Recombinant Alteromonas macleodii GMP synthase [glutamine-hydrolyzing] (guaA), partial

What is Alteromonas macleodii and its significance in marine environments?

Alteromonas macleodii is a free-living heterotrophic marine gammaproteobacterium commonly found in the open ocean. It is characterized as a typical "bloomer" that exhibits rapid growth in response to sporadic inputs of organic matter in relatively nutrient-poor marine environments . This organism plays an important ecological role in marine carbon cycling and nutrient transformation processes.

The genomic analysis of Alteromonas strains has revealed remarkable conservation of synteny (gene order) across the genus, facilitating comparative studies of core and flexible genomic regions . Alteromonas macleodii is distinguished from closely related species such as A. mediterranea by having less than 84% average nucleotide identity (ANI) between them .

What is GMP synthase (guaA) and its role in bacterial metabolism?

GMP synthase, encoded by the guaA gene, is a critical enzyme in the de novo purine biosynthesis pathway that catalyzes the conversion of xanthine monophosphate (XMP) to guanosine monophosphate (GMP) . This enzyme plays a pivotal role in nucleotide metabolism and is essential for bacterial growth and survival, particularly in nutrient-limited environments.

In bacteria, GMP synthase functions through a two-step amidation reaction:

• Activation of XMP using ATP to form an adenylated intermediate

• Replacement of the activated oxygen with an amino group donated by glutamine

The metabolic pathway involving guaA is illustrated in the following table:

What differentiates recombinant GuaA from naturally expressed protein?

Recombinant GuaA refers to the GMP synthase protein produced using recombinant DNA technology rather than isolated from wild-type Alteromonas macleodii. Recombinant DNA technology involves combining DNA from different sources to create sequences that would not normally occur together .

The key differences include:

• The recombinant protein often contains affinity tags (such as His-tag) to facilitate purification

• Expression can occur in non-native host organisms optimized for protein production

• The protein may be engineered for enhanced stability or activity

• Partial recombinant constructs may focus on specific catalytic domains rather than the full-length protein

Recombinant GuaA production typically involves cloning the guaA gene (or a portion of it) from Alteromonas macleodii into an expression vector, transforming this construct into a suitable host (commonly E. coli K12 derivatives), and inducing protein expression .

How do genomic variations in Alteromonas macleodii affect GuaA expression and function?

Alteromonas species demonstrate significant genomic diversity despite maintaining a relatively conserved core genome of approximately 1.4 Mb (representing about 30% of the typical strain genome size) . Analysis of strains with average nucleotide identities ranging from 99.98% to 73.35% shows that recombination rates along the core genome are high among strains belonging to the same species (37.7–83.7% of nucleotide polymorphisms) but decrease significantly between species (18.9–5.1%) .

This genomic diversity affects GuaA in several ways:

• Sequence variations in the guaA coding region can lead to amino acid substitutions that may impact enzyme kinetics or substrate specificity

• Changes in ribosome binding sites or promoter regions can affect expression levels

• In some bacterial species, guaA expression is controlled by riboswitch mechanisms that respond to guanine concentrations

Research has shown that while guaA is part of the core genome in Alteromonas, strain-specific variations may contribute to metabolic adaptations in different marine environments.

What experimental approaches are most effective for studying recombinant A. macleodii GuaA activity?

The enzymatic activity of recombinant A. macleodii GuaA can be studied using several complementary approaches:

Spectrophotometric assays:

• Monitoring AMP formation through coupled enzyme reactions

• Measuring inorganic phosphate release using malachite green

• Following glutamine consumption using glutamate dehydrogenase

Structural studies:

• X-ray crystallography to determine the three-dimensional structure

• Site-directed mutagenesis to identify catalytically important residues

• Hydrogen-deuterium exchange mass spectrometry for conformational dynamics

In vivo functional complementation:

• Transformation of guaA-deficient bacterial strains with recombinant A. macleodii guaA

• Assessment of growth restoration in minimal media lacking guanine

When designing these experiments, researchers should consider that inactivation of guaA typically leads to guanine auxotrophy, meaning the mutant strains require external guanine sources for growth . Studies in Clostridioides difficile have demonstrated that guaA mutants exhibit significantly reduced growth in minimal media and impaired colonization capacity in infection models .

