Zinc- and cadmium-tolerant endophytic bacteria from Murdannia spectabilis (Kurz) Faden. studied for plant growth-promoting properties, in vitro inoculation, and antagonism.

This research aims to isolate and identify Zn- and Cd-tolerant endophytic bacteria from Murdannia spectabilis, identify their properties with and without Zn and Cd stress, and to investigate the effect of bacterial inoculation in an in vitro system. Twenty-four isolates could survive on trypticase soya agar (TSA) supplemented with Zn (250-500 mg L-1) and/or Cd (20-50 mg L-1) that belonged to the genera Bacillus, Pantoea, Microbacterium, Curtobacterium, Chryseobacterium, Cupriavidus, Siphonobacter, and Pseudomonas. Each strain had different indole-3-acetic acid (IAA), 1-aminocyclopropane-1-carboxylate (ACC) deaminase and siderophore production, nitrogen fixation, phosphate solubilization, and lignocellulosic enzyme characteristics. Cupriavidus plantarum MDR5 and Chryseobacterium sp. MDR7 were selected for inoculation into plantlets that were already occupied by Curtobacterium sp. TMIL due to them have a high tolerance for Zn and Cd while showing no pathogenicity. As determined via an in vitro system, Cupriavidus plantarum MDR5 remained in the plants to a greater extent than Chryseobacterium sp. MDR7, while Curtobacterium sp. TMIL was the dominant species. The Zn plus Cd treatment supported the persistence of Cupriavidus plantarum MDR5. Dual and mixed cultivation showed no antagonistic effects between the endophytes. Although the plant growth and Zn/Cd accumulation were not significantly affected by the Zn-/Cd-tolerant endophytes, the inoculation did not weaken the plants. Therefore, Cupriavidus plantarum MDR5 could be applied in a bioaugmentation process.

Novel heavy metal resistance gene clusters are present in the genome of Cupriavidus neocaledonicus STM 6070, a new species of Mimosa pudica microsymbiont isolated from heavy-metal-rich mining site soil.

BACKGROUND: Cupriavidus strain STM 6070 was isolated from nickel-rich soil collected near Koniambo massif, New Caledonia, using the invasive legume trap host Mimosa pudica. STM 6070 is a heavy metal-tolerant strain that is highly effective at fixing nitrogen with M. pudica. Here we have provided an updated taxonomy for STM 6070 and described salient features of the annotated genome, focusing on heavy metal resistance (HMR) loci and heavy metal efflux (HME) systems. RESULTS: The 6,771,773 bp high-quality-draft genome consists of 107 scaffolds containing 6118 protein-coding genes. ANI values show that STM 6070 is a new species of Cupriavidus. The STM 6070 symbiotic region was syntenic with that of the M. pudica-nodulating Cupriavidus taiwanensis LMG 19424T. In contrast to the nickel and zinc sensitivity of C. taiwanensis strains, STM 6070 grew at high Ni2+ and Zn2+ concentrations. The STM 6070 genome contains 55 genes, located in 12 clusters, that encode HMR structural proteins belonging to the RND, MFS, CHR, ARC3, CDF and P-ATPase protein superfamilies. These HMR molecular determinants are putatively involved in arsenic (ars), chromium (chr), cobalt-zinc-cadmium (czc), copper (cop, cup), nickel (nie and nre), and silver and/or copper (sil) resistance. Seven of these HMR clusters were common to symbiotic and non-symbiotic Cupriavidus species, while four clusters were specific to STM 6070, with three of these being associated with insertion sequences. Within the specific STM 6070 HMR clusters, three novel HME-RND systems (nieIC cep nieBA, czcC2B2A2, and hmxB zneAC zneR hmxS) were identified, which constitute new candidate genes for nickel and zinc resistance. CONCLUSIONS: STM 6070 belongs to a new Cupriavidus species, for which we have proposed the name Cupriavidus neocaledonicus sp. nov.. STM6070 harbours a pSym with a high degree of gene conservation to the pSyms of M. pudica-nodulating C. taiwanensis strains, probably as a result of recent horizontal transfer. The presence of specific HMR clusters, associated with transposase genes, suggests that the selection pressure of the New Caledonian ultramafic soils has driven the specific adaptation of STM 6070 to heavy-metal-rich soils via horizontal gene transfer.

