Mercury remediation potential of mercury-resistant strain Rheinheimera metallidurans sp. nov. isolated from a municipal waste dumping site.

A novel mercury-resistant bacterium, designated strain DCL_24T, was isolated from the legacy waste at the Daddu Majra dumping site in Chandigarh, India. It showed resistance up to 300 µM of inorganic mercury (mercuric chloride). The isolate was found to be a Gram-negative, facultative anaerobic, motile, and rod-shaped bacterium that can grow at 4 - 30 °C (optimum 25 °C), pH 6.0 - 12.0 (optimum 7.0), and 0 - 4.0 % (w/v) NaCl (optimum 0.5 - 2.0 %). The 16 S rRNA gene-based phylogenetic analysis showed that DCL_ 24 T shared a 97.53 % similarity with itsºlosest type strain Rheinheimera muenzenbergensis E-49T. Insilico DNA-DNA hybridization and average nucleotide identity values were found to be 18.60 % and 73.77 %, respectively, between the genomes of DCL_24T and R. muenzenbergensis E-49T. The strain DCL_24T has 44.33 DNA G+C content (mol %). Based on the phenotypic, chemotaxonomic, and genotypic data, the strain DCL_24T represents a novel species within the genus Rheinheimera, for which the name Rheinheimera metallidurans sp. nov is proposed. The type strain is DCL_24T (MTCC13203T = NBRC115780T = JCM 35551 T). The isolate was found to volatilize and remove mercury efficiently, as demonstrated by X-ray film and dithizone-based colorimetric methods. Around 92 % of mercury removal was observed within 48 h. The mercury-resistant determinant mer operon consisting of merA, encoding the mercuric reductase enzyme, and transport and regulatory genes (merT, merP, merD, and merR) were found in the isolate. Relative expression analysis of merA at increasing concentrations of HgCl2 was confirmed by quantitative real-time PCR. These data indicate the merA-mediated reduction of toxic Hg2+ into a non-toxic volatile Hg0. The phytotoxicity assay performed using Arabidopsis thaliana seeds further demonstrated the mercury toxicity reduction potential of DCL_24T. The study shows that this novel isolate, DCL_24T, is an interesting candidate for mercury bioremediation. However, further studies are required to assess the bioremediation efficacy of the strain under the harsh environmental conditions prevailing in polluted sites.

Heavy metal resistance in the Yanomami and Tunapuco microbiome

BACKGROUND The Amazon Region hosts invaluable and unique biodiversity as well as mineral resources. Consequently, large illegal and artisanal gold mining areas exist in indigenous territories. Mercury has been used in gold mining, and some has been released into the environment and atmosphere, primarily affecting indigenous people such as the Yanomami. In addition, other heavy metals have been associated with gold mining and other metal-dispersing activities in the region. OBJECTIVE Investigate the gut microbiome of two semi-isolated groups from the Amazon, focusing on metal resistance. METHODS Metagenomic data from the Yanomami and Tunapuco gut microbiome were assembled into contigs, and their putative proteins were searched against a database of metal resistance proteins. FINDINGS Proteins associated with mercury resistance were exclusive in the Yanomami, while proteins associated with silver resistance were exclusive in the Tunapuco. Both groups share 77 non-redundant metal resistance (MR) proteins, mostly associated with multi-MR and operons with potential resistance to arsenic, nickel, zinc, copper, copper/silver, and cobalt/nickel. Although both groups harbour operons related to copper resistance, only the Tunapuco group had the pco operon. CONCLUSION The Yanomami and Tunapuco gut microbiome shows that these people have been exposed directly or indirectly to distinct scenarios concerning heavy metals.

Genomic features of a multidrug-resistant and mercury-tolerant environmental Escherichia coli recovered after a mining dam disaster in South America.

