Recombinant Aliphatic sulfonates import ATP-binding protein SsuB 2 (ssuB2)

How does SsuB2 interact with other components of the SsuABC transporter system?

SsuB2 functions as part of a complex transport system where each component plays a specific role:

• SsuA: The periplasmic aliphatic sulfonate-binding protein that captures the sulfonate substrate in the periplasmic space .

• SsuB: The ATP-binding protein (including SsuB2) that provides energy through ATP hydrolysis.

• SsuC: The transmembrane component that forms the transport channel.

The interaction sequence typically follows:

• SsuA binds the sulfonate substrate in the periplasm

• The substrate-loaded SsuA interacts with the SsuBC complex

• This interaction triggers ATP hydrolysis by SsuB/SsuB2

• Energy from ATP hydrolysis causes conformational changes in the transporter

• These changes facilitate substrate translocation across the membrane

Studies have shown that SsuA undergoes significant conformational changes upon substrate binding, with one domain rotating approximately 17° relative to the other domain . This movement is crucial for proper interaction with the SsuBC complex and subsequent transport.

What conditions regulate the expression of SsuB2 in bacteria?

The expression of SsuB2 and the entire SsuABC transporter system is primarily regulated by sulfur availability. Key regulatory patterns include:

• Sulfur starvation upregulation: The genes encoding SsuABC (including SsuB2) are upregulated during sulfur limitation conditions .

• Repression by preferred sulfur sources: Expression is repressed in the presence of preferred sulfur sources like sulfate or cysteine.

• Regulatory systems: In E. coli and related bacteria, the expression is controlled by:

  • CysB: A LysR-type transcriptional activator

  • Cbl: A secondary transcriptional regulator specifically for sulfonate utilization systems

Experimental evidence shows that multiple gene clusters, including tauABCD and ssuABCDE (which includes SsuB2), are upregulated during sulfur limitation and are capable of sulfur assimilation from organosulfur compounds such as sulfate esters, sulfamates, sulfonates, and alkanesulfonates .

What experimental approaches are most effective for expression and purification of recombinant SsuB2?

Based on successful protocols for similar ABC transporters and specifically SsuA , the following approach is recommended:

Expression system optimization:

Purification protocol:

• Cell harvesting by centrifugation at 6000 g for 10 min

• Resuspension in buffer containing:

  • PBS

  • 1 mg/ml lysozyme

  • 0.1 mg/ml DNAse

  • 0.1 mg/ml Pefablock

  • 5 mM MgCl₂

• Cell disruption using a cell disruptor at 172 MPa

• Clarification of lysate by centrifugation at 30,000 g for 1h

• IMAC purification:

  • Load supernatant supplemented with 20 mM imidazole

  • Wash with 10 column volumes of PBS + 20 mM imidazole

  • Wash with 10 column volumes of PBS + 40 mM imidazole

  • Elute with PBS + 500 mM imidazole

• Size exclusion chromatography on Superdex 75 column

• Final buffer exchange to experimental buffer

This protocol yielded high purity (>95%) for SsuA and should be adaptable for SsuB2 with potential modifications for ATP-binding proteins.

How can researchers design experiments to assess the ATP-binding and hydrolysis activity of SsuB2?

ATP-binding and hydrolysis by SsuB2 can be assessed through multiple complementary approaches:

ATP binding assays:

• Isothermal Titration Calorimetry (ITC):

  • Measures heat changes during ATP binding

  • Provides binding constants (Kd), stoichiometry (n), and thermodynamic parameters (ΔH, ΔS)

  • Typical buffer: 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl₂

• Fluorescent ATP analogs:

  • TNP-ATP fluorescence enhancement upon binding

  • FRET-based assays with fluorescently labeled protein

  • Requires specialized fluorimeter but allows for real-time binding kinetics

ATP hydrolysis assays:

• Malachite green phosphate assay:

  • Measures released inorganic phosphate

  • Standard curve: 0-50 μM phosphate

  • Typical reaction mixture:

    • 0.5-5 μM purified SsuB2

    • 1-5 mM ATP

    • 5 mM MgCl₂

    • 50 mM Tris-HCl pH 7.5

    • 150 mM NaCl

• Coupled enzyme assay:

  • Couples ATP hydrolysis to NADH oxidation via pyruvate kinase and lactate dehydrogenase

  • Continuously monitors ATPase activity by decrease in absorbance at 340 nm

  • Higher sensitivity than malachite green assay

Data analysis considerations:

• Determine Km and Vmax using Michaelis-Menten kinetics

• Assess effects of other SsuABC components on activity

• Evaluate influence of potential substrates on ATPase activity

What strategies can improve soluble expression of recombinant SsuB2 in heterologous systems?

