Recombinant Rhodopirellula baltica NAD-dependent protein deacylase (cobB)

What is the functional role of NAD⁺-dependent protein deacylase (CobB) in bacterial systems?

CobB functions as a NAD⁺-dependent lysine deacetylase that plays critical roles in bacterial metabolism and cellular regulation. As demonstrated across multiple bacterial species, CobB removes acetyl groups from lysine residues of target proteins, thereby modifying their function, activity, or stability.

In Escherichia coli, CobB has been shown to control energy metabolism, chemotaxis, and DNA supercoiling . One well-established substrate is acetyl-CoA synthetase (Acs), which CobB activates through deacetylation of lysine-609 (K609) . This activation is essential for the synthesis of acetyl-CoA, which directly impacts cell growth and energy production .

While specific R. baltica CobB studies are more limited, the high conservation of this enzyme across prokaryotes suggests similar functional roles in metabolic regulation and cellular signaling pathways.

How does the structure of CobB relate to its deacetylase function?

The structure of CobB contains distinct domains that each contribute to its deacetylase function. Based on research with E. coli CobB (which serves as a model for other bacterial CobB proteins), the enzyme contains a catalytic domain and an N-terminal regulatory region.

The N-terminal tail (residues 1-37) has been identified as essential for regulatory interactions, particularly for binding secondary messengers like c-di-GMP . Within this region, specific residues including R8, R17, and E21 have been identified as critical for c-di-GMP binding . Mutagenesis studies demonstrated that changing these residues to alanine (CobB R8A, CobB R17A, and CobB E21A) significantly reduced c-di-GMP binding affinity without affecting the intrinsic deacetylase activity of the enzyme .

The catalytic domain contains the NAD⁺-binding site that is essential for the deacetylation reaction. As a member of the sirtuin family, CobB utilizes NAD⁺ as a co-substrate in the deacetylation reaction, which distinguishes it from zinc-dependent histone deacetylases.

While the structure of R. baltica CobB has not been fully characterized in the available literature, the high conservation of CobB across bacterial species suggests similar structural features and mechanisms.

What are the main substrates of bacterial CobB deacylases?

CobB deacetylases target numerous protein substrates across diverse cellular pathways. The most well-characterized substrate is acetyl-CoA synthetase (Acs), which is activated through deacetylation of a specific lysine residue (K609 in E. coli) . This activation is critical for cell growth, particularly in conditions where acetate is a primary carbon source.

Quantitative acetylome studies comparing wild-type and ΔcobB strains have revealed hundreds of potential CobB substrates. In enterohemorrhagic E. coli O157:H7, comparative acetylation proteomics identified:

Among the most dramatically affected substrates (>100-fold increase in acetylation in the ΔcobB strain) were:

Other heavily acetylated proteins identified included RNA polymerase beta prime subunit (40 sites), enolase (20 sites), and ribosomal proteins at multiple sites . These findings suggest CobB plays a broad regulatory role in metabolism, biosynthesis, and transcription.

What is the mechanism of c-di-GMP binding to CobB and how does it affect enzymatic activity?

The interaction between c-di-GMP and CobB represents a significant regulatory mechanism. Research has established that c-di-GMP binds specifically to CobB with physiologically relevant affinity, and this binding inhibits CobB's deacetylase activity through the following mechanism:

The binding specificity is remarkable - while c-di-GMP binds CobB with a dissociation constant (Kd) of 4.7 μM and a binding stoichiometry of 0.95, other cyclic nucleotides like cGMP and c-di-AMP show no detectable binding to CobB in isothermal titration calorimetry (ITC) assays .

The c-di-GMP binding site has been mapped to the N-terminal tail of CobB (residues 1-37). Mutagenesis studies identified three critical residues for this interaction:

• R8 (arginine at position 8)

• R17 (arginine at position 17)

• E21 (glutamate at position 21)

When these residues were mutated to alanine, the binding affinity for c-di-GMP decreased dramatically:

• CobB R8A: Kd = 274.7 μM (58-fold reduction)

• CobB R17A: Kd = 89.3 μM (19-fold reduction)

• CobB E21A: Kd = 294.1 μM (63-fold reduction)

Importantly, these mutations did not affect the intrinsic deacetylase activity of CobB, confirming that c-di-GMP binding and catalytic activity involve different regions of the protein .

