-protein production, its signal sensory and transduction systems, and its secretion and transport machinery were also considered.

## Results and Discussion

### Genome Structure and General Features

The total genome of *C. metallidurans* CH34, of 6,913,352 bp, comprises four circular replicons: Chromosome 1 (CHR1) (3,928,089 bp); chromosome 2 (CHR2) (2,580,084 bp); and two megaplasmids pMOL28 (171,459 bp), and pMOL30 (233,720 bp). Since the two megaplasmids are detailed by Monchy *et al.* [10] and Mergeay *et al.* [7] this report mainly covers the sequencing and expert annotation of the two large replicons. Nonetheless, in a separate subsection below, we give a brief update on the megaplasmids. Table 1 lists the general features of all four replicons. There are four sets of 5S, 16S, and 23S rRNA genes and 12 structural RNA genes that include one RNAaseP RNA (*mpB*; Rmet_R0004), one tmRNA (*ssr4*; Rmet_R0007), one SRP RNA (*ffs*; Rmet_R0026), and nine riboswitches as predicted by Rfam (http://rfam.sanger.ac.uk/) (*Rmet_R0001*, *Rmet_R0012*, *Rmet_R0043*, *Rmet_R0047*, *Rmet_R0067*, *Rmet_R0068*, *Rmet_R0083*, *Rmet_R0084*, and *Rmet_R0085*). Sixty-two transfer RNA (tRNA) genes with specificities for all 20 amino acids were identified, most located on CHR1. Five tRNA genes are duplicated on CHR2 (two genes once, and three genes twice) (Table 1). Genome analysis using the MaGe system [48] predicts 6,717 coding sequences (CDS). Of those, 4,518 CDS were assigned a function using one of five evidence classes, while 1,274 and 611 CDS remained, respectively, ‘conserved hypothetical’ or ‘hypothetical’. Using the automated COGnitor module [49]

<figure><img src="image_3.png" /></figure>
<table><caption>**Table 1.** General features of the *Cupriavidus metallidurans* CH34 genome.</caption><thead><tr><th rowspan="2">Feature</th><th>Chromosome 1</th><th>Chromosome 2</th><th>Plasmid 1</th><th>Plasmid 2</th></tr><tr><th>(CHR1)</th><th>(CHR2)</th><th>(pMOL28)</th><th>(pMOL30)</th></tr></thead><tbody><tr><td>Size (bp)</td><td>3,928,089</td><td>2,580,084</td><td>171,459</td><td>233,720</td></tr><tr><td>G + C ratio (mol %)</td><td>63.82</td><td>63.60</td><td>60.50</td><td>60.13</td></tr><tr><td>tRNA</td><td>54</td><td>8</td><td>0</td><td>0</td></tr><tr><td>rRNA operons</td><td>2</td><td>2</td><td>0</td><td>0</td></tr><tr><td>IS elements</td><td>30</td><td>19</td><td>3</td><td>5</td></tr><tr><td>Transposable elements</td><td>8</td><td>7</td><td>2</td><td>2</td></tr><tr><td>Genomic Islands</td><td>11</td><td>0</td><td>3</td><td>2</td></tr><tr><td>Total number of CDSs</td><td>3,766</td><td>2,493</td><td>175</td><td>283</td></tr><tr><td>Overlapping CDSs</td><td>104</td><td>92</td><td>2</td><td>13</td></tr><tr><td>CDSs with assigned function</td><td>2,784</td><td>1,468</td><td>100</td><td>166</td></tr><tr><td>Conserved hypothetical</td><td>690</td><td>478</td><td>54</td><td>52</td></tr><tr><td>Hypothetical</td><td>250</td><td>292</td><td>15</td><td>54</td></tr><tr><td>CDSs assigned to COGs</td><td>3,045</td><td>1,888</td><td>77</td><td>123</td></tr><tr><td>Genes involved in transport</td><td>433</td><td>288</td><td>5</td><td>16</td></tr><tr><td>Sigma factors</td><td>10</td><td>7</td><td>1</td><td>0</td></tr><tr><td>bug genes</td><td>37</td><td>87</td><td>0</td><td>1</td></tr></tbody></table>

doi:10.1371/journal.pone.0010433.t001

imbedded in the MaGe system 5,133 CDS were assigned to one or more COG functional classes (COGs are Clusters of Orthologous Groups of proteins generated by comparing the protein sequences of

complete genomes - each cluster contains proteins or groups of paralogs from at least three lineages). (Figure 1; Table 1). The G+C content of the replicons all are very similar to each other, with an

<figure><img src="image_4.png" /><figcaption>**Figure 1.** Circular representation of the two main replicons of CH34. Circles display from the inside outwards: (ring 1) scale in Mb, (ring 2) GC-content using a 2 kb window, (ring 3) GC-skew (G-C/G+C ratio) using a 2 kb window, (rings 4 and 5) predicted CDSs transcribed in a counterclockwise/clockwise direction; genomic islands are indicated in black solid bars and given a numbering; black open triangles represent the IS elements; colored triangles depict transposons: red, Tn6049; blue, Tn6049; and green, Tn6050.
doi:10.1371/journal.pone.0010433.g001</figcaption></figure>

<figure><img src="image_5.png" /></figure>
<table><caption>**Table 1.** General features of the *Cupriavidus metallidurans* CH34 genome.</caption><thead><tr><th rowspan="2">Feature</th><th>Chromosome 1</th><th>Chromosome 2</th><th>Plasmid 1</th><th>Plasmid 2</th></tr><tr><th>(CHR1)</th><th>(CHR2)</th><th>(pMOL28)</th><th>(pMOL30)</th></tr></thead><tbody><tr><td>Size (bp)</td><td>3,928,089</td><td>2,580,084</td><td>171,459</td><td>233,720</td></tr><tr><td>G + C ratio (mol %)</td><td>63.82</td><td>63.60</td><td>60.50</td><td>60.13</td></tr><tr><td>tRNA</td><td>54</td><td>8</td><td>0</td><td>0</td></tr><tr><td>rRNA operons</td><td>2</td><td>2</td><td>0</td><td>0</td></tr><tr><td>IS elements</td><td>30</td><td>19</td><td>3</td><td>5</td></tr><tr><td>Transposable elements</td><td>8</td><td>7</td><td>2</td><td>2</td></tr><tr><td>Genomic Islands</td><td>11</td><td>0</td><td>3</td><td>2</td></tr><tr><td>Total number of CDSs</td><td>3,766</td><td>2,493</td><td>175</td><td>283</td></tr><tr><td>Overlapping CDSs</td><td>104</td><td>92</td><td>2</td><td>13</td></tr><tr><td>CDSs with assigned function</td><td>2,784</td><td>1,468</td><td>100</td><td>166</td></tr><tr><td>Conserved hypothetical</td><td>690</td><td>478</td><td>54</td><td>52</td></tr><tr><td>Hypothetical</td><td>250</td><td>292</td><td>15</td><td>54</td></tr><tr><td>CDSs assigned to COGs</td><td>3,045</td><td>1,888</td><td>77</td><td>123</td></tr><tr><td>Genes involved in transport</td><td>433</td><td>288</td><td>5</td><td>16</td></tr><tr><td>Sigma factors</td><td>10</td><td>7</td><td>1</td><td>0</td></tr><tr><td>bug genes</td><td>37</td><td>87</td><td>0</td><td>1</td></tr></tbody></table>

doi:10.1371/journal.pone.0010433.t001

imbedded in the MaGe system 5,133 CDS were assigned to one or more COG functional classes (COGs are Clusters of Orthologous Groups of proteins generated by comparing the protein sequences of

complete genomes - each cluster contains proteins or groups of paralogs from at least three lineages). (Figure 1; Table 1). The G+C content of the replicons all are very similar to each other, with an

<figure><img src="image_4.png" /><figcaption>**Figure 1.** Circular representation of the two main replicons of CH34. Circles display from the inside outwards: (ring 1) scale in Mb, (ring 2) GC-content using a 2 kb window, (ring 3) GC-skew (G-C/G+C ratio) using a 2 kb window, (rings 4 and 5) predicted CDSs transcribed in a counterclockwise/clockwise direction; genomic islands are indicated in black solid bars and given a numbering; black open triangles represent the IS elements; colored triangles depict transposons: red, Tn6049; blue, Tn6049; and green, Tn6050.
doi:10.1371/journal.pone.0010433.g001</figcaption></figure>

<figure><img src="image_5.png" /></figure>
average of 63.7% for the large chromosomes, and of 60.3% for the two plasmids (Table 1).

The distribution over the replicons of the genome's functional content also was analyzed using the MaGe system. As expected, the main replicon CHR1 carries most of the essential housekeeping genes, including those needed for DNA replication, DNA repair, cell division, transcription and translation, along with sets of genes required for protein processing, folding, and post-translational modification (Figure 2; Table S1). However, a bias towards particular COG classes was also seen for the smaller replicon (classes I, K, N, Q, and T). These functional classes generally point to adaptation and survival in terms of energy storage and responsive cellular behavior. In addition, the CHR2 replicon contains several genes paralogous to CHR1 genes. Most notably are *dbpA* (Rmet_5284) encoding a second ATP-dependent RNA helicase, a second excinuclease gene *uvrA2* (Rmet_4549), a third copy of an exodeoxyribonuclease gene *xthA3* (Rmet_4910), a second glutamate dehydrogenase gene *gdhA2* (Rmet_5114), a second elongation factor G

encoded by *fusA2* (Rmet_5930), a second translation initiation factor IF-1 encoded by *infA2* (Rmet_5176), a second housekeeping sigma-70 factor encoded by *rpoD2* (Rmet_4661) and six genes that code for alternative sigma factors *fliA* (Rmet_3702), *sigJ* (Rmet_3844), *rpoK* (Rmet_4001), *rpoJ* (Rmet_4499), *rpoQ* (Rmet_4686), and *rpoM* (Rmet_5400) - see also dedicated subsection on transcription factors. Furthermore, the CHR2 replicon harbours many genes and gene cassettes involved in specialised metabolic- and biosynthetic-activities and adaptive responses, including those involved in carotenoid biosynthesis (*carX*, Rmet_5644; *crtB*, Rmet_4149), acetone utilisation and tolerance (*acxRABC*, Rmet_4104-07), polyhydroxyalkanoic acid (PHA) synthesis and conversion (*phaZ*, *phaZ3*, *phaB3*, *phaC3*), sulfonate/taurine transport and utilisation (*asfRAB*, *tauABC*, *tauD*, *tauE*), sulfite oxidation (*sorAB*), squalene/hopene synthesis (*sqhC*, Rmet_4148), tannin degradation (*tanA*, Rmet_3841), biofilm formation (*pelABCDEFG*), and exopolysaccharide synthesis (*epsAPB*). It carries most of the genes for motility and chemotaxis (*mot*, *flh*, *fli*, *flg*, *cpa*, *tad*, *pil*, *fim*, and *che* loci), peroxide

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>**Figure 2.** Functional distribution over CHR1 and CHR2 by COG classification. The scale represents the normalised ratio of CDS numbers per replicon (i.e. the ratio of percentages for each class per replicon); a ratio of 1 means that genes classified in a particular COG are, numberwise, evenly distributed over both replicons, taking into account the different total count of genes per replicon. Detailed data are in Table S1.
doi:10.1371/journal.pone.0010433.g002</figcaption></figure>

<figure><img src="image_8.png" /></figure>
average of 63.7% for the large chromosomes, and of 60.3% for the two plasmids (Table 1).

The distribution over the replicons of the genome's functional content also was analyzed using the MaGe system. As expected, the main replicon CHR1 carries most of the essential housekeeping genes, including those needed for DNA replication, DNA repair, cell division, transcription and translation, along with sets of genes required for protein processing, folding, and post-translational modification (Figure 2; Table S1). However, a bias towards particular COG classes was also seen for the smaller replicon (classes I, K, N, Q, and T). These functional classes generally point to adaptation and survival in terms of energy storage and responsive cellular behavior. In addition, the CHR2 replicon contains several genes paralogous to CHR1 genes. Most notably are *dbpA* (Rmet_5284) encoding a second ATP-dependent RNA helicase, a second excinuclease gene *uvrA2* (Rmet_4549), a third copy of an exodeoxyribonuclease gene *xthA3* (Rmet_4910), a second glutamate dehydrogenase gene *gdhA2* (Rmet_5114), a second elongation factor G

encoded by *fusA2* (Rmet_5930), a second translation initiation factor IF-1 encoded by *infA2* (Rmet_5176), a second housekeeping sigma-70 factor encoded by *rpoD2* (Rmet_4661) and six genes that code for alternative sigma factors *fliA* (Rmet_3702), *sigJ* (Rmet_3844), *rpoK* (Rmet_4001), *rpoJ* (Rmet_4499), *rpoQ* (Rmet_4686), and *rpoM* (Rmet_5400) - see also dedicated subsection on transcription factors. Furthermore, the CHR2 replicon harbours many genes and gene cassettes involved in specialised metabolic- and biosynthetic-activities and adaptive responses, including those involved in carotenoid biosynthesis (*carX*, Rmet_5644; *crtB*, Rmet_4149), acetone utilisation and tolerance (*acxRABC*, Rmet_4104-07), polyhydroxyalkanoic acid (PHA) synthesis and conversion (*phaZ*, *phaZ3*, *phaB3*, *phaC3*), sulfonate/taurine transport and utilisation (*asfRAB*, *tauABC*, *tauD*, *tauE*), sulfite oxidation (*sorAB*), squalene/hopene synthesis (*sqhC*, Rmet_4148), tannin degradation (*tanA*, Rmet_3841), biofilm formation (*pelABCDEFG*), and exopolysaccharide synthesis (*epsAPB*). It carries most of the genes for motility and chemotaxis (*mot*, *flh*, *fli*, *flg*, *cpa*, *tad*, *pil*, *fim*, and *che* loci), peroxide

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>**Figure 2.** Functional distribution over CHR1 and CHR2 by COG classification. The scale represents the normalised ratio of CDS numbers per replicon (i.e. the ratio of percentages for each class per replicon); a ratio of 1 means that genes classified in a particular COG are, numberwise, evenly distributed over both replicons, taking into account the different total count of genes per replicon. Detailed data are in Table S1.
doi:10.1371/journal.pone.0010433.g002</figcaption></figure>

<figure><img src="image_8.png" /></figure>
removal and oxidative stress (*yhjA*, *ahpC*, *ahpD*, *katG*, *pcaCD*, *soxR*, *glrX*), DNA adenine methylation (*dam*), DNA repair (*ada*, *alkA*, *alkB1*, *alkB2*, *sbcCD*, *ogt2*, *ogt3*), adaptive mutagenesis (*imuA-imuB-dnaE2*, *Rmet_4128-29-30*), and eight copies of the *uspA* gene (two of which are located on transposons) encoding the universal stress protein [most genes and loci without Rmet numbers are also discussed in separate sections]. The CHR2 replicon also carries an intact master two-component phosphorelay system (*bvgS*A) that, in *Bordetella* species, controls hundreds of genes involved in an extensive variety of responses and physiological functions, including virulence, biofilm formation, chemotaxis, and motility [50].

Both chromosomes display clear GC skew transitions that likely correspond with their replication origin and terminus [51] (Figure 1). For CHR1 the GC skew transition region (at point 3.84 on the map, Figure 1A) is about 90 kb away from the typical *dnaA-dnaN-gyrB* layout (genes *Rmet_0001*, *Rmet_0002*, and *Rmet_0003*, with *dnaA* starting at position 104) although the 400 bp upstream region of *dnaA* contains 1 perfect DnaA-binding box (TTATCCACA) and 3 putative DnaA boxes with one or two mismatches to this consensus (Figure S1A). Interestingly, the GC skew transition region located around coordinate 3,837,500 is preceded immediately by a short region of 213 nt encompassing 3 perfect DnaA boxes. Four more putative DnaA boxes (tightly clustered in a 65 nt region and each with one mismatch to the consensus) occur even further upstream of *dnaA*, at coordinates 3,707,588–3,707,653 (Figure S1A). For CHR2 the switch in GC skew is situated around the *repA-parA-parB* genes (*Rmet_5817*, *Rmet_5819*, and *Rmet_5820*) (point 2.43 of the map, Figure 1B)

indicating that CHR2 partitioning may be plasmid-like. The *repA* upstream region contains 3 putative DnaA boxes with only one mismatch to the consensus, at locations –392, –419, and –1080 with respect to the start codon of *repA* (Figure S1B). This region also contains at least 12 repetitive 17 nt-long elements with a highly conserved motif (consensus CGCAGAA(A/T)(C/T)(A/G)GGTACG(C/T)) that may serve as RepA binding sites. In CHR1, the terminus region as predicted by a shift in GC skew is diametrically opposed to the origin of replication (Figure 1) leading to replicores of approximately equal length. However, the CHR2 replicon is clearly divided in two unequal replicores. The reason for this asymmetry is unclear but may be related to genome dynamics since the *C. metallidurans* CH34 genome harbours numerous Mobile Genetic Elements (MGEs) including many Insertion Sequence (IS) elements, transposons, and Genomic Islands (CMGIs). We further discuss details of the *C. metallidurans* CH34 mobilome in separate subsections.

## Phylogeny and genome comparative analyses

Phylogenetic analyses based on 16S rRNA sequences placed *Cupriavidus metallidurans* CH34 (formerly *Ralstonia* and at one point transferred to *Wautersia*; [34]) firmly into the genus *Cupriavidus*, with its closest relative *C. pauculus* [33,52]. The complete genome sequences of three other *Cupriavidus* species have been published (Table 2): The pollutant degrading *R. eutropha* strain JMP134 (reclassified as *C. pinatubonensis* by Sato *et al.* [52]); the lithoautotrophic “Knallgass” bacterium *C. necator* strain H16 (with the proposed name *C. eutrophus* H16, Vandamme & Coenye, pers. com.); and the beta-rhizobial nitrogen-fixing plant symbiont *C.*

<table><caption>**Table 2.** Genome organisation of representative species (discussed in this contribution) within the order of *Burkholderiales*.</caption><thead><tr><th>Organism<sup>a,b</sup></th><th>Main property</th><th>Family</th><th>Principle habitat</th><th>CHR1</th><th>CHR2</th><th>Replicon 3<sup>c</sup></th><th>Replicon 4</th><th>Year/Ref.</th></tr></thead><tbody><tr><td><i>C. metallidurans</i>* CH34</td><td>heavy metal resistance</td><td><i>Burkholderiaceae</i></td><td>heavy metal-rich sediments and soils</td><td>3.93</td><td>2.58</td><td>0.17 (pMOL28)</td><td>0.23 (pMOL30)</td><td>This report</td></tr><tr><td><i>C. pinatubonensis</i> JMP134</td><td>degrader of xenobiotics</td><td><i>Burkholderiaceae</i></td><td>soil, aquatic; volcanic mudflow deposits</td><td>3.81</td><td>2.74</td><td>0.63 (MPL)</td><td>0.09 (pJP4)</td><td>unpublished</td></tr><tr><td><i>C. eutrophus</i> H16</td><td>hydrogen oxidation, PHA<sup>a</sup> metabolism</td><td><i>Burkholderiaceae</i></td><td>soil, freshwater</td><td>4.05</td><td>2.91</td><td>0.45 (pHG1)</td><td>-</td><td>2006/[45]</td></tr><tr><td><i>C. taiwanensis</i></td><td>N<sub>2</sub>-fixing legume symbiont</td><td><i>Burkholderiaceae</i></td><td>plant root nodules</td><td>3.42</td><td>2.50</td><td>0.56 (pRALTA)</td><td>-</td><td>2008/[46]</td></tr><tr><td><i>R. solanacearum</i> GMI1000</td><td>plant pathogen with wide host range</td><td><i>Burkholderiaceae</i></td><td>soil, rhizosphere</td><td>3.72</td><td>2.09</td><td>-</td><td>-</td><td>2002/[223]</td></tr><tr><td><i>B. xenovorans</i> LB400</td><td>PCB<sup>2</sup> degrader</td><td><i>Burkholderiaceae</i></td><td>rhizosphere, polluted soils, landfills</td><td>4.90</td><td>3.36</td><td>1.47 (MPL)</td><td>-</td><td>2006/[224]</td></tr><tr><td><i>D. acidovorans</i>* SPH-1</td><td>organotrophy, LAS<sup>3</sup> degrader</td><td><i>Comamonadaceae</i></td><td>soil, aquatic, sewage</td><td>6.77</td><td>-</td><td>-</td><td>-</td><td>unpublished</td></tr><tr><td><i>B. petrii</i>* Se-1111R</td><td>degrader of aromatic compounds</td><td><i>Alcaligenaceae</i></td><td>river sediments, TCB<sup>4</sup> contaminated soils</td><td>5.29</td><td>-</td><td>-</td><td>-</td><td>2008/[148]</td></tr><tr><td><i>Janthinobacterium sp.</i></td><td>heavy metal resistance, small sized</td><td><i>Oxalobacteraceae</i></td><td>aquatic, 0.22 µm filtered water</td><td>4.11</td><td>-</td><td>-</td><td>-</td><td>2007/[225]</td></tr><tr><td><i>H. arsenicoxydans</i></td><td>arsenic oxidation and scavenging</td><td><i>Oxalobacteraceae</i></td><td>activated sludge, industrial waste water</td><td>3.42</td><td>-</td><td>-</td><td>-</td><td>2007/[226]</td></tr><tr><td><i>P. necessarius</i> STIR1</td><td>obligate intracellular endosymbiont</td><td><i>Burkholderiaceae</i></td><td>freshwater ciliates</td><td>1.56</td><td>-</td><td>-</td><td>-</td><td>unpublished</td></tr></tbody></table>

<figure><img src="image_9.png" /></figure>

<footnote><sup>a</sup>species that are rarely recovered from clinical samples are indicated with an asterisk (*);</footnote>

<footnote><sup>b</sup>only the propensity for which the species and/or particular strain is best known is given;</footnote>

<footnote><sup>c</sup>MPL, megaplasmid.</footnote>

<footnote n="1"><sup>1</sup>PHA, poly(3-hydroxybutyric acid) (cfr. 'bioplastic');</footnote>

<footnote n="2"><sup>2</sup>PCB, polychlorinated biphenyl (cfr. 'herbicide');</footnote>

<footnote n="3"><sup>3</sup>LAS, linear alkylbenzene sulfonate (cfr. 'laundry surfactant');</footnote>

<footnote n="4"><sup>4</sup>TCB, trichlorobenzene.</footnote>

<footnote>doi:10.1371/journal.pone.0010433.t002</footnote>
removal and oxidative stress (*yhjA*, *ahpC*, *ahpD*, *katG*, *pcaCD*, *soxR*, *glrX*), DNA adenine methylation (*dam*), DNA repair (*ada*, *alkA*, *alkB1*, *alkB2*, *sbcCD*, *ogt2*, *ogt3*), adaptive mutagenesis (*imuA-imuB-dnaE2*, *Rmet_4128-29-30*), and eight copies of the *uspA* gene (two of which are located on transposons) encoding the universal stress protein [most genes and loci without Rmet numbers are also discussed in separate sections]. The CHR2 replicon also carries an intact master two-component phosphorelay system (*bvgS*A) that, in *Bordetella* species, controls hundreds of genes involved in an extensive variety of responses and physiological functions, including virulence, biofilm formation, chemotaxis, and motility [50].

