Recombinant Shewanella sediminis Cysteine desulfurase (iscS)

Basic Research Questions

• What is Recombinant Shewanella sediminis Cysteine desulfurase (iscS) and what is its biological function?

  Recombinant Shewanella sediminis Cysteine desulfurase (iscS) is an enzyme (EC 2.8.1.7) extracted from Shewanella sediminis strain HAW-EB3 that catalyzes the conversion of L-cysteine to L-alanine and sulfane sulfur. This protein consists of 404 amino acids with a specific sequence as detailed in product information sheets . The enzyme serves as a critical sulfur donor for the biosynthesis of iron-sulfur (Fe-S) clusters, which function as essential cofactors for numerous proteins involved in electron transfer, metabolic processes, and gene regulation. Studies with similar cysteine desulfurases in E. coli have demonstrated that IscS plays a major role in the formation of Fe-S clusters in both soluble and membrane-bound proteins .

• How should researchers properly store and reconstitute Recombinant S. sediminis Cysteine desulfurase?

  For optimal stability and activity, Recombinant S. sediminis Cysteine desulfurase should be stored at -20°C, or at -80°C for extended storage periods . Before opening, briefly centrifuge the vial to ensure contents are at the bottom. The protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL. To prevent activity loss during storage, add glycerol to a final concentration of 5-50% (with 50% being the default recommendation) and aliquot for long-term storage at -20°C/-80°C . Repeated freezing and thawing cycles should be avoided. The shelf life in liquid form is approximately 6 months, while the lyophilized form remains stable for up to 12 months when stored properly .

• What experimental approaches can verify the activity of recombinant IscS?

  Multiple complementary approaches can verify IscS activity. The primary assay measures the enzyme's ability to convert cysteine to alanine and release sulfur, detectable through colorimetric sulfide detection methods. Coupled enzyme assays can track the transfer of sulfur to scaffold proteins like IscU and subsequent Fe-S cluster formation using UV-visible spectroscopy to monitor characteristic absorption peaks. Functional complementation assays in iscS-deficient E. coli strains can demonstrate in vivo activity, as these mutants show decreased activities of Fe-S cluster-containing enzymes like aconitase B, 6-phosphogluconate dehydratase, and glutamate synthase . Additionally, researchers can monitor PLP cofactor binding (essential for activity) through absorbance at 390-420 nm. Mass spectrometry can detect the formation of enzyme-bound persulfide intermediates, providing direct evidence of the desulfurase reaction.

• How does IscS deletion affect bacterial growth and metabolism?

  IscS deletion creates profound metabolic deficiencies in bacteria. In E. coli, iscS mutants exhibit a small colony phenotype and significantly slower growth rates (doubling time of 62 minutes versus 31 minutes for wild-type in LB medium) . These mutants cannot grow in glucose minimal medium unless supplemented with thiamine and nicotinic acid, highlighting IscS's role in cofactor biosynthesis . The growth defects stem from decreased activities of Fe-S cluster-containing enzymes critical for central metabolism, including aconitase B, 6-phosphogluconate dehydratase, glutamate synthase, fumarase A, and NADH dehydrogenase I . Notably, the thiamine requirement disappears under anaerobic conditions, suggesting differential metabolic requirements for IscS depending on oxygen availability . These findings indicate that while not absolutely essential for viability in E. coli (unlike in some other organisms), IscS is crucial for normal growth and metabolic function.

• What are the structural features of S. sediminis IscS protein?

  S. sediminis IscS is a PLP-dependent enzyme with several key structural features. The full-length protein consists of 404 amino acids with a sequence beginning with MKLPIYLDYA and ending with WAHH . Like other cysteine desulfurases, it likely adopts a fold consisting of a large PLP-binding domain and a smaller domain containing the active site cysteine residue. The enzyme contains a conserved active site where PLP forms a Schiff base with a lysine residue, and a mobile loop housing the catalytic cysteine that accepts the sulfur from the substrate. Analysis of the amino acid sequence reveals key functional regions including the PLP binding site, substrate binding pocket, and regions involved in protein-protein interactions with Fe-S cluster assembly partners. The enzyme likely functions as a homodimer, with each monomer containing one active site, as observed in related cysteine desulfurases. Its relatively high stability under standard storage conditions suggests a compact, well-folded structure resistant to denaturation .

Advanced Research Questions

• How does S. sediminis IscS contribute to the organism's ecological adaptation in marine environments?

