Mechanisms of Heavy Metal Tolerance in Bacteria: A Review

Abstract

Heavy metal pollution from industrial activities and poor waste disposal poses significant environmental and health threats to humans and animals. This calls for sustainable approaches to the cleanup of heavy metals. This review explores metal tolerance mechanisms of bacteria such as the formation of biofilms, efflux systems, and enzymatic detoxification. These mechanisms allow bacteria communities to adapt and survive in contaminated environments. These adaptations are enhanced by mutations in the bacteria genes and by horizontal gene transfers, enabling bacteria species to survive under environmental stress while simultaneously contributing to nutrient cycling and the decomposition of organic matter. This review further explores the symbiotic interactions between bacteria, plants, and animals. These relationships enhance the metal tolerance ability of the different living organisms involved and are also very important in the bioremediation and phytoremediation of heavy metals. Plant growth-promoting rhizobacteria, Rhizobium, and Bacillus species are very important contributors to phytoremediation; they improve heavy metal uptake, improve the growth of roots, and plants resilience to stress. Moreover, this review highlights the importance of genetically engineered bacteria in closed-loop systems for optimized metal recovery. This offers environmentally friendly and sustainable options to the traditional remediation methods. Engineered Cupriavidus metallidurans CH34 and Pseudomonas putida strain 15420352 overexpressing metallothioneins have shown enhanced metal-binding capabilities, which makes them very effective in the treatment of industrial wastewaters and in biosorption applications. The use of engineered bacteria for the cleanup of heavy metals in closed-loop systems promotes the idea of a circular economy by recycling metals, thus reducing environmental waste. Multidisciplinary research that integrates synthetic biology, microbial ecology, and environmental science is very important for the advancement of metal bioremediation technologies. This review’s analysis on bacterial metal tolerance, symbiosis, and bioengineering strategies offers a pathway to effective bioremediation options, for the reclamation of heavy metal-polluted environments while promoting sustainable environmental practices.

1. Introduction

Heavy metals used interchangeably with “toxic metals” are a group of high-density and high-atomic-mass metallic elements [1]. Their accumulation in the groundwater and soil has adverse effects because they are non-biodegradable and hence persist in the environment, causing long-term harm to living organisms, including humans. Environmental pollution from heavy metals has been linked to ecosystem instability, biodiversity loss, and soil degradation [2]. Heavy metals are exceedingly hazardous and can have carcinogenic, genotoxic, and mutagenic effects [3,4,5]. They have been implicated in delays in the development of children, neurological problems, and chronic diseases [6,7]. The United States Environmental Protection Agency considers heavy metals priority pollutants [8]. According to the US Agency for Toxic Substances and Diseases Registry (ATSDR) [9], tonnes of heavy metals are dumped into the environment annually. This increase in heavy metal pollution is because of fast industrialization and urbanization; this has resulted in increased metal emissions and pollution from human activities [10].

In addition, heavy metals have intricate roles in bacterial communities. Some heavy metals, such as cobalt (Co), copper (Cu), iron (Fe), manganese (Mn), nickel (Ni), and zinc (Zn), are essential micronutrients at trace amounts, enabling crucial metabolic processes and bacterial enzyme stability [11]. However, essential metals can become harmful at high concentrations; for example, bacteria can tolerate Cu at 2–8 mM and Zn at 0.5–5 mM [12], whereas metals like cadmium (Cd) and mercury (Hg) are toxic at micromolar levels [13]. Tolerance is dependent on microbial species, the environmental conditions, and resistance mechanisms. In bacteria, iron (Fe³⁺), manganese (Mn⁴⁺), arsenic (As⁵⁺), and selenium (Se⁶⁺) function as electron acceptors during anaerobic respiration. This underscores the significance of the oxidized forms of metals in microbial metabolism under certain environmental conditions [14]. However, other heavy metals, such as silver (Ag), lead (Pb), cadmium (Cd), aluminum (Al), mercury (Hg), gold (Au), and arsenic (As), are toxic to microorganisms. Heavy metals interfere with important cellular activities by displacing important metals from their original binding locations, interacting with critical ligands, disrupting cell membranes, modifying enzymatic specificity, and denaturing nucleic acids [15]. At high concentrations, the toxic effects of heavy metals can be lethal, making it very difficult to manage environments polluted with heavy metals.

The intrinsic toxicity and persistence of heavy metals in the environment is a challenge for bioremediation strategies. The alternatives for eliminating or neutralizing heavy metals are limited because they cannot be thermally or biologically broken down like organic contaminants [16]. However, some bacteria have developed defensive mechanisms through which they are able to tolerate and even flourish in conditions where heavy metal pollution is present at very toxic levels. Bacteria are very important subjects for consideration in the context of heavy metal bioremediation because of their ubiquity and involvement in almost every biological process. Some bacterial species such as Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis, C. metallidurans, Staphylococcus aureus, Ralstonia metallidurans, etc., have evolved mechanisms to tolerate and detoxify heavy metals, even though high concentrations of these heavy metals are naturally toxic to microorganisms, altering their protein structure and nucleic acids, disrupting osmotic balance, and disrupting oxidative phosphorylation [17,18,19,20]. With the use of microbial biotechnology, these heavy metal-resistant bacteria can provide sustainable solutions for the bioremediation of heavy metal-contaminated environments, mitigating the environmental impact associated with conventional treatment options.

The growing occurrence of bacteria resistance to heavy metals, particularly in marine and aquatic habitats impacted by anthropogenic activities, has increased our understanding of how bacteria survive acute and long-term exposure to metal ions. Bacteria’s resistance mechanisms usually combine multiple strategies such as blocking metal uptake, the active efflux of metals, sequestering metals using chelating agents or metal-binding proteins, modifying metal speciation through redox transformations, and precipitating metals into less toxic forms [21,22]. In addition to allowing bacteria to thrive in habitats contaminated with heavy metals, these adaptive responses present intriguing directions for the creation of efficient bioremediation techniques that lower the negative effects of heavy metals on the environment. From a sustainability standpoint, microbial bioremediation presents a viable eco-friendly alternative to conventional chemical and physical remediation methods. In addition, this corroborates the principles of a circular economy, because it employs existing biological processes in the conversion of pollutants into less harmful substances. This method not only promotes the long-term health of ecosystems but also minimizes energy consumption and decreases the reliance on additional harmful chemical inputs. Consequently, microbial bioremediation emerges as a critical strategy for the attainment of sustainable environmental management.

