Recombinant Magnesium transport protein CorA (corA)

What is CorA and what is its primary function in bacterial cells?

CorA is a ubiquitous magnesium transporter found in many bacterial species, playing a crucial role in maintaining magnesium homeostasis. It facilitates the movement of magnesium ions (Mg²⁺) across the cell membrane, which is essential for various biochemical and physiological processes including protein synthesis, cell membrane integrity, and nucleic acid synthesis. This transporter has been extensively studied in organisms such as E. coli, Salmonella species, Mycobacterium smegmatis, and Thermotoga maritima .

Methodologically, researchers characterize CorA's primary function through:

• Growth complementation assays in magnesium-limited conditions

• Radioactive ⁴⁵Mg²⁺ uptake studies

• Fluorescent magnesium indicator assays to measure intracellular magnesium levels

• Electrophysiological measurements in reconstituted systems

How is CorA structurally organized and what are its key domains?

CorA forms a homopentameric structure with each protomer containing:

• A large intracellular domain (ICD) with magnesium sensing capabilities

• Two transmembrane helices (TMD) that form the transport pore

• A conserved interhelical loop exposed to the periplasmic space that contains the "MPEL" motif

The functional transporter contains key structural elements:

• A symmetric closed conformation and multiple asymmetric open conformations that change dynamically during Mg²⁺ transport

• Conserved hydroxyl-bearing residues (such as S260 and T270 in S. typhimurium or S299 and T309 in M. smegmatis) critical for transport function

• Inter-subunit interfaces that may serve as binding sites for transported substrates

To study CorA structure, researchers typically employ:

• X-ray crystallography

• Cryo-electron microscopy

• Solution NMR spectroscopy

• Small-angle neutron scattering (SANS)

What organisms express CorA and how conserved is it across bacterial species?

CorA is widely distributed across bacterial species with significant sequence and functional conservation. Key organisms studied include:

Experimental approaches to study conservation include:

• Comparative genomics analysis

• Complementation studies across species

• Structural alignments of homologs

• Phylogenetic analysis of CorA families from diverse bacterial genomes

What are the optimal expression systems for recombinant CorA production?

Successful recombinant production of CorA depends on selecting appropriate expression systems based on research objectives:

For functional studies:

• E. coli BL21(DE3) with pET-based vectors is commonly used for high-yield expression

• M. smegmatis expression systems are valuable when studying mycobacterial CorA variants in a native-like environment

• The choice between N-terminal or C-terminal tags significantly affects expression levels and function

For structural studies:

• Specialized strains like E. coli C41(DE3) or C43(DE3) designed for membrane protein expression show improved yields

• Codon-optimized synthetic genes enhance expression levels

• Temperature modulation (typically 18-25°C) during induction improves proper folding

Methodological considerations include:

• Testing multiple promoter systems (T7, arabinose, tetracycline-inducible) to optimize expression levels

• Evaluating tag positions and types (His₆, MBP, GST) for optimal folding and activity

• Considering cell-free expression systems for difficult-to-express variants

What purification strategies yield functional CorA protein suitable for structure-function studies?

Purification of functional CorA requires careful consideration of detergent selection and stabilization conditions:

• Membrane preparation:

  • Differential centrifugation following cell lysis

  • Washing steps to remove peripheral membrane proteins

• Solubilization:

  • Mild detergents (n-dodecyl-β-D-maltoside, DDM; lauryl maltose neopentyl glycol, LMNG)

  • Inclusion of physiological concentrations of Mg²⁺ (1-5 mM) to maintain native conformation

• Chromatography:

  • Initial capture via immobilized metal affinity chromatography (IMAC)

  • Size exclusion chromatography to ensure pentameric assembly

  • Optional ion exchange step for higher purity

• Stabilization for functional studies:

  • Reconstitution into proteoliposomes or nanodiscs

  • Inclusion of 1-5 mM MgCl₂ in all buffers

  • Glycerol (10-15%) for cryostorage

The pentameric assembly should be verified via:

• Blue native PAGE

• Size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS)

• Negative stain electron microscopy

How can isotopic labeling be effectively incorporated for NMR studies of CorA?

