Recombinant Shewanella sp. UPF0060 membrane protein Sputw3181_1172 (Sputw3181_1172)

What is the amino acid sequence of Sputw3181_1172 and what structural features can be predicted?

The full amino acid sequence of Sputw3181_1172 is: MTVITTLGLFIITAIAEIVGCYLPYLWLKKGASAWVLLPAAISLALFAWLLTLHPTAAGRVYAAYGGVYVTIAIAVWLWGVDGIQPHRWDLAGVVLMLAGMAVIMFAPR . Analysis of this sequence suggests it is a highly hydrophobic membrane protein with multiple predicted transmembrane domains. Secondary structure prediction indicates alpha-helical regions that likely span the membrane, consistent with its classification as a UPF0060 family membrane protein. The protein's hydrophobic nature is evident from the prevalence of non-polar amino acids such as leucine, isoleucine, valine, and alanine throughout the sequence.

What expression systems are most effective for producing recombinant Sputw3181_1172?

E. coli-based expression systems have proven effective for the recombinant production of Sputw3181_1172 . When expressing membrane proteins like Sputw3181_1172, several methodological considerations are critical:

• Vector selection: pET vectors with T7 promoters provide controllable, high-level expression

• Host strain: C41(DE3) or C43(DE3) strains specifically designed for membrane protein expression often yield better results than standard BL21(DE3)

• Induction conditions: Lower temperatures (16-20°C) and reduced IPTG concentrations (0.1-0.5 mM) typically improve proper folding

• Membrane extraction: Careful optimization of detergent type and concentration is essential for solubilization without denaturation

The commercially available recombinant Sputw3181_1172 is typically produced with a His-tag to facilitate purification, though the tag type may vary depending on production requirements .

How does the Sputw3181_1172 protein relate to the metabolic versatility of Shewanella species?

While the specific function of Sputw3181_1172 has not been fully characterized, Shewanella species demonstrate remarkable metabolic versatility, particularly in their ability to use diverse electron acceptors. Shewanella species possess genes for extracellular electron transfer (EET), including omcA mtrCAB, enabling them to reduce Fe³⁺ and other metals . They can also reduce NO₃⁻ to NO₂⁻ via napAB genes, produce H₂ via hydAB, and reduce various sulfur compounds through sirA, phsABC, and ttrABC gene products .

As a membrane protein, Sputw3181_1172 may potentially participate in these metabolic pathways, possibly contributing to membrane integrity, transport functions, or signaling processes related to the bacterium's adaptability to various environments. Research methodologies to investigate its role might include:

• Gene knockout studies to observe phenotypic changes

• Protein localization experiments using fluorescent tags or antibodies

• Metabolomic analyses comparing wild-type and mutant strains under various growth conditions

What purification strategies are most effective for Sputw3181_1172?

Purification of membrane proteins like Sputw3181_1172 requires specialized approaches:

• Affinity chromatography: His-tagged Sputw3181_1172 can be purified using immobilized metal affinity chromatography (IMAC) . The inclusion of appropriate detergents throughout the purification process is critical.

• Detergent selection: A systematic screening approach is recommended, testing mild detergents such as n-dodecyl-β-D-maltoside (DDM), n-decyl-β-D-maltoside (DM), or lauryl maltose neopentyl glycol (LMNG).

• Buffer optimization: Typical buffer conditions include:

  • pH 7.5-8.0 Tris or phosphate buffer

  • 150-300 mM NaCl for ionic strength

  • 5-10% glycerol as a stabilizing agent

  • 1-5 mM reducing agent (DTT or β-mercaptoethanol)

• Size exclusion chromatography: As a polishing step to remove aggregates and ensure homogeneity of the protein-detergent complex.

The commercially available recombinant Sputw3181_1172 is supplied in a Tris-based buffer with 50% glycerol, optimized for protein stability .

How can advanced mass spectrometry techniques be applied to study Sputw3181_1172 structural interactions?

Diethylpyrocarbonate (DEPC) covalent labeling mass spectrometry offers a promising approach for studying membrane protein structures and interactions in living cells . This methodology could be applied to Sputw3181_1172 using the following protocol:

• Live cell labeling: Treat Shewanella sp. cultures expressing Sputw3181_1172 with DEPC under controlled conditions.

• Protein extraction and digestion: Extract labeled proteins and digest with proteases (typically trypsin) to generate peptide fragments.

• MS analysis: Analyze the peptides using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify DEPC-modified residues.