Mining dam disasters contribute to the contamination of aquatic environments, impacting associated ecosystems and wildlife. A multidrug-resistant Escherichia coli strain (B2C) was isolated from a river water sample in Brazil after the Mariana mining dam disaster. The genome was sequenced using the Illumina MiSeq platform, and de novo assembled using Unicycler. Resistome, virulome, and plasmidome were predicted using bioinformatics tools. Data analysis revealed that E. coli B2C belonged to sequence type ST219 and phylogroup E. Strikingly, a broad resistome (antibiotics, hazardous heavy metals, and biocides) was predicted, including the presence of the clinically relevant blaCTX-M-2 extended-spectrum ß-lactamase (ESBL) gene, qacE∆1 efflux pump gene, and the mer (mercury resistance) operon. SNP-based analysis revealed that environmental E. coli B2C was clustered along to ESBL-negative E. coli strains of ST219 isolated between 1980 and 2021 from livestock in the United States of America. Acquisition of clinically relevant genes by ST219 seems to be a recent genetic event related to anthropogenic activities, where polluted water environments may contribute to its dissemination at the human-animal-environment interface. In addition, the presence of genes conferring resistance to heavy metals could be related to environmental pollution from mining activities. Antimicrobial resistance genes could be essential biomarkers of environmental exposure to human and mining pollution.

Mercury and Antibiotic Resistance Co-Selection in Bacillus sp. Isolates from the Almadén Mining District.

Antibiotic resistance (AR) in the environment is of great global concern and a threat to public health. Soil bacteria, including Bacillus spp., could act as recipients and reservoirs of AR genes of clinical, livestock, or agricultural origin. These genes can be shared between bacteria, some of which could be potentially human pathogens. This process can be favored in conditions of abiotic stress, such as heavy metal contamination. The Almadén mining district (Ciudad Real, Spain) is one of the environments with the highest mercury (Hg) contamination worldwide. The link between heavy metal contamination and increased AR in environmental bacteria seems clear, due to co-resistance and co-selection phenomena. In the present study, 53 strains were isolated from rhizospheric and bulk soil samples in Almadén. AR was tested using Vitek® 2 and minimum inhibitory concentration (MIC) values were obtained and interpreted based on the criteria of the Clinical and Laboratory Standards Institute (CLSI) guidelines. Based on the resistance profiles, five different antibiotypes were established. The Hg minimum bactericidal concentration (MBC) of each strain was obtained using the plating method with increasing concentrations of HgCl2. A total of 72% of Bacillus spp. showed resistance to two or more commonly used antibiotics. A total of 38 isolates expressed AR to cephalosporins. Finally, the environmental co-selection of AR to cephalosporins and tetracyclines by selective pressure of Hg has been statistically demonstrated.

Characterization of Bacterial and Fungal Assemblages From Historically Contaminated Metalliferous Soils Using Metagenomics Coupled With Diffusion Chambers and Microbial Traps.

The majority of environmental microbiomes are not amenable to cultivation under standard laboratory growth conditions and hence remain uncharacterized. For environmental applications, such as bioremediation, it is necessary to isolate microbes performing the desired function, which may not necessarily be the fast growing or the copiotroph microbiota. Toward this end, cultivation and isolation of microbial strains using diffusion chambers (DC) and/or microbial traps (MT) have both been recently demonstrated to be effective strategies because microbial enrichment is facilitated by soil nutrients and not by synthetically defined media, thus simulating their native habitat. In this study, DC/MT chambers were established using soils collected from two US Department of Energy (DOE) sites with long-term history of heavy metal contamination, including mercury (Hg). To characterize the contamination levels and nutrient status, soils were first analyzed for total mercury (THg), methylmercury (MeHg), total carbon (TC), total nitrogen (TN), and total phosphorus (TP). Multivariate statistical analysis on these measurements facilitated binning of soils under high, medium and low levels of contamination. Bacterial and fungal microbiomes that developed within the DC and MT chambers were evaluated using comparative metagenomics, revealing Chthoniobacter, Burkholderia and Bradyrhizobium spp., as the predominant bacteria while Penicillium, Thielavia, and Trichoderma predominated among fungi. Many of these core microbiomes were also retrieved as axenic isolates. Furthermore, canonical correspondence analysis (CCA) of biogeochemical measurements, metal concentrations and bacterial communities revealed a positive correlation of Chthoniobacter/Bradyrhizobium spp., to THg whereas Burkholderia spp., correlated with MeHg. Penicillium spp., correlated with THg whereas Trichoderma spp., and Aspergillus spp., correlated with MeHg, from the MT approach. This is the first metagenomics-based assessment, isolation and characterization of soil-borne bacterial and fungal communities colonizing the diffusion chambers (DC) and microbial traps (MT) established with long-term metal contaminated soils. Overall, this study provides proof-of-concept for the successful application of DC/MT based assessment of mercury resistant (HgR) microbiomes in legacy metal-contaminated soils, having complex contamination issues. Overall, this study brings out the significance of microbial communities and their relevance in context to heavy metal cycling for better stewardship and restoration of such historically contaminated systems.