Cupriavidus sp. strain Ni-2 resistant to high concentration of nickel and its genes responsible for the tolerance by genome comparison.

The widespread use of metals influenced many researchers to examine the relationship between heavy metal toxicity and bacterial resistance. In this study, we have inoculated heavy metal-contaminated soil from Janghang region of South Korea in the nickel-containing media (20 mM Ni2+) for the enrichment. Among dozens of the colonies acquired from the several transfers and serial dilutions with the same concentrations of Ni, the strain Ni-2 was chosen for further studies. The isolates were identified for their phylogenetic affiliations using 16S rRNA gene analysis. The strain Ni-2 was close to Cupriavidus metallidurans and was found to be resistant to antibiotics of vancomycin, erythromycin, chloramphenicol, ampicillin, gentamicin, streptomycin, and kanamycin by disk diffusion method. Of the isolated strains, Ni-2 was sequenced for the whole genome, since the Ni-resistance seemed to be better than the other strains. From the genome sequence we have found that there was a total of 89 metal-resistance-related genes including 11 Ni-resistance genes, 41 heavy metal (As, Cd, Zn, Hg, Cu, and Co)-resistance genes, 22 cation-efflux genes, 4 metal pumping ATPase genes, and 11 metal transporter genes.

Synergistic Toxicity of Copper and Gold Compounds in Cupriavidus metallidurans.

The bacterium Cupriavidus metallidurans can reduce toxic gold(I/III) complexes and biomineralize them into metallic gold (Au) nanoparticles, thereby mediating the (trans)formation of Au nuggets. In Au-rich soils, most transition metals do not interfere with the resistance of this bacterium to toxic mobile Au complexes and can be removed from the cell by plasmid-encoded metal efflux systems. Copper is a noticeable exception: the presence of Au complexes and Cu ions results in synergistic toxicity, which is accompanied by an increased cytoplasmic Cu content and formation of Au nanoparticles in the periplasm. The periplasmic Cu-oxidase CopA was not essential for formation of the periplasmic Au nanoparticles. As shown with the purified and reconstituted Cu efflux system CupA, Au complexes block Cu-dependent release of phosphate from ATP by CupA, indicating inhibition of Cu transport. Moreover, Cu resistance of Au-inhibited cells was similar to that of mutants carrying deletions in the genes for the Cu-exporting PIB1-type ATPases. Consequently, Au complexes inhibit export of cytoplasmic Cu ions, leading to an increased cellular Cu content and decreased Cu and Au resistance. Uncovering the biochemical mechanisms of synergistic Au and Cu toxicity in C. metallidurans explains the issues this bacterium has to face in auriferous environments, where it is an important contributor to the environmental Au cycle.IMPORTANCEC. metallidurans lives in metal-rich environments, including auriferous soils that contain a mixture of toxic transition metal cations. We demonstrate here that copper ions and gold complexes exert synergistic toxicity because gold ions inhibit the copper-exporting P-type ATPase CupA, which is central to copper resistance in this bacterium. Such a situation should occur in soils overlying Au deposits, in which Cu/Au ratios usually are ≫1. Appreciating how C. metallidurans solves the problem of living in environments that contain both Au and Cu is a prerequisite to understand the molecular mechanisms underlying gold cycling in the environment, and the significance and opportunities of microbiota for specific targeting to Au in mineral exploration and ore processing.

Cupriavidus pinatubonensis AEO106 deals with copper-induced oxidative stress before engaging in biodegradation of the herbicide 4-chloro-2-methylphenoxyacetic acid.

