Complex interactions between diverse mobile genetic elements drive the evolution of metal-resistant bacterial genomes.

In this study, we compared the genomes of three metal-resistant bacteria isolated from mercury-contaminated soil. We identified diverse and novel MGEs with evidence of multiple LGT events shaping their genomic structure and heavy metal resistance. Among the three metal-resistant strains, Sphingobium sp SA2 and Sphingopyxis sp SE2 were resistant to multiple metals including mercury, cadmium, copper, zinc and lead. Pseudoxanthomonas sp SE1 showed resistance to mercury only. Whole genome sequencing by Illumina and Oxford Nanopore technologies was undertaken to obtain comprehensive genomic data. The Sphingobium and Sphingopyxis strains contained multiple chromosomes and plasmids, whereas the Pseudoxanthomonas strain contained one circular chromosome. Consistent with their metal resistance profiles, the strains of Sphingobium and Sphingopyxis contained a higher quantity of diverse metal resistance genes across their chromosomes and plasmids compared to the single-metal resistant Pseudoxanthomonas SE1. In all three strains, metal resistance genes were principally associated with various novel MGEs including genomic islands (GIs), integrative conjugative elements (ICEs), transposons, insertion sequences (IS), recombinase in trio (RIT) elements and group II introns, indicating their importance in facilitating metal resistance adaptation in a contaminated environment. In the Pseudoxanthomonas strain, metal resistance regions were largely situated on a GI. The chromosomes of the strains of Sphingobium and Sphingopyxis contained multiple metal resistance regions, which were likely acquired by several GIs, ICEs, numerous IS elements, several Tn3 family transposons and RIT elements. Two of the plasmids of Sphingobium were impacted by Tn3 family transposons and ISs likely integrating metal resistance genes. The two plasmids of Sphingopyxis harboured transposons, IS elements, an RIT element and a group II intron. This study provides a comprehensive annotation of complex genomic regions of metal resistance associated with novel MGEs. It highlights the critical importance of LGT in the evolution of metal resistance of bacteria in contaminated environments.

Protozoal food vacuoles enhance transformation in Vibrio cholerae through SOS-regulated DNA integration.

Vibrio cholerae, the bacterial pathogen responsible for the diarrheal disease cholera, resides in the aquatic environment between outbreaks. For bacteria, genetic variation by lateral gene transfer (LGT) is important for survival and adaptation. In the aquatic environment, V. cholerae is predominantly found in biofilms associated with chitinous organisms or with chitin "rain". Chitin induces competency in V. cholerae, which can lead to LGT. In the environment, V. cholerae is also subjected to predation pressure by protist. Here we investigated whether protozoal predation affected LGT using the integron as a model. Integrons facilitate the integration of mobile DNA (gene cassettes) into the bacterial chromosome. We report that protozoal predation enhances transformation of a gene cassette by as much as 405-fold. We show that oxidative radicals produced in the protozoal phagosome induces the universal SOS response, which in turn upregulates the integron-integrase, the recombinase that facilitates cassette integration. Additionally, we show that during predation, V. cholerae requires the type VI secretion system to acquire the gene cassette from Escherichia coli. These results show that protozoal predation enhances LGT thus producing genetic variants that may have increased capacity to survive grazing. Additionally, the conditions in the food vacuole may make it a "hot spot" for LGT by accumulating diverse bacteria and inducing the SOS response helping drive genetic diversification and evolution.

The impact of silver nanoparticles on microbial communities and antibiotic resistance determinants in the environment.

Nanosilver (NAg) is currently one of the major alternative antimicrobials to control microorganisms. With its broad-spectrum efficacy and lucrative commercial values, NAg has been used in medical devices and increasingly, in consumer products and appliances. This widespread use has inevitably led to the release and accumulation of the nanoparticle in water and sediment, in soil and even, wastewater treatment plants (WWTPs). This Article describes the physical and chemical transformations of NAg as well as the impact of the nanoparticle on microbial communities in different environmental settings; how the nanoparticle shifts not only the diversity and abundance of microbes, including those that are important in nitrogen cycles and decomposition of organic matters, but also their associated genes and in turn, the key metabolic processes. Current findings on the microbiological activity of the leached soluble silver, solid silver particulates and their respective transformed products, which underpin the mechanism of the nanoparticle toxicity in environmental microbes, is critically discussed. The Article also addresses the emerging evidence of silver-driven co-selection of antibiotic resistance determinants. The mechanism has been linked to the increasing pools of many antibiotic resistance genes already detected in samples from different environmental settings, which could ultimately find their ways to animals and human. The realized ecological impact of NAg calls for more judicial use of the nanoparticle. The generated knowledge can inform strategies for a better 'risks versus benefits' assessment of NAg applications, including the disposal stage.

