Recombinant Bradyrhizobium japonicum 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (ispG)

What is Bradyrhizobium japonicum and why is it significant for ispG research?

Bradyrhizobium japonicum is a gram-negative, rod-shaped, nitrogen-fixing bacteria that forms a symbiotic relationship with Glycine max (soybean plants) . It belongs to the Proteobacteria phylum, specifically the Alphaproteobacteria class, and is classified within the Rhizobiales order and Bradyrhizobiaceae family . This organism is particularly significant for biosynthetic enzyme research due to its unique metabolic adaptations for nitrogen fixation and symbiotic lifestyle.

The bacteria colonize root nodules of soybean plants, where they receive carbon sources such as dicarboxylic acids (succinate, fumarate, and malate) from the plant while providing fixed nitrogen in return . This metabolic specialization makes B. japonicum an interesting source for studying specialized biosynthetic pathways, including the non-mevalonate pathway involving 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (ispG). As a slow-growing heterotrophic bacteria adapted to specific environmental conditions, its enzymes may exhibit unique kinetic properties or substrate specificities compared to homologs from other bacterial species .

What is the function of 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (ispG) in bacterial metabolism?

The 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (ispG) enzyme catalyzes a critical step in the non-mevalonate pathway (also known as the MEP pathway or 2-C-methyl-D-erythritol 4-phosphate pathway) for isoprenoid biosynthesis. This pathway is distinct from the mevalonate pathway found in eukaryotes and some bacteria. In the non-mevalonate pathway, ispG converts 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MEcPP) to 4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HMBPP).

The non-mevalonate pathway is essential for the production of isoprenoids, which are diverse compounds involved in various cellular functions including membrane structure, electron transport, and secondary metabolism. In B. japonicum specifically, isoprenoids play important roles in the symbiotic relationship with host plants, including the synthesis of signaling molecules and components of the bacterial membrane that interface with the plant symbiosome membrane . The ispG enzyme is of particular interest because it represents a potential target for antimicrobial development, as the non-mevalonate pathway is absent in mammals.

How does the genetic context of ispG in B. japonicum compare to other bacterial species?

The genetic context of ispG in Bradyrhizobium japonicum must be considered within the framework of this organism's complex genome. B. japonicum has been found to contain insertion sequence (IS) elements and repeated DNA sequences that can promote genomic rearrangements . These elements are particularly abundant in certain isolates known as highly reiterated sequence-possessing (HRS) isolates, which can contain 86-175 copies of specific repeated sequences .

When examining the ispG gene in B. japonicum, researchers should be aware that these genomic rearrangements might affect the stability and expression of biosynthetic genes. Comparative genomic analyses have shown that plant-associated gram-negative bacteria, including Bradyrhizobium species, often exhibit genomic instability that can affect genes responsible for plant associations . This genetic context is important when designing cloning strategies for ispG, as the presence of repeated sequences might complicate PCR amplification or recombinant expression.

What are the optimal conditions for expressing recombinant B. japonicum ispG in heterologous systems?

When expressing recombinant B. japonicum ispG in heterologous systems, several factors must be considered to achieve optimal expression. First, codon optimization is often necessary due to the significant difference in GC content and codon usage between B. japonicum and common expression hosts like E. coli. B. japonicum is characterized as a slow-growing bacterium , which may reflect differences in translation machinery and efficiency compared to fast-growing expression hosts.

For E. coli-based expression systems, BL21(DE3) strains or derivatives containing additional rare tRNA genes are recommended to address codon bias issues. Expression should be conducted at lower temperatures (16-20°C) after induction to improve protein folding, as ispG is an iron-sulfur cluster-containing enzyme that may require specific chaperones for proper folding. The growth medium should be supplemented with iron (typically 50-100 µM ferric ammonium citrate or ferrous sulfate) added at the time of induction to support iron-sulfur cluster formation.

Additionally, the slow growth characteristics of B. japonicum suggest that its proteins may have evolved for stability rather than rapid turnover . Therefore, longer expression times (24-48 hours) at lower temperatures may yield better results than standard short, high-temperature induction protocols typically used for E. coli proteins.

What purification strategies are most effective for obtaining active recombinant B. japonicum ispG?

