Background
Metals are common in our environment and diet. Rocks and soils are the principal and natural sources of metals in the environment. These sources lead to natural background levels in soils, sediments, waters and organisms that are supplemented by many human activities like agriculture (fertilizers, manure and pesticides) and industrial activities (petrochemical, extractive, metallurgic). Some metals are essential trace elements, however, many are toxic to organisms. The essential elements are acquired from the environment depending on the necessities and their uptake is strictly controlled by homeostasis. Failure of these regulation mechanisms due to metal deficiency or toxicity (excess) results in harmful effects on the organism. Mechanisms to resist metals are abundant and widespread in bacteria, with resistance determinants occurring in a few percent in pristine environments to in nearly all isolates in heavily polluted environments
1
.
Cupriavidus metallidurans is a species characterized by multiple metal-resistances
2
3
4
. For C. metallidurans type strain CH34 a substantial part of the metal resistance mechanisms are carried by its two megaplasmids pMOL28 and pMOL30
5
6
. However, next to these specialized plasmids also chromosomally-encoded metal responsive gene clusters have been identified
7
. The C. metallidurans CH34 genome
8
hosts in addition a large diversity of mobile genetic elements (MGEs) including genomic islands (GIs), integrative and conjugative elements, transposons and Insertion Sequence (IS) elements
9
10
. Both the number and diversity of genes related to MGEs is larger in type strain CH34 than in related strains from other Cupriavidus and Ralstonia genera
8
9
. Multiple GIs, in particular on both plasmids, contain genes for heavy metal resistance
6
9
. Its capacity to degrade toluene, to fix carbon dioxide and to oxidize hydrogen is located on Tn4371-like integrative and conjugative elements
9
11
12
.
C. metallidurans strains were often isolated from industrial sites linked to mining-, metallurgical-, and chemical industries
2
13
14
. Next to this, C. metallidurans strains were isolated from environments not typified by metal contamination like, for instance, from different spacecraft-related environments
15
16
17
18
19
, from patients with cystic fibrosis
20
or even as the causative agent of an invasive human infection
21
.
In this study, we aimed to gain insight in the dispersion and horizontal transfer of genes and the evolutionary forces shaping this species. Therefore, whole-genome oligonucleotide DNA microarrays based on the genome of CH34 were used to compare 16 C. metallidurans strains isolated from diverse metal-contaminated biotopes, from other anthropogenic environments and from human cerebrospinal fluid with type strain CH34.
Results
General comparison
Comparative whole-genome hybridization (CGH) was applied to compare sixteen C. metallidurans strains (Table 1) with type strain CH34 and showed that all strains shared a core of 3387
oing equences (CDS), which represents 54.6% of the 6205 oligonucleotide probes present on the CH34 microarray. This common gene pool represented 58.2%, 53.7%, 16.4% and 31.0% of chromosome 1 (CHR1), chromosome 2 (CHR2), pMOL28 and pMOL30, respectively. Thus, although the main replicon (CHR1) carries most of the housekeeping genes
8
, only a slightly higher percentage of CDS is conserved in CHR1 compared to CHR2. This is different when comparing the sequenced Cupriavidus species (C. metallidurans, C. eutrophus, C. pinatubonensis, C. taiwanensis), for which the percentage of common genes is much larger for CHR1 than for CHR2
8
.
<p>Table 1</p>Strains used in this study
Strain
Isolation site
Isolation place
Reference
AS39
Mine tailings
Likasi-Sud, Congo
14
AS167
Mine tailings
Likasi-Sud, Congo
13
AS168
Mine tailings
Likasi-Sud, Congo
14
KT01
Wastewater treatment plant
Göttingen, Germany
22
KT02
Wastewater treatment plant
Göttingen, Germany
23
KT21
Wastewater treatment plant
Göttingen, Germany
22
SV661
Non-ferrous industry
Beerse, Belgium
14
CH34T
Decantation tank, zinc factory
Liège, Belgium
24
CH42
Polluted sediments, zinc factory
Liège, Belgium
13
CH79
Polluted sediments, zinc factory
Liège, Belgium
13
31A
Galvanization tank, metal factory
Holzminden, Germany
23
NE12
Cleanroom Kennedy Space Center
Florida, USA
25
NA1
Water storage system
International Space Station
26
NA2
Contingency water container
International Space Station
26
NA4
Water recovery system
International Space Station
26
43015
Human cerebrospinal fluid
Göteborg, Sweden
CCUG*
45957
Pharmaceutical industry
Sweden
CCUG
*Culture Collection, University of Göteborg, Sweden
Within this conserved group 2760 CDS were assigned to a Cluster of Orthologous Groups (COG). The most abundant COG functional categories were function unknown (Category S; 12.4%), amino acid transport and metabolism (Cat. E; 9.3%), general function prediction only (Cat. R; 9.0%), transcription (Cat. K; 8.5%) and energy production and conversion (Cat. C; 8.4%), respectively.
