Bacterial Metallothionein Research Articles

Bacterial metallothionein, PmtA, a novel stress protein found on the bacterial surface of Pseudomonas aeruginosa and involved in management of oxidative stress and phagocytosis.

The pathogen Pseudomonas aeruginosa is a highly problematic multidrug-resistant (MDR) pathogen with complex virulence networks. MDR P. aeruginosa infections have been associated with increased clinical visits, very poor healthcare outcomes, and these infections are ranked as critical on priority lists of both the Centers for Disease Control and Prevention and the World Health Organization. Known P. aeruginosa virulence factors have been extensively studied and are implicated in counteracting host defenses, causing direct damage to the host tissues, and increased microbial competitiveness. Targeting virulence factors has emerged as a new line of defense in the battle against MDR P. aeruginosa strains. Bacterial metallothionein is a newly recognized virulence factor that enables evasion of the host immune response. The studies described here identify mechanisms in which bacterial metallothionein (PmtA) plays a part in P. aeruginosa pathogenicity and identifies PmtA as a potential therapeutic target.

Differentiated Zn(II) binding affinities in animal, plant, and bacterial metallothioneins define their zinc buffering capacity at physiological pZn.

Metallothioneins (MTs) are small, Cys-rich proteins present in various but not all organisms, from bacteria to humans. They participate in zinc and copper metabolism, toxic metals detoxification, and protection against reactive species. Structurally, they contain one or multiple domains, capable of binding a variable number of metal ions. For experimental convenience, biochemical characterization of MTs is mainly performed on Cd(II)-loaded proteins, frequently omitting or limiting Zn(II) binding features and related functions. Here, by choosing 10 MTs with relatively well-characterized structures from animals, plants, and bacteria, we focused on poorly investigated Zn(II)-to-protein affinities, stability-structure relations, and the speciation of individual complexes. For that purpose, MTs were characterized in terms of stoichiometry, pH-dependent Zn(II) binding, and competition with chromogenic and fluorescent probes. To shed more light on protein folding and its relation with Zn(II) affinity, reactivity of variously Zn(II)-loaded MTs was studied by (5,5'-dithiobis(2-nitrobenzoic acid) oxidation in the presence of mild chelators. The results show that animal and plant MTs, despite their architectural differences, demonstrate the same affinities to Zn(II), varying from nano- to low picomolar range. Bacterial MTs bind Zn(II) more tightly but, importantly, with different affinities from low picomolar to low femtomolar range. The presence of weak, moderate, and tight zinc sites is related to the folding mechanisms and internal electrostatic interactions. Differentiated affinities of all MTs define their zinc buffering capacity required for Zn(II) donation and acceptance at various free Zn(II) concentrations (pZn levels). The data demonstrate critical roles of individual Zn(II)-depleted MT species in zinc buffering processes.

A metallothionein from an open ocean cyanobacterium removes zinc from the sensor protein controlling its transcription

Bacterial metallothioneins are known for a limited range of phyla including cyanobacteria. We have characterised the BmtA from the marine cyanobacterium Synechococcus sp. WH8102 (SynBmtA). This strain inhabits the open ocean, one of the most nutrient-poor environments on Earth, with very low total and free Zn2+ concentrations. Therefore, the presence of a metallothionein, usually associated with zinc and cadmium tolerance, in this strain is intriguing. Previous transcriptomics work revealed that unprecedentedly, expression of SynBmtA is activated by the Synechococcus sp. WH8102 “zinc uptake regulator” (SynZur) at elevated [Zn2+]. SynBmtA binds four Zn2+ ions, and its first 37 residues adopt the zinc-finger fold characteristic of BmtAs. In contrast, sequence similarity to other BmtAs in the C-terminal stretch is low. This is expected to affect especially the most reactive site in zinc-transfer reactions. Indeed, chelators were unable to extract Zn2+ from SynBmtA, even in the presence of denaturant. This indicates an extremely stable protein fold, with no accessibility to any bound zinc ions in the folded protein. In addition, the zinc-binding affinity of SynBmtA exceeds those of any other metallothioneins. Apo-SynBmtA is capable of removing zinc from the sensory site of SynZur, providing one possible avenue of de-activating transcription of the synbmtA gene. All of these properties are consistent with a role in safely sequestering any excess zinc, to prevent toxic effects. The fact that this strain stores zinc in a metallothionein rather than employing an efflux pump implies that zinc is a valuable resource for Synechococcus sp. WH8102 and related strains.

