Microbial stress response to heavy metals in the environment

Abstract

Heavy metal contamination is a global environmental issue as it poses a significant threat to public health, and exposure to metals above a certain threshold level can cause deleterious effects in all living organisms including microbes. To survive in such harsh environments, some microbes evolved a few defence mechanisms to metabolize and transform heavy metal into a less hazardous form, resulting in the formation of heavy-metal-resistant microbes. Heavy-metal-resistant microbes can be used in bioremediation to remediate the contaminated areas. Bioremediation uses natural biological activities, is relatively low-cost, and has high public acceptance. Herein, we summarize the interactions and mechanisms that occur between the microbes and heavy metal, including stress responses and defence mechanisms, which involve aggregation and biofilm formation, production of extracellular polymeric substances (EPS), and development of resistance genes and signalling pathways against heavy metals.

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Pranesha Prabhakaran

Pranesha Prabhakaran is a Master's student at the Faculty of Science and Technology, UKM. She is working on a study of bacterial aggregation under heavy metal stress.

Muhammad Aqeel Ashraf

Dr Muhammad Aqeel Ashraf is a Professor at the School of Environmental Studies, China University of Geosciences, China. He is working on Environmental Geochemistry.

Wan Syaidatul Aqma

Dr Wan Syaidatul Aqma is a Senior Lecturer at the Faculty of Science and Technology, Universiti Kebangsaan, Malaysia. Her research interest is Environmental Microbiology.

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1. Introduction

Heavy metals can be defined by various criteria including density, atomic weight, atomic number, chemical properties, and Lewis acid behaviour;¹ however, density is considered to be the main defining feature.² The heavy metals have atomic densities exceeding 5 g cm⁻³ and atomic numbers above 20.^3–5 Accumulation of the heavy metals above the threshold level is mainly due to anthropogenic activities, including mining, chemical manufacturing, agriculture,⁶ hospital wastewater,⁷ and electronic waste.⁸ Heavy metals can pose cytotoxic, carcinogenic, and mutagenic effects, and most heavy metals are hazardous to humans, even in low concentrations.⁹ It has been proven that the accumulation of heavy metal in the body has caused severe effects in the heart, brain, kidney, bones, and liver.¹⁰

Heavy metal pollution is considered as the most severe environmental issue since these pollutants are capable of infiltrating deep into the bed of groundwater sources and surface water, and affect public health.^11,12 These heavy metals end up in the food chain and bioaccumulate, transferring from one food chain to another.⁵ Metals are able to exert their toxicity because they are non-degradable and are only transformable via methylation, sorption and complexation, and alteration to a valence state, which influences their bioavailability and mobility.¹² Urban areas with high population density and accelerated anthropogenic activities such as mining are considered as a reservoir of pollution that is commonly made up of heavy metals.^13,14 Mine water pollution can severely impact the biological systems as species diversity and the total biomass composition in aquatic and terrestrial ecosystems can be affected due to acidity and heavy-metal contamination.¹⁵ A recent incident of heavy metal contamination, containing mostly iron, zinc and copper, that occurred in Colarado, US, was reported in August 2015, where a million gallons of wastewater spilled out from an abandoned mine and caused severe heavy metal pollution in the Animas river.¹⁶ It has been reported that heavy-metal contamination due to mining activities involved around 2 million hectares out of 10 million hectares of heavy-metal-contaminated land in China.¹⁴ Another study that was conducted to evaluate the chemical speciation of heavy metals in the sediment from a former tin mining area at Selangor, Malaysia, proved that the sediment was contaminated with chromium, zinc, arsenic, copper, lead, and mainly tin.¹⁵

A bioremediation process that uses biological agents to effectively remove organic and inorganic toxic wastes from the environment, which generally has major public acceptance, could be the key to solving this problem.^17–19 The application of microbial metabolism as an alternative to physicochemical methods to remediate contamination is considered to be safer, more effective, and less expensive.²⁰ Thus, further understanding of the mechanisms involved in heavy metal resistance and the application of resistant bacteria in bioremediation is crucial to meeting this condition.

2. Interaction between microbes, minerals and metals

In the biogeochemical cycling of heavy metals, microbes exhibit an important role in cleaning up the metals.²² Metals are classified into three major groups according to their biological roles and effects: (i) essential metals with a recognized biological role (Na, Ca, K, Mn, Mg, V, Fe, Cu, Co, Mo, Ni, Zn, and W); (ii) toxic metals (Ag, Sn, Cd, Au, Ti, Hg, Pb, Al, and metalloids Ge, Sb, As, and Se); and (iii) non-essential, non-toxic metals with no biological effects (Rb, Sr, Cs, and T).¹⁸ The top most prevalent environmentally toxic metals, such as As, Pb, Cd, and Hg, are dangerous to public health.²³

Heavy metals are grouped into five categories, according to primary accumulation mechanisms in sediments: (i) adsorptive and exchangeable, (ii) bound to reducible phases (Mn oxides and Fe), (iii) bound to carbonate phases, (iv) bound to organic matter and sulphides, and (v) detrital or lattice metals.²² Interaction between microbes, metals, and minerals occurs under both natural and unnatural conditions, with some alteration to their physical and chemical states; at the same time, metals and minerals are also capable of influencing microbial growth, activity, and survival by becoming involved directly or indirectly in all phases of microbial metabolism, growth and differentiation.¹⁸

Metals such as Na, Zn, K, Ca, Cu, Co, Mg, Mn, and Fe, that go beyond the threshold concentrations, will exert toxicity on cells even though they are essential for life.¹⁸ Metals like Cu, Co, Cu, Cr, Ni, Zn, Mg, Fe, Na, K, and Mn are micronutrients that are required by cells and are involved in redox reactions.²⁴ These micronutrients stabilize molecules via electrostatic interactions, regulate osmotic pressure, act as components of various enzymes, and form concentration gradients and charges across cytoplasmic membranes.²⁵ The physicochemical properties of a particular environment and the chemical behaviour of the metal species affect metal toxicity. Some metals even cause microorganisms to flourish, despite their toxicity, in sites that are polluted with metals, with various mechanisms being used to develop resistance toward metals.¹⁸ This condition has caused the development of heavy-metal-resistant bacteria, which have been isolated from various environmental sources globally (Table 1).

Table 1 List of selected heavy-metal-resistant bacteria isolated from the various environmental sources globally

Heavy metal	Microorganism	Location	Reference
As	Enterobacter agglomerans	Cambodia	26
	Acinetobacter lwoffii
Cu, Pb, Cd	Bacillus megaterium X4	Korea	27
Cu	Sphingomonas sp.	Chile	28
	Stenotrophomonas sp.
	Arthrobacter sp.
Cu, Co, Ni, Zn, Cr, Cd, Pb	Pseudomonas aeruginosa ASU 6a	Egypt	29
Pb, Cr, Zn, Cu	Streptomyces	Morocco	30
	Amycolatopsis
Hg, Cr, Ag	Bacillus sp.	Brazil	6
	Pseudomonas aeruginosa
	Enterobacteriaceae strain
Cu, Cd, Pb, Cr, Ni	Pseudomonas putida	China	31
	Cupriavidus necator
	Exiguobacterium sp.
	Bacillus aquimaris
	Bacillus cereus
	Alcaligenes sp.
As, Pb	Bacillus sp.	India	32
As, Hg	Bacillus sp.	Iceland	11
	Lysinibacillus sp.	French Guiana
		Spain
Hg	Pseudomonas sp.	Iran	33
	Escherichia coli
	Serratia marcescens

