Recombinant Bacillus weihenstephanensis Acyl carrier protein (acpP)

What is Bacillus weihenstephanensis Acyl carrier protein (acpP)?

Acyl carrier protein (acpP) from Bacillus weihenstephanensis is a small protein (77 amino acids) with the amino acid sequence MADVLERVTK IVVDRLGVEE TEVVPAASFK EDLGADSLDV VELVMQLEDE FEMEISDEDA EKIATVGDAV TYIESHL . It functions as a carrier of acyl intermediates during fatty acid synthesis, particularly after being converted to its holo-form through phosphopantetheinylation. The protein is identified in the UniProt database with accession number A9VT88 and is derived from Bacillus weihenstephanensis strain KBAB4 .

How does Acyl carrier protein function in bacterial fatty acid synthesis?

Acyl carrier protein functions as a central component in type II fatty acid synthase systems found in bacteria. The protein exists initially as an inactive apo-form that requires post-translational modification to become functional. This conversion is catalyzed by ACP synthase (AcpS), which transfers the 4′-phosphopantetheine moiety from coenzyme A (CoA) onto a specific serine residue of apo-ACP . The resulting holo-ACP contains a flexible phosphopantetheine arm that covalently binds acyl intermediates during fatty acid synthesis, shuttling these intermediates between various enzymes in the biosynthetic pathway. This mechanism is essential for the production of fatty acids and phospholipids required for bacterial cell membrane structure and function.

What distinguishes Bacillus weihenstephanensis from other Bacillus species?

Bacillus weihenstephanensis is a psychrotolerant member of the Bacillus cereus group with several distinctive characteristics:

• Growth capability at temperatures as low as 6°C and inability to grow at 43°C on BHI agar

• Presence of specific genetic markers including the 16S rRNA gene signature for psychrotolerance and the cold shock protein gene cspA

• Some strains (e.g., MC67 and MC118) can produce cereulide, an emetic toxin previously thought to be exclusive to B. cereus

• Distinct genotypic and phenotypic properties that differentiate it from other B. cereus group members (B. anthracis, B. cereus, B. thuringiensis, and B. mycoides)

What is the structural basis for substrate specificity in B. weihenstephanensis acpP?

While specific structural data for B. weihenstephanensis acpP is limited, its function as an acyl carrier protein suggests several key structural features:

• A conserved serine residue that serves as the attachment site for the 4′-phosphopantetheine prosthetic group

• A helical bundle structure typical of ACPs that provides a stable scaffold

• Surface charge distribution that facilitates interactions with various enzymes in the fatty acid synthesis pathway

• Specific recognition elements that enable interaction with AcpS during the conversion to holo-ACP

The amino acid sequence of B. weihenstephanensis acpP would determine its specific structural properties and interaction capabilities within the bacterial fatty acid synthesis machinery.

How does temperature affect acpP function in psychrotolerant B. weihenstephanensis?

B. weihenstephanensis is psychrotolerant, capable of growth at temperatures as low as 6°C . This suggests potential adaptations in its proteins, including acpP, to function efficiently at low temperatures:

Research examining acpP temperature-dependent activity would be valuable for understanding how this essential protein supports the organism's psychrotolerant nature. Studies have shown that some B. weihenstephanensis strains can produce cereulide (an emetic toxin) at temperatures as low as 8°C , suggesting functional metabolic pathways—including fatty acid synthesis—at low temperatures.

What is the relationship between acpP and cereulide production in emetic B. weihenstephanensis strains?

Some B. weihenstephanensis strains (MC67 and MC118) can produce cereulide, a cyclic dodecadepsipeptide toxin that causes food poisoning . While acpP is primarily involved in primary fatty acid metabolism, connections to cereulide biosynthesis may exist:

• Cereulide is synthesized by non-ribosomal peptide synthetases (NRPS)

• NRPS systems often include ACP-like domains (peptidyl carrier proteins)

• Primary metabolism provides precursors and energy for secondary metabolite production

• Fatty acid synthesis pathways may share regulatory mechanisms with toxin production pathways

Research investigating differential expression of acpP in cereulide-producing versus non-producing strains, particularly under different temperature conditions (8-25°C), could reveal potential metabolic connections between primary and secondary metabolism in these bacteria .

What are the optimal expression conditions for producing recombinant B. weihenstephanensis acpP?

Based on the product information , recombinant B. weihenstephanensis acpP is expressed in E. coli. For optimal expression and purification, researchers should consider:

After purification, storage recommendations include keeping the protein at -20°C or -80°C for extended storage, with glycerol added to 5-50% as a cryoprotectant .

How can the phosphopantetheinylation state of recombinant acpP be assessed?

Determining whether recombinant acpP is in its inactive apo-form or functional holo-form is crucial for experimental applications. Several complementary methods can be employed:

• Mass spectrometry analysis:

  • MALDI-TOF or ESI-MS can detect the mass increase (+340 Da) due to phosphopantetheine attachment

  • Can provide quantitative assessment of apo/holo ratio in protein preparations

• Gel-based methods:

  • Conformational differences between apo- and holo-ACP can sometimes be resolved by native PAGE

  • Urea-PAGE can enhance mobility differences

• Functional assays:

  • Only holo-ACP can participate in fatty acid synthesis reactions

  • In vitro acylation assays using purified acyl-ACP synthetase and radiolabeled substrates

  • Coupled enzyme assays that depend on holo-ACP function

• Spectroscopic methods:

  • Holo-ACP often shows subtle differences in circular dichroism or fluorescence spectra

  • These differences can be enhanced by adding specific ligands or dyes

What are the recommended protocols for long-term storage of recombinant B. weihenstephanensis acpP?

