Recombinant Bradyrhizobium japonicum Acyl carrier protein (acpP)

What is Acyl Carrier Protein (acpP) and what role does it play in Bradyrhizobium japonicum?

Acyl Carrier Protein (acpP) in Bradyrhizobium japonicum functions as a central component in fatty acid biosynthesis and serves as an essential cofactor in numerous metabolic pathways. This small, acidic protein contains a 4'-phosphopantetheine prosthetic group that acts as a flexible arm for carrying acyl intermediates during fatty acid synthesis. In B. japonicum, acpP plays a particularly significant role in the biosynthesis of fatty acids required for membrane phospholipids and lipid A components of the bacterial cell envelope.

Evidence indicates that LuxI homologs in proteobacteria prefer fatty acid-acyl carrier protein (ACP) over fatty acyl-CoA as the acyl substrate for signal synthesis in quorum sensing systems . This preference demonstrates the critical importance of acpP in bacterial communication systems, which are essential for coordinating population behaviors during symbiotic interactions with legume hosts like soybean.

How does B. japonicum utilize different sulfur sources and how might this relate to acpP function?

B. japonicum demonstrates remarkable metabolic versatility in sulfur utilization, being able to grow using sulfate, cysteine, sulfonates, and sulfur-ester compounds as sole sulfur sources . This adaptability is particularly important since sulfur in agricultural soils is predominantly present as sulfonates and sulfur esters rather than inorganic forms .

Research has identified two sets of gene clusters (bll6449 to bll6455 and bll7007 to bll7011) that are crucial for the utilization of sulfonate sulfur sources in B. japonicum . The expression of these genes, including bll6451 and bll7010, occurs in symbiotic nodules, indicating their importance during the symbiotic state .

The relationship to acpP function lies in the potential incorporation of sulfur-containing amino acids into the protein structure of acpP and the possible role of sulfur in post-translational modifications that may affect acpP activity. Additionally, adequate sulfur metabolism is essential for the synthesis of iron-sulfur clusters in various proteins that may interact with acpP in metabolic pathways.

What genetic expression patterns are observed for acpP in B. japonicum during free-living versus symbiotic states?

The expression patterns of genes in B. japonicum vary significantly between free-living and symbiotic states. While the search results don't specifically mention acpP expression patterns, we can draw parallels from related gene expression studies.

Transcriptome analyses have shown that various B. japonicum genes, including those involved in quorum sensing like bjaI and bjaR, are expressed both in laboratory culture and during symbiosis . These genes are particularly relevant as they are part of signaling pathways that may involve acpP-dependent fatty acid derivatives.

For researchers investigating acpP expression specifically, quantitative RT-PCR methodologies similar to those used for studying bjaI and bjaR expression would be appropriate . Such analysis would likely reveal differential expression patterns between free-living and bacteroid states, reflecting the metabolic shifts that occur during nodule formation and nitrogen fixation.

What is the relationship between acpP and quorum sensing in B. japonicum?

Quorum sensing in B. japonicum involves a sophisticated system centered around the production and detection of acyl-homoserine lactone (acyl-HSL) signals. The BjaI-BjaR system in B. japonicum USDA110 represents a quorum sensing system that is expressed both in laboratory culture and during symbiosis .

The connection to acpP lies in the biosynthetic pathway of acyl-HSLs. LuxI-type synthases, such as BjaI, utilize acyl-ACP (where the acyl group is carried by the acyl carrier protein) as substrates for synthesizing acyl-HSL signal molecules . Research suggests that LuxI homologs preferentially use fatty acid-acyl carrier protein (ACP) rather than fatty acyl-CoA as the acyl substrate for signal synthesis .

Experimental approaches to study this relationship include:

• Construction of B. japonicum reporter strains with lacZ fusions to monitor gene expression in response to acyl-HSLs

• HPLC separation of cell-free fluid extracts to identify bioactive compounds

• Radio-labeling experiments using [carboxy-14C] to trace acyl group transfer from acpP to acyl-HSLs

These methodologies have successfully identified unique acyl-HSL signals in related systems and can be applied to study the role of acpP in B. japonicum quorum sensing .

How do mutations in acpP affect nitrogen fixation efficiency in B. japonicum-soybean symbiosis?

While the search results don't directly address acpP mutations, we can extrapolate from studies of related mutants. B. japonicum mutants defective in sulfonate utilization operons maintained their symbiotic capability with soybean, suggesting functional redundancy or alternative sulfur source availability in planta .

For acpP mutations, the effects would likely be more pronounced given the central role of fatty acid biosynthesis in bacterial metabolism. Potential impacts include:

• Altered membrane composition affecting bacteroid differentiation

• Disrupted signaling pathways essential for symbiotic development

• Impaired energy metabolism affecting nitrogen fixation efficiency

Experimental approaches to investigate these effects would include:

• Generation of conditional acpP mutants (as complete knockouts might be lethal)

• Measurement of nitrogenase activity using acetylene reduction assays

• Microscopic analysis of nodule development and bacteroid morphology

• Metabolomic profiling to detect changes in fatty acid composition

• Comparative plant growth studies under nitrogen-limited conditions

These methodologies would provide insights into the specific contributions of acpP to symbiotic nitrogen fixation.

What biochemical interactions occur between acpP and other proteins involved in fatty acid synthesis in B. japonicum?