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Bioremediation is carried out in situ or ex situ. In situ bioremediation is executed in the polluted area, which costs less and discharges less pollutants into the environment, whereas in ex situ bioremediation, the contaminated material will be removed to be treated elsewhere and requires shorter treatment time frames. In comparison, the conventional processes that have been used to eliminate heavy metal from industrial wastewater, such as chemical precipitation, oxidoreduction, filtration, electrochemical techniques, and sophisticated separation processes using membranes, are far more expensive.³⁴ The addition of exogenous microorganisms that are genetically modified, or contain natural catabolic genes to enhance and expand the indigenous population, is known as bioaugmentation.³⁵ Engineered bioremediation may speed up the growth of microbes and optimize the detoxification process.³⁶ Reducing the bioavailable concentration and interaction of the toxic metal with the cell helps in boosting the organic bioremediation process.³⁴

Optimizing parameters, such as nutrients, growth, temperature, oxygen level, solute concentrations, and pH, enables microbes to flourish at the peak of their growth rate. Any alteration to these parameters is considered as an environmental stress; thus, microbes need to sense and react to this in order to be sustained in that environment. As a matter of fact, the majority of bacteria that are able to thrive in a constant state of stress with optimum growth conditions, mostly exist only inside a laboratory environment. Bacteria have the potential to sense and react to stress stimuli via a coordinated alteration in gene expression.²¹ Response mechanisms against alterations in the environment are generally available, and changes usually lead to the synthesis of specific molecules that respond to the adverse environmental conditions.²¹ Microbes that develop resistance toward metals can be utilised as bioremediation agents. Biochemical evolution in microbes, occurring in defence against heavy metal toxicity, can be advantageous in the application of bioremediation.³⁷

3. Bacterial resistance towards heavy metals

Microbial inhibition by heavy metal occurs when heavy metals block essential functional groups or interrupt essential metal ion incorporation into biological molecules.³⁴ Heavy metals interrupt the binding of essential metal ions to the cellular structure with their high electrostatic attraction and binding affinities to similar sites. This leads to the destabilization of structures and biomolecules (cell wall enzymes, DNA, and RNA), which triggers defects in the replication process, followed by mutagenesis.³⁵ Three major mechanisms are involved in the attachment of metals to bacterial cell walls: (i) precipitation via nucleation reactions, (ii) complexation with nitrogen and oxygen ligands, and (iii) ion exchange reactions with teichoic acid and peptidoglycan Gram-positive bacteria; in particular, Bacillus sp. possesses a high adsorptive capacity when teichoic acid and peptidoglycan contents in the cell wall are high; on the other hand, Gram-negative bacteria cell membranes, which have a lower amount of these components, are weak metal absorbers.³⁸

There are five mechanisms involved in metal toxicity in microorganisms: (i) substitutive metal–ligand binding, with interruption or destruction of the biological function of the targeted molecules when replacement by another metal ion occurs at the binding sites of specific biomolecules; (ii) covalent and ionic reduction–oxidation (redox) reaction of metal ions with cellular thiols (R–SH), specifically glutathione, with reaction between thiols and oxyanions producing hazardous reactive oxygen species (ROS) as a by-product of reduction; Pinner-type reactions of thiols with metal oxyanions, such as Se and Te oxyanions (SeO₄²⁻, SeO₃²⁻, TeO₄²⁻ and TeO₃²⁻); (iii) Fenton-type reaction, which involves transition metals, such as Cu, Ni and Fe, that produce ROS. ROS are extremely reactive compounds that can oxidize all biological macromolecules; (iv) inhibition of membrane transport processes, specific membrane transporters being inhibited by toxic metals engaging binding sites and/or interrupting the membrane potential conserved for essential substrates; (v) electron siphoning by thiol-disulphide oxidoreductase in the respiratory chain caused destruction of cell membranes' proton motive force.²³ The production of oxygen radicals induced by metals affects DNA as well as other cellular components, such as polyunsaturated fatty acid residues of phospholipids, which are oxidation-sensitive.³⁶

A rapid and effective process for heavy metal elimination from cells is important to avoid toxicity. Typically, there are two types of mechanisms involved in resistance towards heavy metal ions: (i) intracellular complexation of toxic metal ions, mainly in eukaryotes, and (ii) reducing the accumulation of cations based on active efflux in prokaryotes.³⁹ Specifically, heavy metal resistance in bacteria involves five mechanisms: (i) expulsion of metal by a permeability barrier, (ii) extracellular sequestration, (iii) intracellular physical sequestration of metal by binding to protein or other ligands to avoid damage to the metal-sensitive cellular targets, (iv) expulsion by active export of metal from a cell, and (v) transformation and detoxification.^40,41

With a strong ionic nature, metals are able to bind to many cellular ligands and dislocate native essential metals from their regular binding site, which is hazardous to cells. Non-enzymatic detoxification may also occur when microbes release inorganic metabolic products, including carbonate, sulphide, or phosphate ions, through their respiratory metabolism, and through precipitation of toxic metal ions. Cellular sequestration and accumulation or extracellular precipitation is applied by metals to immobilize metals in nature. Metal ions attach to the cell surface through several mechanisms, including van der Waals forces, redox precipitation, covalent bonding, or a combination of these processes. Carboxyl, hydroxyl, and phosphoryl are negatively charged groups in bacterial cell walls that retain metal cations by mineral nucleation after absorbing them.²² Heavy metal toxicity can be reduced by overexpression of metal-binding peptides on the microbial cell surface to increase the capacity of adsorption.⁴²

Enzyme detoxification is the key mechanism of bacterial resistance toward metals. The presence of metal and metalloid-resistant genes in bacteria is an advantage, as observed in Bacillus spp. for Hg²⁺ and Cd²⁺ resistance. The synthesis of various metal-binding peptides and proteins, such as metallothioneins (MTs) and phytochelatins (PCs), aids in the regulation of metal ion homeostasis and affects toxic responses.¹⁸ MTs are low-molecular-weight proteins that are encoded by mt genes and are expressed in bacteria to boost metal resistance through immobilization, while PCs are polypeptides that contain a high number of gammaPCs, dipeptide residues. Both MTs and PCs contain high cysteine (Cys) levels; cysteine is an amino acid that contains sulphur (S) atoms to bind metals.⁴² In bacteria, altering the fatty acid composition of their lipids is one of the defence and/or repair mechanisms used to maintain membrane fluidity. Modifications of the lipid acyl chain structure by altering the ratios of saturation to unsaturation, branched to unbranched formation, and cis to trans unsaturation, as well as acyl chain lengths and forms of branching, are executed as a response to toxic agents.²⁵ Heavy metal stress also causes alteration in fatty acid composition by qualitative and quantitative alteration of lipids, inhibition of biosynthetic pathways, and lipid peroxidation.²⁵

Due to the fact that concentrations of metals above the threshold level are hazardous to microbes in the environment, as they poses a deleterious impact on microbial functional activities, microbes that are present in heavy-metal-contaminated soil have evolved several schemes to develop resistance toward heavy metals.^3,19 Metal-ion-specific physicochemical parameters, including the Pearson softness index, standard reduction potential (ΔE⁰), electron density electronegativity (χ), solubility product of the metal–sulphide complex (pK_SP), and covalent index, are related to the susceptibility of microorganisms towards toxic metal species.²³

Elimination of heavy metals from polluted areas is difficult as, unlike other pollutants, heavy metals cannot be converted into less hazardous, less mobile and/or less bioavailable forms via biodegradation processes. Basically, microbes can either be resistant or tolerant toward a pollutant. Tolerance is described as the ability of a microorganism to survive in a polluted environment through its own intrinsic properties, while resistance is the ability of microbes to survive in a high concentration of a toxic substance via detoxification mechanisms as a direct response toward the existence of a similar contaminant.^3,38 Resistance mechanisms in bacteria are encoded typically on the plasmids and transposons. This might be due to gene transfer or spontaneous mutations that cause these bacteria to eventually gain resistance to heavy metals, as exposure to DNA-damaging agents can result in genetic changes.^19,43 Generally, a gene that is responsible for heavy metal resistance is located in the extrachromosomal circular DNA; for example, a plasmid that is carried by metal-resistant bacteria.¹⁹ Resistant genes will be induced and expressed in the presence of specific metals and regulated when certain concentrations of the metals are reached. Promoters and regulatory genes from the bacterial operon that is responsible for resistance are used as metal-specific biosensors (promoter–reporter gene fusion), regulating metal-resistant gene expressions in the presence of specific metals at specific concentrations.^3,19

