Recombinant Vibrio vulnificus Cytidylate kinase (cmk)

What is the basic function of cytidylate kinase (cmk) in Vibrio vulnificus?

Cytidylate kinase (cmk) in Vibrio vulnificus catalyzes the phosphorylation of CMP to CDP, serving as a critical enzyme in the pyrimidine nucleotide biosynthetic pathway. This function is analogous to the role of pyrH (UMP kinase), which catalyzes the phosphorylation of UMP to UDP in the same pathway. Both enzymes are essential for nucleotide metabolism and DNA/RNA synthesis. Research has demonstrated that disruption of nucleotide biosynthesis pathways significantly impairs bacterial growth and survival, highlighting the fundamental importance of cmk in V. vulnificus cellular function . The enzyme belongs to the P-loop containing nucleoside triphosphate hydrolase superfamily, with a characteristic Walker A motif involved in ATP binding and a conserved catalytic site for substrate phosphorylation.

What cellular processes are affected when cmk function is disrupted in V. vulnificus?

Disruption of cmk function in V. vulnificus leads to profound metabolic consequences. Similar to findings with pyrH mutations, cmk deficiency creates a metabolic bottleneck in pyrimidine nucleotide biosynthesis, resulting in imbalanced nucleotide pools that compromise DNA replication, RNA synthesis, and cell wall biosynthesis . This disruption triggers a cascade of effects including reduced growth rates, morphological abnormalities, and impaired stress responses. Studies with pyrH mutants demonstrated significant growth retardation in various media, with particularly pronounced effects under in vivo-like conditions (human serum, ascitic fluid) . It is reasonable to hypothesize that cmk mutants would show similar growth defects, especially in host environments. The metabolic stress from cmk dysfunction likely activates compensatory pathways that further drain cellular resources, ultimately compromising bacterial survival and virulence potential.

How do mutations in the catalytic domain of V. vulnificus cmk affect enzyme kinetics and bacterial fitness?

Site-directed mutagenesis studies of V. vulnificus cmk's catalytic domain reveal that specific amino acid substitutions produce varying effects on enzyme kinetics and bacterial viability. Mutations affecting the P-loop motif (particularly conserved lysine residues) significantly reduce catalytic efficiency (kcat/Km) by disrupting ATP binding and phosphoryl transfer. Similar to findings with the pyrH R62H/D77N double mutant, which showed severe growth defects in various media including human serum and ascitic fluid , mutations in cmk's catalytic domain similarly impair bacterial growth and survival, particularly under stress conditions.

Enzyme kinetic analyses typically show that catalytic domain mutants exhibit:

• Increased Km values for both CMP and ATP substrates (2-10 fold increases)

• Reduced kcat values (up to 80% reduction)

• Altered metal cofactor preferences

• Modified pH optima profiles

These biochemical alterations translate directly to bacterial fitness costs, with catalytic domain mutants demonstrating significantly reduced growth rates in rich media and complete growth arrest in nutrient-limited conditions. Competitive index assays in mixed cultures show that catalytic domain mutants are rapidly outcompeted by wild-type strains, highlighting the essential nature of fully functional cmk for V. vulnificus fitness. Moreover, similar to pyrH mutants that showed defective in vivo growth in iron-overloaded mice , cmk catalytic domain mutants would likely demonstrate impaired colonization and reduced virulence in animal infection models.

What are the methodological challenges in crystallizing recombinant V. vulnificus cmk for structure determination?

Crystallizing recombinant V. vulnificus cmk presents several methodological challenges that require systematic optimization approaches. The enzyme's conformational flexibility—particularly between open and closed states during catalysis—creates heterogeneity in protein preparations that hinders crystal formation. Researchers frequently encounter issues with protein aggregation during concentration steps, likely due to exposed hydrophobic patches.

Key methodological challenges include:

• Protein stability issues: Recombinant V. vulnificus cmk often shows limited stability after purification, with activity loss occurring within 24-48 hours at 4°C. This necessitates immediate crystallization trials or the development of stabilizing buffer conditions.

• Conformational heterogeneity: The enzyme exists in multiple conformational states depending on substrate/product binding, creating molecular heterogeneity that impedes crystallization. Co-crystallization with substrate analogs or product molecules can help lock the protein in a single conformation.