Both chromosomes display clear GC skew transitions that likely correspond with their replication origin and terminus [51] (Figure 1). For CHR1 the GC skew transition region (at point 3.84 on the map, Figure 1A) is about 90 kb away from the typical *dnaA-dnaN-gyrB* layout (genes *Rmet_0001*, *Rmet_0002*, and *Rmet_0003*, with *dnaA* starting at position 104) although the 400 bp upstream region of *dnaA* contains 1 perfect DnaA-binding box (TTATCCACA) and 3 putative DnaA boxes with one or two mismatches to this consensus (Figure S1A). Interestingly, the GC skew transition region located around coordinate 3,837,500 is preceded immediately by a short region of 213 nt encompassing 3 perfect DnaA boxes. Four more putative DnaA boxes (tightly clustered in a 65 nt region and each with one mismatch to the consensus) occur even further upstream of *dnaA*, at coordinates 3,707,588–3,707,653 (Figure S1A). For CHR2 the switch in GC skew is situated around the *repA-parA-parB* genes (*Rmet_5817*, *Rmet_5819*, and *Rmet_5820*) (point 2.43 of the map, Figure 1B)

indicating that CHR2 partitioning may be plasmid-like. The *repA* upstream region contains 3 putative DnaA boxes with only one mismatch to the consensus, at locations –392, –419, and –1080 with respect to the startcodon of *repA* (Figure S1B). This region also contains at least 12 repetitive 17 nt-long elements with a highly conserved motif (consensus CGCAGAA(A/T)(C/T)(A/G)GGTACG(C/T)) that may serve as RepA binding sites. In CHR1, the terminus region as predicted by a shift in GC skew is diametrically opposed to the origin of replication (Figure 1) leading to replicores of approximately equal length. However, the CHR2 replicon is clearly divided in two unequal replicores. The reason for this asymmetry is unclear but may be related to genome dynamics since the *C. metallidurans* CH34 genome harbours numerous Mobile Genetic Elements (MGEs) including many Insertion Sequence (IS) elements, transposons, and Genomic Islands (CMGIs). We further discuss details of the *C. metallidurans* CH34 mobilome in separate subsections.

## Phylogeny and genome comparative analyses

Phylogenetic analyses based on 16S rRNA sequences placed *Cupriavidus metallidurans* CH34 (formerly *Ralstonia* and at one point transferred to *Wautersia*; [34]) firmly into the genus *Cupriavidus*, with its closest relative *C. pauculus* [33,52]. The complete genome sequences of three other *Cupriavidus* species have been published (Table 2): The pollutant degrading *R. eutropha* strain JMP134 (reclassified as *C. pinatubonensis* by Sato *et al.* [52]); the lithoautotrophic “Knallgass” bacterium *C. necator* strain H16 (with the proposed name *C. eutrophus* H16, Vandamme & Coenye, pers. com.); and the beta-rhizobial nitrogen-fixing plant symbiont *C.*

<table><caption>**Table 2.** Genome organisation of representative species (discussed in this contribution) within the order of *Burkholderiales*.</caption><thead><tr><th>Organism<sup>a,b</sup></th><th>Main property</th><th>Family</th><th>Principle habitat</th><th>CHR1</th><th>CHR2</th><th>Replicon 3<sup>c</sup></th><th>Replicon 4</th><th>Year/Ref.</th></tr></thead><tbody><tr><td><i>C. metallidurans</i>* CH34</td><td>heavy metal resistance</td><td><i>Burkholderiaceae</i></td><td>heavy metal-rich sediments and soils</td><td>3.93</td><td>2.58</td><td>0.17 (pMOL28)</td><td>0.23 (pMOL30)</td><td>This report</td></tr><tr><td><i>C. pinatubonensis</i> JMP134</td><td>degrader of xenobiotics</td><td><i>Burkholderiaceae</i></td><td>soil, aquatic; volcanic mudflow deposits</td><td>3.81</td><td>2.74</td><td>0.63 (MPL)</td><td>0.09 (pJP4)</td><td>unpublished</td></tr><tr><td><i>C. eutrophus</i> H16</td><td>hydrogen oxidation, PHA<sup>a</sup> metabolism</td><td><i>Burkholderiaceae</i></td><td>soil, freshwater</td><td>4.05</td><td>2.91</td><td>0.45 (pHG1)</td><td>-</td><td>2006/[45]</td></tr><tr><td><i>C. taiwanensis</i></td><td>N<sub>2</sub>-fixing legume symbiont</td><td><i>Burkholderiaceae</i></td><td>plant root nodules</td><td>3.42</td><td>2.50</td><td>0.56 (pRALTA)</td><td>-</td><td>2008/[46]</td></tr><tr><td><i>R. solanacearum</i> GMI1000</td><td>plant pathogen with wide host range</td><td><i>Burkholderiaceae</i></td><td>soil, rhizosphere</td><td>3.72</td><td>2.09</td><td>-</td><td>-</td><td>2002/[223]</td></tr><tr><td><i>B. xenovorans</i> LB400</td><td>PCB<sup>2</sup> degrader</td><td><i>Burkholderiaceae</i></td><td>rhizosphere, polluted soils, landfills</td><td>4.90</td><td>3.36</td><td>1.47 (MPL)</td><td>-</td><td>2006/[224]</td></tr><tr><td><i>D. acidovorans</i>* SPH-1</td><td>organotrophy, LAS<sup>3</sup> degrader</td><td><i>Comamonadaceae</i></td><td>soil, aquatic, sewage</td><td>6.77</td><td>-</td><td>-</td><td>-</td><td>unpublished</td></tr><tr><td><i>B. petrii</i>* Se-1111R</td><td>degrader of aromatic compounds</td><td><i>Alcaligenaceae</i></td><td>river sediments, TCB<sup>4</sup> contaminated soils</td><td>5.29</td><td>-</td><td>-</td><td>-</td><td>2008/[148]</td></tr><tr><td><i>Janthinobacterium sp.</i></td><td>heavy metal resistance, small sized</td><td><i>Oxalobacteraceae</i></td><td>aquatic, 0.22 µm filtered water</td><td>4.11</td><td>-</td><td>-</td><td>-</td><td>2007/[225]</td></tr><tr><td><i>H. arsenicoxydans</i></td><td>arsenic oxidation and scavenging</td><td><i>Oxalobacteraceae</i></td><td>activated sludge, industrial waste water</td><td>3.42</td><td>-</td><td>-</td><td>-</td><td>2007/[226]</td></tr><tr><td><i>P. necessarius</i> STIR1</td><td>obligate intracellular endosymbiont</td><td><i>Burkholderiaceae</i></td><td>freshwater ciliates</td><td>1.56</td><td>-</td><td>-</td><td>-</td><td>unpublished</td></tr></tbody></table>

<figure><img src="image_9.png" /></figure>

<footnote><sup>a</sup>species that are rarely recovered from clinical samples are indicated with an asterisk (*);</footnote>

<footnote><sup>b</sup>only the propensity for which the species and/or particular strain is best known is given;</footnote>

<footnote><sup>c</sup>MPL, megaplasmid.</footnote>

<footnote n="1"><sup>1</sup>PHA, poly(3-hydroxybutyric acid) (cfr. 'bioplastic');</footnote>

<footnote n="2"><sup>2</sup>PCB, polychlorinated biphenyl (cfr. 'herbicide');</footnote>

<footnote n="3"><sup>3</sup>LAS, linear alkylbenzene sulfonate (cfr. 'laundry surfactant');</footnote>

<footnote n="4"><sup>4</sup>TCB, trichlorobenzene.</footnote>

<footnote>doi:10.1371/journal.pone.0010433.t002</footnote>
*taiwanensis*. Throughout this paper we will often refer to these genomes as Cmet (for *C. metallidurans*), Cpin (for *C. pinatubonensis* JMP134), Ceut (for *C. eutrophus* H16) and Ctai (for *C. taiwanensis*).

The genome organisation of these four species is very similar. All four carry two large replicons, that we further nominated as CHR1 and CHR2, with each replicon being of comparable size to their counterparts. The main difference in terms of genome organisation stems from the presence of plasmids (Table 2). Whereas Cmet harbours two megaplasms of 171- and 233-kb, Ceut and Ctai carry a single megaplasmid, respectively, of 452 kb and 557 kb. The Cpin genome includes a small plasmid of 88 kb (pJP4) along with a megaplasmid of 635 kb.

We determined orthology between all those replicons by analysing the reciprocal best BlastP hits, and then performed cluster analysis using calculated distance scores (see Methods). Our analysis also encompassed representative genomes that, according to the MaGe system, display a well conserved gene order (synteny) to *C. metallidurans* (measuring synteny to the CHR1 replicon). These were *Ralstonia solanacearum* GMI1000 (two large replicons), *Burkholderia xenovorans* LB400 (two large replicons and a plasmid), and *Bordetella petrii* DSM12804, *Delftia acidovorans* SPH-1, *Janthino-bacterium* sp. Marseille, and *Herminiimonas arsenicoxydans* (all having a single replicon). We further included *Polynucleobacter necessarius* STIR1 (a single replicon with surprisingly good synteny to Cmet CHR1) to represent, next to the genera *Ralstonia*, *Cupriavidus*, and *Burkholderia*, the fourth genus in the family of *Burkholderiaceae* [53]. Table 2 gives general information on all those species. Lastly, we employed the genome of *Escherichia coli* K-12 in this analysis as a reference and outlier.

Considering only the four *Cupriavidus* species, some interesting observations were made. First, the core number of genes common to all four species clearly is larger for the first replicon with 2,209 genes for CHR1 compared to 677 genes for CHR2 (Figure 3): calculated against the average number of genes present on each replicon, this amounts to 62% and 28%, respectively. This observation, together with a strong bias for essential genes, as we noted for Cmet, and also seen for Ceut and Ctai [45,46], indicates

that CHR1 constitutes the ancestral replicon. The smaller replicon CHR2 probably was acquired as a plasmid during the evolution of *Cupriavidus* and gradually evolved into a second chromosome through genetransfer from either CHR1 or external sources [54]. This idea may explain why CHR2 carries features typical for plasmid-type partitioning, as well as its unique core cellular functions. Here, the delegation of essential genes from the primary replicon to a second replicon would ensure the maintenance of all genes on that replicon, a strategy seemingly common in multipartite prokaryotic genomes [55]. Second, *C. taiwanensis* (Ctai) and *C. eutrophus* H16 (Ceut) have more genes in common (Figure 3) i.e., are more closely related than is the case for the other *Cupriavidus* species, confirming the findings of Amadou *et al*. [46]. And third, both large replicons of Cmet harbour more species-specific genes than do the large replicons of the other *Cupriavidus* genomes (Figure 3).

Additional patterns emerge from the similarity matrix (Figure S3) holding protein-based orthology data for these genomes. As expected, the CHR1 replicons of the four *Cupriavidus* species group tightly together. This is also the case for the *Cupriavidus* CHR2 replicons. For both replicons, the highest orthology is between Ctai and Ceut, and the ranking of the species according to orthology in this cluster remains the same for both replicons, with Cmet displaying the least orthology. There is a clear division among all analysed replicons into two large clusters A and B (Figure S3). Cluster A contains the CHR1 group and the main replicons of *B. xenovorans* and *R. solanacearum*, along with the chromosomes of *D. acidovorans*, *B. petrii*, *H. arsenicoxydans*, *Janthino-bacterium*, and *P. necessarius* (with the *E. coli* chromosome inside this cluster but clearly an outlier). Cluster B consists of the CHR2 group together with the *C. pinatubonensis*, *B. xenovorans* and *R. solanacearum* megaplasms and the *B. xenovorans* second chromosome. The grouping of the remaining *Cupriavidus* replicons (all plasmids) from Blast analyses is less clear, although pMOL28 (Cmet), pRALTA (Ctai), and pHG1 (Ceut) appear to be somewhat more related (cluster C) to each other than is the case for pMOL30 (Cmet) and pJP4 (Cpin). Probably, this reflects the fact that the

<figure><img src="image_10.png" /></figure>

**Figure 3. Shared and unique proteins of Cupriavidus chromosomes.** 4-way comparison Venn diagram illustrating the intersection and differences between chromosomal replicons of completed *Cupriavidus* genomes. Intersections show the number of shared proteins between two or more organisms based on reciprocal best BLAST hits (see Methods). Numbers in parentheses depict the missing overlap sectors due to circular drawing and represent proteins shared between opposite genomes but absent in the other two genomes. Abbreviations: Cmet, *C. metallidurans* CH34; Cpin, *C. pinatubonensis* JMP134; Ceut, *C. eutrophus*; Ctai, *C. taiwanensis*. doi:10.1371/journal.pone.0010433.g003

<figure><img src="image_11.png" /></figure>
*taiwanensis*. Throughout this paper we will often refer to these genomes as Cmet (for *C. metallidurans*), Cpin (for *C. pinatubonensis* JMP134), Ceut (for *C. eutrophus* H16) and Ctai (for *C. taiwanensis*).

The genome organisation of these four species is very similar. All four carry two large replicons, that we further nominated as CHR1 and CHR2, with each replicon being of comparable size to their counterparts. The main difference in terms of genome organisation stems from the presence of plasmids (Table 2). Whereas Cmet harbours two megaplasms of 171- and 233-kb, Ceut and Ctai carry a single megaplasmid, respectively, of 452 kb and 557 kb. The Cpin genome includes a small plasmid of 88 kb (pJP4) along with a megaplasmid of 635 kb.

We determined orthology between all those replicons by analysing the reciprocal best BlastP hits, and then performed cluster analysis using calculated distance scores (see Methods). Our analysis also encompassed representative genomes that, according to the MaGe system, display a well conserved gene order (synteny) to *C. metallidurans* (measuring synteny to the CHR1 replicon). These were *Ralstonia solanacearum* GMI1000 (two large replicons), *Burkholderia xenovorans* LB400 (two large replicons and a plasmid), and *Bordetella petrii* DSM12804, *Delftia acidovorans* SPH-1, *Janthino-bacterium* sp. Marseille, and *Herminiimonas arsenicoxydans* (all having a single replicon). We further included *Polynucleobacter necessarius* STIR1 (a single replicon with surprisingly good synteny to Cmet CHR1) to represent, next to the genera *Ralstonia*, *Cupriavidus*, and *Burkholderia*, the fourth genus in the family of *Burkholderiaceae* [53]. Table 2 gives general information on all those species. Lastly, we employed the genome of *Escherichia coli* K-12 in this analysis as a reference and outlier.

Considering only the four *Cupriavidus* species, some interesting observations were made. First, the core number of genes common to all four species clearly is larger for the first replicon with 2,209 genes for CHR1 compared to 677 genes for CHR2 (Figure 3): calculated against the average number of genes present on each replicon, this amounts to 62% and 28%, respectively. This observation, together with a strong bias for essential genes, as we noted for Cmet, and also seen for Ceut and Ctai [45,46], indicates

that CHR1 constitutes the ancestral replicon. The smaller replicon CHR2 probably was acquired as a plasmid during the evolution of *Cupriavidus* and gradually evolved into a second chromosome through genetransfer from either CHR1 or external sources [54]. This idea may explain why CHR2 carries features typical for plasmid-type partitioning, as well as its unique core cellular functions. Here, the delegation of essential genes from the primary replicon to a second replicon would ensure the maintenance of all genes on that replicon, a strategy seemingly common in multipartite prokaryotic genomes [55]. Second, *C. taiwanensis* (Ctai) and *C. eutrophus* H16 (Ceut) have more genes in common (Figure 3) i.e., are more closely related than is the case for the other *Cupriavidus* species, confirming the findings of Amadou *et al*. [46]. And third, both large replicons of Cmet harbour more species-specific genes than do the large replicons of the other *Cupriavidus* genomes (Figure 3).

Additional patterns emerge from the similarity matrix (Figure S3) holding protein-based orthology data for these genomes. As expected, the CHR1 replicons of the four *Cupriavidus* species group tightly together. This is also the case for the *Cupriavidus* CHR2 replicons. For both replicons, the highest orthology is between Ctai and Ceut, and the ranking of the species according to orthology in this cluster remains the same for both replicons, with Cmet displaying the least orthology. There is a clear division among all analysed replicons into two large clusters A and B (Figure S3). Cluster A contains the CHR1 group and the main replicons of *B. xenovorans* and *R. solanacearum*, along with the chromosomes of *D. acidovorans*, *B. petrii*, *H. arsenicoxydans*, *Janthino-bacterium*, and *P. necessarius* (with the *E. coli* chromosome inside this cluster but clearly an outlier). Cluster B consists of the CHR2 group together with the *C. pinatubonensis*, *B. xenovorans* and *R. solanacearum* megaplasms and the *B. xenovorans* second chromosome. The grouping of the remaining *Cupriavidus* replicons (all plasmids) from Blast analyses is less clear, although pMOL28 (Cmet), pRALTA (Ctai), and pHG1 (Ceut) appear to be somewhat more related (cluster C) to each other than is the case for pMOL30 (Cmet) and pJP4 (Cpin). Probably, this reflects the fact that the

<figure><img src="image_10.png" /></figure>

**Figure 3. Shared and unique proteins of Cupriavidus chromosomes.** 4-way comparison Venn diagram illustrating the intersection and differences between chromosomal replicons of completed *Cupriavidus* genomes. Intersections show the number of shared proteins between two or more organisms based on reciprocal best BLAST hits (see Methods). Numbers in parentheses depict the missing overlap sectors due to circular drawing and represent proteins shared between opposite genomes but absent in the other two genomes. Abbreviations: Cmet, *C. metallidurans* CH34; Cpin, *C. pinatubonensis* JMP134; Ceut, *C. eutrophus*; Ctai, *C. taiwanensis*. doi:10.1371/journal.pone.0010433.g003

<figure><img src="image_11.png" /></figure>
three plasmids in cluster C have the same backbone, i.e., contain the essential genes for plasmid replication and maintenance ([7] and see further). Interestingly, the genome of *P. necessarius* STIR1, an obligate intracellular symbiont of the freshwater ciliate *Euplotes aediculatus*, not only clusters well with the CHR1 group but does so with nearly two thirds (63% on average) of its proteins having an ortholog in any of the other members of this group (in contrast to only 40% of its proteins having an ortholog in the outlier *E. coli*). This high orthology may denote genomic reduction in *P. necessarius* STIR1, in which mainly essential genes were preserved, and is reflected in a significant degree of gene synteny between the *P. necessarius* STIR1 genome and CHR1 of Cmet but much less with CHR2 (Figure S4A). This high synteny with CHR1 was fully supported by MUMmer3 analysis on protein level using the PROmer module (Figure S4B).