As some heavy metals are crucial for enzyme function, growth and metabolism, understanding the mechanism of heavy metal uptake in bacterial cells could provide a deeper understanding of the resistance mechanism. Generally, there are two types of heavy metal uptake mechanisms: (i) by osmotic gradient across the cell membrane, which does not require ATP, and (ii) by specific substrates which are dependent upon ATP released from ATP hydrolysis, which is slower when compared to the ATP-independent mechanism.^3,19 Some of the mechanisms involved are highly specific biochemical pathways that act as a protective barrier to protect microbes from toxic heavy metals, which can be favourable in handling metal contamination. The detoxification process by microbes involves altering the chemical properties of the metals, rather than degrading them. Previous studies proved that microbes that belong to a heterotrophic group are capable of mobilizing metals through the production of organic acids, whereas autotrophic bacteria, such as Thiobacillus spp., are capable of producing metal-leaching sulphuric acid by oxidizing elemental sulphur.³⁷

Biosorption is the metabolism-dependent sorption of radionuclides and heavy metals onto biomass. The presence of amine, carboxyl, hydroxyl, sulfhydryl, and phosphate groups leads to a negatively charged cell surface at neutral pH, thus enabling absorption of a considerable amount of positively charged cationic metals.³⁷ The bacterial growth phase, biomass density, and living status of the biomass are directly proportional to the capacity of biosorption.⁴⁴ The cell walls of Gram-positive bacteria possess more affinity than those of Gram-negative bacteria, and are able to attach a higher concentration of metals.³ Microbial detoxification usually requires the efflux or exclusion of metal ions from the cell. This phenomenon results in a high local concentration of metals at the cell surface, which allows reactions with biogenic ligands and precipitates.³⁷ Biosorption of heavy metal by bacteria depends on a non-enzymatic adsorption process, which can be described as the non-specific binding of metal ions to proteins and extracellular/cell surface-associated polysaccharides. Microbial biosorbents rely on the microbial species; the process can be active or passive. Passive uptake of metal ions is rapid, irreversible, independent of cellular metabolism, and non-specific to metal species, physical condition and ionic strength, while in contrast, the active process is slow and dependent on the cellular metabolism.³

The biological reduction of some metals causes significant changes in solubility. For instance, U(VI), a highly soluble and mobile form of uranium, becomes extremely insoluble as U(IV) after undergoing enzymatic reduction by anaerobic bacteria. Anaerobic bacteria use indirect mechanisms to reduce and precipitate some metals, for example, Fe(III)-respiring bacteria, which are capable of catalyzing the formation of Fe(II)-bearing minerals to reduce and precipitate high-valence metals abiotically,³⁷ and Hg, which is more bioavailable to microorganisms in an anaerobic environment.⁴⁵ Another example is Serratia marinorubra, a facultative anaerobic marine bacteria, which is able to transform arsenate to arsenite and methylarsonate under anaerobic conditions.⁴⁶ Biomethylation involves the biotic formation of volatile and non-volatile methylated metal and metalloid compounds⁴⁶ by microorganisms.⁴⁷ For example, during the biotransformation of arsenic, microorganisms, such as bacteria, fungi and algae, transform methylated hazardous inorganic arsenic to form monomethylarsonic acid and dimethylarsinic acid.⁴⁸

3.1 Efflux transporter in heavy-metal-resistant bacteria

In bacteria, the acquisition of essential metal ions from the outside of cells demands consideration. Every Gram-negative bacteria has a periplasmic space, an outer membrane, and inner cytoplasm, which metal ions have to pass through to reach the cytosol. In contrast, Gram-positive bacteria lack periplasm, and the presence of porins on the outer membrane permits metal ions to undergo non-selective passive diffusion across the outer membrane.⁴⁹ Heavy metal elimination relies on an energy-dependent ion efflux from the cell by a membrane protein. This acts as an ATPase or chemiosmotic cation/proton antiporter, rather than performing chemical detoxification.⁵⁰ High-affinity transport systems present in the outer membrane or fixed in the inner membrane aid in the transportation of metal ions into the cytosol. Hydrolysis of ATP on the cytoplasmic side of the membrane drives inner membrane transport systems, for example ATP-binding cassette (ABC) transporters and P-type ATPase, or couples to cation diffusion facilitator (CDF) proteins.⁴⁹ Specific and non-specific transporters help in the transportation of essential metal ions into the cytoplasm. In non-specific transporters, this is conducted by a chemiosmotic gradient across the bacterial cytoplasmic membrane to transport excess metal ions into the cell. This situation, also known as ‘open gate’ causes heavy metal ions to become toxic. For a specific transporter, this requires a specific metabolic situation or starvation, and is only expressed when needed.⁴⁰

There are three main classes of efflux transporters: (i) P-type ATPase, which is incorporated in the inner membrane and uses ATP to transport metal ions from the cytoplasm to the periplasm, (ii) CBA transporters, which exist in Gram-negative bacteria and are three-component transenvelope pumps that play a role as chemiosmotic antiporters, and (iii) cation diffusion facilitator (CDF) transporters that function as chemiosmotic ion-proton exchangers. P-Type ATPase and CDF transporters, which export metal ions from the cytoplasm to the periplasm, are common in many bacterial species, while CBA transporters, resistance-nodulation cell-division (RND) proteins in Gram-positive bacteria, primarily detoxify periplasmic metal (outer membrane efflux), presenting high-level resistance toward heavy metals. CBA transporters eliminate ions that are transported to the periplasm by ATPase and CDF transporters. P-Type ATPase and CDF transporters are functionally identical and can substitute for each other but not CBA transporters (Fig. 1). Each of these transporters has their own mode of action (Table 2). P-Type ATPase transports metal ions from the cytoplasm to the periplasm in the presence of ATP as the energy source; CBA transporters ‘bridge’ the whole cell wall (in Gram-negative bacteria) and transport metal ions from the periplasm and cytoplasm to the cell exterior by using a chemiosmotic gradient; CDF exports ions from the cytoplasm to the periplasm and is driven by a proton motive force.³⁹

Fig. 1 Major transporter families taking part in heavy metal resistance.³⁹

Table 2 Types of efflux transporters and their functions

Transporter	Description and functions
P-Type ATPase	• Involvement of phosphoenzyme intermediate during reaction cycle contributes to the term P-type
	• Driven by energy produced from the removal of γ-phosphate from ATP. Substrates are inorganic substrates, such as H^+, Na^+, K^+, Mg^+, Ca^+, Cu^+, Ag^+, Zn^+, Cd^+, Co^+, and Pb^+
	• ATPases involved in heavy metal translocation are known as CPx-type ATPases, because they contain conserved proline residue (P), followed by cysteine residue (C)
	• Crucial in maintaining homeostasis of vital metals, such as Cu^+, Co^2+, and Zn^2+, and at the same time pose resistance toward toxic metals, Pb^2+, Cd^2+, and Ag^+
	• Metal binding domain (MBD) influences specificity of the heavy metal translocating ATPase
CBA transporter	• RND protein found in the inner membrane is the most essential component, which is linked to the bacterial transport protein, required in nodulation, cell division and heavy metal resistance
	• Known as three-component protein complexes, this is made up of: (i) RND Protein, (ii) membrane fusion protein (MFP), (iii) outer membrane factor (OMF). Formation of efflux protein complex, which functions as a pump that exports substrate from (i) cytoplasm to the periplasm, (ii) periplasm to the outer membrane
	• The presence of RND in this export system shows differences between CBA and ABC transport systems
	• In many protein complexes, the absence of MFP and RND proteins causes lack of resistance, while the loss of OMF usually only has moderate influence
	• RND protein is present in Gram-positive bacteria, but CBA transporter is not functional in the cell walls
CDF transporter	• CDF can be found in both prokaryotes and eukaryotes
	• Mainly involved in Zn^2+ transportation and also in other metals (Fe^2+, Co^2+, Ni^2+, and Cd^2+)
	• Assumed to act as heavy metal buffer when cytoplasmic metal concentration is low due to the fact that this system only exhibits extremely low-level resistance