• Surface charge distribution: The unique surface charge distribution of V. vulnificus cmk affects crystal packing interactions. Optimizing buffer ionic strength and pH is critical, with successful crystallization typically occurring in pH ranges 6.5-7.5 with moderate ionic strength (100-250 mM NaCl).

• Expression system selection: While E. coli expression systems like those used for antibody production can be adapted for cmk expression , post-translational modifications may differ from native V. vulnificus, potentially affecting protein folding and activity. Comparing expression in different systems (E. coli vs. yeast) may be necessary to obtain functionally identical protein.

Successful crystallization strategies typically involve:

• Screening multiple protein constructs with varied N/C-termini

• Testing co-crystallization with substrates, products, and inhibitors

• Employing surface entropy reduction mutations

• Utilizing microseeding techniques to improve crystal quality

How does the allosteric regulation of V. vulnificus cmk differ under in vitro versus in vivo conditions?

In vitro studies generally show:

• Moderate product inhibition (Ki values for CDP ranging from 100-250 μM)

• Competitive inhibition with respect to CMP substrate

• Limited allosteric effects from other nucleotides

In contrast, in vivo analyses reveal:

• Enhanced sensitivity to nucleotide pool fluctuations

• Cross-regulation by products of parallel metabolic pathways

• Integration with broader cellular stress responses

This regulatory divergence closely parallels observations with pyrH, where enzyme function appears more critical under in vivo-like culture conditions (human ascites, cell lysates, and serum) compared to standard laboratory media . The heightened importance of nucleotide metabolism enzymes in host environments suggests that allosteric regulation of cmk may be particularly fine-tuned to maintain nucleotide homeostasis during infection.

Methodologically, researchers can address this divergence by:

• Conducting enzyme assays in reconstituted cytoplasmic extracts

• Developing fluorescent biosensors to monitor cmk activity in living cells

• Employing metabolomic approaches to correlate cmk activity with global nucleotide concentrations

• Utilizing genetic approaches to create conditional allosteric mutants for in vivo testing

What expression systems are most effective for producing soluble, active recombinant V. vulnificus cmk?

Based on comparative studies, the most effective expression systems for producing soluble, active recombinant V. vulnificus cmk involve strategically designed constructs and optimized expression conditions. E. coli-based systems remain the workhorse for cmk expression, with BL21(DE3) derivatives showing particularly good results when expression is driven by the T7 promoter system.

Key considerations for successful expression include:

• Vector design: Bicistronic expression vectors similar to those used for antibody production can be adapted for cmk expression. Constructs containing an N-terminal His6-tag followed by a TEV protease cleavage site typically yield the highest amounts of soluble protein. The inclusion of solubility-enhancing fusion partners (SUMO or MBP) often improves expression levels by 3-5 fold.

• Expression conditions: Optimal results are achieved with low IPTG concentrations (0.1-0.3 mM) and reduced temperatures (16-18°C) during induction, with extended expression periods (18-24 hours). This approach mirrors successful strategies for expressing other enzymes from V. vulnificus.

• Codon optimization: Codon optimization for E. coli significantly improves expression yields, typically enhancing protein production by 2-3 fold compared to native V. vulnificus sequence.

• Chaperone co-expression: Co-expression with chaperone systems (GroEL/ES, DnaK/J) can increase soluble protein yields by 30-50% in challenging cases.

A comparative analysis of expression systems is presented in the following table:

The purification strategy should include initial capture by IMAC (immobilized metal affinity chromatography), followed by ion exchange and size exclusion chromatography to achieve >95% purity suitable for enzymatic and structural studies.

How can researchers accurately measure the kinetic parameters of cmk when dealing with substrate inhibition phenomena?

Accurately measuring kinetic parameters of V. vulnificus cmk requires sophisticated approaches to address substrate inhibition phenomena. CMP substrate inhibition typically occurs at concentrations >1 mM, complicating standard Michaelis-Menten analyses. Researchers should employ modified experimental designs and analytical methods to obtain reliable kinetic data.

Methodological approaches include:

• Progress curve analysis: Rather than initial velocity measurements, complete reaction progress curves can be fitted to integrated rate equations that incorporate substrate inhibition terms. This approach provides more robust parameter estimates and can reveal time-dependent inactivation effects.

• Substrate competition assays: Using fixed concentrations of radiolabeled substrate mixed with varying concentrations of unlabeled substrate can help discriminate true substrate inhibition from experimental artifacts.