Long-lasting effect of mercury contamination on the soil microbiota and its co-selection of antibiotic resistance.

Antibiotic resistance genes (ARGs) in the environment are an exposure risk to humans and animals and is emerging as a global public health concern. In this study, mercury (Hg) driven co-selection of ARGs was investigated under controlled conditions in two Australian non-agricultural soils with differing pH. Soils were spiked with increasing concentrations of inorganic Hg and left to age for 5 years. Both soils contained ARGs conferring resistance to tetracycline (tetA, tetB), sulphonamides (sul1), trimethoprim (dfrA1) and the ARG indicator class 1 integron-integrase gene, intI1, as measured by qPCR. The last resort antibiotic vancomycin resistance gene, vanB and quinolone resistance gene, qnrS were not detected. Hg driven co-selection of several ARGs namely intI1, tetA and tetB were observed in the alkaline soil within the tested Hg concentrations. No co-selection of the experimental ARGs was observed in the neutral pH soil. 16S rRNA sequencing revealed proliferation of Proteobacteria and Bacteriodetes in Hg contaminated neutral and alkaline soils respectively. Multivariate analyses revealed a strong effect of Hg, soil pH and organic carbon content on the co-selection of ARGs in the experimental soils. Additionally, although aging caused a significant reduction in Hg content, agriculturally important bacterial phyla such as Nitrospirae did not regrow in the contaminated soils. The results suggest that mercury can drive co-selection of ARGs in contaminated non-agricultural soils over five years of aging which is linked to soil microbiota shift and metal chemistry in the soil.

Regional and oyster microenvironmental scale heterogeneity in the Pacific oyster bacterial community.

Different organs of a host represent distinct microenvironments resulting in the establishment of multiple discrete bacterial communities within a host. These discrete bacterial communities can also vary according to geographical location. For the Pacific oyster, Crassostrea gigas, the factors governing bacterial diversity and abundance of different oyster microenvironments are poorly understood. In this study, the factors shaping bacterial abundance, diversity and composition associated with the C. gigas mantle, gill, adductor muscle and digestive gland were characterised using 16S (V3-V4) rRNA amplicon sequencing across six discrete estuaries. Both location and tissue-type, with tissue-type being the stronger determinant, were factors driving bacterial community composition. Bacterial communities from wave-dominated estuaries had similar compositions and higher bacterial abundance despite being geographically distant from one another, possibly indicating that functional estuarine morphology characteristics are a factor shaping the oyster bacterial community. Despite the bacterial community heterogeneity, examinations of the core bacterial community identified Spirochaetaceae bacteria as conserved across all sites and samples. Whereas members of the Vulcaniibacterium, Spirochaetaceae and Margulisbacteria, and Polynucleobacter were regionally conserved members of the digestive gland, gill and mantle bacterial communities, respectively. This indicates that baseline bacterial community profiles for specific locations are necessary when investigating bacterial communities in oyster health.

Are the existing guideline values adequate to protect soil health from inorganic mercury contamination?

Currently, data that guide safe concentration ranges for inorganic mercury in the soil are lacking and subsequently, threaten soil health. In the present study, a species sensitivity distribution (SSD) approach was applied to estimate critical mercury concentration that has little (HC5) or no effect (PNEC) on soil biota. Recently published terrestrial toxicity data were incorporated in the approach. Considering total mercury content in soils, the estimated HC5 was 0.6 mg/kg, and the PNEC was 0.12-0.6 mg/kg. Whereas, when only water-soluble mercury fractions were considered, these values were 0.04 mg/kg and 0.008-0.04 mg/kg, respectively.

Toxicity of Inorganic Mercury to Native Australian Grass Grown in Three Different Soils.