• Comparative analysis: Compare labeling patterns under different conditions (e.g., with/without binding partners) to identify:

  • Residues with decreased labeling (indicating burial upon interaction)

  • Residues with increased labeling (indicating conformational changes that expose previously protected sites)

  • Changes in distant regions (suggesting allosteric effects)

This approach has been successfully demonstrated with membrane-bound tumor necrosis factor α (mTNFα) and could reveal structural insights and binding interactions of Sputw3181_1172 in its native cellular environment .

What techniques are available for studying the oligomeric state of Sputw3181_1172?

Determining the oligomeric state of membrane proteins like Sputw3181_1172 requires specialized approaches:

• Blue Native PAGE: Separates native protein complexes while maintaining their oligomeric states

  • Sample preparation: Solubilize membranes with gentle detergents

  • Electrophoresis: Run at 4°C with Coomassie Blue G-250 dye

  • Analysis: Compare migration with known molecular weight standards

• Analytical ultracentrifugation (AUC):

  • Sedimentation velocity experiments: Determine sedimentation coefficients

  • Sedimentation equilibrium: Calculate molecular mass independent of shape

• Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS):

  • Separates proteins based on size

  • MALS detector determines absolute molecular weight

  • Detergent contribution can be accounted for using differential refractive index measurements

• Chemical crosslinking followed by MS analysis:

  • Treat with crosslinkers of defined length

  • Identify crosslinked peptides by MS

  • Map interaction interfaces between subunits

What computational methods can predict the function of Sputw3181_1172?

Several bioinformatic approaches can provide insights into the potential function of Sputw3181_1172:

• Homology-based function prediction:

  • BLAST searches against characterized proteins

  • Hidden Markov Model (HMM) profile analysis against protein family databases

  • Structural homology modeling followed by function inference

• Genomic context analysis:

  • Examine neighboring genes in the Shewanella sp. genome

  • Identify conserved operons across related species

  • Apply guilt-by-association principles for functional prediction

• Protein-protein interaction prediction:

  • Coevolution analysis to identify potential interaction partners

  • Docking simulations with predicted partners

  • Interface conservation analysis across homologs

• Structure-based function prediction:

  • Threading approaches to identify structural templates

  • Active site prediction based on conserved residues

  • Electrostatic surface analysis for potential binding interfaces

These computational predictions should be validated through experimental approaches such as mutagenesis, protein-protein interaction assays, or phenotypic studies.

How might Sputw3181_1172 contribute to Shewanella's metal reduction capabilities?

Shewanella species are known for their ability to reduce metals such as Fe³⁺, contributing to biogeochemical cycling in environments like the Iberian Pyrite Belt . While the direct involvement of Sputw3181_1172 in metal reduction is not established, several experimental approaches could investigate this possibility:

• Gene expression analysis:

  • Compare Sputw3181_1172 expression levels under aerobic vs. anaerobic/metal-reducing conditions

  • Use RT-qPCR or RNA-seq to quantify transcriptional changes

  • Monitor protein levels via western blotting with specific antibodies

• Localization studies:

  • Generate fluorescent protein fusions to determine subcellular localization

  • Use immunogold electron microscopy to visualize the protein's position relative to the cell membrane and metal reduction sites

• Knockout/knockdown experiments:

  • Generate Sputw3181_1172 deletion mutants

  • Assess metal reduction capacity compared to wild-type

  • Complement with recombinant protein to confirm phenotype specificity

• Protein-protein interaction studies:

  • Co-immunoprecipitation with known components of the metal reduction machinery (e.g., MtrC, OmcA)

  • Bacterial two-hybrid assays to identify interaction partners

  • Crosslinking mass spectrometry to map the interaction network

Shewanella species like S. putrefaciens T2.3D-1.1 can reduce Fe³⁺ and promote Fe²⁺ oxidation in the presence of NO₃⁻ under anaerobic conditions , suggesting a complex electron transfer network that Sputw3181_1172 might participate in.

What is known about the potential role of Sputw3181_1172 in antimicrobial resistance?

