Recombinant Bradyrhizobium japonicum Serine hydroxymethyltransferase (glyA)

What is the function of serine hydroxymethyltransferase (glyA) in Bradyrhizobium japonicum?

Serine hydroxymethyltransferase (SHMT), encoded by the glyA gene in Bradyrhizobium japonicum, catalyzes two critical biochemical reactions: the biosynthesis of glycine from serine and the transfer of a one-carbon unit to tetrahydrofolate. This enzyme plays a fundamental role in the carbon metabolism of B. japonicum, particularly in the one-carbon (C1) metabolism pathway. The enzyme is not only important for the bacterium's own metabolic functions but appears to be indispensable for establishing an effective nitrogen-fixing symbiotic relationship with host plants like soybean (Glycine max) .

How does glyA relate to symbiotic nitrogen fixation in the Bradyrhizobium-soybean interaction?

The glyA gene is symbiotically essential for Bradyrhizobium japonicum. Mutations in this gene result in the formation of numerous tiny white nodules dispersed throughout the host soybean's root system. These pseudonodules are ineffective in nitrogen fixation and show disturbed bacteroid and nodule development at very early stages. This phenotype suggests that adequate glycine supply and/or proper functioning of the C1 metabolism pathway are critical requirements for establishing fully effective nitrogen-fixing root nodule symbiosis . In contrast to glyA mutations, mutations in the glycine cleavage system (gcv) in another rhizobial strain (Sinorhizobium fredii USDA257) actually enable nitrogen fixation in certain soybean cultivars that the wild-type strain cannot nodulate effectively .

What is the genomic context of glyA in B. japonicum?

The glyA gene is located within the approximately 8,700 kb genome of Bradyrhizobium japonicum . While the search results don't provide the exact genomic coordinates or neighboring genes, the gene has been successfully mapped using pulsed-field gel electrophoresis (PFGE) techniques combined with rare-cutting restriction enzymes. The gene mapping process involved introducing restriction sites into or near the glyA locus, followed by PFGE analysis to identify altered fragment patterns compared to wild-type .

What are the established methods for creating glyA mutants in Bradyrhizobium japonicum?

The creation of glyA mutants in B. japonicum can be accomplished through several approaches:

• Transposon mutagenesis: Tn5 insertion has been successfully used to disrupt the glyA coding sequence. This approach generated strain 3160, which exhibits the characteristic phenotype of ineffective nodulation .

• Site-directed mutagenesis: More precise mutations can be created using marker exchange methods. This typically involves:

  • Cloning the target region into a suitable vector

  • Introducing a selective marker (such as antibiotic resistance cassettes) into the gene

  • Transforming the construct into E. coli S17-1 for conjugation

  • Mobilizing into B. japonicum through conjugation

  • Selecting for marker exchange mutants through appropriate antibiotic screening

• Cassette insertion: The aphII-PSP cassette system has been used effectively. After introduction of this cassette, selection for kanamycin resistance and screening for tetracycline sensitivity can identify strains with marker exchange mutations .

Verification of the correct genomic structure should be performed using Southern blot hybridization or PCR-based techniques.

What methods are recommended for complementation analysis of glyA mutants?

Complementation analysis of glyA mutants can be conducted through several approaches:

• Heterologous complementation: The B. japonicum glyA region has been shown to fully complement the glycine auxotrophy of an E. coli glyA deletion strain. This cross-species complementation confirms the functional conservation of SHMT activity .

• In situ complementation: For confirming gene function directly in B. japonicum, a wild-type copy of the glyA gene with its native promoter should be cloned into a broad-host-range vector that can replicate in rhizobia.

• Phenotypic rescue assessment: Successful complementation should restore:

  • Normal growth in minimal medium without glycine supplementation

  • The ability to form effective nitrogen-fixing nodules on soybean

  • Normal bacteroid development within nodules

The complementation vector should be maintained under selective pressure throughout the experiment to prevent loss, especially when complementing symbiotic phenotypes.

How can one measure SHMT enzyme activity in B. japonicum extracts?

SHMT enzyme activity can be measured through multiple analytical approaches:

• Spectrophotometric assay: This assay measures the conversion of serine to glycine coupled with the generation of 5,10-methylenetetrahydrofolate, which can be detected spectrophotometrically.

