Recombinant Shigella boydii serotype 4 Magnesium transport protein CorA (corA)

What is Shigella boydii serotype 4 and how is it genetically related to other bacteria?

Shigella boydii serotype 4 is one of the many serotypes of Shigella bacteria that cause shigellosis, a form of bacillary dysentery. Importantly, Shigella strains are not taxonomically distinct species but rather are clones of Escherichia coli that have emerged relatively recently in evolutionary terms. The O-antigen gene clusters for S. boydii serotype 4 have been fully sequenced and characterized, revealing that it has an identical O-antigen to that found in certain E. coli strains (specifically the O53 antigen) . This genetic relationship highlights the close evolutionary connection between these bacterial lineages, with horizontal gene transfer likely playing a significant role in their diversification.

What is the CorA protein and what is its primary function in bacteria?

CorA (Magnesium transport protein CorA) belongs to the 2-TM-GxN family of membrane proteins and plays a major role in magnesium transport in prokaryotes and eukaryotic mitochondria . The primary function of CorA is to facilitate the movement of Mg²⁺ ions across cellular membranes, which is essential since magnesium is involved in numerous metabolic reactions, stabilizes highly charged molecules like ATP, and compensates for the negative charge of phosphate groups in lipid bilayers . The protein forms homo- or hetero-pentamers with large cytoplasmic domains that serve regulatory functions and a transmembrane part consisting of two α-helices per protomer . The signature motif GxN in the loops connecting these helices is believed to be involved in substrate selection .

What are the recommended methods for expressing and purifying recombinant S. boydii serotype 4 CorA protein?

For expressing recombinant S. boydii serotype 4 CorA protein, researchers should consider the following methodological approach:

• Expression system selection: An E. coli expression system (typically BL21(DE3) or derivatives) with a T7 promoter-based vector is recommended due to the prokaryotic origin of the protein.

• Construct design:

  • Include a His-tag (preferably at the C-terminus to avoid interference with the N-terminal ion sensing domain)

  • Consider a TEV protease cleavage site for tag removal

  • Optimize codons for E. coli expression if yields are low

• Expression conditions:

  • Induction with 0.1-0.5 mM IPTG at OD₆₀₀ of 0.6-0.8

  • Lower temperature expression (16-25°C) for 16-20 hours to improve proper folding

  • Supplementation with 5-10 mM MgCl₂ may improve stability

• Purification protocol:

  • Cell lysis in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM MgCl₂, 10% glycerol, 1 mM DTT

  • Membrane fraction isolation by ultracentrifugation

  • Solubilization using mild detergents (DDM or LMNG at 1%)

  • Ni-NTA affinity chromatography

  • Size exclusion chromatography for final purification

This methodology is based on approaches used for related CorA proteins studied in fluorescence-based transport assays, where functional protein was successfully reconstituted into proteoliposomes .

How can researchers effectively reconstitute CorA into proteoliposomes for functional studies?

Reconstitution of CorA into proteoliposomes is critical for functional transport studies. A detailed methodological approach includes:

• Lipid preparation:

  • Use a mixture of E. coli polar lipids and phosphatidylcholine (3:1 ratio)

  • Dissolve lipids in chloroform, dry under nitrogen, and resuspend in reconstitution buffer

  • Create unilamellar vesicles through extrusion through 400 nm filters

• Protein incorporation:

  • Mix purified CorA with lipids at protein:lipid ratios of 1:50 to 1:200 (w/w)

  • Add detergent (Triton X-100) to destabilize liposomes

  • Remove detergent using Bio-Beads or dialysis over 24-48 hours

• Verification of incorporation:

  • Assess protein orientation using protease protection assays

  • Verify incorporation using freeze-fracture electron microscopy

  • Confirm functionality using ion uptake assays

• Transport assays:

  • Load liposomes with fluorescent indicators sensitive to the ion of interest

  • Monitor fluorescence changes upon addition of external ions

  • Use ionophores as positive controls and protein-free liposomes as negative controls

This approach, adapted from successfully established protocols for CorA proteins from T. maritima and M. jannaschii, enables researchers to study transport kinetics and ion selectivity .

