Recombinant Salmonella phage P22 Superinfection exclusion protein B (sieB)

What is the SieB protein of Salmonella phage P22 and how does it function in superinfection exclusion?

SieB is one of four superinfection exclusion systems encoded by the Salmonella phage P22 prophage. Unlike other exclusion systems, SieB specifically causes cellular macromolecular synthesis to cease midway through the lytic cycle during superinfection by P22-like phages, but notably does not affect P22 itself . The SieB protein functions as a late-acting exclusion mechanism, differentiating it from other systems like SieA which blocks at the DNA injection stage. SieB encodes a polypeptide with 192 amino acid residues and has a calculated molecular weight of approximately 22,442 Da . This system represents a sophisticated evolutionary adaptation that allows established prophages to protect their bacterial hosts from competing phage infections.

How does the SieB system differ from other superinfection exclusion systems in P22 phage?

P22 phage employs four distinct superinfection exclusion mechanisms that operate at different stages of phage infection:

• C2 Repressor: Prevents lytic gene expression by homo-immune phages

• GtrABC Proteins: Modify the O-antigen surface polysaccharide to block adsorption by phages that utilize it as a receptor

• SieA Protein: Acts at an early stage by blocking DNA injection into the host cell

• SieB Protein: Functions at a later stage by causing cellular macromolecular synthesis to cease midway through the lytic cycle of superinfecting phages

The key distinction of SieB is its timing - while SieA prevents DNA injection entirely, SieB allows infection to proceed partially before halting the process. This creates a unique temporal protection strategy that complements the other exclusion mechanisms.

What is the genetic structure of the sieB gene and how has it been characterized?

The sieB gene of Salmonella phage P22 has been characterized through detailed genetic and molecular analyses. The gene contains a single open reading frame of 242 codons with six potential translation initiation sites. Through deletion mapping and amber mutant phenotype analysis, researchers determined that the second of these six sites serves as the primary translation initiation site . The gene encodes a polypeptide with 192 amino acid residues.

The transcription start site of sieB was identified using RNase protection assay, which allowed researchers to precisely locate the promoter region. This promoter could be inactivated by introducing a 2-base substitution in its -10 hexamer sequence . Comparative genomic analysis also identified a homologous sieB gene in coliphage lambda, with a promoter sequence similar to that of P22 sieB .

How does the molecular weight of SieB protein correlate with its functional domains?

The SieB protein has a calculated molecular weight of 22,442 Da, which correlates well with estimates obtained from polyacrylamide gel electrophoresis . While the search results don't provide specific information about functional domains within SieB, the correlation between molecular weight calculations and experimental observations suggests that the protein maintains a consistent structural integrity that is likely essential for its function in superinfection exclusion.

Future structural biology research could employ techniques such as X-ray crystallography or cryo-electron microscopy to identify specific functional domains within the SieB protein and correlate them with the protein's ability to halt macromolecular synthesis during superinfection.

What techniques are most effective for mapping the sieB gene in bacteriophage P22?

Researchers have successfully mapped the sieB gene using a combination of complementary approaches:

• Phage Deletion Mutant Analysis: This technique was fundamental in initially mapping the sieB gene of P22. By creating and characterizing phages with specific deletions, researchers could define the boundaries of the functional sieB gene .

• DNA Sequence Analysis: Thorough DNA sequencing of the mapped region allowed researchers to identify open reading frames consistent with the deletion mapping results. This approach revealed discrepancies with previously published sequences and led to a revised sequence with a clear open reading frame .

• Amber Mutant Phenotype Studies: By analyzing the effects of amber mutations at different positions, researchers confirmed the translation initiation site of the sieB gene .

• RNase Protection Assay: This technique precisely identified the transcription start site of sieB, enabling the characterization of its promoter region .

For optimal results, researchers should employ these techniques in combination, as each provides different but complementary information about gene structure and function.

What are the methodological challenges in expressing recombinant SieB protein?

While the search results don't directly address challenges in expressing recombinant SieB, several considerations can be inferred based on similar phage proteins:

• Potential Toxicity: Given that SieB affects cellular macromolecular synthesis, its expression in laboratory strains might be toxic to host cells, requiring carefully regulated expression systems.