For NMR studies of CorA, strategic isotopic labeling approaches include:

• Uniform labeling:

  • Growth in M9 minimal media with ¹⁵N-ammonium chloride and/or ¹³C-glucose

  • Deuteration via growth in D₂O-based media to reduce spectral crowding

  • Expression yields typically decrease 2-3 fold in minimal media

• Amino acid-specific labeling:

  • Selective incorporation of ¹⁵N-labeled alanine (particularly useful as alanine residues are distributed across both TMD and ICD regions)

  • Reverse labeling techniques to simplify spectra

  • SAIL (Stereo-Array Isotope Labeling) for improved resolution

• Segmental labeling:

  • Expressing domains separately and reconstituting via native chemical ligation

  • Split-intein approaches for in vivo segment labeling

Experimental considerations include:

• Optimizing induction conditions (typically lower temperature, longer duration)

• Supplementing media with trace elements for improved growth

• Implementing specialized protocols for membrane protein NMR sample preparation (e.g., using bicelles or nanodiscs instead of detergent micelles)

What are the established assays for measuring CorA-mediated magnesium transport?

Several complementary approaches are used to measure CorA-mediated magnesium transport:

• Radioactive transport assays:

  • ²⁸Mg²⁺ uptake in whole cells or reconstituted proteoliposomes

  • Time-course measurements with rapid filtration

  • Competitive inhibition studies with Co²⁺ and Co(III) hexamine chloride

• Fluorescence-based methods:

  • Mag-fura-2 or similar Mg²⁺-sensitive fluorescent dyes

  • Continuous real-time measurements in proteoliposomes

  • Stopped-flow kinetic analysis for rapid transport events

• Growth-based functional complementation:

  • Rescue of Mg²⁺-auxotrophic strains (such as Salmonella MM281)

  • Dose-dependent growth curves at varying Mg²⁺ concentrations

  • Competition assays between wild-type and mutant strains

• Electrophysiological methods:

  • Patch clamp of giant proteoliposomes

  • Planar lipid bilayer recordings

  • Solid-supported membrane electrophysiology

Data interpretation should consider:

• The bidirectional transport capability of CorA (both influx and efflux)

• Non-linear kinetics resulting from regulatory feedback

• Potential interference from endogenous transporters in cellular systems

How can researchers effectively study CorA's involvement in antimicrobial resistance?

Recent findings suggest CorA may facilitate the efflux of antibiotics in addition to its primary role in magnesium transport . To investigate this function:

• Antibiotic sensitivity testing:

  • Minimum inhibitory concentration (MIC) determination for cells expressing wild-type vs. mutant CorA

  • Checkerboard assays with Mg²⁺ supplementation and antibiotics

  • Time-kill kinetics to assess survival dynamics

• Drug accumulation assays:

  • Fluorescent antibiotics (e.g., norfloxacin, ofloxacin) measurement inside cells

  • Radioisotope-labeled drug uptake studies

  • Effect of efflux pump inhibitors like CCCP on accumulation

• Genetic approaches:

  • Construction of CorA deletion mutants (ΔcorA)

  • Complementation with wild-type or site-directed mutants

  • Dual expression with known efflux systems to assess interaction

Quantitative example from research data:

Based on experimental data from M. smegmatis

What approaches can be used to investigate the relationship between CorA and biofilm formation?