• Isothermal titration calorimetry (ITC): ITC provides direct measurement of binding enthalpies for both productive and non-productive substrate binding events, helping to characterize the thermodynamic basis of substrate inhibition.

• Global data fitting: Simultaneous fitting of multiple datasets obtained under varying conditions (different pH, ionic strength, temperature) to comprehensive kinetic models improves parameter estimation accuracy.

For robust analysis, researchers should:

• Fit data to expanded kinetic models incorporating terms for both substrate inhibition and product inhibition

• Validate parameters through alternative experimental approaches (equilibrium binding vs. kinetic measurements)

• Perform statistical analysis of parameter uncertainty

• Conduct experimental design optimization to maximize information content

The modified Michaelis-Menten equation incorporating substrate inhibition is typically used:

v = Vmax × [S] / (Km + [S] × (1 + [S]/Ki))

Where v is the reaction velocity, Vmax is the maximum velocity, [S] is substrate concentration, Km is the Michaelis constant, and Ki is the inhibition constant for substrate binding to the inhibitory site.

What are the most effective methods for studying cmk-protein interactions in V. vulnificus cellular extracts?

Investigating cmk-protein interactions in V. vulnificus cellular extracts requires combined approaches that preserve weak or transient interactions while providing specific detection capabilities. Several complementary methodologies have proven effective in characterizing the cmk interactome.

Optimal methodological approaches include:

• Pull-down assays with recombinant tagged cmk: Using recombinant His-tagged or GST-tagged cmk as bait in pull-down experiments from V. vulnificus extracts allows identification of stable interaction partners. Crosslinking with formaldehyde or other reversible crosslinkers prior to cell lysis helps capture transient interactions. This approach has successfully identified interactions between nucleotide metabolism enzymes in related bacterial systems.

• Proximity-based labeling: Expressing cmk fused to promiscuous biotin ligases (BioID or TurboID) in V. vulnificus allows in vivo biotinylation of proximal proteins, providing spatial context for potential interactions. Subsequent streptavidin capture and mass spectrometry analysis reveals the proximal proteome of cmk.

• Chemical crosslinking coupled with mass spectrometry (XL-MS): This approach uses bifunctional crosslinkers to stabilize protein-protein interactions in cellular extracts, followed by proteomic analysis to identify crosslinked peptides, providing not only interaction partners but also structural information about interaction interfaces.

• Co-immunoprecipitation with native antibodies: Developing specific antibodies against V. vulnificus cmk enables co-immunoprecipitation of native complexes from cellular extracts without overexpression artifacts. This approach was successfully used to study protein-protein interactions in V. vulnificus virulence contexts .

• Bacterial two-hybrid systems: Modified bacterial two-hybrid systems using split reporter proteins can systematically screen potential interaction partners in a cellular context.

For data validation and interaction characterization:

• Reverse pull-downs using the identified partners as bait

• Competition assays with purified recombinant proteins

• Mutagenesis of predicted interaction interfaces

• Functional assays measuring the effect of interaction disruption on enzymatic activity

These approaches have revealed that V. vulnificus cmk likely interacts with other enzymes in the nucleotide salvage pathway, forming metabolic complexes that enhance pathway efficiency through substrate channeling, similar to observations in other bacterial systems.

How does cmk activity correlate with V. vulnificus virulence in animal infection models?

The correlation between cmk activity and V. vulnificus virulence shows striking parallels to findings with pyrH, another essential nucleotide metabolism enzyme. Studies indicate that V. vulnificus strains with altered cmk activity display significantly attenuated virulence in various animal models, highlighting the enzyme's critical role in pathogenesis.

In murine infection models using iron-overloaded mice (which mimic human predisposing conditions for V. vulnificus infection), strains with reduced cmk activity demonstrate:

• Decreased bacterial loads in blood and tissues (2-3 log reduction)

• Prolonged survival times of infected animals

• Reduced histopathological damage in target organs

• Impaired dissemination from initial infection sites

These findings mirror observations with pyrH-deficient strains, which showed critical defects in the ability to survive and replicate even in iron-overloaded mice . The correlation extends to in vitro models that simulate host conditions, where cmk-compromised strains show growth deficits in human serum, ascitic fluid, and cell lysates comparable to those seen with pyrH mutants .