Cluster analysis based on the pairwise number of overlapping orthologs indicated that all strains can clearly be clustered into two main groups (Figure 1). The strains isolated from the mine tailings in Congo (AS39, AS167 and AS168) grouped together with SV661 isolated from a metal factory in Belgium and with KT01 from a wastewater treatment plant in Germany. This cluster grouped together with a cluster comprising type strain CH34 and the other two isolates KT02 and KT21 from the wastewater treatment plant in Germany forming group I (Figure 1). Group II included the strains isolated from the spacecraft-related environments (NE12, NA1, NA2 and NA4), as well as 31A (Germany) and CH42 (Belgium) from metal factories, the clinical strain 43015 (Sweden), and strain 45957 from pharmaceutics (Sweden). Interestingly, type strain CH34 did not group with the two other strains (CH42 and CH79) isolated from the same site in Belgium.
<p>Figure 1</p>Graphical representation and clustering analysis of C. metallidurans strains
Graphical representation and clustering analysis of C. metallidurans strains. Hierarchically (complete-linkage) clustered heat map based on CGH results of 17 different C. metallidurans strains to a whole-genome oligonucleotide DNA microarray of type strain CH34. The numbers indicated on the heat map are the total number (below diagonal) and percentage (above diagonal) of genes shared between two corresponding strains. The numbers on the diagonal represent the maximal number of genes within one species that gave a detectable signal on the DNA microarray. Bootstrap values (%) from 1,000 times resampling are shown at each dendrogram node.
![]()
Mobile genetic elements
All strains (except CH42) carried one or more megaplasmids (Additional file 1: Figure S1). Concordantly, a good overall conservation of pMOL28 and pMOL30 genes was observed. CGH indicated that for pMOL30 genes in general between 80 and 99% of the probes showed a positive hybridization signal except for CH42 (55.6%), 31A (55.6%), 43015 (51.4%) and 45957 (42.6%) (Figure 2, Table 2). For KT02, 99% or 214 out of 216 gave a positive hybridization signal. For CH42, 31A, 43015 and 45957, a higher percentage of positive hybridization signals was observed for the genomic islands CMGI-30a and CMGI-30b carried by pMOL30 than for the plasmid backbone, indicating that the megaplasmid(s) in these strains probably do not carry a backbone similar to pMOL30. The pMOL28 genes are less conserved than those from pMOL30 but still in general between 71 and 95% with the highest number of positive CGH signals for KT21 (95%) and KT02 (94%) (Figure 2, Table 2). Conservation below 70% was observed for CH42 (29.6%), CH79 (31.6%), NE12 (38.8%) and NA2 (55.9%). More positive hybridization signals were found for the GIs on pMOL28 than the plasmid backbone, indicating that their megaplasmids probably do not carry a backbone similar to pMOL28. Concordantly, strains lacking either the pMOL28 backbone (CH79, NE12 and NA2), the pMOL30 backbone (43015 and 45957) or both (CH42) have only one or no megaplasmid (Additional file 1: Figure S1). Strain 31A, which carries two megaplasmids pTOM8 and pTOM9
27
, showed only a positive hybridization signal with the pMOL28 backbone, indicating that one of both has a backbone related to pMOL28 while the other has a backbone unrelated to pMOL30.
