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Numerical taxonomy of heavy metal-tolerant bacteria isolated from an estuary.

A total of 230 strains of metal-tolerant bacteria from water and sediment samples collected in Chesapeake Bay were isolated on a medium containing cobalt, lead, mercury, or molybdenum. In addition, a set of 71 cultures were simultaneously isolated on glucose tryptone yeast extract agar medium without metals. Twenty-three reference strains were also included in the numerical taxonomy study of these bacteria, bringing the grand total of strains examined to 324. All strains were examined for 112 biochemical, cultural, morphological, and physiological characters. The taxonomic data obtained were analyzed by computer and the simple matching (SSM) and Jaccard (SJ) coefficients were calculated. Clustering achieved by unweighted average linkage is presented and, from sorted similarity matrices and dendrograms, 294 strains, i.e., 97% of the total, were recovered in 12 phenetic groups defined at the 75 to 80% similarity level. Among the strains there were nine phena presumptively identified as Bacillus, Erwinia, Mycobacterium, Pseudomonas, and coryneforms. From the results of the taxonomic study, it is concluded that metal tolerance in estuarine water and sediment bacteria occurs among a restricted range of taxa distributed throughout the estuarine environment.

Actinomycetales

Heavy metal tolerance in the fission yeast requires an ATP-binding cassette-type vacuolar membrane transporter.

In response to heavy metal stress, plants and certain fungi, such as the fission yeast Schizosaccharomyces pombe, synthesize small metal-binding peptides known as phytochelatins. We have identified a cadmium sensitive S. pombe mutant deficient in the accumulation of a sulfide-containing phytochelatin-cadmium complex, and have isolated the gene, designated hmt1, that complements this mutant. The deduced protein sequence of the hmt1 gene product shares sequence identity with the family of ABC (ATP-binding cassette)-type transport proteins which includes the mammalian P-glycoproteins and CFTR, suggesting that the encoded product is an integral membrane protein. Analysis of fractionated fission yeast cell components indicates that the HMT1 polypeptide is associated with the vacuolar membrane. Additionally, fission yeast strains harboring an hmt1-expressing multicopy plasmid exhibit enhanced metal tolerance along with a higher intracellular level of cadmium, implying a relationship between HMT1 mediated transport and compartmentalization of heavy metals. This suggests that tissue-specific overproduction of a functional hmt1 product in transgenic plants might be a means to alter the tissue localization of these elements, such as for sequestering heavy metals away from consumable parts of crop plants.

ATP-Binding Cassette Transporters

Association of metal tolerance with multiple antibiotic resistance of bacteria isolated from drinking water.

Bacterial isolates from the drinking water system of an Oregon coastal community were examined to assess the association of metal tolerance with multiple antibiotic resistance. Positive correlations between tolerance to high levels of Cu2+, Pb2+, and Zn2+ and multiple antibiotic resistance were noted among bacteria from distribution waters but not among bacteria from raw waters. Tolerances to higher levels of Al3+ and Sn2+ were demonstrated more often by raw water isolates which were not typically multiple antibiotic resistant. A similar incidence of tolerance to Cd2+ was demonstrated by isolates of both water types and was not associated with multiple antibiotic resistance. These results suggest that simultaneous selection phenomena occurred in distribution water for bacteria which exhibited unique patterns of tolerance to Cu2+, Pb2+, and Zn2+ and antibiotic resistance.

Anti-Bacterial Agents

Enhanced Heavy Metal Tolerance in Two Strains of Photosynthetic Euglena gracilis by Preexposure to Mercury or Cadmium

The effects of different concentrations of Hg, Cd, and Pb on cell growth, oxygen consumption, and production and contents of chlorophyll a and b of Z and B strains of Euglena gracilis was studied. Cell uptake of Cd and Pb was also determined. A greater cell density reached in the presence of Cd2+ correlated with a lower metal uptake capacity in B-strain cells. Cells of Z strain, preexposed to low concentrations of either Hg2+ (1.5 μM) or Cd2+ (50 μM) for several generations showed improved heavy metal tolerance (greater cell density and viability) together with similar metal uptake capacity. Because Cd2+ preadaptation but not Hg2+ pretreatment induced a significant increase in the content of acid-soluble thiols, the data suggest the existence of different mechanisms of heavy metal detoxification in Euglena. The fact that Z-strain cells showed a greater Cd2+ uptake under all the conditions assayed suggests that Z strain would be more suitable for bioremediation of polluted waters.