Metagenomics-Guided Discovery of Potential Bacterial Metallothionein Genes from the Soil Microbiome That Confer Cu and/or Cd Resistance.

Metallothionein (MT) genes are valuable genetic materials for developing metal bioremediation tools. Currently, a limited number of prokaryotic MTs have been experimentally identified, which necessitates the expansion of bacterial MT diversity. In this study, we conducted a metagenomics-guided analysis for the discovery of potential bacterial MT genes from the soil microbiome. More specifically, we combined resistance gene enrichment through diversity loss, metagenomic mining with a dedicated MT database, evolutionary trace analysis, DNA chemical synthesis, and functional genomic validation to identify novel MTs. Results showed that Cu stress induced a compositional change in the soil microbiome, with an enrichment of metal-resistant bacteria in soils with higher Cu concentrations. Shotgun metagenomic sequencing was performed to obtain the gene pool of environmental DNA (eDNA), which was subjected to a local BLAST search against an MT database for detecting putative MT genes. Evolutional trace analysis led to the identification of 27 potential MTs with conserved cysteine/histidine motifs different from those of known prokaryotic MTs. Following chemical synthesis of these 27 potential MT genes and heterologous expression in Escherichia coli, six of them were found to improve the hosts' growth substantially and enhanced the hosts' sorption of Cu, Cd, and Zn, among which MT5 led to a 13.7-fold increase in Cd accumulation. Furthermore, four of them restored Cu and/or Cd resistance in two metal-sensitive E. coli strains.IMPORTANCE The metagenomics-guided procedure developed here bypasses the difficulties encountered in classic PCR-based approaches and led to the discovery of novel MT genes, which may be useful in developing bioremediation tools. The procedure used here expands our knowledge on the diversity of bacterial MTs in the environment and may also be applicable to identify other functional genes from eDNA.

Bacterial metallothioneins

Abstract Heavy metals are found in all living organisms where, as indispensable microelements (e.g. zinc, iron, copper), are involved in endless metabolic processes. However, living organisms are also at a risk of exposure to highly toxic metals, including cadmium or lead, which do not play any physiological role. Among multiple mechanisms associated with the maintenance of micronutrient homeostasis and detoxification of unwanted metals, there is a family of low-molecular-weight, cysteine-rich proteins, able to chelate multiple metal ions i.e. the metallothioneins (MTs). They are widely distributed among Eucaryota, however, they have also been found in some limited Procaryota, including cyanobacteria, pseudomonads and mycobacteria. These bacterial MTs differ in terms of primary structure, the number and type of metal ions they bind, as well as with regard to their physiological functions. The expression of bacterial MTs is regulated by metals via metalosensors. MTs from cyanobacteria seem to be involved in zinc homeostasis, while in Pseudomonas they are linked to cadmium detoxification. In Mycobacterium, MTs bind copper ions and may play a pivotal role in the virulence of these bacteria. The presence of MTs in other groups of bacteria remains questionable. Problems with identification of new bacterial MTs are mainly associated with low level of homology between MT amino acid sequences of different bacterial groups. Further research is needed to evaluate the physiological functions of metallothioneins in Procaryota. 1. Introduction. 2. The history of discoveries of bacterial metallothioneins. 3. Structure and metal-binding properties of bacterial MTs. 4. Functions of bacterial metallothioneins. 5. Regulation of metallothionein gene expression. 6. Presence of metallothioneins in bacteria. 7. Summary 1. Wstęp. 2. Historia odkryć metalotionein u bakterii. 3. Budowa i sposób wiązania jonów metali ciężkich przez bakteryjne MT. 4. Funkcje metalotionein bakteryjnych. 5. Regulacja ekspresji bakteryjnych metalotionein. 6. Obecność metalotionein u bakterii. 7. Podsumowanie

Unexpected Interactions of the Cyanobacterial Metallothionein SmtA with Uranium.

