Phenotypic and Genetic Characterization of Temperature-Induced Mutagenesis and Mortality in Cupriavidus metallidurans.

Cupriavidus metallidurans strains display a decreased viability when incubated in rich medium at a temperature of 37°C compared to their normal growth temperature of 30°C, a phenomenon coined "temperature-induced mortality and mutagenesis" (TIMM). To scrutinize this aberrant phenotype further, the contributions of specific inducers and protective agents were determined. Different growth media, including lysogeny broth (LB) and Schatz, and components, including casamino acids, in particular amino acids (proline, cysteine, glycine, glutamine, leucine, histidine and phenylalanine) and ammonium, were found to induce TIMM at 37°C. Sorbitol was found to counteract TIMM. Furthermore, although TIMM is well conserved within the C. metallidurans species, multiple and strain-specific TIMM inducers exist. Twenty-nine percent of the TIMM survivors inherited resistance to TIMM. Whole-genome sequencing of two resistant derivatives revealed an important role of an uncharacterized oxidoreductase, indicating putative metabolic poisoning when grown in high-concentration nitrogen-containing media at 37°C.

Adaptation of Cupriavidus metallidurans CH34 to Toxic Zinc Concentrations Involves an Uncharacterized ABC-Type Transporter.

Cupriavidus metallidurans CH34 is a well-studied metal-resistant ß-proteobacterium and contains a battery of genes participating in metal metabolism and resistance. Here, we generated a mutant (CH34ZnR) adapted to high zinc concentrations in order to study how CH34 could adaptively further increase its resistance against this metal. Characterization of CH34ZnR revealed that it was also more resistant to cadmium, and that it incurred seven insertion sequence-mediated mutations. Among these, an IS1088 disruption of the glpR gene (encoding a DeoR-type transcriptional repressor) resulted in the constitutive expression of the neighboring ATP-binding cassette (ABC)-type transporter. GlpR and the adjacent ABC transporter are highly similar to the glycerol operon regulator and ATP-driven glycerol importer of Rhizobium leguminosarum bv. viciae VF39, respectively. Deletion of glpR or the ABC transporter and complementation of CH34ZnR with the parental glpR gene further demonstrated that loss of GlpR function and concomitant derepression of the adjacent ABC transporter is pivotal for the observed resistance phenotype. Importantly, addition of glycerol, presumably by glycerol-mediated attenuation of GlpR activity, also promoted increased zinc and cadmium resistance in the parental CH34 strain. Upregulation of this ABC-type transporter is therefore proposed as a new adaptation route towards metal resistance.

The impact of insertion sequences on bacterial genome plasticity and adaptability.

Transposable elements (TE), small mobile genetic elements unable to exist independently of the host genome, were initially believed to be exclusively deleterious genomic parasites. However, it is now clear that they play an important role as bacterial mutagenic agents, enabling the host to adapt to new environmental challenges and to colonize new niches. This review focuses on the impact of insertion sequences (IS), arguably the smallest TE, on bacterial genome plasticity and concomitant adaptability of phenotypic traits, including resistance to antibacterial agents, virulence, pathogenicity and catabolism. The direct consequence of IS transposition is the insertion of one DNA sequence into another. This event can result in gene inactivation as well as in modulation of neighbouring gene expression. The latter is usually mediated by de-repression or by the introduction of a complete or partial promoter located within the element. Furthermore, transcription and transposition of IS are affected by host factors and in some cases by environmental signals offering the host an adaptive strategy and promoting genetic variability to withstand the environmental challenges.

Zinc-Induced Transposition of Insertion Sequence Elements Contributes to Increased Adaptability of Cupriavidus metallidurans.

Bacteria can respond to adverse environments by increasing their genomic variability and subsequently facilitating adaptive evolution. To demonstrate this, the contribution of Insertion Sequence (IS) elements to the genetic adaptation of Cupriavidus metallidurans AE126 to toxic zinc concentrations was determined. This derivative of type strain CH34, devoid of its main zinc resistance determinant, is still able to increase its zinc resistance level. Specifically, upon plating on medium supplemented with a toxic zinc concentration, resistant variants arose in which a compromised cnrYX regulatory locus caused derepression of CnrH sigma factor activity and concomitant induction of the corresponding RND-driven cnrCBA efflux system. Late-occurring zinc resistant variants likely arose in response to the selective conditions, as they were enriched in cnrYX disruptions caused by specific IS elements whose transposase expression was found to be zinc-responsive. Interestingly, deletion of cnrH, and consequently the CnrH-dependent adaptation potential, still enabled adaptation by transposition of IS elements (ISRme5 and IS1086) that provided outward-directed promoters driving cnrCBAT transcription. Finally, adaptation to zinc by IS reshuffling can also enhance the adaptation to subsequent environmental challenges. Thus, transposition of IS elements can be induced by stress conditions and play a multifaceted, pivotal role in the adaptation to these and subsequent stress conditions.