BACKGROUND: Microbial degradation of phenoxy acid (PA) herbicides in agricultural soils is important to minimize herbicide leaching to groundwater reservoirs. Degradation may, however, be hampered by exposure of the degrader bacteria to toxic metals as copper (Cu) in the soil environment. Exposure to Cu leads to accumulation of intracellular reactive oxygen species (ROS) in some bacteria, but it is not known how Cu-derived ROS and an ensuing oxidative stress affect the degradation of PA herbicides. Based on the previously proposed paradigm that bacteria deal with environmental stress before they engage in biodegradation, we studied how the degradation of the PA herbicide 2-methyl-4-chlorophenoxyacetic acid (MCPA) by the model PA degrader Cupriavidus pinatubonensis AEO106 was affected by Cu exposure. RESULTS: Exposure of C. pinatubonensis in batch culture to sublethal concentrations of Cu increased accumulation of ROS measured by the oxidant sensing probe 2,7-dichlorodihydrofluorescein diacetate and flow cytometry, and resulted in upregulation of a gene encoding a protein belong to the Ohr/OsmC protein family. The ohr/osmC gene was also highly induced by H2O2 exposure suggesting that it is involved in the oxidative stress response in C. pinatubonensis. The increased ROS accumulation and increased expression of the oxidative stress defense coincided with a delay in the catabolic performance, since both expression of the catabolic tfdA gene and MCPA mineralization were delayed compared to unexposed control cells. CONCLUSIONS: The current study suggests that Cu-induced ROS accumulation in C. pinatubonensis activates a stress response involving the product of the ohr/osmC gene. Further, the stress response is launched before induction of the catabolic tfdA gene and mineralization occurs.

Antioxidative enzyme profiling and biosorption ability of Cupriavidus metallidurans CH34 and Pseudomonas putida mt2 under cadmium stress.

Cupriavidus metallidurans CH34 and Pseudomonas putida mt2 were used as cadmium (Cd)-resistant and -sensitive bacteria, respectively, to study their biosorption ability and their antioxidative enzymes. The minimal inhibitory concentration of C. metallidurans CH34 for Cd was found to be 30 mM, and for P. putida mt2 it was 1.25 mM. The tube dilution method revealed the heavy-metal resistance pattern of C. metallidurans CH34 as Ni(2+) (10 mM)>Zn(2+) (4 mM)>Cu(2+) (2 mM)>Hg(2+) (1 mM)>Cr(2+) (1 mM)>Pb(2+) (0 mM), whereas P. putida mt2 was only resistant to Zn(2+) (1 mM). Under Cd stress, the induction of GSH was higher in C. metallidurans CH34 (0.359 ± 0.010 mM g(-1) FW) than in P. putida mt2 (0.286 ± 0.005 mM g(-1) FW). Glutathione reductase was more highly expressed in the mt2 strain, in contrast to non-protein thiols and peroxidase. Unlike dead bacterial cells, live cells of both bacteria showed significant Cd biosorption, i.e. more than 80% at 48 h. C. metallidurans CH34 used only catalase, whereas P. putida mt2 used superoxide dismutase and ascorbate peroxidase to combat Cd stress. This study investigated the Cd biosorption ability and enzymes involved in the Cd detoxification mechanisms of C. metallidurans CH34 and P. putida mt2.

Lead resistance in micro-organisms.

Lead (Pb) is an element present in the environment that negatively affects all living organisms. To diminish its high toxicity, micro-organisms have developed several mechanisms that allow them to survive exposure to Pb(II). The main mechanisms of lead resistance involve adsorption by extracellular polysaccharides, cell exclusion, sequestration as insoluble phosphates, and ion efflux to the cell exterior. This review describes the various lead resistance mechanisms, and the regulation of their expression by lead binding regulatory proteins. Special attention is given to the Pbr system from Cupriavidus metallidurans CH34, which involves a unique mechanism combining efflux and lead precipitation.

Changes in the proteome of the cadmium-tolerant bacteria Cupriavidus taiwanensis KKU2500-3 in response to cadmium toxicity.

