Recombinant Synechococcus sp. Histidine biosynthesis bifunctional protein HisIE (hisI)

What is the role of histidine biosynthesis bifunctional protein HisIE in Synechococcus sp.?

The HisIE bifunctional protein in Synechococcus sp. catalyzes two consecutive steps in the histidine biosynthesis pathway. As a bifunctional enzyme, it contains two distinct domains: the phosphoribosyl-ATP pyrophosphohydrolase (HisI) domain and the phosphoribosyl-AMP cyclohydrolase (HisE) domain. These domains work in concert to convert phosphoribosyl-ATP to phosphoribosyl-formimino-5-aminoimidazole-4-carboxamide ribonucleotide, which are critical steps in histidine production. In cyanobacteria like Synechococcus, this pathway is essential for growth in environments lacking exogenous histidine, making it vital for environmental adaptation and survival .

How does the histidine biosynthesis pathway differ between Synechococcus sp. and other cyanobacteria?

The histidine biosynthesis pathway in Synechococcus sp. shares core components with other cyanobacteria but exhibits distinct characteristics that reflect its evolutionary adaptation to various aquatic environments. While the pathway remains largely conserved, Synechococcus sp. shows variation in gene organization and regulatory elements compared to other cyanobacteria. Synechococcus strains (such as PCC 7942 and PCC 6301) have been used to identify histidine biosynthesis genes through complementation of histidine auxotrophs, revealing the presence of genes like hisS that are involved in this pathway . The unique metabolic adaptations in Synechococcus allow them to thrive in diverse ecological niches, from coastal waters to open oceans, with varying nutrient availability .

What genetic transformation methods are most effective for studying HisIE in Synechococcus sp.?

For studying HisIE in Synechococcus sp., particularly strains PCC 7942 and PCC 6301, an efficient genetic transfer method has been developed that exceeds natural transformation efficiencies by orders of magnitude . This method has successfully been used to complement histidine auxotrophs and identify histidine biosynthesis genes. The procedure typically involves:

• Preparation of recipient cells in exponential growth phase

• Introduction of DNA using enhanced electroporation protocols

• Selection on media lacking histidine to identify successful transformants

• Confirmation through molecular techniques like PCR and sequence analysis

This method has proven particularly valuable for genetic manipulation studies in Synechococcus, offering significantly higher transformation efficiencies than traditional approaches .

What are the optimal conditions for expressing recombinant HisIE protein in E. coli expression systems?

Based on similar protein expression studies with Synechococcus proteins, optimal expression of recombinant HisIE typically requires:

When expressing Synechococcus proteins in E. coli, researchers should be aware that fusion tags like MBP (maltose-binding protein) significantly improve solubility, as demonstrated with other Synechococcus proteins that formed insoluble aggregates when expressed with simple His-tags . Cell growth should be monitored to an OD600 of approximately 0.6 before induction with IPTG .

What purification strategy yields the highest activity for recombinant HisIE protein?

For purifying recombinant HisIE protein while maintaining optimal enzymatic activity, a multi-step approach is recommended:

• Initial capture: Affinity chromatography using the fusion tag (MBP-tag or His-tag)

• Intermediate purification: Ion-exchange chromatography to separate charged variants

• Polishing step: Size-exclusion chromatography to achieve highest purity

When utilizing MBP-fusion proteins, as has been successful with other Synechococcus proteins, purification can be performed using amylose resin followed by tag cleavage with an appropriate protease . For activity preservation, all buffers should contain:

Purified protein should be stored at -80°C in small aliquots to avoid repeated freeze-thaw cycles that reduce activity .

How can you assess the folding and structural integrity of recombinant HisIE protein?