Genome sequence of Pseudomonas aeruginosa PAO1161, a PAO1 derivative with the ICEPae1161 integrative and conjugative element.

BACKGROUND: Pseudomonas aeruginosa is a cause of nosocomial infections, especially in patients with cystic fibrosis and burn wounds. PAO1 strain and its derivatives are widely used to study the biology of this bacterium, however recent studies demonstrated differences in the genomes and phenotypes of derivatives from different laboratories. RESULTS: Here we report the genome sequence of P. aeruginosa PAO1161 laboratory strain, a leu-, RifR, restriction-modification defective PAO1 derivative, described as the host of IncP-8 plasmid FP2, conferring the resistance to mercury. Comparison of PAO1161 genome with PAO1-UW sequence revealed lack of an inversion of a large genome segment between rRNA operons and 100 nucleotide polymorphisms, short insertions and deletions. These included a change in leuA, resulting in E108K substitution, which caused leucine auxotrophy and a mutation in rpoB, likely responsible for the rifampicin resistance. Nonsense mutations were detected in PA2735 and PA1939 encoding a DNA methyltransferase and a putative OLD family endonuclease, respectively. Analysis of revertants in these two genes showed that PA2735 is a component of a restriction-modification system, independent of PA1939. Moreover, a 12 kb RPG42 prophage and a novel 108 kb PAPI-1 like integrative conjugative element (ICE) encompassing a mercury resistance operon were identified. The ICEPae1161 was transferred to Pseudomonas putida cells, where it integrated in the genome and conferred the mercury resistance. CONCLUSIONS: The high-quality P. aeruginosa PAO1161 genome sequence provides a reference for further research including e.g. investigation of horizontal gene transfer or comparative genomics. The strain was found to carry ICEPae1161, a functional PAPI-1 family integrative conjugative element, containing loci conferring mercury resistance, in the past attributed to the FP2 plasmid of IncP-8 incompatibility group. This indicates that the only known member of IncP-8 is in fact an ICE.

Native Plasmid-Encoded Mercury Resistance Genes Are Functional and Demonstrate Natural Transformation in Environmental Bacterial Isolates.

Plasmid-mediated horizontal gene transfer (HGT) is a major driver of genetic diversity in bacteria. We experimentally validated the function of a putative mercury resistance operon present on an abundant 8-kbp native plasmid found in groundwater samples without detectable levels of mercury. Phylogenetic analyses of the plasmid-encoded mercury reductases from the studied groundwater site show them to be distinct from those reported in proximal metal-contaminated sites. We synthesized the entire native plasmid and demonstrated that the plasmid was sufficient to confer functional mercury resistance in Escherichia coli Given the possibility that natural transformation is a prevalent HGT mechanism in the low-cell-density environments of groundwaters, we also assayed bacterial strains from this environment for competence. We used the native plasmid-encoded metal resistance to design a screen and identified 17 strains positive for natural transformation. We selected 2 of the positive strains along with a model bacterium to fully confirm HGT via natural transformation. From an ecological perspective, the role of the native plasmid population in providing advantageous traits combined with the microbiome's capacity to take up environmental DNA enables rapid adaptation to environmental stresses.IMPORTANCE Horizontal transfer of mobile genetic elements via natural transformation has been poorly understood in environmental microbes. Here, we confirm the functionality of a native plasmid-encoded mercury resistance operon in a model microbe and then query for the dissemination of this resistance trait via natural transformation into environmental bacterial isolates. We identified 17 strains including Gram-positive and Gram-negative bacteria to be naturally competent. These strains were able to successfully take up the plasmid DNA and obtain a clear growth advantage in the presence of mercury. Our study provides important insights into gene dissemination via natural transformation enabling rapid adaptation to dynamic stresses in groundwater environments.