Shewanella species have been identified as reservoirs of antimicrobial resistance genes, including blaOXA-48 and qnrA, which can potentially transfer to human pathogens like Enterobacteriaceae . To investigate if Sputw3181_1172 plays a role in antimicrobial resistance:

• Comparative genomics approach:

  • Analyze the genetic context of Sputw3181_1172 in relation to mobile genetic elements

  • Compare presence/absence and sequence variation across resistant and susceptible strains

  • Examine co-occurrence with known resistance determinants

• Gene expression studies:

  • Expose Shewanella cultures to sub-inhibitory concentrations of antibiotics

  • Monitor changes in Sputw3181_1172 expression

  • Correlate expression levels with resistance phenotypes

• Membrane permeability assays:

  • Overexpress or delete Sputw3181_1172 and measure changes in membrane permeability

  • Assess uptake of fluorescent dyes or antibiotics in modified strains

  • Determine minimal inhibitory concentrations (MICs) for various antimicrobials

The genomic analysis revealed that 79 out of 144 Shewanella genomes encoded at least one antimicrobial resistance gene, with highest occurrence in clinical-related lineages , suggesting potential roles for membrane proteins like Sputw3181_1172 in adaptation to clinical environments.

How can CRISPR-Cas systems be applied to study Sputw3181_1172 function in Shewanella?

CRISPR-Cas systems have been identified in Shewanella species, with I-E and I-F being the most frequent types found in 41 of 144 analyzed genomes . These native systems can be leveraged, or heterologous CRISPR systems can be introduced, to study Sputw3181_1172 function:

• Gene knockout strategies:

  • Design sgRNAs targeting the Sputw3181_1172 coding sequence

  • Utilize CRISPR-Cas9 or Cas12a systems with non-homologous end joining (NHEJ) or homology-directed repair (HDR)

  • Screen for complete knockouts using PCR and sequencing

• CRISPRi for conditional repression:

  • Express catalytically inactive dCas9 fused to repressor domains

  • Target sgRNAs to the promoter region of Sputw3181_1172

  • Create tunable repression by modulating inducer concentrations

• CRISPRa for overexpression studies:

  • Use dCas9 fused to activation domains to upregulate Sputw3181_1172

  • Compare phenotypes under varying expression levels

  • Identify potential threshold effects in function

• Base and prime editing:

  • Introduce specific point mutations without double-strand breaks

  • Create systematic variants to map structure-function relationships

  • Engineer protein variants with enhanced or modified functions

Successful CRISPR-based approaches require optimization of transformation protocols for Shewanella species and careful design of sgRNAs to minimize off-target effects.

What structural biology techniques are most promising for resolving the 3D structure of Sputw3181_1172?

Membrane proteins present unique challenges for structural determination. For Sputw3181_1172, consider these methodological approaches:

• Cryo-electron microscopy (cryo-EM):

  • Advantages: Requires less protein, maintains native-like environment

  • Sample preparation: Reconstitute in nanodiscs or amphipols

  • Data collection: Use direct electron detectors and motion correction

  • Processing: Employ 2D classification and 3D reconstruction algorithms

• X-ray crystallography:

  • Protein engineering: Create fusion proteins or remove flexible regions

  • Crystallization: Screen detergents, lipids, and crystallization conditions

  • Optimize: Use lipidic cubic phase or bicelle crystallization methods

  • Data collection: Consider microcrystallography at synchrotron facilities

• Nuclear magnetic resonance (NMR):

  • Solution NMR: For smaller membrane proteins or domains

  • Solid-state NMR: For larger intact membrane proteins

  • Isotopic labeling: Express with ¹⁵N, ¹³C, and ²H incorporation

  • Spectral analysis: Assign resonances and calculate distance constraints

• Integrative structural biology:

  • Combine multiple techniques (SAXS, HDX-MS, crosslinking-MS)

  • Use computational modeling to integrate diverse data types

  • Validate with mutagenesis and functional assays

The appropriate technique depends on protein yield, stability, size, and available equipment. For Sputw3181_1172, a starting point would be optimizing expression and purification, followed by screening structural techniques based on initial biochemical characterization.

How can systems biology approaches integrate Sputw3181_1172 into the broader metabolic network of Shewanella?

