Molecular characterization of cadmium-resistant Cupriavidus spp. and Ralstonia solanacearum isolated from soil and plants in Taiwan
Abstract
Cadmium is a natural heavy-metal element. It is highly toxic and a vital industrial pollutant to the environment. In order to survive in heavy-metal polluted environments, some bacteria can withstand high concentrations of heavy metals in environment due to their specific mechanisms, including the transport of heavy metal ions out of the cell. Three kinds of membrane-bound-proteins are known to participate in this transport. The objective of this study was to determine the cadmium-resistant properties and possible mechanism among strains of Cupriavidus metallidurans (= Ralstonia eutropha), C. taiwanensis (=R. taiwanensis) and Ralstonia solanacearum isolated from soil and plants in Taiwan. Strains tested include six strains, rcd 6, 8, 19 (C. metallidurans) and rcd 12, 14, 21 (C. taiwanensis), from cadmium-polluted soil, seventeen strains, nod 1 to nod 5 and nod s1 to s12 (C. taiwanensis) from root nodules of two Mimosa species (M. pudica and M. diplotricha), and ten strains of R. solanacearum isolated from different diseased plants. Sequence analysis of 16S-23S rDNA ITS and nifH gene regions were used to confirm the taxonomic classification of these bacteria. However, the 624-bp PCR product of nifH gene was only amplified from strains nod 1 to nod 3 and nod s1 to s12, but not from other tested strains of Cupriavidus and Ralstonia spp. These tested strains could tolerate cadmium within the range from 0.67 to 6.70 mM. The gene, czcC, thought to encode the outer membrane factor (OMF) of czc efflux system, was found in fifteen tested strains (rcd 6, 8, 12, 14, 19, 21; nod s1 to s6, s9, s11; PSS161) by PCR. Especially, strains rcd 12, 14, and 21 which could tolerate higher cadmium concentration (6.70 mM) harbored the entire czc operon. In addition, the PCR products revealed that cadmium-tolerant strains contained at least a portion of the czc operon and efflux mechanism was confirmed by cadmium uptake test. The results here indicated that cadmium resistant capability in Cupriavidus spp. and R. solanacearum was related to the presence of czcC or czc operon.
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Introduction
Heavy metals most commonly found at contaminated sites are lead (Pb), chromium (Cr), arsenic (As), zinc (Zn), cadmium (Cd), copper (Cu), mercury (Hg), and nickel (Ni). Soils may become contaminated by the accumulation of heavy metals through emissions from the rapidly expanding industrial areas, disposal of high metal wastes, and sewage sludge (Khan et al., 2008). Some bacteria use heavy metals for respiration, and some have evolved mechanisms to detoxify them. Microbes have evolved several mechanisms to tolerate the presence of heavy metals (by either efflux, complexation, or reduction of metal ions) or to use them as terminal electron acceptors in anaerobic respiration. Under normal conditions, essential and non-essential metals are transported by nonspecific entry systems. However, when metal ions are in excess, specific ion efflux protein complexes may be synthesized to aid in the elimination of non-essential metals. Microbial interactions with metals may have several implications for the environment. Microbes may playa large role in the biogeochemical cycling of toxic heavy metals also in cleaning up or remediating metal-contaminated environments (Nies and Silver, 1995; Spain and Alm, 2003).
The sources of cadmium pollution include industries, such as those producing television screens, lasers, paints, cosmetics, batteries, and zinc refining. It is widely distributed in humans, cigarette smoke, welding, and contaminated food and beverages is also the major source of cadmium contamination (Bernhoft, 2013). Cadmium is highly toxic to animals, plants, microorganisms, and humans even at quite low concentrations (Belimov et al., 2005). It has been widely accepted in the model bacterium Cupriavidus metallidurans (=Ralstonia eutropha, R. metallidurans) that these metal resistance mechanisms appear to be cooperative, not metal specific, and are controlled by a complex regulatory network involving several clusters of genes and functions (Maynaud et al., 2013). Cadmium-resistant bacteria, Cupriavidus metallidurans and C. taiwanensis (=R. taiwanensis) were isolated from cadmium-polluted and tainted soil by a waterink factory in Yunlin, Taiwan. According to cadmium tolerance these bacteria were separated into two groups: lower than 400 mg/kg and ranges from 650 to 900 mg/kg, respectively. These cadmium-resistant strains use czc operon, which is located on plasmid or chromosome, to release cadmium off the organism (Chen et al., 2005).
Ralstonia solanacearum is a devastating, soil-borne plant pathogen with a global distribution and an unusually wide host range. The complete genome sequence of R. solanacearum strain GMI1000 has been recently determined and annotated. It is organized in two replicons: a 3.7 Mbchromosome and a 2.1 Mbmegaplasmid. The megaplasmid appears to encode numerous genes that might playa role in the overall fitness of the bacterium or that may provide advantages in diverse environments (for example: flagellum biosynthesis, many essential pathogenicity genes, catabolism of aromatic compounds, copper and cobalt–zinc–cadmium resistance gene clusters (Salanoubat et al., 2002). The objectives of this study were to examine the cadmium tolerance of Cupriavidus species (C. metallidurans and C. taiwanensis) and Ralstonia solanacearum collected from soil and plants in Taiwan and to determine the possible mechanism of theses bacteria resistance to cadmium.