Recombinant Salmonella schwarzengrund Probable 4-amino-4-deoxy-L-arabinose-phosphoundecaprenol flippase subunit ArnF (arnF)

What is the complete amino acid sequence of Salmonella schwarzengrund ArnF protein?

The full-length Salmonella schwarzengrund ArnF protein consists of 125 amino acids with the following sequence: MGVMWGLISVAIASLAQLSLGFAMMRLPSIAHPLAFISGLGAFNAATLALFAGLAGYLVSVFCWQKTLHTLALSKAYALLSLSYVLVWVASMLLPGLQGAFSLKAMLGVLCIMAGVMLIFPARS. This represents the complete protein from positions 1-125 . The protein's composition includes multiple hydrophobic regions typical of membrane-spanning domains, consistent with its function as a membrane flippase.

What are the alternative nomenclatures for this protein in scientific literature?

While primarily referred to as "Probable 4-amino-4-deoxy-L-arabinose-phosphoundecaprenol flippase subunit ArnF" in databases, it is also documented under synonyms including "L-Ara4N-phosphoundecaprenol flippase subunit ArnF" and "Undecaprenyl phosphate-aminoarabinose flippase subunit ArnF" . The UniProt ID for this specific protein is B4TPI6, which provides standardized reference in proteomics databases .

How does the protein structure of Salmonella schwarzengrund ArnF compare to homologs in other bacterial species?

Based on topological studies of related ArnT proteins, ArnF is predicted to contain multiple transmembrane helices. The homologous ArnT protein has been characterized with 13 transmembrane domains and a large C-terminal region exposed to the periplasm . While specific structural comparisons between S. schwarzengrund ArnF and other species' homologs aren't directly provided in the search results, the Shewanella sediminis ArnF homolog consists of 145 amino acids (compared to the 125 in S. schwarzengrund) and shares similar functional domain organization but with sequence variations reflective of species-specific adaptations .

What is the primary biological function of ArnF in bacterial cells?

ArnF functions as a subunit of the flippase that mediates the transfer of 4-amino-4-deoxy-L-arabinose (L-Ara4N) modifications to bacterial lipopolysaccharide (LPS). As part of this process, ArnF contributes to flipping L-Ara4N-modified lipid carriers across the bacterial inner membrane, facilitating the transport of these modified components to the outer membrane . This modification process is critical for bacterial cell envelope integrity and antimicrobial resistance, particularly against cationic antimicrobial peptides.

What are the key conserved residues essential for ArnF functionality?

While the search results don't specifically identify the critical residues in S. schwarzengrund ArnF, studies on related ArnT proteins have identified four highly conserved periplasmic residues essential for activity: tyrosine-43, lysine-69, arginine-254, and glutamic acid-493 . These residues span two particularly conserved motifs found across bacterial species: 42RYA44 and 66YFEKP70 . These amino acids likely participate in either substrate recognition (undecaprenyl phosphate-L-Ara4N) or the transfer of L-Ara4N to the LPS molecule .

What expression systems yield optimal results for recombinant ArnF production?

The commercially available recombinant ArnF protein is expressed in E. coli expression systems with an N-terminal His-tag for purification purposes . This approach has proven effective in producing full-length, functional protein suitable for research applications. The E. coli expression system offers advantages including rapid growth, high protein yields, and well-established optimization protocols for membrane protein expression.

What buffer conditions are recommended for optimal ArnF stability?

The purified recombinant protein demonstrates optimal stability in Tris/PBS-based buffer with 6% trehalose at pH 8.0 . For long-term storage, it is recommended to add glycerol to a final concentration between 5-50% (typically 50%) and store at -20°C/-80°C in aliquots to prevent repeated freeze-thaw cycles . Working aliquots can be maintained at 4°C for up to one week, though extended storage under these conditions is not recommended .

What protein reconstitution protocol maximizes ArnF activity recovery?

For optimal reconstitution of lyophilized ArnF protein, the recommended protocol includes:

• Brief centrifugation of the vial prior to opening to collect all material at the bottom

• Reconstitution using deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL

• Addition of glycerol (5-50% final concentration) to enhance stability

• Aliquoting for storage to minimize freeze-thaw cycles

This protocol helps maintain protein integrity and functional activity for subsequent experimental applications.

How can researchers effectively study ArnF-mediated LPS modifications in vitro?