<p>Additional file 1</p>
Figure S1. Plasmid patterns of C. metallidurans strains. Agarose gel electrophoresis of plasmid extracts from strains CH34 (1 and 18), KT01 (2), KT02 (3), KT21 (4), CH42 (5), CH79 (6), AS39 (7), AS167 (8), AS168 (9), 31A (10), SV661 (11), 43015 (12), 45957 (13), NE12 (14), NA1 (15), NA2 (16), and NA4 (17). Lower band represents chromosomal DNA (PDF 33 kb).
Click here for file
<p>Figure 2</p>Graphical representation and clustering analysis of MGEs in C. metallidurans strains
Graphical representation and clustering analysis of MGEs in C. metallidurans strains. Hierarchically (complete-linkage) clustered heat map based on CGH results related to plasmids, previously and newly identified genomic islands of 16 different C. metallidurans strains to a whole-genome oligonucleotide DNA microarray of type strain CH34. Bootstrap values (%) from 1,000 times resampling are shown at each dendrogram node.
![]()
<p>Table 2</p>Relative occurrence of C. metallidurans CH34 genomic islands and plasmids in C. metallidurans strains as indicated by CGH*
CMGI
AS39
AS167
AS168
KT01
KT02
KT21
SV661
CH42
CH79
31A
NE12
NA1
NA2
NA4
43015
45957
CMGI-1
90.8
87.5
81.7
81.7
90.8
91.7
86.7
29.2
62.5
23.3
92.5
63.3
65.8
37.5
50.0
41.7
CMGI-2
86.6
86.6
78.6
83.0
86.6
89.3
83.0
27.7
22.3
15.2
30.4
18.8
24.1
12.5
32.1
25.0
CMGI-3
89.5
77.9
47.7
84.9
91.9
87.2
51.2
26.7
22.1
20.9
30.2
26.7
29.1
20.9
30.2
24.4
CMGI-4
88.7
86.8
81.1
84.9
90.6
92.5
86.8
22.6
47.2
13.2
77.4
66.0
64.2
7.5
88.7
86.8
CMGI-5
100
100
100
100
100
100
100
38.9
22.2
16.7
16.7
22.2
27.8
22.2
50.4
44.4
CMGI-6
46.2
46.2
46.2
38.5
46.2
84.6
46.2
23.1
30.8
15.4
7.7
0
7.7
7.7
0
0
CMGI-7
100
100
100
100
100
100
100
100
69.2
100
61.5
53.8
61.5
100
69.2
53.8
CMGI-8
100
100
100
100
100
100
100
42.9
28.6
28.6
28.6
28.6
28.6
28.6
28.6
28.6
CMGI-9
100
100
100
100
100
100
100
20.0
20.0
13.3
6.7
0
13.3
0
0
0
CMGI-10
100
100
100
100
100
100
100
7.1
7.1
0
0
0
7.1
7.1
7.1
0
CMGI-11
100
100
100
100
100
100
100
14.3
57.1
100
57.1
57.1
57.1
28.6
28.6
28.6
CMGI-12§
100
100
100
100
100
100
100
0
0
0
0
0
11.1
0
22.2
0
CMGI-13§
100
100
100
100
100
100
100
100
31.3
25.0
6.3
6.3
12.5
12.5
18.8
18.8
CMGI-A§
89.0
91.8
82.2
87.7
94.5
97.3
91.8
57.5
24.7
84.9
19.2
19.2
21.9
83.6
63.0
61.6
CMGI-B§
100
95.2
85.7
100
100
100
95.2
28.6
19.0
85.7
61.9
14.3
23.8
85.7
71.4
61.9
CMGI-C§
100
100
100
100
100
100
100
14.3
14.3
14.3
14.3
14.3
14.3
0
14.3
14.3
CMGI-D§
96.2
95.2
86.5
87.5
91.3
95.2
91.3
52.9
31.7
91.3
63.5
26.9
32.7
95.2
82.7
77.9
CMGI-E§
99.0
98.0
98.0
96.0
99.0
97.0
99.0
25.7
17.8
9.9
17.8
10.9
20.8
10.9
10.9
8.9
CMGI-28a
91.1
88.9
86.7
86.7
91.1
95.6
88.9
53.3
44.4
73.3
71.1
86.7
66.7
86.7
93.3
95.6
CMGI-28b
100
100
83.3
91.7
100
100
91.7
33.3
100
8.3
100
100
91.7
8.3
25.0
16.7
CMGI-28c
100
83.3
75.0
83.3
83.3
91.7
83.3
25.0
16.7
33.3
8.3
75.0
50.0
75.0
91.7
91.7
pMOL28
93.4
87.5
77.6
86.2
94.1
95.4
82.2
29.6
31.6
72.4
38.8
81.6
55.9
71.7
90.1
88.2
CMGI-30a
98.4
96.8
91.9
88.7
98.4
98.4
93.5
93.5
83.9
83.9
88.7
85.5
85.5
87.1
77.4
72.6
CMGI-30b
98.8
97.6
90.4
73.5
98.8
98.8
92.8
61.4
100
71.1
95.2
95.2
95.2
96.4
66.3
59.0
pMOL30
98.6
97.2
92.6
83.3
99.1
99.1
94.4
55.6
88.0
55.6
88.4
80.1
80.1
86.6
51.4
42.6
*Percentage of genes related to a particular genomic island (CMGI) or plasmid (pMOL) displaying a positive hybridization signal by CGH. §Genomic islands identified in this study.
