Recombinant Bradyrhizobium japonicum Phosphoribosyl-AMP cyclohydrolase (hisI)

What is phosphoribosyl-AMP cyclohydrolase (HisI) and what is its function in bacterial systems?

Phosphoribosyl-AMP cyclohydrolase (HisI) is an enzyme with a phosphoribosyl-AMP-cyclohydrolase (PRA-CH) domain that plays a critical role in histidine biosynthesis. In bacterial systems, this enzyme catalyzes a specific step in the histidine biosynthetic pathway. Research indicates that HisI proteins exhibit significant sequence similarity across different bacterial species, with homologs identified in organisms like Pseudomonas putida (67% amino acid sequence identity) and Pseudomonas syringae (55% amino acid sequence identity) . The conserved nature of this enzyme across bacterial species highlights its fundamental role in cellular metabolism. In Chromohalobacter salexigens, HisI has been shown to be essential for histidine synthesis, which in turn is necessary for siderophore production .

How is the hisI gene typically organized in bacterial genomes?

The organization of the hisI gene varies across bacterial species, but in some cases, it is found in operons with functionally related genes. For instance, in Chromohalobacter salexigens, hisI is part of a six-gene operon (cfuABC-fur-hisI-orf6) located downstream of the ectABC ectoine synthesis genes . This genomic organization suggests a potential functional relationship between histidine biosynthesis and other cellular processes, such as iron regulation (via Fur) and osmotic stress response. The clustering of hisI with genes involved in iron regulation, particularly the fur gene encoding the ferric iron uptake regulator, indicates possible coordinated regulation of histidine biosynthesis with iron homeostasis .

Why is Bradyrhizobium japonicum challenging to work with in genetic studies?

Bradyrhizobium japonicum presents significant challenges for genetic manipulation and mutant screening due to several inherent characteristics. The bacterium exhibits a high incidence of spontaneous antibiotic resistance, which complicates selection strategies using antibiotic markers . Additionally, B. japonicum has exceptionally slow growth rates, making the screening process for site-directed mutants particularly time-consuming and laborious . These challenges have necessitated the development of specialized techniques for generating and selecting recombinant strains, such as the use of multiple antibiotic resistance cassettes and optimized screening methods to efficiently identify true recombinant mutants from spontaneous antibiotic-resistant colonies .

How does the interplay between HisI function and iron homeostasis affect bacterial adaptation to environmental stress?

The relationship between HisI function and iron homeostasis represents a sophisticated adaptive mechanism in bacteria facing environmental stresses. In Chromohalobacter salexigens, research has revealed a decrease in both iron and histidine requirements at high salinity levels, correlating with reduced siderophore synthesis and lower protein content in salt-stressed cells . This adaptation appears to be regulated through the Fur (ferric uptake regulator) system, which can directly interact with DNA in the promoter regions of both the cfuABC-fur-hisI-orf6 and ectABC operons .

Studies with a fur mutant strain demonstrated deregulated siderophore production and delayed growth under iron limitation conditions, confirming Fur's role as a functional iron regulator . The functional connection between histidine synthesis (via HisI) and siderophore production was demonstrated when a mutant strain (CHR100) with interrupted cfuABC-fur genes (causing a polar effect on hisI) showed no siderophore production in the absence of histidine, but restored production when histidine was supplemented in the growth medium . This intricate relationship suggests that bacteria coordinate histidine biosynthesis with iron acquisition systems as part of a broader stress response strategy.

What are the structural and functional variations of HisI across different bacterial species, and how do these variations impact enzymatic activity?

While the search results don't provide comprehensive comparative data on HisI structural variations across bacterial species, they do indicate significant sequence conservation of HisI proteins. The C. salexigens HisI shows highest similarity to the HisI proteins of P. putida (67% amino acid sequence identity) and P. syringae (55% amino acid sequence identity) . These similarity levels suggest conserved catalytic mechanisms but potential variations in regulatory features or protein-protein interactions.

The functional impact of these variations appears to extend beyond the primary catalytic role in histidine biosynthesis. In C. salexigens, HisI function is essential for siderophore production, as demonstrated by the inability of the polar hisI mutant (CHR100) to produce siderophores in the absence of histidine supplementation . This functional connection between histidine biosynthesis and iron acquisition systems may vary across bacterial species depending on their ecological niches and stress adaptation strategies. Further structural studies would be needed to determine precisely how sequence variations translate to functional differences in enzymatic activity and integration with other cellular processes.

How do osmoregulatory mechanisms influence the expression and activity of HisI in Bradyrhizobium japonicum compared to other bacterial species?