Journal Article

Heavy metal tolerance of rice plants (Oryza sativa L.) to some metal oxides at the critical levels.

The toxicity of metal oxides (CdO, ZnO, PbO) were compared with each other and the critical levels of metal uptake by rice plants were determined. The order of metal toxicity to rice plants is CdO greater than ZnO greater than PbO. The highest concentration obtained for unpolished rice was 4.97 micrograms/g at the Cd 10,000 ppm in soil. This values is higher than every other we have seen in the reports for treatment with CdO. We are able to find out that the concentration of 10,000 ppm Cd in the form of CdO in the critical one towards rice plant. By contrast, such damage was not observed at even higher levels of ZnO and PbO were considered to have low toxicity toward rice plant. Also, a negative correlation between Zn or Cu accumulation in rice plants and Cd concentration in soil was found.

Cadmium

Association of metal tolerance with multiple antibiotic resistance of enteropathogenic organisms isolated from coastal region of deltaic Sunderbans.

A survey of microorganisms was conducted in four coastal regions of the deltaic Sunderbans. Among ten different isolates, three were enteropathogenic. They were Vibrio cholerae non-O1 (CT+), enterotoxinogenic Escherichia coli (ETEC) and Pseudomonas aeruginosa. These enteropathogens were able to grow in the presence of zinc (Zn++), cadmium (Cd++), lead (Pb++), cobalt (Co++), copper (Cu++), nickel (Ni++) and silver (Ag+) up to 10 mM concentration. They also showed resistance against 5 to 10 antibiotics. Metal tolerance and drug resistance were not determined by plasmid(s). Synthesis of outer membrane protein among the marine isolates was higher in presence of metal. Enteropathogens isolated from the deltaic Sunderbans were well adapted for growth of the saline environment with higher concentrations of toxic metals.

Drug Resistance, Microbial

[Geomicrobiological studies. XIV. Heavy metal tolerance of desulfurizing bacteria under various ecological conditions].

Tolerance and adaptation to heavy metals of Desulfovibrio- and Desulfotomaculum-strains of different origin have been investigated in enrichment cultures under different conditions of sulphate supply. Three groups of low, medium, and high toxicity were found with As, V, and Mo in the first, Ni, Sb, Co, and Hg in the second, and Cd, Zn, U, and Cu in the third group. If the SO"4-supply was restricted to heavy metal sulphates with a relation of 1:1 of heavy metal- and SO"4 -ions (2:3 with Sb2(SO4)3) the tolerance was somewhat lowered. Adaptation to higher concentrations of heavy metals was possible, if the strains had not yet been exposed and adapted to higher concentration levels at their natural habitats. The tolerance range of desulfurizers has been compared with the tolerance range of different biochemical types of microorganisms.

Bacillaceae

Survey of metal tolerance in moderately halophilic eubacteria.

