ACKA E.Coli

What is the ackA gene and what does it encode in E. coli?

The ackA gene in Escherichia coli encodes the enzyme acetate kinase (AckA), which plays a critical role in the bacterial acetate metabolism. This enzyme works in conjunction with phosphotransacetylase (encoded by the pta gene) to form the AckA-Pta pathway, which is responsible for acetate production and consumption during bacterial growth . Acetate kinase catalyzes the reversible transfer of a phosphate group from ATP to acetate, forming acetyl phosphate (AcP), which serves as a high-energy intermediate with a higher standard free energy of hydrolysis (-43.3 kJ/mol) than ATP (-30.5 kJ/mol in complex with Mg2+) .

What is the primary function of the AckA-Pta pathway in E. coli metabolism?

The AckA-Pta pathway serves dual critical functions in E. coli metabolism:

• Acetogenesis: During overflow metabolism, Pta synthesizes acetyl phosphate (acetyl-P) and coenzyme A (HS-CoA) from acetyl-CoA and inorganic phosphate (Pi). AckA then generates ATP from acetyl-P and ADP while producing acetate, which is excreted into the environment .

• Acetate activation: In the reverse direction, the pathway can activate acetate by converting it back to acetyl-CoA, allowing E. coli to utilize acetate as a carbon source when preferred carbon sources are depleted .

The reversibility of this pathway permits both acetyl-CoA synthesis (acetate activation) and acetate evolution (acetogenesis), with the steady-state concentration of acetyl-P depending on the rate of its formation by Pta and degradation by AckA .

How does the ackA gene influence bacterial antibiotic susceptibility?

The ackA gene significantly impacts bacterial antibiotic susceptibility, particularly to fosfomycin. Research has demonstrated that inactivation of ackA (and pta) genes reduces the expression of GlpT, one of the primary transporters for fosfomycin uptake in E. coli . This decreased expression leads to reduced fosfomycin uptake capacity, rendering the bacteria less sensitive to this antibiotic.

The mechanism involves a cascade effect: mutations in ackA and pta cause a decrease in the expression of fis (a nucleoid-associated protein), which normally enhances glpT expression . With reduced Fis levels, glpT expression declines, limiting fosfomycin transport into bacterial cells. This finding has significant clinical implications as fosfomycin is increasingly important for treating infections caused by multidrug-resistant bacteria, including quinolone-resistant and extended-spectrum β-lactamase (ESBL)-producing strains .

What is the relationship between ackA and acetyl phosphate signaling in E. coli?

Acetyl phosphate (AcP), the intermediate of the AckA-Pta pathway, functions as a global signaling molecule in E. coli, affecting numerous cellular processes . The intracellular concentration of AcP in wild-type cells reaches at least 3 mM, a level sufficient to activate two-component response regulators through direct phosphoryl transfer .

AcP particularly influences gene expression through two-component signal transduction systems. For example, research shows that AcP can regulate flagella biogenesis and capsule biosynthesis via the two-component response regulator RcsB . Since RcsB is known to regulate approximately 5% of the E. coli genome, acetyl phosphate must be considered a global signal with widespread effects on bacterial physiology .

The ackA/pta double deletion mutant exhibits altered expression of various genes, including increased transcription of PhoP-regulated genes, demonstrating AcP's significant role in bacterial gene regulation .

How do mutations in ackA affect E. coli growth and metabolism?

Mutations in the ackA gene have several documented effects on E. coli growth and metabolism:

These effects highlight the complex role of ackA in bacterial physiology beyond its direct enzymatic function.

What experimental approaches can be used to manipulate ackA expression in E. coli?

Several experimental approaches have been successfully employed to manipulate ackA expression in E. coli:

• Gene knockout: Complete deletion of ackA creates mutant strains that lack acetate kinase activity. ackA/pta double deletion mutants are commonly used as negative controls for acetyl phosphate production .

• Antisense RNA technology: This approach involves creating recombinant plasmids harboring antisense sequences targeting ackA (and often pta) mRNA. When expressed, these antisense RNAs hybridize with the target mRNAs, preventing their translation . For example:

  • Antisense cassettes can be constructed and cloned into expression vectors under inducible promoters (like T7)

  • Upon induction (e.g., with IPTG), the antisense RNA is produced, downregulating ackA expression

  • This technique achieved 9-14% of normal ackA mRNA levels and approximately 17% of normal ACK enzymatic activity

• Metabolic manipulation: AcP levels (the product of the AckA-Pta pathway) can be altered by supplementing growth media with glucose or acetate. In wild-type S. typhimurium, AcP concentrations in cells cultured with glucose or acetate addition were 2.10 and 1.66 times higher, respectively, than levels in cells cultured in regular LB medium .