Full length alignment of the main replicons of the four *Cupriavidus* genomes and the closely related *R. solanacearum* genome clearly suggests a common ancestral architecture for CHR1 (Figure 4): overall homology between their respective CHR2 replicons is much lower (Figure S2) probably owing to different evolutionary histories. Such multiple alignments also reveal a large number of DNA rearrangements in the CH34 genome compared to the other analysed genomes (and vice versa), including some large inversions, and multiple sequence duplications causing significant paralogy in both CH34 replicons (discussed further). Pairwise synteny analysis using the LinePlot tool embedded in the MaGe interface fully supported these results (data not shown) while limited synteny was observed between CHR1 of *C. metallidurans* and the single replicons of the other organisms listed

in Table 2. The intragenomic synteny between the CHR1 and CHR2 replicons of *C. metallidurans* was not very apparent. However, choosing a small synton size in the LinePlot analysis tool (with S≤5 e.g considering five or less genes for each synteny group), highlighted a myriad of sequence matches (data not shown). This type of local synteny may be partly explained by evolutionary DNA exchange between the two replicons or by parallel insertion of homologous sequences of incoming DNA.

## Paralogy and protein families

Gene duplications (paralogs) are seen as evolutionary events contributing to the innovative drive of organisms in order to adapt to changing environments. We measured paralogy within the *C. metallidurans* genome at the protein level using the GeneRAGE algorithm that facilitates clustering of large protein sequence sets into families according to their sequence similarity [56]. To allow GeneRage to analyse genome parology, we undertook a basic similarity search by an all-to-all BLAST analysis of all *C. metallidurans* CH34 proteins against each other. Smith-Waterman dynamic programming (see Methods) automatically removed ambiguities in the resulting similarity matrix, assuring the method's high accuracy. Multi-domain proteins detected in the genome were disregarded. Half of the remaining proteins (3,311 or 52%) had one or more paralog, while the other half (3,008 or 48%) remained singletons having no match with any of the other proteins in the genome. Remarkably, 1,358 proteins (21%) grouped together into 43 clusters of 10 or more members, while 4 clusters held more than 110 members. These large paralogous families represented proteins involved in two-component signal

<figure><img src="image_12.png" /><figcaption>**Figure 4.** Comparative DNA analysis of *Cupriavidus* chromosomes. Nucleotide based comparison of the CHR1 replicons of the four *Cupriavidus* species (abbreviations as in Figure 3) and *R. solanacearum* GMI1000 (denoted as Rsol) using the anchor-allignment software Murasaki (http://murasaki.dna.bio.keio.ac.jp/) [218]. Scale in Mb is shown on top. doi:10.1371/journal.pone.0010433.g004</figcaption></figure>

<figure><img src="image_13.png" /></figure>
three plasmids in cluster C have the same backbone, i.e., contain the essential genes for plasmid replication and maintenance ([7] and see further). Interestingly, the genome of *P. necessarius* STIR1, an obligate intracellular symbiont of the freshwater ciliate *Euplotes aediculatus*, not only clusters well with the CHR1 group but does so with nearly two thirds (63% on average) of its proteins having an ortholog in any of the other members of this group (in contrast to only 40% of its proteins having an ortholog in the outlier *E. coli*). This high orthology may denote genomic reduction in *P. necessarius* STIR1, in which mainly essential genes were preserved, and is reflected in a significant degree of gene synteny between the *P. necessarius* STIR1 genome and CHR1 of Cmet but much less with CHR2 (Figure S4A). This high synteny with CHR1 was fully supported by MUMmer3 analysis on protein level using the PROmer module (Figure S4B).

Full length alignment of the main replicons of the four *Cupriavidus* genomes and the closely related *R. solanacearum* genome clearly suggests a common ancestral architecture for CHR1 (Figure 4): overall homology between their respective CHR2 replicons is much lower (Figure S2) probably owing to different evolutionary histories. Such multiple alignments also reveal a large number of DNA rearrangements in the CH34 genome compared to the other analysed genomes (and vice versa), including some large inversions, and multiple sequence duplications causing significant paralogy in both CH34 replicons (discussed further). Pairwise synteny analysis using the LinePlot tool embedded in the MaGe interface fully supported these results (data not shown) while limited synteny was observed between CHR1 of *C. metallidurans* and the single replicons of the other organisms listed

in Table 2. The intragenomic synteny between the CHR1 and CHR2 replicons of *C. metallidurans* was not very apparent. However, choosing a small synton size in the LinePlot analysis tool (with S≤5 e.g considering five or less genes for each synteny group), highlighted a myriad of sequence matches (data not shown). This type of local synteny may be partly explained by evolutionary DNA exchange between the two replicons or by parallel insertion of homologous sequences of incoming DNA.

## Paralogy and protein families

Gene duplications (paralogs) are seen as evolutionary events contributing to the innovative drive of organisms in order to adapt to changing environments. We measured paralogy within the *C. metallidurans* genome at the protein level using the GeneRAGE algorithm that facilitates clustering of large protein sequence sets into families according to their sequence similarity [56]. To allow GeneRage to analyse genome parology, we undertook a basic similarity search by an all-to-all BLAST analysis of all *C. metallidurans* CH34 proteins against each other. Smith-Waterman dynamic programming (see Methods) automatically removed ambiguities in the resulting similarity matrix, assuring the method's high accuracy. Multi-domain proteins detected in the genome were disregarded. Half of the remaining proteins (3,311 or 52%) had one or more paralog, while the other half (3,008 or 48%) remained singletons having no match with any of the other proteins in the genome. Remarkably, 1,358 proteins (21%) grouped together into 43 clusters of 10 or more members, while 4 clusters held more than 110 members. These large paralogous families represented proteins involved in two-component signal

<figure><img src="image_12.png" /><figcaption>**Figure 4.** Comparative DNA analysis of *Cupriavidus* chromosomes. Nucleotide based comparison of the CHR1 replicons of the four *Cupriavidus* species (abbreviations as in Figure 3) and *R. solanacearum* GMI1000 (denoted as Rsol) using the anchor-allignment software Murasaki (http://murasaki.dna.bio.keio.ac.jp/) [218]. Scale in Mb is shown on top. doi:10.1371/journal.pone.0010433.g004</figcaption></figure>

<figure><img src="image_13.png" /></figure>
transduction (n = 138) (predominantly containing GGDEF, EAL, PAS, and HisKA domains), extra-cytoplasmic solute receptors of the tripartite tricarboxylate transporter (TTT) family (n = 123), LysR-type transcriptional regulators (n = 121), and ABC-transporter related proteins (n = 110). Other large families (size > 30) comprised proteins involved in membrane structure and transport (membrane fusion proteins, porins, and major facilitator super-family transporters), or enzymes with general metabolic functions (short-chain dehydrogenases, enoyl-CoA reductases, L-carnitine dehydratases, acyl-CoA dehydrogenases, and acyl-CoA synthetases). These findings are in line with those of Gevers *et al.* [57] who studied paralogy in 106 bacterial genomes, reporting a significant paralog enrichment in functional classes involved in cellular defense mechanisms, metabolism, and transcriptional responses. These authors broadly correlated the number of paralogs in the genomes with the organism's lifestyle, with paranome sizes of 7% for intracellular organisms, such as *Rickettsia conorii*, and up to 41% for free-living highly versatile soil bacteria, such as *Streptomyces coelicolor*. Accordingly, it is not surprising that the survivalist *C. metallidurans* possesses a large, functionally biased paranome. In exploring the smaller clusters holding one- to five-paralogs (656 clusters with a total of 1,724 proteins), it is difficult to discern a functional bias or propose a plausible explanation for paralogy. Many of them contain conserved hypothetical proteins, or proteins similar in function but differing in substrate or cellular roles. Others contain cellular- or transcriptional-regulators specifically directed to heavy-metal resistance (see also Nies *et al.* [58]) or encompass functions related to mobile genetic elements. A quick survey points to several interesting findings. For instance, apparently, there are no less than six copies of 2-nitropropane dioxygenase that catalyzes the enzymatic reaction 2-nitropropane + oxygen <-> acetone + nitrite. This could be an important finding in view of the known acetone tolerance of *C. metallidurans* CH34 (see further in this discussion), or in terms of converting environmental nitroalkanes for bioremediation purposes. Likewise, there are three copies of butyryl-CoA:acetate-CoA transferase, an enzyme that may be involved in acetate detoxification or instrumental in delivering butyrate to produce polyhydroxybuty rates (PHBs), biopolymers that are stored as an energy reserve in the cell, and which have industrial applications as bioplastics (see further, section on polyester synthesis PHA/PHB). Undoubtedly, it will be exciting to follow these leads but a systematic functional breakdown of these clusters is beyond the scope of this report.

## Mobile genetic elements

A striking feature of the *C. metallidurans* CH34 genome is the presence of at least 16 laterally acquired genomic islands and the abundance of transposase- and recombinase-related gene sequences across the four replicons (Table 1). Using Van Houdt *et al*'s criteria [59], 11 GIs were identified on CHR1 but none on CHR2. Defining GIs on CHR2 is not straightforward since its structure is very different from that of CHR1 and it appears to consist of a complicated patchwork of gene clusters and subclusters generated by multiple integration events with the subsequent retention (and occasional loss) of acquired genes. Further, the CHR2 replicon contains fewer tRNA genes (i.e. only eight compared to 54 in CHR1) and only very few *xerC/D*-like or *int*-like recombinase genes. Previously identified GIs of the plasmids pMOL28 and pMOL30, respectively referred to as CMGI-28a, -28b, -28c, and CMGI-30a and -30b, respectively, all carry heavy-metal response genes; discussed in detail elsewhere [7,10] and in the next subsection on megaplasmids.

The largest island CMGI-1 is 109 kb and is almost identical (>99.95% DNA sequence similarity) to the PAGI-2 island in

*Pseudomonas aeruginosa* clone C, and to the highly conserved PAGI-2C-type islands from many other isolates of this species. It also corresponds to several *C. metallidurans* and *Cupriavidus campinensis* strains isolated from environments polluted with sewage and heavy metals [60,61]. The observation that clinical strains of nosocomial infections and environmental strains isolated from industrial sites have such a large segment of genetic material in common is worrying since it suggests a recent breach of the natural and artificial barriers between (industrial) environmental reservoirs and clinical environments. Clearly, *P. aeruginosa* has expanded its genomic repertoire to survive in a wide range of different environments, including the human host, in which lateral gene acquisition might have been enhanced by co-inhabitation and by genetic relatedness [62]. In this respect there is a certain resemblance between the evolutionary forces shaping the genomes of *P. aeruginosa* and *C. metallidurans*, as the latter also acquired and retained many new genetic elements, and consequently new traits, enabling it to survive in many different, often harsh, environments. Moreover, two *C. metallidurans* strains identified by 16S rDNA sequence analysis were recently isolated from the respiratory tract of cystic fibrosis patients [63] warranting concern about the disease-causing potential of *C. metallidurans*.

Three of the *C. metallidurans* GIs (CMGI-2, CMGI-3, and CMGI-4) belong to the Tn4371 family, first described in strain *C. oxalaticus* A5 (formerly *R. eutropha* A5) [64–66]. This family is defined by a quadripartite structure consisting of an integrase module, a region with plasmid/phage/GI maintenance genes, the accessory genes, and the conjugative genes [59] and was recently revisited to describe genomic islands of other environmental bacteria [67].

The 101 kb large CMGI-2 contains at least 25 genes involved in degrading toluene corresponding to CH34's ability to grow with toluene, benzene, or xylene as their sole carbon source (see section on organoautotrophy and degradation of aromatics). Apparently, additional accessory genes are inserted near this catabolic cluster, including genes involved in chemolithotrophy (e.g. *hyp* and *hox* genes – see section on hydrogenotrophy).

The 97 kb CMGI-3 island also shelters accessory genes in chemolithotrophy organized as two gene cassettes flanked by IS1071: one contains the genes for the fixation of CO₂ (*cbb* genes; described in the section on autotrophic metabolism) and the other encompassing additional genes for hydrogenotrophy (*hyp* and *hox* genes). All the genes allowing *C. metallidurans* to grow aerobically at the expense of CO₂ and H₂ are confined to CMGI-2 and CMGI-3 in CHR1: this differs in the closely related H₂-oxidising chemolithotrophic bacterium *C. eutrophus* H16 wherein the *hox* and *hyp* genes are situated on a 452 kb megaplasmid, pHG1 [45,68]. It is unclear whether strains H16 and CH34 inherited these homologous sets of *hyp* and *hox* genes from a common ancestor through vertical descent, or whether they were passed on by lateral gene transfer. In contrast, the *cbb* loci (for CO₂ fixation) in both strains clearly have different origins since the CH34 *cbb* genes are phylogenetically much more closely related to the *cbb* homologues of photosynthetic- and nitrifying-organisms [45].

The 56 kb CMGI-4 island has a mosaic structure and lacks the conjugative module. Instead, its extremity carries genes involved in the biosynthesis of methionine, and the degradation of phospho-nates; the transposon Tn6048 also occurs there. The DNA sequence of the CMGI-4 island is highly similar to a region in the *D. acidovorans* SPH-1 genome [69–71] except that the latter does not contain Tn6048 but carries a complete Tn4371-like structure flanked by Tn6051 [59].

The contribution of the remaining GIs (between 11- and 24-kb in size) towards the particular lifestyle of *C. metallidurans* is unclear.

<figure><img src="image_14.png" /></figure>
transduction (n = 138) (predominantly containing GGDEF, EAL, PAS, and HisKA domains), extra-cytoplasmic solute receptors of the tripartite tricarboxylate transporter (TTT) family (n = 123), LysR-type transcriptional regulators (n = 121), and ABC-transporter related proteins (n = 110). Other large families (size > 30) comprised proteins involved in membrane structure and transport (membrane fusion proteins, porins, and major facilitator super-family transporters), or enzymes with general metabolic functions (short-chain dehydrogenases, enoyl-CoA reductases, L-carnitine dehydratases, acyl-CoA dehydrogenases, and acyl-CoA synthetases). These findings are in line with those of Gevers *et al.* [57] who studied paralogy in 106 bacterial genomes, reporting a significant paralog enrichment in functional classes involved in cellular defense mechanisms, metabolism, and transcriptional responses. These authors broadly correlated the number of paralogs in the genomes with the organism's lifestyle, with paranome sizes of 7% for intracellular organisms, such as *Rickettsia conorii*, and up to 41% for free-living highly versatile soil bacteria, such as *Streptomyces coelicolor*. Accordingly, it is not surprising that the survivalist *C. metallidurans* possesses a large, functionally biased paranome. In exploring the smaller clusters holding one- to five-paralogs (656 clusters with a total of 1,724 proteins), it is difficult to discern a functional bias or propose a plausible explanation for paralogy. Many of them contain conserved hypothetical proteins, or proteins similar in function but differing in substrate or cellular roles. Others contain cellular- or transcriptional-regulators specifically directed to heavy-metal resistance (see also Nies *et al.* [58]) or encompass functions related to mobile genetic elements. A quick survey points to several interesting findings. For instance, apparently, there are no less than six copies of 2-nitropropane dioxygenase that catalyzes the enzymatic reaction 2-nitropropane + oxygen <-> acetone + nitrite. This could be an important finding in view of the known acetone tolerance of *C. metallidurans* CH34 (see further in this discussion), or in terms of converting environmental nitroalkanes for bioremediation purposes. Likewise, there are three copies of butyryl-CoA:acetate-CoA transferase, an enzyme that may be involved in acetate detoxification or instrumental in delivering butyrate to produce polyhydroxybuty rates (PHBs), biopolymers that are stored as an energy reserve in the cell, and which have industrial applications as bioplastics (see further, section on polyester synthesis PHA/PHB). Undoubtedly, it will be exciting to follow these leads but a systematic functional breakdown of these clusters is beyond the scope of this report.

## Mobile genetic elements

A striking feature of the *C. metallidurans* CH34 genome is the presence of at least 16 laterally acquired genomic islands and the abundance of transposase- and recombinase-related gene sequences across the four replicons (Table 1). Using Van Houdt *et al*'s criteria [59], 11 GIs were identified on CHR1 but none on CHR2. Defining GIs on CHR2 is not straightforward since its structure is very different from that of CHR1 and it appears to consist of a complicated patchwork of gene clusters and subclusters generated by multiple integration events with the subsequent retention (and occasional loss) of acquired genes. Further, the CHR2 replicon contains fewer tRNA genes (i.e. only eight compared to 54 in CHR1) and only very few *xerC/D*-like or *int*-like recombinase genes. Previously identified GIs of the plasmids pMOL28 and pMOL30, respectively referred to as CMGI-28a, -28b, -28c, and CMGI-30a and -30b, respectively, all carry heavy-metal response genes; discussed in detail elsewhere [7,10] and in the next subsection on megaplasmids.

The largest island CMGI-1 is 109 kb and is almost identical (>99.95% DNA sequence similarity) to the PAGI-2 island in

*Pseudomonas aeruginosa* clone C, and to the highly conserved PAGI-2C-type islands from many other isolates of this species. It also corresponds to several *C. metallidurans* and *Cupriavidus campinensis* strains isolated from environments polluted with sewage and heavy metals [60,61]. The observation that clinical strains of nosocomial infections and environmental strains isolated from industrial sites have such a large segment of genetic material in common is worrying since it suggests a recent breach of the natural and artificial barriers between (industrial) environmental reservoirs and clinical environments. Clearly, *P. aeruginosa* has expanded its genomic repertoire to survive in a wide range of different environments, including the human host, in which lateral gene acquisition might have been enhanced by co-inhabitation and by genetic relatedness [62]. In this respect there is a certain resemblance between the evolutionary forces shaping the genomes of *P. aeruginosa* and *C. metallidurans*, as the latter also acquired and retained many new genetic elements, and consequently new traits, enabling it to survive in many different, often harsh, environments. Moreover, two *C. metallidurans* strains identified by 16S rDNA sequence analysis were recently isolated from the respiratory tract of cystic fibrosis patients [63] warranting concern about the disease-causing potential of *C. metallidurans*.

Three of the *C. metallidurans* GIs (CMGI-2, CMGI-3, and CMGI-4) belong to the Tn4371 family, first described in strain *C. oxalaticus* A5 (formerly *R. eutropha* A5) [64–66]. This family is defined by a quadripartite structure consisting of an integrase module, a region with plasmid/phage/GI maintenance genes, the accessory genes, and the conjugative genes [59] and was recently revisited to describe genomic islands of other environmental bacteria [67].

The 101 kb large CMGI-2 contains at least 25 genes involved in degrading toluene corresponding to CH34's ability to grow with toluene, benzene, or xylene as their sole carbon source (see section on organoautotrophy and degradation of aromatics). Apparently, additional accessory genes are inserted near this catabolic cluster, including genes involved in chemolithotrophy (e.g. *hyp* and *hox* genes – see section on hydrogenotrophy).

The 97 kb CMGI-3 island also shelters accessory genes in chemolithotrophy organized as two gene cassettes flanked by IS1071: one contains the genes for the fixation of CO₂ (*cbb* genes; described in the section on autotrophic metabolism) and the other encompassing additional genes for hydrogenotrophy (*hyp* and *hox* genes). All the genes allowing *C. metallidurans* to grow aerobically at the expense of CO₂ and H₂ are confined to CMGI-2 and CMGI-3 in CHR1: this differs in the closely related H₂-oxidising chemolithotrophic bacterium *C. eutrophus* H16 wherein the *hox* and *hyp* genes are situated on a 452 kb megaplasmid, pHG1 [45,68]. It is unclear whether strains H16 and CH34 inherited these homologous sets of *hyp* and *hox* genes from a common ancestor through vertical descent, or whether they were passed on by lateral gene transfer. In contrast, the *cbb* loci (for CO₂ fixation) in both strains clearly have different origins since the CH34 *cbb* genes are phylogenetically much more closely related to the *cbb* homologues of photosynthetic- and nitrifying-organisms [45].

The 56 kb CMGI-4 island has a mosaic structure and lacks the conjugative module. Instead, its extremity carries genes involved in the biosynthesis of methionine, and the degradation of phospho-nates; the transposon Tn6048 also occurs there. The DNA sequence of the CMGI-4 island is highly similar to a region in the *D. acidovorans* SPH-1 genome [69–71] except that the latter does not contain Tn6048 but carries a complete Tn4371-like structure flanked by Tn6051 [59].

The contribution of the remaining GIs (between 11- and 24-kb in size) towards the particular lifestyle of *C. metallidurans* is unclear.

<figure><img src="image_14.png" /></figure>
Seemingly, CMGI-5 is composed of plasmid remnants, while CMGI-7 might be involved in resistance to arsenic. The CMG-11 island harbours a putative fimbrial operon of the chaperone-usher-dependent assembly pathway. The islands CMGI-6 and CMGI-8 are cryptic islands holding copies of IS1087 and Tn6049, while CMGI-9 and CMGI-10 consist almost entirely out of hypothetical genes.