Systems biology offers powerful frameworks to understand how Sputw3181_1172 functions within the complex metabolic network of Shewanella:

• Multi-omics integration:

  • Transcriptomics: RNA-seq to identify co-expressed genes

  • Proteomics: MS-based quantification of protein abundances and modifications

  • Metabolomics: Identify metabolic changes associated with Sputw3181_1172 perturbation

  • Integration: Use computational frameworks to correlate across omics layers

• Genome-scale metabolic modeling:

  • Construct or refine existing Shewanella metabolic models

  • Incorporate Sputw3181_1172 functions based on experimental evidence

  • Perform flux balance analysis under different conditions

  • Validate predictions with experimental measurements

• Protein-protein interaction network analysis:

  • Identify first and second-degree interaction partners

  • Map to metabolic pathways and cellular processes

  • Identify network motifs and regulatory patterns

  • Predict emergent properties from network topology

• Comparative systems analysis:

  • Compare systems-level properties across Shewanella species

  • Identify conserved modules and species-specific adaptations

  • Correlate with ecological niches and metabolic capabilities

Shewanella species have evolved diverse metabolic capabilities to thrive in various environments, from deep-sea to clinical settings . Understanding how membrane proteins like Sputw3181_1172 contribute to these adaptations requires integration of molecular details into system-level frameworks.

How conserved is Sputw3181_1172 across different Shewanella species and what does this suggest about its function?

Analyzing the conservation of Sputw3181_1172 across the Shewanella genus can provide insights into its evolutionary importance and functional constraints:

• Sequence conservation analysis:

  • Perform multiple sequence alignment of homologs

  • Calculate position-specific conservation scores

  • Identify highly conserved residues as potential functional sites

  • Map conservation onto predicted structural models

• Phylogenetic distribution:

  • Construct phylogenetic trees of Sputw3181_1172 homologs

  • Compare protein trees with species trees to identify horizontal gene transfer events

  • Correlate presence/absence with ecological niches and metabolic capabilities

• Selective pressure analysis:

  • Calculate dN/dS ratios to identify sites under purifying or positive selection

  • Perform codon-based tests for selection across lineages

  • Identify accelerated evolution in specific clades

The comparative genome analysis of 144 Shewanella genomes revealed significant diversity in accessory genes, particularly in clinical-related lineages . Examining where Sputw3181_1172 fits within this diversity landscape can provide context for its evolutionary history and potential functional significance.

How does Sputw3181_1172 relate to the mobilome and horizontal gene transfer in Shewanella?

Shewanella species possess diverse mobile genetic elements (MGEs) that contribute to their genomic plasticity . To investigate if Sputw3181_1172 has been subject to horizontal gene transfer (HGT):

• Genomic context analysis:

  • Examine flanking regions for MGE signatures (transposases, integrases, etc.)

  • Identify anomalous GC content or codon usage as indicators of HGT

  • Look for synteny breaks across closely related genomes

• Comparative genomics approach:

  • Search for homologs across distantly related bacteria

  • Identify incongruence between gene trees and species phylogeny

  • Analyze distribution patterns inconsistent with vertical inheritance

• Mobile genetic element association:

  • Check for co-occurrence with known MGEs (plasmids, prophages, ICEs)

  • Examine correlation with integron presence and content

  • Assess potential for mobilization based on genomic context

Shewanella species have been shown to acquire various mobile elements, including plasmids, prophages, group II introns, integrons, and integrative and conjugative elements . Understanding whether Sputw3181_1172 has been subject to HGT events can provide insights into its potential role in adaptation to new ecological niches.

What experimental approaches can determine if Sputw3181_1172 contributes to Shewanella's environmental adaptability?

Investigating the role of Sputw3181_1172 in environmental adaptation requires multifaceted experimental approaches:

• Stress response studies:

  • Expose wild-type and Sputw3181_1172 mutant strains to various stressors:

    • Heavy metals (Pb, Cr, Cu, Cd, Co, Ni, Zn)

    • pH extremes

    • Temperature fluctuations

    • Osmotic stress

  • Measure growth rates, survival, and stress response gene expression

• Niche-specific adaptation experiments:

  • Test growth and metabolic activities under conditions mimicking natural habitats:

    • Aerobic vs. anaerobic environments

    • Presence of various electron acceptors (Fe³⁺, NO₃⁻, S₂O₃²⁻)

    • Nutrient limitation scenarios

• Competition assays:

  • Co-culture wild-type and mutant strains under various conditions

  • Track population dynamics using strain-specific markers

  • Determine fitness advantages/disadvantages in different environments

• In situ expression analysis:

  • Design reporter constructs to monitor Sputw3181_1172 expression

  • Measure activity in simulated environmental conditions

  • Correlate expression with specific environmental variables

Shewanella putrefaciens T2.3D-1.1 demonstrates tolerance to various heavy metals (7.5 mM Pb, 5 mM Cr and Cu, 1 mM Cd, Co, Ni, and Zn) , indicating adaptation mechanisms that may involve membrane proteins like Sputw3181_1172 in maintaining cellular homeostasis under stress conditions.