Recombinant Bdellovibrio bacteriovorus Bifunctional protein GlmU (glmU)

What is the bifunctional nature of GlmU in Bdellovibrio bacteriovorus?

GlmU in B. bacteriovorus, similar to its homologs in other bacteria, functions as a bifunctional enzyme with two distinct catalytic activities: N-acetylglucosamine-1-phosphate uridyltransferase and glucosamine-1-phosphate acetyltransferase. These activities are structurally segregated, with the N-terminal domain responsible for uridyltransferase activity and the C-terminal left-handed β-helix (LβH) domain mediating acetyltransferase function . The enzyme catalyzes two sequential reactions in the biosynthetic pathway of UDP-N-acetylglucosamine (UDP-GlcNAc), a crucial precursor for bacterial cell wall peptidoglycan and lipopolysaccharide biosynthesis. This bifunctionality makes GlmU an efficient metabolic checkpoint in cell wall component synthesis.

How does the structure of B. bacteriovorus GlmU compare to GlmU proteins from other bacterial species?

B. bacteriovorus GlmU shares the fundamental trimeric architecture observed in other bacterial GlmU proteins, particularly from E. coli and M. tuberculosis, consisting of:

While core catalytic residues are conserved, B. bacteriovorus GlmU shows unique surface-exposed regions that may reflect adaptation to its predatory lifestyle . Notably, the enzyme forms homotrimers with each active site formed at subunit interfaces, creating three independent active sites per trimer.

What are the optimal conditions for expressing recombinant B. bacteriovorus GlmU in E. coli?

Expression of recombinant B. bacteriovorus GlmU can be optimized using the following protocol:

• Vector selection: pET-based vectors (particularly pET28a with an N-terminal His6-tag) provide high expression levels under T7 promoter control .

• Expression strain: BL21(DE3) or Rosetta(DE3) strains are recommended, with the latter providing additional tRNAs for rare codons that may be present in B. bacteriovorus genes.

• Culture conditions:

  • Growth medium: LB supplemented with appropriate antibiotics

  • Temperature: Initial growth at 37°C until OD600 reaches 0.6-0.8

  • Induction: 0.5 mM IPTG followed by temperature downshift to 18-20°C

  • Post-induction time: 16-18 hours at reduced temperature

This approach typically yields 15-20 mg of purified protein per liter of bacterial culture. The reduced induction temperature is crucial for minimizing inclusion body formation and maintaining proper folding of the trimeric structure.

What purification strategy provides the highest yield and purity of functional B. bacteriovorus GlmU?

A multi-step purification protocol yields highly pure, active GlmU:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

  • Buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol

  • Gradient elution: 20-300 mM imidazole

• Intermediate purification: Ion exchange chromatography (IEX)

  • Q-Sepharose column at pH 8.0

  • Linear gradient of 0-500 mM NaCl

• Polishing step: Size exclusion chromatography (SEC)

  • Superdex 200 column

  • Buffer: 25 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol, 1 mM DTT

• Quality assessment: Purity >95% by SDS-PAGE and trimeric state confirmation by SEC-MALS

The trimeric assembly of GlmU can be stabilized throughout purification by including 5 mM MgCl2 in all buffers, as Mg2+ ions play a crucial role in the uridyltransferase activity and structural integrity of the enzyme.

How can the dual enzymatic activities of B. bacteriovorus GlmU be independently assessed?

The two catalytic activities of GlmU can be measured using the following assays:

Acetyltransferase activity assay:

• DTNB-based spectrophotometric assay

  • Reaction mixture: 50 mM Tris-HCl pH 7.5, 0.2 mM DTNB, 0.1 mM acetyl-CoA, 0.5 mM glucosamine-1-phosphate, and purified enzyme

  • Measure release of CoA-SH by monitoring absorbance at 412 nm

  • Activity calculation: ε412 = 13,600 M-1cm-1 for the thionitrobenzoate anion

Uridyltransferase activity assay:

• Coupled enzyme assay

  • Reaction mixture: 50 mM Tris-HCl pH 7.5, 5 mM MgCl2, 2 mM UTP, 0.5 mM N-acetylglucosamine-1-phosphate, inorganic pyrophosphatase, and purified enzyme

  • Monitor release of inorganic phosphate using malachite green assay

• HPLC-based assay

  • Reaction conditions: 50 mM Tris-HCl pH 7.5, 5 mM MgCl2, 2 mM UTP, 0.5 mM N-acetylglucosamine-1-phosphate, and enzyme

  • Analyze UDP-GlcNAc formation using anion exchange HPLC with UV detection at 260 nm

For B. bacteriovorus GlmU, the optimal temperature for both activities is typically 30°C, which aligns with the physiological temperature range in which this predatory bacterium operates.

What approaches can be used to investigate the role of GlmU in B. bacteriovorus predation and life cycle?

To investigate GlmU's role in B. bacteriovorus predation, researchers can employ:

• Conditional gene expression systems

  • Since complete glmU deletion is likely lethal (as suggested by studies in other bacteria) , a theophylline-responsive riboswitch system can be implemented to regulate GlmU expression

  • The theophylline riboswitch can be inserted into the chromosome to control glmU expression levels

• Predation efficiency assays

  • Predatory capability can be quantified by measuring:

    • Plaque formation on prey bacterial lawns

    • Reduction in prey cell optical density

    • Time-lapse microscopic observation of predation events

  • Compare wild-type B. bacteriovorus with GlmU-modulated strains

• Biofilm formation analysis

  • Crystal violet staining to quantify biofilm formation

  • Confocal microscopy of fluorescently labeled bacteria to assess biofilm architecture

  • Comparison between wild-type and GlmU-altered strains to determine the role of GlmU in biofilm development, which has been implicated in other bacteria

• Growth phase-specific expression analysis

  • qRT-PCR to measure glmU expression during different phases of the predatory life cycle

  • Western blotting with anti-GlmU antibodies to track protein levels during attack phase versus intraperiplasmic growth phase

What are the most effective genetic tools for manipulating the glmU gene in B. bacteriovorus?

Several genetic tools have proven effective for manipulating genes in B. bacteriovorus:

• Markerless deletion system

  • pK18mobsacB-based suicide plasmids containing glmU flanking regions

  • Sucrose counter-selection for second crossover events

  • Particularly useful for creating partial deletions or point mutations in glmU

• Chromosomal insertion of regulatory elements

  • Theophylline-responsive riboswitches can be inserted to control glmU expression

  • Merodiploid selection followed by second crossover selection with sucrose

• Complementation systems

  • pSUP404.2 plasmid system for introducing wildtype or mutant glmU variants

  • Native B. bacteriovorus promoters (P1753, P3184, PAPSRNA5, or PmerRNA) provide stronger expression than heterologous promoters like Plac

• Promoter-reporter fusions

  • mCherry or GFPuv reporters to monitor glmU expression patterns

  • Can be designed with optimized ribosome binding sites (RBS) for enhanced expression in B. bacteriovorus

When designing primers for glmU manipulation, it's important to note that B. bacteriovorus uses a different codon preference than E. coli, with higher GC content in the third codon position.

How can CRISPR-Cas9 technology be adapted for precise editing of glmU in B. bacteriovorus?

While CRISPR-Cas9 systems are not yet widely reported for B. bacteriovorus, a potential protocol can be designed based on successful adaptations in other delta-proteobacteria:

• Vector design considerations:

  • Two-plasmid system: one encoding Cas9 under control of an inducible promoter, and one containing sgRNA and homology arms

  • Codon-optimized Cas9 for B. bacteriovorus

  • Promoters active during the attack phase (such as P1753 or P3184) to drive expression

• sgRNA design parameters:

  • Target unique PAM sites in glmU

  • Verify sgRNA specificity against the B. bacteriovorus genome

  • Design homology arms of 800-1000 bp flanking the cut site

• Transformation protocol:

  • Conjugation from E. coli S17-1 donor strain

  • Sequential introduction of Cas9 and sgRNA plasmids

  • Selection on prey lawns with appropriate antibiotics

• Validation methods:

  • PCR screening of potential mutants

  • Sequencing of the modified glmU locus

  • Phenotypic characterization through predation assays

• Potential challenges:

  • Cytotoxicity of Cas9 expression in B. bacteriovorus

  • Low efficiency of homology-directed repair

  • Need for precise temporal control of Cas9 expression

How do structural features of B. bacteriovorus GlmU contribute to its role in the predatory life cycle?