How does acetyl phosphate accumulation affect global gene regulation in E. coli?

Acetyl phosphate (AcP) accumulation has profound effects on global gene regulation in E. coli through its interaction with two-component signaling systems. The following regulatory effects have been observed:

• Transcriptional regulation of PhoP-dependent genes: Research demonstrates that AcP levels directly correlate with the transcriptional activity of PhoP-regulated genes. When AcP levels increase (after glucose or acetate supplementation), there is reduced transcription of phoP and PhoP-regulated genes . Conversely, in ackA/pta double deletion mutants (where AcP production is defective), transcriptional levels of phoP and other PhoP-regulated genes increase significantly compared to wild-type strains .

• Protein expression levels: Consistent with transcriptional changes, protein levels of PhoP in the ackA/pta double deletion mutant were 2.61 times higher than in the wild-type strain .

• Lysine acetylation: AcP can non-enzymatically acetylate proteins at specific lysine residues, affecting their function. For example, K102 acetylation levels were higher in cells cultured with glucose or acetate supplements, correlating with accumulated AcP levels .

• Broad genomic impacts: Through its regulation of RcsB (a response regulator), acetyl phosphate influences approximately 5% of the E. coli genome, affecting processes such as flagella biogenesis and capsule biosynthesis .

These findings highlight acetyl phosphate's role as a global signal molecule that links bacterial metabolism to gene expression through multiple regulatory mechanisms.

What are the mechanisms of fosfomycin resistance associated with ackA mutations?

The mechanisms of fosfomycin resistance associated with ackA mutations involve a regulatory cascade affecting transporter expression:

• Decreased GlpT transporter expression: Inactivation of ackA and pta genes reduces the expression of GlpT, one of the primary transporters responsible for fosfomycin uptake in E. coli .

• Regulatory pathway impact: The mechanism follows this sequence:

  • ackA and pta mutations cause decreased expression of fis, a nucleoid-associated protein

  • Fis normally enhances glpT expression

  • Reduced Fis levels lead to decreased glpT expression

  • Lower GlpT transporter levels result in reduced fosfomycin uptake capacity

  • Consequently, bacteria become less sensitive to fosfomycin

• Clinical relevance: This mechanism is conserved in multidrug-resistant E. coli isolated from patients with pyelonephritis and enterohemorrhagic E. coli. Deletion of ackA and pta from these clinical strains resulted in decreased susceptibility to fosfomycin .

This represents a novel genetic mechanism leading to fosfomycin resistance, which is particularly concerning as fosfomycin is increasingly important for treating infections caused by drug-resistant bacteria, including quinolone-resistant and ESBL-producing strains .

How can the ackA-pta pathway be targeted to enhance antibiotic efficacy?

Based on current research, several strategies could be employed to target the ackA-pta pathway to enhance antibiotic efficacy, particularly for fosfomycin:

• Upregulation of fosfomycin transporters: Since ackA mutations lead to decreased expression of GlpT (fosfomycin transporter), compounds that increase GlpT expression could potentially restore fosfomycin sensitivity in resistant strains .

• Fis expression enhancement: Because the pathway involves decreased Fis (a nucleoid-associated protein) leading to reduced GlpT expression, strategies to increase Fis levels could indirectly enhance fosfomycin uptake .

• Acetyl phosphate modulation: Manipulating acetyl phosphate levels, potentially through carbon source supplementation (glucose or acetate), might influence bacterial susceptibility to antibiotics by affecting global gene regulation .

• Combination therapy approaches: Understanding the relationship between ackA function and antibiotic resistance provides opportunities for developing combination therapies that target both the primary antibiotic mechanism and resistance pathways.

These approaches would require thorough validation through experimental studies before clinical implementation, but they represent promising avenues for enhancing antibiotic efficacy based on our understanding of the ackA-pta pathway's role in bacterial physiology.

What techniques can be used to measure intracellular acetyl phosphate concentration?