The *C. metallidurans* CH34 genome accommodates 19 transposons of five different types [59] (Table 3). Two ‘mercury transposons’ Tn4378 and Tn4380 (both of the Tn501 family) are located, respectively, on pMOL28 and pMOL30, and were previously reported [10]. We identified three novel types of transposon: Tn6048 (10.4 kbp), containing 8 genes of unknown function but highly induced by zinc and lead, Tn6049 (3.5 kbp), often associated with genomic islands, and Tn6050 (6.8 kbp) found twice in CHR2 in opposite orientations and separated by 143 kb. Remarkably, all 12 copies of Tn6049 carry the same ATPase-encoding gene, putatively named *gsPA* because its product shows strong similarities to putative general secretion pathway ATPases. We also note that although the core region (*tnpA*-*tnpR*) of Tn6050 is very similar to that of the mercury transposons Tn4378 and Tn4380, its accessory gene cassette is uninvolved in mercury resistance and encodes proteins that are only rarely associated with transposons, viz., a sulfate permease, a universal stress protein (UspA), and a DksA-like DnaK suppressor protein. We reason that there is a functional linkage between these genes, as this module, in full or part, also occurs on other Tn3-related transposons i.e., alone, as in Tn1013 from the IncP-1α plasmid pBS228 [72], or in combination with antibiotic resistance determinants, as in Tn6001, Tn1403, and Tn1404* transposons detected in clinical- and environmental-strains of *Pseudomonas* [73,74]. Closer examination of the chromosomal regions flanking the Tn6050 transposons in strain CH34 suggests the possibility of chromosomal inversion through homologous recombination, thereby effecting several loci involved in metal resistance [75,76].

Significantly, the *C. metallidurans* CH34 genome bears no fewer than 57 insertion elements evenly distributed over the four replicons (Table 4). The most abundant are *ISRme3* (an IS3 family member), with 10 copies, and *IS1088* (an IS30 family member), with nine copies. Conceivably, many of those 57 IS elements have played an important role in modulating the architecture of the CH34 genome and in the continuous acquisition of new traits. For instance, the incorporation of *hyp* and *hox* genes into CMGI-3 likely results from active *IS1071* elements, while in contrast, the ability of strain CH34 to grow autotrophically may be lost owing to *IS1071*-mediated excision. Similarly, the two *ISRme5* copies on CMGI-2 are located amidst multiple transposase genes and fragments, and probably were the driving force toward integrating the *hyp/hox* cluster on CMGI-2. In addition, both copies of *ISRme7* flank the genomic island CMGI-11 while two copies of *ISRme3* are associated with the genomic islands in pMOL30, further

<table><caption>**Table 3.** Distribution of transposons in *C. metallidurans* CH34.</caption><thead><tr><th>Element</th><th>Total nr.</th><th>CHR1</th><th>CHR2</th><th>pMOL28</th><th>pMOL30</th></tr></thead><tbody><tr><td>Tn4378</td><td>1</td><td></td><td></td><td>1</td><td></td></tr><tr><td>Tn4380</td><td>1</td><td></td><td></td><td></td><td>1</td></tr><tr><td>Tn6048</td><td>3</td><td>1</td><td>2</td><td></td><td></td></tr><tr><td>Tn6049</td><td>12</td><td>7</td><td>3</td><td>1</td><td>1</td></tr><tr><td>Tn6050</td><td>2</td><td></td><td>2</td><td></td><td></td></tr></tbody></table>

doi:10.1371/journal.pone.0010433.t003

<table><caption>**Table 4.** Distribution of IS elements in *C. metallidurans* CH34.</caption><thead><tr><th>Element</th><th>Family (sub-)</th><th>Total nr.CHR1</th><th>CHR2</th><th>pMOL28</th><th>pMOL30</th></tr></thead><tbody><tr><td>IS1071</td><td>Tn3</td><td>3</td><td>3</td><td></td><td></td></tr><tr><td>IS1086</td><td>IS30</td><td>3</td><td>1</td><td>1</td><td>1</td></tr><tr><td>IS1087</td><td>IS3 (IS2)</td><td>2</td><td>2</td><td></td><td></td></tr><tr><td>IS1088</td><td>IS30</td><td>9</td><td>3</td><td>6</td><td></td></tr><tr><td>IS1090</td><td>IS256</td><td>4</td><td>4</td><td></td><td></td></tr><tr><td>ISRme1</td><td>IS5 (IS427)</td><td>4</td><td>2</td><td>2</td><td></td></tr><tr><td>ISRme3</td><td>IS3 (IS3)</td><td>10</td><td>3</td><td>5</td><td>2</td></tr><tr><td>ISRme4</td><td>IS21</td><td>2</td><td>2</td><td></td><td></td></tr><tr><td>ISRme5</td><td>IS481</td><td>4</td><td>3</td><td>1</td><td></td></tr><tr><td>ISRme6</td><td>IS5 (IS427)</td><td>1</td><td>1</td><td></td><td></td></tr><tr><td>ISRme7</td><td>IS6</td><td>2</td><td>2</td><td></td><td></td></tr><tr><td>ISRme8</td><td>IS4</td><td>2</td><td>1</td><td>1</td><td></td></tr><tr><td>ISRme9</td><td>IS21</td><td>1</td><td></td><td></td><td>1</td></tr><tr><td>ISRme10</td><td>IS30</td><td>1</td><td></td><td></td><td>1</td></tr><tr><td>ISRme11</td><td>IS3</td><td>2</td><td>2</td><td></td><td></td></tr><tr><td>ISRme12</td><td>IS3 (IS150)</td><td>1</td><td>1</td><td></td><td></td></tr><tr><td>ISRme15</td><td>IS3 (IS51)</td><td>2</td><td></td><td>1</td><td>1</td></tr><tr><td>ISRme17</td><td>IS3 (IS150)</td><td>1</td><td>1</td><td></td><td></td></tr><tr><td>ISRme18</td><td>Tn3</td><td>1</td><td></td><td></td><td>1</td></tr><tr><td>ISRme19</td><td>IS66</td><td>1</td><td></td><td></td><td>1</td></tr><tr><td>ISRme20</td><td>IS21</td><td>1</td><td>1</td><td></td><td></td></tr></tbody></table>

doi:10.1371/journal.pone.0010433.t004

strengthening our belief that IS elements principally shaped the CH34 genome.

We also detected in the CH34 genome a series of putative mobile elements that previously were unnoticed (detailed by Van Houdt *et al.* [59]). The first group of such elements contain tyrosine-based site-specific recombinase genes consecutively placed in series of three. We observed such trios in a wide variety of α- and β-proteobacteria and termed them recombinase in trio or *rit* elements. One trio (*ritA*<sub>2</sub>*B*<sub>2</sub>*C*<sub>2</sub>) (Rmet_1239 to Rmet_1241) belongs to the integrase module of CMGI-2 and apparently inactivates a putative DNA repair gene, *radC*. This trio is highly similar to the *B. petrii* *rit* element (Bpet1498 to Bpet1500) of genomic island GI3, having corresponding aa (amino acid) sequence identities of 77% or higher. Intriguingly, the *B. petrii* *rit* element inactivates a *radC* gene (Bpet1497), and all genes in both *rit* elements display ATGA overlaps strongly suggesting translational coupling. A second trio (*ritA*<sub>1</sub>*B*<sub>1</sub>*C*<sub>1</sub>) (Rmet_1271 to Rmet_1273) also lies on CMGI-2 and is part of a complex accessory module; it inactivates a *tnpA* gene of the IS66 family. The *C. metallidurans* *ritA*<sub>1</sub>*B*<sub>1</sub>*C*<sub>1</sub> element exhibits high similarity to the *rit* elements on plasmid pHG1 of *C. eutrophus*, showing very high aa sequence similarities (>92% identity) for *ritA*<sub>1</sub> and *ritB*<sub>1</sub> to either PHG129 and PHG141, or either PHG130 and PHG140, respectively. Further, there are high aa sequence similarities (40–54% identity) between *ritA*<sub>1</sub>*B*<sub>1</sub>*C*<sub>1</sub> and the trio PHG134/135/136. Immediately adjacent to this maze of pHG1 recombinase genes we discerned an IS66 type *tnpA* gene (PHG142) but we are uncertain whether it is interrupted or not. We also noticed four-gene elements, comprising three recombinases close to each other and invariably accompanied by a fourth gene encoding a transcriptional regulator of the TetR family [59]. The last group of mobile

<figure><img src="image_15.png" /></figure>
Seemingly, CMGI-5 is composed of plasmid remnants, while CMGI-7 might be involved in resistance to arsenic. The CMG-11 island harbours a putative fimbrial operon of the chaperone-usher-dependent assembly pathway. The islands CMGI-6 and CMGI-8 are cryptic islands holding copies of IS1087 and Tn6049, while CMGI-9 and CMGI-10 consist almost entirely out of hypothetical genes.

The *C. metallidurans* CH34 genome accommodates 19 transposons of five different types [59] (Table 3). Two ‘mercury transposons’ Tn4378 and Tn4380 (both of the Tn501 family) are located, respectively, on pMOL28 and pMOL30, and were previously reported [10]. We identified three novel types of transposon: Tn6048 (10.4 kbp), containing 8 genes of unknown function but highly induced by zinc and lead, Tn6049 (3.5 kbp), often associated with genomic islands, and Tn6050 (6.8 kbp) found twice in CHR2 in opposite orientations and separated by 143 kb. Remarkably, all 12 copies of Tn6049 carry the same ATPase-encoding gene, putatively named *gsPA* because its product shows strong similarities to putative general secretion pathway ATPases. We also note that although the core region (*tnpA*-*tnpR*) of Tn6050 is very similar to that of the mercury transposons Tn4378 and Tn4380, its accessory gene cassette is uninvolved in mercury resistance and encodes proteins that are only rarely associated with transposons, viz., a sulfate permease, a universal stress protein (UspA), and a DksA-like DnaK suppressor protein. We reason that there is a functional linkage between these genes, as this module, in full or part, also occurs on other Tn3-related transposons i.e., alone, as in Tn1013 from the IncP-1α plasmid pBS228 [72], or in combination with antibiotic resistance determinants, as in Tn6001, Tn1403, and Tn1404* transposons detected in clinical- and environmental-strains of *Pseudomonas* [73,74]. Closer examination of the chromosomal regions flanking the Tn6050 transposons in strain CH34 suggests the possibility of chromosomal inversion through homologous recombination, thereby effecting several loci involved in metal resistance [75,76].

Significantly, the *C. metallidurans* CH34 genome bears no fewer than 57 insertion elements evenly distributed over the four replicons (Table 4). The most abundant are *ISRme3* (an IS3 family member), with 10 copies, and *IS1088* (an IS30 family member), with nine copies. Conceivably, many of those 57 IS elements have played an important role in modulating the architecture of the CH34 genome and in the continuous acquisition of new traits. For instance, the incorporation of *hyp* and *hox* genes into CMGI-3 likely results from active *IS1071* elements, while in contrast, the ability of strain CH34 to grow autotrophically may be lost owing to *IS1071*-mediated excision. Similarly, the two *ISRme5* copies on CMGI-2 are located amidst multiple transposase genes and fragments, and probably were the driving force toward integrating the *hyp/hox* cluster on CMGI-2. In addition, both copies of *ISRme7* flank the genomic island CMGI-11 while two copies of *ISRme3* are associated with the genomic islands in pMOL30, further

<table><caption>**Table 3.** Distribution of transposons in *C. metallidurans* CH34.</caption><thead><tr><th>Element</th><th>Total nr.</th><th>CHR1</th><th>CHR2</th><th>pMOL28</th><th>pMOL30</th></tr></thead><tbody><tr><td>Tn4378</td><td>1</td><td></td><td></td><td>1</td><td></td></tr><tr><td>Tn4380</td><td>1</td><td></td><td></td><td></td><td>1</td></tr><tr><td>Tn6048</td><td>3</td><td>1</td><td>2</td><td></td><td></td></tr><tr><td>Tn6049</td><td>12</td><td>7</td><td>3</td><td>1</td><td>1</td></tr><tr><td>Tn6050</td><td>2</td><td></td><td>2</td><td></td><td></td></tr></tbody></table>

doi:10.1371/journal.pone.0010433.t003

<table><caption>**Table 4.** Distribution of IS elements in *C. metallidurans* CH34.</caption><thead><tr><th>Element</th><th>Family (sub-)</th><th>Total nr.CHR1</th><th>CHR2</th><th>pMOL28</th><th>pMOL30</th></tr></thead><tbody><tr><td>IS1071</td><td>Tn3</td><td>3</td><td>3</td><td></td><td></td></tr><tr><td>IS1086</td><td>IS30</td><td>3</td><td>1</td><td>1</td><td>1</td></tr><tr><td>IS1087</td><td>IS3 (IS2)</td><td>2</td><td>2</td><td></td><td></td></tr><tr><td>IS1088</td><td>IS30</td><td>9</td><td>3</td><td>6</td><td></td></tr><tr><td>IS1090</td><td>IS256</td><td>4</td><td>4</td><td></td><td></td></tr><tr><td>ISRme1</td><td>IS5 (IS427)</td><td>4</td><td>2</td><td>2</td><td></td></tr><tr><td>ISRme3</td><td>IS3 (IS3)</td><td>10</td><td>3</td><td>5</td><td>2</td></tr><tr><td>ISRme4</td><td>IS21</td><td>2</td><td>2</td><td></td><td></td></tr><tr><td>ISRme5</td><td>IS481</td><td>4</td><td>3</td><td>1</td><td></td></tr><tr><td>ISRme6</td><td>IS5 (IS427)</td><td>1</td><td>1</td><td></td><td></td></tr><tr><td>ISRme7</td><td>IS6</td><td>2</td><td>2</td><td></td><td></td></tr><tr><td>ISRme8</td><td>IS4</td><td>2</td><td>1</td><td>1</td><td></td></tr><tr><td>ISRme9</td><td>IS21</td><td>1</td><td></td><td></td><td>1</td></tr><tr><td>ISRme10</td><td>IS30</td><td>1</td><td></td><td></td><td>1</td></tr><tr><td>ISRme11</td><td>IS3</td><td>2</td><td>2</td><td></td><td></td></tr><tr><td>ISRme12</td><td>IS3 (IS150)</td><td>1</td><td>1</td><td></td><td></td></tr><tr><td>ISRme15</td><td>IS3 (IS51)</td><td>2</td><td></td><td>1</td><td>1</td></tr><tr><td>ISRme17</td><td>IS3 (IS150)</td><td>1</td><td>1</td><td></td><td></td></tr><tr><td>ISRme18</td><td>Tn3</td><td>1</td><td></td><td></td><td>1</td></tr><tr><td>ISRme19</td><td>IS66</td><td>1</td><td></td><td></td><td>1</td></tr><tr><td>ISRme20</td><td>IS21</td><td>1</td><td>1</td><td></td><td></td></tr></tbody></table>

doi:10.1371/journal.pone.0010433.t004

strengthening our belief that IS elements principally shaped the CH34 genome.

We also detected in the CH34 genome a series of putative mobile elements that previously were unnoticed (detailed by Van Houdt *et al.* [59]). The first group of such elements contain tyrosine-based site-specific recombinase genes consecutively placed in series of three. We observed such trios in a wide variety of α- and β-proteobacteria and termed them recombinase in trio or *rit* elements. One trio (*ritA*<sub>2</sub>*B*<sub>2</sub>*C*<sub>2</sub>) (Rmet_1239 to Rmet_1241) belongs to the integrase module of CMGI-2 and apparently inactivates a putative DNA repair gene, *radC*. This trio is highly similar to the *B. petrii* *rit* element (Bpet1498 to Bpet1500) of genomic island GI3, having corresponding aa (amino acid) sequence identities of 77% or higher. Intriguingly, the *B. petrii* *rit* element inactivates a *radC* gene (Bpet1497), and all genes in both *rit* elements display ATGA overlaps strongly suggesting translational coupling. A second trio (*ritA*<sub>1</sub>*B*<sub>1</sub>*C*<sub>1</sub>) (Rmet_1271 to Rmet_1273) also lies on CMGI-2 and is part of a complex accessory module; it inactivates a *tnpA* gene of the IS66 family. The *C. metallidurans* *ritA*<sub>1</sub>*B*<sub>1</sub>*C*<sub>1</sub> element exhibits high similarity to the *rit* elements on plasmid pHG1 of *C. eutrophus*, showing very high aa sequence similarities (>92% identity) for *ritA*<sub>1</sub> and *ritB*<sub>1</sub> to either PHG129 and PHG141, or either PHG130 and PHG140, respectively. Further, there are high aa sequence similarities (40–54% identity) between *ritA*<sub>1</sub>*B*<sub>1</sub>*C*<sub>1</sub> and the trio PHG134/135/136. Immediately adjacent to this maze of pHG1 recombinase genes we discerned an IS66 type *tnpA* gene (PHG142) but we are uncertain whether it is interrupted or not. We also noticed four-gene elements, comprising three recombinases close to each other and invariably accompanied by a fourth gene encoding a transcriptional regulator of the TetR family [59]. The last group of mobile

<figure><img src="image_15.png" /></figure>
elements we list here is the *bimAB* module, named after the *bimA* recombinase and *bimB* putative repressor genes first noted on the pMOL28 and pMOL30 plasmids as genepairs Rmet_6192/93 (linked to a *chr* locus) and Rmet_5987/88 (linked to a *cze* locus), respectively [7]. The latter module (21 genes) is fully conserved in *Ralstonia picketti* 12J, indicating a horizontal transfer event. The *bimAB* element has also been associated to HMR loci in other bacteria [59].

An interesting feature of *C. metallidurans* strain CH34, and characteristic for related metallotolerant *C. metallidurans* strains, is the increased activity of transposable elements when cells are grown at elevated temperature i.e. 37°C instead of 30°C. This phenomenon is referred to as temperature induced mutagenesis in which survivor cells appear to have undergone DNA arrangements, insertions, or deletions [6,22,77,78]. One well studied case is the integration of a 44 kb DNA fragment in the pMOL28 plasmid and the identification of IS1086 participating in this event [77,79]. The resulting plasmid pMOL50 has a much higher conjugation frequency then either pMOL28 or pMOL30 and is able to very efficiently (retro)mobilise very large DNA fragments from the CH34 chromosome (of up to 1 Mb), a property that was exploited to construct the genetic map of the CH34 main chromosome, CHR1 [22].

## A brief update on megaplasmids in strain CH34

Both megaplasmids pMOL28 and pMOL30 carry key genes for resistance to heavy metals [9]. Plasmid pMOL28 encodes resistance to nickel and cobalt [80,81], chromate [5], mercury [4,10], and thallium [82,83]. Plasmid pMOL30 specifies resistance to zinc, cadmium, and cobalt [13,84], copper [75], silver [8], and lead [37,85]. The corresponding gene clusters on pMOL28 and pMOL30, and the replication region of pMOL28 were identified subsequently (reviewed by [7,8,10]). Monchy et al. [10] derived detailed genetic maps of both megaplasmids from their full DNA sequence. This distinguished the genes involved in plasmid maintenance, replication, and partition, together constituting the 'backbone' of the plasmid, next to specialized 'accessory' genes that group together on genomic islands i.e., CMGI-28a, -28b, -28c, and CMGI-30a and -30b. In pMOL28 and pMOL30, these GIs contain all the genes involved in metal resistance [7,59]. Notably, the pMOL28 replicon belongs to a family of megaplasmids that share a similar backbone and include the megaplasmids pHG1 (452 kb) of *C. eutrophus* H16 and pRALTA (557 kb) of *C. taïvanensis*. Owing to the distinctly specialized functions on pMOL28 (heavy metal resistance), pHG1 (hydrogenotrophy and chemolithotrophy), and pRALTA (nitrogen fixation and legume symbiosis), these megaplasmids might well be regarded as the decisive factor in fixing their hosts to their ecological niche. A direct relationship of the megaplasmid pMOL30 to other megaplasmids is less certain. Nonetheless, through synteny analysis and advanced annotation using the MaGe platform we noted that the backbone of pMOL30 shares, over a region of approximately 60 kb, many characteristics with the pBVIE01 megaplasmid (398 kb) of *B. vietnamiensis* G4 [7].

Despite detailed annotation of megaplasmids pMOL28 and pMOL30 [10], continuous manual curation of the CH34 genome via updated databases, and earlier functional information from proteomic- and transcriptomic-approaches [5,75,83,86] many enigmatic genes remain in both plasmids (Table 1).

Recent transcriptomic analyses revealed that five such genes, located on CMGI-28a, are induced by submillimolar concentrations of chromate (no data given). All five genes are sited immediately downstream of the chromate-regulated operon *chrIA*, *B1*, *CEF1* (Rmet_6199 through Rmet_6204): we called them

*chrO* (Rmet_6198), *chrN* (Rmet_6197), *chrP* (Rmet_6196, encoding an MFS family permease with ten transmembrane α-helical segments), *chrY* (Rmet_6195, encoding a putative membrane protein), and *chrZ* (Rmet_6194) (Table 5). Synteny analysis using MaGe shows a fair to good resemblance between the pMOL28 *chr* locus of *C. metallidurans* and the *chr* loci of *Methylobacterium* sp. 4-46, *B. vietnamiensis* G4, and *B. pseudomalei*. However, the *chr* locus in *Methylobacterium* sp. 4-46 lacks *chrE*, *chrN*, and *chrP* orthologues, the four *chr* loci of *B. vietnamiensis* G4 all lack *chrP* and *chrZ* orthologues, and the majority of *chr* loci across 21 *B. pseudomallei* strains lack *chrC*, *chrE*, *chrO*, and *chrN* orthologues. This puzzling variation in *chr* locus structure may point to differences among these bacteria in terms of selectivity to chromate or another metal-oxyanion.

