3. RESULTS
3.4. Cupriavidus metallidurans : evolution of a metal-resistant bacterium
0 20 40 60 80 100
ortholog found orthologs common Cupriavidus cluster paralogs
percentage of all proteins
Fig. 19: Distributions of orthologs and paralogs on different replicons.
The bars show the ratios of orthologs and paralogs in C. metallidurans CH34. Blue bars represent the r atios for the chromosomally encoded proteins, The brown bars show the ratios f or the megaplasmid encoded proteins and yellow and green bars show the r atios for plasmids pMOL28 and pMOL30 respectively.
The category on the left side ( ‘ orthologs f ound’ ) show the r atios of proteins in C. metallidurans CH34, which have an ortholog in at least one of the investigated proteobacteria. The next category ( ‘ orthologs common’ ) shows the r atios of proteins that have orthologs in all investigated str ains. The r are orthologs in the Cupriavidus Cluster and the r atios of par alogs are also shown.
3.4.1. The distribution of paralogs
The total ratios of paralogs (9±3%) were nearly equal in all the investigated proteobacteria (figure 20).
The total frequencies of putative paralogous transporters were also within this 9±3% range. Many paralogs were plasmid- or megaplasmid encoded (31% in C. eutrophus H16; 21% in C. eutrophus JMP134 megaplasmid). In C. metallidurans CH34 are nearly the half of the plasmid pMOL30 encoded proteins (41%) and one quarter of plasmid pMOL28 encoded proteins paralogs but only 10.25% of the chromosomal encoded proteins fall into this group.
0 20 40 60
Cme CH34 Ceu H16 Ceu JMP134 Rso GMI1000
paralogous proteins (percentage)
Fig. 20: Distribution of paralogs
The diagr am shows the r atios of par alogous proteins in the Cupriavidus cluster (C. metallidurans CH34 ( Cme CH34) , C. eutrophus H16 ( Ceu H16) , C. eutrophus JMP134 ( Ceu JMP134) ) and in R. solanacearum GMI1000 ( Rso GMI 1000) . The blue bars show the percentages of par alogs within the entire proteomes, red bars show only the fractions of paralogous putative tr anspor ters, green bars show only the fractions of paralogous proteins, that are encoded at the bacterial plasmids and yellow bars show the percentages of paralogs among tr ansport proteins that are encoded at bacter ial plasmids.
In C. metallidurans CH34 are not only one third of all plasmid encoded proteins but even about the half of all plasmid encoded putative transport proteins paralogs (28.9 % in C. eutrophus H16, 2 % in C. eutrophus JMP134). The plasmid encoded proteins of the Cupriavidus cluster exhibit thus an unusual genomic flexibility and in C. metallidurans CH34 is this flexibility specifically directed to the evolution of transport proteins.
The putative function for the bulk of paralogous transport proteins differs in these bacteria. In C.
eutrophus H16 are paralogs more prevalent among transporters of the Major Facilitator Superfamily (MFS) (2.A.1) (Pao et al., 1998) the ATP-binding Cassette (ABC) Superfamily (3.A.1) (Saurin et al., 1999) and the Proton-translocating Transhydrogenase (PTH) Family (3.D.2) (Saier et al., 2000). In contrast to this have many paralogs in C. metallidurans CH34 been found among the transporters of the Type IV Secretory Pathway (IVSP) Family (3.A.7) (Gauthier et al., 2003) and its plasmids encode many paralogous heavy metal ion transporters.
In preceding investigations have sequence similarities between some of these putative heavy metal transport proteins and similarities between sigma factors in proteobacteria been correlated to the phylogenetic relatedness (16S-rRNA identities) of theses strains (Dräger, 2005).
3.4.2. The distribution of recent / middle aged and old paralogs
For further investigations of orthologous and paralogous relationships can the similarities of biological sequences be compared (reconciled) with the species tree (Sonnhammer and Koonin, 2002). Thus have recent paralogs (inparalogs) been formed by a lineage specific duplication which occurred subsequently to the speciation of an organism from related species. In contrast to this are middle aged and old paralogs (outparalogs) the result of a lineage specific duplication preceding this speciation event and this results in orthologous relationships that are restricted to this phylogenetic lineage (Koonin, 2005).