What regulatory considerations apply to research with recombinant A. macleodii GuaA?

Research with recombinant A. macleodii GuaA must comply with institutional and national guidelines governing recombinant DNA work. Key regulatory considerations include:

• All research using recombinant DNA materials must comply with the NIH Guidelines for rDNA Research regardless of funding source

• Non-exempt research with rDNA materials must be registered with the Institutional Biosafety Committee (IBC) before initiation

• The researcher must determine if their work falls under exempt or non-exempt categories based on:

  • The host organism used (e.g., E. coli K12 derivatives may qualify for exemption under certain conditions)

  • The nature of the gene being expressed (guaA is a metabolic gene without toxin properties)

  • The experimental setting (in vitro vs. in vivo studies)

Each investigator working with rDNA is personally responsible for understanding and following NIH guidelines . Special considerations apply if the recombinant proteins will be used in animal models or clinical applications.

What expression systems yield optimal results for recombinant A. macleodii GuaA production?

Optimizing the expression of recombinant A. macleodii GuaA requires careful selection of expression systems based on research objectives. The following table compares common expression systems for bacterial recombinant proteins:

For obtaining catalytically active GuaA, the following protocol has proven effective:

• Clone the A. macleodii guaA gene into pET28a with an N-terminal His-tag

• Transform into E. coli BL21(DE3)

• Grow at 30°C until OD600 reaches 0.6-0.8

• Induce with 0.5 mM IPTG

• Continue expression at 18°C for 16-18 hours

• Purify using immobilized metal affinity chromatography followed by size exclusion chromatography

These conditions minimize inclusion body formation while maintaining high protein yield.

How can researchers generate and validate guaA mutants in Alteromonas macleodii?

Generating guaA mutants in Alteromonas macleodii presents challenges due to the limited genetic tools available for this marine bacterium. The following methodology offers a systematic approach:

Generating guaA mutants:

• Design allelic exchange vectors containing:

  • Homology arms (500-1000 bp) flanking the guaA gene

  • A selectable marker (e.g., kanamycin resistance)

  • A counterselectable marker (e.g., sacB for sucrose sensitivity)

• Introduce the vector via conjugation using triparental mating with an E. coli helper strain

• Select for single crossover events on media containing kanamycin

• Counter-select for double crossover events on sucrose-containing media

• Screen colonies for the desired mutation using PCR

Phenotypic validation:

• Compare growth rates in rich media versus minimal media lacking guanine

• Supplement minimal media with guanine to confirm auxotrophy

• Measure cellular GMP levels using LC-MS/MS

• Perform complementation studies with wild-type guaA

Research with C. difficile has demonstrated that guaA inactivation leads to cell death in minimal growth conditions but not in rich medium, consistent with guanine auxotrophy . Similar phenotypes would be expected in A. macleodii guaA mutants.

What techniques enable structural characterization of recombinant A. macleodii GuaA?

Structural characterization of recombinant A. macleodii GuaA provides crucial insights into enzyme function and potential inhibitor design. The following methodological approaches are recommended:

X-ray crystallography workflow:

• Purify recombinant GuaA to >95% homogeneity with a final concentration of 10-15 mg/ml

• Screen crystallization conditions using sparse matrix screens

• Optimize promising conditions by varying precipitant concentration, pH, and additives

• Collect diffraction data at a synchrotron facility

• Process data and solve structure by molecular replacement using homologous GMP synthase structures as search models

Complementary structural techniques:

• Small-angle X-ray scattering (SAXS) to study solution behavior

• Hydrogen-deuterium exchange mass spectrometry to identify flexible regions

• Cryo-electron microscopy if the protein forms larger complexes

GMP synthase typically consists of two domains: an N-terminal glutaminase domain and a C-terminal synthetase domain . Understanding the specific structural features of A. macleodii GuaA could reveal adaptations to marine environments and provide insights into enzyme evolution.

How does A. macleodii GuaA function differ from that of other marine bacteria?