What methods can be used to assess CorA-mediated ion transport kinetics?

Several complementary methodologies can be employed to characterize CorA-mediated ion transport kinetics:

• Fluorescence-based assays:

  • Mag-Fura-2 for Mg²⁺ transport

  • FluoZin-1 for Zn²⁺ transport

  • Newport Green for Ni²⁺ transport

  • These assays allow real-time monitoring of ion flux into proteoliposomes

• Isotope flux measurements:

  • ²⁸Mg²⁺ or other radioactive ion isotopes

  • Filtration-based separation of proteoliposomes from external media

  • Scintillation counting for quantification

• Patch-clamp electrophysiology:

  • For direct measurement of ion currents

  • Can be performed in giant liposomes or after reconstitution into planar lipid bilayers

• Stopped-flow spectroscopy:

  • For rapid kinetic measurements

  • Allows determination of initial rates of transport

• Factors to measure and analyze:

  • Initial rates of transport at varying ion concentrations (for Km determination)

  • Effects of membrane potential (using K⁺/valinomycin)

  • Proton gradient effects (using pH jumps)

  • Competition between different cations

Research on T. maritima CorA and M. jannaschii CorA demonstrated that transport is stimulated by membrane potential, while the related ZntB protein's transport is stimulated by proton gradients, highlighting the importance of testing these parameters .

How does the GxN motif in S. boydii CorA contribute to ion selectivity compared to other CorA family members?

The GxN motif (where x represents any amino acid) is a signature sequence in the 2-TM-GxN family of membrane proteins that includes CorA . This motif, located in the loops connecting the transmembrane helices, is believed to play a crucial role in ion selectivity.

Comparative analysis of CorA from different organisms reveals variations in the exact sequence of this motif (GxN) that may contribute to differences in ion selectivity profiles. In S. boydii CorA, the specific amino acid at position 'x' likely influences the coordination geometry of bound ions, affecting the protein's preference for Mg²⁺ versus other divalent cations.

Research approaches to investigate this question include:

• Site-directed mutagenesis: Systematically altering the 'x' residue and nearby amino acids to analyze changes in transport specificity

• Structure-function analysis: Comparing transport rates of Mg²⁺, Co²⁺, Ni²⁺, and Zn²⁺ across CorA variants

• Molecular dynamics simulations: Modeling ion coordination and passage through the channel with different GxN configurations

• Ion competition assays: Determining how varying concentrations of competing ions affect transport rates

Experimental data from T. maritima CorA and M. jannaschii CorA transport studies suggest that despite all CorA family members transporting similar ions (Mg²⁺, Co²⁺, Ni²⁺, and Zn²⁺), there may be subtle differences in ion preferences and transport efficiencies that could be attributed to variations in this critical motif and surrounding residues .

What are the structural differences between S. boydii CorA and other bacterial CorA proteins that might impact function?

While specific structural data for S. boydii CorA is limited in the provided sources, comparative analysis with well-characterized CorA proteins suggests several structural features that may differ:

• Cytoplasmic domain variations:

  • The cytoplasmic domain of CorA proteins functions as a regulatory domain

  • Differences in size and amino acid composition could affect magnesium sensing

  • Species-specific variations may influence interactions with other cellular components

• Transmembrane region differences:

  • The arrangement of the inner and outer pentameric transmembrane helices

  • Variations in pore-lining residues that affect ion selectivity and conductance

  • Differences in hydrophobic gating mechanisms

• Loop region variations:

  • Beyond the GxN motif, other residues in the loop regions may influence ion coordination

  • Loop length and flexibility variations could affect the dynamics of channel opening

• Regulatory site differences:

  • Mg²⁺ binding sites in the cytoplasmic domain that regulate channel activity

  • Variations in these sites may result in different sensitivities to intracellular Mg²⁺ levels

A systematic approach to understanding these differences would involve:

• Homology modeling based on existing CorA structures

• Cryo-EM or X-ray crystallography of S. boydii CorA

• Functional comparison of chimeric proteins containing domains from different CorA homologs

The existing research on T. maritima and M. jannaschii CorA proteins provides a foundation for these comparisons, with the observed functional differences in transport mechanisms suggesting underlying structural variations .