Achieving high-yield soluble expression of recombinant SsuB2 requires optimization of multiple parameters. Based on experimental design approaches for similar proteins , the following strategies are recommended:

Multivariate optimization using fractional factorial design:

A systematic approach using a 2^(8-4) fractional factorial design with the following variables at two levels each has proven effective for other recombinant proteins :

Statistical analysis of this design can identify significant factors affecting soluble expression. For example, one study found that induction at mid-exponential phase gave higher cell growth than induction at early exponential phase .

Additional solubility enhancement strategies:

• Fusion partners:

  • Thioredoxin (TrxA)

  • Maltose-binding protein (MBP)

  • N-utilization substance A (NusA)

  • Small ubiquitin-like modifier (SUMO)

• Co-expression with chaperones:

  • GroEL/GroES

  • DnaK/DnaJ/GrpE

• Lysis buffer optimization:

  • Addition of 10% glycerol

  • 0.1-1% non-ionic detergents (Triton X-100, NP-40)

  • 50-300 mM arginine

Through systematic testing of these conditions, researchers have achieved up to 250 mg/L of soluble protein with 75% homogeneity for difficult-to-express proteins .

How do mutations in SsuB2 affect aliphatic sulfonate transport and what techniques can characterize these effects?

Mutations in SsuB2 can significantly impact aliphatic sulfonate transport through various mechanisms. Research approaches to study these effects include:

Site-directed mutagenesis targets:

• Conserved motifs:

  • Walker A motif (P-loop): Typically G-x-x-G-x-G-K-T/S, essential for ATP binding

  • Walker B motif: Contains catalytic glutamate for ATP hydrolysis

  • C-loop (signature motif): L-S-G-G-Q, distinguishes ABC transporters

  • Q-loop: Contains glutamine that coordinates Mg²⁺ in ATP binding

• Transmission interface:

  • Residues interacting with SsuC transmembrane domain

  • Regions involved in conformational changes during transport cycle

Functional characterization techniques:

• Transport assays:

  • Radiolabeled substrate uptake in reconstituted systems

  • Fluorescent substrate analogs for real-time monitoring

  • Growth complementation of sulfur auxotrophs

• ATPase activity correlation:

  • Wild-type vs. mutant ATP hydrolysis rates using methods from FAQ #5

  • Substrate-stimulated ATPase activity measurements

  • ATP hydrolysis:transport coupling efficiency determination

• Structural analysis:

  • X-ray crystallography of mutant proteins (resolution ~1.75Å)

  • Hydrogen/deuterium exchange mass spectrometry

  • Molecular dynamics simulations of conformational changes

Example findings from analogous systems:
For the related ABC transporter PCAT1, cryo-EM studies revealed that ATP binding causes conformational changes that reorient the transmembrane tunnel toward the extracellular space, which releases the core peptide and allows for dissociation of the peptidase domain . Similar mechanisms likely exist for SsuB2, and mutations in key residues would disrupt this conformational cycling.

What approaches can be used to study the regulation of SsuB2 expression under varying environmental conditions?

Understanding the regulation of SsuB2 expression requires a combination of molecular, biochemical, and systems biology approaches:

Transcriptional regulation analysis:

• Promoter analysis tools:

  • Reporter gene fusions (lacZ, gfp)

  • Electrophoretic mobility shift assays (EMSAs) with purified regulators

  • DNase I footprinting to identify protein binding sites

  • ChIP-seq for genome-wide regulator binding profiles

• Transcriptome analysis:

  • RNA-seq to compare expression under varying sulfur conditions

  • qRT-PCR for targeted gene expression quantification

  • Microarray analysis for global expression profiling

Environmental condition parameters to test:

Integration with metabolic networks:

• Metabolomic approaches:

  • Targeted LC-MS/MS analysis of sulfur metabolites

  • Untargeted metabolomics to identify novel regulatory metabolites

  • Flux analysis using labeled sulfur compounds

• Systems biology integration:

  • Construction of genome-scale metabolic models

  • In silico prediction of regulatory effects

  • Validation through gene knockouts and complementation

Recent studies have identified connections between sulfur metabolism, iron uptake, and oxidative stress response pathways , suggesting complex regulatory networks governing SsuB2 expression that can be mapped using these approaches.

How can researchers investigate the role of SsuB2 in bacterial adaptation to sulfur-limited environments?