The tolerance patterns, expressed as MICs, for 250 moderately halophilic eubacteria to 10 heavy metals were surveyed by using an agar dilution method. The moderate halophiles tested included 12 culture collection strains and fresh isolates representative of Deleya halophila (37 strains), Acinetobacter sp. (24 strains), Flavobacterium sp. (28 strains), and 149 moderately halophilic gram-positive cocci included in the genera Marinococcus, Sporosarcina, Micrococcus, and Staphylococcus. On the basis of the MICs, the collection strains showed, overall, similar responses to silver, cobalt, mercury, nickel, lead, and zinc. All were sensitive to silver, mercury, and zinc and tolerant of lead. The response to arsenate, cadmium, chromium, and copper was very heterogeneous. The metal susceptibility levels of the 238 freshly isolated strains were, in general, very heterogeneous among the four taxonomic groups as well as within the strains included in each group. The highest toxicities were found with mercury, silver, and zinc, while arsenate showed the lowest activity. All these strains were tolerant of nickel, lead, and chromium and sensitive to silver and mercury. Acinetobacter sp. strains were the most heavy-metal tolerant, with the majority of them showing tolerance of eight different metal ions. In contrast, Flavobacterium sp. strains were the most metal sensitive. The influence of salinity and yeast extract concentrations of the culture medium on the toxicity of the heavy metals tested for some representative strains was also studied. Lowering the salinity, in general, led to enhanced sensitivity to cadmium and, in some cases, to cobalt and copper. However, increasing the salinity resulted in only a slight decrease in the cadmium, copper, and nickel toxicities.(ABSTRACT TRUNCATED AT 250 WORDS)

Eubacterium

Role of metallothionein in metal detoxification and metal tolerance in protein calorie malnutrition and calcium deficient monkeys (Macaca mulatta).

A monkey model has been set up for protein calorie malnutrition and calcium deficiency. Oral exposure of 5ppm Cd/kg body wt./day for 24 weeks led to increased excretion of Cd, metallothionein (MT) and zinc. Rehabilitation of PCM monkeys for one year resulted in gradual reduction and finally complete disappearance of urinary metallothionein. During Cd exposure, the accumulation of Cd and induction of MT was significantly higher in liver, kidney and intestine. MT was also induced in heart, lung and testis of Cd exposed PCM and calcium deficient monkeys. Metallothionein from liver has been resolved into three isoforms, viz MTa, MTb and MTc on DEAE-Sephadex A 25 ion exchange column. MTc is the major isoform in Cd-treated, normal and protein calorie malnourished monkeys whereas MTb is the major isoprotein in the cadmium treated calcium deficient monkeys. The iso-metallothioneins varied in their metal composition in the nutritional stress conditions and showed different capacities to reactivate apo-enzymes viz. alkaline phosphatase, ceruloplasmin, superoxide dismutase and glutathione peroxidase. Thus, metallothionein plays a key role in metal metabolism during cadmium toxicity under nutritional stress conditions.

Alkaline Phosphatase

Metallothionein gene duplications and metal tolerance in natural populations of Drosophila melanogaster.

A search for duplications of the Drosophila melanogaster metallothionein gene (Mtn) yielded numerous examples of this type of chromosomal rearrangement. These duplications are distributed widely--we found them in samples from four continents, and they are functional--larvae carrying Mtn duplications produce more Mtn RNA and tolerate increased cadmium and copper concentrations. Six different duplication types were characterized by restriction-enzyme analyses using probes from the Mtn region. The restriction maps show that in four cases the sequences, ranging in size between 2.2 and 6.0 kb, are arranged as direct, tandem repeats; in two other cases, this basic pattern is modified by the insertion of a putative transposable element into one of the repeated units. Duplications of the D. melanogaster metallothionein gene such as those that we found in natural populations may represent early stages in the evolution of a gene family.

Animals

Amplification and rearrangement of a prokaryotic metallothionein locus smt in Synechococcus PCC 6301 selected for tolerance to cadmium.