Measuring intracellular acetyl phosphate (AcP) concentration requires specialized techniques due to its unstable nature and rapid turnover. The following methods have been successfully employed:

• Two-dimensional thin-layer chromatography (2D-TLC): This technique has been used to measure the relative concentrations of acetyl phosphate, acetyl coenzyme A, ATP, and GTP throughout the bacterial growth curve . The procedure involves:

  • Labeling cells with radioactive phosphate (32Pi)

  • Extraction of cellular metabolites

  • Separation using 2D-TLC

  • Quantification of labeled compounds

• Enzymatic assays: These methods use specific enzymes that react with acetyl phosphate, coupled with spectrophotometric detection of reaction products.

• Mass spectrometry-based approaches: Modern metabolomic techniques using liquid chromatography coupled with mass spectrometry (LC-MS) can detect and quantify acetyl phosphate in cellular extracts.

• Modified MOPS medium for phosphate labeling: To facilitate uptake of radioactive phosphate for labeling studies, researchers have used a modified MOPS medium with reduced K2HPO4 concentration (0.2 mM final concentration) supplemented with appropriate carbon sources .

For accurate measurements, careful sample preparation is critical due to the high-energy nature of the phosphoanhydride bond in acetyl phosphate.

How can researchers create and verify ackA knockout or mutant strains?

Creating and verifying ackA knockout or mutant strains involves several established molecular biology techniques:

• Creation of ackA knockout strains:

  • Lambda Red recombination system: This method allows for precise deletion of the ackA gene by replacing it with an antibiotic resistance marker

  • P1 phage transduction: Transferring ackA mutations from donor strains to recipient strains

  • CRISPR-Cas9 system: For precise genome editing without leaving marker genes

• Verification of ackA knockout:

  • PCR verification: Using primers flanking the deleted region to confirm successful deletion

  • Sequencing: To verify the exact genetic modifications

  • RT-PCR/qPCR: To confirm absence of ackA mRNA expression

• Functional verification:

  • Enzymatic assays: Measuring acetate kinase activity to confirm functional loss (studies showed approximately 17% residual ACK activity in antisense-regulated strains)

  • Metabolite analysis: Measuring acetate production/consumption and acetyl phosphate levels

  • Growth characteristics: Comparison with wild-type strains (ackA mutants typically show altered growth patterns)

Antisense RNA approaches, as an alternative to complete knockout, require verification of downregulation efficiency:

• Measuring mRNA levels of target genes (9-14% for ackA)

• Measuring protein levels by Western blotting

• Assessing enzymatic activity reduction

What methodologies are effective for studying the impact of ackA on bacterial antibiotic susceptibility?

Several methodologies are effective for studying the impact of ackA on bacterial antibiotic susceptibility:

• Minimum Inhibitory Concentration (MIC) determination:

  • Broth microdilution method

  • Agar dilution method

  • E-test

  • Comparing MIC values between wild-type and ackA mutant strains for various antibiotics, particularly fosfomycin

• Fosfomycin uptake assays:

  • Using radiolabeled fosfomycin to quantify uptake in wild-type versus ackA mutant strains

  • Measuring intracellular accumulation of the antibiotic over time

• Gene expression analysis:

  • RT-qPCR to measure expression levels of transporters (e.g., glpT) and regulatory genes (e.g., fis)

  • RNA-seq for global transcriptomic analysis

  • Western blotting to quantify transporter protein levels

• Genetic complementation studies:

  • Reintroducing functional ackA gene into knockout strains to confirm phenotype reversal

  • Testing complementation with related pathway components

• Time-kill assays:

  • Determining the bactericidal activity of fosfomycin against wild-type and ackA mutant strains over time

• Fitness and competition assays:

  • Mixed culture experiments to assess relative fitness of wild-type versus ackA mutant strains in the presence of antibiotics

  • Evaluating potential survival advantages of ackA mutations under antibiotic pressure

• Clinical isolate analysis:

  • Screening clinical isolates for ackA and pta mutations and correlating with antibiotic susceptibility profiles

  • Creating deletions in multidrug-resistant clinical isolates to assess impacts on fosfomycin susceptibility

These methodologies provide comprehensive insights into how ackA affects antibiotic susceptibility through various mechanisms, particularly those involving transporter expression and metabolic regulation.

What are the implications of ackA research for developing new antibiotic strategies?

