Recombinant Escherichia coli Inner membrane protein yedR (yedR)

What is DrpB and why was it renamed from YedR?

DrpB (Division Ring Protein B) is a small inner membrane protein in Escherichia coli that participates in cell division. It was renamed from YedR following its identification as a division protein in a screen for multicopy suppressors of ΔftsEX mutations. The "Drp" designation follows the systematic naming of division ring proteins, with "B" indicating it was the second in this series (the name DrpA was previously assigned to a protein thought to be involved in DNA replication but later identified as a tRNA synthetase now called ProS) .

What is the structure and topology of DrpB?

DrpB is a small membrane protein of 100 amino acids (when translation initiates at the correct ATG start codon at position 22). The protein has two transmembrane helices separated by a periplasmic loop of approximately 20 amino acids, with both the N and C termini located in the cytoplasm. This topology was confirmed both through bioinformatic predictions and through a large-scale experimental topological analysis of E. coli membrane proteins . The protein is poorly conserved at the sequence level, but its architecture (N-terminus in, C-terminus in, with two transmembrane domains) is conserved among homologs in closely related enteric bacteria .

How widespread is DrpB among bacterial species?

DrpB is not widely conserved across bacterial species. Homologs are found only in a subset of Gammaproteobacteria, including multiple E. coli strains, Shigella flexneri, Salmonella enterica serovar Typhimurium, and Klebsiella pneumoniae. No homologs were identified in other Gammaproteobacteria such as Citrobacter freundii, Pseudomonas aeruginosa, Serratia marcescens, Vibrio cholerae, and Yersinia pestis, nor in more distantly related bacteria commonly used in cell division studies (Caulobacter crescentus, Myxococcus xanthus, Bacillus subtilis, or Staphylococcus aureus) .

How should I design expression constructs for DrpB?

When designing expression constructs for DrpB, it's crucial to consider the correct translational start site. Research has shown that translation primarily initiates at an ATG at position 22 of the annotated sequence rather than the annotated GTG start codon at position 1. When expressed from its native chromosomal locus, only the shorter (M22)DrpB form is detected by Western blotting .

For optimal expression:

• Use the ATG at position 22 as the start codon

• Include a strong Shine-Dalgarno sequence upstream of the start codon

• If tagging the protein, C-terminal tags appear to interfere less with function than N-terminal tags

• Consider including the native promoter (located between positions 1 and 22) for physiological expression levels

What expression systems work best for DrpB membrane protein studies?

For membrane protein studies of DrpB, several expression systems have been successfully employed:

• Chromosomal tagging: In situ fusion of GFP to the C-terminus of DrpB at its native chromosomal locus provides physiological expression levels suitable for functional studies, though fluorescence intensity may be weak .

• Low-copy plasmid expression: pDSW210-derived vectors with weak IPTG-inducible promoters (such as P206) provide moderate expression suitable for localization studies without causing protein aggregation or toxicity .

• Arabinose-inducible system: pBAD33 vectors with the PBAD promoter allow titratable expression and have been successfully used for complementation studies and multicopy suppression assays .

How does medium composition affect DrpB localization?

This osmolarity-dependent localization is not specific to NaCl, as adding 200 mM NaCl, proline, or sucrose to LB0N similarly abolishes septal localization. Thus, DrpB localization is impaired by high osmolarity rather than high salt specifically. This makes DrpB unique among division proteins, as it exhibits almost complete dependence on low osmotic strength for localization .

What visualization techniques are most effective for studying DrpB localization?

Several visualization techniques have been tested for DrpB localization studies:

• C-terminal GFP fusions: Both plasmid-expressed and chromosomally-integrated DrpB-GFP fusions show septal localization, though signals are relatively weak .

• N-terminal GFP fusions: GFP-(M22)DrpB fusions also show septal localization but with similarly weak signals .

• Membrane co-visualization: Combining GFP visualization with FM4-64 membrane staining helps distinguish true septal ring localization from signal artifacts arising from increased membrane density during cell constriction .

For optimal visualization:

• Grow cells in LB0N (low salt/osmolarity) medium

• Optimize the linker between DrpB and GFP (5-amino acid linkers have proven functional)

• Use sensitive microscopy with appropriate exposure settings

• Consider deconvolution techniques to enhance signal-to-noise ratio

What controls should be included in DrpB localization experiments?

When studying DrpB localization, several controls are essential:

• Expression level control: Western blotting to confirm similar expression levels across different conditions (e.g., LB vs. LB0N) .

• Membrane staining control: FM4-64 staining to distinguish true septal localization from artifacts due to increased membrane density at division sites .