Metal-tolerant cyanobacteria have been isolated from metal-polluted aquatic environments and also selected in culture, but no genes which confer metal tolerance have been described. To investigate the possibility that amplification of a prokaryotic metallothionein gene (smtA), or rearrangement of the smt locus, could be involved in the development of Cd tolerance in Synechococcus PCC 6301, Cd-tolerant lines were selected by stepwise adaptation of a Synechococcus culture. An increase in smtA gene copy number and the appearance of unique additional smtA restriction fragments (both larger and smaller) were detected in these tolerant lines (tolerant to 0.8 microM Cd, 1.3 microM Cd and 1.7 microM Cd). Stepwise adaptation was repeated by using a culture of Synechococcus PCC 6301 inoculated from a single plated colony to obtain four new lines (tolerant to 1.4 microM Cd, 1.8 microM Cd, 2.6 microM Cd and 3.2 microM Cd). Amplification of the smtA gene and development of unique smtA restriction fragments (larger and smaller) were once again detected in these tolerant lines. Amplification and rearrangement of the smt locus were only detected in the seven Cd-tolerant lines, with no evidence of amplification or rearrangement in the non-tolerant lines from which they were derived. As a control, another gene, psaE, was also monitored in these cell lines. There was no evidence of amplification or rearrangement of psaE in the non-tolerant or any of the Cd-tolerant lines.

Base Sequence

Role of metallothionein in detoxification and tolerance to transition metals.

Animal tolerance to the transition metals cadmium and zinc is hereditary. The evolution to a high level of resistance can be accelerated through mutation and selective pressure. We have studied inbred strains of mice and mutants of Chinese hamster ovary cells resistant to cadmium to further these understandings. Results with whole animals show that the difference in the rate and level of metallothionein accumulation is at most twofold between sensitive and resistant strains. However, with cadmium resistant CHO mutant cells, there is an over 60 fold increase in metallothionein and its mRNA upon induction. These mutants show over 60 fold amplification in metallothionein genes. These results offer a direct contrast in the correlation between elevation of metal resistance and increases in metallothionein between two genetic systems.

Animals

Adaptation to metals in widespread and endemic plants.

Bryophytes, including the mosses, liverworts, and hornworts, occur in a variety of habitats with high concentrations of metals and have other characteristics that are advantageous for studies of metal tolerance. Mosses may evolve genetically specialized, metal-tolerant races less frequently than flowering plants. Some species of mosses appear to have inherently high levels of metal tolerance even in individuals that have not been subjected to natural selection in contaminated environments. Scopelophila cataractae, one of the so-called copper mosses, not only tolerates extremely high concentrations of metals in its substrates, but requires these substrates for optimum growth. This species should be included in mechanistic studies of tolerance at the cellular and molecular levels.

Adaptation, Biological

Functional homologs of fungal metallothionein genes from Arabidopsis.

Metallothioneins (MTs) are cysteine-rich proteins required for heavy metal tolerance in animals and fungi. Two cDNAs encoding proteins with homology to animal and fungal MTs have been isolated from Arabidopsis. The genes represented by these cDNAs are referred to as MT1 and MT2. When expressed in an MT-deficient (cup1 delta) mutant of yeast, both MT1 and MT2 complemented the cup1 delta mutation, providing a high level of resistance to CuSO4 and moderate resistance to CdSO4. Although the MT-deficient yeast was not viable in the presence of either 300 microM CuSO4 or 5 microM CdSO4, cells expressing MT1 were able to grow in medium supplemented with 3 mM CuSO4 and 10 microM CdSO4, and those expressing MT2 grew in the presence of 3 mM CuSO4 and 100 microM CdSO4. In plants, MT1 mRNA was more abundant in roots and dark-grown seedlings than in leaves. In contrast, MT2 mRNA accumulated more in leaves than in either roots or darkgrown seedlings. MT2 mRNA was strongly induced in seedlings by CuSO4, but only slightly by CdSO4 or ZnSO4. However, MT1 mRNA was induced by CuSO4 in excised leaves that were submerged in medium. These results indicated that Arabidopsis MT genes are involved in copper tolerance. Plants also synthesized metal binding phytochelatins (poly[gamma-glutamylcysteine]glycine) when exposed to heavy metals. The results presented here argue against the hypothesis that phytochelatins are the sole molecules involved in heavy metal tolerance in plants. We conclude that Arabidopsis MT1 and MT2 are functional homologs of yeast MT.