  S. sediminis IscS plays a crucial role in the organism's adaptation to marine environments through its contribution to Fe-S cluster biosynthesis, which supports the species' unique metabolic capabilities. As a benthic species, S. sediminis HAW-EB3 faces distinct challenges in sediment environments, including fluctuating redox conditions and potential metal stress . The IscS enzyme enables proper functioning of Fe-S proteins involved in denitrification and metal reduction, processes that allow Shewanella to utilize alternative electron acceptors when oxygen is limited in marine sediments. In experimental studies, S. sediminis HAW-EB3 demonstrated lower growth rates compared to other Shewanella species and became undetectable in later stages of evolution experiments , suggesting specialized metabolic adaptations that may involve differential regulation of Fe-S cluster assembly. The species' persistence in marine sediments likely depends on IscS function to support respiratory flexibility across environmental gradients, allowing it to occupy ecological niches with variable oxygen and nutrient availability. This adaptation may explain why S. sediminis expresses a functionally optimized IscS variant suited to its specific ecological context.

• What methodologies should be employed to investigate interactions between IscS and partner proteins in the Fe-S cluster assembly pathway?

  A comprehensive investigation of IscS interactions requires multiple complementary approaches. Initially, researchers should conduct pull-down assays using tagged recombinant S. sediminis IscS to identify potential binding partners, followed by validation through co-immunoprecipitation with specific antibodies. Binding kinetics and thermodynamics can be quantified using surface plasmon resonance or isothermal titration calorimetry. Protein-protein interaction surfaces can be mapped using hydrogen-deuterium exchange mass spectrometry or chemical crosslinking coupled with mass spectrometry. For structural characterization, X-ray crystallography or cryo-electron microscopy of IscS-partner complexes would provide atomic-level details of interaction mechanisms. Functional validation can be achieved through enzyme assays measuring sulfur transfer from IscS to scaffold proteins. In vivo approaches include bacterial two-hybrid systems or fluorescence resonance energy transfer to confirm interactions in a cellular context. Site-directed mutagenesis of predicted interface residues can validate structural models and identify key interaction determinants. This multi-method approach would generate a comprehensive interaction map of S. sediminis IscS with its partner proteins in the Fe-S cluster assembly pathway.

• How does IscS function impact denitrification processes in Shewanella species?

  IscS function significantly impacts denitrification in Shewanella species through its essential role in biosynthesizing Fe-S clusters for key denitrification enzymes. Synthetic denitrifying communities (SDCs) composed of Shewanella species show positive correlations between species richness and denitrification rates , suggesting complementary metabolic capabilities dependent on properly functioning Fe-S proteins. Many enzymes in the denitrification pathway (including nitrate reductase, nitrite reductase, and nitric oxide reductase) require Fe-S clusters for electron transfer. In experimental evolution studies with Shewanella SDCs, denitrification capacity changed over time, potentially reflecting altered Fe-S cluster assembly efficiency . S. sediminis HAW-EB3, as a benthic species, showed lower persistence in these communities compared to other Shewanella species , which might indicate differences in its denitrification apparatus, including IscS function. To directly assess IscS's role in denitrification, researchers should compare the activities of Fe-S-containing denitrification enzymes between wild-type and iscS-deficient strains under various oxygen conditions while measuring the conversion rates of nitrate, nitrite, NO, and N₂O along the denitrification pathway.

• What impact does the loss of IscS function have on Fe-S cluster-containing proteins, and how can this be experimentally measured?

  Loss of IscS function profoundly affects Fe-S cluster-containing proteins through impaired cofactor assembly. Studies in E. coli demonstrate that iscS deletion causes decreased activities of multiple [4Fe-4S] cluster-containing enzymes, including aconitase B (87% reduction), 6-phosphogluconate dehydratase (69% reduction), glutamate synthase (56% reduction), and fumarase A (77% reduction) . Membrane-bound Fe-S proteins like NADH dehydrogenase I and succinate dehydrogenase also show significant activity decreases . To experimentally measure these effects, researchers should employ enzyme activity assays specific to each Fe-S protein, comparing wild-type and iscS-deficient strains. The integrity of Fe-S clusters can be directly assessed using electron paramagnetic resonance (EPR) spectroscopy, which provides information about cluster oxidation state and environment. UV-visible spectroscopy can track characteristic absorption peaks of Fe-S clusters (typically 300-500 nm). Iron and sulfide content can be quantified using colorimetric assays. Protein stability and expression should be monitored through western blotting to distinguish between effects on cluster assembly versus protein levels. Critically, researchers should include a control with an Fe-S independent protein variant, as demonstrated with FNR in E. coli studies , to confirm the specificity of observed effects to Fe-S cluster formation.

• How might S. sediminis IscS function be involved in the organism's response to environmental stressors?