The strong conservation of the GIs on pMOL28 (especially CMGI-28a) and pMOL30, which carry all the metal resistance determinants of these plasmids, already indicated a high conservation of the metal responsive clusters (see below).
Next to the megaplasmids and their GIs, CH34 carries 11 previously identified GIs on chromosome 1
6
9
. Based on CGH data analysis for the presence of GIs, the strains could again be divided into two main groups. One group (KT01, KT02, KT21, AS39, AS167, AS168 and SV661) carried almost all GIs identified in CH34, while these GIs were much less abundant in the second group (31A, CH42, CH79, NE12, NA1, NA2, NA4, 43015 and 45957) (Table 2, Figure 2). This clustering (Figure 2) resembled the clustering based on all oligonucleotide probes (Figure 1), which indicated that the presence of GIs is the main source of divergence in these strains. At least 11 strains carried CMGI-1 (or a large part of it). CMGI-1 of CH34 is a 109 kb GI of the PAGI-2 family and is almost 100% identical to PAGI-2 C of P. aeruginosa clone C isolated from a cystic fibrosis patient
9
28
. These data are in agreement with the analysis of the KT and CH strains by Klockgether et al.
28
. The relative occurrence also indicated that CMGI-5 and CMGI-7 to CMGI-11 were highly conserved when present. CMGI-2 to CMGI-4 belong to the Tn4371 family
9
11
12
29
and were previously designated ICETn4371
6054, ICETn4371
6055 and ΔICETn4371
6056, respectively
29
. CMGI-2 (ICETn4371
6054) and CMGI-3 (ICETn4371
6055) are responsible for CH34's ability to grow on aromatic compounds and to fix carbon dioxide, respectively. The presence of these GIs in the C. metallidurans strains is in accordance with their ability to degrade toluene or to grow on hydrogen gas and carbon dioxide (Additional file 2: Table S1). Except for NE12 that apparently lacks the genes involved in degradation of aromatic compounds but is able to degrade toluene and vice versa for AS39 that apparently carries the genes but lacked degradation ability. Therefore, NE12 putatively carries other functional genes, while for AS39 functionality is probably lost. Finally, the presence of other Tn4371-like elements in these strains can not be excluded. Especially since all strains except CH42, 31A and NA4 displayed good conservation of the partial CMGI-4 (ΔICETn4371
6056), which lacks the transfer module
9
, and displayed positive hybridization signals related to the transfer module of CMGI-2.
<p>Additional file 2</p>
Word document containing the Supplementary Tables 1 to 5 (DOC 146 kb).