Cupriavidus taiwanensis KKU2500-3 is a cadmium (Cd)-tolerant bacterial strain that was previously isolated from rice fields contaminated with high levels of Cd. In 500 µmol/L CdCl2, the KKU2500-3 strain grew slower and with a more prolonged lag-phase than when grown in the absence of Cd. A proteomic approach was used to characterize the protein expression in the Cd-tolerant bacteria C. taiwanensis KKU2500-3 during growth under Cd stress. When compared with the untreated cells, a total of 982 differentially expressed protein spots were observed in the CdCl2-treated cells, and 59 and 10 spots exhibited >2- and >4-fold changes, respectively. The level of up- and downregulation varied from 2.01- to 11.26-fold and from 2.01- to 5.34-fold, respectively. Of the 33 differentially expressed protein spots analyzed by MALDI TOF MS/MS, 19 spots were successfully identified, many of which were involved in stress responses. The most highly upregulated protein (+7.95-fold) identified was the chaperone GroEL, which indicated that this factor likely contributed to the bacterial survival and growth in response to Cd toxicity. Detection of the downregulated protein flagellin (-3.52-fold) was consistent with the less effective ATP-mediated and flagella-driven motility. The flagella-losing cells were also observed in the Cd-treated bacteria when analyzed by scanning electron microscopy. Thus, the Cd-stressed cells may downregulate pathways involving ATP utilization in favor of other mechanisms in response to Cd toxicity. When the KKU2500-3 strain was grown in the presence of Cd, H2S was not detected, suggesting a possible role of the sulfur in precipitation with Cd. Apart from a general response, no specific process could be determined using the present proteomic approach. However, the potential role of protein folding-mediated GroEL, flagella-mediated motility and CdS biotransformation in Cd toxicity response observed in this study as well as the extent of Cd-tolerant mechanisms using other methods could facilitate the future application of this strain in addressing Cd environmental contamination.

Swimming, swarming, twitching, and chemotactic responses of Cupriavidus metallidurans CH34 and Pseudomonas putida mt2 in the presence of cadmium.

To use of microorganisms for bioremediation purposes, the study of their motility behavior toward metals is essential. In the present study, Cupriavidus metallidurans CH34 and Pseudomonas putida mt2 were used as cadmium (Cd)-resistant and -sensitive bacteria, respectively, to evaluate the effects of Cd on their motility behaviors. Potassium morpholinopropane sulfonate (MOPS) buffer was used to observe the motility behavior of both isolates. Movement of mt2 was less in MOPS buffer compared with CH34, likely reflecting the mono-flagellated nature of mt2 and the peritrichous nature of CH34. The swimming, swarming, twitching, and chemotaxis behaviors of mt2 were greater in the presence of glucose than that of Cd. mt2 exhibited negative motility behaviors when exposed to Cd, but the opposite effect was seen in CH34. Cd was found to be a chemorepellent for mt2 but a chemoattractant for CH34, suggesting that CH34 is a potential candidate for metal (Cd) bioremediation.

Pyruvic oxime nitrification and copper and nickel resistance by a Cupriavidus pauculus, an active heterotrophic nitrifier-denitrifier.

Heterotrophic nitrifiers synthesize nitrogenous gasses when nitrifying ammonium ion. A Cupriavidus pauculus, previously thought an Alcaligenes sp. and noted as an active heterotrophic nitrifier-denitrifier, was examined for its ability to produce nitrogen gas (N2) and nitrous oxide (N2O) while heterotrophically nitrifying the organic substrate pyruvic oxime [CH3-C(NOH)-COOH]. Neither N2 nor N2O were produced. Nucleotide and phylogenetic analyses indicated that the organism is a member of a genus (Cupriavidus) known for its resistance to metals and its metabolism of xenobiotics. The microbe (a Cupriavidus pauculus designated as C. pauculus strain UM1) was examined for its ability to perform heterotrophic nitrification in the presence of Cu(2+) and Ni(2+) and to metabolize the xenobiotic phenol. The bacterium heterotrophically nitrified well when either 1 mM Cu(2+) or 0.5 mM Ni(2+) was present in either enriched or minimal medium. The organism also used phenol as a sole carbon source in either the presence or absence of 1 mM Cu(2+) or 0.5 mM Ni(2+). The ability of this isolate to perform a number of different metabolisms, its noteworthy resistance to copper and nickel, and its potential use as a bioremediation agent are discussed.

Physicochemical surface properties of Cupriavidus metallidurans CH34 and Pseudomonas putida mt2 under cadmium stress.