Purification of active recombinant B. japonicum ispG requires strategies that preserve the integrity of its iron-sulfur cluster. A typical purification protocol would include the following steps:

• Cell lysis under anaerobic or low-oxygen conditions using a buffer containing:

  • 50 mM Tris-HCl, pH 8.0

  • 300 mM NaCl

  • 10% glycerol (as a stabilizing agent)

  • 1 mM DTT (to maintain reducing conditions)

  • Protease inhibitor cocktail

• Initial purification using immobilized metal affinity chromatography (IMAC) with a His-tag, which is the most common approach for recombinant ispG purification. The elution buffer typically contains 250-300 mM imidazole.

• Size exclusion chromatography as a polishing step, using a buffer containing:

  • 25 mM Tris-HCl, pH 7.5

  • 150 mM NaCl

  • 10% glycerol

  • 1 mM DTT

Throughout the purification process, it's critical to maintain reducing conditions and minimize exposure to oxygen, as the iron-sulfur cluster in ispG is oxygen-sensitive. All buffers should be degassed and, ideally, purification should be conducted in an anaerobic chamber or glove box. The addition of iron and sulfur sources during cell growth can improve the incorporation of the iron-sulfur cluster, thereby enhancing the yield of active enzyme.

How can researchers assess the activity and kinetic parameters of purified recombinant B. japonicum ispG?

To assess the activity and kinetic parameters of purified recombinant B. japonicum ispG, researchers can employ several complementary approaches:

• Spectrophotometric assays: ispG activity can be monitored by coupling the reaction to ferredoxin/ferredoxin reductase and following NADPH oxidation at 340 nm. The reaction mixture typically contains:

  • 100 mM Tris-HCl, pH 7.5

  • 100 mM NaCl

  • 5 mM DTT

  • 200 μM NADPH

  • 10 μM ferredoxin

  • 0.1 μM ferredoxin reductase

  • Varying concentrations of MEcPP substrate (1-200 μM)

  • 0.1-1 μM purified ispG enzyme

• HPLC-based assays: The conversion of MEcPP to HMBPP can be monitored directly using HPLC with UV detection at 270 nm or by LC-MS. This approach allows for direct quantification of both substrate consumption and product formation.

• NMR spectroscopy: For detailed mechanistic studies, 13C or 2H-labeled substrates can be used, and the reaction products analyzed by NMR to elucidate the reaction mechanism and identify potential intermediates.

The kinetic parameters (Km, kcat, kcat/Km) can be determined by varying the substrate concentration and fitting the initial velocity data to the Michaelis-Menten equation. It's important to ensure that the reactions are conducted under linear conditions with respect to both time and enzyme concentration. Given the oxygen sensitivity of the iron-sulfur cluster, all activity assays should be performed in an anaerobic environment or with suitable oxygen-scavenging systems.

What structural features distinguish B. japonicum ispG from homologs in other bacterial species?

B. japonicum ispG belongs to the family of iron-sulfur cluster-containing enzymes that catalyze challenging reductive dehydration reactions. While specific structural data for B. japonicum ispG is limited, comparative analysis with homologs from other bacterial species reveals several distinguishing features that likely impact its function.

The ispG enzyme typically contains a [4Fe-4S] cluster coordinated by three conserved cysteine residues, with the fourth coordination site available for substrate binding. The slow-growing nature of B. japonicum and its adaptation to the symbiotic lifestyle within legume root nodules suggests that its ispG may have evolved structural features optimized for functioning under the microaerobic conditions found in root nodules . These adaptations might include additional stabilizing interactions around the iron-sulfur cluster binding site or modified substrate binding pockets that reflect the specific metabolic profile of B. japonicum.

Additionally, the potential for genomic rearrangements in B. japonicum due to the presence of numerous insertion sequence elements raises the possibility of unique structural variations in its ispG compared to homologs from bacteria with more stable genomes. These structural distinctions may not only affect the enzyme's catalytic efficiency but also its interaction with redox partners and regulatory proteins.

How does the iron-sulfur cluster in B. japonicum ispG affect its catalytic mechanism and sensitivity to environmental conditions?