Research indicates CorA enhances biofilm-forming ability in bacteria . Methods to investigate this include:

• Quantitative biofilm assays:

  • Crystal violet staining with spectrophotometric quantification

  • Calculation of Biofilm Formation Index (BFI)

  • Comparison between wild-type, ΔcorA mutants, and complemented strains

  • Assessment of biofilm viability using resazurin or similar metabolic dyes

• Microscopy techniques:

  • Confocal laser scanning microscopy with live/dead staining

  • Scanning electron microscopy for ultrastructural analysis

  • Time-lapse microscopy to observe biofilm development dynamics

• Molecular analysis of biofilm composition:

  • Extracellular polysaccharide quantification

  • Protein profile analysis of biofilm matrix

  • Gene expression profiling (RNA-seq) of biofilm communities

• Environmental modulators:

  • Effect of Mg²⁺ concentration on biofilm formation

  • Impact of CCCP (which disrupts ionic gradients) on biofilm integrity

  • Influence of antimicrobials on biofilm persistence in CorA-expressing vs. ΔcorA strains

Research data suggests that CorA expression can increase biofilm formation by 2-4 fold in both E. coli and M. smegmatis, with cells expressing CorA showing approximately 26% higher viability in biofilms compared to CorA-deleted strains .

How do mutations in key residues affect CorA's transport function?

Mutational analysis has identified critical residues in CorA that significantly impact transport function:

• Hydroxyl-bearing residues:

  • S299A and T309A mutations in M. smegmatis CorA (corresponding to S260 and T270 in S. typhimurium) completely abolish transport activity

  • These mutations eliminate antimicrobial efflux activity without affecting growth patterns

  • The mechanism appears to involve altered metal binding affinity ("too tight" for transport)

• "MPEL" motif:

  • The glutamic acid residue in this conserved motif forms part of the substrate binding site in the periplasmic loop

  • Mutations affect the electrostatic properties that help concentrate cations near the entrance

• Gating mechanism residues:

  • Mutations at the inter-subunit interfaces alter conformational transitions between symmetric and asymmetric states

  • These can disrupt the coupling between Mg²⁺ binding and channel opening

Experimental approaches include:

• Site-directed mutagenesis targeting conserved residues

• Complementation assays to assess functional rescue

• Direct transport measurements with purified mutant proteins

• Structural studies to identify conformational changes

What is the molecular mechanism of CorA's dual functionality in Mg²⁺ import and antibiotic export?

Recent research proposes that CorA may function as an antiporter that imports Mg²⁺ while exporting antibiotics . The molecular mechanism appears to involve:

• Conformational dynamics:

  • CorA exists in symmetric closed and multiple asymmetric open conformations

  • These states dynamically interchange, particularly when intracellular Mg²⁺ levels are low

  • Different conformational states may facilitate different transport directions

• Binding sites identified through molecular docking:

  • Antibiotics likely bind at inter-subunit interfaces (sites C4 or C5) in the cytoplasmic domain

  • During conformational transitions to asymmetric states, bound antibiotics can be exported

  • The transmembrane pores provide the pathway for both Mg²⁺ import and drug export

• Coupling mechanism:

  • Mg²⁺ import may be electrogenic, creating an electrochemical gradient

  • This gradient could then be used to drive antibiotic export

  • Similar mechanisms have been observed in other metal transporters like NicT and Rv3270

The hypothesized model suggests that proper conformational transitions are required for both Mg²⁺ import and antibiotic export. Hydroxyl-bearing residue mutations that disrupt metal transport also eliminate antibiotic export capabilities, indicating a mechanistic link between these functions .

How does the periplasmic loop of CorA contribute to ion selectivity and transport?

The conserved interhelical loop of CorA, the only portion exposed to the periplasmic space, plays a crucial role in initial ion selection and transport:

• Ligand binding properties:

  • Nuclear magnetic resonance (NMR) spectroscopy reveals that Mg²⁺, Co²⁺, and Co(III) hexamine chloride (a CorA-specific inhibitor) all bind to the same site in this loop

  • The glutamic acid residue from the conserved "MPEL" motif is involved in this binding

  • Binding constants indicate relatively weak interactions (typical for channels)

• Electrostatic funnel function:

  • The negatively charged loop creates an electrostatic ring or funnel

  • This increases local substrate concentration before transport across the membrane

  • Mutations altering the charge distribution affect ion selectivity

• Hydration shell interactions:

  • Mg²⁺ transport requires partial dehydration of the ion

  • The loop participates in coordinating this dehydration process

  • The distinction between D₂O and H₂O appears relevant to selectivity

To study the periplasmic loop experimentally:

• Isolated transmembrane domain preparations for solution NMR

• Titration experiments with various substrates and inhibitors

• Electrophysiological measurements with loop mutations

• Molecular dynamics simulations of ion interactions with the loop

How is CorA expression regulated in response to environmental conditions?