Molecules for remediating or recovering uranium from contaminated environmental resources are of high current interest, with protein-based ligands coming into focus recently. Metallothioneins either bind or redox-silence a range of heavy metals, conferring protection against metal stress in many organisms. Here, we report that the cyanobacterial metallothionein SmtA competes with carbonate for uranyl binding, leading to formation of heterometallic (UO2)(n)Zn4SmtA species, without thiol oxidation, zinc loss, or compromising secondary or tertiary structure of SmtA. In turn, only metalated and folded SmtA species were found to be capable of uranyl binding. (1)H NMR studies and molecular modeling identified Glu34/Asp38 and Glu12/C-terminus as likely adventitious, but surprisingly strong, bidentate binding sites. While it is unlikely that these interactions correspond to an evolved biological function of this metallothionein, their occurrence may offer new possibilities for designing novel multipurpose bacterial metallothioneins with dual ability to sequester both soft metal ions including Cu(+), Zn(2+), Cd(2+), Hg(2+), and Pb(2+) and hard, high-oxidation state heavy metals such as U(VI). The concomitant protection from the chemical toxicity of uranium may be valuable for the development of bacterial strains for bio-remediation.

Cytosolic expression of synthetic phytochelatin and bacterial metallothionein genes in Deinococcus radiodurans R1 for enhanced tolerance and bioaccumulation of cadmium

Due to its exemplary resistance to ionising radiation, oxidative stress, desiccation and several DNA damaging agents, Deinococcus radiodurans R1 (DR1) is considered as one of the most appropriate candidates for the bioremediation of the nuclear waste sites. However, the high sensitivity of this bacterium to heavy metals, which are usually preponderant at nuclear waste dump sites, precludes its application for bioremediation. This study deals with the expression two metal binding peptides in DR1 as an attractive strategy for developing metal tolerance in this bacterium. A synthetic gene (EC20) encoding a phytochelatin analogue with twenty repeating units of glutamate and cysteine was constructed by overlap extension and expressed in DR1. The cyanobacterial metallothionein (MT) gene, smtA was cloned for intracellular expression in DR1. Both the genes were expressed under the native groESL promoter. DR1 strain carrying the recombinant EC20 demonstrated 2.5-fold higher tolerance to Cd(2+) and accumulated 1.21-fold greater Cd(2+) as opposed to the control while the heterologous expression of MT SmtA in DR1 imparted the transformant superior tolerance to Cd(2+) amassing 2.5-fold greater Cd(2+) than DR1 expressing EC20.

Comparison of the effects of silver phosphate and selenium nanoparticles onStaphylococcus aureusgrowth reveals potential for selenium particles to prevent infection

Interactions of silver phosphate nanoparticles (SPNPs) and selenium nanoparticles (SeNPs) with Staphylococcus aureus cultures have been studied at the cellular, molecular and protein level. Significant antibacterial effects of both SPNPs and SeNPs on S.aureus were observed. At a concentration of 300μM, SPNPs caused 37.5% inhibition of bacterial growth and SeNPs totally inhibited bacterial growth. As these effects might have been performed due to the interactions of nanoparticles with DNA and proteins, the interaction of SPNPs or SeNPs with the amplified zntR gene was studied. The presence of nanoparticles decreased the melting temperatures of the nanoparticle complexes with the zntR gene by 23% for SeNPs and by 12% for SPNPs in comparison with the control value. The concentration of bacterial metallothionein was 87% lower in bacteria after application of SPNPs (6.3μgmg(-1) protein) but was increased by 29% after addition of SeNPs (63μgmg(-1) protein) compared with the S.aureus control (49μgmg(-1) protein). Significant antimicrobial effects of the nanoparticles on bacterial growth and DNA integrity provide a promising approach to reducing the risk of bacterial infections that cannot be controlled by the usual antibiotic treatments.

Lessons on the critical interplay between zinc binding and protein structure and dynamics

