The Components of the Unique Zur Regulon of Cupriavidus metallidurans Mediate Cytoplasmic Zinc Handling

ABSTRACT Zinc is an essential trace element, yet it is toxic at high concentrations. In the betaproteobacterium Cupriavidus metallidurans, the highly efficient removal of surplus zinc from the periplasm is responsible for the outstanding metal resistance of the organism. Rather than having a typical Zur-dependent, high-affinity ATP-binding cassette transporter of the ABC protein superfamily for zinc uptake at low concentrations, C. metallidurans has the secondary zinc importer ZupT of the zinc-regulated transporter, iron-regulated transporter (ZRT/IRT)-like protein (ZIP) family. It is important to understand, therefore, how this zinc-resistant bacterium copes with exposure to low zinc concentrations. Members of the Zur regulon in C. metallidurans were identified by comparing the transcriptomes of a Δzur mutant and its parent strain. The consensus sequence of the Zur-binding box was derived for the zupTp promoter-regulatory region by use of a truncation assay. The motif was used to predict possible Zur boxes upstream of Zur regulon members. The binding of Zur to these boxes was confirmed. Two Zur boxes upstream of the cobW 1 gene, encoding a putative zinc chaperone, proved to be required for complete repression of cobW 1 and its downstream genes in cells cultivated in mineral salts medium. A Zur box upstream of each of zur-cobW 2, cobW 3, and zupT permitted both low expression levels of these genes and their upregulation under conditions of zinc starvation. This demonstrates a compartmentalization of zinc homeostasis in C. metallidurans, where the periplasm is responsible for the removal of surplus zinc, cytoplasmic components are responsible for the management of zinc as an essential cofactor, and the two compartments are connected by ZupT. IMPORTANCE Elucidating zinc homeostasis is necessary for understanding both host-pathogen interactions and the performance of free-living bacteria in their natural environments. Escherichia coli acquires zinc under conditions of low zinc concentrations via the Zur-controlled ZnuABC importer of the ABC superfamily, and this was also the paradigm for other bacteria. In contrast, the heavy-metal-resistant bacterium C. metallidurans achieves high tolerance to zinc through sophisticated zinc handling and efflux systems operating on periplasmic zinc ions, so that removal of surplus zinc is a periplasmic feature in this bacterium. It is shown here that this process is augmented by the management of zinc by cytoplasmic zinc chaperones, whose synthesis is controlled by the Zur regulator. This demonstrates a new mechanism, involving compartmentalization, for organizing zinc homeostasis.

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Interplay between the Zur Regulon Components and Metal Resistance inCupriavidus metallidurans

Journal of Bacteriology ◽

10.1128/jb.00192-19 ◽

2019 ◽

Vol 201 (15) ◽

Cited By ~ 2

Author(s):

Lucy Bütof ◽

Cornelia Große ◽

Hauke Lilie ◽

Martin Herzberg ◽

Dietrich H. Nies

Keyword(s):