The [4Fe-4S] cluster in ispG plays a central role in its catalytic mechanism by facilitating electron transfer during the reductive dehydration of MEcPP to HMBPP. This cluster is highly sensitive to oxygen, which can lead to oxidative damage and enzyme inactivation. In B. japonicum, adaptation to the microaerobic environment of root nodules may have led to specific protective mechanisms around the iron-sulfur cluster.

The catalytic mechanism of ispG involves:

• Binding of the substrate MEcPP near the iron-sulfur cluster

• Electron transfer from the reduced [4Fe-4S]1+ cluster to the substrate

• Formation of a radical intermediate

• Dehydration and further reduction to form HMBPP

Environmental factors that can significantly impact the activity of B. japonicum ispG include:

In laboratory settings, maintaining anaerobic conditions during purification and assays is crucial for preserving ispG activity. The use of reducing agents (DTT, β-mercaptoethanol) and oxygen-scavenging systems can help protect the iron-sulfur cluster. Additionally, reconstitution of the iron-sulfur cluster may be necessary if it becomes damaged during purification, typically using iron and sulfide sources under reducing conditions.

What are the challenges and solutions for crystallizing recombinant B. japonicum ispG for structural studies?

Crystallizing recombinant B. japonicum ispG presents several challenges due to its intrinsic properties and the technical difficulties associated with iron-sulfur proteins. These challenges and potential solutions include:

• Protein stability and homogeneity:

  • Challenge: The iron-sulfur cluster in ispG can degrade during purification and crystallization attempts, leading to heterogeneous protein preparations.

  • Solution: Conduct all purification and crystallization steps under strictly anaerobic conditions, preferably in a glove box. Include stabilizing agents such as glycerol (10-20%) and reducing agents (5-10 mM DTT) in all buffers.

• Crystallization conditions:

  • Challenge: Iron-sulfur proteins often require specific conditions to maintain stability during crystallization.

  • Solution: Screen crystallization conditions using commercially available sparse matrix screens designed for metalloproteins. Consider using the microbatch-under-oil method to minimize oxygen exposure. Typical successful conditions often include PEG (polyethylene glycol) precipitants at concentrations of 10-20% and pH ranges of 6.0-8.0.

• Crystal quality and diffraction:

  • Challenge: Crystals of iron-sulfur proteins often diffract poorly due to intrinsic flexibility or degradation during data collection.

  • Solution: Collect diffraction data under cryogenic conditions with minimal exposure to air during crystal mounting. Consider using substrate analogs or inhibitors to stabilize the protein in a defined conformation, potentially improving crystal packing and diffraction quality.

• Phasing strategies:

  • Challenge: Obtaining phase information for structure determination.

  • Solution: Take advantage of the iron atoms in the Fe-S cluster for anomalous dispersion phasing methods. Collect multiple-wavelength anomalous dispersion (MAD) data at synchrotron beamlines, using the iron K-edge for optimal anomalous signal.

Given the genomic complexity of B. japonicum with its numerous insertion sequences and potential for genomic rearrangements , ensuring sequence verification of the crystallized construct is particularly important. Additionally, surface entropy reduction (SER) through site-directed mutagenesis of surface-exposed flexible residues can improve crystallization properties.

How does the ispG from B. japonicum compare functionally to homologs from other nitrogen-fixing bacteria?

The ispG enzyme from B. japonicum likely exhibits functional distinctions when compared to homologs from other nitrogen-fixing bacteria, reflecting the unique evolutionary adaptations of this slow-growing symbiotic organism. Comparative functional analysis reveals several key differences:

B. japonicum, as a slow-growing bacterium , likely possesses an ispG enzyme adapted for sustained activity rather than rapid turnover. This contrasts with faster-growing nitrogen fixers like certain Rhizobium species. The kinetic parameters often reflect these adaptations, with B. japonicum ispG potentially exhibiting lower kcat values but higher substrate affinity (lower Km) and better stability under the microaerobic conditions found in root nodules.

The root nodule environment where B. japonicum resides presents unique challenges, including microaerobic conditions, specific pH ranges, and distinct carbon source availability . These environmental factors have likely shaped the properties of B. japonicum ispG, potentially leading to adaptations in substrate specificity, cofactor requirements, and regulatory mechanisms compared to homologs from free-living nitrogen fixers or those that form different types of symbiotic relationships.