Zinc is one of the most important micronutrients for virtually all living organisms, and hence, it is important to understand the molecular mechanisms for its homeostasis. Besides proteins involved in transmembrane transport, both extra- and intracellular zinc-binding proteins play important roles in the respective metabolic networks. Important examples for extracellular zinc transporters are mammalian serum albumins, and for intracellular zinc handling, certain metallothioneins are of relevance. The availability of protein structures including relevant metal binding sites is a fundamental prerequisite to decipher the mechanisms that govern zinc binding dynamics in these proteins, but their determination can prove to be surprisingly challenging. Due to the spectroscopic silence of Zn2+, combinations of biophysical techniques including electrospray ionisation mass spectrometry (ESI-MS) and multinuclear NMR, isothermal titration calorimetry (ITC) and extended X-ray absorption fine structure (EXAFS) spectroscopy, coupled with site-directed mutagenesis and molecular modelling have proven to be valuable approaches to understand not only the zinc-binding properties of metallothioneins and albumins, but also the influence of other physiologically relevant competing agents. These studies have demonstrated why the bacterial metallothionein SmtA contains a site inert towards exchange with Cd2+, why the plant metallothionein EC from wheat is partially unfolded in the presence of Cd2+, and how fatty acids impact on the zinc-binding ability of mammalian serum albumins.

Proteomic responses of oceanic Synechococcus WH8102 to phosphate and zinc scarcity and cadmium additions

Synechococcus sp. WH 8102 is a motile marine cyanobacterium isolated originally from the Sargasso Sea. To test the response of this organism to cadmium (Cd), generally considered a toxin, cultures were grown in a matrix of high and low zinc (Zn) and phosphate (PO43−) and were then exposed to an addition of 4.4 pM free Cd2+ at mid-log phase and harvested after 24 h. Whereas Zn and PO43− had little effect on overall growth rates, in the final 24 h of the experiment three growth effects were noticed: (i) low PO43− treatments showed increased growth rates relative to high PO43− treatments, (ii) the Zn/high PO43− treatment appeared to enter stationary phase, and (iii) Cd increased growth rates further in both the low PO43− and Zn treatments. Global proteomic analysis revealed that: (i) Zn appeared to be critical to the PO43− response in this organism, (ii) bacterial metallothionein (SmtA) appears correlated with PO43− stress-associated proteins, (iii) Cd has the greatest influence on the proteome at low PO43− and Zn, (iv) Zn buffered the effects of Cd, and (v) in the presence of both replete PO43− and added Cd the proteome showed little response to the presence of Zn. Similar trends in alkaline phosphate (ALP) and SmtA suggest the possibility of a Zn supply system to provide Zn to ALP that involves SmtA. In addition, proteome results were consistent with a previous transcriptome study of PO43− stress (with replete Zn) in this organism, including the greater relative abundance of ALP (PhoA), ABC phosphate binding protein (PstS) and other proteins. Yet with no Zn in this proteome experiment the PO43− response was quite different including the greater relative abundance of five hypothetical proteins with no increase in PhoA or PstS, suggesting that Zn nutritional levels are connected to the PO43− response in this cyanobacterium. Alternate ALP PhoX (Ca) was found to be a low abundance protein, suggesting that PhoA (Zn, Mg) may be more environmentally relevant than PhoX.

Surface Display of Bacterial Metallothioneins and a Chitin Binding Domain on Escherichia coli Increase Cadmium Adsorption and Cell Immobilization

To increase the level of adsorption of cadmium ions to the surface of Escherichia coli, we fused cyanobacterial metallothioneins, SmtA (from Synechococcus elongatus PCC 3601) and MtnA (from Synechococcus vulcanus) to the E. coli cell surface using a Lpp'-OmpA-based display system. E. coli strains expressing Lpp'-OmpA-SmtA-linker-ChBD (chitin-binding domain from Bacillus pumillus SG2 chitinase S; chiS) and Lpp'-OmpA-MtnA-linker-ChBD on their surface adsorbed more cadmium compared to the E. coli cells expressing only the Lpp'-OmpA-linker-ChBD hybrid. These constructs also were bound to chitin through their chitin-binding domain, allowing them to be immobilized on a chitin matrix. We assessed surface presentation of Lpp'-OmpA-SmtA-linker-ChBD, Lpp'-OmpA-MtnA-linker-ChBD, and Lpp'-OmpA-linker-ChBD using immunostaining. The Lpp'-OmpA-SmtA-linker-ChBD chimera adsorbed metal and was bound to chitin with the highest efficiency compared to the other chimeras, suggesting that it is an effective bioadsorbent. This is the first example of coupling metal adsorption with cell immobilization using a whole-cell bioadsorbent.