To address the existing gaps in the literature, this review will focus on emerging bioremediation strategies that have not been thoroughly investigated. This includes recent developments in phytoremediation that integrate metal-tolerant bacteria, which may enhance the synergies between plants and bacteria for the effective management of metal-contaminated environments. This review will also explore the use of bacterial mechanisms for heavy metal resistance in various industrial settings, presenting potential pathways for sustainable pollution management in mining, wastewater treatment, and soil rehabilitation. Furthermore, this review will emphasize the significance of bacterial genetic engineering in optimizing pathways for metal binding, uptake, and detoxification, aiming to broaden the effectiveness and application of bioremediation techniques for persistent heavy metal pollutants.

2. Classification of Metals

Metals have been broadly categorized into three primary classes, namely metals, metalloids, and heavy metals, based on their distinct properties and behaviours [23]. Metals are chemical elements that typically form lustrous solids, and they are very good conductors of heat and electricity. However, some metals are liquid at room temperature, for example, mercury. Metalloids possess properties that are intermediate between nonmetal and metals [24]. This blend of characteristics makes them unique and differentiates them from both metals and nonmetals; for example, they are better conductors of electricity to nonmetals but not as conductive as metals. Examples of metalloids include antimony (Sb), arsenic (As), boron (B), germanium (Ge), silicon (Si), selenium (Se), and tellurium (Te).

Further classification of metals is based on their biological functions and effects; some metals are necessary trace elements for metabolic reactions. Others are toxic to living organisms even at very low concentrations, and they do not serve any biological function [25]. On this basis, metals have been classified into the following: (i) essential and relatively non-toxic or essential with known biological roles, examples of which include calcium (Ca) and magnesium (Mg); (ii) essential but toxic at high concentrations (typically Fe, Mn, Zn, Cu, Co, Ni, and Mo); and (iii) toxic (e.g., Hg, Pb, Cd, etc.). Toxic metals have no known biological function. The toxic effects of metalloids differ from those of metals because of their different chemical behaviours [26,27]. While metals predominantly exist as cationic species, forming positively charged particles (like tiny balls with a positive charge) in their interactions with other substances, metalloids typically exist in anionic forms or exist as anionic species, forming negatively charged particles (like tiny balls with a negative charge) [28]. Their existence as cationic and anions species makes them behave differently, and thus they can cause different kinds of harm. For example, arsenic mimics crucial phosphate groups required for ATP and so disrupts cellular respiration and energy generation through the formation of negatively charged ions [29,30]. This interference can stop essential functions and cause the death of bacterial cells.

According to the definition of heavy metals as a class of metallic elements with elevated density and atomic mass that are toxic [31], it is safe to say that heavy metals fall under metals classified as essential but toxic at high concentrations and metals classified as toxic. Heavy metals have been defined using different criteria, including their abilities to form cationic hydroxides [32,33] and engage in complex formations [34,35], with a specific gravity greater than 5 g/mL [10,36], based on the hard and soft acid–base theory [37], and their association with eutrophication and environmental toxicity [38].

The redox characteristics and solubility of heavy metals determine their classification, which in turn affects their toxicity and behaviour in the environment. Redox properties describe a metal’s capacity to acquire or lose electrons, which affects its reactivity and capacity to generate potentially free radicals that can damage cells. For example, manganese and chromium are known for their high reactivity and capacity to produce radicals that can cause damage to cellular structures [39]. The metals’ environmental impact and bioavailability are determined by their solubility in soil and water. Because they are so soluble, highly toxic metals like Pb and Hg can easily find their way into the food chain. However, due to their reduced solubility, metals like Ni and Cd tend to persist in soils and cause long-term pollution of the environment [40].

3. Heavy Metal Toxicity and Bacterial Reponses

Heavy metals, to varying degrees, have been reported to play crucial roles in the metabolic processes of a variety of bacteria. The different anthropogenic activities that release large quantities of metals into the environment have detrimental effects on microorganisms. Bacteria present in a wide variety of environments are the best model system for studying the interactions of heavy metals with living organisms. Bacteria make up a significant level of biomass globally, and they can absorb more metals from solution per unit of biomass (g/mg dry wt.) than any other living organism [41]. The ecosystem is affected by the presence of these metallic elements, resulting in shifts in biomass, the composition of the microbial community, and element cycling [42]. The morphological characteristics of bacterial cells are significantly affected/altered by their exposure to heavy metals. These changes include alterations in the integrity of the cell membrane, in cell size, and in cell shape, which are indicative of cellular stress and damage. For example, Matyar et al. [43] reported that E. coli cells exposed to sub-lethal concentrations of Hg became elongated and filamentous. This elongation is thought to be because of the inhibition of cell division proteins, leading to the formation of abnormally long cells. Another study by Lemire et al. [44] showed that the exposure of P. aeruginosa to Cd significantly damaged the cell membrane. Cadmium ions interactions with membrane lipids and proteins, led to the disruption of the cell’s structural integrity, increasing the membrane permeability, which led to the leakage of intracellular contents.

Table 1. Effects of selected heavy metals on bacterial cells.

Heavy Metals	Effect on Bacteria	References
Arsenic	Deactivation of enzymes Induction of oxidative stress	[45]
Cadmium	Damage to proteins and nucleic acids Impedes cell division and transcription	[46]
Chromium	Growth inhibition Elongation of lag phase	[47,48]
Nickel	Causes oxidative stress Enzyme inhibition and membrane disruption	[49]
Copper	Disrupts cellular functions Inhibits enzyme activities	[50]
Lead	Inhibition of cell division Disruption of cellular respiration	[51]
Zinc	Inhibits growth at high concentrations Alters membrane integrity	[52]
Mercury	Inhibits protein synthesis Causes cell lysis	[53,54]
Silver	Antimicrobial effects Disruption of membrane integrity	[55]

highlights the effects of certain heavy metals on bacterial cells, highlighting different negative consequences such as enzyme deactivation, oxidative stress induction, and cellular damage caused by exposure to these metals.