Investigating SsuB2's role in adaptation to sulfur limitation requires a multifaceted approach combining genetics, physiology, and evolutionary perspectives:

Genetic approaches:

• Knockout studies:

  • Generation of ΔssuB2 deletion mutants

  • Complementation with wild-type and mutant alleles

  • Construction of conditional expression systems (inducible promoters)

• Competition assays:

  • Co-culture of wild-type and mutant strains under sulfur limitation

  • Quantification of relative fitness over multiple generations

  • Whole genome sequencing to identify compensatory mutations

Physiological characterization:

• Growth kinetics:

  • Measurement of growth rates in varying sulfur sources

  • Determination of sulfonate uptake rates

  • Lag phase analysis during adaptation to new sulfur sources

• Sulfur uptake profiling:

  • Comparative analysis of various aliphatic sulfonates as substrates

  • Kinetic parameters (Km, Vmax) for different substrates

  • Transport rates under various environmental conditions

Evolutionary approaches:

• Experimental evolution:

  • Long-term cultivation under sulfur limitation

  • Sequencing of evolved populations

  • Characterization of SsuB2 mutations that emerge

• Comparative genomics:

  • Analysis of SsuB2 conservation across bacterial species

  • Correlation with ecological niches and sulfur availability

  • Identification of co-evolving components in the sulfur utilization pathway

Proteomics integration:

Proteomic analysis has revealed that when bacteria encounter sulfur limitation, they express a suite of proteins related to aliphatic sulfonate utilization. In a study examining Pseudomonas putida exposed to different carbon sources, researchers identified differential expression of proteins involved in:

• Oxidation-reduction processes

• Metabolic pathways

• Aldehyde dehydrogenase activity

• Carbon storage

• Chemotaxis

• Amino acid synthesis

This suggests that SsuB2 functions as part of a coordinated response to nutrient limitation that extends beyond simply importing alternative sulfur sources.

What techniques are most effective for studying the interaction between SsuB2 and potential inhibitors for antimicrobial development?

Investigating SsuB2 as an antimicrobial target requires robust methods to characterize protein-inhibitor interactions:

High-throughput screening approaches:

• ATPase activity inhibition assays:

  • Adaptation of ATP hydrolysis assays from FAQ #5 for 96/384-well format

  • Z-factor determination for assay quality

  • Counterscreening against human ABC transporters for selectivity

• Fragment-based screening:

  • Differential scanning fluorimetry (thermal shift)

  • Surface plasmon resonance (SPR)

  • NMR-based fragment screening

Structure-based design methods:

• Computational approaches:

  • Molecular docking to nucleotide-binding domain

  • Molecular dynamics simulations of protein-inhibitor complexes

  • Virtual screening of compound libraries

• Structural characterization:

  • X-ray crystallography of SsuB2-inhibitor complexes

  • Cryo-EM studies of full transporter with bound inhibitors

  • Hydrogen/deuterium exchange mass spectrometry

Inhibitor optimization tools:

• Medicinal chemistry approaches:

  • Structure-activity relationship (SAR) studies

  • Binding pocket analysis for rational design

  • Fragment growing/linking/merging strategies

• Mechanism of action studies:

  • Competitive vs. non-competitive inhibition determination

  • ATP binding vs. hydrolysis inhibition discrimination

  • Conformational trapping mechanisms

Antimicrobial evaluation:

• Microbiological testing:

  • Minimum inhibitory concentration (MIC) determination

  • Activity in sulfur-limited media

  • Resistance development monitoring

• Target validation:

  • Activity against SsuB2 overexpression strains

  • Reduced efficacy in ΔssuB2 strains

  • Synergy with other antibiotics targeting sulfur metabolism

Targeting SsuB2 could be particularly effective against bacteria in sulfur-limited environments, such as those found in certain infection sites. The essentiality of the SsuABC system increases in these environments, potentially providing a selective antibacterial strategy that exploits specific niche conditions.

Bibliography

• Stratigopoulos, G., et al. "Structure of the aliphatic sulfonate-binding protein SsuA from Escherichia coli." Protein Science (2010): 1.75 Å resolution structure in substrate-free state.

• AAA Biotech. "Aliphatic sulfonates import ATP-binding protein SsuB 2 (ssuB2) Recombinant Protein." Product information for recombinant Pseudomonas syringae pv. syringae protein.

• Eichhorn, E., et al. "Identification of novel bacterial sulfur-regulated genes." Journal of Bacteriology (2000): Gene expression under sulfur limitation.

• Knerr, P.J., et al. "Proteases Involved in Leader Peptide Removal during RiPP Biosynthesis." ACS Bio & Med Chem Au (2023): ABC transporter domain functions.

• Maunders, E.A. "The impact of a single nucleotide polymorphism in fusA1 on biofilm formation and virulence in Pseudomonas aeruginosa." Doctoral dissertation (2022).

• Experimental design approaches in recombinant protein expression. Protein Expression and Purification (2014): Statistical strategies for optimizing protein expression.

• Castro, D.P. "Biodegradation studies of petroleum compounds by bacteria isolated from contaminated sites in the Amazon." Doctoral thesis, Federal University of Amazonas (2022).

• Paulo, A. "Anaerobic degradation of anionic surfactants by denitrifying bacteria." PhD thesis, Wageningen University (2014): Bacterial adaptation to alternative sulfur sources.