Recombinant Gloeobacter violaceus Sulfate/thiosulfate import ATP-binding protein CysA (cysA)

What is the function of CysA protein in Gloeobacter violaceus?

CysA in G. violaceus functions as the ATP-binding subunit of the sulfate transport system (SulT family). This protein couples the transport of sulfate with ATP hydrolysis, working together with the periplasmic sulfate binding protein (Sbp) and permease components CysT and CysW to form a complete sulfate transport channel across the cytoplasmic membrane . The CysA protein is especially significant in G. violaceus due to its primitive evolutionary position among cyanobacteria and the absence of thylakoid membranes, where photosynthesis occurs instead in the cytoplasmic membrane .

How is the CysA gene organized in the Gloeobacter violaceus genome?

The CysA gene in G. violaceus is part of its 4.66 Mb circular chromosome . While specific genomic context of G. violaceus CysA isn't directly detailed in the search results, comparable sulfate transporter systems like those in Synechococcus show that adjacent to cysA and transcribed in the opposite direction is a gene encoding the sulfate-binding protein (sbpA). Two other genes, cysT and cysW, encode proteins that form a channel for sulfate transport across the cytoplasmic membrane . Unlike some other organisms where CysP transporters are located in operons with genes involved in sulfate assimilation, the majority of sulfate transporter genes in sulfate-reducing microorganisms appear to be located randomly in the examined genomes, often hundreds of genes away from the sulfate reduction pathway genes .

What methods are commonly used to express recombinant CysA protein?

For expressing recombinant CysA from G. violaceus, researchers typically employ standard molecular cloning techniques:

• Gene amplification by PCR with primers containing appropriate restriction sites

• Cloning into expression vectors compatible with p15A replicon or similar plasmids

• Use of promoters like tac promoter, modified by introducing restriction sites at both ends

• Expression in host organisms such as E. coli

• Purification using affinity chromatography based on fusion tags

For example, in a similar study of carotenoid biosynthesis genes in G. violaceus, researchers constructed an expression vector by digesting the broad host range plasmid RSF1010 with XmnI and PstI, and ligating to fragments of pBSL130 containing a streptomycin-resistance gene cassette . Similar approaches could be applied to CysA, with genes generated by PCR and containing appropriate restriction sites for cloning into expression vectors.

How can single recombination and double recombination techniques be used to create CysA mutants in G. violaceus?

Creating CysA mutants in G. violaceus requires sophisticated genetic manipulation techniques:

Single Recombination Approach:

• Clone a fragment of the cysA gene into a vector containing a neomycin-resistant cassette (npt)

• Transfer the construct to G. violaceus using standard conjugation procedures

• Select single recombinant mutants based on neomycin resistance

• Confirm single recombination using Southern blot analysis with npt- and cysA-specific probes

• Evaluate phenotype in nitrogen-free media supplemented with neomycin

Double Recombination Method:

• Use the sacB method of positive selection for double recombinant mutants

• Create a fragment of the cysA gene using PCR with appropriate primers

• Disrupt the gene fragment with a neomycin-resistance cassette (npt)

• Clone the disrupted gene fragment into a suicide vector containing the sacB gene

• Transfer into G. violaceus using standard conjugation procedures

• Select ex-conjugants by resistance to neomycin, followed by selection with 8% sucrose to identify double recombinant mutants

• Confirm double recombination by Southern blot analysis

These methodologies are vital for creating stable mutants for functional characterization of CysA in G. violaceus.

What are the structural and functional differences between CysA in G. violaceus and other cyanobacteria?

The structural and functional differences of CysA in G. violaceus compared to other cyanobacteria reflect its phylogenetic distance and primitive nature:

G. violaceus represents an evolutionary branch that diverged early from the main cyanobacterial lineage, as demonstrated by molecular phylogenetic analyses . This is reflected in the unique features of many proteins, including CysA. The thylakoid-less nature of G. violaceus means the CysA protein functions in a different cellular context compared to other cyanobacteria, potentially affecting its structural requirements and interactions .

What analytical techniques are appropriate for studying the ATP binding and hydrolysis activities of recombinant CysA?

