Genomic and Physiological Investigation of Heavy Metal Resistance from Plant Endophytic Methylobacterium radiotolerans MAMP 4754, Isolated from Combretum erythrophyllum

Combretum erythrophyllum is an indigenous southern African tree species, a metal hyperaccumulator that has been used as a phytoextraction option for tailing dams in Johannesburg, South Africa. In hyperaccumulators, metal detoxification has also been linked or attributed to the activities of endophytes, and, in this regard, metal detoxification can be considered a form of endophytic behavior. Therefore, we report herein on the identification of proteins that confer heavy metal resistance, the in vitro characterization of heavy metal resistance, and the production of plant growth-promoting (PGP) volatiles by Methylobacterium radiotolerans MAMP 4754. Multigenome comparative analyses of M. radiotolerans MAMP 4754 against eight other endophytic strains led to the identification of zinc, copper, and nickel resistance proteins in the genome of this endophyte. The maximum tolerance concentration (MTC) of this strain towards these metals was also investigated. The metal-exposed cells were analyzed by transmission electron microscopy (TEM). The ethyl acetate and chloroform extracts (1:1 v/v) of heavy metal untreated M. radiotolerans MAMP 4754 were also screened for the production of PGP compounds by Gas Chromatography–Mass Spectroscopy (GC/MS). The MTC was recorded at 15 mM, 4 mM, and 12 mM for zinc, copper, and nickel, respectively. The TEM analysis showed the accumulation of metals in the intracellular environment of M. radiotolerans MAMP 4754, while the GC/MS analysis revealed several plant growth-promoting compounds, including alcohols, phthalate esters, alkenes, ketones, sulfide derivatives, phenols, and thiazoles. Our findings suggest that the genetic makeup of M. radiotolerans MAMP 4754 encodes heavy metal resistant proteins that indicate hyperaccumulator-specific endophytic behavior and the potential for application in bioremediation. The production of plant growth-promoting volatiles in pure culture by M. raditotolerans MAMP 4754 is a characteristic feature for plant growth-promoting bacteria.

Antibiotic-Selected Gene Amplification Heightens Metal Resistance

ABSTRACT The increasing frequency of antibiotic resistance poses myriad challenges to modern medicine. Environmental survival of multidrug-resistant bacteria in health care facilities, including hospitals, creates reservoirs for transmission of these difficult to treat pathogens. To prevent bacterial colonization, these facilities deploy an array of infection control measures, including bactericidal metals on surfaces, as well as implanted devices. Although antibiotics are routinely used in these health care environments, it is unknown whether and how antibiotic exposure affects metal resistance. We identified a multidrug-resistant Enterobacter clinical isolate that displayed heteroresistance to the antibiotic colistin, where only a minor fraction of cells within the population resist the drug. When this isolate was grown in the presence of colistin, a 9-kb DNA region was duplicated in the surviving resistant subpopulation, but surprisingly, was not required for colistin heteroresistance. Instead, the amplified region included a three-gene locus (ncrABC) that conferred resistance to the bactericidal metal, nickel. ncrABC expression alone was sufficient to confer nickel resistance to E. coli K-12. Due to its selection for the colistin-resistant subpopulation harboring the duplicated 9-kb region that includes ncrABC, colistin treatment led to enhanced nickel resistance. Taken together, these data suggest that the use of antibiotics may inadvertently promote enhanced resistance to antimicrobial metals, with potentially profound implications for bacterial colonization and transmission in the health care environment. IMPORTANCE To inhibit bacterial transmission and infection, health care facilities use bactericidal metal coatings to prevent colonization of surfaces and implanted devices. In these environments, antibiotics are commonly used, but their effect on metal resistance is unclear. The data described here reveal that exposure of a human isolate of Enterobacter cloacae to a last-line antibiotic, colistin, resulted in a DNA amplification that does not confer antibiotic resistance but instead facilitates resistance to the toxic metal nickel. This highlights a novel aspect of antibiotic and metal interplay. Concerningly, these data suggest the use of antibiotics could in some cases promote bacterial survival and colonization in the health care environment and ultimately increase transmission and infection of patients.

Effect of aluminum in Bacillus megaterium nickel resistance and removal capability

The increasing water pollution by heavy metals is considered an alarming situation worldwide, due to the adverse impact they cause in ecosystems and human health. Although conventional techniques are available to diminish the metal concentration present in water bodies, they offer disadvantages, like inefficient metal removal, toxic sludge generation, and high operating costs. In contrast, biotechnological approaches may render a viable alternative, since they offer lower environmental impacts and operating costs, and also higher removal efficiencies when metals are present in small concentrations. It has been shown that the simultaneous presence of more than one metal can generate synergistic, additive or antagonistic effects, thus affecting their removal, and it has been previously demonstrated that B. megaterium strain MNSH1-9K-1 possesses the ability to remove metals present in liquid and solid wastes. Therefore, the goal of the present work was to study B. megaterium MNSH1-9K-1 Ni resistance and removal properties in liquid medium, and to evaluate the variation of these abilities in the presence of another toxic metal, namely Al, which is also commonly found in liquid wastes. To this end, B. megaterium was grown in LB medium with the addition of Ni and/or Al at diverse concentrations, and both metal resistance and Ni removal capabilities were assayed by viable count, and Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES), respectively. The results obtained strongly suggest that B. megaterium MNSH1-9K-1 presents more susceptibility to Ni than to Al, and that Ni removal is enhanced by the presence of Al.

Regulation of the Cobalt/Nickel Efflux OperondmeRFin Agrobacterium tumefaciens and a Link between the Iron-Sensing Regulator RirA and Cobalt/Nickel Resistance

ABSTRACTTheAgrobacterium tumefaciensC58 genome harbors an operon containing thedmeR(Atu0890) anddmeF(Atu0891) genes, which encode a transcriptional regulatory protein belonging to the RcnR/CsoR family and a metal efflux protein belonging to the cation diffusion facilitator (CDF) family, respectively. ThedmeRFoperon is specifically induced by cobalt and nickel, with cobalt being the more potent inducer. Promoter-lacZtranscriptional fusion, an electrophoretic mobility shift assay, and DNase I footprinting assays revealed that DmeR repressesdmeRFtranscription through direct binding to the promoter region upstream ofdmeR. A strain lackingdmeFshowed increased accumulation of intracellular cobalt and nickel and exhibited hypersensitivity to these metals; however, this strain displayed full virulence, comparable to that of the wild-type strain, when infecting aNicotiana benthamianaplant model under the tested conditions. Cobalt, but not nickel, increased the expression of many iron-responsive genes and reduced the induction of the SoxR-regulated genesodBII. Furthermore, control of iron homeostasis via RirA is important for the ability ofA. tumefaciensto cope with cobalt and nickel toxicity.IMPORTANCEThe molecular mechanism of the regulation ofdmeRFtranscription by DmeR was demonstrated. This work provides evidence of a direct interaction of apo-DmeR with the corresponding DNA operator siteinvitro. The recognition site for apo-DmeR consists of 10-bp AT-rich inverted repeats separated by six C bases (5′-ATATAGTATACCCCCCTATAGTATAT-3′). Cobalt and nickel cause DmeR to dissociate from thedmeRFpromoter, which leads to expression of the metal efflux genedmeF. This work also revealed a connection between iron homeostasis and cobalt/nickel resistance inA. tumefaciens.