Metal Resistance ◽

Zinc Homeostasis ◽

Transport Systems ◽

Starvation Response ◽

Content Type ◽

Resistance Factors ◽

Cupriavidus Metallidurans ◽

Dependent Protein ◽

Zinc Storage ◽

Physiological And Biochemical

ABSTRACTThe Zur regulon is central to zinc homeostasis in the zinc-resistant bacteriumCupriavidus metallidurans. It comprises the transcription regulator Zur, the zinc importer ZupT, and three members of the COG0523 family of metal-chaperoning G3E-type GTPases, annotated as CobW1, CobW2, and CobW3. The operon structures of thezurandcobW1loci were determined. To analyze the interplay between the Zur regulon components and metal resistance, deletion mutants were constructed from the wild-type strain CH34 and various other strains. The Zur regulon components interacted with the plasmid-encoded and chromosomally encoded metal resistance factors to acquire metals from complexes of EDTA and for homeostasis of and resistance to zinc, nickel, cobalt, and cadmium. The three G3E-type GTPases were characterized in more detail. CobW1 bound only 1 Zn atom per mol of protein with a stability constant slightly above that of 2-carboxy-2′-hydroxy-5′-sulfoformazylbenzene (Zincon) and an additional 0.5 Zn with low affinity. The CobW1 system was necessary to obtain metals from EDTA complexes. The GTPase CobW2 is a zinc storage compound and bound 0.5 to 1.5 Zn atoms tightly and up to 6 more with lower affinity. The presence of MgGTP unfolded the protein partially. CobW3 had no GTPase activity and equilibrated metal import by ZupT with that of the other metal transport systems. It sequestered 8 Zn atoms per mol with decreasing affinity. The three CobWs bound to the metal-dependent protein FolEIB2, which is encoded directly downstream ofcobW1. This demonstrated an important contribution of the Zur regulon components to metal homeostasis inC. metallidurans.IMPORTANCEZinc is an important transition metal cation and is present as an essential component in many enzymes, such as RNA polymerase. As with other transition metals, zinc is also toxic at higher concentrations so that living cells have to maintain strict control of their zinc homeostasis. Members of the COG0523 family of metal-chaperoning GE3-type GTPases exist in archaea, bacteria, and eucaryotes, including humans, and they may be involved in delivery of zinc to thousands of different proteins. We used a combination of molecular, physiological, and biochemical methods to demonstrate the important but diverse functions of COG0523 proteins inC. metallidurans, which are produced as part of the Zur-controlled zinc starvation response in this bacterium.

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CodY-Mediated Regulation of Guanosine Uptake in Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.05899-11 ◽

2011 ◽

Vol 193 (22) ◽

pp. 6276-6287 ◽

Cited By ~ 13

Author(s):

Boris R. Belitsky ◽

Abraham L. Sonenshein

Keyword(s):

Bacillus Subtilis ◽

Binding Sites ◽

Consensus Sequence ◽

Regulatory Region ◽

Binding Motif ◽

Downstream Site ◽

Upstream Site ◽

Binding Motifs ◽

Content Type ◽

A Minor

CodY is a global transcriptional regulator known to control expression of more than 100 genes and operons inBacillus subtilis. Some of the most strongly repressed targets of CodY, thenupNOPQ(formerly,yufNOPQ) genes, were found to encode a guanosine transporter. Using DNase I footprinting experiments, we identified two high-affinity CodY-binding sites in the regulatory region of thenupNgene. The two sites are located 50 bp upstream and 163 bp downstream of the transcription start site. The downstream site was responsible for 6- to 8-foldnupNrepression in the absence of the upstream site. When the upstream site was intact, however, only a minor contribution of the downstream site tonupNregulation could be detected under the conditions tested. Both sites contained 15-bp CodY-binding motifs with two mismatches each with respect to the consensus sequence, AATTTTCWGTTTTAA. However, the experimentally determined binding sites included additional sequences flanking the 15-bp CodY-binding motifs. An additional version of the 15-bp CodY-binding motif, with 5 mismatches with respect to the consensus but essential for efficient regulation by CodY, was found within the upstream site. The presence of multiple 15-bp motifs may be a common feature of CodY-binding sites.

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Genome Sequence and Mutational Analysis of Plant-Growth-Promoting Bacterium Agrobacterium tumefaciens CCNWGS0286 Isolated from a Zinc-Lead Mine Tailing

Applied and Environmental Microbiology ◽

10.1128/aem.01200-12 ◽

2012 ◽

Vol 78 (15) ◽

pp. 5384-5394 ◽

Cited By ~ 45

Author(s):

Xiuli Hao ◽

Pin Xie ◽

Laurel Johnstone ◽

Susan J. Miller ◽

Christopher Rensing ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Plant Growth ◽