We also identified hypothetical genes on CMGI-28b that encode membrane proteins rich in YD (tyrosyl-aspartyl) motifs and named them *rhsA* (Rmet_6263), *rhsB* (Rmet_6261), and *rhsC* (Rmet_6258) because such YD-repeat proteins apparently are linked to RHS (Recombination Hot Spot) regions [87,88]. Noteworthy, these genes are centered around the *tnpA* and *tnpB* genes (respectively Rmet_6260 and Rmet_6259) of the *ISR* gene9 mobile element.

The pMOL28 backbone region covered by Rmet_6288 through Rmet_6297 provisionally was named the *pil* locus because of some resemblance in its sequence and gene order with other *pil* loci [7,10]. However, although this region and the flanking ones display good synteny with the corresponding backbone regions on the *C. taïvanensis* pRALTA and *C. eutrophus* pHG1 megaplasmids, the individual sequence similarity of genes Rmet_6288 to Rmet_6294 to known *pil* genes is very low. Either these genes are not involved in conjugation (but rather in secretion), or pMOL28 has evolved a *sui generis* conjugative system. For now, we will retain the *pil* epitaph. We note that the Rmet_6291 gene, formerly annotated as *pilQ* [10], now is annotated as *pilB* because its product shows no aa sequence similarity to the *PilQ* usher protein encoded in the *pilMNOPQ* locus on CHR1 (Rmet_3268 to Rmet_3272); rather, it probably acts as an ATPase.

A fair number of plasmid-born genes are seemingly involved in either gene exchange or membrane processing. For instance, the entire region between the *pbr* and *cze* loci on CMGI-30a is speckled by very small hypothetical genes, transposases (*tnp*), site-specific recombinases (*int*), or remnants thereof, some of which are upregulated by heavy metals [10]. In addition, both genomic islands on pMOL30 contain a *gtrMAB* locus encoding proteins that are required for translocating oligosaccharide precursors across the cytoplasmic membrane and the subsequent glucosylation of lipopolysaccharide (LPS). Several genes of both *gtr* loci are upregulated by heavy metals [10] while the *gtr* loci are centrally placed between the *pbr* and *mer* clusters on CMGI-30a (as genes Rmet_5966/67/68) and between the *cop* and *sil* clusters on CMGI-30b (as genes Rmet_6128/29/30). A possible reason for recruiting *gtr* loci and embedding them in metal resistance clusters may be that modified (i.e., shortened) LPS chains could positively affect heavy-metal extrusion. Alternatively, *GtrMAB* proteins may act on other components outside of the inner membrane thereby limiting heavy-metal damage inflicted on membranes.

## Heavy metal resistance (HMR) genes in CH34

Metal detoxification in *C. metallidurans* CH34 mainly occurs via ion efflux although other systems are present (Table 5; Table S2). A variety of export systems that enlist members of three protein families assure ion efflux: The RND (for Resistance, Nodulation, Cell Division) family of permeases [74], the CDF (for Cation Diffusion Factor) family of heavy metal transporters [89,90], and

<figure><img src="image_16.png" /></figure>
elements we list here is the *bimAB* module, named after the *bimA* recombinase and *bimB* putative repressor genes first noted on the pMOL28 and pMOL30 plasmids as genepairs Rmet_6192/93 (linked to a *chr* locus) and Rmet_5987/88 (linked to a *cze* locus), respectively [7]. The latter module (21 genes) is fully conserved in *Ralstonia picketti* 12J, indicating a horizontal transfer event. The *bimAB* element has also been associated to HMR loci in other bacteria [59].

An interesting feature of *C. metallidurans* strain CH34, and characteristic for related metallotolerant *C. metallidurans* strains, is the increased activity of transposable elements when cells are grown at elevated temperature i.e. 37°C instead of 30°C. This phenomenon is referred to as temperature induced mutagenesis in which survivor cells appear to have undergone DNA arrangements, insertions, or deletions [6,22,77,78]. One well studied case is the integration of a 44 kb DNA fragment in the pMOL28 plasmid and the identification of IS1086 participating in this event [77,79]. The resulting plasmid pMOL50 has a much higher conjugation frequency then either pMOL28 or pMOL30 and is able to very efficiently (retro)mobilise very large DNA fragments from the CH34 chromosome (of up to 1 Mb), a property that was exploited to construct the genetic map of the CH34 main chromosome, CHR1 [22].

## A brief update on megaplasmids in strain CH34

Both megaplasmids pMOL28 and pMOL30 carry key genes for resistance to heavy metals [9]. Plasmid pMOL28 encodes resistance to nickel and cobalt [80,81], chromate [5], mercury [4,10], and thallium [82,83]. Plasmid pMOL30 specifies resistance to zinc, cadmium, and cobalt [13,84], copper [75], silver [8], and lead [37,85]. The corresponding gene clusters on pMOL28 and pMOL30, and the replication region of pMOL28 were identified subsequently (reviewed by [7,8,10]). Monchy et al. [10] derived detailed genetic maps of both megaplasmids from their full DNA sequence. This distinguished the genes involved in plasmid maintenance, replication, and partition, together constituting the 'backbone' of the plasmid, next to specialized 'accessory' genes that group together on genomic islands i.e., CMGI-28a, -28b, -28c, and CMGI-30a and -30b. In pMOL28 and pMOL30, these GIs contain all the genes involved in metal resistance [7,59]. Notably, the pMOL28 replicon belongs to a family of megaplasmids that share a similar backbone and include the megaplasmids pHG1 (452 kb) of *C. eutrophus* H16 and pRALTA (557 kb) of *C. taïivanensis*. Owing to the distinctly specialized functions on pMOL28 (heavy metal resistance), pHG1 (hydrogenotrophy and chemolithotrophy), and pRALTA (nitrogen fixation and legume symbiosis), these megaplasmids might well be regarded as the decisive factor in fixing their hosts to their ecological niche. A direct relationship of the megaplasmid pMOL30 to other megaplasmids is less certain. Nonetheless, through synteny analysis and advanced annotation using the MaGe platform we noted that the backbone of pMOL30 shares, over a region of approximately 60 kb, many characteristics with the pBVIE01 megaplasmid (398 kb) of *B. vietnamiensis* G4 [7].

Despite detailed annotation of megaplasmids pMOL28 and pMOL30 [10], continuous manual curation of the CH34 genome via updated databases, and earlier functional information from proteomic- and transcriptomic-approaches [5,75,83,86] many enigmatic genes remain in both plasmids (Table 1).

Recent transcriptomic analyses revealed that five such genes, located on CMGI-28a, are induced by submillimolar concentrations of chromate (no data given). All five genes are sited immediately downstream of the chromate-regulated operon *chrIA*, *B1*, *CEF1* (Rmet_6199 through Rmet_6204): we called them

*chrO* (Rmet_6198), *chrN* (Rmet_6197), *chrP* (Rmet_6196, encoding an MFS family permease with ten transmembrane α-helical segments), *chrY* (Rmet_6195, encoding a putative membrane protein), and *chrZ* (Rmet_6194) (Table 5). Synteny analysis using MaGe shows a fair to good resemblance between the pMOL28 *chr* locus of *C. metallidurans* and the *chr* loci of *Methylobacterium* sp. 4-46, *B. vietnamiensis* G4, and *B. pseudomalei*. However, the *chr* locus in *Methylobacterium* sp. 4-46 lacks *chrE*, *chrN*, and *chrP* orthologues, the four *chr* loci of *B. vietnamiensis* G4 all lack *chrP* and *chrZ* orthologues, and the majority of *chr* loci across 21 *B. pseudomallei* strains lack *chrC*, *chrE*, *chrO*, and *chrN* orthologues. This puzzling variation in *chr* locus structure may point to differences among these bacteria in terms of selectivity to chromate or another metal-oxyanion.

We also identified hypothetical genes on CMGI-28b that encode membrane proteins rich in YD (tyrosyl-aspartyl) motifs and named them *rhsA* (Rmet_6263), *rhsB* (Rmet_6261), and *rhsC* (Rmet_6258) because such YD-repeat proteins apparently are linked to RHS (Recombination Hot Spot) regions [87,88]. Noteworthy, these genes are centered around the *tnpA* and *tnpB* genes (respectively Rmet_6260 and Rmet_6259) of the *ISR* gene9 mobile element.

The pMOL28 backbone region covered by Rmet_6288 through Rmet_6297 provisionally was named the *pil* locus because of some resemblance in its sequence and gene order with other *pil* loci [7,10]. However, although this region and the flanking ones display good synteny with the corresponding backbone regions on the *C. taïivanensis* pRALTA and *C. eutrophus* pHG1 megaplasmids, the individual sequence similarity of genes Rmet_6288 to Rmet_6294 to known *pil* genes is very low. Either these genes are not involved in conjugation (but rather in secretion), or pMOL28 has evolved a *sui generis* conjugative system. For now, we will retain the *pil* epitaph. We note that the Rmet_6291 gene, formerly annotated as *pilQ* [10], now is annotated as *pilB* because its product shows no aa sequence similarity to the *PilQ* usher protein encoded in the *pilMNOPQ* locus on CHR1 (Rmet_3268 to Rmet_3272); rather, it probably acts as an ATPase.

A fair number of plasmid-born genes are seemingly involved in either gene exchange or membrane processing. For instance, the entire region between the *pbr* and *cze* loci on CMGI-30a is speckled by very small hypothetical genes, transposases (*tnp*), site-specific recombinases (*int*), or remnants thereof, some of which are upregulated by heavy metals [10]. In addition, both genomic islands on pMOL30 contain a *gtrMAB* locus encoding proteins that are required for translocating oligosaccharide precursors across the cytoplasmic membrane and the subsequent glucosylation of lipopolysaccharide (LPS). Several genes of both *gtr* loci are upregulated by heavy metals [10] while the *gtr* loci are centrally placed between the *pbr* and *mer* clusters on CMGI-30a (as genes Rmet_5966/67/68) and between the *cop* and *sil* clusters on CMGI-30b (as genes Rmet_6128/29/30). A possible reason for recruiting *gtr* loci and embedding them in metal resistance clusters may be that modified (i.e., shortened) LPS chains could positively affect heavy-metal extrusion. Alternatively, *GtrMAB* proteins may act on other components outside of the inner membrane thereby limiting heavy-metal damage inflicted on membranes.

## Heavy metal resistance (HMR) genes in CH34

Metal detoxification in *C. metallidurans* CH34 mainly occurs via ion efflux although other systems are present (Table 5; Table S2). A variety of export systems that enlist members of three protein families assure ion efflux: The RND (for Resistance, Nodulation, Cell Division) family of permeases [74], the CDF (for Cation Diffusion Factor) family of heavy metal transporters [89,90], and

<figure><img src="image_16.png" /></figure>
<table><caption>**Table 5.** Metal detoxification in C. metallidurans CH34 occurs via a variety of export systems encoded by 24 HMR gene clusters on 4 replicons.</caption><thead><tr><th>Type<sup>a</sup></th><th>Replicon</th><th>Gene cluster<sup>b</sup></th><th>Rmet_number</th><th>Metal(s)</th><th>GI/transposon<sup>c</sup></th><th>Mechanism<sup>d</sup></th><th>Fc<sup>e</sup></th><th>Refs.</th></tr></thead><tbody><tr><td rowspan="10">partially plasmid-redundant</td><td>CHR1</td><td>cdfX pbrR<sub>2</sub> cadA pbrC<sub>2</sub>, pbrR<sub>3</sub> (§)</td><td>2299, 2302–2304, 3456</td><td>Pb<sup>2+</sup></td><td>CMGI-1 (P)</td><td>ExP, pCDF</td><td>+</td><td>[59]</td></tr><tr><td>pMOL30</td><td>pbrUbUaTR pbrABCD (#)</td><td>6180, 5944–5949</td><td></td><td>CMGI-30a</td><td>ExP, seq</td><td>+</td><td>[85]</td></tr><tr><td>CHR1</td><td>merRTPA' A"</td><td>2312–2315</td><td>Hg<sup>2+</sup></td><td>CMGI-1 (P)</td><td>HgRed</td><td>?</td><td>[59]</td></tr><tr><td>pMOL28</td><td>merRTPADE urf-2</td><td>6344–6346, 6183–6186</td><td></td><td>CMGI-28a/Tn4378</td><td>HgRed</td><td>+</td><td>[4]</td></tr><tr><td>pMOL30</td><td>merRTPADE urf-2</td><td>6171–6177</td><td></td><td>CMGI-30a/Tn4380</td><td>HgRed</td><td>+</td><td>[4]</td></tr><tr><td>pMOL30</td><td>merRTΔP</td><td>5990–5992</td><td></td><td>CMGI-30a</td><td>HgTra</td><td>?</td><td>[10]</td></tr><tr><td>CHR2</td><td>chrBAF</td><td>3866–3864</td><td>CrO<sub>4</sub><sup>2-</sup></td><td>-</td><td>ExChr</td><td>?</td><td>[5]</td></tr><tr><td>pMOL28</td><td>chrIBACEF ONPYZ</td><td>6204–6194</td><td></td><td>CMGI-28a</td><td>ExChr, FS, MFS</td><td>+</td><td>[12]</td></tr><tr><td>CHR2</td><td>cusDCBAF</td><td>5030–5034</td><td>Ag<sup>+</sup>, Cu<sup>+</sup>, Cu<sup>2+</sup></td><td>-</td><td>ExHME4</td><td>+</td><td>[8]</td></tr><tr><td>pMOL30</td><td>silDCBA cusΔF</td><td>6133–6136, 5953</td><td></td><td>CMGI-30b</td><td>ExHME4</td><td>(+)</td><td>[10]</td></tr><tr><td>CHR2</td><td>copSRABCD</td><td>5673–5668</td><td>Cu<sup>2+</sup></td><td>-</td><td>ExCop, seq</td><td>+</td><td>[8]</td></tr><tr><td>pMOL30</td><td>copVTKMNRSABCDIJGFOLQHEW</td><td>6105–6119, 6382, 6120–6124</td><td></td><td>CMGI-30b</td><td>ExCop, ExP, seq</td><td>+</td><td>[75]</td></tr><tr><td rowspan="10">not plasmid-redundant</td><td>CHR2</td><td>zntA czcICΔBA ubiG czc SRL hns mmmQ (†)</td><td>4594–4597, 4469–4461</td><td>Cd<sup>2+</sup>, Zn<sup>2+</sup>, Co<sup>2+</sup></td><td>-</td><td>ExHME1, ExP</td><td>(+)</td><td>[59]</td></tr><tr><td>pMOL30</td><td>czcMNICBADRSEJ ompP czcP</td><td>5985–5974, 5970</td><td></td><td>CMGI-30a</td><td>ExHME1, ExP</td><td>+</td><td>[10]</td></tr><tr><td>CHR2</td><td>nimBA|AC (*)</td><td>5682–5677</td><td>Ni<sup>2+</sup>, Co<sup>2+</sup></td><td>-</td><td>ExHME3b</td><td>-</td><td>[8]</td></tr><tr><td>pMOL28</td><td>cnrYXHCBAT</td><td>6205–6211</td><td></td><td>CMGI-28a</td><td>ExHME2, CDF</td><td>+</td><td>[80,81]</td></tr><tr><td>pMOL30</td><td>nccCB<sup>2</sup>B'A nreB mmrQ</td><td>6148–6143</td><td></td><td>CMGI-30b</td><td>ExHME2, MFS</td><td>(+)</td><td>[10]</td></tr><tr><td>CHR1</td><td>arsPHC<sub>1</sub>BC<sub>2</sub>IRM</td><td>0327–0334</td><td>HAsO<sub>4</sub><sup>2-</sup>, AsO<sub>2</sub><sup>-</sup></td><td>CMGI-7</td><td>AsRed (ExAs)</td><td>+</td><td>[94]</td></tr><tr><td>CHR1</td><td>cupric</td><td>3523–3525</td><td>Ag<sup>+</sup>, Cu<sup>+</sup>, Cu<sup>2+</sup></td><td>-</td><td>ExP</td><td>+</td><td>[75]</td></tr><tr><td>CHR1</td><td>agrCBARS</td><td>1748–1752</td><td></td><td>-</td><td>ExHAE</td><td>?</td><td>[8]</td></tr><tr><td>CHR1</td><td>hmzRS yodB hmzBΔA</td><td>3016–3011</td><td>divalent metals</td><td>CMGI-4</td><td>ExHME3b</td><td>-</td><td>[59]</td></tr><tr><td>CHR2</td><td>hmvCBΔA</td><td>3836–3838</td><td></td><td>-</td><td>ExHME3a</td><td>-</td><td>[8]</td></tr><tr><td>CHR2</td><td>zniABCSR znePRSCAB</td><td>5319–5323, 5325–5330</td><td></td><td>-</td><td>ExHME3a, MSF</td><td>?</td><td>[58]</td></tr><tr><td>CHR2</td><td>hmyCBhmyA (‡)</td><td>4120–4123</td><td></td><td>-</td><td>ExHME3b</td><td>+</td><td>[8]</td></tr></tbody></table>

<sup>a</sup>chromosomal HMR resistance loci with genes that have a counterpart with a homologous function on a plasmid are called partially redundant, other loci are not-redundant.

<sup>b</sup>Underlined genes are regulators; bold genes are new in respect to an earlier report [8] (see also Table S2); geneparts created by a frameshift are indicated by ‘and’; IS- or Tn-insertions are indicated by a pipe symbol (|); truncated genes are indicated with a delta (Δ) symbol; (§), pbrR<sub>3</sub> acts on zntA (Rmet_4594); (‡), insertion of Tn6049 in pbrU; (†), czcB truncated by Tn6050-mediated rearrangement (inversion) and insertion of IS1088 between czcL and hns; (*), insertion of ISRme3 in nimA; (‡), insertion of IS1088 between hmyB and hmyA.

<sup>c</sup>CMGI, Cupriavidus metallidurans CH34 Genomic Island; (P), PAGI-2 family.

<sup>d</sup>Mechanisms of HM detoxification: ExP, efflux (P1-ATPase); pCDF, putative CDF-like; seq, periplasmic sequestration; HgRed, reduction of Hg<sup>2+</sup> into Hg<sup>0</sup> which then volatilizes out of the cell; HgTra, tentative transport of Hg<sup>2+</sup>; ExChr, transport of chromate; FS, Fe-SOD (chrC); MFS, major facilitator superfamily permease (i.e., chrP); CDF, cation diffusion facilitator; ExHME, efflux by RND-HME type 1 to 4 (see text); ExHAE, efflux by HAE-RND (unknown substrate); ExCop, extrusion of Cu<sup>2+</sup>; AsRed (ExAs), reduction of HAsO<sub>4</sub><sup>2-</sup> into AsO<sub>2</sub> and efflux of AsO<sub>2</sub>.

<sup>e</sup>Functionality; +, active; (+), partly active; ?, unknown; -, inactive.

doi:10.1371/journal.pone.0010433.t005

The P-type ATPase family of ion pumps [91]. These export systems may act on a range of cations, including Cu<sup>+</sup>, Ag<sup>+</sup>, Cu<sup>2+</sup>, Co<sup>2+</sup>, Cd<sup>2+</sup>, Ni<sup>2+</sup>, Pb<sup>2+</sup> and Zn<sup>2+</sup> [8,10,59,75,80,83,85,90,92,93]. Alternative mechanisms with specificity to other cations or oxyanions include the chromate CrO<sub>4</sub><sup>2-</sup> efflux system [5], the reduction of arsenate HAsO<sub>4</sub><sup>2-</sup> by ArsC reductases followed by the extrusion of the formed arsenite AsO<sub>2</sub><sup>-</sup> by the ArsB permease [94], the CopABCD/PcoABCD mediated periplasmic detoxification for Cu<sup>2+</sup> [95,96], and the reduction of Hg<sup>2+</sup> into metallic mercury [4,59,97,98] (Table 5; Table S2). However, the function and mode of action of many proteins encoded by putative heavy-metal resistance genes remain undetermined. Structural analysis of

proteins at the molecular level offered some mechanistic information (i.e., via protein crystallization, nuclear magnetic resonance analysis, and cryo electron microscopy) for the C. metallidurans proteins CopK (Rmet_6108), CopH (Rmet_6122), and CzcE (Rmet_5976) expressed by pMOL30, and CnrX (Rmet_6206) expressed by pMOL28 [99–102]. In other cases, such data were gained from aa sequence based comparison of closely related proteins, such as for the CzcD protein (Rmet_5979) that greatly resembles the recently studied E. coli YiiP zinc transporter protein [103,104].

