Recombinant Vibrio vulnificus Sulfate adenylyltransferase subunit 1 (cysN)

Basic Research Questions

• What is the genomic context of cysN in Vibrio vulnificus and how does it compare between pathogenic and non-pathogenic strains?

  Vibrio vulnificus cysN is part of the sulfate assimilation pathway, encoding the GTP-binding subunit of ATP sulfurylase, which catalyzes the first step in sulfate reduction for cysteine biosynthesis. While the search results don't specifically address cysN, genomic analyses of V. vulnificus reveal that the organism contains two circular chromosomes with 4,389 potential coding sequences as observed in strain VV2018 .

  When comparing pathogenic and non-pathogenic strains, research has identified approximately 3,016 core genes present in ≥99% of V. vulnificus strains, which likely includes essential metabolic genes like cysN . Genomic comparisons between clinical (C1) and environmental (C2) strains show that they primarily differ in only ~2% of their persistent genome content, with differences mainly associated with virulence-related genes rather than core metabolic functions .

  Methodology for comparative genomics analysis of cysN:

  1. Perform whole genome sequencing of multiple V. vulnificus isolates from clinical and environmental sources

  2. Use comparative genomics tools to identify the cysN gene and its flanking regions

  3. Analyze sequence conservation, potential recombination events, and regulatory elements

  4. Correlate any variations with ecological niche or pathogenic potential

• What role might sulfur metabolism play in Vibrio vulnificus pathogenesis?

  While the search results don't directly link cysN to virulence, sulfur metabolism potentially contributes to V. vulnificus pathogenesis in several ways. Cysteine biosynthesis is critical for protein structure, redox homeostasis, and stress response - all important during host infection.

  V. vulnificus is an opportunistic pathogen that naturally inhabits warm brackish and saltwater environments, causing deadly septicemia primarily in individuals with underlying conditions such as liver disease, hemochromatosis, diabetes, or immune dysfunction . The pathogen's genome contains numerous virulence factors, with 115 core virulence factors identified across 26 representative genomes .

  Experimental approaches to investigate cysN's role in pathogenicity:

  1. Generate cysN knockout mutants and assess virulence in appropriate models

  2. Perform transcriptomics to measure cysN expression during infection

  3. Test growth under sulfur limitation to simulate host conditions

  4. Compare cysN expression between clinical and environmental isolates

• What methods are best suited for purifying recombinant V. vulnificus cysN protein for biochemical studies?

  For recombinant V. vulnificus cysN protein purification, several methodological approaches can be considered based on protein characteristics and experimental goals:

  Recommended purification protocol:

  1. Clone the cysN gene from V. vulnificus genomic DNA (ideally from well-characterized strains like VV2018 )

  2. Express in E. coli with appropriate affinity tags (His6 or GST)

  3. Use immobilized metal affinity chromatography (IMAC) for initial purification

  4. Follow with size exclusion chromatography to remove aggregates

  5. Validate protein activity using ATP sulfurylase assays

  6. Confirm purity through SDS-PAGE and mass spectrometry

  For optimal expression, consider codon optimization for the host system and testing multiple expression conditions (temperature, IPTG concentration, culture media) to maximize soluble protein yield.

Advanced Research Questions

• How might genetic variation in cysN correlate with V. vulnificus population structure and ecological adaptation?

  Analysis of V. vulnificus population structure reveals distinct genetic clusters with ecological differentiation. Genomic studies have identified two primary genotypes (C1 and C2), with C1 strains being more frequently associated with clinical cases .

  While cysN isn't specifically mentioned in the available research, its variation could potentially follow patterns similar to other metabolic genes. Genomic analyses show that V. vulnificus strains exhibit considerable genetic diversity, as demonstrated by ANI (Average Nucleotide Identity) values ranging from 95.41% to 98.45% between strains .

  Methodological approach for analyzing cysN variation:

  Analysis Type	Method	Expected Outcome
  Sequence Variation	Multi-locus sequence typing including cysN	Identification of cysN sequence types correlated with lineages
  Selection Analysis	dN/dS ratio calculation	Evidence of selection pressure on cysN
  Recombination Assessment	Phylogenetic network analysis	Detection of horizontal gene transfer events
  Expression Profiling	qRT-PCR across strain panels	Identification of expression differences between lineages
  Environmental Correlation	Statistical correlation of variants with habitat parameters	Insight into ecological adaptation through cysN

  This approach would build on known phylogenetic relationships, where strains like VV2018 group with other human-isolated strains (FORC_009 and FORC_016) , to determine if cysN variation follows similar patterns.