Identification of A Novel Arsenic Resistance Transposon Nested in A Mercury Resistance Transposon of Bacillus sp. MB24.

A novel TnMERI1-like transposon designated as TnMARS1 was identified from mercury resistant Bacilli isolated from Minamata Bay sediment. Two adjacent ars operon-like gene clusters, ars1 and ars2, flanked by a pair of 78-bp inverted repeat sequences, which resulted in a 13.8-kbp transposon-like fragment, were found to be sandwiched between two transposable genes of the TnMERI1-like transposon of a mercury resistant bacterium, Bacillus sp. MB24. The presence of a single transcription start site in each cluster determined by 5'-RACE suggested that both are operons. Quantitative real time RT-PCR showed that the transcription of the arsR genes contained in each operon was induced by arsenite, while arsR2 responded to arsenite more sensitively and strikingly than arsR1 did. Further, arsenic resistance complementary experiments showed that the ars2 operon conferred arsenate and arsenite resistance to an arsB-knocked out Bacillus host, while the ars1 operon only raised arsenite resistance slightly. This transposon nested in TnMARS1 was designated as TnARS1. Multi-gene cluster blast against bacteria and Bacilli whole genome sequence databases suggested that TnMARS1 is the first case of a TnMERI1-like transposon combined with an arsenic resistance transposon. The findings of this study suggested that TnMERI1-like transposons could recruit other mobile elements into its genetic structure, and subsequently cause horizontal dissemination of both mercury and arsenic resistances among Bacilli in Minamata Bay.

Genomic Sequence Analysis of the Multidrug-Resistance Region of Avian Salmonella enterica serovar Indiana Strain MHYL.

A series of human and animal diseases that are caused by Salmonella infections pose a serious threat to human health and huge economic losses to the livestock industry. We found antibiotic resistance (AR) genes in the genome of 133 strains of S. Indiana from a poultry production site in Shandong Province, China. Salmonella enterica subsp. enterica serovar Indiana strain MHYL had multidrug-resistance (MDR) genes on its genome. Southern blot analysis was used to locate genes on the genomic DNA. High-throughput sequencing technology was used to determine the gene sequence of the MHYL genome. Areas containing MDR genes were mapped based on the results of gene annotation. The AR genes blaTEM, strA, tetA, and aac(6')-Ib-cr were found on the MHYL genome. The resistance genes were located in two separate MDR regions, RR1 and RR2, containing type I integrons, and Tn7 transposons and multiple IS26 complex transposons with transposable functions. Portions of the MDR regions were determined to be highly homologous to the structure of plasmid pAKU_1 in S. enterica serovar Paratyphi A (accession number: AM412236), SGI11 in S. enterica serovar Typhimurium (accession number: KM023773), and plasmid pS414 in S. Indiana (accession No.: KC237285).

Bacterial community rather than metals shaping metal resistance genes in water, sediment and biofilm in lakes from arid northwestern China.

Lakes in arid northwestern China are valuable freshwater resources that drive socioeconomic development. Environmental pollution can significantly influence the composition of microbial communities and the distribution of functional genes in lakes. This study investigated heavy metal pollution to identify possible correlations with metal resistance genes (MRGs) and bacterial community composition in water, sediment and biofilm samples from Bosten Lake and Ebi Lake in northwestern China. High levels of zinc were detected in all samples. However, the metals detected in the sediment samples of both lakes were determined to be at low risk levels according to an ecological index. The mercury resistance gene subtype merP had the greatest average abundance (4.61 × 10-3 copies per 16S rRNA) among all the samples, followed by merA and merC. The high abundance of merA in the pelagic zone rather than in benthic sediment suggests that the pelagic microbial community was important in mercury reduction. Proteobacteria were the main phylum found in the microbial communities in all samples. However, microbial communities in most of the water, sediment and biofilm samples had different compositions, indicating that the habitat niche plays an important role in shaping the bacterial communities in lakes. The microbial community, rather than the heavy metals, was the main driver of MRG distribution. The abundances of some bacterial genera involved in the decomposition of organic matter and the terrestrial nitrogen cycle were negatively correlated with heavy metals. This result suggests that metal pollution can adversely affect the biogeochemical processes that occur in lakes.