Additionally, the presence of highly reiterated DNA sequences and insertion elements in B. japonicum genomes suggests a capacity for genomic plasticity that might extend to the ispG gene, potentially resulting in strain-specific variations in enzyme properties that could be advantageous under different environmental conditions.

What insights can comparative genomics provide about the evolution of ispG in B. japonicum strains?

Comparative genomics offers valuable insights into the evolution of ispG in B. japonicum strains, particularly in the context of this organism's complex genome structure and capacity for genomic rearrangements. The search results indicate that B. japonicum strains, especially those designated as highly reiterated sequence-possessing (HRS) isolates, can contain numerous copies of repeated sequences (RS elements) that promote genomic rearrangements .

These genomic characteristics have several implications for ispG evolution:

• Potential for gene duplication and divergence: The presence of numerous insertion sequence elements increases the likelihood of gene duplication events, which could lead to the emergence of ispG variants with modified functions or expression patterns.

• Selection pressure in symbiotic contexts: Different B. japonicum strains have been isolated from various field sites , suggesting adaptation to specific environmental conditions. These adaptations may extend to metabolic enzymes like ispG, with selection favoring variants that function optimally under specific symbiotic conditions.

• Horizontal gene transfer potential: The genomic plasticity evidenced by the presence of insertion sequences and repeated elements suggests that horizontal gene transfer might have played a role in the evolution of ispG in B. japonicum, potentially incorporating beneficial variations from other bacterial species.

Analysis of 16S rRNA gene sequences has confirmed that even the HRS isolates with numerous genomic rearrangements remain classified as B. japonicum , indicating that these genomic changes represent intraspecies variation rather than speciation events. This genomic flexibility may contribute to the adaptability of B. japonicum to different symbiotic partners and environmental conditions.

How might the genomic context of ispG in HRS isolates affect its expression and function?

The genomic context of ispG in highly reiterated sequence-possessing (HRS) isolates of B. japonicum could significantly impact its expression and function through several mechanisms. HRS isolates contain extremely high numbers of repeated sequence elements (RSα and RSβ), with copy numbers ranging from 86 to 175 in some cases . These repetitive elements can drive genomic rearrangements that affect gene structure and regulation.

Potential effects on ispG expression and function in HRS isolates include:

• Altered gene expression: The search results indicate that HRS isolates show shifts and duplications of nif- and hup-specific hybridization bands , suggesting genomic rearrangements affecting symbiosis-related genes. Similar rearrangements affecting the ispG locus could alter its expression patterns through changes in promoter sequences or regulatory elements.

• Modified enzyme structure: If transposition events occur within or near the ispG gene, they could lead to modifications in the enzyme's structure, potentially altering its catalytic properties or stability. The tandem repeat structures observed in some HRS isolates are similar to those shown to cause transpositional rearrangements in E. coli, suggesting a mechanism for such modifications.

• Growth rate implications: HRS isolates exhibit slower growth than normal isolates , which could affect the cellular context in which ispG functions. These growth differences might reflect broader metabolic adaptations that indirectly impact isoprenoid biosynthesis pathways.

It's worth noting that despite these genomic rearrangements, the search results indicate no difference in symbiotic properties between HRS and normal isolates . This suggests that essential metabolic functions, potentially including the MEP pathway involving ispG, remain sufficiently conserved to maintain symbiotic capabilities.

What are the most promising applications of recombinant B. japonicum ispG in enzyme engineering?

Recombinant B. japonicum ispG offers several promising applications in enzyme engineering, leveraging the unique properties of this iron-sulfur enzyme from a symbiotic nitrogen-fixing bacterium. Key opportunities include:

• Development of oxygen-tolerant variants: Given that B. japonicum naturally functions in the microaerobic environment of root nodules , its ispG may possess features that could inform the engineering of oxygen-tolerant variants. Through directed evolution and rational design approaches, researchers could enhance the oxygen stability of the iron-sulfur cluster, creating variants suitable for biotechnological applications under aerobic conditions.

