Torsten von Rozycki Æ Dietrich H. Nies
Cupriavidus metallidurans CH34 is a gram-negative bacterium that is frequently found in soils and sediments with a high content of heavy metals and is therefore resistant to multiple heavy metals such as Zn, Cd, Co, Pb, Cu, Hg, Ni and Cr. C. metallidurans CH34 has a large mega-plasmid and two large plasmids (pMOL28 and pMOL30) that have low copy numbers and can be maintained in the cell even without selective pressure. Resistance to these metals is mediated by transmembrane protein complexes, which export cations from the cytoplasm to the exterior of the cell. The main question this work is attempting to answer is: “ How did C. metallidurans CH34 acquire so many metal resistance genes? ”
To answer this question, the authors decided to analyze the genomes of seven bacteria to study the occurrence of orthologous and paralogous proteins coding for metal resistance. All seven bacteria belong to the β-proteobacterial family Burkholderiaceae of the order Burkholderiales. These include the hydrogen oxidizing C. eutrophus strain H16 (Pohlmann et al. 2006) and the xenobiotic degrader C. eutrophus JMP134 along with 2 phytopathogenic bacteria Ralstonia solanacearum strain GMI1000 (Salanoubat et al. 2002) and strain UW551. The last two organisms that were included were Burkholderia xenovorans strain LB400 and Burkholderi cepacia strain AMMD and were taxonomically distinct from Ralstonia and Cupriavidus. For this analysis, a standardized database for transporter proteins TCDB (http://www.tcdb.org/) was used as a reference. The latest releases of protein sequences of all seven strains were obtained from JGI and NCBI. These were then blasted against the TCDB database (Busch and Saier 2002). A total of seven transporter protein classes (channels/pore, electrochemical potential-driven
transporters, primary active transporters, PTS-group translocators, transport electron carriers, accessory factors involved in transport, incompletely characterized transport
systems) were found in all of the seven genomes. These transporter proteins differed from each other based on the method of transport and also on the mechanism of energy utilization (Saier 2000; Saier et al. 2006). It was also seen that the number of transporter proteins per Mb was similar in all of the strains (and most of the plasmids) analyzed. Thus, the authors conclude that metal resistance in C. metallidurans is not due to a higher number of transport proteins.
In the next step the authors analyzed the paralogs in all seven strains. Paralogs arise by gene duplication in an organism. A high percentage of protein coding paralogs were found on the plasmids of CH34 (34%), H16 (31%) and JMP134 (21%). Moreover, half of the transport proteins found on plasmids of CH34 were paralogs. For instance, the plasmid pMOL30 had a higher percentage of paralogous proteins than any of the other plasmids or chromosomes. The authors surmise that evolution of CH34 has been due to the duplication of transport proteins on its plasmids. The same mechanism may have been responsible for the evolution of the strains H16 and JMP134. Orthologs were investigated next. Here also, pMOL30 exhibited an unusually low percentage (17%) of orthologous proteins. This fact along with the high number of paralogs on plasmid pMOL30 may indicate that gene duplication and horizontal gene transfer played important roles in the evolution of this plasmid.
A total of 700 transport proteins were common among the three Cupriavidus strains. The transport proteins of CH34 could be assigned to twenty protein families based on the classification of the TCDB database. The twenty protein families had orthologs in all strains, however; some protein families were present more than once in CH34. Examples include the Mot/Exb complex components that energize active transport across the outer membrane, ABC transport systems, and metal inorganic transport (MIT) systems, RND, MFP and OMF protein families, P-type ATPases, proteins of the major facilitator superfamily (MFS), and components of the type III (TTS) and the type IV (TFS) secretion systems. Since all of the above proteins export cations, this shows that CH34 has twice as many of these proteins as the other six strains.
Next, the number of protein families involved in the transport of transition metals such as CDF, MerTP, MFP, MIT, NiCoT, OMF, OMR, P-type ATPase, CHR, HME/RND, and ZIP protein families was studied in the seven strains. The authors found that CH34 had a much higher number of the above protein families than the other bacterial strains (i.e., 83 compared to between 44 and 69). When genome size was taken into consideration it was shown that CH34 had 12 transition metal transport proteins per Mb while all the other six bacteria had 6–8 such proteins per Mb. Thus CH34 seems to have evolved its metal resistance by horizontal gene transfer and gene duplication.
RND proteins are a superfamily of proteins that are part of multi subunit protein complexes involved in efflux reactions (Tseng et al. 1999). A subgroup of this family called the HME-RND proteins are involved in the efflux of metals. CH34 has twelve HME-RND operons (Nies 2003), while the other six bacteria have fewer than twelve. This means that the number of operons has steadily increased in CH34 probably by horizontal gene transfer. Three of these twelve operons were vigorously expressed in CH34 and code for the following: the chromosomal copper/silver HME4-RND system, cnr for cobalt/nickel resistance on plasmid pMOL28, and czc for cobalt/zinc/cadmium resistance on plasmid pMOL30. The cobalt/zinc/cadmium resistance operon is czcICBA (Nies 2003) which is found not only on pMOL30, but also on CH34 chromosome 2 and has homologs on chromosome 2 of both C. eutrophus strains. Thus the authors conclude that all three strains might have inherited a czcICBA-like operon on chromosome 2 from an ancestral Cupriavidus strain and in CH34 this operon was duplicated onto plasmid pMOL30. Another operon, czcDRSE, (Große et al. 1999, 2004) is located downstream of the czcICBA operon on pMOL30 and encodes the CDF protein CzcD which transports divalent cations. The authors suggest that this operon was probably assembled by the horizontal transfer of czcD and regulatory genes czcRS along with the duplication of the copH gene (from the copper resistance cluster on pMOL30) to form czcE. Since czcE binds copper, it may form a link between the czcDRSE and czcICBA operons. Similarly, nickel/cobalt resistance is encoded on pMOL28 by the cnrYXHCBA operon (Liesegang et al. 1993), which has no homologs on any of the other bacterial strains. This operon, too, may have been acquired by horizontal gene transfer. P-type ATPases form a family of membrane-bound primary transport systems (Fagan and Saier 1994). Strain CH34 contains a high number of 13 predicted P-type ATPases. The other two Cupriavidus strains 7 or 8 orthologs including Ca2+ and Zn2+/Cd2+/Pb2+ exporting enzymes. Cupriavidus metallidurans contains four CHR proteins that export chromate from the cytoplasm (Nies 2003; Nies et al. 1998). This too could have been a result of gene duplication after speciation from the ancestral Cupriavidus strain. During speciation of C. metallidurans CH34 two MerT proteins duplicated into four, yielding three active mercury-detoxification systems. The authors summarize by saying that “ the ancestral Cupriavidus strain might have been a facultatively hydrogen-oxidizing, moderately metal-resistant degrader of aromatic compounds and organic acids rather than a dweller on sugars .” This strain evolved by the acquisition of plasmids such as those that carry hydrogen-oxidizing genes, metal resistance genes such as nickel, cobalt, chromate, and mercury, as well as genes coding for degradation of organic compounds such as 2,4-D. CH34 in particular probably evolved by a combination of horizontal gene transfer and gene duplication events along with rearrangements on Pmol30 which lead to adaptation of this strain to a wide range of metals.
It is now a known fact that horizontal gene transfer plays a crucial role in prokaryotic evolution. Studies such as these are important since they provide evidence of the role of horizontal gene transfer in the evolution of a complex strain such as CH34. Detailed analysis of each operon on the CH34 strain made it possible to trace its origin from the ancestral strain.
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University of Idaho