Pseudomonas aeruginosa strain WI-1 from Mandovi estuary possesses metallothionein to alleviate lead toxicity and promotes plant growth

A bacterial isolate from Mandovi estuary Goa, India, which can resist 0.6mM lead nitrate in Tris-buffered minimal medium was identified as Pseudomonas aeruginosa and designated as strain WI-1. PCR amplification clearly revealed presence of bmtA gene encoding bacterial metallothionein responsible for metal sequestration and AAS analysis proved intracellular bioaccumulation of 26.5mg lead/gram dry weight of cells. SDS-PAGE analysis confirmed lead induced bacterial metallothionein with molecular weight 11kDa, which corresponds to the predicted bmtA gene. Significant growth inhibition of phytopathogenic fungi Fusarium oxysporum NCIM 1008 by siderophore-rich culture supernatant was also observed. Pot experiment with Pisum sativum L inoculated with this strain revealed higher seed germination percentage and significant growth promotion than uninoculated seeds in a soil amended with 7.704g/kg lead, which indicates amelioration of lead toxicity. This lead resistant strain showed cross tolerance to cadmium, mercury and Tributyltin chloride (TBTC) along with resistance to multiple antibiotics.

Bacterial metallothioneins: past, present, and questions for the future

Bacterial metallothioneins (MTs) have been known since the mid-1980s. The only family known until recently was the BmtA family, exemplified by the zinc- and cadmium-binding SmtA from the cyanobacterium Synechococcus PCC 7942, for which a structure was determined in 2001. Only in 2008 was a second type of bacterial MT identified in mycobacteria, and the copper-binding gene product was called MymT. Many of the features of SmtA either have been unexpected or are otherwise "unusual", for example the presence of a zinc finger fold and the kinetic inertness of one of the four zinc ions bound to the protein. The unpredictability of molecular properties of this protein exemplified the need for continued biophysical studies of novel proteins. Homologues for SmtA have been identified in a limited number of bacterial genomes from cyanobacteria, pseudomonads, alphaproteobacteria, gammaproteobacteria, and firmicutes. Except for the residues defining the zinc finger fold, these homologous protein sequences display an intriguing variety, especially in terms of metal ligand position and identity. The increased number of homologues has allowed use of hidden Markov models to look for more remote relatives of SmtA, leading to the identification of a novel family of putative hybrid LIM domain MTs. However, database searches based on sequence similarity are of limited use for mining for further "overlooked" bacterial MTs, as so far undiscovered bacterial MTs may be too diverse from any other known MTs, and other approaches are required.

Cyanobacteria MT gene SmtA enhance zinc tolerance in Arabidopsis

Zinc is essential but toxic in excess. A bacterial metallothionein, SmtA from Synechococcus PCC 7942, has high affinity for Zn2+ and the intracellular exclusively handling of Zn2+. In this study, we report a functional analysis of SmtA in Arabidopsis thaliana and its response to zinc stress. After high zinc stress, the transgenic plants over-expressing SmtA showed higher survival rate than the wild type. We also found that over-expression of SmtA in Arabidopsis increased the activities of SOD and POD, and enhanced the tolerance to zinc stress. Together, our results indicate that SmtA may play an important role in the response to zinc stress in Arabidopsis.

A bacterial copper metallothionein

Metallothioneins sequester cadmium and surplus atoms of copper and zinc to prevent aberrant metal-catalyzed reactions. The identification of a 'cryptic' copper metallothionein in the bacterium that causes tuberculosis suggests that bacterial metallothioneins may be more widespread than previously suspected.

Differential reactivity of individual zinc ions in clusters from bacterial metallothioneins

The bacterial metallothionein SmtA binds four zinc ions with high affinity and specificity in a Zn 4S 9N 2 cluster. We have explored the reactivity of these zinc ions towards the metal-chelator EDTA. Under pseudo-first-order conditions, initial break-down of zinc-thiolate bonds is rapid, followed by several slower phases. The reaction with stoichiometric amounts of EDTA is relatively slow and has been followed by 1H NMR and mass spectrometry. Both methods reveal that partially metallated intermediates occur during the reaction. Three- and two-metal species are observed in only minor amounts, whereas the Zn 1 species is dominant during the mid stages of the reaction, before complete metal depletion occurs. These results suggest that the zinc finger site in SmtA is not only inert towards metal exchange, but also more resilient towards chelating agents. The greater inertness of this site may help to maintain the protein fold during metal depletion, and allow subsequent facile metal uptake. Conversely, it is likely that the protein fold is the major contributor to the observed persistence of Zn 1SmtA in this reaction. Mass spectrometric studies with His-to-Cys mutants of SmtA reveal that the primary site for EDTA attack is the His49-containing zinc site C, and that His40 has a major influence on the reactivity of three sites.