Click here for file
The mosaic structure of the second chromosome of C. metallidurans CH34, but also other Cupriavidus, Ralstonia and Burkholderia strains, made it complicated to clearly identify GIs on this replicon. Consequently, no GIs on chromosome 2 of C. metallidurans CH34 were defined in a previous study
9
. Here we took advantage of the clear pattern of GI distribution in chromosome 1 over the 16 different C. metallidurans strains to scan chromosome 2 for similar patterns (Additional file 3: Figure S2), which could be an indication for the presence of a genomic island. Five different GIs could be identified (Tables 2 and 3, Figure 2). Interestingly, in CH34 both CMGI-B and CMGI-D flank a copy of the Tn6050 transposon. It was previously shown that in CH34 a chromosomal inversion occurred by recombination between the pair of Tn6050 transposons
9
. Therefore, CMGI-B and CMGI-D are in fact two parts of the same genomic island of 160.7 kb with at one extremity multiple genes coding for phage-related proteins (associated to phages of Ralstonia solanacearum) and at the other extremity a cluster of 22 genes in synteny with R. solanacearum. Accordingly, the relative occurrence of CMGI-B was comparable to that of CMGI-D for each strain, and both were conserved (> 60%) in 13 strains (Table 2, Figure 2). In CH34, the 120 kb CMGI-E carries at one extremity an ISRme3, three Tn7-related genes (tnsABC) and an ISRme11 inserted into tnsC. In fact a very small gene cluster (located approximately 1.89 Mb upstream or +/- 687 kb downstream) in chromosome 2 carried a ISRme3 and two Tn7-related genes (tnsD1 tnsD2). Furthermore, for each strain the presence of these genes coincides with the presence of tnsABC. To determine the relation between these distant Tn7-related genes, the STRING database (version 8.3; http://string.embl.de) was used to find genomes where these genes occur as immediate neighbors. This identified with high confidence (based on STRING parameters) orthologous groups of TnsABCD2 in Anabaena variabilis and of TnsABCD1 in Hahella chejuensis, Shigella sonnei and Idiomarina loihiensis. Additional BLASTP searches indicated the presence of this cluster also in strains more closely related to CH34 such as Burkholderia phymatum and Herminiimonas arsenicoxydans (both β-Proteobacteria). These results evidenced that a putative chromosomal inversion of a very large region (+/- 687 kb) occurred in CH34 by recombination between the pair of ISRme3 elements. In fact, before inversion the region formed a genomic island with a set of Tn7-encoded proteins (TnsABCD1D2) at one extremity and accessory genes putatively involved in the degradation of aromatic compounds.
<p>Additional file 3</p>
Figure S2. Cartographic map of chromosome 2 of the different C. metallidurans strains. Negative hybridization signals are highlighted red. Newly identified putative genomic islands are indicated by blue bars (PDF 23 kb).
Click here for file
<p>Table 3</p>Newly identified putative genomic islands on chromosome 1 and chromosome 2 of C. metallidurans CH34
GI
Replicon
Size (kb)
Coordinates
Features
CMGI-12
CHR1
9.1
2,895,950-2,905,048
Direct repeats (3' end sequence of a
boundary tRNA gene), genes coding for
hypothetical proteins
CMGI-13
CHR1
15.9
2,957,898-2,973,796
Genes involved in polysaccharide
biosynthesis
CMGI-A
CHR2
87.1
813,173-900,292
ISRme1 at one extremity
CMGI-B
CHR2
19.5
1,075,185-1,094,655
Multiple genes coding for phage-related
proteins (associated to phages of Ralstonia
solanacearum)
CMGI-C
CHR2
7.1
1,160,750-1,167,881
Fragment of tyrosine-based site-specific
recombinase at one extremity, direct repeats
(41 bp), gene coding for mannose-binding
lectin
CMGI-D
CHR2
141.2
1,217,667-1,358,908
At one extremity 22 genes in synteny with R.
solanacearum
CMGI-E
CHR2
120.2
2,202,142-2,322,293
Tn7-related genes at one extremity, genes
putatively involved in degradation of
aromatic compounds
The above described observations also encouraged us to scrutinize once more chromosome 1 of CH34 for putative genomic islands (Additional file 4: Figure S3). In addition to the previously identified islands
9
, at least two other putative GIs could be identified (Tables 2 and 3, Figure 2). CMGI-12 carries genes coding for hypothetical proteins and is flanked by direct repeats (3' end sequence of a boundary tRNA gene). CMGI-13 carries genes involved in polysaccharide biosynthesis, however, no genes putatively involved in mobility could be identified.