It is worth mentioning that RND sytems for HME (Heavy Metal Efflux) were first discovered in C. metallidurans CH34 with

<figure><img src="image_17.png" /></figure>
<table><caption>**Table 5.** Metal detoxification in C. metallidurans CH34 occurs via a variety of export systems encoded by 24 HMR gene clusters on 4 replicons.</caption><thead><tr><th>Type<sup>a</sup></th><th>Replicon</th><th>Gene cluster<sup>b</sup></th><th>Rmet_number</th><th>Metal(s)</th><th>GI/transposon<sup>c</sup></th><th>Mechanism<sup>d</sup></th><th>Fc<sup>e</sup></th><th>Refs.</th></tr></thead><tbody><tr><td rowspan="10">partially plasmid-redundant</td><td>CHR1</td><td><i>cdfX pbrR<sub>2</sub> cadA pbrC<sub>2</sub>, pbrR<sub>3</sub> (§)</i></td><td>2299, 2302–2304, 3456</td><td>Pb<sup>2+</sup></td><td>CMGI-1 (P)</td><td>ExP, pCDF</td><td>+</td><td>[59]</td></tr><tr><td>pMOL30</td><td><i>pbrUbUaTR pbrABCD (#)</i></td><td>6180, 5944–5949</td><td></td><td>CMGI-30a</td><td>ExP, seq</td><td>+</td><td>[85]</td></tr><tr><td>CHR1</td><td><i>merRTPA' A''</i></td><td>2312–2315</td><td>Hg<sup>2+</sup></td><td>CMGI-1 (P)</td><td>HgRed</td><td>?</td><td>[59]</td></tr><tr><td>pMOL28</td><td><i>merRTPADE urf-2</i></td><td>6344–6346, 6183–6186</td><td></td><td>CMGI-28a/Tn4378</td><td>HgRed</td><td>+</td><td>[4]</td></tr><tr><td>pMOL30</td><td><i>merRTPADE urf-2</i></td><td>6171–6177</td><td></td><td>CMGI-30a/Tn4380</td><td>HgRed</td><td>+</td><td>[4]</td></tr><tr><td>pMOL30</td><td><i>merRTΔP</i></td><td>5990–5992</td><td></td><td>CMGI-30a</td><td>HgTra</td><td>?</td><td>[10]</td></tr><tr><td>CHR2</td><td><i>chrBAF</i></td><td>3866–3864</td><td>CrO<sub>4</sub><sup>2-</sup></td><td>-</td><td>ExChr</td><td>?</td><td>[5]</td></tr><tr><td>pMOL28</td><td><i>chrIBACEF ONPYZ</i></td><td>6204–6194</td><td></td><td>CMGI-28a</td><td>ExChr, FS, MFS</td><td>+</td><td>[12]</td></tr><tr><td>CHR2</td><td><i>cusDCBAF</i></td><td>5030–5034</td><td>Ag<sup>+</sup>, Cu<sup>+</sup>, Cu<sup>2+</sup></td><td>-</td><td>ExHME4</td><td>+</td><td>[8]</td></tr><tr><td>pMOL30</td><td><i>silDCBA cusΔF</i></td><td>6133–6136, 5953</td><td></td><td>CMGI-30b</td><td>ExHME4</td><td>(+)</td><td>[10]</td></tr><tr><td>CHR2</td><td><i>copSRABCD</i></td><td>5673–5668</td><td>Cu<sup>2+</sup></td><td>-</td><td>ExCop, seq</td><td>+</td><td>[8]</td></tr><tr><td>pMOL30</td><td><i>copVTKMNRSABCDIJGFOLQHEW</i></td><td>6105–6119, 6382, 6120–6124</td><td></td><td>CMGI-30b</td><td>ExCop, ExP, seq</td><td>+</td><td>[75]</td></tr><tr><td rowspan="10">not plasmid-redundant</td><td>CHR2</td><td><i>zntA czcICΔBA ubiG czc SRL hns mmmQ (†)</i></td><td>4594–4597, 4469–4461</td><td>Cd<sup>2+</sup>, Zn<sup>2+</sup>, Co<sup>2+</sup></td><td>-</td><td>ExHME1, ExP</td><td>(+)</td><td>[59]</td></tr><tr><td>pMOL30</td><td><i>czcMNICBADRSEJ ompP czcP</i></td><td>5985–5974, 5970</td><td></td><td>CMGI-30a</td><td>ExHME1, ExP</td><td>+</td><td>[10]</td></tr><tr><td>CHR2</td><td><i>nimBA|AC (*)</i></td><td>5682–5677</td><td>Ni<sup>2+</sup>, Co<sup>2+</sup></td><td>-</td><td>ExHME3b</td><td>-</td><td>[8]</td></tr><tr><td>pMOL28</td><td><i>cnrYXHCBAT</i></td><td>6205–6211</td><td></td><td>CMGI-28a</td><td>ExHME2, CDF</td><td>+</td><td>[80,81]</td></tr><tr><td>pMOL30</td><td><i>nccCB<sup>+</sup>B'A nreB mmrQ</i></td><td>6148–6143</td><td></td><td>CMGI-30b</td><td>ExHME2, MFS</td><td>(+)</td><td>[10]</td></tr><tr><td>CHR1</td><td><i>arsPHC<sub>1</sub>BC<sub>2</sub>IRM</i></td><td>0327–0334</td><td>HAsO<sub>4</sub><sup>2-</sup>, AsO<sub>2</sub><sup>-</sup></td><td>CMGI-7</td><td>AsRed (ExAs)</td><td>+</td><td>[94]</td></tr><tr><td>CHR1</td><td><i>cupric</i></td><td>3523–3525</td><td>Ag<sup>+</sup>, Cu<sup>+</sup>, Cu<sup>2+</sup></td><td>-</td><td>ExP</td><td>+</td><td>[75]</td></tr><tr><td>CHR1</td><td><i>agrCBARS</i></td><td>1748–1752</td><td></td><td>-</td><td>ExHAE</td><td>?</td><td>[8]</td></tr><tr><td>CHR1</td><td><i>hmzRS yodB hmzBΔA</i></td><td>3016–3011</td><td>divalent metals</td><td>CMGI-4</td><td>ExHME3b</td><td>-</td><td>[59]</td></tr><tr><td>CHR2</td><td><i>hmvCBΔA</i></td><td>3836–3838</td><td></td><td>-</td><td>ExHME3a</td><td>-</td><td>[8]</td></tr><tr><td>CHR2</td><td><i>zniABCSR znePRSCAB</i></td><td>5319–5323, 5325–5330</td><td></td><td>-</td><td>ExHME3a, MSF</td><td>?</td><td>[58]</td></tr><tr><td>CHR2</td><td><i>hmyCB<sub>1</sub>hmyA (‡)</i></td><td>4120–4123</td><td></td><td>-</td><td>ExHME3b</td><td>+</td><td>[8]</td></tr></tbody></table>

<sup>a</sup>chromosomal HMR resistance loci with genes that have a counterpart with a homologous function on a plasmid are called partially redundant, other loci are not-redundant.

<sup>b</sup>Underlined genes are regulators; bold genes are new in respect to an earlier report [8] (see also Table S2); geneparts created by a frameshift are indicated by ‘and’; IS- or Tn-insertions are indicated by a pipe symbol (|); truncated genes are indicated with a delta (Δ) symbol; (§), pbrR<sub>3</sub> acts on zntA (Rmet_4594); (‡), insertion of Tn6049 in pbrU; (†), czcB truncated by Tn6050-mediated rearrangement (inversion) and insertion of IS1088 between czcL and hns; (*), insertion of ISRme3 in nimA; (‡), insertion of IS1088 between hmyB and hmyA.

<sup>c</sup>CMGI, Cupriavidus metallidurans CH34 Genomic Island; (P), PAGI-2 family.

<sup>d</sup>Mechanisms of HM detoxification: ExP, efflux (P1-ATPase); pCDF, putative CDF-like; seq, periplasmic sequestration; HgRed, reduction of Hg<sup>2+</sup> into Hg<sup>0</sup> which then volatilizes out of the cell; HgTra, tentative transport of Hg<sup>2+</sup>; ExChr, transport of chromate; FS, Fe-SOD (chrC); MFS, major facilitator superfamily permease (i.e., chrP); CDF, cation diffusion facilitator; ExHME, efflux by RND-HME type 1 to 4 (see text); ExHAE, efflux by HAE-RND (unknown substrate); ExCop, extrusion of Cu<sup>2+</sup>; AsRed (ExAs), reduction of HAsO<sub>4</sub><sup>2-</sup> into AsO<sub>2</sub> and efflux of AsO<sub>2</sub>.

<sup>e</sup>Functionality; +, active; (+), partly active; ?, unknown; -, inactive.

doi:10.1371/journal.pone.0010433.t005

The P-type ATPase family of ion pumps [91]. These export systems may act on a range of cations, including Cu<sup>+</sup>, Ag<sup>+</sup>, Cu<sup>2+</sup>, Co<sup>2+</sup>, Cd<sup>2+</sup>, Ni<sup>2+</sup>, Pb<sup>2+</sup> and Zn<sup>2+</sup> [8,10,59,75,80,83,85,90,92,93]. Alternative mechanisms with specificity to other cations or oxyanions include the chromate CrO<sub>4</sub><sup>2-</sup> efflux system [5], the reduction of arsenate HAsO<sub>4</sub><sup>2-</sup> by ArsC reductases followed by the extrusion of the formed arsenite AsO<sub>2</sub><sup>-</sup> by the ArsB permease [94], the CopABCD/PcoABCD mediated periplasmic detoxification for Cu<sup>2+</sup> [95,96], and the reduction of Hg<sup>2+</sup> into metallic mercury [4,59,97,98] (Table 5; Table S2). However, the function and mode of action of many proteins encoded by putative heavy-metal resistance genes remain undetermined. Structural analysis of

proteins at the molecular level offered some mechanistic information (i.e., via protein crystallization, nuclear magnetic resonance analysis, and cryo electron microscopy) for the C. metallidurans proteins CopK (Rmet_6108), CopH (Rmet_6122), and CzcE (Rmet_5976) expressed by pMOL30, and CnrX (Rmet_6206) expressed by pMOL28 [99–102]. In other cases, such data were gained from aa sequence based comparison of closely related proteins, such as for the CzcD protein (Rmet_5979) that greatly resembles the recently studied E. coli YiiP zinc transporter protein [103,104].

It is worth mentioning that RND sytems for HME (Heavy Metal Efflux) were first discovered in C. metallidurans CH34 with

<figure><img src="image_17.png" /></figure>
the archytypes CzcCBA, conferring resistance to Co, Zn and Cd [9,105], and CnrCBA, conferring resistance to Co and Ni [80,106], with their genes, respectively, located on megaplasmids pMOL30 and pMOL28. Such RND-HME complexes comprise three components spanning the complete cell wall and mediating cation/proton antiporter efflux via a chemiosmotic gradient from the cytoplasm towards the periplasm and into the exterior of the cell. The main component of these complexes (i.e. CzcA, CnrA) is associated with the inner membrane and typically contains 12 transmembrane domains. An outer-membrane efflux protein (i.e. CzcC, CnrC) functions as an auxiliary element to export metals and is characterized by a channel formed by 12 beta-sheets extending further into the periplasm by alpha-helices. The third component (i.e. CzcB, CnrB) is a membrane fusion protein (MFP) bridging the two membrane-bound export proteins. This configuration effectively avoids the release of metal ions in the periplasmic space, and ensures their full disposal by the cell. Other chemiosmotic antiporter efflux systems include the NccCBA complex (Ni, Co, Cd) of *C. metallidurans* 31A (formerly known as *Alcaligenes/Achromobacter xylosoxydans*) [107], the CzrCBA complex (Cd and Zn) of *Pseudomonas aeruginosa* [108], and the CusCBA complex (Cu and Ag) of *E. coli* [109]. Remarkably, *C. metallidurans* CH34 possesses 12 such transenvelope RND-HME export devices (Table 5).

In contrast, the cation diffusion facilitator (CDF) family transporters, which act as chemiosmotic ion-proton exchangers, and the P-type ATPases, that use ATP-energy to pump metal ions out of the cytoplasm, are single-subunit systems located in the cytoplasmic membrane. Sometimes, all three types of metal-ion-efflux systems, e.g. RND-HME, P-ATPase, and CDF, occur in the same locus. For instance the *czc* cluster of pMOL30 contains besides the RND-HME module *czcCBA* also *czcD* (Rmet_5979; a CDF protein) and *czcP* (Rmet_5970; a cation efflux P1-ATPase). We and others [8,37,75,76,110,111] suggested that this combination entails much higher resistance to heavy metals and better detoxification of cellular compartments, especially the cytoplasm (thiol pool). Together, there are at least 30 heavy-metal-efflux systems in *C. metallidurans* CH34 encoded by 155 genes in 25 loci (Table 5; Table S2). To our knowledge no other soil bacterium has acquired so many ion efflux systems specialized in removing heavy metals. We think that genomic islands likely played an important role in this procurement. This is most notable for CHR1 (viz. *cdfX pbrR cadA pbrC* in CMGI-1, *hmz* in CMGI-4, and *ars* in CMGI-7), and for the large genomic islands on the megaplasmids pMOL28 and pMOL30 encoding intertwined nested HMR loci.

In addition to this interplay of HMR determinants there is a redundancy of HMR genes across the four replicons of *C. metallidurans* CH34 (Table 5). For instance the two chromosomes of CH34 contain a number of HMR loci, i.e., *chrBAF*, *copSRABCD*, *pbrR-cadA/pbrA-pbrC*, and *czcICBA-RS* that have counterparts on either pMOL28 (*chr*) or pMOL30 (*cop*, *pbr*, *czo*); furthermore their products display a high sequence similarity (i.e., for the products of the *copSRABCD* locus this ranges between 50 and 87% identity over their entire length). Such duplicates may reflect multiple acquisition of the same HMR locus (possibly derived from different lineages), or may represent internal rearrangements generated by DNA mobility. However, not all of these loci (or genes within loci) are functional, as some HMR loci appear to be affected by either a frameshift mutation (e.g. the *merA* gene of CMGI-1 and the *nccB* gene of CMGI-30b) or by partial deletion (e.g. *hmzA* of CMGI-4 and *merP* of CMGI-30a). A number of HMR loci could be extended or updated via MaGe-based annotation (Tables 5 and S2, gene names in bold face). Some of those novel genes are strongly induced by heavy metals (not

shown), most notably *czoJ* (Rmet_5975) and *mmrQ* (Rmet_6143; formerly *orf-167*), and to a lesser extent *copQ* (Rmet6121). We noted previously that *CczJ*, *MmrQ* and *CopQ* have a signal sequence (well defined by SignalP embedded in MaGe) and have a common domain motif, RDXXTDG, repeated 3 to 4 times [10]. A closer look learns that these domains are connected with spacers of defined length and sequence. Three other genes that are highly induced in the presence of metals, *mmmQ* (Rmet_4461), *mmtQ* (Rmet_4187), and *mmsQ* (Rmet_4908), encode proteins with apparent signal sequences and similar domains and spacers. Note that *mmsQ* has an almost identical paralog *mmsQI* (Rmet_5594) thus both genes are covered by the same probe in transcriptomic analysis. The *mmmQ* gene is linked to the *czo* locus on CHR2 (Table 5) displaying excellent synteny with metal efflux loci in the three other *Cupriavidus* genomes (in spite of an *IS1088* insertion between the *mmmQ* and the *czo* genes). In contrast, the other genes *mmtQ*, *mmsQ* and *mmsQI* appear to be unlinked to any HMR locus. The metal induction of these seven genes (*czoJ*, *mmrQ*, *copQ*, *mmmQ*, *mmtQ* and *mmsQ/QI*) follows a varied and overlapping response, with very high induction by some metals and no induction by other metals. For example, *czoJ* and *mmrQ* are strongly induced by *Cd<sup>2+</sup>*, *Cu<sup>2+</sup>*, *Ni<sup>2+</sup>*, and *Zn<sup>2+</sup>*, moderately induced by *Pb<sup>2+</sup>*, and not induced by *Hg<sup>2+</sup>*, while the *mmtQ* gene is highly induced by *Cd<sup>2+</sup>*, *Hg<sup>2+</sup>*, and *Pb<sup>2+</sup>*, only moderately by *Zn<sup>2+</sup>* and not by *Cu<sup>2+</sup>* or *Ni<sup>2+</sup>* (data not shown). Much to our surprise, according to BLAST analyses against GenBank, these genes (with *copQ* as prototype) and their orthologs are restricted to the genera *Cupriavidus* and *Ralstonia*. We are currently extending this analysis using pattern matching software to search for CopQ-like protein sequence motifs across multiple genomes. We are also performing additional transcriptomic studies on these genes induced by a wider range of metals and other types of stress, and are investigating their gene upstream regions for regulatory DNA sequences.

Through genome annotation, we also found additional candidate HMR genes that warrant further investigation. They are genes *dmeF* (Rmet_0198 on CHR1) and *fieF* (Rmet_3406 on CHR2) encoding CDF-type transporters, and the *atmA* gene (Rmet_0391 on CHR1) coding for an ABC-type transporter. According to MaGe data, these genes have orthologues in the three other *Cupriavidus* species and are well-conserved in other bacteria. Interestingly, disrupting *dmeF* in CH34 causes the Cnr and Czc systems of pMOL28 and pMOL30, respectively, to become ineffective against *Co<sup>2+</sup>*, indicating that *dmeF* is essential in cobalt homoeostasis [112]. Correspondingly, *fieF* mutants of strain CH34 showed decreased resistance to *Ni<sup>2+</sup>*, *Co<sup>2+</sup>*, *Cd<sup>2+</sup>* and *Zn<sup>2+</sup>* [112] and *atmA* mutants of strain CH34 exhibit lower resistance to *Ni<sup>2+</sup>* and *Co<sup>2+</sup>* [113].

Clearly, *C. metallidurans* CH34 has a history of colonizing biotopes with moderate to high-levels of bioavailable metals and, via the uptake and reshuffling of HMR loci, adapted very well to such extreme conditions. This adaptation required the dedicated management of incoming HMR systems to avoid functional redundancy (i.e., under stress when resources are scarce), the optimization of plasmid-born genes to counter elevated levels of metal toxicity, and the economical interplay between plasmid- and chromosomal-genes, supported by a large diversity of sigma subunits of RNA polymerase [114]. We expect that whole-genome transcriptomic studies using a wide range of metal cations and oxyanions will reveal additional HMR genes and highlight new interplays between chromosomal and plasmid borne genes. Such analyses also will refine our knowledge about the intricate regulation patterns apparently governing the response to high concentrations of heavy metals.

<figure><img src="image_18.png" /></figure>
the archytypes CzcCBA, conferring resistance to Co, Zn and Cd [9,105], and CnrCBA, conferring resistance to Co and Ni [80,106], with their genes, respectively, located on megaplasmids pMOL30 and pMOL28. Such RND-HME complexes comprise three components spanning the complete cell wall and mediating cation/proton antiporter efflux via a chemiosmotic gradient from the cytoplasm towards the periplasm and into the exterior of the cell. The main component of these complexes (i.e. CzcA, CnrA) is associated with the inner membrane and typically contains 12 transmembrane domains. An outer-membrane efflux protein (i.e. CzcC, CnrC) functions as an auxiliary element to export metals and is characterized by a channel formed by 12 beta-sheets extending further into the periplasm by alpha-helices. The third component (i.e. CzcB, CnrB) is a membrane fusion protein (MFP) bridging the two membrane-bound export proteins. This configuration effectively avoids the release of metal ions in the periplasmic space, and ensures their full disposal by the cell. Other chemiosmotic antiporter efflux systems include the NccCBA complex (Ni, Co, Cd) of *C. metallidurans* 31A (formerly known as *Alcaligenes/Achromobacter xylosoxydans*) [107], the CzrCBA complex (Cd and Zn) of *Pseudomonas aeruginosa* [108], and the CusCBA complex (Cu and Ag) of *E. coli* [109]. Remarkably, *C. metallidurans* CH34 possesses 12 such transenvelope RND-HME export devices (Table 5).

In contrast, the cation diffusion facilitator (CDF) family transporters, which act as chemiosmotic ion-proton exchangers, and the P-type ATPases, that use ATP-energy to pump metal ions out of the cytoplasm, are single-subunit systems located in the cytoplasmic membrane. Sometimes, all three types of metal-ion-efflux systems, e.g. RND-HME, P-ATPase, and CDF, occur in the same locus. For instance the *czc* cluster of pMOL30 contains besides the RND-HME module *czcCBA* also *czcD* (Rmet_5979; a CDF protein) and *czcP* (Rmet_5970; a cation efflux P1-ATPase). We and others [8,37,75,76,110,111] suggested that this combination entails much higher resistance to heavy metals and better detoxification of cellular compartments, especially the cytoplasm (thiol pool). Together, there are at least 30 heavy-metal-efflux systems in *C. metallidurans* CH34 encoded by 155 genes in 25 loci (Table 5; Table S2). To our knowledge no other soil bacterium has acquired so many ion efflux systems specialized in removing heavy metals. We think that genomic islands likely played an important role in this procurement. This is most notable for CHR1 (viz. *cdfX pbrR cadA pbrC* in CMGI-1, *hmz* in CMGI-4, and *ars* in CMGI-7), and for the large genomic islands on the megaplasmids pMOL28 and pMOL30 encoding intertwined nested HMR loci.