CorA regulation involves multiple layers of control:

• Transcriptional regulation:

  • The corA gene is regulated at transcription initiation through the stringent response pathway

  • In Salmonella, RpoS (σᵈ) positively regulates corA expression, as demonstrated by reduced expression in ΔrpoS mutants

  • Additional transcription elongation control occurs through an unknown mechanism

• Post-transcriptional regulation:

  • Evidence suggests potential riboswitch-like elements may respond to Mg²⁺ levels

  • Translation efficiency may be modulated by Mg²⁺ concentration

  • mRNA stability could be affected by metal-responsive elements

• Post-translational regulation:

  • Conformational changes in response to intracellular Mg²⁺ levels

  • Potential protein-protein interactions affecting localization or activity

  • Possible modification of key residues under specific stress conditions

Experimental approaches include:

• Transcriptional fusions (e.g., corA-lacZ) to monitor promoter activity

• Reporter assays under various environmental stresses

• qRT-PCR to measure transcript levels

• Western blotting to assess protein expression

• Chromatin immunoprecipitation to identify regulatory factors

What compensatory mechanisms exist when CorA is absent or non-functional?

Bacteria have evolved redundant systems to maintain magnesium homeostasis when CorA is absent:

• Upregulation of alternative transporters:

  • In a ΔcorA mutant of Salmonella, MgtA (but not MgtB) is produced even in high extracellular Mg²⁺ conditions

  • This MgtA production becomes PhoP-independent in the ΔcorA background

  • Consistent with this, a ΔmgtA mutation slightly reduces magnesium content in a ΔcorA mutant

• Regulatory network rewiring:

  • The PhoP-PhoQ two-component system plays an important role in compensating for CorA absence

  • Synthetic phenotypes appear when both ΔphoP and ΔcorA mutations are combined

  • These include reduced growth and motility, even with sufficient magnesium

• Proteome adaptations:

  • Altered abundance of proteins involved in flagella formation, chemotaxis, and secretion occurs specifically when both ΔcorA and ΔphoP mutations are combined

  • Metabolic pathways may be restructured to minimize magnesium requirements

How does CorA interact with other cellular systems like the PhoP-PhoQ regulatory network?

CorA functions within a complex network of magnesium homeostasis systems:

This sophisticated network ensures magnesium homeostasis through redundant and compensatory systems, with CorA functioning as a central hub in both Mg²⁺ transport and regulatory signaling. The network becomes particularly important during environmental transitions, such as exiting stationary phase or adapting to magnesium-limited conditions .

What are the key considerations when designing site-directed mutagenesis studies for CorA?

Effective mutagenesis studies of CorA require careful planning:

• Target selection strategy:

  • Focus on conserved residues identified through multiple sequence alignments

  • Prioritize hydroxyl-bearing residues in transmembrane domains (e.g., S299, T309 in M. smegmatis)

  • Consider the "MPEL" motif in the periplasmic loop

  • Target residues at inter-subunit interfaces that may form antibiotic binding sites

• Mutation type selection:

  • Conservative substitutions (e.g., S→A, T→A) to eliminate hydroxyl groups without major structural disruption

  • Charge reversals (E→K) for evaluating electrostatic interactions

  • Introduction of bulky side chains to probe spatial constraints

  • Cysteine substitutions for subsequent labeling studies

• Expression and functional validation:

  • Confirm protein expression levels match wild-type

  • Verify proper membrane localization using fractionation or fluorescent tagging

  • Assess pentamer formation through native PAGE or crosslinking

  • Perform complementation assays in ΔcorA backgrounds

• Comprehensive phenotypic analysis:

  • Evaluate both Mg²⁺ transport and antibiotic efflux functions

  • Test biofilm formation capabilities

  • Assess growth kinetics under varying Mg²⁺ concentrations

  • Measure competitive fitness in mixed cultures

Special considerations include designing mutations that specifically affect one function (Mg²⁺ transport or antibiotic efflux) to dissect the mechanistic relationship between these activities.