Assessment of folding and structural integrity of recombinant HisIE protein requires multiple complementary techniques:

• Circular Dichroism (CD) Spectroscopy:

  • Far-UV CD (190-260 nm): Determines secondary structure content

  • Near-UV CD (250-350 nm): Evaluates tertiary structure organization

• Thermal Shift Assays:

  • Monitors protein unfolding with temperature using fluorescent dyes

  • Provides melting temperature (Tm) data to assess stability

• Limited Proteolysis:

  • Partially digests protein with proteases like trypsin or chymotrypsin

  • Well-folded proteins show resistance to proteolysis at low protease concentrations

• Size Exclusion Chromatography:

  • Evaluates oligomeric state and aggregation tendency

  • Should be combined with multi-angle light scattering for accurate molecular weight determination

• Enzymatic Activity Assays:

  • Direct measurement of both HisI and HisE activities

  • Comparison with activity levels from native enzyme sources

The combination of these methods provides a comprehensive assessment of whether the recombinant protein maintains its native structure and catalytic properties.

What enzymatic assays can be used to measure the bifunctional activity of HisIE protein?

The bifunctional HisIE protein requires distinct assays to measure each of its catalytic activities:

HisI Activity (Phosphoribosyl-ATP pyrophosphohydrolase):

• Substrate: Phosphoribosyl-ATP

• Direct method: Monitor decrease in phosphoribosyl-ATP by HPLC

• Coupled method: Measure pyrophosphate release using pyrophosphatase and malachite green for phosphate detection

HisE Activity (Phosphoribosyl-AMP cyclohydrolase):

• Substrate: Phosphoribosyl-AMP

• Direct method: Spectrophotometric measurement of phosphoribosyl-formimino-5-aminoimidazole-4-carboxamide ribonucleotide formation at 290 nm

• Coupled method: Use downstream enzymes to convert the product to a more easily detectable compound

Combined Bifunctional Assay:

• Substrate: Phosphoribosyl-ATP

• Method: Measure the final product phosphoribosyl-formimino-5-aminoimidazole-4-carboxamide ribonucleotide by HPLC or spectrophotometry

• Control: Compare with individual domain activities to assess functional coupling

Reaction conditions should mimic physiological conditions of Synechococcus sp., with temperature at 30°C and pH 7.0-7.5 in a buffer system like HEPES .

How does temperature and pH affect the catalytic activity of HisIE from Synechococcus sp.?

The catalytic activity of HisIE from Synechococcus sp. shows temperature and pH dependence that reflects the organism's environmental adaptation:

Temperature Effects:

pH Effects:

These activity profiles are consistent with the physiological conditions of marine Synechococcus, which typically grow optimally at temperatures between 22-30°C and pH values near neutral . The temperature sensitivity profile also aligns with the adaptation of different Synechococcus strains to various ecological niches from polar to tropical waters .

What are the kinetic parameters of the HisIE protein and how do they compare to homologs from other organisms?

The kinetic parameters of HisIE protein from Synechococcus sp. reflect its adaptation to marine environments:

Kinetic Parameters for HisI Activity:

Kinetic Parameters for HisE Activity:

The slightly higher Km values observed in Synechococcus HisIE compared to E. coli counterparts suggest adaptation to potentially higher substrate concentrations in their native environment. The catalytic efficiency (kcat/Km) falls within the range observed for other cyanobacteria, indicating evolutionary conservation of function while adapting to specific ecological niches from polar to tropical waters .

What genetic strategies can be used to create histidine auxotrophs in Synechococcus sp. for studying HisIE function?

Creating histidine auxotrophs in Synechococcus sp. requires strategic genetic manipulation approaches:

• Targeted Gene Disruption:

  • Insert antibiotic resistance cassette into the hisI gene through homologous recombination

  • Use efficient genetic transfer methods developed specifically for Synechococcus sp. strains PCC 7942 and PCC 6301 that exceed natural transformation efficiencies

  • Selection on media supplemented with histidine and appropriate antibiotics

• CRISPR-Cas9 Genome Editing:

  • Design sgRNA targeting hisI gene region

  • Introduce Cas9 and sgRNA delivery systems optimized for cyanobacteria

  • Include repair template with antibiotic marker flanked by homology arms

• Transposon Mutagenesis:

  • Use transposon libraries to generate random insertions

  • Screen for histidine auxotrophy on selective media

  • Confirm disruption of hisI gene through PCR and sequencing

Researchers should note that complete knockout of essential genes like hisI may not be achievable in all chromosomal copies due to Synechococcus often having multiple chromosomes per cell, similar to what was observed with prxI gene knockouts in Synechococcus sp. PCC7002 . Partial knockouts (where some chromosome copies retain the intact gene) may still show distinct phenotypic responses compared to wild type .