Research on the ackA gene and its associated pathways presents several promising implications for developing novel antibiotic strategies:

• Overcoming fosfomycin resistance: Understanding that inactivation of ackA reduces GlpT expression and consequently fosfomycin susceptibility opens new avenues for combination therapies . Potential approaches include:

  • Developing adjuvants that maintain GlpT expression even when ackA is mutated

  • Creating fosfomycin derivatives with alternative uptake mechanisms

  • Designing drugs that target both the ackA-pta pathway and bacterial cell wall synthesis

• Targeting metabolic vulnerabilities: The dual role of the AckA-Pta pathway in both acetate production and consumption suggests that manipulating this pathway could create metabolic imbalances lethal to bacteria . This could involve:

  • Developing inhibitors that target specific steps in the pathway

  • Creating conditions that force reliance on this pathway, then disrupting it

• Reducing acetyl phosphate-mediated gene regulation: Since acetyl phosphate acts as a global signal affecting approximately 5% of the E. coli genome, interfering with this signaling could disrupt various essential bacterial processes . Strategies might include:

  • Compounds that mimic acetyl phosphate but block downstream signaling

  • Agents that prevent acetyl phosphate interaction with two-component response regulators

• Biofilm prevention: Research on the role of ackA in biofilm formation could lead to anti-biofilm strategies that enhance antibiotic penetration and efficacy against persistent infections.

These approaches represent promising directions for antibiotic development based on our understanding of ackA's multifaceted roles in bacterial physiology and antibiotic resistance.

How does ackA function differ across pathogenic versus non-pathogenic E. coli strains?

The function of ackA across pathogenic versus non-pathogenic E. coli strains shows both conservation and divergence:

• Conservation of basic function: The ackA and pta genes are conserved in both laboratory strains and clinical isolates, including multidrug-resistant E. coli from patients with pyelonephritis and enterohemorrhagic E. coli . This suggests the fundamental metabolic role of the AckA-Pta pathway is maintained across E. coli strains.

• Impact on virulence: Differences may exist in how ackA function affects virulence factors specific to pathogenic strains. The global regulatory role of acetyl phosphate likely influences virulence gene expression differently in pathogenic strains compared to laboratory strains.

• Antibiotic susceptibility effects: Research has demonstrated that deletion of ackA and pta from pathogenic strains, including multidrug-resistant clinical isolates, resulted in decreased susceptibility to fosfomycin, similar to the effect observed in laboratory strains . This suggests that the mechanism linking ackA function to antibiotic susceptibility is conserved across strain types.

• Metabolic adaptations: Pathogenic strains may have evolved different regulatory controls over the AckA-Pta pathway to better adapt to host environments, potentially affecting their growth characteristics, stress responses, and metabolic flexibility compared to non-pathogenic strains.

Further comparative studies are needed to fully elucidate strain-specific differences in ackA function and regulation, which could reveal important insights for targeted antimicrobial development.

What emerging technologies are advancing our understanding of ackA-mediated cellular processes?

Several emerging technologies are significantly advancing our understanding of ackA-mediated cellular processes:

• CRISPR-Cas9 genome editing: Enabling precise manipulation of ackA and related genes without antibiotic markers, allowing clean genetic modifications for functional studies.

• Single-cell analyses: Technologies such as single-cell RNA-seq and time-lapse microscopy with fluorescent reporters are revealing cell-to-cell variability in ackA expression and function within bacterial populations.

• Metabolic flux analysis: Advanced fluxomics methods using stable isotope labeling and mass spectrometry enable tracking of carbon flow through the AckA-Pta pathway in different conditions.

• Protein-protein interaction mapping: Techniques such as bacterial two-hybrid systems, proximity labeling, and co-immunoprecipitation coupled with mass spectrometry are identifying novel interaction partners of AckA.

• Structural biology advances: Cryo-electron microscopy and X-ray crystallography are providing detailed insights into the structure of AckA and how modifications affect its function.

• Multi-omics integration: The integration of transcriptomics, proteomics, and metabolomics data is generating comprehensive views of how ackA influences global cellular processes.

• Biosensors for acetyl phosphate: Development of genetically encoded biosensors allows real-time monitoring of acetyl phosphate levels in living cells.

• Microfluidics and real-time monitoring: These technologies enable precise control of environmental conditions while monitoring bacterial responses, particularly useful for studying how ackA function changes during environmental transitions.

These technological advances are collectively enhancing our understanding of the complex roles of ackA in bacterial physiology, antibiotic resistance, and global cellular regulation.