The influence of osmoregulation on HisI expression and activity is incompletely characterized in Bradyrhizobium japonicum specifically, but insights can be drawn from studies in other bacterial systems. In Chromohalobacter salexigens, a halophilic bacterium, there is clear evidence of osmoregulated inhibition of the cfuABC-fur-hisI-orf6 operon . Under high salinity conditions, C. salexigens exhibits decreased requirements for both iron and histidine, correlating with lower siderophore synthesis levels .

The regulatory mechanisms appear to involve Fur-mediated repression, as Fur boxes were identified in the promoters of both the cfuABC-fur-hisI-orf6 and ectABC operons . The presence of these regulatory elements suggests that Fur directly interacts with DNA in these regions to coordinate iron homeostasis, histidine biosynthesis, and osmotic stress responses. This coordination is likely species-specific, with adaptation to particular ecological niches driving the evolution of different regulatory networks. In B. japonicum, which forms symbiotic relationships with soybean plants, the regulatory connections between osmoregulation and histidine biosynthesis may reflect the unique demands of the rhizosphere environment and symbiotic lifestyle.

What are the most effective techniques for generating site-directed mutants of hisI in Bradyrhizobium japonicum?

Creating site-directed mutants in B. japonicum requires specialized approaches due to the organism's high spontaneous antibiotic resistance rates and slow growth. An efficient method involves using antibiotic resistance cassettes (such as kanamycin or spectinomycin) to replace DNA fragments in the chromosome through homologous recombination . The technique follows these key steps:

• Construction of a recombinant plasmid carrying the desired mutation, typically flanked by regions homologous to the target site in the B. japonicum chromosome

• Introduction of the plasmid into B. japonicum cells through triparental mating or other transformation methods

• Simple plate selection for antibiotic-resistant mutants following recombination

• Colony streaking to isolate pure cultures

• Direct identification of recombinant site-directed mutants through DNA hybridization on nitrocellulose filters, eliminating the need to first isolate genomic DNA from each mutant for Southern hybridization

This streamlined approach has been demonstrated to quickly and efficiently identify large numbers of positive recombinant mutants from numerous individual colonies, with tested site-directed mutants exhibiting the expected mutant phenotypes .

How can researchers effectively distinguish between spontaneous antibiotic-resistant mutants and true recombinant Bradyrhizobium japonicum mutants?

Distinguishing true recombinant mutants from spontaneous antibiotic-resistant mutants is a critical challenge when working with B. japonicum. An effective approach combines selective media, molecular verification, and phenotypic analysis:

• Dual selection strategy: Using a combination of antibiotic selection and counter-selection markers (such as sucrose sensitivity conferred by the sacB gene) can help eliminate spontaneous antibiotic-resistant mutants .

• Direct colony hybridization: After initial antibiotic selection, colonies can be streaked and lysed directly on nitrocellulose filters for DNA hybridization, allowing rapid identification of colonies carrying the desired recombination event without isolating genomic DNA from each candidate .

• PCR verification: Amplification of the target region using primers that flank the expected insertion or deletion site, followed by size analysis or sequencing of the PCR product, can confirm the presence of the intended genetic modification .

• Phenotypic confirmation: Testing the putative mutants for the expected phenotype provides functional verification of the genetic modification. For example, a hisI mutant would be expected to show histidine auxotrophy and impaired siderophore production in the absence of histidine supplementation .

This multi-faceted approach minimizes false positives and ensures that the isolated mutants truly carry the desired genetic modifications.

What are the optimal conditions for expressing and purifying recombinant HisI protein from Bradyrhizobium japonicum?

While the search results don't provide specific protocols for HisI purification from B. japonicum, general principles for expressing and purifying recombinant proteins from bacterial systems can be applied, with modifications to address the specific characteristics of this enzyme:

• Expression system selection: Due to the slow growth of B. japonicum, heterologous expression in faster-growing hosts like E. coli is often preferred. The hisI gene should be cloned into an appropriate expression vector with a strong, inducible promoter (such as T7) and suitable affinity tags (His6 or GST) to facilitate purification.

• Optimization of induction conditions: Parameters including temperature (typically lowered to 16-25°C during induction to improve protein folding), inducer concentration, and induction time should be systematically optimized to maximize soluble protein yield.

• Buffer optimization: Since HisI is involved in histidine biosynthesis, buffers containing appropriate cofactors and maintaining optimal pH (typically 7.0-8.0) are important for preserving enzyme activity during purification.

• Purification strategy: A multi-step purification approach is recommended, typically including:

  • Initial capture using affinity chromatography (e.g., Ni-NTA for His-tagged proteins)

  • Intermediate purification using ion exchange chromatography

  • Polishing step using size exclusion chromatography

• Activity assessment: Following purification, enzyme activity should be assessed using a phosphoribosyl-AMP cyclohydrolase assay to confirm that the purified protein retains its catalytic function.

