Max Mergeay

Physiological changes induced in four bacterial strains following oxidative stress.

In order to study the behavior and resistance of bacteria under extreme conditions, physiological changes associated with oxidative stress were monitored using flow cytometry. The study was conducted to assess the maintenance of membrane integrity and potential as well as the esterase activity, the intracellular pH and the production of superoxide anions in four bacterial strains (Ralstonia metallidurans, Escherichia coli, Shewanella oneidensis and Deinococcus radiodurans). The strains were chosen for their potential use in bioremediation. Suspensions of R. metallidurans, E. coli, S. oneidensis and D. radiodurans were submitted to 1 h of oxidative stress (H2O2 at various concentrations from 0 to 880 mM). Cell membrane permeability (propidium iodide) and potential (rhodamine-123,3,3’-dihexyloxacarbocyanine iodide), intracellular esterase activity (fluorescein diacetate), intracellular-reactive oxygen species concentration (hydroethidine) and intracellular pH (carboxy-fluorescein diacetate succinimidyl ester 5-(6)) were monitored to evaluate the physiological state and the overall fitness of individual bacterial cells under oxidative stress. The four bacterial strains exhibited varying sensitivities towards H2O2. However, for all the bacterial strains, some physiological damage could already be observed from 13.25 mM H2O2 onwards, in particular with regard to their membrane permeability. Depending on the bacterial strains, moderate to high physiological damage could be observed between 13.25 mM and 220 mM H2O2. The membrane potential, esterase activity, intracellular pH and production of superoxide anion production were in all four strains considerably modified at high H2O2 concentrations. In conclusion, we show that a range of significant physiological alterations occur when bacteria are challenged with H2O2 and fluorescent staining methods coupled with flow cytometry are used for monitoring the changes induced not only by oxidative stress, but also by other stresses like temperature, radiation, pressure, pH, etc.

Microarray analysis of a microbe–mineral interaction

The weathering of volcanic minerals makes a significant contribution to the global silicate weathering budget, influencing carbon dioxide drawdown and long-term climate control. Basalt rocks may account for over 30% of the global carbon dioxide drawdown in silicate weathering. Micro-organisms are known to play a role in rock weathering yet the genomics and genetics of biological rock weathering are unknown. We apply DNA microarray technology to determine putative genes involved in weathering using the heavy metal-resistant organism, Cupriavidus metallidurans CH34; in particular we investigate the sequestering of iron. The results show that the bacterium does not depend on siderophores. Instead, the up-regulation of porins and transporters which are employed concomitantly with genes associated with biofilm formation suggests that novel passive and active iron uptake systems are involved. We hypothesize that these mechanisms induce rock weathering by changes in chemical equilibrium at the microbe-mineral interface, reducing the saturation state of iron. We also demonstrate that low concentrations of metals in the basalt induce heavy metal-resistant genes. Some of the earliest environments on the Earth were volcanic. Therefore, these results not only elucidate the mechanisms by which micro-organisms might have sequestered nutrients on the early Earth but also provide an explanation for the evolution of multiple heavy metal resistance genes long before the creation of contaminated industrial biotopes by human activity.

Adaptation to Xenobiotics and Toxic Compounds by Cupriavidus and Ralstonia with Special Reference to Cupriavidus metallidurans CH34 and Mobile Genetic Elements

The purpose of this book chapter is to give a view of the relationships between mobile genetic elements and genes related to environmental adaptations, and the development of new catabolic properties but also of other properties linked to the survival in anthropogenic (mostly industrial) environments. The insights and knowledge in the genetics and genomics of the β-proteobacteria belonging to the closely related Cupriavidus and Ralstonia genera, with some focus on the genome of Cupriavidus metallidurans CH34, will be central.

Genomic Context of Metal Response Genes in Cupriavidus metallidurans with a Focus on Strain CH34

Cupriavidus metallidurans CH34 has been studied for over 30 years, mostly because of its resistance to numerous heavy metals. Many of these metal resistance determinants were rapidly associated with native megaplasmids. However, its genome sequencing and whole genome expression profiling not only revealed the complex structure of its multiple replicons and complex responses to metals, but also revealed the presence of unnoticed/unstudied metal resistance determinants on the different replicons. In this chapter, the genomic context of the metal response genes in C. metallidurans CH34 will be described with a focus on its mobilome including insertion sequence elements, transposons, integrative and conjugative elements and genomic islands.

Metal Response in Cupriavidus metallidurans

Cupriavidus metallidurans CH34 displays resistance to a plethora of metals. Its response and underlying genetic determinants are dissected and detailed metal by metal (from arsenic to zinc). An important role for its megaplasmids pMOL28 and pMOL30 is shown, with high level resistance to cadmium, chromate, cobalt, copper, mercury, nickel, lead and zinc mediated by well-known genes for detoxification often accompanied by other functions linked to acute or chronic stress. Nevertheless, metal resistance determinants are also found on the chromid (e.g. to chromate, copper and zinc) as well as on a large genomic island integrated in the chromosome (e.g. to cadmium, lead and mercury), even the core genome participates in certain responses such as to gold or selenium. Next, we summarized the environmental applications, which were developed based on the knowledge gained by studying these different determinants, and in particular biosensors and soil and water bioremediation. Finally, the general transcriptional response of C. metallidurans to sixteen different metals supplied at different concentrations (including acute stress) is discussed within the framework of its intricate regulatory network.The multiple metal-resistant Cupriavidus metallidurans strain CH34 is quite a latecomer on the scene of transposonand plasmid-mediated resistance to heavy metals. Yet, straightaway it was remarkable because of its combined resistance to cadmium, cobalt, zinc, copper, nickel and lead, and the first genetic determinants for tripartite chemiosmotic cation/proton efflux systems and cation diffusion factors. Later, other resistances and interesting satellite genes attracted the attention and the presence of orthologs for most of the metal resistance genes already described in other bacteria became apparent. A long time designated as Alcaligenes eutrophus, this β-proteobacterium belongs to the Burkholderiaceae and got its definitive name after diverse taxonomic vicissitudes. The first isolates of this species were obtained from Belgian industrial sites in the Meuse basin for which the historical and geographical context is briefly sketched to illustrate the interactions between bacteria and human activities, and the possible evolutionary consequences on bacterial genomes especially as far as the association of metal resistance genes with mobile genetic elements is concerned. Other C. metallidurans isolates were found in a variety of industrial sites around the world up to China and Australia and up to the International Space station, and even in clinical isolates. Besides the C. metallidurans CH34 genome, other genomes of C. metallidurans and Ralstonia pickettii strains containing metal resistance genes are available. The C. metallidurans CH34 genome displays a peculiar richness in mobile genetic elements (genomic islands, transposons and insertion sequences spread on the four replicons: the chromosome, the chromid (formerly second chromosome) and the megaplasmids). Genetic and ecological studies on mobile genetic elements and the metal resistance genes carried by them are also significant in the general context of horizontal gene transfer and the dispersion of epidemiological important genetic determinants as those conferring antibiotic resistance or pathogenicity.

Metal Response in Cupriavidus metallidurans : Insights into the Structure-Function Relationship of Proteins

Bacteria such as Cupriavidus metallidurans have developed different strategies for tolerating toxic levels of metal ions. Metal ion resistance requires the contribution of multiple layers of mechanisms, the most efficient being the efflux of the noxious cations out of the cell regulated by transport systems. Structural and functional data from bacterial primary and secondary transporters are outlined and detailed for the corresponding C.metallidurans proteins. Next, the available high-resolution three-dimensional structures of C. metallidurans proteins involved in metal resistance mechanisms are reviewed and their structure-function relationship is discussed.