Organisation of heavy metal resistance genes in the four replicons of Ralstonia metallidurans

The species Ralstonia metallidurans belongs to the phylum -Proteobacteria and includes various isolates of soil bacteria adapted to harsh industrial biotopes. The genome of strain CH34 has been sequenced and revealed the presence of four replicons: two large plasmids pMOL28 (171 Kb) and pMOL30 (234kb), and two very large replicons, one of approx. 3.9Mb that is especially rich in biosynthetic genes, and one of approx. 2.6 Mb, displaying substantial identity with the 2.1 Mb megaplasmid of Ralstonia solanacearum in the region around the replication origin. The organisation in two megareplicons was also found in R. eutropha and R. taiwanensis: these latter two species also harbour a large plasmid that may reflect their adaptation to specific niches via hydrogenotrophy and nitrogen fixation, respectively. In R. metallidurans, the presence of pMOL28 and pMOL30 carrying a variety of metal resistance genes could also be correlated with a specific adaptation to heavy metals. Comparative analysis on the genomic level (against existing databases and through phylogenetic approaches), the transcriptomic level (using RT-PCR e.g. after induction by different metals), and the proteomic level (using 2-D gel electrophoresis and mass spectrometry) indicate that Ralstonia metallidurans carries around 140 genes possibly involved in metal resistance or -detoxification. Although the content in metal resistance genes is above average in other genomes such as those of R. solanacearum or P seudomonas putida, but it is clearly the highest in the genome of R.metallidurans. Most mechanisms for cytoplasm detoxification (via P1-ATPase efflux) and for cytoplasm/periplasm detoxification (via tricomponent chemoosmotic efflux of cations: HME-RND) belong to patrimonium shared by many procaryotic genomes.These genes seem to be associated with the smaller megareplicon of R. metallidurans. Other broadly represented genes include the genes for the detoxification of periplasmic copper (first described as pcoABCD in an E.coli plasmid) or genes involved in the reduction of mercury that are linked to a Tn21 family of transposon. Yet, plasmid pMOL28 and pMOL30 contain some interesting genes (for detoxification) that have not been observed elsewhere and which could help R. metallidurans to cope with heavy metal toxicity in permanently toxic biotopes. Plasmid pMOL28 contains 19 genes in three clusters conferring resistance to nickel and cobalt (cnr), and to chromate (chr) and mercury (mer in Tn4378). The whole group constitutes a 35 kb block flanked by IS1071 on the mer side and a deleted form of IS1071 on the cnr side. In plasmid pMOL30, around 47 genes for heavy metal resistance are clustered in a 90 kb segment also flanked by IS elements and located opposite to the replication origin of the plasmid. Among other minor determinants, this region contains a cluster of nine genes conferring resistance to Cd++, Zn++ and Co++ (czc), a mercury transposon, a cluster of six genes conferring resistance to lead (pbr), three genes (silCBA) that respond to Cu++ and Ag+ in both transcriptomic and proteomic studies, and a large cluster of 14 genes conferring resistance to copper (cop). The great majority of the other 75 genes involved in heavy metal resistance are located in the smaller megareplicon (which also appears to be especially rich in IS elements). Some of these genes are associated to representatives of at least two families of integrative genomic islands: one found in clinical isolates of P. aeruginosa RMGI-2(C) and one qualified by the biphenyl catabolic transposon Tn4371.