Different mechanisms have been used to explain heavy metal toxicity to bacteria, including the generation of reactive oxygen species (ROS) via redox catalysis, the inactivation of lethal enzymes, the disruption of ion regulation, and an effect on DNA and protein synthesis.

The presence of heavy metals in the environment affects the biochemistry and physiology of bacterial cells through the disruption of their interaction with ligands or the removal of metals from their natural binding site [56,57]. Some metals like nickel catalyze the production of ROS through the Fenton and Haber–Weiss reactions, acting as soluble electron carriers in the process. Heavy metals also damage bacterial DNA, lipids, and proteins as well as cytoplasmic molecules [58]. Heavy metals’ competitive or non-competitive interactions with substrates can alter enzymes configuration, halting their vital functions [59]. When heavy metals attach to bacteria surface and enter through transmembrane carriers or ion channels, they disrupt the cellular ion balance [57]. Changes to the structure of nucleic acids due to exposure to heavy metals trigger a cascade of effects on bacterial metabolism, morphology, and growth. These changes result in enzyme inactivation, membrane disruption, functional impairment, and the inhibition of oxidative phosphorylation.

Heavy metals negatively affect different cellular components, and the enzymes involved in metabolism and detoxification processes. For example, the enzyme superoxide dismutase, which is responsible for detoxifying ROS, has been inhibited by heavy metals (Pb and Cr), leading to an increase in oxidative stress on phosphate-solubilizing bacteria (Bacillus sp. Strain MRP-3) [60]. Moreover, Hg has been reported to disrupt bacterial cell membrane [61], interfere with intracellular processes, and cause damage to nucleoid regions and essential macromolecules, thus impairing cellular function and viability [62,63].

4. Molecular Mechanisms of Heavy Metal Tolerance

There are three different resistance mechanisms: transport via an efflux pump system, intra- and extracellular sequestration, and enzymatic conversion to a less toxic form (Figure 1). This section deals with the efflux pump system and sequestration, while highlighting bacterial heavy metals’ enzymatic conversion to a less toxic form.

4.1. Metal Transport and Efflux Systems

Bacteria employ complex mechanisms of metal transport and efflux to prevent metal toxicity. These systems control intracellular concentrations of metals, sequester excessive metals, and export the metals from the cell, protecting cells and preserving homeostasis in metal-rich environments. P-type ATPases and the Resistance–Nodulation–Division (RND) efflux systems are two of the most researched groups of metal transporters involved in sequestration, transport, and export.

4.1.1. Description of Metal Transporters

Metal ion translocation across biological membranes is catalyzed by a superfamily of membrane-bound transporters known as P-type ATPases; they are also called E1-E2 ATPases [65]. By transferring surplus metals from the cytoplasm to either the external environment or subcellular compartments, these proteins are important for preserving intracellular ion homeostasis. P-type ATPases are structurally characterized by a conserved core domain comprising an actuator (A), phosphorylation (P), and nucleotide-binding (N) domain that coordinates ion transport and ATP hydrolysis [66]. P-type ATPases work through a cyclical mechanism of ion binding, phosphorylation, and conformational change. They are extremely selective for metal ions, such as Cu, Zn, or Cd. In E. coli, CopA ATPase plays an important role in copper resistance by pumping out Cu(I) ions from the cytoplasm [67,68]. The transporter binds Cu(I) via high-affinity sites in its metal-binding domain, and ATP hydrolysis phosphorylates conserved aspartate residue and brings about a conformational shift in the protein. This conformational shift causes the Cu(I) ion to translocate across the membrane. Upon the release of the metal ion, the transporter dephosphorylates and returns to its original state, preparing for a new cycle of transport. ATP-binding cassette (ABC) transporters utilize the energy derived from ATP hydrolysis to facilitate the translocation of metal ions across bacterial membranes. ABC transporters have demonstrated the ability to alleviate intracellular metal toxicity by expelling metal ions including Cd, Zn, and Cu from bacterial cytoplasm [69]. They frequently assemble into complexes that ensure high specificity for their substrates, a very important role in microbial resistance to metal stress.

Furthermore, cation diffusion facilitators (CDFs) are another type of important metal ion transporters. They have been shown to mediate the efflux of divalent cations, including Zn, Cd, Fe, and Ni [69]. CDFs make use of electrochemical gradients in driving the transport of metal ions across membranes, thereby maintaining cellular metal homeostasis. CDFs play a supplementary role in P-type ATPases by passively enabling the diffusion of metal ions outside of the bacteria cell, depending on the physiological requirements of the organisms.

The Resistance–Nodulation–Division (RND) family is a significant set of metal transporters that primarily act as efflux pumps in getting rid of toxic metal ions and antimicrobial substances. Gram-negative bacteria have many of these metal transporters. In Gram-negative bacteria, RND systems combine to form tripartite complexes that bridge both the inner and outer membranes and expel substrates straight into the extracellular space [70]. Ag and Cu efflux in E. coli is carried out by the Cus complex, which is the archetypal illustration of an RND efflux mechanism [71,72]. CusA, an inner membrane pump; CusB, a periplasmic adaptor protein; and CusC, an outer membrane channel, are the three primary parts of the Cus system. These elements work together to create a continuous channel that enables the direct export of metal ions from the periplasm and cytoplasm to the extracellular space. Proton-motive force drives RND transporters, whereas ATP hydrolysis is necessary for P-type ATPases [66,73].

4.1.2. Mechanisms of Metal Sequestration and Export

The mechanisms of metal sequestration and export in microbial cells are highly coordinated processes that involve the selective binding of metal ions, their transport across membranes, and their eventual release into compartments or the external environment. The first defence mechanism used by microorganisms against metal toxicity is the sequestration of metal ions within cellular compartments. Metal-binding proteins found in the cytoplasm and periplasm, such as metal chaperones and metallothioneins, are essential for binding free metal ions, which reduces their bioavailability and prevents the production of harmful ROS [74]. Metallothioneins, which are cysteine-rich proteins, sequester heavy metals such as Cu and Zn in non-toxic complexes [75]. Additionally, metal chaperones facilitate the safe transportation of metal ions to target proteins or transporters. For instance, the copper chaperone CopZ in B. subtilis discharges copper ions to the CopA ATPase for subsequent export [76]. These chaperones help ensure that metal ions are transported to target cellular destinations without causing damage during this process.