To characterize the ATP binding and hydrolysis activities of recombinant CysA from G. violaceus, researchers can employ multiple complementary techniques:

• ATP Binding Assays:

  • Isothermal Titration Calorimetry (ITC) to measure binding thermodynamics

  • Fluorescence-based assays using TNP-ATP or other fluorescent ATP analogs

  • Surface Plasmon Resonance (SPR) to determine binding kinetics

  • Filter binding assays with radiolabeled ATP

• ATP Hydrolysis Assays:

  • Colorimetric phosphate release assays (malachite green or molybdate-based)

  • Coupled enzyme assays linking ATP hydrolysis to NADH oxidation

  • Thin-layer chromatography to separate ATP from ADP and Pi

  • High-Performance Liquid Chromatography (HPLC) to quantify ATP/ADP ratios

• Structural Analysis:

  • X-ray crystallography of CysA with and without bound nucleotides

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to detect conformational changes

  • Cryo-electron microscopy (cryo-EM) of the complete transporter complex

  • Nuclear Magnetic Resonance (NMR) spectroscopy for dynamic studies

These methods would help elucidate the catalytic mechanism and the effect of mutations on CysA function, important for understanding the primitive sulfate transport system in G. violaceus.

How do single-case experimental designs apply to functional studies of G. violaceus CysA?

Single-case experimental designs (SCEDs) provide a flexible methodological framework for studying CysA function with limited resources:

Application to CysA research:

• Reversal Designs (A-B-A):

  • Phase A: Baseline measurement of sulfate uptake in wild-type G. violaceus

  • Phase B: Introduction of recombinant CysA with specific mutations

  • Return to Phase A: Complementation with wild-type CysA

  • This approach allows evaluation of causality between specific CysA mutations and altered sulfate transport

• Multiple Baseline Designs:

  • Implement CysA mutations across different strains or conditions at staggered timepoints

  • Measure multiple dependent variables (ATP hydrolysis, sulfate transport, growth rate)

  • This controls for time-dependent variables affecting CysA function

• Combined Designs:

  • Integrate multiple baseline and reversal designs for robust experimental control

  • Use different CysA variants across multiple experimental preparations

  • This approach increases internal validity of findings on structure-function relationships

SCEDs are particularly valuable for CysA studies because they accommodate the challenges of working with the primitive cyanobacterium G. violaceus, where growth conditions and genetic manipulation may be difficult to standardize across large sample sizes.

What is known about the interaction between CysA and drug resistance markers in genetic manipulation of G. violaceus?

The interaction between CysA and drug resistance markers is crucial for genetic manipulation of G. violaceus:

When creating CysA mutants in cyanobacteria, neomycin resistance cassettes (npt) are commonly used as selectable markers. These markers can be inserted into the cysA gene through single or double recombination events . The position and orientation of the resistance cassette relative to the cysA gene can affect:

• Transcriptional polarity - The resistance gene may disrupt downstream gene expression if inserted in the same orientation

• Protein integrity - Depending on insertion site, truncated or fusion proteins may be produced

• Regulatory effects - Promoters in resistance cassettes may influence nearby gene expression

When studying CysA function in sulfate transport, researchers must consider how the resistance marker might alter cellular physiology beyond simply disrupting CysA. In related studies with cysA mutants in Synechococcus, strains in which cysA was interrupted by a drug resistance marker were not viable when grown with sulfate as the sole sulfur source and exhibited essentially no sulfate uptake . This indicates that proper experimental design must account for the markers' metabolic burden and potential polar effects.

What are the best approaches for homology modeling of G. violaceus CysA protein structure?

Homology modeling of G. violaceus CysA requires a systematic approach:

• Template Selection:

  • Identify ATP-binding cassette (ABC) proteins with solved structures in the Protein Data Bank

  • Prioritize templates with high sequence identity to G. violaceus CysA

  • Consider both bacterial sulfate transporters and related ABC transporters

  • Evaluate the resolution and quality of potential template structures

• Sequence Alignment and Model Building:

  • Perform multiple sequence alignments using CLUSTAL W or similar tools

  • Pay special attention to conserved motifs in ABC transporter proteins

  • Generate multiple models using software like MODELLER, SWISS-MODEL, or I-TASSER

  • Apply knowledge-based constraints focusing on ATP-binding residues

• Model Validation:

  • Evaluate stereochemical quality using PROCHECK or MolProbity

  • Perform energy minimization to resolve structural clashes

  • Validate with tools like ERRAT, VERIFY3D, and ProSA

  • Compare models to experimental data when available

• Refinement and Analysis:

  • Refine models through molecular dynamics simulations

  • Identify key functional residues like SER41, GLY42, ARG50, GLN85, HIS86, LYS91, ARG142, and ASP161 that are crucial for ATP binding

  • Analyze protein-protein interactions through docking methods to understand conformational changes

  • Predict functional effects of mutations based on the structural model

This approach has been successfully applied to predict structures of related CysA proteins, as demonstrated in research on Mycobacterium tuberculosis CysA .

