Recombinant Bradyrhizobium japonicum Sulfate/thiosulfate import ATP-binding protein CysA (cysA)

What is the function of CysA in Bradyrhizobium japonicum?

CysA is part of the ABC transporter complex CysAWTP involved in sulfate/thiosulfate import. It functions as the ATP-binding protein component responsible for energy coupling to the transport system . This ABC transporter is essential for sulfur acquisition in B. japonicum, particularly in environments where sulfur is limited. As evidenced in comparative transcriptomic studies, genes involved in sulfur uptake and metabolism show increased expression in bacteroids compared to free-living cells, highlighting their importance in symbiotic conditions .

How does the CysAWTP complex function in sulfate/thiosulfate import?

The CysAWTP complex operates as a canonical ABC importer with distinct functional components:

• CysA (ATP-binding protein): Provides energy through ATP hydrolysis

• CysP: Substrate-binding protein with high affinity for thiosulfate

• Sbp: Substrate-binding protein specific for sulfate

• CysW/T: Transmembrane components forming the translocation pathway

The transport mechanism follows a typical ABC importer cycle:

• Substrate binding by the appropriate periplasmic binding protein (CysP or Sbp)

• Interaction of the loaded binding protein with the transmembrane domains

• ATP binding and hydrolysis by CysA, inducing conformational changes

• Substrate translocation across the membrane

• Release of inorganic phosphate and ADP, returning the transporter to its resting state

This cycle enables the energy-dependent uptake of essential sulfur compounds required for bacterial metabolism .

What are optimal conditions for expressing recombinant B. japonicum CysA?

While specific conditions for B. japonicum CysA aren't detailed in the search results, effective expression protocols can be designed based on related ABC transporters:

Recommended Expression Protocol:

• Vector selection: pET-based expression vectors with T7 promoter systems have shown success for ATP-binding proteins from the ABC transporter family

• Host strain: E. coli BL21(DE3) or its derivatives, particularly strains optimized for membrane-associated proteins

• Growth conditions:

  • Medium: LB or 2×YT supplemented with appropriate antibiotics

  • Temperature: Initial growth at 37°C to OD₆₀₀ of 0.6-0.8

  • Induction: IPTG concentration of 0.1-0.5 mM

  • Post-induction temperature: 16-20°C for 16-20 hours (to enhance solubility)

• Co-expression considerations: For functional studies, consider co-expressing with CysW and CysT components

For challenging expression scenarios, consider using specialized E. coli strains like Rosetta(DE3) to address codon bias issues, as B. japonicum uses a different codon preference than E. coli.

What purification strategies work best for recombinant CysA protein?

For ABC transporter ATPase components like CysA, the following purification strategy is recommended:

• Cell lysis: French press or sonication in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM DTT, and protease inhibitors

• Initial purification: Immobilized metal affinity chromatography (IMAC) using His-tagged protein

• Secondary purification: Size exclusion chromatography to remove aggregates and improve purity

• Buffer optimization: Final buffer typically contains 20 mM Tris-HCl pH 7.5, 100-150 mM NaCl, 5% glycerol, and 1-5 mM MgCl₂ (essential for maintaining ATPase activity)

Expected Purity: Greater than 85% as determined by SDS-PAGE , with typical yields of 3-10 mg per liter of culture.

For long-term storage, the protein should be maintained in a buffer containing 50% glycerol at -20°C/-80°C, with a typical shelf life of 6 months .

How can I assess CysA ATPase activity in vitro?

Effective methods for measuring CysA ATPase activity include:

• Malachite green phosphate assay:

  • Principle: Quantifies released inorganic phosphate from ATP hydrolysis

  • Reaction conditions: 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl₂, 1 mM DTT

  • ATP concentration: 0.1-5 mM ATP

  • Protein concentration: 50-200 nM purified CysA

  • Assay time: 10-30 minutes at 37°C

  • Detection: Absorbance at 620-650 nm

• Coupled enzyme assay:

  • Principle: Links ATP hydrolysis to NADH oxidation through pyruvate kinase and lactate dehydrogenase

  • Detection: Continuous monitoring of NADH absorbance decrease at 340 nm

  • Advantage: Real-time kinetics measurement

Important considerations:

• ATPase assays with purified CysA should include both transmembrane partners (CysW/T) as the ATP hydrolysis activity of CysA is typically dependent on these interactions

• Studies with similar ABC transporters have shown that "BioM is capable of hydrolyzing ATP only in the presence of a transmembrane partner" , suggesting this may also apply to CysA

What methods are suitable for studying CysA-substrate interactions?