Metagenomic insights into the abundance and composition of resistance genes in aquatic environments: Influence of stratification and geography.

A global survey was performed with 122 aquatic metagenomic DNA datasets (92 lake water and 30 seawater) obtained from the Sequence Read Archive (SRA). Antibiotic resistance genes (ARGs) and metal resistance genes (MRGs) were derived from the dataset sequences via bioinformatic analysis. The relative abundances of ARGs and MRGs in lake samples were in the ranges ND (not detected)-1.34 × 100 and 1.22 × 10-3-1.98 × 10-1 copies per 16S rRNA, which were higher than those in seawater samples. Among ARGs, multidrug resistance genes and bacitracin resistance genes had high relative abundances in both lake and sea water samples. Multi-metal resistance genes, mercury resistance genes and copper resistance genes had the greatest relative abundance for MRGs. No significant difference was found between epilimnion and hypolimnion in abundance or the Shannon diversity index for ARGs and MRGs. Principal coordinates analysis and permutational multivariate analysis of variance (PERMANOVA) test showed that stratification and geography had significant influence on the composition of ARGs and MRGs in lakes (p < 0.05, PERMANOVA). Coastal seawater samples had significantly greater relative abundance and a higher Shannon index for both ARGs and MRGs than deep ocean and Antarctic seawater samples (p < 0.05, Kruskal-Wallis one-way ANOVA), suggesting that human activity may exert more selective pressure on ARGs and MRGs in coastal areas than those in deep ocean and Antarctic seawater.

Large Circular Plasmids from Groundwater Plasmidomes Span Multiple Incompatibility Groups and Are Enriched in Multimetal Resistance Genes.

Naturally occurring plasmids constitute a major category of mobile genetic elements responsible for harboring and transferring genes important in survival and fitness. A targeted evaluation of plasmidomes can reveal unique adaptations required by microbial communities. We developed a model system to optimize plasmid DNA isolation procedures targeted to groundwater samples which are typically characterized by low cell density (and likely variations in the plasmid size and copy numbers). The optimized method resulted in successful identification of several hundred circular plasmids, including some large plasmids (11 plasmids more than 50 kb in size, with the largest being 1.7 Mb in size). Several interesting observations were made from the analysis of plasmid DNA isolated in this study. The plasmid pool (plasmidome) was more conserved than the corresponding microbiome distribution (16S rRNA based). The circular plasmids were diverse as represented by the presence of seven plasmid incompatibility groups. The genes carried on these groundwater plasmids were highly enriched in metal resistance. Results from this study confirmed that traits such as metal, antibiotic, and phage resistance along with toxin-antitoxin systems are encoded on abundant circular plasmids, all of which could confer novel and advantageous traits to their hosts. This study confirms the ecological role of the plasmidome in maintaining the latent capacity of a microbiome, enabling rapid adaptation to environmental stresses.IMPORTANCE Plasmidomes have been typically studied in environments abundant in bacteria, and this is the first study to explore plasmids from an environment characterized by low cell density. We specifically target groundwater, a significant source of water for human/agriculture use. We used samples from a well-studied site and identified hundreds of circular plasmids, including one of the largest sizes reported in plasmidome studies. The striking similarity of the plasmid-borne ORFs in terms of taxonomical and functional classifications across several samples suggests a conserved plasmid pool, in contrast to the observed variability in the 16S rRNA-based microbiome distribution. Additionally, the stress response to environmental factors has stronger conservation via plasmid-borne genes as marked by abundance of metal resistance genes. Last, identification of novel and diverse plasmids enriches the existing plasmid database(s) and serves as a paradigm to increase the repertoire of biological parts that are available for modifying novel environmental strains.

Thinking outside the (pill) box: Does toxic metal exposure thwart antibiotic stewardship best practices?