Cupriavidus metallidurans CH34 and Pseudomonas putida mt2 were used as cadmium (Cd) resistant and sensitive bacteria, respectively to study the effect of Cd on physicochemical surface properties which include the study of surface charge and hydrophobicity which are subjected to vary under stress conditions. In this research work, effective concentration 50 (EC50 ) was calculated to exclude the doubt that dead cells were also responding and used as reference point to study the changes in cell surface properties in the presence of Cd. EC50 of C. metallidurans CH34 was found to be 2.5 and 0.25 mM for P. putida mt2. The zeta potential analysis showed that CH34 cells were slightly less unstable than mt2 cells as CH34 cells exhibited -8.5 mV more negative potential than mt2 cells in the presence of Cd in growth medium. Cd made P. putida mt2 surface to behave as intermediate hydrophilic (θw = 25.32°) while C. metallidurans CH34 as hydrophobic (θw = 57.26°) at their respective EC50 . Although belonging to the same gram-negative group, both bacteria behaved differently in terms of changes in membrane fluidity. Expression of trans fatty acids was observed in mt2 strain (0.45%) but not in CH34 strain (0%). Similarly, cyclopropane fatty acids were observed more in mt2 strain (0.06-0.14%) but less in CH34 strain (0.01-0.02%). Degree of saturation of fatty acids decreased in P. putida mt2 (36.8-33.75%) while increased in C. metallidurans CH34 (35.6-39.3%). Homeoviscous adaptation is a survival strategy in harsh environments which includes expression of trans fatty acids and cyclo fatty acids in addition to altered degree of saturation. Different bacteria show different approaches to homeoviscous adaptation.

Mobile genetic elements associated with utilization of dichloromethane and methanol as energy sources in Cupriavidus metallidurans.

Cupriavidus metallidurans strain PD11 isolated from laboratory waste drainage can use C1 compounds, such as dichloromethane (DCM) and methanol, as a sole carbon and energy source. However, strain CH34 (a type-strain) cannot grow in the medium supplemented with DCM. In the present study, we aimed to unravel the genetic elements underlying the utilization of C1 compounds by strain PD11. The genome subtraction approach indicated that only strain PD11 had several genes highly homologous to those of Herminiimonas arsenicoxydans strain ULPAs1. Moreover, a series of polymerase chain reaction (PCR) to detect the orthologs of H. arsenicoxydans genes and the comparative study of the genomes of three strains revealed that the 87.9 kb DNA fragment corresponding to HEAR1959 to HEAR2054 might be horizontally transferred to strain PD11. The 87.9 kb DNA fragment identified was found to contain three genes whose products were putatively involved in the metabolism of formaldehyde, a common intermediate of DCM and methanol. In addition, reverse transcription PCR analysis showed that all three genes were significantly expressed when strain PD11 was cultivated in the presence of DCM or methanol. These findings suggest that strain PD11 can effectively utilize the C1 compounds because of transfer of the mobile genetic elements from other bacterial species, for instance, from H. arsenicoxydans.

Characterization of the survival ability of Cupriavidus metallidurans and Ralstonia pickettii from space-related environments.

Four Cupriavidus metallidurans and eight Ralstonia pickettii isolates from the space industry and the International Space Station (ISS) were characterized in detail. Nine of the 12 isolates were able to form a biofilm on plastics and all were resistant to several antibiotics. R. pickettii isolates from the surface of the Mars Orbiter prior to flight were 2.5 times more resistant to UV-C(254nm) radiation compared to the R. pickettii type strain. All isolates showed moderate to high tolerance against at least seven different metal ions. They were tolerant to medium to high silver concentrations (0.5-4 µM), which are higher than the ionic silver disinfectant concentrations measured regularly in the drinking water aboard the ISS. Furthermore, all isolates survived a 23-month exposure to 2 µM AgNO(3) in drinking water. These resistance properties are putatively encoded by their endogenous megaplasmids. This study demonstrated that extreme resistance is not required to withstand the disinfection and sterilization procedures implemented in the ISS and space industry. All isolates acquired moderate to high tolerance against several stressors and can grow in oligotrophic conditions, enabling them to persist in these environments.

Metal-induced conformational changes in ZneB suggest an active role of membrane fusion proteins in efflux resistance systems.

Resistance nodulation cell division (RND)-based efflux complexes mediate multidrug and heavy-metal resistance in many Gram-negative bacteria. Efflux of toxic compounds is driven by membrane proton/substrate antiporters (RND protein) in the plasma membrane, linked by a membrane fusion protein (MFP) to an outer-membrane protein. The three-component complex forms an efflux system that spans the entire cell envelope. The MFP is required for the assembly of this complex and is proposed to play an important active role in substrate efflux. To better understand the role of MFPs in RND-driven efflux systems, we chose ZneB, the MFP component of the ZneCAB heavy-metal efflux system from Cupriavidus metallidurans CH34. ZneB is shown to be highly specific for Zn(2+) alone. The crystal structure of ZneB to 2.8 A resolution defines the basis for metal ion binding in the coordination site at a flexible interface between the beta-barrel and membrane proximal domains. The conformational differences observed between the crystal structures of metal-bound and apo forms are monitored in solution by spectroscopy and chromatography. The structural rearrangements between the two states suggest an active role in substrate efflux through metal binding and release.