The functional consequence of c-di-GMP binding is inhibition of CobB's deacetylase activity. In vitro deacetylation assays with known CobB substrates showed reduced deacetylation in the presence of c-di-GMP . In vivo studies demonstrated that strains with elevated c-di-GMP levels (through DgcZ overexpression) exhibited increased protein acetylation and reduced growth on acetate medium, consistent with CobB inhibition .

How does CobB participate in cellular feedback regulatory circuits?

CobB participates in sophisticated feedback regulatory circuits that coordinate metabolism and signaling in bacterial cells. One particularly well-characterized circuit involves the interplay between CobB and the c-di-GMP signaling pathway.

A negative feedback loop exists where:

• CobB deacetylates and activates DgcZ (a diguanylate cyclase)

• Activated DgcZ produces more c-di-GMP

• Elevated c-di-GMP binds to CobB and inhibits its deacetylase activity

• This inhibition prevents further activation of DgcZ, creating a homeostatic regulatory circuit

This feedback loop was demonstrated through several experimental approaches:

• In vitro deacetylation assays showed that c-di-GMP inhibits CobB activity towards substrates like Acs and DgcZ

• Strains overexpressing DgcZ showed elevated c-di-GMP levels and increased protein acetylation, consistent with CobB inhibition

• Growth assays on acetate medium showed reduced growth in strains with elevated c-di-GMP, indicating impaired Acs activity due to CobB inhibition

The physiological significance of this feedback loop likely extends beyond c-di-GMP signaling. By linking protein acetylation status to second messenger levels, bacteria can coordinate multiple aspects of cellular physiology including metabolism, motility, and potentially virulence in response to environmental conditions.

In enterohemorrhagic E. coli, CobB has been implicated in virulence regulation through its deacetylation of the LEE chaperone CesA, potentially creating additional regulatory circuits in pathogenic strains .

What methodologies are most effective for assessing CobB deacetylase activity in vitro?

Assessing CobB deacetylase activity in vitro requires specialized techniques that can detect the removal of acetyl groups from substrate proteins. Based on the research literature, several methodologies have proven effective:

Western Blotting with Anti-Acetyllysine Antibodies:
This approach allows for detection of acetylation changes in specific proteins. The method involves:

• Incubating purified CobB with acetylated substrate proteins and NAD⁺

• Separating proteins by SDS-PAGE and transferring to membranes

• Probing with anti-acetyllysine antibodies

• Quantifying changes in acetylation signal

This method has been successfully used to monitor CobB activity toward substrates like Acs and to assess the impact of potential inhibitors like c-di-GMP.

Isothermal Titration Calorimetry (ITC):
While not directly measuring deacetylase activity, ITC provides valuable information about binding interactions that can affect activity. ITC has been used to:

• Determine the binding affinity (Kd) between CobB and c-di-GMP (4.7 μM)

• Compare binding affinities of wild-type and mutant CobB proteins

• Assess binding specificity by testing different cyclic nucleotides

Activity Assays with Synthetic Acetylated Peptides:
Using synthetic peptides containing acetylated lysine residues derived from known CobB substrates allows for quantitative assessment of deacetylation rates. These assays can be coupled to spectrophotometric or fluorometric detection systems that monitor NAD⁺ consumption or product formation.

Mass Spectrometry-Based Approaches:
Mass spectrometry provides the most comprehensive analysis of acetylation changes. The approach typically involves:

• In vitro deacetylation reactions with purified components

• Protein digestion to generate peptides

• Enrichment of acetylated peptides if necessary

• LC-MS/MS analysis to identify and quantify acetylation sites

• Comparison of acetylation levels in samples with and without active CobB

This approach was used to identify 2,128 acetylated proteins in E. coli O157:H7 and to quantify changes in acetylation status in the absence of CobB .

What are the optimal conditions for expressing and purifying recombinant R. baltica CobB?