  S. sediminis IscS likely plays a pivotal role in the organism's response to environmental stressors through its central position in Fe-S cluster homeostasis. Under oxidative stress, Fe-S clusters are primary targets for damage, necessitating efficient repair mechanisms dependent on IscS activity. In temperature fluctuations common in marine environments, IscS maintains the functionality of temperature-sensitive metabolic pathways containing Fe-S proteins. During metal stress, prevalent in sediments where Shewanella species thrive, IscS contributes to the assembly of Fe-S clusters in detoxification systems. The metabolic flexibility of Shewanella across varying redox conditions depends on properly functioning electron transport chains rich in Fe-S clusters . To investigate these roles, researchers should measure IscS expression and activity under defined stressors, correlating these with cellular Fe-S protein function and stress response outcomes. Particularly informative would be comparisons of wild-type and iscS-deficient strains for survival rates, growth kinetics, and transcriptomic profiles under relevant stressors. These experiments should incorporate environmentally realistic conditions reflecting the sediment habitats where S. sediminis naturally occurs .

• What role might IscS play in antibiotic resistance mechanisms observed in Shewanella species?

  IscS may significantly influence antibiotic resistance in Shewanella species through several mechanisms linked to Fe-S cluster biosynthesis. Recent studies have identified Shewanella as both a vehicle and potential progenitor of antibiotic resistance genes, with some species harboring multidrug-resistant plasmids containing resistance genes associated with mobile genetic elements . Fe-S cluster-containing proteins are critical components of several processes that impact antibiotic susceptibility, including efflux pump function, DNA repair, and stress response. Many antibiotics generate oxidative stress as part of their killing mechanism, which damages Fe-S clusters and may trigger compensatory responses requiring IscS activity. Additionally, certain antibiotics directly target processes dependent on Fe-S proteins, such as DNA replication and protein synthesis. To investigate this connection, researchers should compare antibiotic susceptibility profiles of wild-type and iscS-deficient Shewanella strains across different drug classes, correlate Fe-S protein activities with resistance phenotypes, and examine whether IscS inhibition potentiates antibiotic effects. The research should also investigate whether horizontal gene transfer of resistance determinants is affected by IscS function, given Shewanella's emerging role in resistance gene dissemination .

• How do different Shewanella species compare in terms of IscS sequence, structure, and function?

  Comparative analysis of IscS across Shewanella species reveals informative variations reflecting evolutionary adaptations to diverse ecological niches. While the catalytic core and PLP-binding regions show high conservation consistent with the enzyme's fundamental role, peripheral regions display greater variability that may influence substrate specificity, protein-protein interactions, or regulatory mechanisms. Shewanella species occupy various aquatic environments ranging from deep-sea to freshwater, with corresponding metabolic adaptations. S. sediminis, as a benthic species isolated from sediments, displays different growth characteristics compared to other Shewanella species in experimental settings . This ecological specialization likely correlates with functional optimizations in its Fe-S cluster assembly machinery, including IscS. In synthetic communities, S. sediminis HAW-EB3 showed lower persistence compared to species like S. marisflavi EP1 and S. loihica PV-4, which dominated the communities by the 180th day of experiments . This competitive disadvantage may reflect differences in metabolic efficiency potentially linked to variations in Fe-S protein function. To experimentally characterize these differences, researchers should conduct comprehensive enzyme kinetic analyses of IscS from multiple Shewanella species under standardized conditions, coupled with structural studies and in vivo complementation experiments.

• What experimental design would best elucidate the specific role of IscS in biodiversity-ecosystem functioning relationships in microbial communities?

  An optimal experimental design to elucidate IscS's role in biodiversity-ecosystem functioning (BEF) relationships would build upon established synthetic community approaches while incorporating genetic manipulation of iscS. Researchers should construct synthetic denitrifying communities (SDCs) with wild-type and iscS-modified Shewanella strains at varying richness levels (1, 2, 4, 8, and 12 species), similar to previous studies . The iscS modifications should include complete knockouts, conditional expression systems, and variants with site-directed mutations affecting activity. Community function should be assessed through multiple metrics including growth (OD600), denitrification rates (nitrate reduction), and carbon utilization patterns . The experiment should track community composition over time using metagenomic sequencing or species-specific qPCR to monitor relative abundances, as previous work showed significant shifts with species like S. marisflavi EP1 becoming dominant (99.3%) by day 180 . Key innovations should include: 1) measuring activities of specific Fe-S proteins across community members to link IscS function directly to ecosystem processes; 2) incorporating environmental stressors to test how IscS affects community resilience; and 3) analyzing meta-transcriptomics to identify compensatory mechanisms when IscS function is compromised. This comprehensive approach would mechanistically connect Fe-S cluster assembly to the positive BEF relationships observed in microbial communities .

Data adapted from research on E. coli iscS mutants