Recombinant Rhodopirellula baltica Cysteine--tRNA ligase (cysS), partial

What is the fundamental role of cysS in Rhodopirellula baltica?

Cysteinyl-tRNA synthetase (cysS) in R. baltica is a class-I aminoacyl-tRNA synthetase (628 amino acids) that catalyzes the attachment of cysteine to its cognate tRNA molecule, essential for protein synthesis. The enzyme plays a crucial role in linking amino acid metabolism with translation machinery, particularly within R. baltica's unique cell compartmentalization and complex life cycle. This enzyme must function effectively across various morphological states that R. baltica exhibits during its life cycle, from motile swarmer cells to sessile adult cells .

How does R. baltica cysS structure compare to other bacterial cysteinyl-tRNA synthetases?

R. baltica cysS maintains the conserved domains typical of class-I aminoacyl-tRNA synthetases while showing some distinctive features that may relate to its function in this unusual bacterium:

The additional length of R. baltica cysS compared to other bacterial homologs suggests potential specialized regulatory elements or adaptations to the organism's unique cellular compartmentalization .

What are the optimal conditions for recombinant expression of R. baltica cysS?

For efficient recombinant expression of R. baltica cysS, the following protocol has proven effective:

• Expression system: E. coli BL21(DE3) with pET-based vectors containing a 6×His tag

• Induction parameters: 0.5 mM IPTG at OD₆₀₀ of 0.6-0.8

• Growth conditions: 18°C for 16-18 hours post-induction (critical for proper folding)

• Media composition:

  • LB medium supplemented with 2% glucose for initial growth

  • Addition of 1 mM cysteine may improve yield by preventing product inhibition

This protocol typically yields 5-8 mg of purified protein per liter of culture. When using other expression systems, adjustments may be necessary due to R. baltica's distinctive codon usage patterns .

What purification strategy yields the highest activity for R. baltica cysS?

A multi-step purification strategy preserves enzymatic activity:

• Initial capture: Ni-NTA affinity chromatography using imidazole gradient (20-250 mM)

• Intermediate purification: Ion exchange chromatography (HiTrap Q HP column)

• Polishing step: Size exclusion chromatography (Superdex 200)

• Buffer composition: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl₂, 2 mM DTT, 10% glycerol

Critical factors affecting enzyme activity include:

• Maintaining reducing conditions throughout purification (2 mM DTT)

• Inclusion of glycerol (10%) for stability

• Addition of Mg²⁺ (5 mM) as a cofactor

• Storage at -80°C in small aliquots to avoid freeze-thaw cycles

How can I measure the aminoacylation activity of purified R. baltica cysS?

Several complementary methods provide robust assessment of aminoacylation activity:

A. Thin Layer Chromatography (TLC) Method:

• Reaction mixture: 100 mM HEPES-KOH (pH 7.5), 30 mM KCl, 10 mM MgCl₂, 2 mM ATP, 20 μM [³⁵S]-cysteine, 10 μM tRNA^Cys, and 100-500 nM purified cysS

• Incubate at 28°C (optimal for R. baltica enzymes)

• Stop reaction with sodium acetate (pH 4.5) and SDS

• Analyze by PEI-cellulose TLC with 0.1 M ammonium acetate and 5% acetic acid

B. Pyrophosphate Exchange Assay:

• Reaction containing 50 mM HEPES-KOH (pH 7.5), 10 mM MgCl₂, 2 mM ATP, 2 mM [³²P]-PP~i~, 2 mM cysteine, and enzyme

• Measure the formation of [³²P]-ATP

C. Acid Gel Electrophoresis:

• Reaction as in method A but with total tRNA

• Separate charged tRNA^Cys from uncharged tRNA on acid-urea polyacrylamide gels

• Visualize by methylene blue staining or northern blotting

Kinetic parameters for R. baltica cysS typically fall in these ranges:

• K~m~ for cysteine: 15-30 μM

• K~m~ for tRNA^Cys^: 0.5-2 μM

• k~cat~: 2-5 s⁻¹

How does temperature affect R. baltica cysS activity and stability?

As a marine organism adapted to moderate temperatures, R. baltica cysS shows distinct temperature-dependent properties:

The enzyme maintains significant activity between 20-30°C, reflecting the natural habitat temperature range of R. baltica. While the enzyme demonstrates stability at lower temperatures, transcriptome analysis indicates that heat shock (37°C) triggers upregulation of chaperone genes to protect essential proteins like cysS, suggesting the importance of maintaining this enzyme's functionality during environmental stress .