In addition to this interplay of HMR determinants there is a redundancy of HMR genes across the four replicons of *C. metallidurans* CH34 (Table 5). For instance the two chromosomes of CH34 contain a number of HMR loci, i.e., *chrBAF*, *copSRABCD*, *pbrR-cadA/pbrA-pbrC*, and *czcICBA-RS* that have counterparts on either pMOL28 (*chr*) or pMOL30 (*cop*, *pbr*, *czo*); furthermore their products display a high sequence similarity (i.e., for the products of the *copSRABCD* locus this ranges between 50 and 87% identity over their entire length). Such duplicates may reflect multiple acquisition of the same HMR locus (possibly derived from different lineages), or may represent internal rearrangements generated by DNA mobility. However, not all of these loci (or genes within loci) are functional, as some HMR loci appear to be affected by either a frameshift mutation (e.g. the *merA* gene of CMGI-1 and the *nccB* gene of CMGI-30b) or by partial deletion (e.g. *hmzA* of CMGI-4 and *merP* of CMGI-30a). A number of HMR loci could be extended or updated via MaGe-based annotation (Tables 5 and S2, gene names in bold face). Some of those novel genes are strongly induced by heavy metals (not

shown), most notably *czeJ* (Rmet_5975) and *mmrQ* (Rmet_6143; formerly *orf-167*), and to a lesser extent *copQ* (Rmet6121). We noted previously that *CczJ*, *MmrQ*, and *CopQ* have a signal sequence (well defined by SignalP embedded in MaGe) and have a common domain motif, RDXXTDG, repeated 3 to 4 times [10]. A closer look learns that these domains are connected with spacers of defined length and sequence. Three other genes that are highly induced in the presence of metals, *mmmQ* (Rmet_4461), *mmtQ* (Rmet_4187), and *mmsQ* (Rmet_4908), encode proteins with apparent signal sequences and similar domains and spacers. Note that *mmsQ* has an almost identical paralog *mmsQI* (Rmet_5594) thus both genes are covered by the same probe in transcriptomic analysis. The *mmmQ* gene is linked to the *cze* locus on CHR2 (Table 5) displaying excellent synteny with metal efflux loci in the three other *Cupriavidus* genomes (in spite of an *IS1088* insertion between the *mmmQ* and the *cze* genes). In contrast, the other genes *mmtQ*, *mmsQ* and *mmsQI* appear to be unlinked to any HMR locus. The metal induction of these seven genes (*czeJ*, *mmrQ*, *copQ*, *mmmQ*, *mmtQ* and *mmsQ/QI*) follows a varied and overlapping response, with very high induction by some metals and no induction by other metals. For example, *czeJ* and *mmrQ* are strongly induced by *Cd<sup>2+</sup>*, *Cu<sup>2+</sup>*, *Ni<sup>2+</sup>*, and *Zn<sup>2+</sup>*, moderately induced by *Pb<sup>2+</sup>*, and not induced by *Hg<sup>2+</sup>*, while the *mmtQ* gene is highly induced by *Cd<sup>2+</sup>*, *Hg<sup>2+</sup>*, and *Pb<sup>2+</sup>*, only moderately by *Zn<sup>2+</sup>* and not by *Cu<sup>2+</sup>* or *Ni<sup>2+</sup>* (data not shown). Much to our surprise, according to BLAST analyses against GenBank, these genes (with *copQ* as prototype) and their orthologs are restricted to the genera *Cupriavidus* and *Ralstonia*. We are currently extending this analysis using pattern matching software to search for CopQ-like protein sequence motifs across multiple genomes. We are also performing additional transcriptomic studies on these genes induced by a wider range of metals and other types of stress, and are investigating their gene upstream regions for regulatory DNA sequences.

Through genome annotation, we also found additional candidate HMR genes that warrant further investigation. They are genes *dmeF* (Rmet_0198 on CHR1) and *fieF* (Rmet_3406 on CHR2) encoding CDF-type transporters, and the *atmA* gene (Rmet_0391 on CHR1) coding for an ABC-type transporter. According to MaGe data, these genes have orthologues in the three other *Cupriavidus* species and are well-conserved in other bacteria. Interestingly, disrupting *dmeF* in CH34 causes the Cnr and Czc systems of pMOL28 and pMOL30, respectively, to become ineffective against *Co<sup>2+</sup>*, indicating that *dmeF* is essential in cobalt homoeostasis [112]. Correspondingly, *fieF* mutants of strain CH34 showed decreased resistance to *Ni<sup>2+</sup>*, *Co<sup>2+</sup>*, *Cd<sup>2+</sup>* and *Zn<sup>2+</sup>* [112] and *atmA* mutants of strain CH34 exhibit lower resistance to *Ni<sup>2+</sup>* and *Co<sup>2+</sup>* [113].

Clearly, *C. metallidurans* CH34 has a history of colonizing biotopes with moderate to high-levels of bioavailable metals and, via the uptake and reshuffling of HMR loci, adapted very well to such extreme conditions. This adaptation required the dedicated management of incoming HMR systems to avoid functional redundancy (i.e., under stress when resources are scarce), the optimization of plasmid-born genes to counter elevated levels of metal toxicity, and the economical interplay between plasmid- and chromosomal-genes, supported by a large diversity of sigma subunits of RNA polymerase [114]. We expect that whole-genome transcriptomic studies using a wide range of metal cations and oxyanions will reveal additional HMR genes and highlight new interplays between chromosomal and plasmid borne genes. Such analyses also will refine our knowledge about the intricate regulation patterns apparently governing the response to high concentrations of heavy metals.

<figure><img src="image_18.png" /></figure>
## Small-molecule transport and protein secretion systems

The four *Cupriavidus* genomes were submitted to the TransportDB [115,116] website (http://www.membranetransport.org/) to identify efflux systems and transporter families by the TransAAP (Transporter Automatic Annotation Pipeline) tool. The TransportDB relational database holds the complete membrane transport complements of 288 bacteria classified into protein families according to the Transport Classification (TC) system [117,118]. The recently updated TC system organizes nearly 5,000 representative transporters in more than 500 families [118] and is freely accessible at http://www.tcdb.org/. From the mode of transport and the mechanisms of energy coupling, TCDB discerns four major classes of transporters: Channels; secondary carriers; primary transporters; and group translocators. Each class is divided in families and subfamilies according to their function, phylogeny, and substrate specificity. In our analysis with TransAAP we consider only five trans-membrane families: 1.A: alpha-Type channels; 2.A: Porters (uniporters, symporters, anti-porters); 3.A: P-P-bond-hydrolysis-driven transporters; 4.A: Phosphotransfer-driven group translocators; and 9.A: Recognized transporters of unknown biochemical mechanism. A more extensive analysis using all 9 TC classes was reported albeit on the unfinished genome sequence [119]. The numbers of transport proteins predicted by our TransAAP analysis were about the same in all four *Cupriavidus* species (Table S3), amounting to 12 to 14% of their total number of proteins. This appears to be typical for β-proteobacteria [120]. In addition the distribution of transporter classes by species is very similar (Table S3). The largest group of transporters (ca. 400 per species) is presented by the porters (TC2.A), closely followed by the primary transporters (TC3.A). The largest families herein are the Major Facilitator Superfamily (MSF) (TC2.A.1) and the ATP-binding Casette (ABC) Superfamily (TC3.A.1) that are involved in the uptake of various nutrients, such as amino acids, sugars, nucleosides and various organic and inorganic anions. They also remove antibiotics, metabolites, and toxic byproducts. A third large group is the Tricarboxylate Transporter (TTT) Family of solute receptors (TC2.A.80) that carry a typical carboxylate-binding motif (the “Bug domain”), and probably is involved in diverse metabolic and transport activities (discussed further under ‘Signal Transduction’). These three large families constitute up to 70% of all predicted transporter proteins in the four *Cupriavidus* strains. Of immediate interest to our research on *C. metallidurans* are the systems involved in transporting transition metals: MIT (1.A.35), CDF (2.A.4), ZIP (2.A.5), RND (2.A.6), CHR (2.A.51), NiCoT (2.A.52), P-type ATPase (3.A.3), MerT (9.A.2), and TerC (9.A.30) (with TC class in parentheses). These systems, their genes, and their regulation were discussed in detail elsewhere [8, 10, 75, 76]. Evidently, the cellular household of *C. metallidurans* CH34 is tuned towards adapting to a wide range of heavy-metal species (see our brief update on HMR genes earlier in this discussion) or to surviving in biotopes that are either occasionally or constitutively rich in heavy metals. Unsurprisingly therefore, *C. metallidurans* and its related strains most often are found in heavily polluted anthropogenic- or industrial-environments [6,8].

Only a few transporter systems are unique to *C. metallidurans* CH34 compared to the other three *Cupriavidus* species (Table S3): they are the 2-Hydroxycarboxylate Transporter (2-HCT) Family (TC2.A.24), and the Iron/Lead Transporter (ILT) Family (TC9.A.10). The former is represented in *C. metallidurans* by gene Rmet_4998 (situated on CHR2 and listed in MaGe as encoding a citrate carrier) while the latter is represented by two members Rmet_5945 and Rmet_6106 (both located on plasmid pMOL30 as part of the *czc* and *pbr* operons, respectively).

Nine transport systems not present in *C. metallidurans* were found in at least one of the other *Cupriavidus* species (indicated in gray in Table S3) but apparently this observation lacks physiological relevance. However, no member of the Monovalent Cation (K+ or Na+):Proton Antiporter-3 (CPA3) Family (TC2.A.63) was detected in *C. metallidurans*, while the other three *Cupriavidus* species have seven to nine predicted transport proteins belonging to this family. A closer examination reveals that these predicted proteins are subunits of a large multicomponent antiporter with their encoding genes organized as a single operon (e.g., denoted as the *mnhABCDEFG* operons in *C. eutrophus* H16 and *C. taivanensis*). Such antiporters were linked in *Bacillus* species with cellular responses to short-term changes in salinity or pH [121]. Interestingly, MaGe synteny maps show that *D. acidovorans* and *B. petrii* have exactly the same set of genes in one single locus and so does *Sinorhizobium meliloti* and *Bacillus halodurans* but not *Ralstonia pickettii* nor any of the three *R. solanacearum* species listed in the MaGe databases.

*C. metallidurans* CH34 possesses fully intact Sec-dependent and Sec-independent (twin-arginine translocation “TAT”) protein export pathways for secreting proteins across the inner/periplasmic membrane. The Sec-dependent pathway consists of a protein translocation channel made by the contact of two heterotrimeric complexes SecYEG (encoded by Rmet_3297, Rmet_3340, and Rmet_0926) and SecDFYajC (encoded by Rmet_2945-47). The SecA and SecB proteins (encoded by Rmet_3118 and Rmet_0254, respectively) cooperatively bind with the signal sequence of secretory proteins that, after translocation, is cleaved off by signal peptidases SPaseI (Rmet_2420) or SPaseII (of which there are three candidate enzymes in *C. metallidurans* i.e. encoded by Rmet_2304, Rmet_2886, and Rmet_5948). Here, an important accessory component is the membrane insertase YidC (Rmet_3613) that may act independently from the Sec translocase and is involved in folding the inner membrane proteins and the biogenesis of respiratory-chain complexes (listed under TC class 2.A.9 in Table S3). Genome analysis of *C. metallidurans* also identified Rmet_3094 encoding a signal recognition particle (SRP54) that acts with FtsY (Rmet_0281) as an alternative to the SecA-SecB pair for targeting protein precursors. The TAT secretion pathway is specialized to translocate proteins that already have undergone folding in the cytoplasm [122]. It consists of the three subunits TatA, TatB and TatC (encoded by Rmet_3235-27; listed under TC class 2.A.64 in Table S3) that form distinct multimeric TatA or TatABC complexes acting together at the point of translocation.

Extracellular secretion of folded proteins across the outer membrane depends on the Type II Secretion System (T2SS), also often referred to as the “general secretion pathway” (gsp). In proteobacteria this apparatus comprises 12 to 16 different gene products forming a multiprotein complex spanning the periplasmic compartment; it shares many features with the type IV pilus biogenesis system, including the ability to assemble a pilus-like structure. There are two clusters of *gsp* genes in *C. metallidurans* CH34. The largest cluster of 11 genes *gspGHIJKLMNDEF* (Rmet_3384-94) is situated on CHR1 and is well conserved in the four *Cupriavidus* species, while CHR2 (Rmet_4704 through Rmet_4713) supports another smaller cluster consisting of *gspD*, *gspEFG*, two additional copies of *gspG*, and four conserved hypothetical genes (three of which predictably code for trans-membrane proteins). Lateral gene transfer may have allowed the acquisition of this genomic region, which does not display any synteny with the genomes of the other three *Cupriavidus* species. Interestingly, this cluster of genes is flanked by two IS elements in *R. solanacearum* GMI1000. We detected around 30 genes required

<figure><img src="image_19.png" /></figure>
## Small-molecule transport and protein secretion systems

The four *Cupriavidus* genomes were submitted to the TransportDB [115,116] website (http://www.membranetransport.org/) to identify efflux systems and transporter families by the TransAAP (Transporter Automatic Annotation Pipeline) tool. The TransportDB relational database holds the complete membrane transport complements of 288 bacteria classified into protein families according to the Transport Classification (TC) system [117,118]. The recently updated TC system organizes nearly 5,000 representative transporters in more than 500 families [118] and is freely accessible at http://www.tcdb.org/. From the mode of transport and the mechanisms of energy coupling, TCDB discerns four major classes of transporters: Channels; secondary carriers; primary transporters; and group translocators. Each class is divided in families and subfamilies according to their function, phylogeny, and substrate specificity. In our analysis with TransAAP we consider only five trans-membrane families: 1.A: alpha-Type channels; 2.A: Porters (uniporters, symporters, anti-porters); 3.A: P-P-bond-hydrolysis-driven transporters; 4.A: Phosphotransfer-driven group translocators; and 9.A: Recognized transporters of unknown biochemical mechanism. A more extensive analysis using all 9 TC classes was reported albeit on the unfinished genome sequence [119]. The numbers of transport proteins predicted by our TransAAP analysis were about the same in all four *Cupriavidus* species (Table S3), amounting to 12 to 14% of their total number of proteins. This appears to be typical for β-proteobacteria [120]. In addition the distribution of transporter classes by species is very similar (Table S3). The largest group of transporters (ca. 400 per species) is presented by the porters (TC2.A), closely followed by the primary transporters (TC3.A). The largest families herein are the Major Facilitator Superfamily (MSF) (TC2.A.1) and the ATP-binding Casette (ABC) Superfamily (TC3.A.1) that are involved in the uptake of various nutrients, such as amino acids, sugars, nucleosides and various organic and inorganic anions. They also remove antibiotics, metabolites, and toxic byproducts. A third large group is the Tricarboxylate Transporter (TTT) Family of solute receptors (TC2.A.80) that carry a typical carboxylate-binding motif (the “Bug domain”), and probably is involved in diverse metabolic and transport activities (discussed further under ‘Signal Transduction’). These three large families constitute up to 70% of all predicted transporter proteins in the four *Cupriavidus* strains. Of immediate interest to our research on *C. metallidurans* are the systems involved in transporting transition metals: MIT (1.A.35), CDF (2.A.4), ZIP (2.A.5), RND (2.A.6), CHR (2.A.51), NiCoT (2.A.52), P-type ATPase (3.A.3), MerT (9.A.2), and TerC (9.A.30) (with TC class in parentheses). These systems, their genes, and their regulation were discussed in detail elsewhere [8, 10, 75, 76]. Evidently, the cellular household of *C. metallidurans* CH34 is tuned towards adapting to a wide range of heavy-metal species (see our brief update on HMR genes earlier in this discussion) or to surviving in biotopes that are either occasionally or constitutively rich in heavy metals. Unsurprisingly therefore, *C. metallidurans* and its related strains most often are found in heavily polluted anthropogenic- or industrial-environments [6,8].

Only a few transporter systems are unique to *C. metallidurans* CH34 compared to the other three *Cupriavidus* species (Table S3): they are the 2-Hydroxycarboxylate Transporter (2-HCT) Family (TC2.A.24), and the Iron/Lead Transporter (ILT) Family (TC9.A.10). The former is represented in *C. metallidurans* by gene Rmet_4998 (situated on CHR2 and listed in MaGe as encoding a citrate carrier) while the latter is represented by two members Rmet_5945 and Rmet_6106 (both located on plasmid pMOL30 as part of the *czc* and *pbr* operons, respectively).

Nine transport systems not present in *C. metallidurans* were found in at least one of the other *Cupriavidus* species (indicated in gray in Table S3) but apparently this observation lacks physiological relevance. However, no member of the Monovalent Cation (K+ or Na+):Proton Antporter-3 (CPA3) Family (TC2.A.63) was detected in *C. metallidurans*, while the other three *Cupriavidus* species have seven to nine predicted transport proteins belonging to this family. A closer examination reveals that these predicted proteins are subunits of a large multicomponent antiporter with their encoding genes organized as a single operon (e.g., denoted as the *mnhABCDEFG* operons in *C. eutrophus* H16 and *C. taivanensis*). Such antiporters were linked in *Bacillus* species with cellular responses to short-term changes in salinity or pH [121]. Interestingly, MaGe synteny maps show that *D. acidovorans* and *B. petrii* have exactly the same set of genes in one single locus and so does *Sinorhizobium meliloti* and *Bacillus halodurans* but not *Ralstonia pickettii* nor any of the three *R. solanacearum* species listed in the MaGe databases.

*C. metallidurans* CH34 possesses fully intact Sec-dependent and Sec-independent (twin-arginine translocation “TAT”) protein export pathways for secreting proteins across the inner/periplasmic membrane. The Sec-dependent pathway consists of a protein translocation channel made by the contact of two heterotrimeric complexes SecYEG (encoded by Rmet_3297, Rmet_3340, and Rmet_0926) and SecDFYajC (encoded by Rmet_2945-47). The SecA and SecB proteins (encoded by Rmet_3118 and Rmet_0254, respectively) cooperatively bind with the signal sequence of secretory proteins that, after translocation, is cleaved off by signal peptidases SPaseI (Rmet_2420) or SPaseII (of which there are three candidate enzymes in *C. metallidurans* i.e. encoded by Rmet_2304, Rmet_2886, and Rmet_5948). Here, an important accessory component is the membrane insertase YidC (Rmet_3613) that may act independently from the Sec translocase and is involved in folding the inner membrane proteins and the biogenesis of respiratory-chain complexes (listed under TC class 2.A.9 in Table S3). Genome analysis of *C. metallidurans* also identified Rmet_3094 encoding a signal recognition particle (SRP54) that acts with FtsY (Rmet_0281) as an alternative to the SecA-SecB pair for targeting protein precursors. The TAT secretion pathway is specialized to translocate proteins that already have undergone folding in the cytoplasm [122]. It consists of the three subunits TatA, TatB and TatC (encoded by Rmet_3235-27; listed under TC class 2.A.64 in Table S3) that form distinct multimeric TatA or TatABC complexes acting together at the point of translocation.

Extracellular secretion of folded proteins across the outer membrane depends on the Type II Secretion System (T2SS), also often referred to as the “general secretion pathway” (gsp). In proteobacteria this apparatus comprises 12 to 16 different gene products forming a multiprotein complex spanning the periplasmic compartment; it shares many features with the type IV pilus biogenesis system, including the ability to assemble a pilus-like structure. There are two clusters of gsp genes in *C. metallidurans* CH34. The largest cluster of 11 genes *gspGHIJKLMNDEF* (Rmet_3384-94) is situated on CHR1 and is well conserved in the four *Cupriavidus* species, while CHR2 (Rmet_4704 through Rmet_4713) supports another smaller cluster consisting of *gspD*, *gspEFG*, two additional copies of *gspG*, and four conserved hypothetical genes (three of which predictably code for trans-membrane proteins). Lateral gene transfer may have allowed the acquisition of this genomic region, which does not display any synteny with the genomes of the other three *Cupriavidus* species. Interestingly, this cluster of genes is flanked by two IS elements in *R. solanacearum* GMI1000. We detected around 30 genes required

<figure><img src="image_19.png" /></figure>
for type-IV pilus biogenesis and assembly in *C. metallidurans*, some of which occurred in multiple copies across the genome (see also section on motility).

There is no direct evidence for a functional pathogenicity-
related Type III Secretion System (T3SS) in *C. metallidurans*.
However, all the *fli* and *flh* genes encoding the structural proteins
for a basal flagella body, i.e., those that display homology to T3SS
genes through common ancestry, are present (Rmet_3689-99,
Rmet_5258, Rmet_5261, Rmet_5263-64, Rmet_5299, and
Rmet_5301-03) together with all additional *fli*, *flh*, *mot*, and *flg*
genes required for assembling full flagella (see section on motility).