The outparalogs in the investigated proteobacteria have been subdivided into old paralogs which were duplicated before R. solanacearum GMI1000 has separated from other Burholderiacea and into middle aged paralogs which evolved during the divergence of the Cupriavidus cluster. To identify outparalogs and to shed light into the probable evolutionary timescale of the gene duplication were certain proteomes stepwise removed from consideration. When certain bacteria are taken out of consideration will the corresponding orthologous relationships to their proteins be ignored. This might then result in the occurrence of new paralogous relationships in the remaining organisms (outparalogs). However might not only gene duplication events, but also gene losses and horizontal gene transfer result in the occurrence of outparalogs. The iteration process started with the strain which exhibits the lowest degree of phylogenetic relatedness to C. metallidurans CH34 and was concluded with the most closely related strain. The phylogenetic relationships between the investigated proteobacteria were revealed by the Tree Builder (RDPII) program (Maidak et al., 2001).
The phylogenetic tree (figure 21) was based on multiple sequence alignments of the corresponding 16S-rRNA sequences.
Fig. 21: Phylogenetic tree of the investigated proteobacteria
The Tree Builder ( RDPI I ) progr am was used to create a phylogenetic tree that was derived from 16SrRNA sequences ( Neighbour Joining with bootstr apping). I t indicates the evolutionary relationship between the investigated proteobacteria. The 16SrRNA sequence of E. coli K12 was used as the taxonomic outgroup to find the root f or this tree.
3.4.3. The evolution of metal ion transporters
The CDF (TC 2.A.4), MerTP (TC 9.A.2), MFP (TC 8.A.1), MIT (TC 1.A.35), NiCoT (TC 2.A.52), OMF (1.B.17), OMR (1.B.14), P-ATPase (TC 3.A.3), CHR (2.A.51), HME (RND) (TC 2.A.6.1) and ZIP (2.A.5) transport protein families are specific for heavy metal ions (outlined in table 4).
Tab 4: Important protein families for heavy-metal transport.
TC-Family TC-Family TC # references
OMR The outer membrane receptor Family 1.B.14 ( Locher et al. 1998) OMF The Outer Membrane Factor Family 1.B.17 ( Dong and Mergeay, 1994), MIT The CorA Metal I on Transporter Family 1.A.35 ( Smith and Maguire, 1998) MFS The Major Facilitator Superfamily 2.A.1 ( Saier, 2000)
CDF The Cation Diffusion Facilitator Family 2.A.4 ( Nies et al., 1995) ZIP The Zinc ( Zn
2+
) - Iron ( Fe
2+
) Per mease Family 2.A.5 ( Grotz et al., 1998) RND Resistance, Nodulation, Cell division Family 2.A.6 ( Dinh et al., 1994) CHR The Chromate I on Transporter Family 2.A.51 ( Nies and Brown, 1998) NiCoT The Ni
2+-Co
2+
Tr anspor ter Family 2.A.52 ( Eitinger and Friedr ich, 1991) ABC ATP- Binding Cassette Tr anspor ters 3.A.1 ( Saurin et al., 1999)
P-type ATPases
The P- type ATPase Family 3.A.3 ( Solioz and Vulpe, 1996) MFP The Membr ane Fusion Protein Family 8.A.1 ( Dinh et al., 1994 MerTP The Mer TP Mercuric I on ( Hg
2+
) Per mease Family 9.A.2 Qian et al., 1998 FeoB The Fer rous Iron Uptake Family 9.A.8 ( Kammler et al., 1993).