• Dependency controls: Test localization in strains lacking key division proteins (e.g., ΔftsZ) to confirm requirement of the division machinery .

• Functionality control: Confirm the fusion protein is functional by complementation assays (e.g., rescue of the ΔdrpB ΔdedD synthetic phenotype) .

• Osmolarity controls: Test different osmolytes (NaCl, sucrose, proline) to confirm osmolarity-dependent effects versus ion-specific effects .

How can I design experiments to study synthetic phenotypes involving DrpB?

To investigate synthetic phenotypes involving DrpB, the following experimental approach is recommended:

• Gene deletion construction: Create a clean ΔdrpB deletion mutant using lambda Red recombineering. Replace the drpB coding sequence with a selectable marker (e.g., cat cassette), then remove the marker if desired .

• Candidate gene selection: Select genes for testing synthetic interactions based on:

  • Known division genes (e.g., dedD, ftsEX)

  • Genes with similar localization patterns

  • Genes encoding proteins with similar topology

  • Genes with similar expression patterns or regulation

• Double mutant construction: Generate double mutants using P1 transduction to transfer marked deletions into the ΔdrpB background .

• Phenotypic characterization:

  • Test viability on different media (LB, LB0N) and at different temperatures (30°C, 37°C, 42°C)

  • Perform growth curve analysis in liquid media

  • Measure cell morphology changes using phase contrast microscopy

  • Quantify cell length distributions

  • Assess division frequency and septation using membrane staining

• Complementation tests: Verify phenotypes by complementation with plasmid-expressed DrpB or candidate gene .

Notable example: The ΔdrpB ΔdedD double mutant exhibits a striking synthetic phenotype with a 3-log drop in plating efficiency on LB at 42°C and severe filamentation in LB broth at 42°C, while single mutants show minimal defects .

How does DrpB function as a multicopy suppressor of ΔftsEX mutants?

DrpB was discovered as a multicopy suppressor of ΔftsEX mutants, which normally cannot form colonies on LB0N (low salt medium). The mechanism of suppression involves:

• Improved divisome assembly: Overproduction of DrpB in a ΔftsEX background improves recruitment of downstream division proteins, particularly the septal peptidoglycan synthase FtsI, as demonstrated by fluorescence microscopy with GFP-FtsI fusions .

• Mass action effects: Similar to other multicopy suppressors of ΔftsEX (FtsN, FtsP, DapE), overproduction of DrpB likely drives divisome assembly through mass action, compensating for the absence of FtsEX in the recruitment pathway .

• Medium-specific effect: Suppression is observed specifically on LB0N medium, where the ΔftsEX mutant has a profound viability defect. The suppression improves viability by approximately 5 logs compared to control plasmids .

• Division improvement: In addition to improving viability, DrpB overexpression reduces average cell length of ΔftsEX mutants from ~29 μm to ~17 μm when grown in LB0N broth, indicating more efficient division .

This suppression mechanism aligns with a network model of divisome assembly rather than a strictly linear recruitment pathway, as overproduction of various divisome components can compensate for the absence of others.

What protein-protein interactions have been identified for DrpB?

Protein-protein interactions for DrpB have been investigated using the Bacterial Adenylate Cyclase Two-Hybrid (BACTH) system, revealing several potential interaction partners:

Strong interactions were observed with:

• DamX (cell division protein)

• FtsI (septal peptidoglycan synthase)

• FtsN (essential late divisome protein)

• FtsQ (early divisome protein)

• YmgF (divisome protein, one configuration only)

Weaker interactions were observed with:

• DedD (cell division protein)

• FtsA (cytoplasmic division protein)

Little to no interaction was observed with:

• Blr (small membrane protein)

• DrpB itself (no homo-oligomerization)

• FtsB (divisome protein)

• FtsL (divisome protein)

• FtsX (divisome protein)

• FtsZ (cytoskeletal division protein)

What is the relationship between DrpB and DedD?

DrpB and DedD exhibit a synthetic lethal relationship that provides important insights into their functions:

• Synthetic phenotype: While a ΔdrpB single mutant shows no significant division defects, and a ΔdedD mutant shows only mild defects, the ΔdrpB ΔdedD double mutant exhibits a severe division defect with extensive filamentation and a 3-log reduction in viability when grown in LB medium at 42°C .

• Medium-specific effect: Intriguingly, the synthetic phenotype is observed in LB (high osmolarity) but not in LB0N (low osmolarity), despite DrpB localizing primarily in low osmolarity conditions .

• Functional relationship: DedD is known to activate septal peptidoglycan synthesis, suggesting DrpB may play a complementary or redundant role in this process, possibly by improving FtsN activity or another aspect of divisome function .