The Type IV Secretion System (T4SS) is a multifunctional
family of translocation pathways that mediate the transfer of
macromolecules (proteins, DNA or DNA-protein complexes)
across the bacterial cell envelope either to the outside medium
or to other cells (prokaryotic or eukaryotic). These systems,
widespread in nature, are evolutionary related to bacterial
conjugation systems and largely contribute to the rapid dissem-
ination among bacterial populations of a range of catabolic- and
metabolic-genes and of antibiotic resistance genes and other
virulence traits. Moreover, because often they are sited on
plasmids or other mobile genetic elements, many T4SS are
laterally transferred between species. We discerned two T4SS gene
clusters in the *C. metallidurans* genome, each with nine genes
homologous to the *vir* genes of the Ti-plasmid of *Agrobacterium
tumefaciens* (Rmet_1336 through _1350 and Rmet_1546 through
_1559). These clusters are located on CHR1 as part of Tn4371-
like mobile genetic elements (MGEs) and may be involved in GI
maintenance or conjugational transfer [66]. We denoted these
MGEs as genomic islands CMGI-2 and CMGI-3; they carry large
sets of genes required for chemolithotrophic growth and degrading
aromatic compounds (addressed elsewhere in this discussion). A
third homologous cluster of ten genes, mostly denoted as *trb* or *tra*
genes (Rmet_6299 through _6309), is present on plasmid
pMOL28 preceeding the *wrd-parAB* genes. These *trb/tra* genes
probably contribute to the maintenance and conjugal transfer of
the pMOL28 plasmid and are part of the plasmid backbone
common to a range of megaplasmids, including pHG1 of *C.
eutrophus* strain H16 and pRALTA of *C. taiwanensis* [7,10].

Sigma factors and transcriptional regulators

An important strategy of bacteria is to use alternative sigma
factors to cope with environmental changes; *C. metallidurans* CH34,
with eighteen sigma factors, appears to be particularly poised to
combat stress. We generally adopted the earlier proposed
description [76,114,123] for annotating these eighteen sigma
factors, except for the housekeeping sigma-70 factor. In the initial
annotation [123], gene Rmet_4661 located on CHR2 was named
as *rpoD* while a second RpoD-like protein (encoded by Rmet_2606
situated on CHR1) was named as RpoG [114]. However, this
latter annotation was cumbersome. In updating the genome
sequence, it became evident that this ambiguity reflected a
sequencing error by which only the carboxyterminal part of RpoG
displays conserved sigma factor domains (regions 1.1 and 1.2),
while the erroneous aminoterminal part does not reveal any
features typical of sigma factors. In this updated version, the
Rmet_2606 protein now displays all proper characteristics of a
sigma-70 housekeeping factor, and has a high homology with the
*E. coli* RpoD (56% aa identity, minLrap 0.99). Consequently we
propose to rename RpoG as RpoD1 and its gene Rmet_2606 as
*rpoD1*. In addition, since the Rmet_4661 protein displays a good
similarity with the *E. coli* RpoD (34% aa identity, minLrap 0.99)
and clearly contains all necessary domains to be a functional

housekeeping sigma factor, we propose renaming RpoD as RpoD2
with its gene Rmet_4661 as *rpoD2* (i.e. replacing *rpoD*).

The presence of multiple sigma-70 housekeeping factors is not unique to *C. metallidurans*: *C. eutrophus* H16 and *C. taiwanensis* both contain two sigma-70 factors (albeit both located on the main replicon) while *C. pinatubonensis* JMP134 contains three copies of this sigma factor (one on each chromosome and one on the megaplasmid) (Table S4). In our study, only *B. xenovorans* contained four sigma-70 genes, all on its second chromosome. Only one copy was detected in the other genomes we analyzed (of *R. solanacearum*, *H. arsenicoxydans*, *Janthinobacterium*, *Polynucleobacter*, *B. petrii*, *D. acidovorans*, and *E. coli*).

Pairwise Smith-Waterman alignments of RpoD protein sequences for all analysed genomes (represented in Figure S5 as a heatmap) showed that *C. metallidurans* RpoD1 (Rmet_2606) forms a tight group with orthologous RpoD proteins from the three other *Cupriavidus* species (Reut_A0888, RALTA_A2226 and H16_A2725) whose genes all sit on CHR1. In contrast, in this same analysis, *C. metallidurans* RpoD2 (Rmet_4661) did not group with any of the other RpoD orthologs in the *Cupriavidus* family (Figure S5); indeed, it had more significant BlastP hits with the RpoD proteins of *Bordetella* and *Burkholderia* species. Therefore we believe that the *C. metallidurans* RpoD2 protein originates from another lineage. The co-existence of multiple sigma-70 factors may be useful for regulatory intricacy. Assuming that these factors would recognize the same set of promoters, slight nuances in factor binding and/or DNA interaction support finely tuned expression patterns such as when cells enter a different growth mode, or at the onset of a particular stress condition. It is unclear how this complexity in transcriptional regulation is attained. It would be interesting to undertake mutational analysis on either one of the *C. metallidurans* rpoD genes (Rmet_2606, Rmet_4661) to check their functionality, to follow cell viability of mutants, and subsequently to measure whole-genome gene expression under a variety of conditions.

Grosse et al. [114] described at least ten sigma factors in *C. metallidurans* CH34 that belong to the extracytoplasmic function (ECF) family, most of which were implicated in HMR. These authors also showed that one particular sigma factor, RpoI (Rmet_1120) is essential for producing siderophores in CH34 (see following section) and that this sigma factor and two other related ones, RpoJ (Rmet_4001) and RpoK (Rmet_4499), were induced by iron depletion. Consequently, the researchers classified these three factors as belonging to the ECF:FeC1 group (type 1) to indicate a similar function in iron assimilation as FeC1 in *E. coli*. Interestingly, the other three *Cupriavidus* species (Cpin, Ceut, and Ctai) do not possess RpoJ or RpoK factors while only *C. taiwanensis* has an RpoI ortholog (RALTA_B1239) albeit with weak similarity (37% aa identity, minLrap 0.98) (Table S4).

The complex transcriptional behavior of *C. metallidurans* not only
is manifested by the many alternative sigma factors, but also its
high count of transcriptional regulators. Using MaGe-based data
for CH34 at least 543 transcription factors were discerned,
corresponding to 9% of all genes in the genome, while the average
percentage transcription factors is only 2.4% for the 673 bacteria
in the DBD database (http://www.transcriptionfactor.org/)
[124,125]. This large difference partially may be explained by
the facts that the relative number of transcriptional regulators
increases with genome size [126], and generally depends on the
organism's life-style [127,128]. Nonetheless, our analysis shows
that only *Bordetella pertussis* strain Tohama I, six *Burkholderia* species,
and *Streptomyces coelicor* display a higher density of transcriptional
regulators. Based on Pfam predictions [129], roughly a quarter of
all regulators in *C. metallidurans* belong to the LysR family (n = 126)

<figure><img src="image_20.png" /></figure>
for type-IV pilus biogenesis and assembly in *C. metallidurans*, some of which occurred in multiple copies across the genome (see also section on motility).

There is no direct evidence for a functional pathogenicity-
related Type III Secretion System (T3SS) in *C. metallidurans*.
However, all the *fli* and *flh* genes encoding the structural proteins
for a basal flagella body, i.e., those that display homology to T3SS
genes through common ancestry, are present (Rmet_3689-99,
Rmet_5258, Rmet_5261, Rmet_5263-64, Rmet_5299, and
Rmet_5301-03) together with all additional *fli*, *flh*, *mot*, and *flg*
genes required for assembling full flagella (see section on motility).

The Type IV Secretion System (T4SS) is a multifunctional
family of translocation pathways that mediate the transfer of
macromolecules (proteins, DNA or DNA-protein complexes)
across the bacterial cell envelope either to the outside medium
or to other cells (prokaryotic or eukaryotic). These systems,
widespread in nature, are evolutionary related to bacterial
conjugation systems and largely contribute to the rapid dissem-
ination among bacterial populations of a range of catabolic- and
metabolic-genes and of antibiotic resistance genes and other
virulence traits. Moreover, because often they are sited on
plasmids or other mobile genetic elements, many T4SS are
laterally transferred between species. We discerned two T4SS gene
clusters in the *C. metallidurans* genome, each with nine genes
homologous to the *vir* genes of the Ti-plasmid of *Agrobacterium
tumefaciens* (Rmet_1336 through _1350 and Rmet_1546 through
_1559). These clusters are located on CHR1 as part of Tn4371-
like mobile genetic elements (MGEs) and may be involved in GI
maintenance or conjugational transfer [66]. We denoted these
MGEs as genomic islands CMGI-2 and CMGI-3; they carry large
sets of genes required for chemolithrophic growth and degrading
aromatic compounds (addressed elsewhere in this discussion). A
third homologous cluster of ten genes, mostly denoted as *trb* or *tra*
genes (Rmet_6299 through _6309), is present on plasmid
pMOL28 preceeding the *wrd-parAB* genes. These *trb/tra* genes
probably contribute to the maintenance and conjugal transfer of
the pMOL28 plasmid and are part of the plasmid backbone
common to a range of megaplasmids, including pHG1 of *C.
eutrophus* strain H16 and pRALTA of *C. taiwanensis* [7,10].

Sigma factors and transcriptional regulators

An important strategy of bacteria is to use alternative sigma
factors to cope with environmental changes; *C. metallidurans* CH34,
with eighteen sigma factors, appears to be particularly poised to
combat stress. We generally adopted the earlier proposed
description [76,114,123] for annotating these eighteen sigma
factors, except for the housekeeping sigma-70 factor. In the initial
annotation [123], gene Rmet_4661 located on CHR2 was named
as *rpoD* while a second RpoD-like protein (encoded by Rmet_2606
situated on CHR1) was named as RpoG [114]. However, this
latter annotation was cumbersome. In updating the genome
sequence, it became evident that this ambiguity reflected a
sequencing error by which only the carboxyterminal part of RpoG
displays conserved sigma factor domains (regions 1.1 and 1.2),
while the erroneous aminoterminal part does not reveal any
features typical of sigma factors. In this updated version, the
Rmet_2606 protein now displays all proper characteristics of a
sigma-70 housekeeping factor, and has a high homology with the
*E. coli* RpoD (56% aa identity, minLrap 0.99). Consequently we
propose to rename RpoG as RpoD1 and its gene Rmet_2606 as
*rpoD1*. In addition, since the Rmet_4661 protein displays a good
similarity with the *E. coli* RpoD (34% aa identity, minLrap 0.99)
and clearly contains all necessary domains to be a functional

housekeeping sigma factor, we propose renaming RpoD as RpoD2
with its gene Rmet_4661 as *rpoD2* (i.e. replacing *rpoD*).

The presence of multiple sigma-70 housekeeping factors is not unique to *C. metallidurans*: *C. eutrophus* H16 and *C. taiwanensis* both contain two sigma-70 factors (albeit both located on the main replicon) while *C. pinatubonensis* JMP134 contains three copies of this sigma factor (one on each chromosome and one on the megaplasmid) (Table S4). In our study, only *B. xenovorans* contained four sigma-70 genes, all on its second chromosome. Only one copy was detected in the other genomes we analyzed (of *R. solanacearum*, *H. arsenicoxydans*, *Janthinobacterium*, *Polynucleobacter*, *B. petrii*, *D. acidovorans*, and *E. coli*).

Pairwise Smith-Waterman alignments of RpoD protein sequences for all analysed genomes (represented in Figure S5 as a heatmap) showed that *C. metallidurans* RpoD1 (Rmet_2606) forms a tight group with orthologous RpoD proteins from the three other *Cupriavidus* species (Reut_A0888, RALTA_A2226 and H16_A2725) whose genes all sit on CHR1. In contrast, in this same analysis, *C. metallidurans* RpoD2 (Rmet_4661) did not group with any of the other RpoD orthologs in the *Cupriavidus* family (Figure S5); indeed, it had more significant BlastP hits with the RpoD proteins of *Bordetella* and *Burkholderia* species. Therefore we believe that the *C. metallidurans* RpoD2 protein originates from another lineage. The co-existence of multiple sigma-70 factors may be useful for regulatory intricacy. Assuming that these factors would recognize the same set of promoters, slight nuances in factor binding and/or DNA interaction support finely tuned expression patterns such as when cells enter a different growth mode, or at the onset of a particular stress condition. It is unclear how this complexity in transcriptional regulation is attained. It would be interesting to undertake mutational analysis on either one of the *C. metallidurans* rpoD genes (Rmet_2606, Rmet_4661) to check their functionality, to follow cell viability of mutants, and subsequently to measure whole-genome gene expression under a variety of conditions.

Grosse et al. [114] described at least ten sigma factors in *C. metallidurans* CH34 that belong to the extracytoplasmic function (ECF) family, most of which were implicated in HMR. These authors also showed that one particular sigma factor, RpoI (Rmet_1120) is essential for producing siderophores in CH34 (see following section) and that this sigma factor and two other related ones, RpoJ (Rmet_4001) and RpoK (Rmet_4499), were induced by iron depletion. Consequently, the researchers classified these three factors as belonging to the ECF:FeC1 group (type 1) to indicate a similar function in iron assimilation as FeC1 in *E. coli*. Interestingly, the other three *Cupriavidus* species (Cpin, Ceut, and Ctai) do not possess RpoJ or RpoK factors while only *C. taiwanensis* has an RpoI ortholog (RALTA_B1239) albeit with weak similarity (37% aa identity, minLrap 0.98) (Table S4).

The complex transcriptional behavior of *C. metallidurans* not only
is manifested by the many alternative sigma factors, but also its
high count of transcriptional regulators. Using MaGe-based data
for CH34 at least 543 transcription factors were discerned,
corresponding to 9% of all genes in the genome, while the average
percentage transcription factors is only 2.4% for the 673 bacteria
in the DBD database (http://www.transcriptionfactor.org/)
[124,125]. This large difference partially may be explained by
the facts that the relative number of transcriptional regulators
increases with genome size [126], and generally depends on the
organism's life-style [127,128]. Nonetheless, our analysis shows
that only *Bordetella pertussis* strain Tohama I, six *Burkholderia* species,
and *Streptomyces coelicor* display a higher density of transcriptional
regulators. Based on Pfam predictions [129], roughly a quarter of
all regulators in *C. metallidurans* belong to the LysR family (n = 126)

<figure><img src="image_20.png" /></figure>
(Table S5). Members of this family typically repress transcription from their own promotor, but activate transcription from a divergent promotor. The products of these activated genes perform a wide range of functions [130] while their activation is mediated by co-inducers, such as extracellular signaling molecules, metabolites, or ions. Other important one-component systems in *C. metallidurans* were GntR (n = 43), TetR (n = 35), AraC (n = 25), MarR (n = 16), and IclR (n = 15) (Table S5), all of which contribute to the transcriptional control of stress responses, carbon metabolism, catabolic pathways, multidrug efflux pumps, biosynthesis of antibiotics, response to osmotic stress and toxic chemicals, differentiation processes, and virulence and pathogenicity.

In terms of one-component metalloregulatory proteins in CH34, three families MerR (n = 10), ArsR (n = 6), and Fur (n = 2) deserve our attention. Although these regulators are evolutionary related in their DNA binding properties, their mode of action is different [131]. While MerR-type regulators nearly exclusively function as transcriptional activators, ArsR-type regulators generally act as repressors that upon metal binding dissociate from the DNA giving way to the RNA polymerase (coined as transcriptional derepression). Fur proteins although being typical repressors can also activate gene expression, either directly, or indirectly via the Fur-dependent repression of a small regulatory antisense RNA. Importantly, there is a wide range in metal selectivity within these three families. Metal sensing in MerR regulators is governed by the effector binding domain in their C-terminal region. Structural diversity in this domain allows MerR family proteins to sense not only various metal ions, e.g., Zn²⁺ by ZntR, Au⁺ by GolS, Cd²⁺ by CadR, Pb²⁺ by PbrR, or Hg²⁺ by MerR, but also oxidative stress, e.g., by SoxR via an [2Fe-2S]²⁺ cluster (reviewed in [131]. Preferential sensing by MerR regulators of one metal ion over another, or monovalent ions over divalent ions, is defined by the positioning of Cys and His but also other conserved residues, mainly in the DNA binding loop. The stringency of this preference probably varies among MerR regulators as some respond to more then one metal. In strain CH34 there are four MerR proteins and three PbrR proteins encoded by the *mer* and *pbr* loci (regulators are underlined in Table 5), and one CupR (Rmet_3523) which responds to Cu⁺ and Au⁺ [35]. The remaining MerR-type regulators in CH34, including SoxR (Rmet_4538), are not physically linked to any structural HMR gene.

The metal sensing in ArsR regulators in contrast is not defined by a single metal binding pocket but is distributed over distinct locations of the repressor involving a number of alpha helices containing key Cys or His residues [131]. Members of this family sense, next to As(III) and Sb(III), a wide variety of divalent metal ions including Zn²⁺, Ni²⁺, Co²⁺, Pb²⁺, and Cd²⁺. Some ArsR family members display sensitivity to multiple metal ions and are able to respond to either monovalent or multivalent ions, probably as a result of evolutionary adaptations in their protein structure particularly in the α3N and α5 helical regions. The CH34 genome enlists only one bona fide ArsR regulator (Rmet_0333 of the *ars* locus on CHR1; Table 5). This repressor has been shown to dissociate from its operator DNA in a metal and valence qualifying manner [94], with three groups of heavy metals displaying decreasing degrees of ArsR dissociation: As(III)/Bi(III)/Co²⁺/Cu²⁺/Ni²⁺; Cd²⁺; and Pb²⁺/Zn²⁺. No ArsR dissociation was observed in the presence of As(V). The other five ArsR family proteins of CH34 appear to be unlinked to any HMR gene cluster except Rmet_5676 which is located adjacent to the defective *nim* locus on CHR2 (Table 5). The two Fur proteins in CH34 are discussed in the next section on siderophores. Besides the MerR, ArsR, and Fur families, no other structural families of one-

component metal responsive regulators are currently recognized in the CH34 genome. Although also LysR, TetR, and MarR families reportedly contain members that are directly involved in metal homeostasis or metal-regulated responses [131], only very few well-documented cases are known sofar. Nonetheless, these three families are represented in CH34 by a total of a few hundred proteins so it may be worthwhile to look also within this pool for further candidate metal response regulators.

Among two-component systems the OmpR system constituted the largest group (n = 33) while the LuxR family (best known for its quorum sensing regulators) also was well represented (n = 41) (Table S5). According to the MiST2 database [132], there are 56 two-component pairs of adjacent genes in the CH34 genome. Several of those have been implicated in HMR, notably two pairs of copRS and czcRS, and one pair each of agrRS, hmzRS, zniRS, and zneRS (underlined in Table 5). From MaGe data and the DBD and MiST2 databases we discovered that the second replicon of *C. metallidurans* (CHR2) carries proportionally almost twice the number of regulatory genes as compared to the main replicon CHR1. This enrichment in regulatory genes agrees well with the earlier notion that CHR2 carries many functions relevant to the survival and responsive behavior of *C. metallidurans* CH34 (Figure 2).

Clearly, the wide diversity of sigma factors and transcriptional regulators in *C. metallidurans* CH34 points to intricate regulatory mechanisms for activating genes in response to environmental stimuli. For instance, microarray analyses of genome-wide gene expression studies on strain CH34 pointed to layered multiple responses when cells are challenged with heavy metals [10]. Similar complex expression patterns were shown for CH34 cells grown under other harsh conditions, including simulated microgravity, growth on basalt, and spaceflight (data not shown). To unravel these regulatory mechanisms and better understand the molecular basis of *C. metallidurans*' ability to survive prolonged periods of stress, we now are focussing on reconstructing the regulatory networks by systematically identifying regulatory motifs in the upstream regions of stress-induced and co-regulated genes.

## Siderophores

At neutral pH and oxic conditions, bacteria cannot readily utilize low-solubility Fe(III) minerals. Accordingly, they synthesise and secrete soluble iron-complexing compounds, called siderophores. *C. metallidurans* CH34 grown under iron-limiting conditions produces a unique phenolate-type siderophore containing neither hydroxamate nor catecholate groups [133]. This siderophore, originally referred to as “alcaligen E”, eventually was classified as a polycarboxylate staphyloferrin B [134] because it resembled the siderophores typically produced by staphylococci. It since was recognized that other organisms produce staphyloferrin B, such as *R. solanacearum* [135]. Notably, *C. metallidurans* staphyloferrin B can interact with Cd²⁺ thereby decreasing the metal’s bioavailability and hence lowering its cellular toxicity in the bacterium. Also, siderophore-producing *C. metallidurans* tested in bioaugmentation-assisted phytoextraction increased, by a factor of 5, the accumulation of Cr(VI) in plant shoots [136]. Future studies on siderophore production in *C. metallidurans* CH34 might thus open up exciting lines of research in heavy metal tolerance and bioremediation.

Genes for staphyloferrin B biosynthesis (Rmet_1109-1118) and two genes encoding ferric/ferrichrome siderophore TonB-dependent receptors BfrC and FcuA (Rmet_1104 and Rmet_1108, respectively) are located on CHR1 within a 52 kb region that lacks synteny with Cpin, Ceut, and Cai. Although a tRNA-Ser gene precedes this region, we could not identify any tyrosine-based site-

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