How do membrane potential and proton gradients differentially affect CorA function compared to other ion transporters?

Research comparing CorA proteins from T. maritima and M. jannaschii with ZntB from E. coli has revealed fundamental differences in their energy coupling mechanisms . These findings provide a framework for understanding how S. boydii CorA might function:

• Membrane potential dependence:

  • CorA proteins from T. maritima and M. jannaschii show transport stimulation by membrane potential

  • This suggests that the electrical gradient across the membrane is a primary driving force for CorA-mediated transport

  • The positive-outside membrane potential likely facilitates the movement of divalent cations through the CorA channel

• Proton gradient effects:

  • Unlike CorA, ZntB (a related family member) shows transport stimulation by proton gradients

  • This indicates a divergence in transport mechanisms within the same protein scaffold

  • CorA proteins appear less dependent on or unaffected by pH gradients

• Experimental approaches to investigate these effects:

  • Manipulation of membrane potential using valinomycin/K⁺ gradients

  • Creation of pH gradients across proteoliposome membranes

  • Simultaneous monitoring of ion flux and membrane potential

  • Site-directed mutagenesis of charged residues that might be involved in sensing membrane potential

• Mechanistic implications:

  • The dependence on membrane potential suggests CorA may function primarily as a channel rather than an active transporter

  • The protein structure likely contains voltage-sensing domains that undergo conformational changes in response to membrane potential

This fundamental difference in energy coupling mechanisms represents a significant divergence in how these related transport proteins have evolved to move similar sets of ions across membranes .

What experimental techniques can be used to study the regulatory mechanisms of S. boydii CorA activity?

Several sophisticated experimental approaches can be employed to elucidate the regulatory mechanisms controlling S. boydii CorA activity:

• Intracellular Mg²⁺ sensing studies:

  • Fluorescent Mg²⁺ indicators (Mag-Fura-2) in whole cells

  • Patch-clamp electrophysiology with controlled cytoplasmic Mg²⁺ concentrations

  • Isothermal titration calorimetry to measure Mg²⁺ binding to purified cytoplasmic domains

• Structural dynamics investigations:

  • Hydrogen-deuterium exchange mass spectrometry to identify regions with altered conformational dynamics

  • FRET-based sensors incorporated into strategic positions to monitor conformational changes

  • Single-molecule fluorescence studies to capture transitional states

• Mutational analysis platforms:

  • Alanine-scanning mutagenesis of putative regulatory sites

  • Creation of Mg²⁺-binding site mutants

  • Introduction of cysteine pairs for disulfide cross-linking studies

• Proteomic interactions:

  • Pull-down assays to identify protein interaction partners

  • Bacterial two-hybrid screening for regulatory protein interactions

  • Chemical cross-linking followed by mass spectrometry (XL-MS)

• In vivo regulation studies:

  • Gene expression analysis under varying Mg²⁺ conditions

  • Reporter gene fusions to monitor corA expression

  • Proteomics to identify changes in the CorA interactome under different conditions

These approaches should be integrated to develop a comprehensive model of how S. boydii CorA activity is regulated in response to environmental conditions and cellular needs. Based on studies of other CorA proteins, regulation likely involves sensing of intracellular Mg²⁺ concentrations by the cytoplasmic domain, resulting in conformational changes that affect channel opening .

How can researchers investigate the physiological role of CorA in S. boydii in the context of pathogenesis?