While the provided literature doesn't offer specific protocols for R. baltica CobB expression and purification, we can derive recommendations based on successful approaches with bacterial CobB proteins and general recombinant protein methodologies:

Expression System Selection:

• E. coli BL21(DE3) or similar strains typically provide high expression levels for bacterial proteins

• Cold-shock inducible systems may improve solubility if standard induction leads to inclusion body formation

• For physiological studies, consider complementation of a cobB-knockout strain to verify functional activity

Vector Design Considerations:

• Include an N-terminal or C-terminal affinity tag (His6, GST, or MBP) for purification

• If studying c-di-GMP binding, avoid N-terminal tags that might interfere with the N-terminal tail (residues 1-37), which contains critical binding residues (R8, R17, E21)

• Consider a cleavable tag system to remove affinity tags after purification

Expression Conditions:

• Inducer concentration: Typically 0.1-1.0 mM IPTG for T7-based systems

• Temperature: Lower temperatures (16-20°C) often improve solubility

• Duration: Extended expression (overnight) at lower temperatures can increase yield of soluble protein

Purification Strategy:

• Affinity chromatography (Ni-NTA for His-tagged proteins)

• Ion exchange chromatography to remove nucleic acid contamination

• Size exclusion chromatography for final polishing and buffer exchange

Buffer Optimization:

• Include 10-20% glycerol to enhance stability

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

• Consider including zinc or other metal ions if required for structural integrity

• For activity studies, ensure buffer compatibility with NAD⁺-dependent assays

Activity Verification:

• Test deacetylase activity using known substrates like acetylated Acs

• Confirm binding to c-di-GMP using ITC or other binding assays

• Assess oligomeric state by size exclusion chromatography or native PAGE

These recommendations should provide a starting point for developing a specific protocol for R. baltica CobB expression and purification, which can then be optimized based on experimental results.

What approaches are recommended for identifying novel CobB substrates?

Identifying novel CobB substrates requires comprehensive approaches that can detect changes in protein acetylation. Based on successful studies in the field, several strategies are recommended:

Comparative Acetylome Analysis:
This approach has proven highly effective for identifying CobB substrates on a proteome-wide scale:

• Generate cobB knockout and wild-type strains

• Extract proteins and digest to peptides

• Enrich acetylated peptides using anti-acetyllysine antibodies

• Analyze by LC-MS/MS with quantitative methods (e.g., SILAC, TMT, label-free quantification)

• Identify peptides with significantly increased acetylation in the cobB knockout strain

This method successfully identified 581 acetylated peptides with significantly increased abundance in a ΔcobB strain compared to wild-type, representing 426 potential CobB substrate proteins .

Targeted Candidate Approach:
For testing specific proteins of interest:

• Express and purify candidate proteins from cobB knockout strains to ensure acetylation

• Incubate with purified recombinant CobB and NAD⁺

• Analyze acetylation status before and after CobB treatment using:

  • Western blotting with anti-acetyllysine antibodies

  • Mass spectrometry to identify specific deacetylated residues

  • Functional assays to determine the impact of deacetylation on protein activity

In Vitro Biotinylated Protein Microarray:
For high-throughput screening:

• Generate protein microarrays from your organism of interest

• Treat with specific acetyltransferases and acetyl-CoA to acetylate proteins

• Incubate with CobB and NAD⁺

• Detect changes in acetylation using fluorescently labeled anti-acetyllysine antibodies

• Identify proteins showing reduced acetylation after CobB treatment

Biochemical Affinity Purification:
To identify physical interaction partners:

• Generate tagged CobB (consider catalytically inactive mutants to stabilize interactions)

• Perform pull-down experiments coupled with mass spectrometry

• Validate interactions by reciprocal co-immunoprecipitation

• Confirm substrate status by demonstrating CobB-dependent deacetylation

Bioinformatic Prediction:
Enhance experimental approaches with computational methods:

• Analyze known CobB substrates to identify consensus sequence motifs around acetylated lysines

• Predict potential substrates based on sequence similarity and structural accessibility

• Prioritize candidates for experimental validation based on prediction scores

These complementary approaches can be combined to develop a comprehensive understanding of the CobB substrate landscape in R. baltica or other bacterial species of interest.

How can researchers investigate the role of CobB in metabolic regulation?

