Recombinant Kocuria rhizophila Acyl carrier protein (acpP)

What is the genomic context of acpP in Kocuria rhizophila?

Kocuria rhizophila acpP is encoded in its single circular chromosome (2,697,540 bp; G+C content of 71.16%) containing 2,357 predicted protein-coding genes . Unlike specialized acyl carrier proteins, the constitutive AcpP is typically located in a region involved with primary metabolism. The genome shows fairly good conservation of synteny with taxonomically related actinobacterial genomes, suggesting similar genomic organization of fatty acid biosynthesis genes. When conducting experiments, researchers should reference the complete genome sequence of K. rhizophila DC2201 (NBRC 103217) for accurate primer design and genetic manipulation .

What is the role of AcpP in K. rhizophila metabolism compared to specialized ACPs?

AcpP in K. rhizophila functions as the constitutive acyl carrier protein involved in primary fatty acid biosynthesis and transfer . Unlike specialized ACPs (such as NodF in rhizobia that is flavonoid-inducible, or RkpF that participates in capsular polysaccharide biosynthesis), AcpP is involved in the biosynthesis of common fatty acids essential for cell membrane formation and cellular function . K. rhizophila genome analysis reveals limited presence of secondary metabolism genes (only one each of nonribosomal peptide synthetase and type III polyketide synthase) , suggesting that its AcpP primarily serves essential metabolic functions rather than specialized biosynthetic pathways.

What expression system is most efficient for recombinant K. rhizophila AcpP production?

The T7 polymerase expression system in Escherichia coli, particularly with BL21(DE3) strain, has proven effective for overproduction of bacterial ACPs . For K. rhizophila AcpP, researchers should:

• Clone the acpP gene into a pET vector with a His-tag for purification

• Transform into E. coli BL21(DE3)

• Induce expression with IPTG (typically 0.5-1.0 mM)

• Grow cultures at lower temperatures (16-25°C) after induction to enhance solubility

• Harvest cells and purify using affinity chromatography

This approach has successfully been used for other actinobacterial ACPs and should be applicable to K. rhizophila AcpP with appropriate optimization of growth conditions and induction parameters.

How can post-translational modification of recombinant K. rhizophila AcpP be verified?

The critical post-translational modification of AcpP is the attachment of the 4'-phosphopantetheine prosthetic group. To verify this modification:

• Mass Spectrometry Analysis: Compare the molecular weight of the expressed protein with the theoretical weight to detect the mass shift (+339 Da) corresponding to the prosthetic group.

• Radioactive Labeling: Add radioactive β-alanine to the E. coli growth medium during expression, as β-alanine is incorporated into the 4'-phosphopantetheine group, allowing detection through autoradiography .

• Functional Assays: Test the ability of the purified AcpP to participate in fatty acid biosynthesis reactions in vitro, as only the modified form will be functional.

• Conformational Analysis: Use circular dichroism to detect structural changes associated with the attachment of the 4'-phosphopantetheine group.

The radioactive labeling approach has been specifically demonstrated with rhizobial ACPs and can be adapted for K. rhizophila AcpP verification .

How can K. rhizophila AcpP be utilized in studies of organic solvent tolerance mechanisms?

K. rhizophila shows notable organic solvent tolerance, making its AcpP potentially valuable for investigating membrane adaptations . Researchers can:

• Express recombinant AcpP in a solvent-sensitive host to evaluate its contribution to solvent tolerance

• Study the fatty acid profile changes mediated by AcpP under solvent stress conditions

• Investigate interactions between AcpP and membrane proteins that may contribute to solvent resistance

• Create chimeric AcpPs between K. rhizophila and solvent-sensitive species to identify domains responsible for solvent tolerance

This research direction is particularly relevant given that K. rhizophila has demonstrated utility as a biocatalyst in organic solvent-water biphasic reaction systems, suggesting unique adaptations in its fatty acid metabolism machinery .

What approaches can be used to study the interaction between K. rhizophila AcpP and fatty acid synthase components?

To investigate AcpP-enzyme interactions:

• Bacterial Two-Hybrid System: Construct fusion proteins of AcpP and fatty acid synthase components to detect protein-protein interactions in vivo.

• Surface Plasmon Resonance (SPR): Immobilize purified AcpP on a sensor chip and measure binding kinetics with various enzymes from the fatty acid synthesis pathway.

• Cross-linking Studies: Use chemical cross-linkers followed by mass spectrometry to identify proteins that interact with AcpP in vivo.

• Co-immunoprecipitation: Express tagged versions of AcpP and use antibodies to pull down protein complexes for identification.

• Crystallography of Protein Complexes: Attempt to co-crystallize AcpP with known interacting enzymes to determine the structural basis of interactions.