How can researchers resolve contradictory data regarding CorA's role in magnesium homeostasis?

Some studies report contradictory findings about CorA's contribution to cellular magnesium levels. Strategies to resolve these include:

• Methodological standardization:

  • Use multiple complementary techniques to measure magnesium content:

    • Atomic absorption spectroscopy

    • ICP-MS for total cellular magnesium

    • Mag-fura-2 for free cytosolic magnesium

    • ²⁸Mg²⁺ uptake for transport kinetics

  • Carefully define growth conditions (media composition, growth phase)

  • Control for strain background differences

• Addressing compensatory mechanisms:

  • Generate multiple transporter knockout combinations (ΔcorA, ΔmgtA, ΔmgtB)

  • Employ inducible expression systems to control timing of CorA production

  • Use transcriptomics/proteomics to identify upregulated alternative systems

• Differentiating pools of magnesium:

  • Separate analyses of bound vs. free Mg²⁺

  • Subcellular fractionation to measure compartmentalization

  • Consider magnesium binding to ribosomes, ATP, and membrane components

• Temporal dynamics consideration:

  • Measure kinetics rather than steady-state levels

  • Assess adaptation responses over time

  • Evaluate fitness during transitions between environmental conditions

For example, in Salmonella, a ΔcorA mutation shows no effect on total and free cellular magnesium contents in steady state, yet confers a competitive disadvantage when exiting stationary phase . This apparent contradiction is resolved by considering temporal dynamics and specific growth transitions.

What approaches can reconcile in vitro and in vivo findings about CorA function?

Discrepancies between in vitro and in vivo studies of CorA can be addressed through:

• Bridging experimental systems:

  • Reconstituted proteoliposomes with controlled lipid composition

  • Spheroplast patch-clamp recordings

  • Giant unilamellar vesicles with incorporated CorA

  • Nanodiscs maintaining native-like membrane environment

• Controlling for cellular context:

  • Expression of CorA in heterologous systems lacking endogenous magnesium transporters

  • Complementation studies with chimeric transporters

  • Generation of conditional knockouts for temporal control

  • Single-cell imaging to capture population heterogeneity

• Replicating physiological conditions:

  • Maintaining physiological Mg²⁺ gradients

  • Including relevant intracellular components (ATP, polyamines)

  • Considering membrane potential effects

  • Accounting for molecular crowding

• Advanced structural approaches:

  • In-cell NMR to observe conformational dynamics in native environment

  • Cryo-electron tomography of native membranes

  • Mass spectrometry of intact membrane complexes

  • FRET-based conformational sensors for live-cell imaging

For example, while isolated transmembrane domain studies by NMR suggest specific binding sites in the periplasmic loop , the full pentameric structure in lipid bilayers shows complex conformational equilibria that may not be captured in fragmentary analyses . Reconciling these findings requires integrating structural data across different resolution scales and experimental conditions.

What emerging technologies might advance our understanding of CorA structure and function?