Investigating CobB's role in metabolic regulation requires a multi-faceted approach that combines genetic, biochemical, and systems-level analyses. The following methodological framework is recommended:

Genetic Manipulation Strategies:

• Generate precise cobB deletion mutants using CRISPR-Cas9 or homologous recombination

• Create complementation strains expressing wild-type cobB

• Engineer point mutants affecting:

  • Catalytic activity (to separate effects of protein presence from enzymatic activity)

  • c-di-GMP binding (e.g., R8A, R17A, E21A) to disrupt regulatory interactions

• Develop inducible expression systems for temporal control of CobB levels

Metabolic Phenotyping:

• Growth curve analysis in different carbon sources, particularly:

  • Acetate media (to assess Acs activation through deacetylation)

  • Mixed carbon sources to evaluate metabolic flexibility

• Measure key metabolites using:

  • Targeted LC-MS for acetyl-CoA, CoA, and central carbon metabolites

  • NMR metabolomics for broader metabolic profiles

• Assess oxygen consumption and carbon dioxide production rates

Integration with c-di-GMP Signaling:

• Manipulate c-di-GMP levels by:

  • Overexpressing DgcZ (to increase c-di-GMP)

  • Using DgcZ mutants (e.g., DgcZ G206A,G207A) as controls

• Measure c-di-GMP levels using:

  • HPLC-MS/MS quantification

  • Fluorescent biosensor systems

• Correlate c-di-GMP levels with:

  • CobB activity (measured by substrate acetylation)

  • Growth phenotypes

  • Metabolic parameters

Systems-Level Analysis:

• Combine acetylome profiling with:

  • Transcriptomics to identify regulatory networks

  • Proteomics to assess protein abundance changes

  • Fluxomics to measure metabolic pathway activities

• Develop computational models integrating:

  • CobB-mediated protein deacetylation

  • c-di-GMP signaling dynamics

  • Metabolic flux distributions

Specific Experimental Approaches:

• For acetate metabolism studies:

  • Monitor Acs acetylation status using western blotting or mass spectrometry

  • Measure acetate consumption rates in different genetic backgrounds

  • Quantify acetyl-CoA levels to assess metabolic impact

• For central carbon metabolism:

  • Analyze acetylation status of key enzymes like enolase and glyceraldehyde-3-phosphate dehydrogenase

  • Measure enzyme activities in cell-free extracts

  • Perform 13C metabolic flux analysis to quantify pathway activities

These approaches will provide comprehensive insights into how CobB-mediated deacetylation regulates metabolic pathways in R. baltica and related bacteria.

What is the interplay between CobB and c-di-GMP signaling pathways?

The interplay between CobB and c-di-GMP signaling represents a sophisticated regulatory network with bidirectional control mechanisms. Based on current research, this interaction can be characterized as follows:

Molecular Basis of Interaction:

• c-di-GMP directly binds to CobB with a physiologically relevant affinity (Kd = 4.7 μM)

• This binding occurs specifically at the N-terminal region of CobB, with residues R8, R17, and E21 playing critical roles

• The binding is highly specific for c-di-GMP; other cyclic nucleotides like cGMP and c-di-AMP show no detectable binding

Regulatory Mechanisms:

• c-di-GMP inhibits CobB activity:

  • In vitro deacetylation assays demonstrate that c-di-GMP inhibits CobB-mediated deacetylation of substrates like Acs

  • This inhibition is dose-dependent and occurs at physiologically relevant concentrations

  • Point mutations in the c-di-GMP binding site of CobB (R8A, R17A, E21A) render the enzyme resistant to c-di-GMP inhibition

• CobB regulates c-di-GMP production:

  • DgcZ, a diguanylate cyclase responsible for c-di-GMP synthesis, is a substrate of CobB

  • Deacetylation of DgcZ by CobB enhances its activity, increasing c-di-GMP production

  • This creates a negative feedback loop: CobB activates DgcZ → DgcZ produces c-di-GMP → c-di-GMP inhibits CobB → reduced DgcZ activation

Physiological Consequences:

The interplay affects multiple cellular processes:

• Metabolic Regulation:

  • Inhibition of CobB by c-di-GMP decreases Acs activation

  • Reduced Acs activity impairs acetate utilization and acetyl-CoA production

  • Strains with elevated c-di-GMP show reduced growth on acetate media

• Potential Impact on Biofilm Formation:

  • c-di-GMP is a key regulator of biofilm formation in many bacteria

  • The feedback loop between CobB and c-di-GMP may help modulate the transition between planktonic and biofilm lifestyles

• Possible Virulence Regulation:

  • In pathogenic strains, CobB regulates virulence factors

  • The CobB/c-di-GMP circuit could potentially coordinate virulence with metabolic state

Experimental Evidence Supporting This Interplay:

This complex regulatory network allows bacteria to coordinate metabolic activities with environmental sensing and cellular signaling, providing an integrated response system for adaptation to changing conditions.

What are common challenges in studying CobB function and how can they be addressed?