Multi-antibiotic resistant (MAR) bacteria cost billions in medical care and tens of thousands of lives annually but perennial calls to limit agricultural and other misuse of antibiotics and to fund antibiotic discovery have not slowed this MAR deluge. Since mobile genetic elements (MGEs) stitch single antibiotic resistance genes into clinically significant MAR arrays, it is high time to focus on how MGEs generate MAR and how disabling them could ameliorate the MAR problem. However, to consider only antibiotics as the drivers of MAR is to miss the significant impact of exposure to non-antibiotic toxic chemicals, specifically metals, on the persistence and spread of MAR. Toxic metals were among the earliest discovered targets of plasmid-encoded resistance genes. Recent genomic epidemiology clearly demonstrated the co-prevalence of metal resistances and antibiotic multi-resistance, uniquely in humans and domestic animals. Metal resistances exploit the same, ancient "transportation infrastructure" of plasmids, transposons, and integrons that spread the antibiotic resistance genes and will continue to do so even if all antibiotic misuse were stopped today and new antibiotics were flowing from the pipeline monthly. In a key experiment with primates, continuous oral exposure to mercury (Hg) released from widely used dental amalgam fillings co-selected for MAR bacteria in the oral and fecal commensal microbiomes and, most importantly, when amalgams were replaced with non-metal fillings, MAR bacteria declined dramatically. Could that also be happening on the larger public health scale as use of amalgam restorations is curtailed or banned in many countries? This commentary covers salient past and recent findings of key metal-antibiotic resistance associations and proposes that the shift from phenotyping to genotyping in surveillance of resistance loci will allow a test of whether declining exposure to this leading source of Hg is accompanied by a decline in MAR compared to countries where amalgam is still used. If this hypothesis is correct, the limited success of antibiotic stewardship practices may be because MAR is also being driven by continuous, daily exposure to Hg, a non-antibiotic toxicant widely used in humans.

A plasmid-encoded mobile genetic element from Pseudomonas aeruginosa that confers heavy metal resistance and virulence.

Mobile plasmid-encoded elements are DNA segments that are transferred for horizontal gene transfer and that confer adaptive proprieties, as well as virulence and antibiotic and heavy metal resistance to bacteria. The conjugative plasmid pUM505, isolated from a clinical strain of Pseudomonas aeruginosa, possesses a putative 31.292 kb mobile element (denominated Mpe: Mobile plasmid- encoded element) that, in addition to possessing chr genes that confer chromate resistance to Pseudomonas, contains two putative mer operons that could confer mercury resistance. Moreover, the Mpe contains genes related previously with the virulence of both P. aeruginosa and Escherichia coli strains. In this work, we determined that Mpe from pUM505 was able to independently move to another DNA molecule, conferring chromate and mercury resistance to P. aeruginosa PAO1 and mercury resistance to E. coli JM101, suggesting that its transference might be beneficial to bacteria under certain environmental conditions. Additionally, the transference of Mpe increased the virulence of P. aeruginosa PAO1 against the nematode Caenorhabditis elegans, suggesting its contribution to the pathogenicity of P. aeruginosa. In this work, we describe a new mobile plasmid-encoded element that possesses the potential to be transferred by horizontal gene transference, which could provide bacteria with a wide variety of adaptive traits such as heavy metal resistance and virulence, which can be selective factors for the distribution and prevalence of this plasmid in diverse environments, including hospitals and heavy metal contaminated soils.

Mercury bioremediation by mercury resistance transposon-mediated in situ molecular breeding.

Mercury-resistant (HgR) bacteria occur in various bacterial species from a wide variety of environmental sources. Resistance is conferred by a set of operon genes termed the mer operon. Many HgR bacteria have been isolated from diverse environments and clinical samples, and it is recognized that mer operons are often localized on transposons. Previous research reports have suggested that HgR transposons participate in the horizontal gene transfer of mer operons among bacteria. This was confirmed by a study that found that mer operons were distributed worldwide in Bacilli with dissemination of TnMERI1-like transposons. In this mini review, possible strategies for transposon-mediated in situ molecular breeding (ISMoB) of HgR bacteria in their natural habitat are discussed. In ISMoB, the target microorganisms for breeding are indigenous bacteria that are not HgR but that are dominant and robust in their respective environments. Additionally, we propose a new concept of bioremediation technology for environmental mercury pollution by applying transposon-mediated ISMoB for environmental mercury pollution control.