• Suppression potential: The severe phenotype of the double mutant provides a useful tool for studying DrpB function through suppressor screens and for testing the functionality of DrpB variants and fusions .

How do osmotic conditions affect DrpB function and localization?

The relationship between osmotic conditions and DrpB function/localization is complex:

• Localization pattern: DrpB-GFP localizes to the division site in approximately 30% of cells in low osmolarity medium (LB0N) but in only about 1% of cells in standard LB or when LB0N is supplemented with 200 mM NaCl, proline, or sucrose .

• Expression levels: The dramatic difference in localization is not due to differences in expression levels, as Western blotting shows similar quantities of fusion protein under both conditions .

• Functional paradox: Despite primarily localizing in low osmolarity conditions, the functional importance of DrpB (revealed by the ΔdrpB ΔdedD synthetic phenotype) is most evident in high osmolarity conditions .

• Molecular mechanisms: The molecular basis for this osmolarity-dependent localization remains unknown but could involve:

  • Conformational changes in DrpB structure

  • Altered interactions with other divisome components

  • Changes in membrane properties affecting protein-membrane interactions

  • Differential recruitment mechanisms

This makes DrpB unique among division proteins, as most divisome components show enhanced localization in high osmolarity conditions, not reduced localization .

What are the correct translation start sites for DrpB expression?

Determining the correct translation start site for DrpB required detailed molecular analysis:

• Annotated start: DrpB was initially annotated as starting with a GTG codon (position 1) and predicted to encode a 121-amino-acid protein .

• Experimental evidence: Several lines of evidence demonstrated that translation primarily initiates at an ATG annotated as codon 22:

  • Mutational analysis: Changing the codon for Ile 10 to a stop codon did not abolish function, but stop codons at positions 23 and 30 did eliminate function .

  • Western blotting: Analysis of GFP-tagged DrpB expressed from its native chromosomal locus revealed only the shorter (M22)DrpB-GFP protein (~38.8 kDa) and not the longer (M1)DrpB-GFP (~41.3 kDa) .

  • Both GTG-1 and ATG-22 had to be changed to alanine codons simultaneously to completely abolish function, indicating that translation can initiate at either position, though ATG-22 is strongly preferred .

• Promoter location: RNA-seq data and experimental evidence indicate that the promoter for DrpB is located between GTG-1 and ATG-22, further supporting ATG-22 as the primary start codon .

The correct identification of the translation start site is critical for proper expression of functional recombinant DrpB in research applications.

How can I design experiments to study the effects of multicopy suppression by DrpB?

To study multicopy suppression effects of DrpB on ΔftsEX mutants, the following experimental design is recommended:

• Strain construction:

  • Create a clean ΔftsEX deletion strain (using lambda Red recombineering)

  • Prepare control strains (wild-type, single gene deletions)

• Plasmid construction:

  • Clone drpB into expression vectors (e.g., pBAD33 for arabinose-inducible expression)

  • Create appropriate control plasmids (empty vector, other known suppressors)

  • Consider testing various truncations or mutations of DrpB to identify functional domains

• Viability assays:

  • Plate serial dilutions on permissive (LB) and non-permissive (LB0N) media

  • Quantify colony formation to measure suppression efficiency (log improvement)

  • Test suppression at different temperatures (30°C, 37°C, 42°C)

• Divisome assembly assessment:

  • Introduce GFP-tagged division proteins (e.g., GFP-FtsI) on compatible plasmids

  • Use fluorescence microscopy to quantify localization with and without DrpB overexpression

  • Analyze cell morphology and division site formation

• Controls and variables to include:

  • Empty vector controls

  • Known suppressors (FtsN, FtsP) as positive controls

  • Varying inducer concentrations to establish dose-response relationships

  • Media with different osmolyte compositions

This experimental design will allow quantitative assessment of DrpB's suppression effects and provide insights into its mechanism of action.

What methods can be used to create and verify drpB gene deletions?

For creating and verifying clean drpB gene deletions:

• Lambda Red Recombineering:

  • Design primers with 50-bp homology to regions flanking drpB and 20-bp homology to a selectable marker (e.g., cat cassette conferring chloramphenicol resistance)

  • Amplify the marker by PCR

  • Transform the PCR product into a strain expressing lambda Red recombination functions (e.g., strain DY330)

  • Select for marker integration

  • Optionally remove the marker using FLP recombinase if flanked by FRT sites

• Verification methods:

  • PCR verification: Design primers flanking the deletion site and within the gene to confirm deletion

  • Sequencing: Verify the deletion junction to ensure clean removal

  • Phenotypic testing: Confirm expected phenotypes (e.g., normal growth, synthetic phenotype with ΔdedD)

  • Complementation: Test if the phenotype can be rescued by plasmid-expressed DrpB

• Recommended controls:

  • Include wild-type and appropriate deletion controls in all experiments

  • For functional studies, complementation with plasmid-expressed DrpB is essential to confirm phenotypes are specifically due to drpB deletion

Example PCR verification strategy:

How can apparent contradictions in DrpB localization and function be reconciled?