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4. Bacterial stress response

Microorganisms in soil are exposed to changes in the environment. To survive these unfavourable conditions, soil microbes developed adaptive defence mechanisms or physiological and structural adaptations, which resulted from evolution. The metabolic reaction known as stress response is included in the adaptive mechanism.⁵¹ Microbial stress responses induced by changes in the metabolic activity of cells lead to repression of the syntheses of most proteins that are found under normal physiological conditions, and the synthesis of specific proteins for cell survival in the new environment.⁵¹ Meanwhile, changes that occur in gene expressions are linked to alterations that involve different sigma protein factors and the catalytic core of RNA polymerase. RNA polymerase is needed to identify genes that are required under particular environmental conditions, and produces mRNA transcripts that will later be translated into a protein.²¹ Table 3 represents the general stress responses that can be found in bacteria.

Table 3 General stress responses in bacteria

Type of stress	Response
Chaotropic solutes^52	• Up-regulating proteins for lipid metabolism protein stabilization, membrane structure, energy metabolism, and protein synthesis. Accumulation of compatible solutes
Osmotic stress^53	High osmolality
	• Increase in K^+ ion influx i.e. uptake systems: trk, kdp, and kup
	• Increased excretion results in a drop in intracellular putrescine levels
	• Synthesis of glutamate (i) glutamate dehydrogenase (gdh) and (ii) glutamate synthase (gs)
	• Accumulation of disaccharide trehalose
	Low osmolality
	• Elongation of the cell envelope and trigger of stretch-activated channels
	• Increase in the membrane's permeability
	• Complex sugar synthesis; membrane-derived oligosaccharides (mdos)
Nutritional stress^53	• Inducing the expression of proteins involved in starvation-stress response (SSR)
	• Collection of cellular nucleotides: (i) cyclic 3,5-adenosine monophosphate (cAMP) and (ii) guanosine 3,5-bis(diphosphate)
	• Major SSR regulators: two alternative σ factors and σ^E encoded by the rpoS and rpoE genes
	• Activation of nutrient utilization systems, which are novel or higher-affinity
Temperature^54	High temperature
	• Increased synthesis of heat shock proteins (hsps)
	• Proteins dnaK and dnaJ, the RNA polymerase σ^70 subunit (rpod), groEL, groES, protease, and lysU are induced
	• Heat shock increases expression of σ^h target genes
	Low temperature
	• Involves two signal transduction cascades: the σ^E and cpx systems
	• Increased stability of DNA secondary structure and RNA. Reduced efficiency of transcription, replication, and translation
pH and acid stress^55	• Induces the acid tolerance response (ATR)
	• Results in increased expression of synthesized or existing acid shock proteins
	• Mg^2+-dependent proton translocating ATPase system crucial in some organisms for acid tolerance utilizing arginine deiminase (ADI) pathway to produce ATP under acid stress
	• Production of urease (nickel-containing metalloenzyme) to convert urea to carbon dioxide and ammonia
Oxidative stress^53	• Controlled by two major transcriptional regulators OxyR and SoxRS (Cabiscol, Tamarit et al. 2010). The OxyR regulon is induced by H2O2 and the SoxRs are induced by superoxide
	• In Escherichia coli, cytoplasmic Mn-SOD (SodA) and Fe-SOD (SodB) are produced during oxidation to protect protein and DNA
	• A periplasmic Cu/Zn-SOD (SodC) defends the periplasmic and membrane constituents from exogenous superoxide
	• No molecular oxygen produced during elimination of superoxide via superoxide reductase
Heavy metal stress^41	• In Caulobacter crescentus, genes regulating against oxidative stress and efflux pumps, including metal ion efflux membrane fusion protein and outer membrane efflux protein, are up-regulated
	• Sulphate transporters were down-regulated to reduce non-specific uptake of the metal

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Biochemical changes occur, followed by physiological changes, such as temporary slowing or stopping of the cell division cycle, morphological changes in the cell, or the development of resistance to stress factors. Activation of defence mechanisms becomes impossible when unfavourable stimuli are prolonged, and components of cellular structures may be damaged. These severe environmental stresses can lead to cell death and evacuation of susceptible cells. Microbes that have resistance towards these conditions enable themselves to tolerate stress factors without activation of adaptive mechanisms, whereas some microbes require adaptive mechanisms, which can delay the synthesis of defence molecules. Microorganisms will enter the stationary phase of growth, and cell division will stop when the nutrient supply is depleted and the microorganisms are unable to sustain stable growth. Most of the earth's biomass consists of resting microbes, which are normally present in a stationary phase due to limited nutrients and harsh conditions that are common in the natural environment.⁵¹

4.1 Aggregation and biofilm

Ecological processes, such as competition, adaptation, epidemics, and succession, involve bacterial aggregation. Microbes developed survival skills against harsh conditions, such as temporal and spatial changes in stimuli through motility, which is an unavoidable part of the life cycle of most microbes.^56,57 Aggregate formation resulted in enhanced efficiency in bioremediation.⁵⁸ Aggregation leads to the formation of biofilm, which depends on distinct interactions, including synergistic, antagonistic, mutualistic, competitive, and commensal.

Autoaggregation is defined as the adhesion of the same bacterial species, while coaggregation is the adhesion of two or more different species of bacteria.⁵⁹ Coaggregation is a highly specific adhesion process, which happens between two genetically different bacteria via specific molecules, generally mediated by ‘adhesin’ proteins on one bacteria and a complementary saccharide ‘receptor’ on the other. Coaggregation between bacteria from distinct taxonomies is known as intergeneric coaggregation, while interaction between strains that belong to the same species is intraspecies coaggregation. Molecules associated with surfaces, such as proteins and sugars, are observed mediating the coaggregation of bacteria, and this interaction led to the development of multispecies.⁵⁹ Adhesion, and capsules with surface hydrophobicity, enable bacteria to adhere to abiotic and biotic surfaces, thus leading to the formation of biofilms. Adhesiveness increases with hydrophobicity. Contradictorily, there are studies that show no relationship between the extent of initial binding, either to a hydrophobic or hydrophilic substrate, and the surface hydrophobicity of bacteria. Autoaggregation interactions are stronger than coaggregation, which is enhanced by the presence of surface hydrophobicity.^59–61 Physicochemical properties of surfaces influence autoaggregation phenomena.⁶² Cell-to-cell aggregation leads to biogranulation, the self-immobilization of microorganisms, and formation of dense aggregates.⁶¹

Bacterial biofilm involves cell-surface and cell–cell interactions as part of the development process. Bacterial aggregation is the interaction of microbes from cell to cell to form a stable and multi-cellular cluster.⁵⁸ Microbial aggregates, known as biofilm, can consist of a single species or multiple species,⁵¹ and are surrounded by self-produced extracellular polymeric substances (EPS).⁵⁷ Based on an assay that depends on time and dosage, biofilm consists of subpopulations of cells. These cells tend to die at different rates upon exposure of the whole community in the biofilm to metal ions.²³