Several cutting-edge technologies show promise for CorA research:

• Cryo-electron microscopy advancements:

  • Time-resolved cryo-EM to capture transport intermediates

  • Correlative light and electron microscopy to link structure and function

  • Microcrystal electron diffraction for challenging structural variants

  • In situ structural studies in native membrane environments

• Advanced spectroscopic approaches:

  • Single-molecule FRET to observe conformational dynamics

  • EPR distance measurements with site-specific spin labels

  • Solid-state NMR of membrane-embedded CorA

  • Hydrogen-deuterium exchange mass spectrometry for dynamics

• Computational methods:

  • Molecular dynamics simulations at longer timescales

  • Machine learning for predicting transport properties from sequence

  • Quantum mechanical calculations of ion coordination

  • Multiscale modeling linking structural dynamics to transport kinetics

• Genetic engineering tools:

  • CRISPR-Cas9 genome editing for precise chromosomal modifications

  • Optogenetic control of CorA expression or conformation

  • Expanded genetic code to incorporate non-canonical amino acids

  • Single-cell tracking of transporter activity with fluorescent sensors

These technologies would particularly advance our understanding of the conformational transitions between symmetric and asymmetric states and how these are coupled to transport function.

What are the critical unresolved questions about CorA's role in antibiotic resistance?

Several key questions remain about CorA's involvement in antimicrobial resistance:

• Mechanistic uncertainties:

  • Is CorA directly transporting antibiotics or indirectly affecting other efflux systems?

  • What is the molecular basis for substrate promiscuity (how can it transport structurally diverse antibiotics)?

  • Does antibiotic binding occur at the same site as Mg²⁺ or at distinct locations?

  • Is there a mandatory coupling between Mg²⁺ import and antibiotic export?

• Clinical relevance questions:

  • Do clinical isolates with altered CorA expression show changed antimicrobial susceptibility profiles?

  • Could CorA inhibitors serve as adjuvants to enhance antibiotic efficacy?

  • Does CorA contribute to persistence during antibiotic treatment?

  • How does CorA-mediated resistance interact with other resistance mechanisms?

• Regulatory aspects:

  • How is CorA expression altered during antibiotic exposure?

  • Are there post-translational modifications affecting transport selectivity?

  • Does biofilm formation enhancement by CorA contribute significantly to antibiotic tolerance?

• Structural biology questions:

  • What structural features determine antibiotic binding specificity?

  • How do conformational changes facilitate antibiotic transport?

  • Can we identify specific binding pockets for different antibiotic classes?

Research approaches to address these questions would include:

• Direct measurement of labeled antibiotic transport in reconstituted systems

• High-resolution structures with bound antibiotics

• Comprehensive mutational analysis targeting predicted antibiotic binding sites

• Pharmacological studies with CorA inhibitors in combination with antibiotics

How might CorA research contribute to novel antimicrobial development strategies?

CorA research offers several promising avenues for antimicrobial development:

• CorA as a direct drug target:

  • Developing specific inhibitors targeting the periplasmic loop binding site

  • Designing compounds that lock the transporter in non-conducting conformations

  • Creating molecules that disrupt the pentameric assembly

  • Exploiting species-specific structural differences for selective targeting

• CorA inhibitors as resistance-breaking adjuvants:

  • Combining CorA inhibitors with conventional antibiotics to prevent efflux

  • Targeting the Mg²⁺-dependent enhancement of antibiotic resistance

  • Disrupting biofilm formation through CorA inhibition

  • Preventing compensatory responses to antibiotic exposure

• Structure-based approaches:

  • Using the antibiotic binding sites identified in CorA to design drugs that evade efflux

  • Developing compounds that exploit the ion selectivity filter

  • Creating molecular "plugs" for the periplasmic entrance

  • Engineering peptides that mimic critical inter-subunit interfaces

• Bacterial physiology manipulation:

  • Targeting magnesium homeostasis networks to create synergistic antimicrobial effects

  • Exploiting the CorA-PhoP-PhoQ relationship to disrupt adaptation responses

  • Developing strategies to prevent biofilm formation enhancement by CorA

  • Creating synthetic lethality by simultaneously targeting redundant transport systems

Methodological approaches would include:

• High-throughput screening for CorA inhibitors

• Structure-based drug design targeting key functional sites

• Whole-cell phenotypic screening with CorA-overexpressing strains

• in vivo infection models to assess clinical relevance

These research directions could lead to novel therapeutic strategies that overcome antimicrobial resistance mechanisms and enhance the efficacy of existing antibiotics.