Genomic plasticity of Cupriavidus metallidurans in response to environmental stressors

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    Bacteria continuously evolve to survive different environmental challenges and it has always been a point of discussion if selective pressure could precede or promote mutations. Currently, there is increasing evidence that stress can indeed induce genomic plasticity. This increased mutagenesis could facilitate adaptation as, presumably, an increased mutation rate enhances the likelihood that mutations permitting adaptation to the stressor will arise. However, this claim that mutation rate might be promoted by stress is still under debate. One frequently encountered type of mutation results from the hopping of mobile genetic elements (MGEs). It was believed for some time that mobile DNA elements were exclusively deleterious parasites unable to exist outside of a host genome. However, mobile elements are important bacterial mutagenic agents, enabling the host to adapt to new environmental challenges and to colonize new niches. Insertion Sequence (IS) elements, the smallest autonomous MGEs, are of interest as they constitute an important driving force for genomic evolution. Transposition of IS elements have an important impact on the bacterial genome plasticity and concomitant adaptability and phenotypic traits. Furthermore, IS transposition can be modulated by environmental signals and stressors, and can have different outcomes, from simple gene inactivation to constitutive expression or repression of adjacently located genes by delivering IS-specified (partial) promoter or terminator sequences, respectively.
    The soil bacterium Cupriavidus metallidurans CH34, which harbors a multitude of MGEs, including 21 different Insertion Sequence (IS) elements, 22 genomic islands (GI), 19 transposons, conjugative and integrative elements and two plasmids, appears to be well-equipped to adapt to and survive harsh and anthropogenic environments. In this study, three cases were used to evaluate the adaptation potential of C. metallidurans.
    First, the adaptation capability of C. metallidurans CH34 to toxic zinc concentrations was evaluated by performing a directed evolution experiment. Derivatives resistant to 24 mM Zn2+ were isolated and whole-genome sequencing of one mutant, which also exhibited an increased resistance to cadmium, revealed several mutations, including insertion of IS1088 into Rmet_2235, encoding the GlpR repressor. Furthermore, constitutive derepression of the adjacently encoded ABC-type sugar transporter was detected. Next, the mutation conferring resistance was confirmed by complementing and reconstructing the zinc-resistant genotype. Deletion of the ABC-type sugar transporter verified the essential role that the transporter played as the deletion mutant displayed a drastically decreased zinc resistance approximating the parental level. Furthermore, addition of glycerol confirmed that inhibition of GlpR function will lead to increased zinc resistance. It is proposed that GlpR is the transcriptional regulator of this ABC-type sugar transporter and that inhibition of GlpR function will result in derepression of the neighboring transporter enabling the cell to cope with high zinc and cadmium toxicity.
    Second, the interplay between stress and generation of mutations was evaluated by challenging a C. metallidurans AE126, a zinc-sensitive derivate of CH34, to toxic zinc concentrations. C. metallidurans AE126 could readily evolve resistance to 0.8 mM Zn2+ and two populations of zinc-resistant derivatives, differing in the timing of their mutation responsible for acquiring zinc resistance, were observed. For acquiring this zinc resistance, inactivation of the anti-sigma factor CnrYX complex is essential to liberate CnrH and increase cnrCBAT expression and concomitant nonspecific zinc efflux. Derivatives belonging to the second population most probably originated from zinc selection in contrast with those from the first population that probably already existed in the cell population before the selective condition. Screening a large quantity of CnrH-dependent zinc-resistant derivatives revealed active transposition of seven different IS elements, including ISRme5, IS1088, IS1087B, IS1086, IS1090, ISRme3 and ISRme15. Furthermore, zinc/cadmium-induced transcription of the transposase promoters of ISRme5, IS1088 and IS1087B was demonstrated and it was shown that the genomic context of the insertion site had an important influence on promoter activity. To further analyze the genomic plasticity of AE126 to toxic zinc concentrations, a cnrH deletion mutant was constructed and zinc-resistant derivatives were isolated and characterized. In these mutants, expression of cnrCBAT is driven by an outward-directed promoter situated in reallocated ISRme5 or IS1086 elements. Thus, transposition of IS elements can mediate the adaptation of C. metallidurans AE126 to toxic concentrations of zinc ions via insertional inactivation of regulators or by providing outward-directed promoter sequences and, moreover, transposase expression can be specifically induced by metal ions.
    Third, C. metallidurans CH34 is not only known for its resistance to a wide range of metallic elements but also for its unique behavior when exposed to a growth temperature shift from 30 °C to 37 °C on nutrient-rich medium, coined as temperature-induced mortality and mutagenesis (TIMM). This phenotype was further scrutinized by comparing different growth media. Seven different amino acids were found to induce TIMM at 37 °C and moreover, excess of inorganic nitrogen also induced TIMM. In addition to supplementation with particular molecules to a mineral salt medium, a second TIMM induction pathway existed as TIMM could also be promoted by cultivation on a Schatz minimal salt medium. All 21 C. metallidurans strains tested positive for TIMM induction, although they displayed different patterns of amino acid sensitivity, indicating strain-specific pathways. Furthermore, it was demonstrated that different factors could mitigate TIMM induction. Increasing the salt concentration of the minimal salt medium Schatz or addition of sodium azide, inhibiting the enzymatic activity of nitrate reductase, and addition of sorbitol to all TIMM-inducing media mitigated the temperature effect, representing an important role for (i) the cell membrane and (ii) the nitrate reduction pathway. In addition, stable TIMM-survivors could be evolved and it was demonstrated that they displayed a reduced fitness in non-restrictive conditions compared to their parent, indicating that detrimental mutations occurred throughout the TIMM-selection and were subsequently co-selected thanks to a beneficial mutation. Whole-genome sequencing of two TIMM-survivors showed multiple mutations including a gain-of-function missense mutation in a porin and the effect on TIMM resistance was confirmed by complementation assays. Finally, comparing whole-genome expression profiles of a TIMM-resistant strain and its wild type parent cultivated in TIMM-inducing and non-inducing media indicated that (i) the nitrate pathway, (ii) the reductive pentose phosphate pathway and (iii) aberrant amino acid transport and metabolism are probably involved in TIMM induction. Thus, this study further demonstrated that C. metallidurans strains can readily adapt to fluctuating environmental conditions, including environments typified by toxic heavy metal concentrations, and that this adaptation is often mediated by redistribution of IS elements, highlighting the impact of IS elements on the evolution of their hosts. Moreover, it was found that transcription of at least some transposases is stress-inducible, putatively facilitating bacterial adaptation. This study further discovered a new zinc and cadmium resistance determinant, whose overexpression enables C. metallidurans CH34 to survive in high concentration of zinc. Finally, additional pieces of the TIMM puzzle were solved and TIMM-resistant derivatives were characterized.
    Original languageEnglish
    Awarding Institution
    • KU Leuven
    • Van Houdt, Rob, SCK CEN Mentor
    • Aertsen, Abram, Supervisor, External person
    Date of Award2 Jun 2016
    StatePublished - 2 Jun 2016

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