B. bacteriovorus GlmU likely shares key structural features with GlmU enzymes from other bacteria, but with adaptations specific to its predatory lifestyle:

• Trimeric architecture: Like other bacterial GlmU proteins, B. bacteriovorus GlmU likely forms a homotrimer with three active sites at subunit interfaces . This structure maximizes catalytic efficiency during the rapid remodeling of cell wall components required during prey invasion and intraperiplasmic growth.

• Domain-specific functions:

  • N-terminal domain: Contains the uridyltransferase active site with conserved metal-binding residues for Mg2+ coordination

  • C-terminal domain: Forms a left-handed β-helix with acetyltransferase activity and CoA binding site

  • Interdomain flexibility: Likely allows for conformational changes during the predatory life cycle

• Potential predation-specific adaptations:

  • Surface charge distribution may be optimized for function within the prey's periplasmic space

  • Substrate binding pockets might show modifications for rapid catalysis during the compressed timeframe of the predatory cycle

  • Regulatory sites might exist for rapid modulation of activity during transition between attack and growth phases

Structural alignment of B. bacteriovorus GlmU with characterized GlmU structures from E. coli and M. tuberculosis would reveal conserved catalytic residues and highlight potential predator-specific features that could be targeted for further investigation.

What structural insights can inform the design of catalytic site mutations to study GlmU function in B. bacteriovorus?

Based on structural studies of GlmU from other bacteria, the following catalytic site mutations would be informative for B. bacteriovorus GlmU functional studies:

Uridyltransferase domain mutations:

• Metal coordination site:

  • Mutations in the conserved DXD motif (likely Asp105 and Asp107 based on E. coli homology)

  • Expected outcome: Complete loss of uridyltransferase activity while preserving acetyltransferase function

• UTP binding residues:

  • Conservative substitutions in lysine residues that interact with UTP phosphates

  • Expected outcome: Altered substrate affinity without complete loss of function

Acetyltransferase domain mutations:

• Catalytic histidine:

  • Mutation of the conserved histidine (likely His377 based on E. coli homology) to alanine

  • Expected outcome: Significant reduction in acetyltransferase activity while maintaining uridyltransferase function

• CoA binding site:

  • Mutations in residues interacting with the adenosine portion of CoA

  • Expected outcome: Altered CoA binding kinetics with potential effects on catalytic efficiency

Interface mutations:

• Trimer interface residues:

  • Conservative substitutions at subunit interfaces

  • Expected outcome: Destabilized trimer formation leading to altered catalytic efficiency

These structure-guided mutations would allow for domain-specific functional analysis and help elucidate the relative importance of each catalytic activity during different stages of the B. bacteriovorus life cycle.

How does B. bacteriovorus GlmU differ from homologs in its prey bacteria, and what are the functional implications?

A comparative analysis of B. bacteriovorus GlmU with homologs from common prey bacteria reveals several distinctions:

These differences suggest B. bacteriovorus GlmU may have evolved specific adaptations that:

• Enable rapid cell wall remodeling during the transition from attack phase to intraperiplasmic growth

• Support efficient utilization of prey-derived metabolites for UDP-GlcNAc synthesis

• Coordinate cell wall biosynthesis with the unique bidirectional growth pattern observed during intraperiplasmic elongation

• Potentially contribute to predator-specific cell wall modifications that protect against prey hydrolytic enzymes

Sequence alignment studies would be valuable to identify predator-specific amino acid substitutions that could be experimentally investigated to determine their contribution to the predatory lifestyle.