The ability to synthesize EPS, proteins, and nucleic acids that surround the cell surface, to form a biofilm matrix, is a unique characteristic of cells living in the form of biofilm.⁵¹ Mechanisms of toxicity for biofilm and planktonic cells are different. The physiological states of microorganisms in biofilm are different, even when they are separated by only 10 μm, due to non-uniform distribution of extracellular pH and redox poise. Immature biofilm composed of layers of cells in the early stages of growth shows increased resistance to metal and antibiotics compared to planktonic cells.²³ Compared to planktonic bacteria, the formation of biofilm boosts microbial resistance toward hydrogen peroxide, heavy metals, bacteriophages or amoebae,⁵¹ and toward antibiotic by up to 1000 times.⁵⁷ Biofilm matrix is composed of water (nearly 97%), microbial cells, secreted polymer, nutrients, metabolites, products of cell lysis, particulate materials, and detritus from cell environments.⁶¹ Dead cells in a biofilm community might defend the living cells against the toxicity of a metal by precipitating or sequestering the reactive metal species, as dead cells are chemically reactive and contain biosorption sites that cause the formation of metal precipitates and chelates. Dead cells are also able to affect physiological microenvironments and pH discontinuities in biofilm.²³

The formation of biofilm occurs when suboptimal growth conditions (including a lack of easily assimilable nutrients), hazardous stress factors (such as the presence of metals or antibiotics), or the presence of specific low-molecular-weight compounds excreted by plants exist. Resistance of cells towards several environmental stress factors is due to the activation of various stress response mechanisms during the formation of biofilm and in mature biofilm.⁵¹ Biofilm alters their physiological characteristics to defend sensitive chemical targets of the reactive metal species, in order to decrease metal toxicity.²³ Carbohydrates and proteins are the major players in the process of metal elimination.⁶¹ The presence of enzymes, such as peptidase, polysaccharides, and phosphatase, within the biofilm proved that it helps to boost the bioavailability of nutrients in the environment. The physiological properties of cells within biofilm are unlike those of free-floating planktonic cells. Genes that are involved in adhesion, gene clusters, and autoaggregation are highly expressed in biofilm cells, or are induced during the transition process of the biofilm growth phase.⁵⁸

Both natural and engineered microbial biofilm can be applied to handle heavy metal pollution by the accumulation of toxic metal ions and/or biochemical modification. Natural processes of phenotypic diversification that occur inside a biofilm population are related to reducing the susceptibility of biofilm to toxic metals. An interruption in the metabolic processes can be avoided when the biosorption of metal ions to components of the biofilm (cell membrane, extracellular polymers, and cell walls) sequesters these compounds. Metabolic end products produced by microorganisms also react with metals and cause precipitation of bioinorganic metal complexes. For example, the co-precipitation of heavy metals, such as Ni, Cu, U, Zn, Cd, and Pb, with sulphide (S²⁻) in a biofilm of sulphur-reducing bacteria and archaea, and co-precipitation of heavy metal with carbonates (HCO₃⁻ and CO₃²⁻) produced during microbial respiration, caused the elimination of toxic metals from the aqueous phase.²³

Bacteria attached to each other (aggregates) and/or on a surface (biofilm) act as a pool, creating sessile communities that are capable of adapting to alterations in the environment or executing extremely specialised tasks, similarly to multi-cellular organisms.⁵⁸ Quorum sensing (QS) is involved in the information, development, and susceptibility to metal toxicity in biofilm by regulating genes that are involved in the different developmental stages in the biofilm.^23–63 QS is a mechanism of cell-to-cell signalling through the excretion of extracellular compounds that are recognized as autoinducers. The accumulation of autoinducers in an extracellular medium regulates gene expression and amplification of various types of phenotypes. Throughout the growth, bacteria produce autoinducers that activate the QS system when they achieve a threshold concentration.

The formation of biofilm involves five stages: (i) initial and reversible adhesion, (ii) initial irreversible attachment with production of EPS, (iii) initial maturation and acquisition of a biofilm structure, (iv) mature biofilm, and (v) dispersion (Fig. 2).⁶³ The primary attachment of a bacterium to a particular surface leads to the formation of microcolonies, which mature into three-dimensional structures enwrapped and braced by EPS. Based on the analysis of biofilm, cell surface structures, including fimbriae, pili, EPS, flagellae, and outer membrane proteins (OMPs) allow primary attachment to a surface, leading to the formation of biofilms.⁶⁰

Fig. 2 Steps involved in the formation of biofilm.

The rate and extent of attachment of microbial cells are determined by cell surface hydrophobicity, the presence of fimbriae and flagellae, and the yield of EPS.⁶⁴ Non-motile mutant bacteria showed a lack of ability to form biofilm compared to wild-type cells. Hydrophobicity and the ability to coaggregate and autoaggregate can increase bacterial adhesiveness. Surface hydrophobicity is usually related to bacterial adhesiveness and is different among organisms and strains, being affected by bacterial age, growth medium, and bacterial surface.⁶⁰ This initialization of biofilm involves regulatory processes that indirectly activate genetic and biochemical pathways that are used as a response toward antibiotic and metal exposure by microorganisms. This suggests that microorganisms are able to form biofilm that is multidrug resistant and tolerant to exposure to metals in the environment or in clinical circumstances.²³

4.2 Extracellular polymeric substances (EPS)

EPS are metabolic products containing high-molecular-weight polysaccharides (10–30 kDa) with homopolymeric and heteropolymeric compositions,⁶⁵ which are made up of macromolecules, including polysaccharides, proteins, nucleic acids and lipids.⁵⁸

EPS have various biological uses, such as prevention of dehydration, preservation against environmental stresses, including antibiotics and toxins, adherence to surfaces, symbiosis, and pathogenesis under oligotrophic circumstances. EPS play a role in the microbial survival scheme by separating nutrient materials from the environment and act as a protective layer by restricting the diffusion of some antimicrobial agents into the biofilm by acting as ion exchangers. Normally, EPS-producing bacteria can be found in environments rich in organic substances, in capsular materials, or as dispersed slime with no connection to one particular cell. Factors that affect EPS production are composition medium (carbon and nitrogen source, pH, temperature), bacterial growth phase,⁶⁵ and microbial species.⁶¹ EPS production demands many activated nucleotide sugars as energy sources for building the repeating units, transmembrane translocation, and for polymerization; thus, the production of EPS is predicted to occur during active sugar consumption.⁵⁸

EPS are classified as homopolysaccharides and heteropolysaccharides. Homopolysaccharides possess neutral charge, while most heteropolysaccharides are polyanionic due to uronic acids (glucuronic acids, mannuronic acids, and galacturonic acids) or ketal-linked pyruvate. Only in a few cases can EPS be polycationic. EPS are required in flocculation and binding of metal ions from solutions, thus are relevant to bioremediation processes.⁶¹ The major categories of macromolecules in biofilm EPS are anionic because of uronic acids or ketal-linked pyruvates, and ionisable functional groups that communicate with other molecules, minerals, and heavy metals.⁶¹ Uronic acids that influence the anionic characteristics of EPS present potential in biotechnology applications as they can be used in the biodetoxification of heavy metals and waste water, considering the heavy metal-binding properties of these polymers.^66,67

Factors that affect metal binding to biofilm EPS are determined by environmental pH, metal concentration, and availability of organic material and biomass. EPS act as a protective layer against heavy metal stress by metal-ion binding or by delaying their diffusion within the biofilm.⁶¹ The ability of EPS to sequester heavy metal is mainly due to the presence of ionisable functional groups, including carboxyl, amine, phosphoric, and hydroxyl groups.³⁴ The capability of microorganisms to catalyse changes in the oxidation states of metals, thereby affecting their solubility, is applicable in the bioremediation of heavy metal.⁶¹