The DrpB system presents several apparent contradictions that require careful experimental design to reconcile:

• Localization vs. function paradox: DrpB localizes primarily in low osmolarity conditions (LB0N) but shows its strongest phenotype (with ΔdedD) in high osmolarity conditions (LB) .

• Possible explanations and experimental approaches:

  a) Different detection thresholds:

  • Hypothesis: DrpB may localize in high osmolarity media but below detection limits

  • Test: Use super-resolution microscopy or more sensitive detection methods

  • Test: Try different fixation methods to preserve weak signals

  b) Transient localization:

  • Hypothesis: DrpB may localize briefly or at specific cell cycle stages in high osmolarity

  • Test: Time-lapse microscopy under different conditions

  • Test: Cell synchronization followed by microscopy at defined intervals

  c) Different functional states:

  • Hypothesis: DrpB may have different conformations/interactions in different media

  • Test: FRET analysis with interaction partners under different conditions

  • Test: Photo-crosslinking studies to capture transient interactions

  d) Redundant mechanisms:

  • Hypothesis: Other proteins may substitute for DrpB in high osmolarity media

  • Test: Suppressor screens of the ΔdrpB ΔdedD phenotype

  • Test: Transcriptome/proteome analysis under different conditions

• Experimental controls:

  • Always compare wild-type, single mutants, and double mutants in parallel

  • Test multiple media conditions and temperatures simultaneously

  • Include appropriate internal standards for microscopy to allow direct comparisons

By systematically testing these hypotheses with robust experimental designs, researchers can resolve the apparent contradictions in DrpB behavior and gain insight into its physiological role.

What approaches can identify the molecular mechanism of DrpB function?

Several approaches can help elucidate the molecular mechanism of DrpB function:

• Structure-function analysis:

  • Create site-directed mutations in conserved residues, particularly in the transmembrane domains

  • Test truncated versions to determine minimal functional units

  • Attempt protein crystallization or NMR studies of purified DrpB

  • Use computational modeling to predict structure and interactions

• Interaction mapping:

  • Employ pull-down assays with tagged DrpB to identify binding partners

  • Use crosslinking approaches to capture transient interactions

  • Perform systematic BACTH or split-GFP analysis with divisome components

  • Map interaction sites through targeted mutations and truncations

• In vivo dynamics:

  • Employ FRAP (Fluorescence Recovery After Photobleaching) to assess protein mobility

  • Use single-molecule tracking to observe DrpB behavior during division

  • Perform time-lapse microscopy under various conditions

  • Test DrpB recruitment kinetics in reconstituted systems

• Functional assays:

  • Develop biochemical assays for potential enzymatic activities

  • Test effects on peptidoglycan synthesis or remodeling

  • Examine membrane properties in DrpB mutants

  • Assess the impact on divisome assembly kinetics

These approaches, especially when used in combination, can provide significant insights into DrpB's molecular function in the divisome.

How might DrpB research inform our understanding of bacterial division networks?

Research on DrpB provides valuable insights into bacterial division networks:

• Network robustness: The ability of DrpB overexpression to suppress ΔftsEX mutations supports a network model of divisome assembly rather than a strictly linear pathway. This explains how bacteria can adapt to the loss of seemingly essential components .

• Conditional essentiality: DrpB exemplifies how proteins may be dispensable under standard laboratory conditions but critical under specific environmental stresses or genetic backgrounds, highlighting the importance of testing multiple conditions .

• Divisome discovery approaches: The identification of DrpB through a multicopy suppressor screen validates this approach for finding additional division proteins, especially those with redundant or condition-specific functions .

• Evolutionary insights: The limited phylogenetic distribution of DrpB suggests recent evolutionary acquisition in enteric bacteria, providing an opportunity to study how division machinery evolves and incorporates new components .

• Research implications:

  • Test divisome components under diverse conditions to reveal cryptic functions

  • Look for synthetic phenotypes between seemingly dispensable proteins

  • Consider how osmotic conditions and other environmental factors influence divisome assembly

  • Explore the potential for condition-specific divisome configurations in different bacterial species