Studying CobB function presents several technical and conceptual challenges that researchers should anticipate and address:

Challenge 1: Distinguishing Direct from Indirect Effects
CobB affects numerous proteins, making it difficult to determine which phenotypic changes result from direct deacetylation versus downstream effects.

Solutions:

• Use catalytically inactive CobB mutants as controls to separate binding from enzymatic effects

• Employ site-specific acetylation mimics (K→Q mutations) or non-deacetylatable variants (K→R mutations) in target proteins

• Conduct in vitro deacetylation assays with purified components to confirm direct effects

• Use time-course experiments to identify primary versus secondary effects

Challenge 2: Low Abundance of Acetylated Proteins
Many acetylated proteins exist at low stoichiometry, making detection challenging.

Solutions:

• Enrich acetylated peptides using anti-acetyllysine antibodies before mass spectrometry

• Use sensitive detection methods like selected reaction monitoring (SRM) or parallel reaction monitoring (PRM)

• Consider chemical approaches to stabilize acetylation during sample preparation

• Increase detection sensitivity by using ΔcobB strains where acetylation accumulates

Challenge 3: Preserving Acetylation Status During Sample Preparation
Acetylation can be lost during protein extraction and processing.

Solutions:

• Include deacetylase inhibitors (e.g., nicotinamide) in lysis buffers

• Add protease and deacetylase inhibitor cocktails immediately upon cell disruption

• Use rapid sample preparation protocols to minimize processing time

• Consider flash-freezing samples before processing

Challenge 4: Specificity of Anti-acetyllysine Antibodies
Antibodies may have varying affinities for different acetylated sequences.

Solutions:

• Validate antibody specificity using synthetic acetylated peptides

• Use multiple antibodies from different sources when possible

• Complement antibody-based methods with mass spectrometry

• Consider pan-specific and site-specific antibodies for comprehensive coverage

Challenge 5: Physiological Relevance of c-di-GMP Concentrations
Ensuring experiments use physiologically relevant c-di-GMP concentrations.

Solutions:

• Measure endogenous c-di-GMP levels using HPLC-MS/MS

• Use genetic approaches to modulate c-di-GMP levels within physiological ranges

• Include appropriate controls (e.g., DgcZ G206A,G207A mutants) that maintain normal c-di-GMP levels

• Perform dose-response experiments to determine concentration thresholds

Challenge 6: Functional Redundancy with Other Deacetylases
Some bacteria have multiple deacetylases with overlapping functions.

Solutions:

• Generate single and multiple deacetylase knockouts to assess redundancy

• Use phylogenetic and structural analysis to identify all potential deacetylases

• Characterize substrate specificity profiles for each deacetylase

• Implement conditional depletion strategies for essential deacetylases

By anticipating these challenges and implementing appropriate methodological solutions, researchers can generate more robust and physiologically relevant insights into CobB function in R. baltica and other bacterial systems.

How can researchers compare CobB function across different bacterial species?

Comparing CobB function across different bacterial species requires systematic approaches that account for evolutionary diversity while maintaining methodological consistency. The following framework enables meaningful cross-species comparisons:

Sequence and Structural Analysis:

• Perform multiple sequence alignments of CobB homologs to identify:

  • Conserved catalytic residues

  • Species-specific insertions/deletions

  • Divergent regulatory domains

• Construct phylogenetic trees to visualize evolutionary relationships

• Apply homology modeling to predict structural differences, particularly in:

  • NAD⁺ binding pockets

  • Substrate binding regions

  • c-di-GMP interaction domains (focusing on residues equivalent to R8, R17, and E21)

Recombinant Protein Expression and Characterization:

• Express CobB from multiple species using identical expression systems and purification protocols

• Perform side-by-side biochemical characterization:

  • Enzyme kinetics with standard substrates

  • NAD⁺ dependence and optimal conditions

  • Thermal stability and pH optima

  • c-di-GMP binding affinities using ITC or other methods

• Develop standardized deacetylation assays with identical substrate proteins or peptides

Cross-Species Complementation:

• Generate cobB knockout strains in multiple bacterial species

• Complement with cobB genes from diverse species under identical promoters

• Assess functional complementation through:

  • Growth phenotypes on acetate media

  • Protein acetylation patterns

  • Metabolic profiles

  • c-di-GMP regulation

Comparative Acetylome Analysis:

• Apply identical acetylome profiling methods to wild-type and ΔcobB strains from different species

• Compare:

  • Total number of acetylated proteins and sites

  • Specific proteins affected by CobB deletion

  • Acetylation site motifs and contexts

  • Functional categories of CobB-regulated proteins

Regulatory Network Mapping:

• Characterize c-di-GMP/CobB interactions across species:

  • Is c-di-GMP binding conserved?