Comparative analysis of GuaA from various marine bacteria reveals adaptive features that may reflect ecological specialization. While core catalytic mechanisms are conserved, several distinctions in A. macleodii GuaA have been observed:

• Amino acid composition shows higher proportion of acidic residues compared to terrestrial bacterial homologs, potentially contributing to halotolerance

• Kinetic parameters suggest optimization for function in nutrient-limited marine environments

• Substrate specificity may be tuned for efficient function at lower temperatures typical of marine settings

The evolutionary relationship between Alteromonas species, with average nucleotide identities ranging from 99.98% (nearly identical strains) to 73.35% (borderline genus level) , provides an excellent framework for studying how GuaA has evolved within this genus.

What is the significance of guaA in bacterial pathogenicity studies?

Although Alteromonas macleodii is not a human pathogen, research on guaA in pathogenic bacteria provides valuable insights that may apply to A. macleodii:

• In Clostridioides difficile, guaA inactivation significantly reduced colonization capacity in mouse gut infection models

• The impaired colonization demonstrates the importance of de novo GMP biosynthesis during infection

• Guanine riboswitches, which control guaA expression in some bacteria, have been proposed as antimicrobial targets

These findings suggest that GuaA functions are critical not only for basic metabolism but also for bacterial fitness in competitive ecological niches. For A. macleodii, this may translate to competitive advantages in marine environments with fluctuating nutrient availability.

How can recombinant A. macleodii GuaA be used to study riboswitch mechanisms?

Guanine riboswitches represent sophisticated gene regulation mechanisms that control guaA expression in response to guanine concentrations. Research methodologies for studying these interactions include:

• In-line probing assays to study riboswitch conformational changes upon guanine binding

• Reporter gene assays (e.g., GusA) to quantify riboswitch-mediated gene regulation

• Binding affinity measurements to determine Kd values for guanine and related metabolites

Studies in C. difficile have demonstrated that guanine riboswitches exhibit high affinity for guanine (Kd values in the low nanomolar range) and can also bind xanthine and guanosine with lower affinity . These riboswitches cause premature transcription termination upon binding guanine .

Similar mechanisms may regulate guaA expression in A. macleodii, offering opportunities to study how marine bacteria balance de novo nucleotide synthesis with salvage pathways in response to environmental conditions.

What strategies address poor solubility of recombinant A. macleodii GuaA?

Researchers frequently encounter solubility issues when expressing recombinant GMP synthase. The following methodological solutions have proven effective:

Expression optimization strategies:

• Reduce induction temperature to 16-18°C

• Decrease inducer concentration (e.g., 0.1-0.2 mM IPTG)

• Co-express with molecular chaperones (GroEL/GroES, DnaK/DnaJ)

• Use solubility-enhancing fusion partners (SUMO, MBP, thioredoxin)

Buffer optimization for purification:

• Include stabilizing agents (5-10% glycerol, 0.5-1 M NaCl)

• Add cofactors or substrate analogs (ATP, XMP)

• Test different pH ranges (typically pH 7.5-8.5 works best)

• Include reducing agents (5 mM β-mercaptoethanol or 1-2 mM DTT)

These approaches should be systematically tested to identify optimal conditions for obtaining soluble, active enzyme.

How can researchers verify the functional integrity of purified recombinant GuaA?

Confirming that purified recombinant A. macleodii GuaA maintains its native functional properties is essential for reliable research outcomes. A comprehensive validation approach includes:

Enzymatic activity assays:

• Direct assay: Monitor conversion of XMP to GMP using HPLC or coupled enzyme systems

• ATP hydrolysis: Measure ATPase activity associated with the synthetase domain

• Glutaminase activity: Assess glutamine to glutamate conversion

Structural integrity assessment:

• Circular dichroism spectroscopy to confirm secondary structure composition

• Thermal shift assays to evaluate protein stability

• Size exclusion chromatography to verify quaternary structure

Functional complementation:

• Transform guaA-deficient bacterial strains with an expression construct containing the recombinant guaA

• Evaluate growth restoration in minimal media lacking guanine supplements

A functionally intact GuaA enzyme should demonstrate concentration-dependent activity with kinetic parameters consistent with other characterized GMP synthases.