How mutagenesis and laboratory conditions affect the genome of the metal-resistant bacterium Cupriavidus metallidurans strain CH34.

Insights into transition metal homeostasis of Cupriavidus metallidurans have relied heavily on the construction and characterization of deletion mutants. To reveal the genetic consequences of these deletions, the genomes of C. metallidurans strain CH34 wild type and 34 of its mutants were analyzed. The genome of C. metallidurans wild type remained stable when the strain was strictly kept under the appropriate conditions. Omission of selection pressure or construction of mutants, however, resulted in three different kinds of mutations. Large deletions affected genomic islands or regions in the vicinity of transposon-associated genes. Some of these large deletions could be assigned to groups of overlapping deletions associated with groups of mutants, for instance, those with deletions in metal-efflux systems. Second, single-nucleotide polymorphisms (SNPs), such as point mutations, small insertions, or deletions, with a high variant frequency, were candidates for suppressor mutations, for example, in the corA1 or yidC genes. The third and novel kind of mutations comprised hypervariable regions, groups of SNPs with low variant frequencies in a small region of up to 70 base pairs that were located upstream or within genes. In the well-studied example involving the central zinc uptake regulator Zur, the hypervariable regions in the zur gene enabled the production of a GTP cyclohydrolase, restoring folate biosynthesis in double mutants. These data demonstrate that mutations de-stabilize the genome of C. metallidurans, accumulating changes that result in adaptation to the absence of the respective deleted gene, in some cases via hypervariable regions.IMPORTANCETo determine how bacteria thrive, they are often isolated from their natural environment and maintained in a laboratory. Mutations are introduced, and these derivatives are characterized phenotypically. Understanding how bacteria and their derivatives adapt to the effects of site-directed mutations, curing of plasmids, or just the laboratory environment is important. We show here that changes in the genomes of Cupriavidus metallidurans mutants occurred in all instances except when the wild type was maintained under selection conditions. The secondary mutations identified may be neutral, but some may affect the outcome of subsequent experiments performed to analyze the phenotypes. Our findings indicate that all generated mutations should undergo complete genomic sequencing. The information gained may deepen our understanding of bacterial life processes.