How can researchers effectively compare G. violaceus CysA with homologs from other cyanobacteria?

Effective comparison of G. violaceus CysA with homologs requires a multi-faceted approach:

• Sequence-Based Analysis:

  • Generate comprehensive multiple sequence alignments using CLUSTAL W

  • Perform BLASTP searches against databases like GenBank and Swiss-Prot

  • Calculate conservation scores for each amino acid position

  • Identify signature motifs unique to G. violaceus CysA versus other cyanobacteria

  • Construct phylogenetic trees to visualize evolutionary relationships

• Structural Comparison:

  • Generate homology models for all CysA proteins being compared

  • Superimpose structures to identify conserved and divergent regions

  • Analyze ATP-binding pockets for differences in geometry and electrostatics

  • Examine protein surface properties that might affect interaction with CysT and CysW

• Functional Assays:

  • Design complementation experiments where G. violaceus CysA is expressed in other cyanobacterial strains with cysA mutations

  • Measure sulfate transport rates with radioactive sulfate uptake assays

  • Compare ATP hydrolysis kinetics (Km, Vmax, catalytic efficiency)

  • Evaluate protein-protein interactions with CysT, CysW, and Sbp components

• Genomic Context Analysis:

  • Map the organization of sulfate transport genes across cyanobacterial genomes

  • Analyze promoter regions for differential regulation

  • Determine operon structures and potential co-regulated genes

  • Identify horizontal gene transfer events that might explain unique features

This comprehensive approach would highlight the evolutionary adaptations of CysA in the primitive G. violaceus compared to more evolved cyanobacteria.

How should researchers interpret discrepancies in CysA function between in vivo and in vitro studies?

When confronting discrepancies between in vivo and in vitro studies of G. violaceus CysA, researchers should:

• Consider Cellular Context:

  • G. violaceus lacks thylakoid membranes, with photosynthesis occurring in the cytoplasmic membrane

  • This unique cellular architecture may create a different microenvironment for CysA function

  • In vitro studies might not replicate the specific membrane composition and protein interactions

• Evaluate Experimental Conditions:

  • Compare buffer compositions, pH, ionic strength, and temperature between studies

  • Assess whether the recombinant CysA contains the correct post-translational modifications

  • Consider if the protein's oligomeric state matches the native form

• Analyze Protein-Protein Interactions:

  • CysA functions as part of a complex with CysT, CysW, and Sbp components

  • In vitro studies might lack essential interacting partners

  • Perform pull-down assays or co-immunoprecipitation to identify missing interaction partners

• Apply Complementary Methodologies:

  • Use reconstituted systems like proteoliposomes to bridge in vitro and in vivo approaches

  • Conduct site-directed mutagenesis to validate specific mechanistic hypotheses

  • Employ single-cell techniques to observe heterogeneity in cellular responses

• Statistical Framework for Reconciliation:

  • Develop statistical models that account for both in vitro and in vivo observations

  • Consider Bayesian approaches to update hypotheses based on combined evidence

  • Perform meta-analysis when multiple studies are available

This systematic approach helps distinguish between true biological differences and experimental artifacts in CysA function.

What bioinformatic tools are most appropriate for analyzing G. violaceus CysA evolution?

For evolutionary analysis of G. violaceus CysA, researchers should employ specialized bioinformatic tools:

• Sequence Retrieval and Alignment:

  • Database resources: NCBI, CyanoBase, UniProt, and specialized cyanobacterial databases

  • Alignment tools: MUSCLE, MAFFT, or T-Coffee for improved alignment of divergent sequences

  • Visualization software: Jalview or AliView for alignment inspection and editing

• Phylogenetic Analysis:

  • Maximum Likelihood methods: RAxML or IQ-TREE for robust phylogenetic inference

  • Bayesian approaches: MrBayes or BEAST for estimating divergence times

  • Model selection tools: ProtTest or ModelFinder to identify appropriate substitution models

  • Tree visualization: FigTree, iTOL, or EvolView for presenting evolutionary relationships

• Detection of Selection and Functional Divergence:

  • Selection analysis: PAML, HyPhy, or MEME for detecting positive selection

  • Coevolution analysis: CAPS or PSICOV to identify co-evolving residues

  • Rate-shift analysis: DIVERGE to detect functional divergence between CysA clades