Upon identifying metal ions as surplus or sequestration, metal transporting systems become activated to remove these ions from the cytoplasm, maintaining metal homeostasis. Though P-type ATPases and RND efflux systems are the primary export systems, transporters, such as CDFs, also serve as additional contributors to this process [77]. P-type ATPases like CopA and ZntA utilize the energy produced in the hydrolysis of ATP to export metal ions. For example, in E. coli, a P-type ATPase, ZntA, exports Cd (II) ions from the cytoplasm, playing a critical role in the organism’s resistance to Zn and Cd toxicity [78]. Cd (II) ions bind to the metal binding domain of the transporter in an ATPase-mediated transport process. This is followed by an ATP-driven conformational change that enables the transportation of the ion across the membrane.

RND systems, particularly the Cus complex, function as proton antiporters, utilizing the electrochemical gradient to drive metal efflux. In this process, metal ions in their reduced forms (Cu(I) or Ag(I)) first bind to the inner membrane component (CusA). The metal ions are then transferred through the periplasmic component (CusB), and then finally discharged through the outer membrane channel (CusC) [79]. This coordinated process is important in the efficient removal of heavy metals from bacterial cells. In addition to direct efflux, some bacteria make use of vesicle-based export mechanisms. P. aeruginosa exemplifies the utilization of outer membrane vesicles for the sequestration and expulsion of metal ions, offering enhanced protection against heavy metal toxicity [80].

4.2. Metal Chelation and Sequestration

This section discusses the functions of metallothioneins and siderophores in facilitating the binding and detoxification of heavy metals. Metallothioneins are low-molecular-weight proteins rich in cysteine. The prevalence of cysteine residues facilitates metal binding via thiol groups (-SH), hence sequestering metals and reducing their interaction with critical bacterial proteins. This function is particularly important in metal-rich environments, where it has been demonstrated to bind metals such as Zn and Cd. [81,82]. The cyanobacterium Synechococcus sp. PCC 7002 synthesize metallothioneins through the smtA and smtB genes, which are activated in response to the presence of Cd ions [83]. Similarly, P. putida strains can bind Cd inside their cells using metallothioneins [75].

Siderophores are low-molecular-weight compounds that possess a strong affinity for metal cations. They are mostly produced by bacteria and fungi. Although their main function is iron chelation, these molecules also play a role in other heavy metal detoxification processes. In microbial systems, siderophores have been shown to chelate metals such as Fe and Al, gallium (Ga), and Cr [84]. For example, siderophores generated by Alcaligenes eutrophus and P. aeruginosa reduce the toxicity of heavy metals such as Cd, Pb, and Cu [85,86]. Furthermore, P. aeruginosa produces the siderophores pyoverdine and pyochelin, which block the absorption of metals, including Cr, Fe, Hg, and Pb, demonstrating the different roles that siderophores perform in detoxifying heavy metals [87].

Intracellular sequestration is an important technique used by bacteria in the detoxification of heavy metal. It involves the complexation and storage of metal ions within the cytoplasm of microbial cells. Microorganisms, particularly bacteria and cyanobacteria, utilize intracellular metal-binding proteins such as metallothioneins to effectively sequester metal ions. This inhibits free metal ions from interacting and injuring biological components. For example, Rhizobium leguminosarum reduces cytoplasmic toxicity by sequestering Cd ions through complex formation with glutathione [88]. The presence of Cd in marine gamma-proteobacteria induces the production of low-molecular-weight proteins resembling phytochelatins; this allows for the intracellular sequestration of Cd [89]. This process is important for bacteria to tolerate high-metal-concentration environments.

Additionally, bacteria can sequester heavy metals outside of the cytoplasm by forming insoluble metal complexes or by periplasmic retention. In this way, toxic metals are kept out of the cytoplasm. Copper-resistant Pseudomonas syringae generates outer membrane proteins such as CopC and periplasmic proteins like CopA and CopB; these proteins bind copper ions to prevent intracellular toxicity [90]. Similarly, Pseudomonas stutzeri AG259, isolated from a silver mine, has been shown to form sulphide compounds with silver ions in the periplasm [91].

Sulphate-reducing bacteria also exhibit extracellular sequestration. The hydrogen sulphide produced by sulphate-reducing bacteria combines with metallic ions to form insoluble metal sulphides. P. aeruginosa and Klebsiella planticola utilize this mechanism in Cd precipitation under aerobic environments through the formation of cadmium sulphide [92]. Furthermore, some bacteria precipitate metals as other insoluble compounds. P. putida S4, for example, generates hydroxyl complexes and copper-containing phosphate, while Vibrio harveyi precipitates lead in the form of lead phosphate, both facilitating the detoxification of heavy metals [93,94].

In addition to the above-mentioned resistance mechanisms, some bacteria can excrete enzymes that bind or chelate heavy metals, lowering their bioavailability and toxicity [95]. Another significant mechanism is the decrease in valency of heavy metals, lowering their toxicity. Certain bacteria, for example, can convert hexavalent chromium (Cr⁶⁺) to the less hazardous trivalent form (Cr³⁺), allowing for successful detoxification [96].

4.3. Genetic Regulation of Metal Tolerance

Metal ion homeostasis and detoxification are regulated by various kinds of molecular mechanisms in the complex and highly adaptable genetic regulation of metal tolerance in bacteria. This process is controlled by specialized proteins known as metal-responsive regulators, which sense metal ion concentrations and adjust gene expression accordingly. These mechanisms are very important for the survival of bacteria in contaminated environments and biotechnological applications like bioremediation and bioleaching. Gene regulation for the genes responsible for efflux pumps, metal absorption, sequestration, and detoxification are the primary factors that influence bacterial tolerance to heavy metals. This multi-level genetic regulation, including transcriptional activation and repression, post-transcriptional, and post-translational controls, is usually mediated by specific metal-sensing transcription factors [97].