Whole-genome sequence of Cupriavidus sp. strain BIS7, a heavy-metal-resistant bacterium.

Cupriavidus sp. strain BIS7 is a Malaysian tropical soil bacterium that exhibits broad heavy-metal resistance [Co(II), Zn(II), Ni(II), Se(IV), Cu(II), chromate, Co(III), Fe(II), and Fe(III)]. It is particularly resistant to Fe(II), Fe(III), and Zn(II). Here we present the assembly and annotation of its genome.

Variation in genomic islands contribute to genome plasticity in Cupriavidus metallidurans.

BACKGROUND: Different Cupriavidus metallidurans strains isolated from metal-contaminated and other anthropogenic environments were genotypically and phenotypically compared with C. metallidurans type strain CH34. The latter is well-studied for its resistance to a wide range of metals, which is carried for a substantial part by its two megaplasmids pMOL28 and pMOL30. RESULTS: Comparative genomic hybridization (CGH) indicated that the extensive arsenal of determinants involved in metal resistance was well conserved among the different C. metallidurans strains. Contrary, the mobile genetic elements identified in type strain CH34 were not present in all strains but clearly showed a pattern, although, not directly related to a particular biotope nor location (geographical). One group of strains carried almost all mobile genetic elements, while these were much less abundant in the second group. This occurrence was also reflected in their ability to degrade toluene and grow autotrophically on hydrogen gas and carbon dioxide, which are two traits linked to separate genomic islands of the Tn4371-family. In addition, the clear pattern of genomic islands distribution allowed to identify new putative genomic islands on chromosome 1 and 2 of C. metallidurans CH34. CONCLUSIONS: Metal resistance determinants are shared by all C. metallidurans strains and their occurrence is apparently irrespective of the strain's isolation type and place. Cupriavidus metallidurans strains do display substantial differences in the diversity and size of their mobile gene pool, which may be extensive in some (including the type strain) while marginal in others.

Cysteine coordination of Pb(II) is involved in the PbrR-dependent activation of the lead-resistance promoter, PpbrA, from Cupriavidus metallidurans CH34.

BACKGROUND: The pbr resistance operon from Cupriavidus metallidurans CH34 plasmid pMOL30 confers resistance to Pb(II) salts, and is regulated by the Pb(II) responsive regulator PbrR, which is a MerR family activator. In other metal sensing MerR family regulators, such as MerR, CueR, and ZntR the cognate regulator binds to a promoter with an unusually long spacer between the -35 and -10 sequences, and activates transcription of resistance genes as a consequence of binding the appropriate metal. Cysteine residues in these regulators are essential for metal ion coordination and activation of expression from their cognate promoter. In this study we investigated the interaction of PbrR with the promoter for the structural pbr resistance genes, PpbrA, effects on transcriptional activation of altering the DNA sequence of PpbrA, and effects on Pb(II)-induced activation of PpbrA when cysteine residues in PbrR were mutated to serine. RESULTS: Gel retardation and footprinting assays using purified PbrR show that it binds to, and protects from DNase I digestion, the PpbrA promoter, which has a 19 bp spacer between its -35 and -10 sites. Using ß-galactosidase assays in C. metallidurans, we show that when PpbrA is changed to an 18 bp spacer, there is an increase in transcriptional activation both in the presence and absence of Pb(II) salts up to a maximum induction equivalent to that seen in the fully-induced wild-type promoter. Changes to the -10 sequence of PpbrA from TTAAAT to the consensus E. coli -10 sequence (TATAAT) increased transcriptional activation from PpbrA, whilst changing the -10 sequence to that of the Tn501 mer promoter (TAAGGT) also increased the transcriptional response, but only in the presence of Pb(II). Individual PbrR mutants C14S, C55S, C79S, C114S, C123S, C132S and C134S, and a double mutant C132S/C134S, were tested for Pb(II) response from PpbrA, using ß-galactosidase assays in C. metallidurans. The PbrR C14S, C79S, C134S, and C132S/C134S mutants were defective in Pb(II)-induced activation of PpbrA. CONCLUSIONS: These data show that the metal-dependent activation of PbrR occurs by a similar mechanism to that of MerR, but that metal ion coordination is through cysteines which differ from those seen in other MerR family regulators, and that the DNA sequence of the -10 promoter affects expression levels of the lead resistance genes.