Genome Sequence ◽

Metal Resistance ◽

Zinc Homeostasis ◽

Dominant Factor ◽

Content Type ◽

Iaa Production ◽

Plant Growth Promoting ◽

Growth Promoting

ABSTRACTThe plant-growth-promoting bacteriumAgrobacterium tumefaciensCCNWGS0286, isolated from the nodules ofRobinia pseudoacaciagrowing in zinc-lead mine tailings, both displayed high metal resistance and enhanced the growth ofRobiniaplants in a metal-contaminated environment. Our goal was to determine whether bacterial metal resistance or the capacity to produce phytohormones had a larger impact on the growth of host plants under zinc stress. Eight zinc-sensitive mutants and one zinc-sensitive mutant with reduced indole-3-acetic acid (IAA) production were obtained by transposon mutagenesis. Analysis of the genome sequence and of transcription via reverse transcriptase PCR (RT-PCR) combined with transposon gene disruptions revealed that ZntA-4200 and the transcriptional regulator ZntR1 played important roles in the zinc homeostasis ofA. tumefaciensCCNWGS0286. In addition, interruption of a putative oligoketide cyclase/lipid transport protein reduced IAA synthesis and also showed reduced zinc and cadmium resistance but had no influence on copper resistance. In greenhouse studies,R. pseudoacaciainoculated withA. tumefaciensCCNWGS0286 displayed a significant increase in biomass production over that without inoculation, even in a zinc-contaminated environment. Interestingly, the differences in plant biomass improvement amongA. tumefaciensCCNWGS0286,A. tumefaciensC58, and zinc-sensitive mutants 12-2 (zntA::Tn5) and 15-6 (low IAA production) revealed that phytohormones, rather than genes encoding zinc resistance determinants, were the dominant factor in enhancing plant growth in contaminated soil.

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Behind the shield of Czc: ZntR controls expression of the gene for the zinc-exporting P-type ATPase ZntA in Cupriavidus metallidurans

Journal of Bacteriology ◽

10.1128/jb.00052-21 ◽

2021 ◽

Author(s):

Vladislava Schulz ◽

Christopher Schmidt-Vogler ◽

Phillip Strohmeyer ◽

Stefanie Weber ◽

Daniel Kleemann ◽

...

Keyword(s):

Cadmium Concentration ◽

Zinc Content ◽

Metal Resistance ◽

Zinc Uptake ◽

Uptake System ◽

Zinc Ions ◽

Cupriavidus Metallidurans ◽

Zinc Concentrations ◽

Main Regulator ◽

Cellular Zinc

In the metallophilic beta-proteobacterium Cupriavidus metallidurans, the plasmid-encoded Czc metal homeostasis system adjusts the periplasmic zinc, cobalt and cadmium concentration, which influences subsequent uptake of these metals into the cytoplasm. Behind this shield, the PIB2-type APTase ZntA is responsible for removal of surplus cytoplasmic zinc ions, thereby providing a second level of defense against toxic zinc concentrations. ZntA is the counterpart to the Zur-regulated zinc uptake system ZupT and other import systems; however, the regulator of zntA expression was unknown. The chromid-encoded zntA gene is adjacent to the genes czcI2C2B2’, which are located on the complementary DNA strand and transcribed from a common promoter region. These genes encode homologs of plasmid pMOL30-encoded Czc components. Candidates for possible regulators of zntA were identified and subsequently tested: CzcI, CzcI2, and the MerR-type gene products of the locus tags Rmet_2302, Rmet_0102, Rmet_3456. This led to the identification of Rmet_3456 as ZntR, the main regulator of zntA expression. Moreover, both CzcIs decreased Czc-mediated metal resistance, possibly to avoid “over-excretion” of periplasmic zinc ions, which could result in zinc starvation due to diminished zinc uptake into the cytoplasm. Rmet_2302 was identified as CadR, the regulator of the cadA gene for an important cadmium-exporting PIB2-type ATPase, which provides another system for removal of cytoplasmic zinc and cadmium. Rmet_0102 was not involved in regulation of the metal resistance systems examined here. Thus, ZntR forms a complex regulatory network with CadR, Zur and the CzcIs. Moreover, these discriminating regulatory proteins assign the efflux systems to their particular function. Importance Zinc is an essential metal for numerous organisms from humans to bacteria. The transportome of zinc uptake and efflux systems controls the overall cellular composition and zinc content in a double feed-back loop. Zinc starvation mediates, via the Zur regulator, an up-regulation of the zinc import capacity via the ZIP-type zinc importer ZupT and an amplification of zinc storage capacity, which together raise the cellular zinc content again. On the other hand, an increasing zinc content leads to ZntR-mediated up-regulation of the zinc efflux system ZntA, which decreases the zinc content. Together, the Zur regulon components and ZntR/ZntA balance the cellular zinc content under both high external zinc concentrations and zinc starvation conditions.