• Membrane Association: If SieB associates with cellular membranes like SieA (which is an inner membrane protein ), this could complicate purification protocols and necessitate specialized detergent-based extraction methods.

• Functional Assays: Developing assays to confirm the activity of recombinant SieB would require systems that can measure the cessation of macromolecular synthesis in response to phage superinfection.

• Structural Integrity: Ensuring that recombinant SieB maintains its native conformation might require co-expression of chaperones or specific folding conditions.

Researchers working with recombinant SieB should consider employing inducible expression systems, testing various solubilization strategies, and developing robust functional assays to overcome these challenges.

How does SieB from P22 compare with similar proteins in other bacteriophages?

The SieB protein from Salmonella phage P22 shares similarities with the SieB protein found in coliphage lambda, as both were identified through comparative analysis . While detailed functional comparisons aren't provided in the search results, the identification of lambda sieB through homology to P22 sieB suggests conservation of this superinfection exclusion mechanism across different phage species.

In contrast to SieB, the SieA system has been more extensively characterized across different phages. There are three sequence types of phage-encoded SieA proteins that are less than 30% identical to one another, yet comparison of two of these types found no differences in target specificity . This suggests that superinfection exclusion proteins can maintain functional conservation despite significant sequence divergence.

The diversity of superinfection exclusion systems across phages reflects the evolutionary arms race between phages and their competitors, with different phages developing varied mechanisms to protect their bacterial hosts.

How do the mechanisms of SieB and SieA complement each other in superinfection exclusion?

SieB and SieA represent two distinct but complementary approaches to superinfection exclusion in P22 phage:

• SieA Mechanism: Acts as an early barrier by blocking DNA injection. SieA is an inner membrane protein that interferes with the assembly or function of the periplasmic gp20 and membrane-bound gp16 DNA delivery conduit . This prevents the DNA of superinfecting phages from entering the host cell cytoplasm, as evidenced by the lack of potassium ion release during infection attempts .

• SieB Mechanism: Functions as a secondary defense by causing cellular macromolecular synthesis to cease midway through the lytic cycle of superinfecting phages . This means that if a phage manages to bypass the SieA system and inject its DNA, SieB can still prevent the completion of the infection cycle.

This two-tiered defense system provides robust protection against superinfection:

• SieA presents a physical barrier at the cell membrane

• SieB offers a backup mechanism that disrupts the replication process if DNA injection occurs

The complementary nature of these systems ensures that prophage-carrying bacteria are well-protected against competing phages, even those that might evolve to overcome one of the exclusion mechanisms.

What molecular mechanisms allow some phages to overcome SieB-mediated exclusion?

While the search results don't specifically address mechanisms for overcoming SieB exclusion, parallels can be drawn from studies on SieA escape. Researchers isolated P22 mutants that could overcome SieA-mediated exclusion by acquiring amino acid changes in the C-terminal regions of the gene 16 and 20 encoded ejection proteins . Three different single amino acid changes in these proteins were required to obtain nearly full resistance to SieA .

For SieB, potential escape mechanisms might involve:

• Mutations in key phage proteins that interact with the SieB system

• Alternative pathways for macromolecular synthesis that are not inhibited by SieB

• Production of inhibitors that neutralize SieB function

Understanding these escape mechanisms would require isolation of SieB-resistant phage mutants, whole-genome sequencing to identify mutations, and functional studies to confirm the resistance mechanism.

How might the structural biology of SieB inform the development of novel antimicrobial strategies?

Understanding the structural biology of SieB could reveal insights into bacterial exclusion mechanisms that might be harnessed for antimicrobial development:

• Targeted Bacterial Killing: Knowledge of how SieB halts macromolecular synthesis could inform the design of synthetic antimicrobials that mimic this action but target specific bacterial species.

• Phage Therapy Enhancement: Engineering phages with modified exclusion systems could create more effective phage therapies that overcome bacterial resistance mechanisms.

• Novel Screening Platforms: Structural understanding of SieB could enable the development of high-throughput screening platforms to identify small molecules that mimic or enhance its activity.

• Synthetic Biology Applications: The exclusion mechanism could be engineered into synthetic microbial communities to regulate population dynamics and prevent invasion by unwanted species.

Future research combining structural biology approaches (X-ray crystallography, cryo-EM) with functional assays would be necessary to fully realize these potential applications.