<p>Additional file 4</p>
Figure S3. Cartographic map of chromosome 1 of the different C. metallidurans strains. Negative hybridization signals are highlighted red. Previously identified genomic islands are indicated by dark blue bars. Newly identified putative genomic islands are indicated by green bars (PDF 33 kb).
Click here for file
The occurrence of insertion sequences in the different C. metallidurans strains followed the same trend as that of the GIs. Based on CGH the same two main clusters could be derived. One group (KT01, KT02, KT21, AS39, AS168, AS169 and SV661) displayed positive hybridization signals for 90 to 98% of the 42 probes related to IS elements, while the second group (31A, CH42, CH79, NE12, NA1, NA2, NA4, 43015 and 45957) only displayed 43 to 62% positive signals (data not shown).
The occurrence of transposon Tn6048 displayed the same pattern as for the IS elements and GIs, however, Tn6049 and the mercury transposons (Tn4378 and Tn4380) appear to be present in all strains (data not shown).
Heavy metal resistance genes
The presence of all gene clusters, which are identified in CH34 as involved in heavy metal detoxification, was evaluated in the genomes of the other strains (Table 4). Almost all metal responsive clusters where found in the other isolates, however, some clusters did not fully correspond to those from CH34. The metal resistance arsenal of CH34 is most conserved in KT21 followed by KT02, AS39, AS167 and SV661. These strains also grouped into a separate cluster based on total genome comparison (Figure 1).
<p>Table 4</p>Occurrence of C. metallidurans CH34 metal resistance gene clusters in C. metallidurans strains as indicated by CGHa, b
Gene cluster in CH34
c, d
AS39
AS167
AS168
KT01
KT02
KT21
SV661
CH42
CH79
31A
NE12
NA1
NA2
NA4
43015
45957
cdfX, pbr2cadA pbrC2,
XAC2
XAC2
XAC2
XAC2
XAC2
XAC2
XAC2
XAC2
pbr3
pbrUb|UaTpbrABCD
R
UbUa
UbRAD
UbRABCD
merTPA'A″
R
R
R
R
R
RPA
RPA
R
R
RA
merTPADE urf-2
merTPADE urf-2
merTΔP
RTΔP
RTΔP
RTΔP
RTΔP
RTΔP
RTΔP
RTΔP
RTΔP
RTΔP
ChrBAF
F
F
BF
BF
BF
BF
chrIBACEONPYZ
IBACEOY
IBACEFOY
Z
BO
O
cusDCBAF
D
D
D
DB
D
D
D
D
silDCBA cusΔF
C
F
F
copABCD
S
S
SC
SC
C
C
SC
SC
C
SC
C
C
C
SC
C
copV-W(*)
IJF
KCIJFOH
FH
czcLubiG czcBa|BbCI
I
I
I
I
I
zntA
czcMNICBADEJ ompP
B
BS
ompP
S
S ompP
S
czcP
ompP
nimBAa|AbC
C
C
BC
BC
C
C
C
BC
BC
C
C
cnrCBAT
HC
HC
YHC
HC
HC
C
HC
YXHCBA
YXHCBAT
C
HC
HC
YXHCA
C
C
nccCB″B'A nreB
CB'
CB
B'
CB"B'A
B"
B"
CB'
CB"B'A
arsPHC1BC2IRM
cupRAC
R
R
agrCBA
S
S
B
hmzhmzBΔA
RB
RB
RBA
RBA
R
R
B
RSBΔA
RSBΔA
RSBΔA
R
R
B
RSBΔA
R
R
hmvCBΔA
CB
C
BA
BA
BA
BA
A
zniABCznePCAB
(#)
Ci
Ci
Ci
Ci
Ci
Ci
Ci
Ci
Ci
Ci
Ci
CiCe
CiCe
Ci
hmyCB|hmyA
C
C
A
A
aOnly genes that did not display positive hybridization signals are shown. bSee ref. 8 for the extensive and descriptive table with metal detoxifications in C. metallidurans CH34. cUnderlined genes are regulators; dInsertion (IS or Tn), frameshift and truncation are indicated by (|), (','') and (Δ), respectively. *Complete cluster is copVTKMNABCDIJGFOLQHEW; #zniC (Ci) and zneC (Ce).