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Adaptation of Cupriavidus metallidurans CH34 to Toxic Zinc Concentrations Involves an Uncharacterized ABC-Type Transporter

Microorganisms ◽

10.3390/microorganisms9020309 ◽

2021 ◽

Vol 9 (2) ◽

pp. 309

Author(s):

Rob Van Houdt ◽

Joachim Vandecraen ◽

Natalie Leys ◽

Pieter Monsieurs ◽

Abram Aertsen

Keyword(s):

Abc Transporter ◽

Rhizobium Leguminosarum ◽

Constitutive Expression ◽

Metal Resistance ◽

Cadmium Resistance ◽

Gene Encoding ◽

Cupriavidus Metallidurans Ch34 ◽

Cupriavidus Metallidurans ◽

High Zinc ◽

Zinc Concentrations

Cupriavidus metallidurans CH34 is a well-studied metal-resistant β-proteobacterium and contains a battery of genes participating in metal metabolism and resistance. Here, we generated a mutant (CH34ZnR) adapted to high zinc concentrations in order to study how CH34 could adaptively further increase its resistance against this metal. Characterization of CH34ZnR revealed that it was also more resistant to cadmium, and that it incurred seven insertion sequence-mediated mutations. Among these, an IS1088 disruption of the glpR gene (encoding a DeoR-type transcriptional repressor) resulted in the constitutive expression of the neighboring ATP-binding cassette (ABC)-type transporter. GlpR and the adjacent ABC transporter are highly similar to the glycerol operon regulator and ATP-driven glycerol importer of Rhizobium leguminosarum bv. viciae VF39, respectively. Deletion of glpR or the ABC transporter and complementation of CH34ZnR with the parental glpR gene further demonstrated that loss of GlpR function and concomitant derepression of the adjacent ABC transporter is pivotal for the observed resistance phenotype. Importantly, addition of glycerol, presumably by glycerol-mediated attenuation of GlpR activity, also promoted increased zinc and cadmium resistance in the parental CH34 strain. Upregulation of this ABC-type transporter is therefore proposed as a new adaptation route towards metal resistance.

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Zinc and Sepsis

Nutrients ◽

10.3390/nu10080976 ◽

2018 ◽

Vol 10 (8) ◽

pp. 976 ◽

Cited By ~ 22

Author(s):

Wiebke Alker ◽

Hajo Haase

Keyword(s):

Defense Mechanism ◽

Acute Phase Proteins ◽

Innate Immune ◽

Zinc Homeostasis ◽

Major Health ◽

Nutritional Immunity ◽

Life Threatening ◽

Zinc Concentrations ◽

Severity Of The Disease ◽

Major Health Issue

Sepsis, defined as a “life-threatening organ dysfunction caused by a dysregulated host-response to infection” is a major health issue worldwide and still lacks a fully elucidated pathobiology and uniform diagnostic tests. The trace element zinc is known to be crucial to ensure an appropriate immune response. During sepsis a redistribution of zinc from serum into the liver has been observed and several studies imply a correlation between zinc and sepsis outcome. Therefore the alterations of zinc concentrations in different tissues might serve as one part of the host’s defense mechanism against pathogens during sepsis by diverse mechanisms. It has been suggested that zinc is involved in nutritional immunity, acts as a hepatoprotective agent, or a differentiation signal for innate immune cells, or supports the synthesis of acute phase proteins. Further knowledge about these events could help in the evaluation of how zinc could be optimally applied to improve treatment of septic patients. Moreover, the changes in zinc homeostasis are substantial and correlate with the severity of the disease, suggesting that zinc might also be useful as a diagnostic marker for evaluating the severity and predicting the outcome of sepsis.