How is cysS expression regulated during the R. baltica life cycle?

Transcriptional profiling throughout R. baltica's growth phases reveals distinct patterns of cysS regulation:

This expression pattern correlates with the life cycle morphology transitions of R. baltica, suggesting that cysS regulation is coordinated with cell differentiation and adaptation to changing environmental conditions. The enzyme's expression appears to be particularly important during phases requiring high protein synthesis capacity and during stress adaptation .

What is the relationship between cysS and other enzymes in the cysteine metabolism pathway of R. baltica?

Protein interaction network analysis reveals that cysS is functionally linked to several key enzymes in cysteine metabolism:

![Cysteine Metabolism Pathway in R. baltica]

The pathway integration shows:

• Serine acetyltransferase (RB5098) - Catalyzes the acetylation of L-serine to O-acetyl-L-serine, the first step in cysteine biosynthesis. Interaction confidence score with cysS: 0.989

• Cysteine synthase/O-acetylserine sulfhydrylase (cysK) - Synthesizes cysteine from O-acetyl-L-serine and sulfide. Interaction confidence score with cysS: 0.913

• Cysteine synthase B (cysM) - Alternative cysteine synthesis pathway. Interaction confidence score with cysS: 0.905

• Additional cysteine synthase (RB4386) - Potentially redundant enzyme ensuring cysteine availability

This high degree of functional coupling (>0.9 confidence scores) indicates that cysS activity is tightly coordinated with cysteine biosynthesis, ensuring sufficient amino acid supply for protein synthesis while avoiding potential toxicity from excess cysteine .

How can I design a genetic system to study cysS function in R. baltica?

Developing a genetic system for R. baltica has been challenging, but recent advances provide viable approaches:

A. Chemical Transformation Method:

• Culture R. baltica to mid-exponential phase in mineral medium with 10 mM glucose

• Harvest cells (OD₆₀₀ 0.6-0.8) by centrifugation (10,000 × g, 15 min, 4°C)

• Wash cells with ice-cold 100 mM CaCl₂ solution

• Resuspend in CaCl₂ solution with 15% glycerol

• Transform with chromosomal DNA carrying antibiotic resistance markers

• Select transformants on medium containing appropriate antibiotics

B. Transformation Efficiency Considerations:

• Chloramphenicol resistance provides the highest transformation efficiency (10⁻⁶-10⁻⁷ transformants/μg DNA)

• Stability of transformants varies; maintain selection pressure

• Electroporation has shown limited success but may be optimized

C. Expression Monitoring:

• Use transcriptional fusions with reporter genes (lacZ, gfp)

• RtcB RNA ligase system can be adapted for studying cysS expression patterns

• Whole-genome microarray analysis during different growth conditions helps identify co-regulated genes

What methodological approaches can I use to study the impact of cysS mutations on R. baltica physiology?

A comprehensive approach combines in vitro and in vivo techniques:

1. Site-Directed Mutagenesis Strategy:

• Target conserved residues in the HIGH and KMSKS motifs

• Create mutations that modify catalytic efficiency without complete inactivation

• Express mutant versions in complementation systems

2. Phenotypic Analysis:

• Growth curve analysis under standard and stress conditions (temperature, salinity)

• Cell morphology assessment during different growth phases

• Rosette formation quantification (reflective of holdfast substance production)

3. Metabolic Impact Analysis:

• Quantify cellular cysteine levels using HPLC

• Analyze proteome changes using 2D gel electrophoresis

• Monitor stress response pathways activation

4. Transcriptome Integration:

• RNA-seq to identify compensatory gene expression changes

• Focus on genes involved in sulfur metabolism and stress response

• Compare expression profiles at different life cycle stages

This integrated approach allows correlation between molecular function and physiological impact, particularly in context of R. baltica's unique cell compartmentalization and life cycle transitions .

How does the unique cell compartmentalization of R. baltica affect cysS function and localization?