According to the product information , the recommended storage conditions are:

• Store at -20°C, or at -80°C for extended storage

• For liquid preparations, add glycerol to a final concentration of 5-50% (with 50% recommended)

• Divide into small single-use aliquots to avoid repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

• Expected shelf life: 6 months for liquid form at -20°C/-80°C; 12 months for lyophilized form

For reconstitution of lyophilized protein:

• Briefly centrifuge the vial to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

How can researchers investigate acpP involvement in cold adaptation mechanisms?

As a psychrotolerant bacterium capable of growth at temperatures as low as 6-8°C , B. weihenstephanensis must maintain functional membrane biosynthesis at low temperatures. Research approaches to investigate acpP's role include:

• Comparative transcriptomics/proteomics:

  • Measure acpP expression levels at different temperatures (8°C, 15°C, 25°C)

  • Identify potential temperature-responsive regulatory elements in the acpP promoter region

• Biochemical characterization:

  • Compare enzyme kinetics of AcpS-mediated phosphopantetheinylation at different temperatures

  • Measure acpP-dependent fatty acid synthesis rates across temperature ranges

  • Analyze structural flexibility using hydrogen-deuterium exchange mass spectrometry

• Lipidomic analysis:

  • Correlate changes in fatty acid composition with acpP expression at different temperatures

  • Measure membrane fluidity in relation to fatty acid composition and growth temperature

• Genetic approaches:

  • Create temperature-sensitive mutations in acpP and evaluate their effects on growth

  • Perform complementation studies with acpP variants from psychrotolerant versus mesophilic bacteria

How does pH affect acpP stability and function in B. weihenstephanensis?

Research indicates that B. weihenstephanensis strains can grow and produce metabolites at various pH values, with acid resistance mechanisms being important for survival . For acpP function, relevant pH considerations include:

Experimental approaches to investigate pH effects include:

• Circular dichroism spectroscopy to assess structural changes across pH gradient

• Activity assays at different pH values to determine optimal conditions

• Gene expression analysis to determine if acpP is differentially regulated under acid stress

• Investigation of potential post-translational modifications induced by pH stress

What methods can distinguish between acpP from B. weihenstephanensis and other Bacillus species?

Distinguishing between closely related acpP proteins from different Bacillus species is important for ecological and diagnostic studies. Approaches include:

• PCR-based methods:

  • Species-specific primers targeting unique regions of the acpP gene

  • Real-time PCR with species-specific probes for quantitative detection

  • High-resolution melting analysis to distinguish closely related sequences

• Protein-based methods:

  • Mass spectrometry to identify species-specific peptides

  • Immunological approaches using antibodies against species-specific epitopes

  • 2D-gel electrophoresis combined with western blotting

• Sequence analysis:

  • While acpP is generally conserved, species-specific variations can be identified through comparative genomics

  • Analysis of flanking regions may provide additional discriminatory power

This discrimination is particularly relevant given that B. weihenstephanensis strains possess distinctive genetic markers that differentiate them from other members of the B. cereus group.

How might acpP be exploited as a target for antimicrobial development?

ACP synthase (AcpS), which activates acpP, has been described as "an attractive target for therapeutic intervention" . Research directions include:

• Inhibitor development:

  • Structure-based design of small molecules targeting the AcpS-acpP interaction

  • Peptidomimetics that compete with acpP for binding to AcpS

  • Natural product screening for inhibitors of acpP function

• Species selectivity:

  • Comparative analysis of acpP and AcpS across bacterial species to identify selective targeting opportunities

  • Focus on unique structural features of B. weihenstephanensis acpP

• Targeting psychrotolerant-specific features:

  • Cold-adapted enzymes often show unique structural features that could be exploited for selective inhibition

  • Temperature-dependent inhibitors could provide selective activity against psychrotolerant pathogens

What is the relationship between acpP sequence variation and metabolic diversity in B. weihenstephanensis strains?

B. weihenstephanensis strains show metabolic diversity, including variation in cereulide production . Investigating acpP's role in this diversity could involve:

• Comparative genomics:

  • Sequence analysis of acpP across multiple B. weihenstephanensis strains

  • Correlation of sequence variations with specific metabolic phenotypes

  • Identification of potential selective pressures on acpP evolution

• Functional characterization:

  • Heterologous expression and biochemical characterization of acpP variants

  • Complementation studies in acpP-deficient backgrounds

  • Investigation of substrate specificity differences between variants

• Ecological context:

  • Analysis of acpP variations in relation to strain isolation sources

  • Correlation with adaptation to specific environmental conditions

Understanding how sequence variation in core metabolic proteins like acpP contributes to strain-specific adaptations could provide insights into bacterial evolution and niche adaptation.

How does acpP interact with the wider metabolic network in B. weihenstephanensis?

Acyl carrier protein functions within a complex metabolic network, potentially influencing multiple pathways beyond fatty acid synthesis:

• Systems biology approaches:

  • Metabolic flux analysis to trace carbon flow through acpP-dependent pathways

  • Network modeling to predict the effects of acpP perturbations on global metabolism

  • Integration of transcriptomic, proteomic, and metabolomic data

• Protein-protein interaction studies:

  • Affinity purification-mass spectrometry to identify acpP interaction partners

  • Bacterial two-hybrid screening for novel interactions

  • Cross-linking studies to capture transient interactions in vivo

• Regulatory network mapping:

  • Identification of transcription factors controlling acpP expression

  • Investigation of potential feedback regulation by fatty acid intermediates

  • Analysis of coordination between primary and secondary metabolism

Understanding these interactions could provide insights into how B. weihenstephanensis adapts its metabolism to different environmental conditions, particularly low temperatures and varying pH levels .