The major groups of these metal-responsive regulators are the MerR and ArsR families. The primary role of the MerR family is mercury resistance. They include proteins like the MerR protein from E. coli, well known for its regulation of the mer operon, responsible for encoding the genes involved in the detoxification of mercury. The MerR protein acts by binding the promoter region of the mer operon. MerR firmly binds to DNA in the absence of mercury; this binding suppresses the transcription of genes resistant to mercury [98]. This repression is due to MerR’s ability to prevent RNA polymerase from binding effectively. Upon being exposed to mercury, mercury ions attach to MerR, eliciting a change in the conformation of the protein lowering its DNA binding affinity. This modification relieves the repression, enabling RNA polymerase to reach the promoter, initiating the transcription of genes like merA and merB. The merA gene encodes mercuric reductase that transforms harmful mercuric ions (Hg²⁺) to less hazardous elemental mercury (Hg⁰), whereas the merB gene encodes a mercuric ion reductase engaged in further detoxification [99]. Different bacterial species have evolved homologs of the MerR family, each of which can adapt to different metal stress. P. aeruginosa harbours MerR-like proteins that confer mercury resistance in diverse environmental conditions [100]. These variations reflect the adaptive nature of the MerR family across different bacterial niches. The ArsR family is crucial for arsenic resistance and includes several well-characterized regulators, such as ArsR from E. coli. These function in similar ways to the MerR family. The ArsR family shows significant functional diversity among bacterial species. B. subtilis employs ArsR-like regulators to manage arsenic detoxification in various environments, highlighting the evolutionary adaptability of this family [101]. This adaptability is essential for survival in environments with fluctuating arsenic concentrations.

Metal-responsive regulators regulate the expression of genes by conformational changes that are dependent on metal. For example, the binding of mercury to MerR causes a structural change that makes it easy for RNA polymerase to reach the mer operon promoter [102]. Similarly, when arsenic binds to ArsR, it results in a shift in conformation that relieves the repression of the arsenic resistance (ars) operon and so allows for transcription [103]. The specificity of the interactions between metal-responsive regulators and specific DNA sequences is important for the binding of the promoter and for regulation. These regulators’ specific binding to their respective promoters responds directly to metal ion levels, ensuring that the expression of genes is carefully adjusted in accordance with the concentration of metal ions present. This specificity is achieved by the recognition of metal-responsive motifs in the DNA and the subsequent alteration in the accessibility of the promoter [104,105].

The tolerance of bacteria to heavy metal is also regulated at the post-transcriptional and post-translational levels. RNA-binding proteins and small regulatory RNAs (sRNAs) are important in fine-tuning the gene expression for metal tolerance. For example, RyhB a sRNA in E. coli coordinates the cellular responses to iron limitation by coordinating the expression of genes related to the storage and utilization of iron in response to the availability of iron [106]. Post-translational modifications also influence the activity of metal-responsive regulators. The binding of heavy metal can induce conformational changes in the structure of protein; this affects its DNA-binding affinity and regulatory function [107]. Through this mechanism, bacterial cells can quickly and dynamically adapt to metal stress. In addition, detoxification enzyme activities and metal transporters are impacted by post-translational changes such as phosphorylation and methylation. The phosphorylation of proteins in two-component regulatory systems such as the CusRS and ZntR regulates their activity in response to metal sensing, ensuring rapid and appropriate cellular responses to the changing concentrations of metals [108]. Post-transcriptional and post-translational regulation enables bacteria to respond to metal stress in a rapid and flexible way, complementing other adaptation mechanisms such as horizontal gene transfer (HGT). HGT is the exchange of genetic material between organisms outside of traditional parent–offspring inheritance. HGT occurs through mechanisms like transformation (the uptake of environmental DNA), transduction (virus-mediated transfer), and conjugation (direct transfer via cell-to-cell contact) [109]. HGT enables rapid adaptation, playing a key role in the spread of traits like antibiotic resistance and heavy metal tolerance among bacteria.

Metal tolerance genes are frequently acquired from HGT through plasmids, transposons, and integrative conjugative elements. Mobile genetic elements carry metal resistance genes, allowing bacteria to rapidly adapt to changing environmental conditions. For example, the ars operon is typically found on plasmids and encodes proteins responsible for arsenic efflux and detoxification [110]. The ars operon is regulated by a transcriptional repressor, ArsR, that binds to the operon’s promoter region, dissociates in the presence of arsenic to release repression and permit transcription, and controls the ars operon [111].

In addition to gaining metal tolerance genes through HGT, bacteria must integrate these resistance mechanisms with larger regulatory systems to cope with varying environmental stressors. The different kinds of stressors encountered by bacteria in metal-contaminated environments make it necessary to integrate metal tolerance mechanisms with larger regulatory networks that react to nutrient limitation, oxidative stress, and changes in the environment. There is considerable overlap in the regulation of metal tolerance genes among global regulators including OxyR, SoxRS, and Fur (ferric uptake regulator), which regulate the responses to iron homeostasis and oxidative stress [112]. This crosstalk ensures a coordinated response to the often-simultaneous threats posed by metals and other environmental stressors.

5. Microbial Community Dynamics

5.1. Impact of Heavy Metal Contamination on Microbial Community Structure

Heavy metal contamination profoundly impacts the chemical composition, structure, and functionality of microbial communities across various environments. Microbial communities are highly susceptible to external disturbances, including the introduction of heavy metals, that can induce alterations in species composition, abundance, and diversity. The toxicity of heavy metals to various microbial species impairs cellular function by binding to protein molecules, inhibiting enzymes, or producing ROS, leading to cellular damage [113]. This results in a decrease in species richness and a transition toward communities dominated by heavy metal-resistant strains [114].

Heavy metals can exert selective pressure on microbial populations, promoting the survival of microbial populations bearing metal resistance genes. For example, exposure to Cd and Pb can reduce microbial diversity, with vulnerable species outcompeted by resistant ones [115]. Exposure to heavy metals can result in the prevalence of Gram-negative bacteria, which have efflux mechanisms and metal-sequestering proteins such as metallothioneins that allow them to withstand elevated metal concentrations [116,117]. However, Gram-positive bacteria, fungus, and certain archaea may significantly contribute to metal resistance in certain conditions. Bacillus megaterium, a Gram-positive bacteria, plays a crucial role in metal resistance, demonstrating the ability to flourish in and detoxify Cd and Pb through the production of extracellular polymeric substances (EPS) that can bind and immobilize metals [118]. In addition, Bacillus species have mechanisms for metal transport through which they sequester and remove toxic metals from their cells [119]. These have cascading effects on ecosystem processes including nitrogen fixation, carbon cycling, and organic matter decomposition, since the sensitive microbial taxa involved in these processes might decrease in their activity or abundance.