To study interactions between CysA and its binding partners:

• Surface Plasmon Resonance (SPR):

  • Immobilize CysA on a sensor chip

  • Flow various concentrations of potential binding partners (ATP, other complex components)

  • Measure association/dissociation kinetics

  • Expected KD values: Low μM range for ATP binding

• Isothermal Titration Calorimetry (ITC):

  • Directly measure thermodynamic parameters of binding

  • No labeling required

  • Provides complete thermodynamic profile (ΔH, ΔS, ΔG, and binding stoichiometry)

• Fluorescence-based assays:

  • Intrinsic tryptophan fluorescence quenching upon ligand binding

  • Similar ATP-binding proteins have shown nanomolar to micromolar affinities for their substrates

  • Typical fluorescence quenching of 20-50% at saturation

For all interaction studies, include magnesium as a cofactor (typically 5 mM MgCl₂) as it's essential for ATP binding to ABC transporters.

How can I design site-directed mutagenesis experiments for CysA?

When planning site-directed mutagenesis of B. japonicum CysA, consider these strategic approaches:

• Target selection:

  • Walker A motif (G-X-X-G-X-G-K-S/T): Essential for ATP binding; mutate lysine to alanine to abolish ATP binding

  • Walker B motif (hhhhD, where h is hydrophobic): Critical for ATP hydrolysis; mutate aspartate to asparagine to allow binding but prevent hydrolysis

  • Q-loop: Important for transmembrane communication; identify conserved glutamine residues

  • Signature motif (LSGGQ): Characteristic of ABC transporters; mutations affect ATP hydrolysis

• Methodology:

  • PCR-based site-directed mutagenesis using complementary primers containing desired mutations

  • Selection method should account for high spontaneous antibiotic resistance in B. japonicum

  • Use a rapid screening method involving "simple plate selection for antibiotic resistant mutants, then colony streaking, and lysis for DNA hybridization on a nitrocellulose filter"

• Validation:

  • Confirm mutations by DNA sequencing

  • Express and purify mutant proteins

  • Compare ATP binding/hydrolysis activities with wild-type CysA

Considering that "screening for site-directed mutants is cumbersome and time-consuming" due to "high incidence of spontaneous antibiotic resistance and slow growth of Bradyrhizobium japonicum strains" , implementing an efficient screening strategy is crucial.

How does environmental stress affect CysA expression in B. japonicum?

Research on related transporters in bacteroids suggests that CysA expression is likely regulated by environmental conditions, particularly sulfur availability and symbiotic status:

• Transcriptomic evidence:

  • Genes involved in sulfur uptake and metabolism show increased expression in bacteroids compared to free-living cells

  • Similar to thiamine biosynthesis genes that showed higher expression in bacteroids (WAD 0.63 to 1.22) and cells grown in minimal medium (WAD 1.01 to 2.52) compared to those grown in rich medium

• Environmental signals affecting expression:

  • Sulfur limitation likely upregulates CysA expression

  • Iron availability may influence expression as "iron regulation of gene expression in the Bradyrhizobium japonicum/soybean symbiosis" has been documented

  • pH tolerance may affect expression as B. japonicum "can tolerate the low pH but cannot tolerate the high pH"

• Methodological approach to study expression:

  • Real-time PCR (qPCR) with efficiency-corrected quantification

  • Use dilution-replicate approach rather than identical replicates for more accurate results

  • Example of dilution schema: "Two samples diluted two-, ten- and 50-fold, and two other samples diluted five-, 50-, and 500-fold"

How does CysA from B. japonicum compare with homologs in other species?

CysA is highly conserved among prokaryotes, with distinctive features in different bacterial lineages:

The B. japonicum CysA shares structural features with other ABC transporter ATP-binding proteins like BioM in the biotin transporter BioMNY of R. capsulatus, where "BioM and BioN form stable bipartite complexes and tripartite complexes together with BioY" . This suggests similar complex formation patterns may exist for CysA.

What is the evolutionary significance of CysA in rhizobia-legume symbiosis?

The evolutionary importance of CysA in rhizobia-legume symbiosis can be understood through several lines of evidence:

• Upregulation during symbiosis:

  • Transcriptomic analyses show that "genes involved in sulfur uptake and metabolism were highly expressed in bacteroids compared to the expression levels in free-living cells"

  • This suggests a critical role for sulfate/thiosulfate import during the nitrogen-fixing symbiotic state

• Contribution to metabolic adaptation:

  • The efficient uptake of sulfur compounds is essential for the synthesis of sulfur-containing amino acids and iron-sulfur clusters in nitrogenase

  • The CysA transport system likely supports the high metabolic demands of bacteroids during symbiosis

• Co-evolution with host plants:

  • The presence of specialized sulfur transport mechanisms may represent adaptation to the particular microenvironment of root nodules

  • Similar to how "genes involved in thiamine-related genes" show differential expression patterns between free-living cells and bacteroids

CysA contributes to the sophisticated metabolic integration between rhizobia and legume hosts that has evolved over millions of years, enabling the efficient exchange of nutrients in this mutualistic relationship.