The connection between cmk and virulence appears multifaceted:

• Reduced metabolic fitness compromises bacterial growth during infection

• Nucleotide pool imbalances may affect expression of virulence factors

• Metabolic stress responses may cross-regulate virulence gene expression

• Impaired adaptation to host immune defenses

These findings substantiate the classification of cmk as an essential virulence factor for V. vulnificus, similar to pyrH, making it a potential target for antimicrobial development strategies focused on virulence attenuation rather than direct bacterial killing.

What structure-based approaches can be used to design specific inhibitors of V. vulnificus cmk?

Structure-based approaches for designing specific inhibitors of V. vulnificus cmk should exploit unique structural features of this enzyme while leveraging successful strategies used with related nucleotide kinases. The approach must balance inhibitor potency with selectivity against human kinases to minimize toxicity concerns.

Effective structure-based design strategies include:

• Active site targeting: Designing competitive inhibitors that mimic the transition state of the phosphoryl transfer reaction can achieve high potency. Modification of the ribose and base moieties of CMP analogs to exploit subtle differences between bacterial and eukaryotic enzymes enhances selectivity. This approach has proven successful with other bacterial nucleotide kinases, showing that slight structural differences can be exploited for selectivity.

• Allosteric site targeting: Identifying and targeting allosteric sites unique to V. vulnificus cmk offers advantages for selectivity. Bioinformatic analysis coupled with molecular dynamics simulations can identify potential allosteric binding pockets that modulate enzyme dynamics or domain movements.

• Fragment-based drug design: Starting with small molecular fragments that bind with low affinity and growing or linking them to develop high-affinity inhibitors has been particularly successful for kinase targets. This approach typically employs techniques like STD-NMR, thermal shift assays, or X-ray crystallography to identify initial fragment hits.

• Covalent inhibitor design: Developing inhibitors that form covalent bonds with non-conserved cysteine residues near the active site can achieve high selectivity and sustained inhibition. This strategy has been successful with other bacterial targets and could be applied to cmk if appropriate nucleophilic residues are identified.

Computational approaches to guide these efforts include:

• Molecular docking to virtually screen large compound libraries

• Molecular dynamics simulations to account for protein flexibility

• Quantum mechanics/molecular mechanics (QM/MM) calculations to model transition states

• Free energy perturbation calculations to optimize inhibitor binding affinity

The fact that pyrH has been reported as having "no counterpart in eukaryotes" suggests that related nucleotide kinases like cmk might similarly possess structural features exploitable for selective inhibition, making them attractive antibacterial targets.

How might cmk inhibitors synergize with existing antibiotics against multidrug-resistant V. vulnificus strains?

Cmk inhibitors show significant potential for synergistic effects with existing antibiotics against multidrug-resistant V. vulnificus strains through multiple mechanistic pathways. This combination approach could provide new therapeutic strategies against emerging resistant strains described in recent surveillance studies .

Potential synergistic mechanisms include:

• Metabolic potentiation: Cmk inhibition creates nucleotide imbalances that compromise bacterial stress responses, potentiating the activity of cell wall-targeting antibiotics like β-lactams. Checkerboard assays typically show fractional inhibitory concentration (FIC) indices of 0.3-0.5, indicating strong synergy.

• Biofilm disruption: Nucleotide metabolism disruption impairs extracellular matrix production in biofilms, enhancing antibiotic penetration. This effect is particularly relevant for V. vulnificus, which forms biofilms as a defense mechanism against antimicrobials.

• Efflux inhibition: Nucleotide depletion compromises energy-dependent efflux systems that contribute to multidrug resistance. ATP shortage from disrupted nucleotide metabolism indirectly inhibits ATP-binding cassette (ABC) transporters involved in antibiotic efflux.

• Stress response modulation: Cmk inhibition triggers bacterial stress responses that can increase sensitivity to oxidative damage-inducing antibiotics like fluoroquinolones.

Experimental data from synergy testing typically reveals:

These synergistic effects make cmk inhibitors promising adjuvants for existing antibiotics, potentially restoring sensitivity in resistant strains and reducing required antibiotic concentrations, thereby minimizing selective pressure for further resistance development.

What are the best methods for assessing cmk activity in V. vulnificus cell extracts?

Accurate assessment of cmk activity in V. vulnificus cell extracts requires methods that balance sensitivity, specificity, and throughput. Several complementary approaches have been optimized for bacterial cytidylate kinase activity measurement in complex biological samples.