Amino Acid Sequence

Evidence for the involvement of vacuolar activity in metal(loid) tolerance: vacuolar-lacking and -defective mutants of Saccharomyces cerevisiae display higher sensitivity to chromate, tellurite and selenite.

The responses of Saccharomyces cerevisiae towards the oxyanions tellurite, selenite and chromate were investigated in order to establish the involvement of the yeast vacuole in their detoxification. Three mutants of S. cerevisiae with defective vacuolar morphology and function were used; mutant JSR180 delta 1 is devoid of any vacuolar-like structure while ScVatB and ScVatC are deficient in specific protein subunits of the vacuolar (V)-H(+)-ATPase. All the mutant strains showed increased sensitivity to tellurite and chromate compared to their parental strains. Such sensitivity of the mutants was associated with increased accumulation of tellurium and chromium. These results indicate that accumulation of both tellurium and chromium occurred mainly in the cytosolic compartment of the cell, with detoxification influenced by the presence of a functionally-active vacuole which may play a role in compartmentation as well as regulation of the cytosolic compartment for optimal expression of a detoxification mechanism, e.g. reduction. In contrast, the vacuolar-lacking mutant, JSR180 delta 1, and the defective V-H+ATPase mutant ScVatB displayed lower selenium accumulation than their parental strains. Additionally, the mutant strain ScVatB displayed a higher tolerance to selenite than the parental strain. This result suggests that accumulation of selenium occurs mainly in the vacuolar compartment of the cell with tolerance depending on the ability of the cytosolic component to reduce selenite to elemental selenium, which might, in turn, be related to activity of the V-H(+)-ATPase. These results are discussed in relation to vacuolar compartmentation and the significance of the vacuolar H(+)-ATPase in cytosolic homeostasis of H+ both of which may affect the accumulation, reduction, and tolerance to the tested metal(loids).

Chromates

Integrated metagenomic, culture-based, and whole genome sequencing analyses of antimicrobial resistance in wastewater and drinking water treatment plants in Barcelona, Spain.

The misuse and overuse of antimicrobials drive the emergence of antimicrobial resistance (AMR), a critical global health concern. While wastewater treatment plants (WWTPs) are essential for removing microorganisms and contaminants, they also serve as hotspots for antibiotic-resistant bacteria (ARB) and antimicrobial resistance genes (ARGs), facilitating their persistence and dissemination. This study investigated AMR in two WWTPs and one drinking water treatment plant (DWTP) in the Baix Llobregat area of Barcelona, Spain. Four sampling campaigns were conducted during winter and summer 2023 across different treatment stages. Due to drought conditions, reclaimed water from the Baix Llobregat WWTP was discharged upstream of the DWTP intake to supplement water resources for indirect potable reuse. A total of 991 cultivable ARB were obtained, enabling phenotypic and genotypic characterisation. The most prevalent included Aeromonas spp. (44.3 %), Enterobacterales (27.9 %), Pseudomonas spp. (19.1 %), Acinetobacter spp. (4.8 %), Shewanella spp. (2.2 %), Stenotrophomonas spp. (1 %), and others (0.7 %). Among these, 57.3 % were multidrug-resistant and 2.7 % were extensively drug-resistant. Furthermore, 34.6 % produced extended-spectrum beta-lactamases, 14.1 % harboured carbapenemase genes, and 2.9 % exhibited colistin resistance. Shotgun metagenomic analysis revealed high taxonomic diversity, without dominant genera across treatment stages. The resistome was dominated by ARGs conferring resistance to beta-lactams, aminoglycosides, and macrolides, alongside genes linked to biocide resistance and heavy metal tolerance. Spearman correlation analysis of selected sequenced strains suggested a weak to moderate co-occurrence between ARGs and biocide or heavy metal tolerance genes. These findings underline WWTPs as AMR hotspots and reinforce the need to monitor DWTP source water within the One Health framework.

Wastewater