Investigating the physiological role of CorA in S. boydii pathogenesis requires a multifaceted approach combining molecular genetics, cellular microbiology, and infection models:

• Genetic manipulation strategies:

  • Creation of corA deletion mutants in S. boydii

  • Complementation studies with wild-type and mutant corA variants

  • Construction of conditional expression systems for controlled CorA expression

• Phenotypic characterization:

  • Growth kinetics under varying Mg²⁺ concentrations

  • Bacterial survival under environmental stresses (pH, antimicrobials, oxidative stress)

  • Biofilm formation capabilities

  • Motility and chemotaxis assays

• Host-pathogen interaction studies:

  • Invasion and intracellular survival in epithelial cell lines

  • Macrophage survival and inflammatory response induction

  • Transcriptional profiling during host cell interaction

  • Competitive index assays with wild-type bacteria in infection models

• Mg²⁺ dynamics during infection:

  • Development of genetically encoded Mg²⁺ sensors

  • Real-time imaging of Mg²⁺ levels during host cell invasion

  • Quantification of Mg²⁺ in various cellular compartments during infection

• Connection to virulence factors:

  • Impact of corA deletion on Type III secretion system function

  • Effects on lipopolysaccharide synthesis and outer membrane integrity

  • Influence on expression of other virulence-associated genes

What is the relationship between CorA function and antibiotic resistance in Shigella species?

The relationship between CorA function and antibiotic resistance in Shigella species represents an emerging area of research with significant implications for therapeutic approaches:

• Mg²⁺-dependent antimicrobial resistance mechanisms:

  • Many antibiotics require Mg²⁺ for uptake or activity

  • Altered Mg²⁺ homeostasis via CorA dysfunction may affect antibiotic efficacy

  • Aminoglycosides particularly require cation-dependent uptake systems

• Membrane potential alterations:

  • CorA function is linked to membrane potential

  • Membrane potential disruption can affect the activity of numerous antibiotics

  • CorA dysfunction could alter membrane electrochemistry and subsequent drug susceptibility

• Experimental approaches to investigate this relationship:

  • Minimum inhibitory concentration (MIC) determination for corA mutants

  • Time-kill assays under varying Mg²⁺ conditions

  • Antibiotic uptake studies in corA mutants

  • Membrane potential measurements in wild-type vs. corA mutant strains

• Potential interaction with efflux systems:

  • Many efflux pumps are energy-dependent

  • Altered ion gradients may impact efflux pump efficiency

  • Co-regulation of corA and efflux pump genes could occur

• Clinical implications:

  • Rising antimicrobial resistance is particularly concerning in Shigella species

  • Understanding the role of CorA could identify novel drug targets

  • Potential for combination therapies targeting both CorA and existing resistance mechanisms

This research direction is particularly relevant given the documented ability of Shigella sonnei to acquire antimicrobial resistance genes, including extended-spectrum beta-lactamase (ESBL)-mediated resistance from other Enterobacteriaceae , raising questions about whether S. boydii may have similar capacities and how CorA function might influence these processes.

How does S. boydii CorA compare to CorA proteins in other Shigella species and E. coli?

Comparing S. boydii CorA to its counterparts in other Shigella species and E. coli provides valuable insights into evolutionary relationships and functional adaptation:

• Sequence conservation analysis:

  • Given that Shigella strains are clones of E. coli that have emerged relatively recently , CorA proteins across these species likely share high sequence identity

  • Critical functional domains including the GxN motif are expected to be highly conserved

  • Variations may exist in regulatory regions that respond to different environmental niches

• Functional differences across species:

  • Transport kinetics for various ions may differ subtly

  • Regulatory mechanisms might be adapted to species-specific physiological requirements

  • Expression patterns and cellular abundance might vary

• Evolutionary insights:

  • Analysis of synonymous vs. non-synonymous mutations can reveal selective pressures