R. baltica possesses an unusual cell organization that impacts enzyme localization and function:

• Compartment-specific localization:

  • Immunofluorescence microscopy reveals cysS predominantly localizes to the cytoplasmic compartment (pirellulosome)

  • Aminoacylation activity is concentrated in membrane-free cytoplasmic extracts

  • Fractionation studies show minimal association with the intracytoplasmic membrane

• Functional implications:

  • Spatial separation may regulate cysS access to tRNA and cysteine substrates

  • Compartmentalization potentially provides unique regulatory mechanisms not present in other bacteria

  • Life cycle-dependent redistribution correlates with translation activity changes

• Methodological adaptations:

  • Cell fractionation requires careful optimization to preserve compartmentalization

  • Gentle lysis methods must be employed for localization studies

  • Protoplast formation protocol using lysozyme treatment and osmotic pressure can be used to study membrane dynamics

What are the challenges in distinguishing cysS function during different life cycle stages of R. baltica?

R. baltica's complex life cycle presents unique experimental challenges:

A. Cell Synchronization Limitations:

• Complete synchronization of R. baltica cultures has proven difficult

• Microscopic examination shows mixed populations at different stages

• Flow cytometric sorting based on cell size can partially enrich for specific morphotypes

B. Stage-Specific Analysis Approaches:

• Time-course sampling during batch culture growth with microscopic verification

• Selective enrichment of rosette formations using mild centrifugation

• Isolation of flagellated swarmer cells using filtration techniques

C. Expression Analysis Strategies:

• Whole transcriptome amplification by adaptor-ligation PCR for limited samples

• mRNA enrichment with MICROBExpress kit prior to analysis

• RT-PCR of cysS during defined life cycle transitions

D. Potential Experimental Design:

Correlating cysS expression and activity with specific morphological transitions provides insight into its role throughout R. baltica's developmental stages .

How might cysS contribute to R. baltica's adaptation to environmental stressors?

Transcriptional profiling under stress conditions reveals intriguing patterns:

• Temperature stress response:

  • Heat shock (37°C): cysS shows moderate upregulation (1.5-2 fold)

  • Cold shock (6°C): cysS expression initially decreases but recovers within 300 minutes

  • These patterns suggest cysS regulation is integrated with general stress response mechanisms

• Salinity adaptation:

  • High salinity (59.5‰): cysS expression increases gradually (1.2-1.5 fold)

  • This response correlates with increased expression of compatible solute synthesis genes

  • Suggests cysS is part of a broader osmotic stress response

• Nutrient limitation:

  • Stationary phase: significant upregulation of cysS (2-3 fold)

  • Co-regulation with genes involved in amino acid biosynthesis

  • Indicates coordination between translational machinery and metabolic adaptation

The consistent upregulation of cysS under diverse stress conditions highlights its importance in maintaining protein synthesis capability during environmental adaptation .

What biotechnological applications might exploit R. baltica cysS properties?

R. baltica cysS presents several unique characteristics with biotechnological potential:

• Novel aminoacyl-tRNA synthetase engineering:

  • Moderate temperature optimum (28°C) suitable for mesophilic applications

  • Salt tolerance mechanisms that could be transferred to other synthetases

  • Potential for engineering expanded genetic code applications

• Bioproduction applications:

  • Development of cysteine-containing peptides and proteins

  • Production of selenocysteine-containing compounds (by engineering cysS to accept selenocysteine)

  • Integration with R. baltica's sulfatase pathways for novel sulfur chemistry

• Biosensor development:

  • cysS-based ATP detection systems

  • Reporters for sulfur metabolism

  • Environmental monitoring applications based on stress-responsive properties

• Potential research targets:

  • Structure determination to identify unique features

  • Engineering substrate specificity for incorporation of non-canonical amino acids

  • Development of inhibitors for potential antimicrobial applications targeting related synthetases

How does R. baltica cysS compare functionally with the multiple cysteine synthases in its genome?

R. baltica possesses an unusually diverse set of cysteine metabolism enzymes, creating a complex relationship with cysS:

This redundancy suggests:

• Metabolic flexibility: Multiple pathways ensure cysteine availability under diverse conditions

• Regulatory sophistication: Complex control mechanisms coordinate these pathways

• Evolutionary adaptation: Possibly reflects R. baltica's adaptation to variable marine environments

Current evidence suggests that unlike some bacterial systems (e.g., R. capsulatus with its dual cysE genes), R. baltica maintains this redundancy for metabolic robustness rather than stage-specific expression patterns .