5. Mechanisms of gene regulation under heavy metal toxicity stress

Signalling proteins require two-component signalling (TCS) systems, which constitute the major signal transduction system in bacteria.⁶⁸ Signal transduction systems, which are part of the pathways of intracellular information-processing, act as a bridge between external stimuli and specific adaptive responses. Other proteins, such as sigma factors, cyclic-di-guanosine monophosphate (c-di-GMP)-related proteins, and methyl-accepting chemotaxis with flagella proteins are also involved in signal transduction.⁶⁹

5.1 Two-component signalling (TCS) systems

The resistance of bacteria towards heavy metal is linked with cellular signalling pathways. TCS pathway transduction systems in bacteria allow them to sense, react, and adjust to changes that occur in their environment, or in an intracellular state, by responding to signals and stimuli, such as nutrients, changes in osmolarity, quorum signals, cellular redox states, and antibiotics, and are mediated by TCS pathways.⁷⁰

TCS systems react to various environmental signals and regulate functions, such as sporulation, division, metabolism, motility, virulence, communication, and stress adaptation.⁷¹ Simple phosphotransfers are generally used by prokaryotes, while phosphorelays and hybrid kinases are TCS systems in eukaryotes.⁷² Typical prokaryotic TCS systems constitute a membrane-bound histidine kinase (HK) and a response regulator (RR). Briefly, HK contains a variable sensing domain and a conserved kinase domain. When sensing a stimulus, the HK sensor is activated and autophosphorylates at a conserved histidine (His), effecting gene expression by phosphorylating its cognate RR at a conserved aspartate (Asp). The response regulator is usually a DNA-binding transcription factor that undergoes conformational changes due to phosphorylation, which controls the expression of the target genes.⁷³

TCS begins once a stimulus is detected that leads to autophosphorylation of conserved histidine residue on HK protein,⁷⁴ followed by transfer of a phosphoryl group to an RR. The attached output domain will be activated after phosphorylation of the RR on a conserved aspartate residue in its receiver domain. Phosphorylation of RR is linked to changes in the transcription level as DNA-binding domains act as output domains,⁷⁰ giving a physiological response via repression or activation of genes.⁷⁴ Generally, HKs are bifunctional, as they are able to catalyse both phosphorylation and dephosphorylation of their related RR. Bifunctional HKs are able to regulate either the kinase or phosphatase activity.⁷⁰ One of the properties of TCS systems is that gene transcription demands both the RR and the signal that triggers its activation, which is sensed by the cognate HK. This indicates that TCS is controlled by another TCS system transcriptionally, where a gene is regulated by a system that will be only expressed when a signal that activates both systems is present.

TCS systems have unique properties compared to other pathways. The sensor is usually placed at the cytoplasmic membrane and receives periplasmic and/or cytoplasmic signals.⁷⁵ Many TCS systems regulate their own expression. Autoregulation allows bacteria to have a ‘memory’ of a previous incident with a signal due to abundant amounts of sensors and the presence of RR proteins after the signal disappears. Autoregulation is important for TCS, whereby RR controls target binding sites that are relatively too large to RR made from the constitutive promoter. Furthermore, autoregulation provides a threshold level for gene activation where, when a signal remains, it will promote adequate levels of phosphorylated RR for gene regulation.⁷⁶

TCS is a functional and accurate system of regulation and is expanded by mutation and gene duplication to play roles from gene regulation to chemotaxis.⁷⁷ In E. coli, over 40 different TCS systems that respond to different environmental stimuli have been identified.⁷⁸ The TCS pathways sense changes in the environment and initiate regulatory factors for the formation of biofilm. For example, the attachment of E. coli cells onto a hydrophobic surface activates Cpx TCS, which is known as the general stress response. Activation of the Cpx system induces genes that code for periplasmic protein folding and protein degradation factors.⁶³ In Bacillus subtilis, transcription of the ResD/ResE system that regulates genes needed for anaerobic respiration is manipulated by the PhoP/PhoR system, which reacts to phosphate starvation.⁷⁶ Phosphotransfer networks in TCS systems also incorporate components of C-di-GMP signalling pathways. A subfraction of GGDEF/EAL domain proteins are connected to TCS systems. Genes that encode EAL domain proteins are co-expressed with RR and sensor kinase genes in some bacteria. Many GGDEF/EAL domain proteins consist of N-terminal receiver domains that are phosphorylated by cognate sensor kinase.⁷⁹

Phosphorelay is a common version of a TCS pathway, which is started by a hybrid HK that autophosphorylates and transfers its phosphoryl group intermolecularly to an RR-like domain (Fig. 3).⁷⁰ Phosphorelay is a more complex variant of TCS systems,⁷⁷ which is applied in complex cellular processes, such as development and cell cycle control, in bacteria.⁸⁰ Phosphorelay was first discovered in B. subtilis to initiate sporulation.⁸¹ The phosphoryl group will be moved to a histidine phosphotransferase (HPT) and then to the terminal response regulator, which then will arouse related responses.⁷⁰ In phosphorelay, the first regulatory domain phosphorylated by sensor kinase passes its phosphoryl group to a second phosphotransferase domain that assists as the primary phosphoryl donor to response regulators or transcription factors.⁷⁷ Phosophorelay contains sensor kinase, terminal response regulator, intermediate response regulator lacking an output domain, and His-containing phosphotransfer protein.⁸²

Fig. 3 Signal transduction in TCS and phosphorelay.

5.1.1 Examples of TCS in selected heavy metal resistance.
(i) Cadmium resistance. Microbial resistance toward Cd²⁺ is generally based on energy-dependent efflux mechanisms. ColRS, which is known for metal resistance, or the homeostasis ColRS operon, is a type of TCS transduction system. ColR and ColS act as response regulators and HK, respectively. A lack of ColRS causes about a fivefold reduction in resistance to Mn²⁺. This proves that the ColRS signal transduction system is important for regulating resistance or homeostasis of Mn²⁺.⁸³
(ii) Zinc resistance. Metal-inducible mechanisms that are based on active efflux of metal ions to avoid hazardous effects on cells took place in the presence of excess Zn²⁺, Pb²⁺, and Cd²⁺.³⁹ P-Type ATPase transports only zinc across the cytoplasmic membrane, while the resistance-nodulation-cell division (RND) system transports zinc across the complete cell wall of Gram-negative bacteria. Zinc resistance in Saccharomyces cerevisiae was mediated via CzcD, which is from the cation diffusion facilitator (CDF) family, including ZRC-1 protein. The czc regulatory genes are ordered upstream and downstream of structural genes czcCBA. TCS systems are formed between the downstream regulatory regions that contain czcD, czcR, and czcS, with czcS (HK) and czcR (RR).⁴⁰
(iii) Copper resistance. In copper homeostasis, two copper-responsive regulatory systems involve genes like cutC, cutF, and ndh. A sensor–regulator pair formed by cusRS triggers the adjacent, and at the same time, transcribed gene, cusCFBA. The cusCBA genes, which are homologous to a family of proton-cation antiporter complexes, are required in the export of metal ions, xenobiotics, and drugs, while cusF is a putative periplasmic copper-binding protein. CueR, a copper-activated homologue to MerR, regulates two genes: cueO and copA. CopA is recognized as a Cu(I)-translocating P-type ATPase, while CueO is a putative multi-copper oxidase. In two chromosomal copper-responsive determinants for copper homeostasis, cus determinants are regulated by TCS transduction systems that are encoded by cusRS genes.⁸⁴
(iv) Silver resistance. In silver resistance, the gene silE encodes SilE, a periplasmic Ag(I) binding protein. SilE are 47% identical to PcoE in the E. coli plasmid copper resistance system. SilCBA and Silp, which are two parallel membrane Ag(I) efflux pumps, are encoded. Upstream from silE is silRS, a TCS signal transduction pair, which contains transcriptional regulatory responder, SilR, and membrane kinase sensor, SilS, which is homologous to other two-component family pairs. The silCBA genes resemble a cadmium, zinc, and cobalt resistance system (Czc) of Ralstonia sp. and a multi-drug resistance system of E. coli. SilA forms a pore cavity for substrates, for example, Ag⁺, from the cytoplasmic region straight to the outer membrane protein, SilC. This ensures movement across the periplasmic space of Gram-negative bacteria and directly to the outside of cells without release into the periplasmic space. SilB, which is also known as a membrane fusion protein, anchors into the inner membrane and connects to the outer membrane protein, SilC. SilP is homologous to membrane P-type ATPase and pumps Ag(I) from the cell cytoplasm to the periplasmic space.⁵⁰

5.2 Sigma factors

Changes in gene expression are manipulated transcriptionally via alteration in interactions between different sigma factors and catalytic core RNA polymerase in bacteria. Sigma factors are dissociable subunits of prokaryotic RNA polymerase that manipulate several iron uptake pathways, tolerance to several stresses, alginate biosynthesis, expression of outer-membrane porins, and expression of virulence factors.