These approaches will help elucidate the specific molecular mechanisms by which K. rhizophila AcpP participates in fatty acid biosynthesis pathways.

How does K. rhizophila AcpP contribute to the rhizosphere adaptation of this bacterium?

K. rhizophila was originally isolated from the rhizoplane of narrow-leaved cattail (Typha angustifolia) , suggesting specific adaptations to this environment. To study AcpP's role in rhizosphere adaptation:

• Compare fatty acid profiles of wild-type and acpP mutants grown in rhizosphere-mimicking conditions

• Investigate transcriptional regulation of acpP in response to plant root exudates

• Analyze the role of AcpP-dependent fatty acid modifications in bacterial attachment to plant surfaces

• Study the production of specialized lipids that may mediate plant-microbe interactions

This research direction connects to K. rhizophila's ecological niche and may reveal specialized roles of AcpP beyond primary metabolism, potentially in producing lipid compounds that facilitate plant-microbe symbiosis or competition in the rhizosphere.

What strategies can overcome low solubility of recombinant K. rhizophila AcpP?

Researchers frequently encounter solubility issues with recombinant ACPs. To improve solubility:

• Expression Temperature Optimization: Lower induction temperatures (16-20°C) often improve proper folding

• Solubility Tags: Fuse MBP (maltose-binding protein) or SUMO tags to enhance solubility

• Co-expression with Chaperones: Co-express with molecular chaperones like GroEL/GroES

• Buffer Optimization: Screen various buffers with different pH values and salt concentrations

• Refolding Protocols: Develop refolding protocols from inclusion bodies if soluble expression fails

How can researchers address incomplete post-translational modification of K. rhizophila AcpP in heterologous expression systems?

In E. coli expression systems, AcpP may not be fully modified with the 4'-phosphopantetheine group. To improve modification rates:

• Co-express with PPTase: Co-express a compatible phosphopantetheinyl transferase (PPTase) such as Sfp from Bacillus subtilis

• In vitro Modification: Purify the apo-AcpP and perform in vitro modification with purified PPTase and CoA

• Expression Host Selection: Use expression hosts with higher endogenous PPTase activity

• Optimization of Culture Conditions: Supplement growth media with pantothenic acid precursors

Researchers should verify modification status by mass spectrometry to confirm the percentage of holo-AcpP (modified form) versus apo-AcpP (unmodified form) in their preparations.

How does K. rhizophila AcpP compare functionally with ACPs from other actinobacteria?

To perform comparative functional analysis:

• Clone and express ACPs from related actinobacteria (e.g., Micrococcus luteus) and K. rhizophila

• Evaluate their ability to complement ACP-deficient mutants

• Compare substrate specificities using in vitro assays with various fatty acid precursors

• Analyze structural differences and relate them to functional variations

• Perform phylogenetic analysis to understand evolutionary relationships

This comparative approach can reveal unique aspects of K. rhizophila AcpP function that may relate to the bacterium's ecological niche or metabolic capabilities. The resuscitation-promoting factor (Rpf) from Micrococcus luteus, a related actinobacterium, has been shown to influence bacterial culturability , suggesting possible connections between primary metabolism (involving AcpP) and cell growth regulation that could be explored.

Can K. rhizophila AcpP be engineered to improve biocatalytic applications?

K. rhizophila has demonstrated potential in biocatalytic applications, particularly in organic solvent-water biphasic systems . To engineer AcpP for enhanced performance:

• Site-directed Mutagenesis: Modify key residues involved in substrate binding or protein-protein interactions

• Domain Swapping: Create chimeric ACPs by swapping domains with ACPs from other solvent-tolerant bacteria

• Directed Evolution: Apply directed evolution approaches to select for AcpP variants with desired properties

• Expression Level Modulation: Optimize expression levels to balance metabolic burden with production capacity

Engineered AcpP variants could potentially enhance K. rhizophila's performance in biocatalytic applications such as the production of (S)-styrene oxide, where the bacterium has already shown promise .

How do host-pathogen interactions influence the function of K. rhizophila AcpP in clinical isolates?

Given reports of K. rhizophila involvement in human infections , researchers studying clinical isolates should:

• Compare acpP sequences from environmental versus clinical isolates to identify potential adaptations

• Investigate whether AcpP contributes to survival within host environments (e.g., altered fatty acid profiles)

• Examine the role of AcpP in biofilm formation, which may contribute to medical device colonization

• Study potential inhibitors of AcpP as antimicrobial candidates

This research direction bridges environmental microbiology with clinical microbiology, potentially revealing adaptation mechanisms of K. rhizophila as it transitions from an environmental organism to a human opportunistic pathogen.