A noticeable phenotypic difference was observed for nickel, chromate and lead resistance compared to CH34 (Additional file 2: Table S2). For most isolates, including CH34, the maximum tolerable nickel concentration is around 4 mM, however, this concentration is much higher for NA4, KT01, KT02, KT21 and 31A (Additional file 2: Table S2). For strains 31A and KT02, it has been shown that the nccYXHCBAN locus is responsible for the resistance to 40 mM nickel
30
. The nccCBA locus in CH34 bears a frameshift in nccB indicating that the ncc genes probably are not functional
5
. The pMOL28-encoded chromate cluster (chrIchrBACEFONPYZ) is almost completely absent in strains CH42 and CH79 (Table 4). Concordantly, the maximum tolerable chromate concentration for these strains is around 0.1 mM, while this concentration is around 0.2 mM for the other strains (Additional file 2: Table S2). Similarly, the pMOL30-encoded lead cluster (pbrABCD) is almost completely absent in strains 43015 and 45957 (Table 4), resulting in a lower MTC compared to the other strains (Additional file 2: Table S2).
Strain CH34 carries several metal resistance genes that are putatively inactivated by frame shift mutation (like nccB, see above), truncation or insertion by an IS element or transposon (Table 4). The presence of three different insertions was evaluated for all isolates, specifically (i) insertion of IS1088 between hmyA and hmyB (coding for part of a resistance-nodulation-cell division (RND) transporter), (ii) insertion of ISRme3 in nimA (coding part of system), and (iii) insertion of Tn6049 in pbrU (encoding a Major Facilitator Superfamily permease).
The presence of these insertions followed the same trend as the occurrence of insertion sequences (described above). Insertions of IS1088, ISRme3 and Tn6049 were present in strains KT01, KT02, KT21, AS39, AS167, AS168 and SV661 but not in strains CH42, CH79, 31A, NE12, NA1, NA2, NA4, 43015 and 45957 (data not shown). The occurrence of an insertion in nimBAC, which codes for a RND transporter putatively involved in the resistance to Ni2+ and Co2+
3
8
, does not clearly influence the MTC of nickel or cobalt, however, the presence of other (plasmid-encoded) Ni2+ and Co2+ resistance determinants could mask this. Similarly, the occurrence of an insertion in pbrU is not univocally related to a lower MTC (Additional file 2: Table S2). Finally, it should be noted that potentially additional metal resistance determinants could be present in the other strains, which are undetectable with a CGH array based on the CH34 genome.
Genes encoding sigma factors and small stress responsive proteins
No less than 18 different sigma factors were identified in C. metallidurans CH34, which enable specific binding of RNA polymerase to promoters, and are activated in response to different environmental changes
8
. Generally, the number and diversity of sigma factor genes in a certain genome relates to the environmental variation allowing growth
31
, which thus indicates that C. metallidurans CH34 is able to respond to a broad range of environmental changes. CGH showed for all C. metallidurans strains positive signals for 12 sigma factors (Additional file 2: Table S3). Hybridization signals below the threshold were found for the housekeeping sigma-70 factors RpoD1 and RpoD2 for strain AS168, KT01 and CH79, and NE12, NA1 and 45957, respectively. Sigma factor CnrH carried by plasmid pMOL28 and encoded by the cnr gene cluster, which is involved in nickel resistance, was only observed for KT21, 31A, NA4, 43015 and 45957. Next to these, differences were observed for RpoR (for strains KT01, CH79, AS39, AS167, AS168, 31A, SV661), RpoP (for strains CH79, NE12, NA1, NA2, 43015 and 45957) and RpoJ (for strain NA4).
Another interesting group of genes in CH34 consists of 19 homologous genes encoding for putative small (between 69 and 165 amino acids) stress responsive proteins in CH34, which are likely to be secreted since all have a distinctive signal peptide and are apparently only found in Cupriavidus and Ralstonia species
8
. Ten of these genes were found to be induced in response to different heavy metals
7
like the pMOL30-encoded copQ
32
and czcJ
33
, while three others were induced by hydrogen peroxide (Saiful Islam Muhammed, pers. comm.). CGH indicated that these genes are well conserved among C. metallidurans strains (Additional file 2: Table S4).