Sigma factors are classed into two major protein categories, σ⁵⁴ and σ⁷⁰ families (Fig. 4), based on literature regarding P. aeruginosa.^21,85 Subunits containing the σ⁵⁴ family are typically known as σ^N, whereas σ^N-dependent genes not only regulate nitrogen metabolism in many organisms, but at the same time also contribute to a wide range of metabolic processes. P. aeruginosa and P. putida KT2440 specify 22 σ⁵⁴-dependent transcriptional regulators. Various σ⁵⁴-dependent regulators in KT2440 belong to TCS and exhibit a domain that could be phosphorylated by a sensor-kinase protein in the N-terminal section.

Fig. 4 Sigma factors in Pseudomonas aeruginosa.

The sigma 70 family has two subcategories: (i) the primary sigma factor RpoD (σ⁷⁰), which is involved in the transcription of housekeeping genes and coordinates transcription of genes that are essential for bacterial metabolism and growth, and (ii) the alternative sigma factors, which play important roles in the transcription of stress-related genes, which, based on conservation of their primary structures and sequences, can be grouped into four different classes;

• RpoS (σ³²) activates expression of multiple genes that are needed to sustain cell viability as the cell exits the exponential growth conditions and proceeds into stationary phases,

• FliA (σ²⁸) controls flagellin synthesis in P. aeruginosa. The mechanism of fliA transcription is still unclear but is suggested to be constitutive,⁸⁶

• RpoH (σ³²) manipulates heat shock regulation in E. coli. The role of RpoH in P. putida is not completely understood.

• Extracytoplasmic function (ECF) is involved in sensing and responding to conditions in the periplasm, the membrane, or the extracellular environment. P. putida KT2440 is reported to have 19 ECF sigma factors.^21,87

Bacteria have different single stress-induced responses to aid in adaptation to specific stress situations by removing the hazardous substances. The general stress response is usually regulated by a single master regulator. For example, the master regulator in E. coli is σ^S (RpoS).⁸⁸ Sigma regulatory proteins are crucial in the transition to the stationary phase in both Gram negative and Gram positive bacteria.⁵¹ Sigma factors link up with RNA polymerase to create an RNA polymerase holoenzyme that allows the holoenzyme to identify the promoter site in DNA. The σ^B from group III sigma factors that are found in B. subtilis regulates σ^B-dependent general stress regulators, which are expressed upon exposure of the bacterial cell to ethanol, heat, salt stress, acid, moving to the stationary phase, or starvation for oxygen, glucose, or phosphate. While σ^E from group IV, which can be found in E. coli, is an extracytoplasmic function sigma protein responsible for heat-shock stress.²¹ In Caulobacter crescentus, ECF sigma factor σ^F is one of the regulatory proteins that are required in the regulation of the transcriptional response to chromium and cadmium, and controls eight genes under chromium stress.⁸⁹

In harsh environments, reductive division and dwarfing cause bacterial cells to shrink and acquire a spherical shape compared to their log-phase counterparts. Reductive division enhances the surface-area-to-volume ratio, producing a spherical shape, while dwarfing is a type of self-digestion caused by the degradation of endogenous cell materials, especially cytoplasm and the outer membrane.⁵¹

Reorganization causes the cell envelope (outer membrane, periplasm, peptidoglycan, and inner membrane) to become stiff and resistant to chemical and physical agents. The nucleoid undergoes condensation, in which DNA-binding proteins from starved cells (Dsp) defend the DNA from several damaging agents. Dsp are triggered by OxyR in oxidative stress conditions as a result of an expression dependent on housekeeping transcription factor σ⁷⁰, whereas in starvation conditions, this occurs by the σ^S transcription factor. Dimerization of ribosomes into an inactive form occurs as a form of preservation, as some ribosomes are degraded, which explains the low translation levels observed under these conditions. Modifications at the metabolic level require inhibition of transcription of gene coding for rRNA, tRNA, and ribosomal proteins, which causes a decrease in cellular protein synthesis. The synthesis of cell wall components and lipids is also reduced. Protease and peptidase synthesis increases in the early stages of starvation, with an increase in protein turnover (as much as five fold in E. coli).⁵¹

5.3 C-di-GMP

C-di-GMP influences complex biological processes, such as virulence and biofilm formation, in many bacteria.⁹⁰ It has been proven that C-di-GMP signaling is involved in cell aggregation and biofilm formation in P. aeruginosa and other bacteria,⁵⁶ while in C. crescentus, GGDEF domain protein PleD manipulates flagellum ejection and cell morphology.⁷⁹ C-di-GMP-metabolizing proteins, phosphodiesterase and di-guanylate cyclase, each possess one GGDEF and EAL domain as common domains. GGDEF domains are involved in synthesis and EAL domains are involved in the hydrolysis of C-di-GMP. The amino acid sequences and protein structures in both domains share high similarity, even though the proteins catalyze opposite biochemical reactions.⁵⁶ Inactivation of gene-encoding GGDEF and EAL domain proteins regulates the amplitude of a phenotype or retrieval of a function under dissimilar environmental conditions, but rarely causes major phenotype changes.⁷⁹ C-di-GMP affects biofilm formation and virulence in Staphylococcus aureus and fimbrial expression on P. aeruginosa, despite the fact that it is an intracellular second messenger.⁹⁰

Levels of C-di-GMP are involved in many cellular processes, including conversion between motile and sessile lifestyles in bacteria (Fig. 5). The saturation of C-di-GMP-directed processes was attained by the expression of diguanylate cyclase, leading to high C-di-GMP production triggering the sessile lifestyle, favoring phenotypes, including extended biofilm formation, which linked with the fimbriae, adhesive matrix components, and exopolysaccharides. C-di-GMP depletion was accomplished by the overexpression of a cytoplasmic phosphodiesterase, which led to motility activities, such as swimming, swarming, and twitching.⁷⁹ In P. putida, EAL and GGDEF domain proteins suppressed the biosynthesis of flagella in the early growth phase.⁹⁰ In hns mutant E. coli, which loses swimming motility due to loss of flagella motion, this was recovered by the production of an EAL domain protein. This proves that down-regulation of the C-di-GMP concentration leads to functional activation of structural components that decoupled from the synthesis of respective structures. The structure of EAL and/or GGDEF domain proteins (sensor output domain) is the same as that of the sensor HKs and methyl-carrier chemotaxis proteins. This is due to the existence of an amino acid that is able to regulate the turnover of C-di-GMP in a similar manner to the regulation of HKs.⁷⁹

Fig. 5 Known input and output signals of C-di-GMP metabolism.