Plasmid stability is enhanced by higher-frequency pulses of positive selection.

Plasmids accelerate bacterial adaptation by sharing ecologically important traits between lineages. However, explaining plasmid stability in bacterial populations is challenging owing to their associated costs. Previous theoretical and experimental studies suggest that pulsed positive selection may explain plasmid stability by favouring gene mobility and promoting compensatory evolution to ameliorate plasmid cost. Here we test how the frequency of pulsed positive selection affected the dynamics of a mercury-resistance plasmid, pQBR103, in experimental populations of Pseudomonas fluorescens SBW25. Plasmid dynamics varied according to the frequency of Hg2+ positive selection: in the absence of Hg2+ plasmids declined to low frequency, whereas pulses of Hg2+ selection allowed plasmids to sweep to high prevalence. Compensatory evolution to ameliorate the cost of plasmid carriage was widespread across the entire range of Hg2+ selection regimes, including both constant and pulsed Hg2+ selection. Consistent with theoretical predictions, gene mobility via conjugation appeared to play a greater role in promoting plasmid stability under low-frequency pulses of Hg2+ selection. However, upon removal of Hg2+ selection, plasmids which had evolved under low-frequency pulse selective regimes declined over time. Our findings suggest that temporally variable selection environments, such as those created during antibiotic treatments, may help to explain the stability of mobile plasmid-encoded resistance.

Mercury remediation potential of a mercury resistant strain Sphingopyxis sp. SE2 isolated from contaminated soil.

A mercury resistant bacterial strain SE2 was isolated from contaminated soil. The 16s rRNA gene sequencing confirms the strain as Sphingopyxis belongs to the Sphingomonadaceae family of the α-Proteobacteria group. The isolate showed high resistance to mercury with estimated concentrations of Hg that caused 50% reduction in growth (EC50) of 5.97 and 6.22mg/L and minimum inhibitory concentrations (MICs) of 32.19 and 34.95mg/L in minimal and rich media, respectively. The qualitative detection of volatilized mercury and the presence of mercuric reductase enzyme proved that the strain SE2 can potentially remediate mercury. ICP-QQQ-MS analysis of the remaining mercury in experimental broths indicated that a maximum of 44% mercury was volatilized within 6hr by live SE2 culture. Furthermore a small quantity (23%) of mercury was accumulated in live cell pellets. While no volatilization was caused by dead cells, sorption of mercury was confirmed. The mercuric reductase gene merA was amplified and sequenced. Homology was observed among the amino acid sequences of mercuric reductase enzyme of different organisms from α-Proteobacteria and ascomycota groups.

Finished genome sequence and methylome of the cyanide-degrading Pseudomonas pseudoalcaligenes strain CECT5344 as resolved by single-molecule real-time sequencing.

Pseudomonas pseudoalcaligenes CECT5344 tolerates cyanide and is also able to utilize cyanide and cyano-derivatives as a nitrogen source under alkaline conditions. The strain is considered as candidate for bioremediation of habitats contaminated with cyanide-containing liquid wastes. Information on the genome sequence of the strain CECT5344 became available previously. The P. pseudoalcaligenes CECT5344 genome was now resequenced by applying the single molecule, real-time (SMRT(®)) sequencing technique developed by Pacific Biosciences. The complete and finished genome sequence of the strain consists of a 4,696,984 bp chromosome featuring a GC-content of 62.34%. Comparative analyses between the new and previous versions of the P. pseudoalcaligenes CECT5344 genome sequence revealed additional regions in the new sequence that were missed in the older version. These additional regions mostly represent mobile genetic elements. Moreover, five additional genes predicted to play a role in sulfoxide reduction are present in the newly established genome sequence. The P. pseudoalcaligenes CECT5344 genome sequence is highly related to the genome sequences of different Pseudomonas mendocina strains. Approximately, 70% of all genes are shared between P. pseudoalcaligenes and P. mendocina. In contrast to P. mendocina, putative pathogenicity genes were not identified in the P. pseudoalcaligenes CECT5344 genome. P. pseudoalcaligenes CECT5344 possesses unique genes for nitrilases and mercury resistance proteins that are of importance for survival in habitats contaminated with cyano- and mercury compounds. As an additional feature of the SMRT sequencing technology, the methylome of P. pseudoalcaligenes was established. Six sequence motifs featuring methylated adenine residues (m6A) were identified in the genome. The genome encodes several methyltransferases, some of which may be considered for methylation of the m6A motifs identified. The complete genome sequence of the strain CECT5344 now provides the basis for exploitation of genetic features for biotechnological purposes.