• Radiometric assay: Using 14C-labeled serine as substrate and measuring the transfer of the labeled methylene group to tetrahydrofolate.

• HPLC-based methods: Quantifying the conversion of substrate to products through high-performance liquid chromatography.

Activity should be expressed as specific activity (μmol/min/mg protein) and should include appropriate controls (heat-inactivated enzyme, reactions without substrate, etc.) to ensure specificity.

How does the expression of glyA in B. japonicum compare to the expression of the glycine cleavage system (gcv)?

The glycine cleavage system (gcv) and glyA (SHMT) serve complementary but distinct roles in one-carbon metabolism. While specific expression data comparing these systems in B. japonicum is not directly provided in the search results, some key observations can be made:

• Regulatory patterns: The gcvTHP operon in Sinorhizobium fredii USDA257 is shown to be inducible by glycine, with approximately seven-fold increase in expression in the presence of glycine . This induction pattern suggests differential regulation between basal and glycine-rich conditions.

• Functional relationship: Both pathways are involved in C1 metabolism, with glyA typically serving as the primary source of C1 units, while the gcv system functions as a secondary source .

• Contrasting symbiotic effects:

  • glyA mutation in B. japonicum leads to ineffective nodulation

  • gcv mutation in S. fredii USDA257 expands host range, enabling nitrogen fixation in soybean cultivars not nodulated by the wild-type strain

This contrasting effect suggests that these pathways interact in complex ways to influence symbiotic relationships.

What is known about the regulation of glyA expression during different stages of symbiosis?

While the search results don't provide direct data on glyA expression during different symbiotic stages, several inferences can be made:

• Early nodule development: The formation of ineffective pseudonodules in glyA mutants suggests that SHMT activity is critical during the early stages of the symbiotic interaction. The disturbance observed "at a very early step of bacteroid and nodule development" indicates that glyA expression is likely important from the onset of the symbiotic process .

• Bacteroid differentiation: Since glyA mutation disrupts bacteroid development, the gene's expression is likely regulated during the bacteroid differentiation phase.

• Stress response considerations: B. japonicum undergoes significant gene expression changes in response to oxidative stress, including during the plant infection process. While glyA was not specifically mentioned among the differentially expressed genes in oxidative stress conditions studied, other metabolic pathways show significant changes .

For definitive analysis, targeted expression studies using techniques such as RT-qPCR, RNA-seq, or reporter gene fusions during different stages of the symbiotic interaction would be necessary.

How does one-carbon metabolism in B. japonicum differ from other rhizobial species, and what implications does this have for glyA function?

One-carbon metabolism shows both conservation and divergence across rhizobial species:

• Pathway conservation: Core components of one-carbon metabolism, including SHMT (glyA) and the glycine cleavage system (gcvTHP), are conserved across rhizobial species. The glycine cleavage system components show high sequence similarity (80-93%) among Sinorhizobium medicae, Sinorhizobium meliloti, Rhizobium leguminosarum bv. trifolii, and Rhizobium etli .

• Functional divergence: Despite sequence conservation, functional differences are evident:

  • In B. japonicum, glyA mutation results in ineffective nodulation

  • In S. fredii USDA257, gcv mutation expands host range

• Metabolic redundancy: B. japonicum with glyA mutation is described as a "bradytroph" (leaky auxotroph), suggesting the existence of an alternative pathway for glycine biosynthesis . This metabolic redundancy may vary across rhizobial species.

These differences may be related to varying metabolic demands during symbiosis with different host plants or adaptation to specific environmental niches.

What is the detailed nodulation phenotype of B. japonicum glyA mutants?

B. japonicum glyA mutants exhibit a distinctive nodulation phenotype characterized by:

• Nodule morphology and distribution:

  • Numerous tiny white nodules

  • Nodules dispersed over the entire root system of soybean

  • Classified as ineffective, nitrogen non-fixing pseudonodules

• Developmental defects:

  • Disturbed at very early stages of bacteroid development

  • Impaired nodule development

  • Unable to progress to mature nitrogen-fixing nodules

• Physiological limitations:

  • Absence of nitrogen fixation activity

  • Presumed inability to properly differentiate into bacteroids

  • Insufficient C1 metabolism capabilities that appear critical for symbiotic development

This phenotype contrasts with that of gcv mutants in USDA257, which can form nitrogen-fixing nodules on soybean cultivars that are not nodulated by the wild-type strain .