How can CysA be used as a target for enhancing symbiotic nitrogen fixation?

CysA offers potential for improving symbiotic nitrogen fixation through several research approaches:

• Overexpression strategies:

  • Designing expression constructs with stronger promoters to enhance sulfur uptake

  • Methodology: Create a recombinant B. japonicum strain overexpressing CysA under a symbiosis-specific promoter

  • Expected outcome: Improved sulfur metabolism supporting higher nitrogenase activity

• Protein engineering:

  • Develop CysA variants with enhanced ATP hydrolysis efficiency

  • Target sites for mutagenesis include the ATP-binding pocket and interfaces with transmembrane partners

  • Screening method: Use the "rapid method for selection of recombinant site-directed mutants" followed by symbiotic performance assays

• Field application design:

  • Integration with encapsulation techniques similar to those used for B. japonicum where "beads were prepared from cultured broths which were viable for more than 190 days"

  • Formulation optimization: Include appropriate sucrose concentration as "beads of Bradyrhizobium japonicum prefers the lower concentration of the sucrose"

What novel experimental approaches could advance our understanding of CysA function?

Several cutting-edge techniques could provide deeper insights into CysA function:

• Cryo-electron microscopy:

  • Determine the structure of the complete CysAWTP complex in different conformational states

  • Visualize substrate translocation pathway

  • Resolution target: <3Å to identify key interaction residues

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Map conformational changes during the transport cycle

  • Identify regions with altered solvent accessibility upon ATP binding

  • Detect interfaces between CysA and other complex components

• Single-molecule FRET:

  • Monitor real-time conformational changes during substrate transport

  • Observe transport kinetics at the single-molecule level

  • Design: Label CysA and transmembrane partners with appropriate fluorophore pairs

• Systems biology approaches:

  • Integrate transcriptomic, proteomic, and metabolomic data to create comprehensive models of sulfur metabolism

  • Similar to the approaches used in studying "efficient experimental design and analysis of real-time PCR assays" to understand gene regulation networks

These advanced approaches would provide unprecedented insights into the structural dynamics and regulatory mechanisms governing CysA function in B. japonicum.

What strategies can address low expression or insolubility of recombinant CysA?

Researchers frequently encounter expression and solubility issues with ABC transporter proteins. Consider these approaches:

• Optimization of expression conditions:

  • Reduce induction temperature to 16-18°C

  • Lower IPTG concentration to 0.1-0.2 mM

  • Extend expression time to 18-24 hours

  • Add 5-10% glycerol to growth medium to improve protein folding

• Solubility enhancement strategies:

  • Fusion tags: MBP (maltose-binding protein) tag often improves solubility more effectively than His-tag alone

  • Co-expression with chaperones: GroEL/GroES system or DnaK/DnaJ/GrpE

  • Addition of stabilizing agents: 1-5 mM ATP, 5 mM MgCl₂, and 10% glycerol in lysis buffer

• Alternative expression systems:

  • Cell-free protein synthesis for difficult-to-express proteins

  • Bacillus-based expression systems which may better accommodate GC-rich genes from B. japonicum

Success metric: Aim for purity "greater than 85% as determined by SDS-PAGE" , with homogeneous protein population confirmed by size exclusion chromatography.

How can I resolve inconsistent results in CysA functional assays?

When facing variability in CysA functional studies, implement these troubleshooting approaches:

• ATPase activity inconsistency:

  • Verify Mg²⁺ concentration (5 mM optimal)

  • Check for contaminating phosphate in reagents

  • Ensure protein stability by adding 10% glycerol and 1 mM DTT

  • Consider that CysA may require transmembrane partners for full activity, as observed with BioM which "is capable of hydrolyzing ATP only in the presence of a transmembrane partner"

• Transport assay variability:

  • Standardize vesicle/proteoliposome preparation

  • Control protein:lipid ratios precisely

  • Verify orientation of reconstituted protein

  • Perform activity controls with each batch

• Experimental design improvements:

  • When using qPCR for expression analysis, implement the constrained fit approach as it "is less influenced by individual Cq values than the independent fit approach"

  • For studying CysA expression, consider that "the proposed experimental design uses dilution-replicates instead of identical replicates" for more accurate results