Genomic Plasticity in Ralstonia eutropha and Ralstonia pickettii: Evidence for Rapid Genomic Change and Adaptation

Terence L. MArsh

关键词: CONFORMATIONAL CHANGES;    COPPER;    DNA;    GENES;    PLASTICITY Genomes to Life;    genomic plasticity;    metal resistance;    filamentous phage;   

DOI  :  10.2172/909163
RP-ID  :  DOE/ER/63141-FinalReport
PID  :  OSTI ID: 909163
Others  :  TRN: US200722%%1021

学科分类：环境科学（综合）

美国|英语

来源: SciTech Connect

【 摘 要 】

The proposed foci of our investigations were on Ralstonia eutropha and Rasltonia pickettii. We have 18 derived lineages of the former as well as their progenitor and eleven isolates of the latter. Our goal was to measure the level of plasticity in these strains and attempt to derive a mechanistic understanding of how genomic plasticity formed. Extensive attempts to reproducibly induce conformational changes in the genome of R. eutropha were unsuccessful. We thought that we had a reasonable lead on this inasmuch as we had shown that the ancestral strain along with many of the derivative lineages exhibited “temperature induced mutation and mortality akin to R. metallodurans. However we were unable to get subtractive hybridization working to the degree that it revealed differences between the lineages. During this time the R. pickettii analysis was proving quite fruitful and so we concentrated our efforts on our analyses of R. pickettii. These strains were isolated from a copper-contaminated lake sediment and were resistant to copper at 800 µg/ml (CuSO4). Our results in the investigation of R. pickettii permitted a view into the adaptation of a beta-proteobacteria to an extreme environment. Our worked revealed that within the same ecosystem two genomovars with structurally different genomes and genome sizes were present and apparently filling similar if not identical niches. The genomovars were detected with REP & BOX-PCR, pulse field gel electrophoresis, and DNA:DNA hybridizations. Moreover there were different metal resistance patterns associated with the different genomovars, one showing resistance to Zn and Cd while the other had resistance to Ni. Five of the isolates had a high-copy number extrachromosomal element that was identified as the replicative form of a filamentous phage. Mature virions were isolated from culture broth using PEG precipitation and CsCl density centrifugation. The DNA associated with the filamentous particles was single stranded and had a sequence identical to the intracellular replicative form. Using PCR targeting phage-specific genes we showed that seven out of the eleven isolates carried the phage. The seven isolates positive for phage were formed one of the genomovar groups, hence the presence of the phage may have generated the divergent lineage. One representative of each of these genomovars was sequence by JGI. While the genomes have not been closed completely, the results so far are provocative. Strain 12J, which carries the phage, revealed four integrated copies of the phage genome, each copy at a different level of divergence from the active replicative form. A comparative analysis of the common genes found in these integrated phage copies revealed that the gene complements were incongruent with one another within a phage copy, suggesting that the copies had become sites of recombination or that the cell had recruited genes for different functions. One of the integrated copies had the exact sequence as the replicative form and we assume this to be the most recent integration event. Evidence for a recent integration was revealed in a repeated sequence element found on each terminus of the phage genome. Finally, these isolates are of interest in bioremediation and reclamation of metals from waste streams. We have determined that each Ralstonia cell is capable of binding 6 x 107 Cu (II) ions and that 90% of the binding occurs within 12 hours of exposure to Cu(II). Our studies have revealed a species with a uniquely fluid genome. This Ralstonia population has been under extreme selective pressure over the past hundred years as it responded to the accumulation of copper in the lake sediment as a result of unregulated mining practices. In addition to the selective pressure of copper, the population was repeatedly infected with a filamentous phage that may have contributed to the divergence of the genomovars. This dynamic population could help reveal the selective forces and their consequences on genome structure in a proteobacterium that has potential for remediation.