  • Horizontal gene transfer events affecting corA genes or their regulatory elements

  • Phylogenetic analysis to trace the evolution of CorA across Enterobacteriaceae

• Experimental approaches:

  • Heterologous expression of CorA variants from different species in a common background

  • Cross-complementation studies in corA deletion mutants

  • Detailed biochemical characterization of purified proteins from multiple species

This comparative analysis is particularly relevant given the understanding that Shigella strains have evolved from E. coli through acquisition of virulence factors and other adaptive changes , raising questions about whether CorA function has been specifically adapted to support the pathogenic lifestyle of Shigella species.

What insights can be gained from studying CorA proteins across different bacterial phyla?

Studying CorA proteins across diverse bacterial phyla reveals fundamental insights into ion transport evolution and adaptation:

• Structural conservation and divergence:

  • Core functional elements (GxN motif, transmembrane domains) show conservation across phyla

  • Regulatory domains exhibit greater divergence, reflecting adaptation to different ecological niches

  • Emergence of specialized variants in extremophiles and other specialized bacteria

• Functional adaptation patterns:

  • Species from magnesium-limited environments may show higher affinity transport

  • Extremophiles demonstrate adaptations for function under high/low temperatures or extreme pH

  • Pathogen-specific adaptations may relate to host environment sensing

• Evolutionary relationships:

  • Ancient origin of CorA as a fundamental ion transport mechanism

  • Horizontal gene transfer vs. vertical inheritance patterns

  • Co-evolution with other magnesium homeostasis systems

• Comparative functional data:

  • Transport specificities vary across different bacterial phyla

  • Energy coupling mechanisms differ between related transporters like CorA and ZntB

  • Regulatory mechanisms may have evolved independently

• Methodological approaches:

  • Phylogenetic analysis across diverse bacterial genomes

  • Structural modeling of distant CorA homologs

  • Heterologous expression of diverse CorA variants in model organisms

  • Comparative biochemistry of purified proteins

The findings regarding distinct energy coupling mechanisms between CorA proteins from T. maritima and M. jannaschii versus ZntB from E. coli exemplify the valuable insights gained from cross-phyla comparisons, revealing how similar protein scaffolds have evolved different transport mechanisms .

How have CorA proteins evolved in response to different environmental pressures?

The evolution of CorA proteins in response to environmental pressures represents a fascinating case study in adaptive molecular evolution:

• Adaptation to magnesium availability:

  • Bacteria from magnesium-limited environments show adaptations in CorA affinity and regulation

  • Species experiencing fluctuating magnesium levels demonstrate more sophisticated regulatory mechanisms

  • Correlation between environmental magnesium concentration and CorA expression systems

• Host-pathogen co-evolution:

  • Pathogenic bacteria like S. boydii face host-imposed magnesium restriction as an antimicrobial strategy

  • CorA adaptations may have evolved to overcome host sequestration of essential ions

  • Potential specialization for function within specific host niches or cell types

• Adaptation to physiological requirements:

  • Species with high magnesium demands (due to metabolic specializations) show corresponding CorA adaptations

  • Growth rate correlations with CorA transport efficiency

  • Environmental bacteria vs. host-associated bacteria may show different optimization patterns

• Research approaches:

  • Comparative genomics across bacteria from diverse environments

  • Experimental evolution under controlled magnesium limitation

  • Reconstruction of ancestral CorA sequences

  • Ecological correlation studies linking CorA variants to environmental parameters

• Case study: CorA vs. ZntB evolution:

  • Despite structural similarities, CorA and ZntB have evolved different energy coupling mechanisms

  • CorA proteins are stimulated by membrane potential, while ZntB is stimulated by proton gradients

  • This divergence represents adaptation to different cellular needs within a conserved protein scaffold

This evolutionary perspective provides valuable context for understanding S. boydii CorA function and may inform targeted approaches for antimicrobial development that exploit unique aspects of this essential transport system.