To study ArnF-mediated LPS modifications, researchers should consider a comprehensive approach involving:

• Membrane protein reconstitution into liposomes containing appropriate lipid composition mimicking bacterial inner membranes

• Incorporation of purified ArnF protein into these model membrane systems

• Development of assays measuring the translocation of L-Ara4N-modified lipid carriers

• Analysis of modified LPS products using techniques such as mass spectrometry and nuclear magnetic resonance

Key controls should include systems lacking ArnF protein, systems with mutated non-functional ArnF variants, and positive controls using well-characterized homologous proteins .

What mutagenesis strategies can elucidate structure-function relationships in ArnF?

Based on studies of related ArnT proteins, a strategic approach to ArnF mutagenesis would target:

• The highly conserved residues equivalent to tyrosine-43, lysine-69, arginine-254, and glutamic acid-493 in related proteins

• The conserved motifs 42RYA44 and 66YFEKP70 (with appropriate sequence alignment to S. schwarzengrund ArnF)

• Predicted transmembrane domains to assess their role in membrane integration

• The C-terminal region believed to be exposed to the periplasm

Site-directed mutagenesis followed by functional assays would provide valuable insights into which residues are critical for substrate binding, catalysis, and membrane integration .

How can interactions between ArnF and other components of the LPS modification machinery be characterized?

To characterize protein-protein interactions within the LPS modification pathway:

• Employ co-immunoprecipitation studies using tagged ArnF variants

• Utilize bacterial two-hybrid systems to screen for interaction partners

• Apply chemical crosslinking followed by mass spectrometry analysis

• Develop fluorescence resonance energy transfer (FRET) assays using fluorescently labeled components

• Perform proximity labeling techniques like BioID or APEX to identify proteins in close proximity to ArnF in vivo

These approaches would help establish the complete interactome of ArnF and elucidate its role within the larger LPS modification complex.

What is the epidemiological significance of ArnF-mediated modifications in Salmonella schwarzengrund?

Salmonella schwarzengrund has been identified as an international concern with isolates recovered from persons, food, and food animals across multiple countries including Thailand, Denmark, and the United States . Research indicates potential transmission patterns from chickens to humans in Thailand and from imported Thai food products to consumers in Denmark and the United States . The LPS modifications facilitated by ArnF contribute to antimicrobial resistance in these strains, complicating treatment options and potentially contributing to increased virulence.

How does ArnF function correlate with clinical antimicrobial resistance patterns?

Studies examining antimicrobial resistance in Salmonella isolates have found that resistance to nalidixic acid and fluoroquinolones is frequent among isolates from various sources . While direct experimental evidence linking specific ArnF variants to resistance profiles is not provided in the search results, the general mechanism of L-Ara4N addition to LPS has been established as a significant contributor to resistance against multiple antibiotic classes. The presence of specific mutations in the quinolone resistance-determining regions has been identified in ciprofloxacin-resistant isolates, suggesting complex resistance mechanisms that may work in concert with LPS modifications .

What comparative analyses between different Salmonella serovars reveal about ArnF conservation and variability?

A comprehensive comparison examining 581 Salmonella enterica serotype Schwarzengrund isolates using pulsed-field gel electrophoresis (PFGE) typing revealed 183 distinct patterns . This diversity suggests genetic variability across isolates, though specific analysis of ArnF sequence conservation was not detailed in the search results. Future comparative genomic studies specifically examining the arnF gene and its promoter regions across Salmonella isolates with varying resistance profiles would provide valuable insights into how sequence variations might correlate with functional differences and resistance phenotypes.

What are common challenges in working with recombinant ArnF and how can they be addressed?

As a membrane protein, common challenges when working with ArnF include:

• Protein aggregation during expression and purification

  • Solution: Optimize detergent selection and concentration during extraction and purification steps

• Loss of activity during storage

  • Solution: Maintain strict storage conditions (-20°C/-80°C) with appropriate glycerol concentration and minimize freeze-thaw cycles

• Difficulty in measuring flippase activity

  • Solution: Develop specialized assays using fluorescently labeled lipid substrates that can track translocation across membrane bilayers

• Protein misfolding during reconstitution

  • Solution: Carefully control the rate of detergent removal during proteoliposome formation and ensure appropriate lipid composition

What quality control measures ensure functional integrity of purified ArnF preparations?

To ensure recombinant ArnF preparations maintain their functional integrity:

• Verify protein purity (>90%) using SDS-PAGE analysis

• Confirm proper folding using circular dichroism spectroscopy

• Assess membrane integration capability using liposome association assays

• Validate substrate binding through direct binding assays with labeled L-Ara4N-containing compounds

• Perform activity assays measuring lipid flipping in reconstituted systems

These quality control measures help ensure that experimental findings accurately reflect native ArnF function rather than artifacts from compromised protein preparations.