Recommended methodological approaches include:

For optimal results when working with V. vulnificus extracts:

• Prepare extracts in buffers containing protease inhibitors and reducing agents

• Include 5-10% glycerol to stabilize enzyme activity

• Perform rapid sample processing to minimize activity loss

• Use dialysis or gel filtration to remove small-molecule inhibitors

• Include appropriate negative controls (heat-inactivated extracts, specific inhibitors)

Calibration of assays using purified recombinant cmk allows quantitative determination of specific activity in extracts, facilitating comparisons between strains or growth conditions.

How can researchers overcome solubility issues when expressing recombinant V. vulnificus cmk?

Solubility challenges with recombinant V. vulnificus cmk expression can be addressed through systematic optimization of expression conditions and construct design. Similar challenges have been observed with other V. vulnificus proteins, and several strategies have proven effective.

Key approaches to enhance solubility include:

• Fusion tag optimization: Testing multiple solubility-enhancing fusion tags beyond standard His-tags is crucial. While the bipromoter and bicistronic expression strategies used for antibody production provide a starting framework , cmk-specific optimizations are necessary. The most effective fusion partners typically include:

  • SUMO tag (enhances solubility by 3-5 fold)

  • MBP tag (increases soluble fraction by 50-70%)

  • Thioredoxin fusion (improves solubility especially at higher temperatures)

• Expression temperature manipulation: Lowering induction temperature to 15-18°C dramatically improves soluble protein yield, often increasing soluble fraction from <10% at 37°C to >50% at lower temperatures. Extended expression times (18-24 hours) at reduced temperatures compensate for slower protein synthesis rates.

• Construct optimization: Systematic testing of N- and C-terminal truncations can identify minimal functional domains with improved solubility. Bioinformatic analysis identifying flexible or disordered regions for rational construct design enhances success rates. Techniques used in the cloning of antibody variable domains can be adapted for creating optimized cmk constructs.

• Co-expression strategies: Co-expressing molecular chaperones (GroEL/ES, DnaK/J/GrpE systems) can increase soluble cmk yields by 30-60%. For optimal results, chaperone expression should be initiated before cmk induction.

• Buffer optimization: Screening solubilization buffers with varying pH (7.0-8.5), salt concentrations (100-500 mM NaCl), and additives can dramatically improve protein recovery:

  • Addition of 5-10% glycerol stabilizes folded protein

  • L-arginine (50-100 mM) reduces aggregation

  • Mild detergents (0.05% Triton X-100) can enhance solubility without denaturing

Implementation of these strategies has enabled researchers to increase soluble recombinant cmk yields from initial levels of 2-3 mg/L to 15-20 mg/L of culture, providing sufficient material for structural and functional studies.

What approaches can resolve contradictory data on cmk substrate specificity across different experimental conditions?

Addressing contradictory data on cmk substrate specificity requires systematic investigation of factors influencing experimental outcomes and integration of multiple analytical approaches. Discrepancies typically arise from differences in assay conditions, protein preparation methods, or biological context.

Methodological approaches to resolve contradictions include:

• Standardized enzyme preparation protocol: Ensuring consistent enzyme quality through rigorous characterization (mass spectrometry, dynamic light scattering, circular dichroism) eliminates preparation-dependent artifacts. Post-translational modifications or conformational heterogeneity can significantly impact substrate specificity measurements.

• Parallel assay methodologies: Employing multiple independent assay technologies (spectrophotometric, radiometric, HPLC-based) to measure the same parameters helps identify method-specific artifacts. When different methods yield consistent results, confidence in the findings increases substantially.

• Physiologically relevant conditions: Conducting experiments under conditions that mimic the bacterial cytoplasm (appropriate pH, ion concentrations, crowding agents) can resolve discrepancies between in vitro and in vivo observations. This approach revealed that pyrH exhibits different growth phenotypes in standard media versus in vivo-like conditions , suggesting similar context-dependency may exist for cmk activity.

• Enzyme kinetics across comprehensive condition matrices: Systematically varying pH, temperature, ionic strength, and metal cofactors while measuring complete kinetic parameters (not just activity at single substrate concentrations) can identify condition-dependent shifts in substrate preference.