  • Ancestral sequence reconstruction: FastML or PAML to infer ancestral CysA sequences

• Comparative Genomics:

  • Synteny analysis: SynMap or Mauve to compare genomic context of cysA genes

  • Pan-genome analysis: GET_HOMOLOGUES or Roary to define core and accessory genes

  • Horizontal gene transfer detection: IslandViewer or Alien_Hunter

  • Genome visualization: CGView or GenoPlotR for contextual visualization

G. violaceus has been shown to branch off from the main cyanobacterial tree at an early stage of evolution, making it a key organism for understanding the evolution of various cellular processes, including sulfate transport mechanisms .

How should researchers design experiments to study the effect of G. violaceus CysA mutations on sulfate transport?

A comprehensive experimental design for studying CysA mutations should include:

• Mutation Strategy:

  • Structure-guided mutations targeting:

    • ATP binding pocket residues (e.g., SER41, GLY42, ARG50)

    • Transmembrane interface regions for interaction with CysT/CysW

    • Conserved motifs identified through multiple sequence alignments

  • Scanning mutagenesis (alanine scanning) of regions with unknown function

  • Conservative and non-conservative substitutions to establish structure-function relationships

• Expression Systems:

  • Homologous expression in G. violaceus using methods similar to those used for gene inactivation

  • Heterologous expression in model cyanobacteria with cysA knockouts

  • E. coli-based expression systems for high-yield protein production

  • Cell-free protein synthesis for problematic mutants

• Functional Assays:

  • In vivo measurements:

    • Growth rates with sulfate as sole sulfur source

    • Radioactive sulfate (35S) uptake assays

    • Membrane potential measurements during transport

  • In vitro characterization:

    • ATP binding affinity using isothermal titration calorimetry

    • ATPase activity with malachite green phosphate assays

    • Reconstitution in proteoliposomes for transport studies

• Controls and Validation:

  • Wild-type CysA as positive control

  • Known non-functional ABC transporter mutants as negative controls

  • Complementation assays to verify phenotype rescue

  • Western blotting to confirm expression levels

  • Structural integrity verification through circular dichroism or thermal shift assays

• Data Analysis Framework:

  • Statistical comparison of transport rates and kinetic parameters

  • Structure-activity relationship modeling

  • Classification of mutations by phenotypic severity

  • Integration with homology models and molecular dynamics simulations

This design allows for robust characterization of how specific residues contribute to CysA function in G. violaceus.

What considerations are important when developing a high-throughput screening system for G. violaceus CysA inhibitors?

Developing an effective high-throughput screening system for G. violaceus CysA inhibitors requires careful consideration of multiple factors:

• Assay Development:

  • Primary screening assays:

    • ATP hydrolysis assays using colorimetric phosphate detection

    • Fluorescence-based nucleotide binding assays (e.g., FRET)

    • Bioluminescent ADP detection assays (e.g., ADP-Glo)

  • Secondary confirmation assays:

    • Sulfate transport assays in proteoliposomes

    • Thermal shift assays to confirm direct binding

    • Surface plasmon resonance for binding kinetics

• Protein Production Strategy:

  • Optimized expression in E. coli with suitable fusion tags

  • Purification protocols maintaining native conformation

  • Quality control metrics for batch consistency

  • Stability assessment under screening conditions

• Compound Library Design:

  • Focused libraries targeting ATP-binding pockets

  • Natural product collections, particularly from cyanobacteria

  • Fragment-based approaches for novel scaffolds

  • Virtual screening pre-selection based on docking to CysA homology models

• Screening Parameters Optimization:

  • Buffer composition mimicking physiological conditions

  • DMSO tolerance assessment

  • Signal-to-background ratio optimization

  • Z-factor determination for assay quality control

  • Positive and negative controls for normalization

• Data Analysis and Hit Selection:

  • Statistical methods for hit identification

  • Dose-response curve analysis for potency determination

  • Structure-activity relationship studies

  • Chemoinformatic clustering of active compounds

  • Counter-screening against mammalian ABC transporters for selectivity

• Follow-up Studies:

  • Mechanism of action studies

  • Cellular activity verification

  • Antimicrobial activity assessment

  • Structure optimization of promising scaffolds

  • In silico ADME prediction for lead compounds

This approach has been successfully applied to related ABC transporters, as demonstrated in research on Mycobacterium tuberculosis CysA as a drug target .