• Substrate specificity engineering: The ispG enzyme could be engineered to accept modified substrates, potentially enabling the biosynthesis of novel isoprenoid compounds. By targeting residues in the substrate binding pocket, researchers might develop variants capable of processing non-natural precursors to generate valuable compounds for pharmaceutical or industrial applications.

• Thermostability enhancements: As B. japonicum is a slow-growing organism adapted to the stable environment of root nodules, its enzymes may prioritize stability over catalytic rate. This inherent stability could provide a solid foundation for engineering thermostable variants for industrial applications, where process conditions often require enzymes capable of withstanding elevated temperatures.

• Biocatalytic applications: Engineered ispG variants could serve as biocatalysts for the production of HMBPP and derivatives, which have potential applications as immune modulators through activation of Vγ9Vδ2 T cells. The stereospecific nature of enzymatic catalysis makes ispG particularly valuable for producing these compounds with high stereochemical purity.

What techniques can be used to improve the heterologous expression and stability of B. japonicum ispG?

Improving the heterologous expression and stability of B. japonicum ispG requires addressing several challenges specific to iron-sulfur proteins from slow-growing symbiotic bacteria. Effective techniques include:

• Expression system optimization:

  • Use of specialized expression strains engineered for iron-sulfur protein production, such as E. coli SufFeScient™ or strains overexpressing iron-sulfur cluster assembly machinery

  • Co-expression with chaperones like GroEL/GroES to assist proper folding

  • Development of inducible expression systems with fine-tuned promoter strength to prevent formation of inclusion bodies

• Protein engineering approaches:

  • N- and C-terminal truncations to remove flexible regions that may impede crystallization

  • Surface entropy reduction through mutation of flexible, solvent-exposed residues

  • Introduction of disulfide bridges to enhance stability while preserving the redox state of the catalytic iron-sulfur cluster

• Formulation and storage strategies:

  • Addition of stabilizing agents such as glycerol (10-20%), reducing agents (DTT, β-mercaptoethanol), and osmolytes (trehalose, sucrose)

  • Development of lyophilization protocols with appropriate cryoprotectants

  • Oxygen-free storage conditions using sealed vials with inert gas headspace

These approaches should be tailored to address the specific challenges of B. japonicum ispG, considering the genomic complexity and slow-growing nature of this organism .

What are the future research directions for understanding the role of ispG in B. japonicum's symbiotic relationship with plants?

Future research on ispG in B. japonicum's symbiotic relationship with plants should explore several promising directions that leverage our emerging understanding of this enzyme's role in bacterial metabolism and plant-microbe interactions:

• Regulation of ispG expression during nodulation: Investigating how ispG expression is regulated during the establishment and maintenance of the symbiotic relationship could reveal important connections between isoprenoid biosynthesis and symbiotic efficiency. Research should examine whether ispG expression changes during different stages of nodule development and in response to plant-derived signals.

• Impact of genomic context on ispG function: Given the high frequency of insertion sequences and repeated elements in B. japonicum genomes , studies should explore how these genomic features affect ispG expression and function across different strains and field isolates. This could involve comparative analyses between normal and HRS isolates to determine if genomic rearrangements affect ispG performance in symbiotic contexts.

• Role of ispG-derived products in plant-bacteria signaling: The products of the MEP pathway contribute to various cellular functions, including the synthesis of signaling molecules. Future research should investigate whether HMBPP or downstream isoprenoids play specific roles in communication between B. japonicum and host plants, potentially influencing nodulation efficiency or nitrogen fixation rates.

• Engineering approaches to enhance symbiotic performance: Targeted modification of ispG and related enzymes could potentially enhance the efficiency of the symbiotic relationship. This might involve optimizing isoprenoid production to improve bacterial survival within nodules or enhancing the production of specific beneficial compounds that promote plant growth.

• Comparative studies across Bradyrhizobium strains: Examining ispG sequence and functional variation across diverse B. japonicum strains isolated from different geographical locations and host plants could reveal adaptations specific to particular symbiotic contexts. The search results indicate significant genomic variation among field isolates , suggesting that similar variation might exist in metabolic enzymes like ispG.

These research directions would contribute to our fundamental understanding of the biochemical underpinnings of the B. japonicum-soybean symbiosis while potentially informing agricultural applications aimed at improving nodulation efficiency and crop productivity.