Acyl carrier protein interacts with numerous enzymes in the fatty acid synthesis pathway, including:

• AcpS (ACP synthase) - responsible for the attachment of the 4'-phosphopantetheine prosthetic group

• FabD (malonyl-CoA:ACP transacylase) - transfers malonyl groups to ACP

• FabH (β-ketoacyl-ACP synthase III) - initiates fatty acid elongation

• FabG (β-ketoacyl-ACP reductase) - catalyzes the first reduction step

• FabZ (β-hydroxyacyl-ACP dehydratase) - catalyzes dehydration

• FabI (enoyl-ACP reductase) - catalyzes the final reduction step

In B. japonicum, these interactions may have unique characteristics due to the specialized membrane requirements of bacteroids. Particularly interesting would be potential interactions with enzymes involved in the biosynthesis of specialized fatty acids used for signaling or membrane adaptation during symbiosis.

Experimental methods to study these interactions include:

• Protein co-immunoprecipitation followed by mass spectrometry

• Bacterial two-hybrid screening

• Surface plasmon resonance to measure binding kinetics

• X-ray crystallography of protein complexes

• Cross-linking studies followed by proteomic analysis

What are optimal expression systems for producing recombinant B. japonicum acpP?

When expressing recombinant B. japonicum acpP, researchers should consider the following expression systems and optimization strategies:

It is critical to validate that recombinantly produced acpP maintains its biological activity. As Dr. Michael Fiebig notes regarding recombinant proteins, "Just because you can make 50 μg of [a protein] in a bacterial system and perform sufficient testing with it does not mean you will be able to manufacture hundreds or thousands of mg of whole [protein] in a mammalian expression system" . This highlights the importance of scalability considerations in expression system selection.

For purification, a common approach utilizes a His6-tag that can be later removed by specific proteases. The purification protocol typically includes:

• Immobilized metal affinity chromatography (IMAC)

• Tag removal by TEV protease

• Reverse IMAC to remove the cleaved tag

• Size exclusion chromatography for final polishing

This strategy yields highly pure, native-like acpP suitable for structural and functional studies.

How can researchers effectively study the interaction between acpP and acyl-HSL synthases in B. japonicum?

To investigate the interactions between acpP and acyl-HSL synthases (like BjaI) in B. japonicum, researchers can employ several complementary approaches:

• In vitro reconstitution assays: Purified recombinant acpP and BjaI can be combined in the presence of necessary cofactors (ATP, SAM) and acyl substrates to measure acyl-HSL production. Products can be analyzed by LC-MS/MS.

• Acyl-ACP preparation: Different acyl groups can be loaded onto purified acpP using either chemical acylation or enzymatic loading with purified acyl-ACP synthetase. These acyl-ACPs can then be tested as substrates for BjaI.

• Binding studies: Isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR) can quantify binding affinities between acpP and BjaI, revealing how different acyl chains affect protein-protein interactions.

• Genetic approaches: Construction of reporter strains similar to the bjaI-lacZ fusion described in search result can help monitor how modifications to acpP affect signaling.

• Structural biology: X-ray crystallography or cryo-EM studies of the acpP-BjaI complex can reveal critical interaction interfaces.

The experimental design should consider that "antibodies discovered by recombinant methods require additional validation as they do not benefit from the tolerizing effect of an immune system" . Similarly, recombinant proteins used in these assays should be thoroughly validated for proper folding and activity.

What techniques can be used to study post-translational modifications of acpP in B. japonicum?

Post-translational modifications (PTMs) of acpP, particularly the attachment of the 4'-phosphopantetheine prosthetic group and subsequent acylation, are crucial for its function. The following methodologies can effectively characterize these modifications:

• Mass Spectrometry-Based Approaches:

  • LC-MS/MS analysis following tryptic digestion

  • Top-down proteomics using high-resolution MS

  • Phosphopantetheine ejection assays for acyl chain identification

  • Multiple reaction monitoring (MRM) for quantitative analysis

• Gel-Based Methods:

  • Conformationally-sensitive gel electrophoresis (holo- vs. apo-ACP)

  • Urea-PAGE for separation based on acyl chain length

  • Radioactive labeling with [14C]-labeled acyl substrates

• Spectroscopic Techniques:

  • Circular dichroism to monitor conformational changes upon acylation

  • NMR spectroscopy for structural characterization of modified acpP

Researchers should follow the GRADE approach for assessing certainty of evidence from these experiments, considering "risk of bias, inconsistency, indirectness, imprecision and publication bias" .

How can researchers design experiments to investigate the role of acpP in B. japonicum's adaptation to different environmental conditions?

To study how acpP contributes to B. japonicum's environmental adaptations, researchers can implement these experimental designs:

• Conditional Expression Systems:

  • Create strains with acpP under control of inducible promoters

  • Regulate acpP expression levels under different environmental stresses

  • Monitor growth, survival, and metabolic parameters

• Environmental Stress Experiments:

  • Subject cultures to varying pH, temperature, osmotic stress, and nutrient limitations

  • Measure acpP expression using qRT-PCR and reporter fusions

  • Analyze fatty acid profiles using GC-MS to correlate with acpP activity

• Soil Microcosm Studies:

  • Introduce wild-type and acpP-modified strains into soil microcosms

  • Monitor population dynamics using selective plating and qPCR

  • Analyze competition with indigenous soil bacteria

• Plant Interaction Studies:

  • Similar to the field trials performed with modified B. japonicum strains , examine how acpP modifications affect:

    • Nodulation efficiency

    • Nitrogen fixation rates

    • Competitiveness against native rhizobia

    • Plant growth parameters

• Sulfur Utilization Connection:

  • Investigate how acpP expression and activity change when B. japonicum utilizes different sulfur sources (sulfate, cysteine, sulfonates, and sulfur-esters)

  • Analyze whether acpP is involved in the regulatory networks controlling sulfur metabolism

These methodologies should be designed with consideration for the "functional redundancy" that often exists in bacterial systems, as observed with sulfonate utilization operons in B. japonicum .