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Transposase-Mediated Excision, Conjugative Transfer, and Diversity of ICE6013 Elements in Staphylococcus aureus

Journal of Bacteriology ◽

10.1128/jb.00629-16 ◽

2017 ◽

Vol 199 (8) ◽

Cited By ~ 11

Author(s):

Emily A. Sansevere ◽

Xiao Luo ◽

Joo Youn Park ◽

Sunghyun Yoon ◽

Keun Seok Seo ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Sequence Analysis ◽

Integration Site ◽

Consensus Sequence ◽

Open Reading Frames ◽

Conjugative Plasmid ◽

Site Preference ◽

Conjugative Transfer ◽

Content Type ◽

Integrative Conjugative Elements

ABSTRACT ICE6013 represents one of two families of integrative conjugative elements (ICEs) identified in the pan-genome of the human and animal pathogen Staphylococcus aureus. Here we investigated the excision and conjugation functions of ICE6013 and further characterized the diversity of this element. ICE6013 excision was not significantly affected by growth, temperature, pH, or UV exposure and did not depend on recA. The IS30-like DDE transposase (Tpase; encoded by orf1 and orf2) of ICE6013 must be uninterrupted for excision to occur, whereas disrupting three of the other open reading frames (ORFs) on the element significantly affects the level of excision. We demonstrate that ICE6013 conjugatively transfers to different S. aureus backgrounds at frequencies approaching that of the conjugative plasmid pGO1. We found that excision is required for conjugation, that not all S. aureus backgrounds are successful recipients, and that transconjugants acquire the ability to transfer ICE6013. Sequencing of chromosomal integration sites in serially passaged transconjugants revealed a significant integration site preference for a 15-bp AT-rich palindromic consensus sequence, which surrounds the 3-bp target site that is duplicated upon integration. A sequence analysis of ICE6013 from different host strains of S. aureus and from eight other species of staphylococci identified seven divergent subfamilies of ICE6013 that include sequences previously classified as a transposon, a plasmid, and various ICEs. In summary, these results indicate that the IS30-like Tpase functions as the ICE6013 recombinase and that ICE6013 represents a diverse family of mobile genetic elements that mediate conjugation in staphylococci. IMPORTANCE Integrative conjugative elements (ICEs) encode the abilities to integrate into and excise from bacterial chromosomes and plasmids and mediate conjugation between bacteria. As agents of horizontal gene transfer, ICEs may affect bacterial evolution. ICE6013 represents one of two known families of ICEs in the pathogen Staphylococcus aureus, but its core functions of excision and conjugation are not well studied. Here, we show that ICE6013 depends on its IS30-like DDE transposase for excision, which is unique among ICEs, and we demonstrate the conjugative transfer and integration site preference of ICE6013. A sequence analysis revealed that ICE6013 has diverged into seven subfamilies that are dispersed among staphylococci.

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Short repeated elements in the upstream regulatory region of the SUC2 gene of Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.6.7.2324-2333.1986 ◽

1986 ◽

Vol 6 (7) ◽

pp. 2324-2333

Author(s):

L Sarokin ◽

M Carlson

Keyword(s):

Carbon Catabolite Repression ◽

Consensus Sequence ◽

Regulatory Region ◽

Lacz Fusion ◽

Upstream Region ◽

Upstream Regulatory Region ◽

Heterologous Promoter ◽

Suc2 Gene ◽

High Level ◽

Repeated Motif

Expression of secreted invertase from the SUC2 gene is regulated by carbon catabolite repression. Previously, an upstream regulatory region that is required for derepression of secreted invertase was identified and shown to confer glucose-repressible expression to the heterologous promoter of a LEU2-lacZ fusion. In this paper we show that tandem copies of a 32-base pair (bp) sequence from the upstream regulatory region activate expression of the same LEU2-lacZ fusion. The level of expression increased with the number of copies of the element, but was independent of their orientation; the expression from constructions containing four copies of the sequence was only twofold lower than that when the entire SUC2 upstream regulatory region was present. This activation was not significantly glucose repressible. The 32-bp sequence includes a 7-bp motif with the consensus sequence (A/C)(A/G)GAAAT that is repeated at five sites within the upstream regulatory region. Genetic evidence supporting the functional significance of this repeated motif was obtained by pseudoreversion of a SUC2 deletion mutant lacking part of the upstream region, including two copies of the 7-bp element. In three of five pseudorevertants, the mutations that restored high-level SUC2 expression altered one of the remaining copies of the 7-bp element.