ILT The Iron/Lead Transporter Family 9.A.10 ( Cao, et al., 2007) 0.1095
0.0298 0.0187
0.0207 0.009
0.008 0.009 0.0032 0.0118 0.0282
0.0178 0.0593
Cupriavidus eutrophus JMP134 Cupriavidus eutrophus H16
Cupriavidus metallidurans CH34 Ralstonia solanacearum GMI1000 Burkholderia cepacia AMMD Burkholderia xenovorans LB400
Escherichia coli K12
Several putative transporters of the MerT, and KUP families and many lead, cadmium and calcium specific P-type ATPases (3.A.3) in C. metallidurans CH34 are probably the product of a recent gene duplication event. Only few (6 to 23%) of the components of CBA-type efflux systems (RND, MFP and OMF) in this organism have orthologs. The most important components of CBA-type-efflux systems are heavy metal efflux (HME) transporters (TC 2.A.6.1) of the RND superfamily (Saier et al., 1994, Dinh et al., 1994). These can be subcategorized into HME1 (Zn
2+
/Co
2+
/Cd
2+
), HME2 (Ni
2+
/Co
2+
), HME3a (Zn
2+
), HME3b (Co
2+
) and HME4 (Cu
+
/Ag
+
) transporters (Nies, 2003). The proteins of HME3/HME4 subfamilies of RND transporters have not recently been duplicated in this organism. Not only the HME4 RND transporters SilA and CusB (Gupta et al., 1999) but also the corresponding MFPs of their respective CBA-type efflux complex are middle aged paralogs and orthologs were most widely distributed in C. eutrophus JMP134 and C. eutrophus H16. These proteins may have thus been formed by a gene duplication event that occurred prior to the divergence of the Cupriavidus cluster. Components of the CBA-type efflux complex CzcCBA for cobalt zinc and cadmium resistance are however old paralogs. The periplasmic metal binding protein CzcE that is also encoded at the czc-operon is a recent paralog (inparalog). The two cobalt and nickel specific homologs of the HME2 subfamily of RND transporters are also recent paralogs. The high genomic flexibility of the HME RND families has obviously been an important factor for the unusual heavy metal resistance in C. metallidurans CH34. The CDF (CzcD, DmeF and FieF), CHR (ChrA
1
/ChrA
3
and ChrA
4N
/ChrA
4c
), MIT transporters and P-ATPases of C. metallidurans CH34 have not extensively been duplicated and the most of these transporters are thus old paralogs.
3.4.4. The conservation of regulators
Many metal ion transporters extrude not only toxic, but also essential cations and their expression is often tightly regulated (van der Lelie et al., 1997). Particularly the group of ECF (extracytoplasmatic function) sigma factors (Raivio and Silhavy, 2001) plays a crucial role in the response to transition metal ions and other cellular stressors (Grosse et al., 2007). Putative regulatory proteins in C.
metallidurans CH34 and the related proteobacteria where identified by applying keyword searches in NCBI and SWISSPROT (Bairoch et al., 1997) databases. The information and sequences from the supplementary material of recent publications (e.g. Grosse et al., 2007; Nies et al., 2006) and the information about the proximity to determinants coding for the efflux system was also used.
An unusual high degree of evolutionary flexibility has already been reported for the sigma factor RpoN (Dräger, 2005). RpoN (nitrogen metabolism) controls operons that have to remain absolutely silent when not needed (Reitzer and Schneider 2001; Nies, 2004).
Several plasmid encoded ECF-sigma factors are unique to C. metallidurans CH34 (e.g. CnrH) and C.
metallidurans has triplicated its main sigma factor for iron homeostasis namely RpoI (Sig680), RpoJ (Sig611) and RpoK (Sig697) (Große et al. 2007). RpoK and RpoJ are recent paralogs. This flexibility
of ECF sigmafactors contrasts to a largely conserved set of chromosomal encoded sigma factors of the Sigma70 and Sigma54 family. C. metallidurans CH34, C. eutrophus H16 and C. eutrophus JMP134 have a nearly equal number of putative two-component-systems. About two third of these regulators in the three organisms are orthologs. Extensive recent gene duplications have not occurred in this family.