Mechanisms of gold biomineralization in the bacterium Cupriavidus metallidurans.

While the role of microorganisms as main drivers of metal mobility and mineral formation under Earth surface conditions is now widely accepted, the formation of secondary gold (Au) is commonly attributed to abiotic processes. Here we report that the biomineralization of Au nanoparticles in the metallophillic bacterium Cupriavidus metallidurans CH34 is the result of Au-regulated gene expression leading to the energy-dependent reductive precipitation of toxic Au(III)-complexes. C. metallidurans, which forms biofilms on Au grains, rapidly accumulates Au(III)-complexes from solution. Bulk and microbeam synchrotron X-ray analyses revealed that cellular Au accumulation is coupled to the formation of Au(I)-S complexes. This process promotes Au toxicity and C. metallidurans reacts by inducing oxidative stress and metal resistances gene clusters (including a Au-specific operon) to promote cellular defense. As a result, Au detoxification is mediated by a combination of efflux, reduction, and possibly methylation of Au-complexes, leading to the formation of Au(I)-C-compounds and nanoparticulate Au(0). Similar particles were observed in bacterial biofilms on Au grains, suggesting that bacteria actively contribute to the formation of Au grains in surface environments. The recognition of specific genetic responses to Au opens the way for the development of bioexploration and bioprocessing tools.

Hospital sink traps as a potential source of the emerging multidrug-resistant pathogen Cupriavidus pauculus: characterization and draft genome sequence of strain MF1.

Introduction. Cupriavidus pauculus is historically found in soil and water but has more recently been reported to cause human infection and death. Hospital sink traps can serve as a niche for bacterial persistence and a platform for horizontal gene transfer, with evidence of dissemination of pathogens in hospital plumbing systems driving nosocomial infection.Gap Statement. This paper presents the first C. pauculus strain isolated from a hospital sink trap. There are only six genome assemblies available on NCBI for C. pauculus; two of these are PacBio/Illumina hybrids. This paper presents the first ONT/Illumina hybrid assembly, with five contigs. The other assemblies available consist of 37, 38, 111 and 227 contigs. This paper also presents data on biofilm formation and lethal dose in Galleria mellonella; there is little published information describing these aspects of virulence.Aim. The aims were to identify the isolate found in a hospital sink trap, characterize its genome, and assess whether it could pose a risk to human health.Methodology. The genome was sequenced, and a hybrid assembly of short and long reads produced. Antimicrobial susceptibility was determined by the broth microdilution method. Virulence was assessed by measuring in vitro biofilm formation compared to Pseudomonas aeruginosa and in vivo lethality in Galleria mellonella larvae.Results. The isolate was confirmed to be a strain of C. pauculus, with a 6.8 Mb genome consisting of 6468 coding sequences and an overall G+C content of 63.9 mol%. The genome was found to contain 12 antibiotic resistance genes, 8 virulence factor genes and 33 metal resistance genes. The isolate can be categorized as resistant to meropenem, amoxicillin, amikacin, gentamicin and colistin, but susceptible to cefotaxime, cefepime, imipenem and ciprofloxacin. Clear biofilm formation was seen in all conditions over 72 h and exceeded that of P. aeruginosa when measured at 37 °C in R2A broth. Lethality in G. mellonella larvae over 48 h was relatively low.Conclusion. The appearance of a multidrug-resistant strain of C. pauculus in a known pathogen reservoir within a clinical setting should be considered concerning. Further work should be completed to compare biofilm formation and in vivo virulence between clinical and environmental strains, to determine how easily environmental strains may establish human infection. Infection control teams and clinicians should be aware of the emerging nature of this pathogen and further work is needed to minimize the impact of contaminated hospital plumbing systems on patient outcomes.