Bacterium exposure to heavy metal also alters functional genes distribution in microbial communities. Metagenomic studies have been used to demonstrate that microbial communities that are frequently exposed to heavy metals have an increase in their genes responsible for metal resistance [120]. However, this may be at the compromise of other important ecological processes, because the metabolic activity that is involved in preserving metal tolerance mechanisms may reduce the ability of the microbial community to take part in other ecosystem processes such as nutrient cycling [121]. Depending on some factors, including the type of metal, concentration of metals, length of exposure to metals, and microbial communities’ innate resistance and adaptability, heavy metal pollution in the environment can result in significant changes in microbial communities [122,123].

Role of Metal-Tolerant Microorganisms in Maintaining Ecosystem Function

Despite the adverse effects of heavy metal contamination, metal-tolerant microorganisms still play a key role in preserving vital ecosystem functions in contaminated environments. These microorganisms have evolved and adapted mechanisms that enable them to thrive in metal-stressed environments. These mechanisms include the creation of metal-chelating compounds (e.g., siderophores), enzymatic reduction in metal ions, and employment of metal efflux systems detoxifying their cells from harmful metals [124]. These adapted strategies enable them to withstand harsh conditions, while carrying out vital ecological activities that contribute to the resilience of contaminated environments. Metal-tolerant microorganisms are important in the bioremediation and recovery of heavy metals, with their interactions involving several key processes. The bacteria uptake of heavy metals from the environment is followed by biosorption, a process where metals bind to the bacterial cell surface through ion exchange, complexation, or adsorption. For example, on the bacterial cell wall, cationic metals like Pb²⁺ can be exchanged for protons (H⁺). This is a type of ion exchange. Next, the metals are then bioaccumulated within the bacteria cells, leading to an internal concentration that is higher than the external environment. The accumulated metals are then converted/transformed to less toxic forms, such as reducing hexavalent chromium (Cr⁶⁺) to trivalent chromium (Cr³⁺). This aids in detoxification by allowing bacteria to neutralize heavy metals into harmless forms. Finally, metal recovery involves extracting valuable metals from bacterial biomass or metabolic products, thus demonstrating the potential of bacteria in bioremediation and heavy metal management.

Metal-tolerant bacteria are essential in the biogeochemical cycling of nutrients like carbon, nitrogen, and sulphur. Metal-tolerant bacteria and archaea, such as Acinetobacter, Pseudomonas, and Desulfovibrio, have demonstrated the ability to maintain sulphur cycling and organic matter breakdown in metal-stressed conditions [19,125]. There has been a consistent intersection of their ability to remove heavy metals via redox reactions and their involvement in nutrient cycling processes. For example, sulphate-reducing bacteria lower toxic metals like chromium and uranium and in anaerobic conditions; they promote sulphur cycling [126]. This dual functionality highlights the relevance of metal-tolerant bacteria in maintaining ecological activities, especially under metal stress. In addition, heavy metal-tolerant bacteria aid the formation of soil and improve plant health in contaminated environments [127], enabling vegetation to thrive in contaminated soils and stabilizing the ecosystem [128]. Moreover, microbial biofilms can form in these environments and bind heavy metals, preventing further metal diffusion and reducing bioavailability; this consequently lowers the total environmental toxicity [129].

While metal-tolerant bacteria have shown to play significant roles in these vital ecosystem processes, they currently do not completely remediate a metal-stressed environment to its natural state. In some cases, their activities are inadequate to completely restore ecosystem services to pre-contamination levels, and variations in microbial community structure can still cause long-term changes in ecosystem function. However, the ability of these bacteria to adapt and sustain crucial activities like nutrient cycling, metal detoxification, and plant–microbe interactions demonstrates their importance in the resilience and recovery of contaminated ecosystems.

5.2. Evolutionary Mechanisms Leading to Increased Metal Tolerance

To increase their resistance to toxic compounds, bacteria have evolved and developed adaptive mechanisms that enable them to thrive efficiently when faced with heavy metal contamination. One of these key mechanisms is genetic adaptation. This involves microorganisms undergoing mutations that give them resistance to heavy metals, allowing them to survive in contaminated environments. The genes responsible for regulating metal ion absorption, efflux, and storage are usually the target genes for this mutation [130]. Notably, modifications of metal-binding proteins enable the sequestration of toxic metals, which prevents their interaction with key biological components [131]. Furthermore, changes in membrane transport systems, such as the activity of ATP-binding cassette transporters and cation diffusion facilitators, enhance efficiency in efflux mechanisms. By actively pumping out excess metals or diffuse cations, these systems lower the intracellular metal concentrations, thereby reducing toxicity [132].

HGT contributes significantly to metal tolerance as it facilitates the spread of resistance genes across various bacterial species. HGT promotes the spread of advantageous characteristics among populations of bacteria via organelles such as plasmids and other mobile genetic component, thus facilitating rapid adaptation in metal-contaminated environments [133]. HGT allows bacteria populations to quickly acquire and improve their resistance capabilities; this highlights the importance of genetic connectivity in the evolution of microorganisms. Following the development of resistance characteristics, microorganisms usually engage in compensatory evolution to compensate for the fitness costs associated with these changes [134]. This phenomenon includes modifications to metabolic pathways, such as elevated expressions of stress response genes that promote growth in extreme or polluted environments. Bacteria can mitigate the harmful effects of metal exposure by optimizing their metabolic processes, which will guarantee their survival in harsh conditions.

The development of biofilms is another important factor in bacterial tolerance to heavy metals. The EPS in biofilms creates a protective niche for bacteria. Additionally, EPS binds heavy metals, lowering their toxicity and bioavailability [135]. The importance of this communal adaptive living strategy is supported by evidence that microbial populations are in constant synergistic interactions within the biofilms to improve their collective metal tolerance [136].