Mercury resistance transposons in Bacilli strains from different geographical regions.

A total of 65 spore-forming mercury-resistant bacteria were isolated from natural environments worldwide in order to understand the acquisition of additional genes by and dissemination of mercury resistance transposons across related Bacilli genera by horizontal gene movement. PCR amplification using a single primer complementary to the inverted repeat sequence of TnMERI1-like transposons showed that 12 of 65 isolates had a transposon-like structure. There were four types of amplified fragments: Tn5084, Tn5085, Tn(d)MER3 (a newly identified deleted transposon-like fragment) and Tn6294 (a newly identified transposon). Tn(d)MER3 is a 3.5-kb sequence that carries a merRETPA operon with no merB or transposase genes. It is related to the mer operon of Bacillus licheniformis strain FA6-12 from Russia. DNA homology analysis shows that Tn6294 is an 8.5-kb sequence that is possibly derived from Tn(d)MER3 by integration of a TnMERI1-type transposase and resolvase genes and in addition the merR2 and merB1 genes. Bacteria harboring Tn6294 exhibited broad-spectrum mercury resistance to organomercurial compounds, although Tn6294 had only merB1 and did not have the merB2 and merB3 sequences for organomercurial lyases found in Tn5084 of B. cereus strain RC607. Strains with Tn6294 encode mercuric reductase (MerA) of less than 600 amino acids in length with a single N-terminal mercury-binding domain, whereas MerA encoded by strains MB1 and RC607 has two tandem domains. Thus, Tn(d)MER3 and Tn6294 are shorter prototypes for TnMERI1-like transposons. Identification of Tn6294 in Bacillus sp. from Taiwan and in Paenibacillus sp. from Antarctica indicates the wide horizontal dissemination of TnMERI1-like transposons across bacterial species and geographical barriers.

The effect of aqueous speciation and cellular ligand binding on the biotransformation and bioavailability of methylmercury in mercury-resistant bacteria.

Mercury resistant bacteria play a critical role in mercury biogeochemical cycling in that they convert methylmercury (MeHg) and inorganic mercury to elemental mercury, Hg(0). To date there are very few studies on the effects of speciation and bioavailability of MeHg in these organisms, and even fewer studies on the role that binding to cellular ligands plays on MeHg uptake. The objective of this study was to investigate the effects of thiol complexation on the uptake of MeHg by measuring the intracellular demethylation-reduction (transformation) of MeHg to Hg(0) in Hg-resistant bacteria. Short-term intracellular transformation of MeHg was quantified by monitoring the loss of volatile Hg(0) generated during incubations of bacteria containing the complete mer operon (including genes from putative mercury transporters) exposed to MeHg in minimal media compared to negative controls with non-mer or heat-killed cells. The results indicate that the complexes MeHgOH, MeHg-cysteine, and MeHg-glutathione are all bioavailable in these bacteria, and without the mer operon there is very little biological degradation of MeHg. In both Pseudomonas stutzeri and Escherichia coli, there was a pool of MeHg that was not transformed to elemental Hg(0), which was likely rendered unavailable to Mer enzymes by non-specific binding to cellular ligands. Since the rates of MeHg accumulation and transformation varied more between the two species of bacteria examined than among MeHg complexes, microbial bioavailability, and therefore microbial demethylation, of MeHg in aquatic systems likely depends more on the species of microorganism than on the types and relative concentrations of thiols or other MeHg ligands present.