How does the plant host respond to B. japonicum glyA mutants compared to wild-type bacteria?

While the search results don't provide direct measurements of plant host responses to glyA mutants, several aspects can be inferred:

• Nodule initiation: The plant clearly permits initial infection and nodule initiation, as evidenced by the formation of numerous nodules. This suggests that early recognition factors and infection processes remain functional despite the glyA mutation .

• Developmental arrest: The host-bacteria interaction appears to stall at an early developmental stage, preventing normal bacteroid and nodule maturation.

• Defense responses: Though not specifically documented for glyA mutants, B. japonicum encounters oxidative bursts during infection as part of the plant's defense system. The search results show that B. japonicum has complex transcriptional responses to H₂O₂-mediated oxidative stress .

• Metabolic exchange: The disruption in C1 metabolism likely affects metabolic exchange between the host and bacteria, potentially contributing to the developmental arrest.

A comprehensive analysis would require comparative transcriptomic or proteomic studies of plant tissues infected with wild-type versus glyA mutant strains.

What correlation exists between glyA function and oxidative stress responses during plant infection?

The relationship between glyA function and oxidative stress response can be considered in several contexts:

• General stress responses: B. japonicum shows differential expression of 439 genes under prolonged exposure to H₂O₂ and 650 genes under fulminant shock conditions . While glyA was not specifically mentioned among these differentially expressed genes, pathways related to central metabolism are often affected by oxidative stress.

• C1 metabolism and redox balance: One-carbon metabolism interfaces with cellular redox processes. The generation and utilization of tetrahydrofolate derivatives have implications for redox homeostasis.

• Potential indirect connections: The search results indicate that during oxidative stress:

  • Transport and binding proteins, particularly ABC transporter systems, are upregulated

  • Sigma factors and global stress response proteins show increased expression

  • The isocitrate lyase gene (aceA) is induced under fulminant H₂O₂ shock

These changes in cellular physiology could indirectly affect glyA expression or function, possibly contributing to the symbiotic defects observed in glyA mutants.

What expression systems are most effective for producing recombinant B. japonicum SHMT?

Based on the information available and general principles of recombinant protein production:

• E. coli expression systems: The successful complementation of an E. coli glyA deletion strain with the B. japonicum glyA gene suggests that the protein can fold properly and function in E. coli. This makes E. coli-based expression systems like BL21(DE3) or its derivatives promising candidates for recombinant production.

• Expression vector considerations:

  • Controlled induction systems (T7, tac, or arabinose-inducible promoters)

  • Fusion tags for purification (His-tag, GST, MBP)

  • Codon optimization may be beneficial given the high G+C content (61-65 mol%) of B. japonicum DNA

• Alternative hosts: For applications requiring post-translational modifications or when E. coli expression proves challenging, consider:

  • Yeast expression systems (Pichia pastoris or Saccharomyces cerevisiae)

  • Insect cell expression systems

  • Homologous expression in rhizobial hosts

Expression should be verified through Western blotting and enzymatic activity assays to confirm proper folding and function.

What purification strategies yield the highest activity for recombinant B. japonicum SHMT?

Effective purification strategies for recombinant SHMT should consider:

• Initial purification:

  • Affinity chromatography using His-tag, GST-tag, or other fusion partners

  • Immobilized substrate analogs or cofactors

  • Ion exchange chromatography (IEX) based on the protein's theoretical isoelectric point

• Secondary purification:

  • Size exclusion chromatography to remove aggregates and obtain homogeneous protein

  • Hydrophobic interaction chromatography as a complementary method to IEX

• Stability considerations:

  • Include tetrahydrofolate or analogs during purification

  • Add reducing agents (DTT or β-mercaptoethanol) to prevent oxidation of critical cysteine residues

  • Consider adding glycerol (10-20%) to enhance stability

  • Test various buffer compositions and pH values for optimal activity preservation

Throughout purification, monitor enzymatic activity using the assays described in FAQ 2.3. Calculate specific activity at each step to track purification efficiency and identify steps that may cause activity loss.

What are the optimal reaction conditions for measuring recombinant B. japonicum SHMT activity in vitro?