In this study, three native Australian grasses namely Iseilema membranaceum (Barcoo), Dichanthium sericeum (Queensland Blue) and Sporobolus africanus (Tussock) were grown in three different soils spiked with different concentrations of inorganic mercury and the root elongation was monitored up to 28 days following the germination. Results showed that mercury at certain concentrations significantly inhibited the root growth of all three tested native grasses grown in three soils, however, the toxicity was less in the soil with high organic carbon content and acidic pH. The calculated EC50 values ranged from 10 to 224 mg/kg total Hg in soil. However, the EC10 values indicated that existing guideline values for mercury may be of protective to the native Australian vegetation. Considering their tolerance to soil mercury, these grass species have the potential for their use in rehabilitation of mercury contaminated sites.

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.

Bioremediation of mercury: not properly exploited in contaminated soils!

Contamination of land and water caused by heavy metal mercury (Hg) poses a serious threat to biota worldwide. The seriousness of toxicity of this neurotoxin is characterized by its ability to augment in food chains and bind to thiol groups in living tissue. Therefore, different remediation approaches have been implemented to rehabilitate Hg-contaminated sites. Bioremediation is considered as cheaper and greener technology than the conventional physico-chemical means. Large-scale use of Hg-volatilizing bacteria are used to clean up Hg-contaminated waters, but there is no such approach to remediate Hg-contaminated soils. This review focuses on recent uses of Hg-resistant bacteria in bioremediation of mercury-contaminated sites, limitation and advantages of this approach, and identifies the gaps in existing research.

Mercury alters the bacterial community structure and diversity in soil even at concentrations lower than the guideline values.

This study evaluated the effect of inorganic mercury (Hg) on bacterial community and diversity in different soils. Three soils-neutral, alkaline and acidic-were spiked with six different concentrations of Hg ranging from 0 to 200 mg kg-1 and aged for 90 days. At the end of the ageing period, 18 samples from three different soils were investigated for bacterial community structure and soil physicochemical properties. Illumina MiSeq-based 16s ribosomal RNA (rRNA) amplicon sequencing revealed the alteration in the bacterial community between un-spiked control soils and Hg-spiked soils. Among the bacterial groups, Actinobacteria (22.65%) were the most abundant phyla in all samples followed by Proteobacteria (21.95%), Bacteroidetes (4.15%), Firmicutes (2.9%) and Acidobacteria (2.04%). However, the largest group showing increased abundance with higher Hg doses was the unclassified group (45.86%), followed by Proteobacteria. Mercury had a considerable negative impact on key soil functional bacteria such as ammonium oxidizers and nitrifiers. Canonical correspondence analysis (CCA) indicated that among the measured soil properties, Hg had a major influence on bacterial community structure. Furthermore, nonlinear regression analysis confirmed that Hg significantly decreased soil bacterial alpha diversity in lower organic carbon containing neutral and alkaline soils, whereas in acidic soil with higher organic carbon there was no significant correlation. EC20 values obtained by a nonlinear regression analysis indicated that Hg significantly decreased soil bacterial diversity in concentrations lower than several guideline values.

Mercury toxicity to Eisenia fetida in three different soils.

Three different soils were spiked with 12 different concentrations of inorganic mercury (Hg). Sub-chronic Hg toxicity tests were carried out with Eisenia fetida in spiked soils by exposing the worms for 28 days following standard procedures. The toxicity studies revealed that Hg exerted less lethal effect on earthworms in acidic soil with higher organic carbon (S-3 soil) where water soluble Hg recovery was very low compared to the water soluble Hg fractions in soils with less organic carbon and higher pH (S-1 and S-2 soils). The concentrations of total Hg that caused 50 % lethality to E. fetida (LC50) after 28 days of exposure in S-1, S-2 and S-3 soils were 152, 294 and 367 mg kg-1, respectively. The average weight loss of E. fetida in three soils ranged from 5 to 65 %. The worms showed less weight loss in the organic carbon-rich soil (S-3) compared to less organic carbon containing soils (S-1 and S-2). The bioconcentration of Hg in E. fetida increased with increased Hg concentrations. The highest bioaccumulation took place in the acidic soil with higher organic carbon contents with estimated bioaccumulation factors ranging from 2 to 7.7. The findings of this study will be highly useful for deriving a more robust soil ecological guideline value for Hg.

Bio-augmentation and nutrient amendment decrease concentration of mercury in contaminated soil.