Discussion
Comparative whole-genome hybridization (CGH) was used to compare sixteen C. metallidurans strains isolated from different biotopes with type strain CH34. Although the nature of these sites differs, ranging from pharmaceutical and space industry to metal mining and metal industries, waste treatment plants and even human infection, at least the oligotrophic aspect is common.
The global comparison indicated that while chromosome 1 is the ancestral replicon of the Cupriavidus genus, chromosome 2 appears to be more specific to and conserved within the C. metallidurans species. These results are in agreement with the study of Bavishi and colleagues
34
, which indicated that chromosome 2 evolves faster than chromosome 1, leading to different conservation on inter- and intraspecies level. This global comparison also indicated that strain CH34 is more closely related to strains isolated from Congo than with the two other strains (CH42 and CH79) isolated in Belgium. This supports an old assumption that strain CH34 was transported into Belgium by the import of ores from Congo, which was a Belgian colony from 1908 until 1960.
The incidence of MGEs showed a clear pattern as well as evident phenotypes carried by them like the degradation of toluene or the ability to grow on H2 and CO2. No clear correlation was found between the occurrence of some MGEs and isolation site characteristics and location (geographic). However, it was apparent that strains carried either most of the MGEs or only a few. Interestingly, strains isolated from more hygienic settings like clinical, pharmaceutical or spacecraft environments carried almost no MGEs contrary to strains isolated from sites related to raw materials or environmental sources. This could indicate that either acquisition or loss of these MGEs was advantageous at one point enabling their spread in numerous environments and locations. For example, the MGEs related to hydrogenotrophy and degradation of toluene may recall to the volcanic origin
35
36
37
and such MGEs may have been dispensable and lost in technologized environments. Whereas MGEs with a putative link (however without direct evidence at this time) to opportunistic infection were maintained, such as CMGI-1, which is almost identical to the PAGI-2 C island identified in Pseudomonas aeruginosa clone C (isolated from a cystic fibrosis patient), and CMGI-11, which carries fimbrial genes.
The clear presence or absence of a multitude of MGEs in this group of C. metallidurans strains allowed us to further scrutinize the genome of CH34 for MGEs, especially in the second chromosome. At least five additional regions could be identified with one island formed by a Tn7-like transposon carrying accessory genes putatively involved in the degradation of certain aromatic compounds. Genomic islands formed by Tn7-like transposons have been identified in H. chejuensis, S. sonnei and I. loihiensis, in which Tn7 inserted into the attTn7 site adjacent to a glmS gene
38
. In C. metallidurans CH34 this Tn7-like element is not found within the attTn7 site and in addition lacks the tnsE gene and possesses two distinct tnsD genes. Parks and Peters
39
showed that the presence of two distinct tnsD genes is common in Tn7-like elements that are not found within the attTn7 site and hypothesized that one of the TnsD proteins might actually allow non-specific target site recognition.
Both the CGH and physiological data indicated that the heavy metal resistance determinants identified in C. metallidurans CH34 are well conserved among other C. metallidurans strains. This strong conservation was also observed for genes encoding small stress responsive proteins and sigma factors, of which at least a part are involved in metal resistance
7
40
.
The incidence of these metal resistance determinants could, however, not be directly related to their isolation source (biotype) nor location (geographic). This indicates that these resistance determinants are probably acquired earlier in evolution, which is consistent with the hypothesis that toxic metal resistance systems are preexistent to the recent anthropogenic activities and arose soon after life began, in a world already polluted by volcanic activities
41
. However, taking into account that most of the metal determinants are on the native megaplasmids and the GIs thereon, it could be argued that anthropogenic activities and technologized environments provided a selective pressure for the conservation of these determinants or even the acquisition of some, considering both the arsenal of determinants as well as the level of resistance to metals. Putatively, these determinants may even render a higher fitness in infections as these megaplasmids, despite their fitness cost, are also present in strains isolated from human infections. The structural and functional characteristics that metal resistance systems share with antibiotic resistance systems could be significant for this
42
.