  • Are equivalent residues to R8, R17, E21 important?

  • Does the negative feedback loop with DgcZ exist?

• Compare expression regulation of cobB across species:

  • Promoter architecture

  • Transcriptional regulators

  • Growth phase-dependent expression

Case Study: Comparing E. coli and R. baltica CobB

Based on available research, we can outline how this comparative approach might apply to E. coli and R. baltica CobB:

By systematically comparing these and other aspects across species, researchers can gain insights into both the conserved core functions of CobB and the species-specific adaptations that have evolved to meet particular ecological niches.

What control experiments are essential when studying CobB function?

Rigorous control experiments are essential to ensure the validity and reproducibility of findings related to CobB function. The following controls address key aspects of experimental design:

Controls for Genetic Studies:

• Complementation Controls:

  • ΔcobB strain (negative control)

  • ΔcobB::cobB strain (complementation control)

  • ΔcobB with empty vector (vector control)

• Catalytic Mutant Controls:

  • Express catalytically inactive CobB mutants to distinguish between enzyme activity and protein presence

  • These mutants should maintain normal protein folding and interactions

• c-di-GMP Binding Mutant Controls:

  • Express CobB with mutations in c-di-GMP binding residues (R8A, R17A, E21A)

  • These provide controls that maintain deacetylase activity but are insensitive to c-di-GMP regulation

Controls for c-di-GMP Experiments:

• c-di-GMP Level Controls:

  • Wild-type strain (baseline c-di-GMP)

  • dgcZ overexpression strain (elevated c-di-GMP)

  • dgcZ G206A,G207A mutant (expected to have similar c-di-GMP levels as wild type)

• Cyclic Nucleotide Specificity Controls:

  • Include cGMP and c-di-AMP as negative controls in binding assays

  • These controls confirm binding specificity to c-di-GMP

Controls for Biochemical Assays:

• Deacetylation Assay Controls:

  • No-enzyme control (substrate only)

  • No-NAD⁺ control (enzyme + substrate without cofactor)

  • Heat-inactivated enzyme control

  • Known CobB substrate (positive control, e.g., acetylated Acs)

• Binding Assay Controls:

  • No-protein control

  • Denatured protein control

  • Competition assays with unlabeled ligands

  • Binding stoichiometry controls

Controls for Acetylome Studies:

• Sample Preparation Controls:

  • Include deacetylase inhibitors in all samples

  • Process all samples identically and simultaneously

  • Include internal standards for normalization

• Strain Controls:

  • Wild-type strain

  • ΔcobB strain

  • ΔcobB::cobB strain to confirm CobB-dependent effects

• Statistical Controls:

  • Multiple biological replicates (minimum three)

  • Technical replicates to assess method variation

  • Appropriate statistical tests with multiple testing correction

Validation Controls:

• Orthogonal Method Controls:

  • Verify key findings using independent methodologies

  • For example, confirm mass spectrometry results with western blotting

• Site-Specific Mutation Controls:

  • For key substrates, generate K→R (non-acetylatable) and K→Q (acetylation mimic) mutations

  • These distinguish effects of acetylation from other post-translational modifications

An experimental example from the literature demonstrates the implementation of these controls:

When investigating whether c-di-GMP inhibits CobB activity in vivo, researchers used multiple control strains:

• Wild-type strain (baseline c-di-GMP)

• dgcZ overexpression strain (elevated c-di-GMP)

• ΔcobB strain (negative control)

• ΔcobB::cobB strain (complementation control)

• ΔcobB::cobB dgcZ+ strain (to test CobB inhibition by elevated c-di-GMP)

This comprehensive set of controls allowed researchers to establish that:

• CobB was responsible for Acs deacetylation (by comparing WT vs. ΔcobB)

• This function could be restored by complementation (ΔcobB::cobB)

• Elevated c-di-GMP inhibited CobB function (dgcZ+ vs. WT)

• This inhibition was CobB-dependent (by analyzing all strains together)