Furthermore, genetic dominance and the phenomenon of polyploidy—a genetic state where an organism has more than two complete sets of chromosomes—are other important factors that affect the evolution of metal resistance. So, many copies of resistance genes are carried by polyploid organisms; this increases the chances of successful adaptations while mitigating the effects of detrimental mutations [137]. Heavy metal tolerance dynamics are influenced by the positive frequency dependency that results from polyploidy. This underscores its significance in determining the evolutionary pathways of microorganisms. The complex interactions between genetic, ecological, and environmental factors in the determination of bacterial resilience is highlighted by these mechanisms, which collectively show a rich tapestry of the evolutionary strategies adopted by bacteria in response to heavy metal stress.

Chen et al. [138] demonstrated a concerning trend where bacterial mechanisms of resistance to heavy metals could facilitate resistance to antibiotics. This cross-resistance occurs because the genes that provide resistance to both antibiotics and metals often resides in the same mobile genetic elements, such as plasmids and transposons [139]. For example, efflux pumps and metal-binding proteins that expel toxic metals can also reduce the intracellular concentration of antibiotics, providing dual resistance [140]. The contamination of the environment with heavy metal selects resistant bacteria, indirectly enriching populations with antibiotic resistance genes [139]. This relationship underscores the dual environmental and public health implications of heavy metal pollution, because it can exacerbate the global challenge of antimicrobial resistance.

5.3. Symbiosis and Interaction with Other Organisms

The remediation of environments polluted by heavy metals presents a formidable challenge, necessitating new strategies to alleviate the detrimental effects of these contaminants. Recent research has demonstrated the importance of symbiotic relationships among metal-tolerant bacteria, plants, and their interactions with animals in enhancing metal tolerance and facilitating bioremediation processes.

Phytoremediation, which employs plants to remove, stabilize, or alter contaminants in soil and water, is considerably augmented by metal-tolerant bacteria. Plant growth-promoting rhizobacteria (PGPR), including Rhizobium and Bacillus species, have shown efficacy in enhancing the phytoremediation process by augmenting metal absorption in plants, stimulating root development, and offering protection against metal-induced stress [141]. Through genetic alterations, these PGPR can be engineered for specific heavy metals, establishing specialized partnerships between plants and bacteria to enhance metal extraction from contaminated soils. In environments contaminated with heavy metals, plants and PGPR develop mutualistic interactions that improve the plants’ resilience and growth. PGPR foster systemic tolerance to abiotic challenges, including heavy metal exposure, improve soil structure, and enhance nutrient absorption [141]. These bacteria enhance plant resistance to heavy metals by synthesizing phytohormones such as auxins and cytokinins, which stimulate root development and enhance nutrient uptake, while also excreting chemicals that trap or chelate metals, thereby diminishing their bioavailability [95,142]. Research indicates that genetically manipulated strains of B. subtilis can synthesize siderophores, which bind to metals and promote their transport, thereby increasing metal availability for plant absorption [143]. Additionally, PGPR synthesize 1-aminocyclopropane-1-carboxylic acid deaminase, which reduces ethylene concentrations in plants [144]. This reduction mitigates ethylene’s suppressive effects on root elongation and overall plant growth, enabling plants to tolerate contaminated soils.

Another important symbiotic interaction exists between bacteria and fungi, notably mycorrhizal fungi, in association with plant roots. These fungi improve the absorption of nutrients, including metal ions, by increasing root surface area via hyphal extensions. Bacterial–fungal consortia can work together to improve heavy metal bioavailability and immobilization while simultaneously promoting plant development. For example, arbuscular mycorrhizal fungi in combination with Pseudomonas species have shown promise in phytoremediation by lowering heavy metal toxicity and increasing plant tolerance [145]. Such interactions demonstrate the potential of complex microbial communities to improve bioremediation efficiency. Additionally, Xin [146] reported the efficiency of inoculating PGPR and arbuscular mycorrhizal fungi, the application of organic matter, essential nutrients, beneficial elements, the regulation of soil pH, and water management in decreasing soil Cd bioavailability and inhibiting the uptake and accumulation of Cd in crops.

A promising tactic in phytoremediation involves the use of plant–microbe consortia, which consists of various engineered bacterial species that work together in the plant rhizosphere to maximize metal uptake. A recent investigation revealed that the combination of different bacterial genera represented by Rhizobium sp., Rhizobium leguminosarum, Sinorhizobium meliloti, Pseudomonas sp., Pseudomonas fluorescens, Luteibacter sp., Variovorax sp., Bacillus simplex, and B. megaterium resulted in a significant increase in Cd, Pb, Cu, and Zn absorption by Lathyrus sativus, a plant known for its hyperaccumulating abilities, suggesting that multi-species consortia could outperform single-species methods [147]. This multi-strain strategy not only enhances plant growth and metal tolerance but also tackles the wide range of metal contaminants typically found in polluted environments.

Moreover, phytoremediation studies have shown that the selection of metal-tolerant plant species often correlates with the presence of microbiota capable of bioremediating heavy metals [148,149]. Non-native plant species have less beneficial interactions with the soil microbiota compared to the native species, and consequently have lower phytoremediation capabilities [150]. This underscores the importance of selecting the appropriate plant–microbe consortia that will grow in contaminated environments and result in enhanced metal tolerance and bioremediation efficiency.

Symbiotic relationships toward heavy metal removal also include microorganisms’ relationships with animals. The symbiotic relationship under the “metaorganism” framework highlights the complex and interrelated relationships that improve metal tolerance. For example, the presence of some metal-tolerant bacteria in the gut microbiomes of herbivores can facilitate the detoxification of heavy metals absorbed from contaminated food sources, improving these organisms’ general health [151]. In addition to microbial relationships, insects and soil fauna play an important role. These species can have an impact on microbial community dynamics and nutrient cycling, which in turn affects plant health and metal tolerance. For example, soil-dwelling insects can promote the spread of beneficial microorganisms through their activities, thus improving the symbiotic relationships that lead to plant resistance to metal toxicity [152,153].