What evolutionary insights can be gained from analyzing GlmU sequences across different Bdellovibrio and Like Organisms (BALOs)?

Phylogenetic analysis of GlmU across the BALOs group can provide valuable evolutionary insights:

• Evolutionary conservation patterns:

  • Core catalytic residues show high conservation across all BALOs

  • The highest sequence divergence typically occurs in surface-exposed regions

  • The N-terminal uridyltransferase domain tends to be more conserved than the C-terminal acetyltransferase domain

• Predation-specific adaptations:

  • BALOs with broader prey ranges may show more flexible substrate binding pockets

  • Epibiotic predators (attaching to prey surface) versus periplasmic invaders show distinct patterns in cell wall biosynthesis enzymes

  • Host-independent variants often display mutations affecting cell wall modification pathways

• Horizontal gene transfer assessment:

  • Analysis of GC content and codon usage in glmU can reveal potential horizontal gene transfer events

  • Comparison with prey bacteria GlmU sequences can identify potential genetic exchange

A comprehensive phylogenetic tree of BALOs GlmU proteins would likely show clustering according to predatory strategy (periplasmic vs. epibiotic) rather than strictly following taxonomic relationships, highlighting the importance of GlmU in the evolution of bacterial predation mechanisms.

How can B. bacteriovorus GlmU be leveraged for designing inhibitors against pathogenic bacteria?

B. bacteriovorus GlmU offers unique advantages for developing antimicrobial strategies:

• Structural template for inhibitor design:

  • The highly conserved active sites of GlmU across bacterial species make B. bacteriovorus GlmU a valuable structural template

  • Comparative analysis with pathogen GlmU structures can reveal subtle differences for selective targeting

  • The naturally predatory role of B. bacteriovorus may provide insights into vulnerable aspects of bacterial cell wall biosynthesis

• High-throughput screening approaches:

  • Recombinant B. bacteriovorus GlmU can be used in activity-based assays to screen compound libraries

  • Fluorescence-based assays monitoring either acetyltransferase or uridyltransferase activity

  • Fragment-based drug discovery utilizing the solved crystal structure

• Bifunctional inhibitor development:

  • Design of molecules that simultaneously target both enzymatic activities

  • Potential for reduced resistance development due to the essential nature of both functions

• Domain-specific targeting:

  • C-terminal acetyltransferase domain is absent in eukaryotes, making it a selective antimicrobial target

  • N-terminal domain inhibitors could be developed with structural features that prevent human cellular uptake

The unique evolutionary adaptations in B. bacteriovorus GlmU may reveal novel inhibitory mechanisms that could be applied to combat antibiotic-resistant pathogens.

What methodological approaches can be used to study the role of GlmU in B. bacteriovorus biofilm interactions?

To investigate GlmU's role in B. bacteriovorus biofilm interactions, researchers can employ:

• Experimental biofilm models:

  • Flow cell systems to establish prey bacterial biofilms

  • Confocal laser scanning microscopy with fluorescently-labeled B. bacteriovorus (wild-type and GlmU-modified strains)

  • Time-lapse imaging to track predator penetration, movement, and predation within biofilms

• Quantitative biofilm predation assays:

  • Crystal violet staining to quantify biofilm biomass before and after predation

  • Live/dead staining to assess predation efficiency within biofilm structures

  • qPCR-based quantification of prey versus predator cells in mixed biofilms

• GlmU activity manipulation approaches:

  • Riboswitch-controlled expression of GlmU during biofilm predation

  • Chemical inhibition using established GlmU inhibitors

  • Expression of wild-type versus catalytic mutants

• Biofilm matrix analysis:

  • Comparison of exopolysaccharide composition in biofilms with and without predation

  • Correlation of UDP-GlcNAc levels with predation efficiency

  • Investigation of potential B. bacteriovorus glycosidases that may work synergistically with GlmU-dependent processes

These methodological approaches would help elucidate whether GlmU plays a direct role in biofilm penetration, prey cell wall degradation, or predator propagation within biofilm structures, insights that could inform new biofilm control strategies.