While the search results don't provide specific conditions for B. japonicum SHMT, optimal assay conditions typically include:

• Buffer composition:

  • pH range: 7.0-8.0 (phosphate or Tris-based buffers)

  • Ionic strength: 50-100 mM

  • Reducing agents: 1-5 mM DTT or β-mercaptoethanol

• Cofactor requirements:

  • Tetrahydrofolate: 0.2-1.0 mM

  • Pyridoxal phosphate (PLP): 0.1-0.5 mM (SHMT is a PLP-dependent enzyme)

• Substrate concentrations:

  • L-Serine: 0.5-5.0 mM

  • Determine Km values through kinetic analysis to identify optimal concentrations

• Temperature and time:

  • Typical range: 25-37°C

  • Linear reaction range should be determined empirically

• Detection methods:

  • Spectrophotometric detection of 5,10-methylenetetrahydrofolate formation

  • HPLC-based quantification of glycine formation

  • Coupled enzyme assays that link product formation to a readily measurable output

A systematic approach using factorial design of experiments (DoE) would be ideal for optimizing multiple parameters simultaneously.

How conserved is SHMT across rhizobial species, and what does this reveal about its evolutionary importance?

SHMT shows significant conservation across bacterial species, reflecting its essential metabolic role:

• Sequence conservation in rhizobia:

  • While direct SHMT (glyA) sequence comparisons aren't provided in the search results, related enzymes in the glycine metabolism pathway show high conservation

  • The glycine cleavage system components (GcvT, GcvH, GcvP) show 80-93% sequence similarity among Sinorhizobium medicae, Sinorhizobium meliloti, Rhizobium leguminosarum bv. trifolii, and Rhizobium etli

• Cross-kingdom conservation:

  • USDA257 GcvT shows 35-38% amino acid sequence similarity to GcvT from eukaryotic organisms (Arabidopsis, bovine, and human proteins)

  • This suggests fundamental conservation of one-carbon metabolism across diverse life forms

• Functional conservation:

  • B. japonicum glyA successfully complements E. coli glyA deletion mutants

  • This indicates functional conservation despite evolutionary distance between these bacterial genera

The high conservation across diverse species underscores the evolutionary importance of SHMT in central metabolism and suggests it has been under strong selective pressure throughout bacterial evolution.

What structural differences exist between B. japonicum SHMT and other bacterial or eukaryotic SHMTs?

While the search results don't provide specific structural information about B. japonicum SHMT, some inferences can be made based on general knowledge and the available information about related proteins:

• Sequence divergence as a structural indicator:

  • The glycine cleavage system component GcvT from USDA257 shows only 23.8% amino acid sequence similarity to E. coli GcvT

  • This suggests potentially significant structural differences between rhizobial and enterobacterial enzymes involved in glycine metabolism

• Expected structural features based on other SHMTs:

  • SHMT typically functions as a homodimer or homotetramer

  • Contains a PLP binding site

  • Has distinct domains for substrate binding and catalysis

• Adaptation to cellular environment:

  • Given the high G+C content of B. japonicum DNA (61-65 mol%) , the codon usage and potentially amino acid composition may differ from lower G+C content organisms

  • These differences could influence protein stability and folding properties

Determination of the actual structural differences would require X-ray crystallography or cryo-EM studies of B. japonicum SHMT compared with other bacterial and eukaryotic SHMTs.

How does B. japonicum SHMT compare functionally to the glycine cleavage system (GCV) in rhizobial metabolism?

SHMT and the glycine cleavage system have complementary but distinct roles in one-carbon metabolism:

• Directional differences:

  • SHMT (glyA) primarily catalyzes the biosynthesis of glycine from serine and transfers a one-carbon unit to tetrahydrofolate

  • The glycine cleavage system (gcvTHP) operates in the opposite direction, breaking down glycine and generating C1 units

• Metabolic priorities:

  • glyA is considered the primary source of C1 units

  • gcv is believed to function as a secondary source of C1 units

• Symbiotic impact contrast:

  • glyA mutation in B. japonicum leads to ineffective nodulation

  • gcv mutation in S. fredii USDA257 enables nitrogen fixation on soybean cultivars not nodulated by wild-type

• Regulatory differences:

  • The gcvTHP operon in USDA257 is inducible by glycine, showing approximately seven-fold higher expression in the presence of glycine

  • Similar information about glyA regulation isn't provided in the search results

This functional comparison highlights the complex interplay between these pathways in rhizobial metabolism and symbiotic interactions.