High concentrations of C-di-GMP in Salmonella typhimurium triggered the formation of biofilm, with the production of adhesive surface organelles, including curli fimbriae and cellulose, and suppressed motility. In low concentrations of C-di-GMP, the production of adhesive surface organelles and biofilm formation is inhibited, with the induction of swarming and swimming motility. The adhesion of cells to a surface results in several C-di-GMP concentrations, depending on whether the cells show twitching motility or produce an adhesive extracellular matrix.⁹⁰

5.4 Chemotaxis and flagellae

Chemotaxis is a process whereby motile unicellular organisms coordinate their movement away from or towards gradients of specific substances, which are either attractants or repellents.⁹¹ Bacterial chemotaxis, which involves a chemosensory pathway, is part of the TCS superfamily of receptor-regulated phosphorylation pathways. When a cell swims through different regions of concentration, the chemosensory pathway monitors local concentrations of chemical species that vary with time as the cell swims. When a cell swims up an attractant gradient, the chemosensory pathways detect an increasing attractant concentration with time and deliver a signal to the propulsion motor, which will lower the chances of a tumble event, thus prolonging the average run up the gradient and vice versa.⁹² The chemotaxis process requires two separate systems, including the chemo-receptors situated in the bacterial cell membrane, which are crucial for detecting the binding compounds, and the transduction proteins, which are needed in the downstream signal transduction to respond to the stimuli.⁹¹ Molecular mechanisms of bacterial chemotaxis consist of cytoplasmic chemotaxis proteins (Che proteins), methyl-accepting chemotaxis proteins (MCPs), and flagellae (Fig. 6).

Fig. 6 Chemotaxis pathway in bacteria.

MCPs are reversibly methylated transmembrane chemosensory proteins for environmental stimuli and function as homodimers. A cluster of chemotaxis genes has been located, comprising cheB, cheJ, cheA, cheY, and cheZ. MCPs, together with CheW, regulate the autophosphorylation activity of CheA as a response to temporal changes in stimulus intensity. Methyltransferase CheB and methyltransferase CheR, which receive the phosphoryl group from phosphorylated CheA, reversibly methylated MCPs at several glutamate residues. Methylesterase activity will increase as phosphorylation of CheB occurs, and the level of methylation of MCPs is regulated in response to environmental stimuli. This occurrence, which is known as reversible methylation of MCPs, is important for chemical gradient sensing.

Alterations in repellent or attractant concentrations are detected by a protein complex comprising transmembrane receptors (Tar, Tap, Tsr, Aer, and Trg), an adaptor protein CheW, and an HK CheA. The autophosphorylation activity of CheA is manipulated by attractant binding (inhibited) and repellent binding (raised) to receptors. The phosphoryl group is immediately transferred from CheA to the response regulator CheY. Phosphorylated CheY (CheYp) alters the direction of motor rotation from counterclockwise (CCW) to clockwise (CW) to allow tumbling by diffusing to flagellar motors. CheZ phosphatase, localized to sensory complexes through binding to CheA, assures a rapid turnover of CheYp, which is crucial to rapidly readjust bacterial behavior. Receptor modification boosts CheA activity and reduces sensitivity to attractants. The response is provided by CheB phosphorylation through CheA, which raises CheB activity.⁹¹

Flagellae are complex organelles generating motility, which enable bacteria to propel through liquids (swimming) and through highly viscous environments or along surfaces (swarming).⁹³ Six different types of bacterial surface motility are involved in bacteria, including swarming, swimming, twitching, darting, gliding, and sliding. Among these, swimming and swarming are flagella-dependent.⁹⁴ Flagellar rotation, and the number of flagellae, may differ depending on the species. For example E. coli and S. typhimurium can have up to 10 peritrichous flagellae,⁹⁴ while P. aeruginosa has a single polar flagellum,⁸⁶ and an exception is Burkholderia mallei, which are permanently immotile.⁹⁵ P. putida has been proven to have multiple polar flagellae and typically has between five and seven flagellae inserted at one end to form a tuft. Flagellar filaments are typically 2 to 3 wavelengths long and are able to change the direction of movement within 20 to 30 milliseconds.⁹⁶

Flagellar rotations are in a CCW or CW direction. When flagellae rotate in a CCW direction, the cell moves forward in a straight line, which is recognized as a run. Meanwhile, when some of the flagellae rotate CW and others rotate CCW, cells start to tumble. Cells coordinate their movement by alternating between runs and tumbles, and the alternation is believed to be random. In exhibiting chemotactic behaviour, i.e., sensing the gradients of an attractant or repellent substrate, they change the frequency of tumbles and runs. When the cells sense increasing concentrations of attractants, they tumble less frequently and swim for longer times, whereas when they sense decreasing concentrations of attractants, they tumble more and decrease the run times.⁹⁷

In the C. crescentus strain CB15N (ATCC 19089), chemotaxis protein McpJ, cell motility proteins, and other additional chemotaxis proteins were down-regulated under all metal stresses. Exposure to uranium most significantly down-regulated the proteins involved in cell motility and chemotaxis proteins, such as flagellin protein FljM. Down-regulation in transcription and/or translation of chemotaxis and cell motility proteins can also be observed in Shewanella oneidensis MR-1 under Cr exposure and in Camplylobacter jejuni and P. putida under Cd exposure. This indicates that reduction in cell motility and chemotaxis is a common mechanism in bacterial heavy-metal stress responses. Interference of heavy metals with chemoreceptors, and the ability of cells to sense a non-conducive environment, may reduce chemotactic activities and cause down-regulation of these proteins.⁹⁸

According to a study on chromium(VI) exposure on S. oneidensis MR-1, the abundance levels of 7 proteins, including 2 chemotaxis proteins (SO1144 and SO3207), were reduced upon exposure to Cr(VI) for 24 hours compared to control conditions. The prevalence of non-motile cells upon prolonged exposure to Cr(VI) caused down-regulation of proteins involved in motility and chemotaxis. This was proved by confocal laser scanning microscopy observation. Chemotaxis genes, cheY1, cheA, cheW, and cheB1, experienced 0.4-fold, 0.5-fold, 0.3-fold, and 0.5-fold transcriptional repression, respectively.⁹⁹ Transcriptomic analysis of B. cereus ATCC 14579 showed that most of the hook-associated genes (flgE, flgE, and fliL), chemotaxis-related genes (cheV, cheY, and cheA), flagellar biosynthesis genes (fliO and flip), motor switch genes (fliN and fliG), and basal body rod genes (flgG and flgB) were down-regulated after exposure to silver nitrate. There were no changes in flagellar motor switch (fliR) expression, indicating that this may not be influenced by ionic stress response. A prolonged introduction of silver nitrate has slowed cell motility, based on a study on B. cereus related to the chemotactic response to silver stress.¹⁰⁰

6. Conclusion

Heavy metal contamination is not only hazardous to humans but also to microbes that are present in the environment. Anthropogenic contamination by heavy metal is harmful when it exceeds certain threshold levels. The non-degradable properties of metals contribute to their toxicity. In order to survive, defence mechanisms were developed by some microbes to adapt to their conditions. Adaptations of microbes in such adverse conditions cause not only physiological but also genetic changes. The presence of ion-selective ATPase pumps enhances the efflux transport of heavy metals to reduce toxicity in microbes. The formation of aggregations, biofilm, and EPS contributes to the application of heavy-metal-resistant bacteria in bioremediation. Genetic manipulation has been approached to create engineered microbes to be used in bioremediation. Research over the past decade has provided better understanding of the mechanisms and signalling pathways required in response to heavy metal stress in microbes. However, further understanding of these mechanisms and signalling pathways is crucial in coping with heavy metal contamination, which is increasing alarmingly.

Acknowledgements

This study was supported by the Ministry of Higher Education of Malaysia, MOHE (FRGS/2/2014/SG05/UKM/03/1). The authors would like to thank Mohd Faiz Mohd Yusoff for his assistance in illustration editing.

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