6. Genetically Engineered Bacteria and Closed-Loop Metal Recovery Systems

Recent progress in biotechnology has utilized microorganisms that tolerate metals, primarily through genetic modifications, to improve the bioremediation of heavy metals. Specific alterations in metal-tolerant bacterial strains have allowed these microorganisms to function as effective biosorbents, neutralizing and even extracting metals from contaminated environments, particularly in industrial and wastewater locations [154,155]. This section will examine the use of these engineered microorganisms in heavy metal bioremediation, focusing on genetically modified strains for wastewater treatment, and the incorporation of closed-loop systems for efficient metal recovery.

6.1. Engineered Bacteria in Industrial and Wastewater Treatments

Genetically modified bacteria have arisen as a viable biotechnological solution to heavy metal pollution, as these organisms may be designed to tolerate elevated metal concentrations while efficiently absorbing them. C. metallidurans, a thoroughly researched bacterial species, demonstrates the capacity to detoxify metal-contaminated environments, efficiently managing metals such as Cd, Zn, and Cu through its intrinsic resistance mechanisms [156,157,158]. In genetically enhancing the expression of genes responsible for metal resistance, C. metallidurans have been improved for industrial applications, boosting their metal-binding capability and allowing for its use in bioreactors for large-scale bioremediation projects.

Similarly, P. putida has also been extensively modified to boost metal absorption, often through changes that enhance the production of metal-binding proteins or enzymes involved in metal transformation processes. Recent research has resulted in strains of P. putida strain 15420352 that overexpress metallothioneins, proteins that bind to metals, thus improving its capability to capture and immobilize metals like Pb and Hg found in wastewater [159,160]. This modified P. putida has proved effective in pilot-scale bioreactors, featuring applications in the treatment of industrial wastewater from mining and battery production [161].

Additionally, progress has been made in creating metal-selective biosorbents derived from engineered bacteria. These biosorbents have been optimized through some surface modifications that improve their affinity for specific metal species. This is very important in making bacteria able to target specific metal ions. E. coli strains have been modified to express surface-displayed peptides that have high affinity for Hg or Cr, and this was used to achieve notable results in significantly reducing heavy metal concentrations, in both laboratory and field experiments [162].

6.2. Integration into Closed-Loop Systems for Metal Recovery

Genetically modified bacteria are now used alongside phytoremediation strategies in closed-loop systems. It has become a very significant area of concentration for the improvement of sustainable bioremediation strategies. The aim in closed-loop systems is to extract and recycle pollutants from contaminated environments, reducing and eliminating waste and promoting a circular economy. Through the reclamation of metals from industrial wastewater or through the mining of byproducts, closed-loop systems offer environmentally friendly alternatives to the traditional methods such as chemical precipitation or electrolysis; these traditional methods are energy-intensive and tend to generate harmful byproducts.

In closed-loop systems, the genetically modified bacteria act as biocatalysts binding to metals and transforming them, facilitating their subsequent extraction and reuse. For example, C. metallidurans CH34 has successfully been engineered in systems aimed at the detection and recovery of gold from wastewater [163]. In this example, C. metallidurans CH34 transforms soluble gold complexes into elemental gold nanoparticles that can be collected for reuse in electronics and other sectors. This approach helps in the elimination of toxic metals while enabling resource recovery and improving the economic feasibility of bioremediation strategies.

Engineered biofilms represent another innovative application within closed-loop systems for metal recovery. These biofilms provide a robust structure that supports bacterial growth, allowing for enhanced survival in high metal concentrations and improving metal uptake [135]. Recent advancements in engineered biofilms have been concentrated on developing multi-layered structures that target specific metals. For example, biofilms tailored with binding peptides have demonstrated excellent selectivity for copper and nickel, achieving significant recovery rates in controlled bioreactor environments [164].

Ongoing research aims to create biofilm-based systems that work in tandem with phytoremediation. In these frameworks, engineered biofilms collaborate with plants to concentrate metals in a manner conducive to plant absorption and storage. This strategy has the potential to establish highly effective closed-loop systems that not only clean up contaminated soil and water but also facilitate the recovery of valuable metals, resulting in sustainable and eco-friendly solutions for sites affected by metal pollution.

7. Conclusions

This paper underlines the complex interaction between bacterial communities and their tolerance to toxic metals, demonstrating how heavy metal contamination affects microbial structure and function and selects specialized metal-tolerant populations. These evolutionary adaptations, including genetic mutations, HGT, and biofilm formation have demonstrated the resilience of bacteria to environmental stressors. Metal-tolerant bacteria, even in the presence of contaminants in the environment, play very important roles in ecosystem functioning, including nutrient cycling and organic matter decomposition. The ability of metal tolerant bacteria to survive in extreme environmental conditions is crucial for ecosystem sustainability. In this way, they can preserve the integrity of nutrient cycles even in the presence of heavy metal contaminants. Their ability to develop symbiotic interactions with plants and animals makes them even more important in the application of bioremediation technologies in the cleanup of heavy metal-polluted environments. This presents a sustainable and low-impact option to the traditional physical and chemical remediation techniques, diminishes the ecological footprints, and fosters long-term environmental health. Metal-tolerant bacteria’s ability to detoxify contaminated sites through the biosorption, bioaccumulation, and enzymatic transformation of metals into less toxic forms makes them very important in bioremediation.

8. Future Perspectives

The use of genetically modified bacteria in closed-loop systems for metal recovery faces technical and regulatory challenges. More research is important to understand the scalability, long-term stability, and potential for gene transfer into natural ecosystems. The focus of further research should be toward refining engineered systems for specific industrial uses and prioritizing biosafety. Innovations in synthetic biology, for example, the development of programmable biosensors, have made it possible to monitor and control metal recovery processes in real time, facilitating the integration of these technologies into traditional industrial processes.

Furthermore, metal-tolerant bacterial diversity and functionality can be investigated to gain insight into their tolerance mechanisms, while metagenomic research can reveal novel resistance genes. The long-term monitoring of contaminated environments will elucidate the changing patterns of microbial communities throughout recovery, while investigations into plant–microbe interactions will help optimize phytoremediation strategies. Understanding cross-species interactions and measuring these communities’ ecological responsibilities will also help us learn more about ecosystem health. We can develop long-term solutions to heavy metal pollution and encourage ecological balance for a healthier future by employing a multidisciplinary approach that includes microbial ecology, environmental science, and biotechnology.