What experimental designs are most effective for studying the impact of SieB on host cell physiology?

To effectively study SieB's impact on host cell physiology, researchers should consider several experimental approaches:

• Comparative Transcriptomics:

  • Compare gene expression profiles of cells with and without SieB expression

  • Analyze changes during normal growth versus during phage superinfection

  • Time-course studies to capture the dynamics of the SieB response

• Metabolic Profiling:

  • Measure key metabolites in cells expressing SieB versus control cells

  • Monitor ATP levels, redox status, and central carbon metabolism

  • Correlate metabolic changes with the cessation of macromolecular synthesis

• Real-time Visualization:

  • Employ fluorescent reporters to visualize macromolecular synthesis in real-time

  • Use time-lapse microscopy to monitor cellular responses to superinfection

  • Implement FRET-based sensors to detect protein-protein interactions

• Genetic Interaction Mapping:

  • Construct bacterial strains with SieB expression in various genetic backgrounds

  • Screen for synthetic lethal or synthetic rescue interactions

  • Identify host factors required for SieB function

These approaches, especially when used in combination, would provide comprehensive insights into how SieB affects host cell physiology during superinfection exclusion.

What are the methodological considerations for studying interactions between SieB and host cell components?

Studying interactions between SieB and host cell components requires careful methodological considerations:

• Protein-Protein Interaction Methods:

  • Co-immunoprecipitation with tagged SieB variants

  • Bacterial two-hybrid systems for screening potential interactors

  • Proximity labeling approaches (BioID, APEX) to identify proteins in close proximity to SieB in vivo

  • Cross-linking mass spectrometry to capture transient interactions

• Localization Studies:

  • Fluorescent protein fusions to determine subcellular localization

  • Fractionation approaches to determine membrane association

  • Immunogold electron microscopy for high-resolution localization

• Functional Reconstitution:

  • In vitro reconstitution of SieB activity with purified components

  • Liposome-based systems to study membrane interactions

  • Cell-free expression systems to avoid toxicity issues

• Structural Analysis:

  • Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

  • Cryo-electron microscopy to visualize SieB in complex with interaction partners

  • NMR studies for dynamic interactions

When designing these experiments, researchers should consider potential artifacts from protein tags, ensure appropriate controls for non-specific interactions, and validate key findings using multiple complementary approaches.

What evolutionary pressures have shaped the development of SieB in P22 and related phages?

The evolution of superinfection exclusion systems like SieB likely reflects several selective pressures:

• Competition for Host Resources: Bacteria can only support a limited number of productive phage infections. By preventing superinfection, established prophages ensure they don't have to compete for cellular resources .

• Prophage Stability: Superinfection by related phages could trigger induction of the resident prophage. Exclusion systems like SieB protect the lysogenic state by blocking competing infections .

• Co-evolution with Escape Mutants: The emergence of phages that can overcome exclusion creates selective pressure for more effective exclusion systems, driving an evolutionary arms race. This is evident in the SieA system, where multiple amino acid changes are required for phages to overcome exclusion .

• Horizontal Gene Transfer: The identification of similar sieB genes in different phages (P22 and lambda) suggests horizontal gene transfer may have played a role in the dissemination of these systems .

The presence of multiple exclusion systems in P22 (c2 repressor, gtrABC, sieA, and sieB) that act at different stages of infection demonstrates the importance of these defense mechanisms in phage ecology and evolution .

How has the sequence and function of SieB been conserved across different Salmonella phages?

While the search results don't provide specific information about SieB conservation across different Salmonella phages, the identification of sieB in both Salmonella phage P22 and coliphage lambda suggests some degree of conservation across different phage types .

By comparison, the SieA system shows interesting evolutionary patterns, with three sequence types of extant phage-encoded SieA proteins that are less than 30% identical to one another. Despite this sequence divergence, comparison of two of these types found no differences in target specificity . This suggests that superinfection exclusion systems can maintain functional conservation despite significant sequence divergence.

Future research addressing SieB conservation could include:

• Comparative genomic analysis of sieB genes across Salmonella phages

• Functional testing of SieB proteins from different phages

• Identification of conserved domains critical for function

• Analysis of selection pressures on different regions of the sieB gene

Such studies would provide valuable insights into the evolutionary history and functional constraints of this important superinfection exclusion system.