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Repeated CT elements bound by zinc finger proteins control the absolute and relative activities of the two principal human c-myc promoters

Molecular and Cellular Biology ◽

10.1128/mcb.13.9.5710-5724.1993 ◽

1993 ◽

Vol 13 (9) ◽

pp. 5710-5724

Author(s):

E DesJardins ◽

N Hay

Keyword(s):

Zinc Finger ◽

Tandem Repeats ◽

Zinc Finger Protein ◽

Consensus Sequence ◽

Regulatory Region ◽

Maximal Activity ◽

Single Copy ◽

Promoter Usage

Transcription of the human proto-oncogene c-myc is governed by two tandem principal promoters, termed P1 and P2. In general, the downstream promoter, P2, is predominant, which is in contrast to the promoter occlusion phenomenon usually observed in genes containing tandem promoters. A shift in human c-myc promoter usage has been observed in some tumor cells and in certain physiological conditions. However, the mechanisms that regulate promoter usage are not well understood. The present studies identify regulators which are required to promote transcription from both human c-myc promoters, P1 and P2, and have a role in determining their relative activities in vivo. A novel regulatory region located 101 bp upstream of P1 was characterized and contains five tandem repeats of the consensus sequence CCCTCCCC (CT element). The integrity of the region containing all five elements is required to promote transcription from P1 and for maximal activity from P2 in vivo. A single copy of this same element, designated CT-I2, also appears in an inverted orientation 53 bp upstream of the P2 transcription start site. This element has an inhibitory effect on P1 transcription and is required for P2 transcription. The transcription factor Sp1 was identified as the factor that binds specifically to the tandem CT elements upstream of P1 and to the CT-I2 element upstream of P2. In addition, the recently cloned zinc finger protein ZF87, or MAZ, was also able to bind these same elements in vitro. The five tandem CT elements can be functionally replaced by a heterologous enhancer that only in the absence of CT-I2 reverses the promoter usage, similar to what is observed in the translocated c-myc allele of Burkitt's lymphoma cells.

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Target (MexB)- and Efflux-Based Mechanisms Decreasing the Effectiveness of the Efflux Pump Inhibitor D13-9001 in Pseudomonas aeruginosa PAO1: Uncovering a New Role for MexMN-OprM in Efflux of β-Lactams and a Novel Regulatory Circuit (MmnRS) Controlling MexMN Expression

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01718-18 ◽

2018 ◽

Vol 63 (2) ◽

pp. e01718-18 ◽

Cited By ~ 1

Author(s):

Srijan Ranjitkar ◽

Adriana K. Jones ◽

Mina Mostafavi ◽

Zachary Zwirko ◽

Oleg Iartchouk ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Efflux Pump ◽

Response Regulator ◽

Heavy Metal Resistance ◽

Metal Resistance ◽

Rna Seq ◽

Efflux Pump Inhibitor ◽

Content Type ◽

Regulatory Circuit ◽

Outer Membrane Channel

ABSTRACT Efflux pumps contribute to antibiotic resistance in Gram-negative pathogens. Correspondingly, efflux pump inhibitors (EPIs) may reverse this resistance. D13-9001 specifically inhibits MexAB-OprM in Pseudomonas aeruginosa. Mutants with decreased susceptibility to MexAB-OprM inhibition by D13-9001 were identified, and these fell into two categories: those with alterations in the target MexB (F628L and ΔV177) and those with an alteration in a putative sensor kinase of unknown function, PA1438 (L172P). The alterations in MexB were consistent with reported structural studies of the D13-9001 interaction with MexB. The PA1438L172P alteration mediated a >150-fold upregulation of MexMN pump gene expression and a >50-fold upregulation of PA1438 and the neighboring response regulator gene, PA1437. We propose that these be renamed mmnR and mmnS for MexMN regulator and MexMN sensor, respectively. MexMN was shown to partner with the outer membrane channel protein OprM and to pump several β-lactams, monobactams, and tazobactam. Upregulated MexMN functionally replaced MexAB-OprM to efflux these compounds but was insusceptible to inhibition by D13-9001. MmnSL172P also mediated a decrease in susceptibility to imipenem and biapenem that was independent of MexMN-OprM. Expression of oprD, encoding the uptake channel for these compounds, was downregulated, suggesting that this channel is also part of the MmnSR regulon. Transcriptome sequencing (RNA-seq) of cells encoding MmnSL172P revealed, among other things, an interrelationship between the regulation of mexMN and genes involved in heavy metal resistance.

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