How to Hide Zinc in a Small Protein

AbstractFor Abstract see ChemInform Abstract in Full Text.

How to Hide Zinc in a Small Protein

Small cysteine-rich proteins (metallothioneins) and related domains of some large proteins (e.g., lysine methyltransferases) bind tri- and tetranuclear zinc clusters with topologies resembling fragments of Zn(II) sulfide minerals. These clusters are ubiquitous in animals, plants, and bacteria. Bacterial metallothioneins can also contain histidines as cluster ligands and embed Zn(II) with a "treble-clef"-like finger fold. This unusual embedded Zn(II) is "hidden" and surprisingly inert toward Zn or Cd exchange. Clearly, proteins can exert fine control over both the thermodynamics and kinetics of zinc binding in thiolate clusters. Genome sequences suggest that related zinc-finger sites are common in a variety of bacteria.

Multiple bacteria encode metallothioneins and SmtA-like zinc fingers.

Zinc is essential but toxic in excess. Bacterial metallothionein, SmtA from Synechococcus PCC 7942, sequesters and detoxifies four zinc ions per molecule and contains a zinc finger structurally similar to eukaryotic GATA. The dearth of other reported bacterial metallothioneins has been surprising. Here we describe related bacterial metallothioneins (BmtA) from Anabaena PCC 7120, Pseudomonas aeruginosa and Pseudomonas putida that bind multiple zinc ions with high stability towards protons. Thiol modification demonstrates that cysteine coordinates zinc in all of these proteins. Additionally, (111)Cd-NMR, and (111)Cd-edited (1)H-NMR, identified histidine ligands in Anabaena PCC 7120 BmtA, analogous to SmtA. A related Escherichia coli protein bound only a single zinc ion, via four cysteine residues, with low stability towards protons; (111)Cd-NMR and (111)Cd-edited (1)H-NMR confirmed exclusive cysteine-coordination, and these cysteine residues reacted rapidly with 5,5'-dithiobis-(2-nitrobenzoic acid). (1)H-NMR of proteins from P. aeruginosa, Anabaena PCC 7120 and E. coli generated fingerprints diagnostic for the GATA-like zinc finger fold of SmtA. These studies reveal first the existence of multiple bacterial metallothioneins, and second proteins with SmtA-like lone zinc fingers, devoid of a cluster,and designated GatA. We have identified 12 smtA-like genes in sequence databases including four of the gatA type.

Microbial metallothioneins

Bacterial metallothioneins bind, sequester and buffer excess intracellular zinc. At present, the vast majority of the available experimental data relate to cyanobacterial metallothionein, SmtA, from Synechococcus PCC 7942. SmtA is required for normal resistance to zinc and smtA-mediated zinc resistance has been used as a selectable marker. The imidazole groups of histidine residues, in addition to the thiol groups of cysteine residues, co-ordinate zinc in bacterial metallothioneins. The structure of bacterial metallothionein must facilitate some discrimination between 'adventitious' and 'adventageous' zinc-binding sites such that under excess zinc conditions metal is predominantly scavenged from the former. It remains unclear whether or not bacterial metallothionein also acts as a zinc store that supplies zinc-requiring proteins or if under some conditions it deactivates a subset of proteins via zinc removal. Expression of smtA is induced in response to elevated concentrations of zinc via the action of SmtB. SmtB has some sequence similarity to the arsenic responsive repressor ArsR and genes encoding related proteins are present in many bacterial genomes. Metal perception by SmtB differs from ArsR. The latter contains a characteristic Cys-Val-Cys motif associated with a DNA-binding helix-turn-helix (the ArsR motif), while the former contains metal-binding motifs associated with a carboxyl-terminal alpha-helix that forms the interface between SmtB dimers (the SmtB motif). Some SmtB-ArsR family proteins, including the zinc sensor ZiaR from the cyanobacterium Synechocystis PCC 6803, have the metal-sensory motifs of both SmtB and ArsR. The mechanisms of action, and the features that allow discrimination between different metal ions by these sensors, are discussed.