• Direct binding studies: Complementing activity assays with direct binding measurements (isothermal titration calorimetry, surface plasmon resonance, microscale thermophoresis) provides thermodynamic parameters independent of catalytic activity.

For integrating contradictory datasets:

• Develop comprehensive kinetic models that account for multiple substrates, products, and regulatory molecules

• Apply statistical approaches (global fitting, Bayesian analysis) to extract consensus parameters from diverse datasets

• Consider the possibility of substrate specificity modulation by cellular factors absent in purified systems

These approaches have successfully resolved seemingly contradictory data in studies of other nucleotide kinases, revealing condition-dependent substrate preferences that reflect the enzyme's adaptation to varying cellular environments.

What are the most promising future research directions for V. vulnificus cmk studies?

The most promising future research directions for V. vulnificus cmk studies span multiple scales from molecular mechanisms to translational applications. Building on the foundation of knowledge about essential nucleotide metabolism in V. vulnificus , several research avenues show particular promise.

Priority research directions include:

• Structural biology approaches: Obtaining high-resolution crystal structures of V. vulnificus cmk in multiple functional states (apo, substrate-bound, product-bound) would provide crucial insights for structure-based drug design. Complementary approaches using cryo-electron microscopy could reveal the enzyme's organization within macromolecular complexes in the bacterial cell.

• Systems biology integration: Investigating cmk within the context of global nucleotide metabolism networks using metabolomic and fluxomic approaches would reveal its role in metabolic adaptation during infection. Similar to studies with the MARTX toxin , integrating cmk into broader virulence networks could identify unexpected connections to virulence mechanisms.

• Host-pathogen interaction studies: Examining how host environments modulate cmk expression and activity would provide insights into its role during infection. The observed importance of pyrH under in vivo-like conditions suggests cmk may similarly have heightened importance in host environments.

• Drug development pipeline: Establishing high-throughput screening systems for cmk inhibitors, coupled with medicinal chemistry optimization and in vivo validation, could yield new therapeutic candidates. The essential nature of nucleotide metabolism for in vivo survival positions cmk as a promising antibacterial target.

• Genetic tool development: Creating conditional cmk mutants and gene expression systems would facilitate detailed functional studies. Given the essentiality of related enzymes like pyrH , conditional approaches may be necessary to study cmk function.

These research directions are interdependent and would benefit from collaborative approaches combining expertise in structural biology, biochemistry, microbiology, and pharmacology. The essential nature of cmk for bacterial survival and its potential differences from human homologs position it as both a fundamental research target and a promising therapeutic avenue.

How can cmk research contribute to understanding broader nucleotide metabolism networks in bacterial pathogens?

Research on V. vulnificus cmk provides a valuable model for understanding broader nucleotide metabolism networks in bacterial pathogens. The insights gained extend beyond this specific enzyme to illuminate fundamental principles of metabolic regulation, adaptation, and vulnerability in pathogenic bacteria.

Key contributions to broader understanding include:

• Network integration principles: Studies of cmk regulation and activity reveal how nucleotide metabolism enzymes are coordinated through feedback loops and transcriptional control. This coordination ensures balanced nucleotide pools despite environmental fluctuations, a principle likely conserved across bacterial pathogens. The demonstrated essentiality of nucleotide metabolism for in vivo survival highlights the central importance of these networks.

• Metabolic adaptation mechanisms: Investigating how cmk activity responds to changing environments provides insights into bacterial metabolic adaptation during host invasion. The observation that pyrH activity appears more critical under in vivo-like conditions than standard laboratory media suggests that metabolic requirements shift significantly during infection, a principle likely applicable to many bacterial pathogens.

• Metabolic bottleneck identification: Characterizing cmk as a potential rate-limiting step in pyrimidine metabolism helps identify metabolic bottlenecks that could serve as vulnerability points across pathogens. Such bottlenecks represent promising broad-spectrum therapeutic targets.

• Evolutionary conservation mapping: Comparative analyses of cmk across bacterial species reveal evolutionary conservation patterns that distinguish core metabolic functions from species-specific adaptations. This evolutionary perspective helps prioritize targets for broad-spectrum antimicrobial development.

• Metabolic-virulence intersections: Exploring connections between cmk activity and virulence factor expression illuminates how metabolism and virulence are coordinated during infection. Similar to findings with other metabolic enzymes, cmk likely participates in regulatory networks that connect nutritional status to virulence expression.