How is the expression of HisIE regulated in Synechococcus sp. under different environmental conditions?

The expression of HisIE in Synechococcus sp. is regulated in response to various environmental factors:

The regulation likely involves complex interplay between specific transcription factors, global regulators, and small regulatory RNAs. The metabolic diversity observed in Synechococcus across different ecological niches suggests that regulation of histidine biosynthesis genes, including hisI, may vary between clades and strains to optimize survival in their specific environments . Like other metabolic pathways in Synechococcus, histidine biosynthesis regulation would be integrated with the organism's environmental adaptation strategies, including nutrient uptake systems and stress responses .

What complementation strategies can verify the function of recombinant HisIE in Synechococcus sp.?

To verify the function of recombinant HisIE in Synechococcus sp., several complementation strategies can be employed:

• In vivo Complementation in Synechococcus:

  • Transform histidine auxotroph mutants with plasmids expressing recombinant HisIE

  • Assess restoration of growth on histidine-free media

  • Compare growth rates between complemented strains and wild type

• Heterologous Complementation:

  • Express Synechococcus HisIE in E. coli or yeast histidine auxotrophs

  • Test growth on selective media lacking histidine

  • Compare with complementation using known functional HisIE genes from other organisms

• Domain-Specific Complementation:

  • Create constructs expressing only HisI or HisE domains

  • Complement corresponding single-function auxotrophs

  • Assess whether individual domains maintain catalytic activity

• Site-Directed Mutagenesis Complementation:

  • Generate HisIE variants with mutations in catalytic residues

  • Test complementation efficiency compared to wild-type protein

  • Map critical residues for enzyme function

Researchers have successfully used complementation approaches with histidine auxotrophs to identify histidine biosynthesis genes in Synechococcus sp. strains PCC 7942 and PCC 6301, demonstrating the efficacy of this approach . The versatile genetic transfer method developed for these strains provides an efficient platform for such complementation studies .

How can recombinant HisIE be used to study the evolution of histidine biosynthesis across different clades of Synechococcus?

Recombinant HisIE provides a powerful tool for evolutionary studies of histidine biosynthesis across Synechococcus clades:

• Comparative Biochemistry Approach:

  • Express and purify HisIE from representative strains of the 18 distinct Synechococcus clades identified in metagenomic analyses

  • Compare enzyme kinetics, substrate specificity, and thermostability

  • Correlate biochemical differences with ecological distribution and adaptation

• Phylogenetic Analysis:

  • Sequence hisI genes from diverse Synechococcus strains

  • Construct phylogenetic trees to track evolutionary relationships

  • Identify signatures of selection and adaptation in different environments

• Domain Architecture Analysis:

  • Compare organization of HisI and HisE domains across clades

  • Assess fusion events versus separate genes across evolutionary history

  • Determine if bifunctional architecture is conserved across all Synechococcus clades

• Horizontal Gene Transfer Assessment:

  • Analyze genomic context and codon usage of hisI genes

  • Identify potential horizontal gene transfer events

  • Determine if prophage regions containing metabolic genes may have contributed to HisIE evolution

This research would provide insights into how the histidine biosynthesis pathway has adapted across the diverse ecological niches occupied by Synechococcus, from polar to tropical waters, and in coastal versus open ocean environments .

What experimental approaches can elucidate the structural basis for the bifunctional nature of HisIE?

Elucidating the structural basis for HisIE bifunctionality requires multiple advanced structural biology techniques:

These approaches would reveal how the two catalytic domains coordinate their activities and whether substrate channeling occurs between the active sites for enhanced catalytic efficiency.

How might phage infection affect histidine biosynthesis and HisIE function in Synechococcus sp.?

Phage infection can significantly impact histidine biosynthesis and HisIE function in Synechococcus sp. through several mechanisms:

Given that cyanophages are known to affect 0.005-30% of Synechococcus populations daily , their influence on metabolism, including histidine biosynthesis, represents an important but understudied aspect of marine microbial ecology.