Protein yield and purity should be monitored at each step using SDS-PAGE and Western blotting to ensure successful purification of the target protein.

How should researchers design experiments to investigate the relationship between HisI activity and siderophore production in Bradyrhizobium japonicum?

Investigating the relationship between HisI activity and siderophore production requires a well-designed experimental approach that addresses both genetic and biochemical aspects of this connection. Based on findings from C. salexigens, where HisI is essential for histidine synthesis and subsequent siderophore production , a comprehensive experimental design should include:

• Genetic manipulation strategies:

  • Construction of a conditional hisI mutant using inducible promoters or partial deletions to avoid complete growth inhibition

  • Creation of a hisI complementation strain to verify phenotype restoration

  • Development of a hisI overexpression strain to assess the effects of increased histidine biosynthesis capacity

• Growth conditions variation:

  • Testing multiple iron concentrations using iron chelators like 2,2'-dipyridyl at various concentrations (e.g., 36 μM) to create iron-limiting conditions

  • Assessing growth under different salt concentrations (0.75-2.5 M NaCl) to evaluate osmotic stress effects

  • Supplementation experiments with histidine (1 mM) to determine if exogenous histidine can rescue siderophore production in hisI mutants

• Analytical methods:

  • Quantitative assessment of siderophore production using Chrome Azurol S (CAS) assays on agar plates and in liquid medium

  • Measurement of histidine levels in cells using HPLC or LC-MS/MS analysis

  • Monitoring HisI enzyme activity using specific biochemical assays

This experimental approach would enable researchers to establish the causal relationship between HisI activity, histidine biosynthesis, and siderophore production in B. japonicum, while also elucidating potential regulatory mechanisms.

What controls and validation steps are essential when studying the genetic regulation of hisI expression in response to environmental stressors?

Studying the genetic regulation of hisI expression in response to environmental stressors requires rigorous controls and validation steps to ensure reliable and interpretable results:

• Transcriptional fusion controls:

  • When constructing reporter gene fusions (such as hisI promoter::gfp), both positive controls (constitutive promoters) and negative controls (promoterless vectors) should be included

  • Multiple independent transformants should be tested to account for position effects or copy number variations

• Environmental condition controls:

  • Experiments should include carefully controlled stress gradients (e.g., increasing salt concentrations from 0.75 to 2.5 M NaCl)

  • Time-course experiments should be performed to distinguish between transient and sustained regulatory responses

  • Combined stressors (e.g., iron limitation plus osmotic stress) should be tested alongside individual stressors to identify potential interactions

• Validation approaches:

  • Transcriptional analysis using qRT-PCR to confirm reporter gene fusion results

  • Protein-level validation using western blotting with specific antibodies

  • In vivo DNA-protein interaction studies (ChIP) to confirm direct regulatory interactions

  • Complementation experiments to verify that observed phenotypes are specifically due to the targeted genetic modifications

• Statistical analysis:

  • All experiments should be performed with appropriate biological and technical replicates

  • Statistical methods should be applied to determine significance of observed differences

  • Data normalization approaches should account for differences in growth rates under different stress conditions

How can researchers effectively analyze the interplay between iron regulation, histidine biosynthesis, and osmotic stress response in bacterial systems?

Analyzing the complex interplay between iron regulation, histidine biosynthesis, and osmotic stress response requires an integrated experimental approach that combines genetic, biochemical, and systems biology techniques:

• Multi-omics approach:

  • Transcriptomic analysis (RNA-seq) to identify co-regulated genes under different stress conditions

  • Proteomic profiling to assess changes in protein abundance and post-translational modifications

  • Metabolomic analysis to measure fluctuations in relevant metabolites including histidine, iron-binding compounds, and osmoprotectants

• Genetic interaction studies:

  • Construction of double and triple mutants affecting key regulatory components (e.g., fur-hisI double mutant) to identify epistatic relationships

  • Suppressor mutant screening to identify additional components of the regulatory network

  • Targeted CRISPR interference to modulate gene expression levels and identify threshold effects

• In vivo DNA-protein interaction analysis:

  • Chromatin immunoprecipitation followed by sequencing (ChIP-seq) to identify global binding patterns of regulators like Fur under different stress conditions

  • DNA footprinting to precisely map binding sites within promoter regions

  • Electrophoretic mobility shift assays (EMSAs) to validate specific DNA-protein interactions

• Reporter systems for simultaneous monitoring:

  • Development of multiplexed reporter systems using different fluorescent proteins to simultaneously track expression of genes involved in different pathways

  • Time-lapse microscopy to observe dynamic responses at the single-cell level

  • Flow cytometry analysis to quantify population heterogeneity in stress responses

This integrated approach would allow researchers to construct comprehensive models of how these interconnected pathways respond to changing environmental conditions and regulate bacterial adaptation to stress.