• What structural and functional adaptations might be present in V. vulnificus cysN compared to other bacterial homologs?

  V. vulnificus, as an aquatic bacterium capable of transitioning between environmental reservoirs and human hosts, likely has adaptations in key metabolic enzymes including cysN. While specific structural information about V. vulnificus cysN isn't provided in the search results, functional adaptations might include:

  1. Temperature-dependent activity profiles suited to both marine environments and human body temperature

  2. Salt tolerance mechanisms for function in brackish water habitats

  3. Allosteric regulation responsive to host-specific signals

  4. Potential roles in stress response during host infection

  Research methodology for structural-functional analysis:

  1. Perform homology modeling using solved structures of bacterial cysN proteins

  2. Identify unique sequence motifs in V. vulnificus cysN through multiple sequence alignment

  3. Express and crystallize the recombinant protein for structural determination

  4. Conduct site-directed mutagenesis of unique residues to assess functional impacts

  5. Compare enzymatic parameters (Km, Vmax, substrate specificity) with homologs from other pathogens

• How can cysN be targeted for potential antimicrobial development against V. vulnificus infections?

  Given the high mortality rate of V. vulnificus infections (close to 50% fatality in foodborne cases) , novel therapeutic targets are urgently needed. The cysN protein could represent a viable drug target if:

  1. It's essential for V. vulnificus survival or virulence

  2. It possesses structural features distinguishable from human homologs

  3. It's accessible to inhibitory compounds

  Strategic approach for antimicrobial development:

  Research Phase	Methodology	Expected Outcome
  Target Validation	Gene knockout and complementation studies	Confirmation of cysN essentiality
  Structural Analysis	X-ray crystallography or cryo-EM	Identification of druggable pockets
  Virtual Screening	In silico docking of compound libraries	Identification of potential inhibitors
  Biochemical Validation	Enzyme inhibition assays	Confirmation of inhibitory activity
  Cellular Testing	Growth inhibition of V. vulnificus cultures	Validation of cellular activity
  Specificity Assessment	Testing against human cell lines	Confirmation of selective toxicity

  This approach acknowledges the growing concern about V. vulnificus infections, which can progress very rapidly, especially in patients with underlying conditions like liver disease or diabetes .

• What interactions might exist between cysN and virulence factor regulation in V. vulnificus?

  While direct interactions between cysN and virulence factors aren't specified in the search results, V. vulnificus possesses numerous virulence determinants that could potentially be influenced by sulfur metabolism. Genomic analyses have identified 115 core virulence factors across V. vulnificus strains .

  Key virulence factors in V. vulnificus include the capsular polysaccharide (CPS) cluster, which exhibits high diversity due to recombination . Additionally, clinical strains often possess sialic acid metabolism genes that contribute to pathogenicity .

  Experimental design to investigate cysN-virulence interactions:

  1. Perform transcriptomic analysis comparing wild-type and cysN-deficient mutants under infection-mimicking conditions

  2. Use chromatin immunoprecipitation (ChIP-seq) to identify potential regulatory interactions

  3. Assess virulence factor production in media with varying sulfur availability

  4. Investigate potential protein-protein interactions between CysN and virulence regulators

  5. Analyze the effect of cysN mutation on capsular polysaccharide synthesis, which is a primary virulence determinant

• How does environmental sulfur availability influence cysN expression and V. vulnificus ecology?

  V. vulnificus naturally inhabits warm brackish and saltwater environments, with higher concentrations occurring in filter-feeding shellfish during warmer months . While the search results don't specifically address cysN regulation, environmental factors likely influence sulfur metabolism and consequently cysN expression.

  Methodological approaches for ecological studies:

  1. Measure cysN expression in V. vulnificus grown in water samples with varying sulfur content

  2. Correlate sulfur availability with V. vulnificus population density in natural habitats

  3. Perform competition experiments between wild-type and cysN mutants in environmental microcosms

  4. Analyze seasonal variation in cysN expression corresponding to changes in environmental conditions

  5. Investigate potential horizontal gene transfer of sulfur metabolism genes between environmental strains

  Understanding these ecological relationships is crucial since V. vulnificus is found in coastal waters and can reach high concentrations in oysters, which are the primary vector for human infections .