Four mercury (Hg) contaminated soils with different pH (7.6, 8.5, 4.2 and 7.02) and total organic carbon contents (2.1, 2.2, 4 and 0.9%) were subjected to bioremediation utilizing a Hg volatilizing bacterial strain Sphingobium SA2 and nutrient amendment. In a field with ~280mg/kgHg, 60% of Hg was removed by bio-augmentation in 7days, and the removal was improved when nutrients were added. Whereas in artificially spiked soils, with ~100mg/kgHg, removal due to bio-augmentation was 33 to 48% in 14days. In the field contaminated soil, nutrient amendment alone without bio-augmentation removed 50% of Hg in 28days. Nutrient amendment also had an impact on Hg remediation in the spiked soils, but the best results were obtained when the strain and nutrients both were applied. The development of longer root lengths from lettuce and cucumber seeds grown in the remediated soils confirmed that the soil quality improved after bioremediation. This study clearly demonstrates the potential of Hg-reducing bacteria in remediation of Hg-contaminated soils. However, it is desirable to trap the volatilized Hg for enhanced bioremediation.

Predicting plant uptake and toxicity of lead (Pb) in long-term contaminated soils from derived transfer functions.

Regulatory assessment of lead (Pb) in contaminated soils is still expressed primarily as total Pb concentrations in soil. In this study, we estimated effective concentrations (ECx) of Pb to Cucumis sativa L. (cucumber) focusing primarily on pore-water Pb data from 10 different soils after 12 weeks ageing. Phytotoxicity expressed in terms of Pb(2+) was observed to occur in the nanomolar range in neutral to alkaline soils (EC50 values 90 to 853 nM) and micromolar levels for acidic soils (EC50 values 7.35 to 9.66 µM). Internal Pb concentrations relating to toxicity (PT50) in roots and shoots also decreased with increasing pore-water pH (R (2) = 0.52 to 0.53). From a series of dose-response studies, we developed transfer functions predicting Pb uptake in C. sativa and we validated these functions with long-term Pb contaminated soils. The significant independent parameters were pore-water Pb(2+) and dissolved Pb plus dissolved organic carbon (DOC). The observed RMSE for the Pb-DOC model and Pb(2+) were 2.6 and 8.8, respectively. The Pb-DOC model tended to under-predict Pb, whilst Pb(2+) tended to over-predict accumulation despite reasonable RMSE values. Further validation is needed in soils with higher pore-water Pb solubility.

Bioremediation potential of a highly mercury resistant bacterial strain Sphingobium SA2 isolated from contaminated soil.

A mercury resistant bacterial strain, SA2, was isolated from soil contaminated with mercury. The 16S rRNA gene sequence of this isolate showed 99% sequence similarity to the genera Sphingobium and Sphingomonas of α-proteobacteria group. However, the isolate formed a distinct phyletic line with the genus Sphingobium suggesting the strain belongs to Sphingobium sp. Toxicity studies indicated resistance to high levels of mercury with estimated EC50 values 4.5 mg L(-1) and 44.15 mg L(-1) and MIC values 5.1 mg L(-1) and 48.48 mg L(-1) in minimal and rich media, respectively. The strain SA2 was able to volatilize mercury by producing mercuric reductase enzyme which makes it potential candidate for remediating mercury. ICP-QQQ-MS analysis of Hg supplemented culture solutions confirmed that almost 79% mercury in the culture suspension was volatilized in 6 h. A very small amount of mercury was observed to accumulate in cell pellets which was also evident according to ESEM-EDX analysis. The mercuric reductase gene merA was amplified and sequenced. The deduced amino acid sequence demonstrated sequence homology with α-proteobacteria and Ascomycota group.

Mercury Inhibits Soil Enzyme Activity in a Lower Concentration than the Guideline Value.

Three soil types - neutral, alkaline and acidic were experimentally contaminated with nine different concentrations of inorganic mercury (0, 5, 10, 50, 100, 150, 200, 250, 300 mg/kg) to derive effective concentrations of mercury that exert toxicity on soil quality. Bioavailability of mercury in terms of water solubility was lower in acidic soil with higher organic carbon. Dehydrogenase enzyme activity and nitrification rate were chosen as indicators to assess soil quality. Inorganic mercury significantly inhibited (p < 0.001) microbial activities in the soils. The critical mercury contents (EC10) were found to be less than the available safe limits for inorganic mercury which demonstrated inadequacy of existing guideline values.