Recombinant Acinetobacter baumannii Succinyl-CoA ligase [ADP-forming] subunit beta (sucC)

What is the role of Succinyl-CoA ligase in A. baumannii metabolism?

Succinyl-CoA ligase [ADP-forming] subunit beta (sucC) catalyzes a key step in the tricarboxylic acid (TCA) cycle, converting succinyl-CoA to succinate while generating ATP. In A. baumannii, this enzyme is part of the core genome containing many genes important for diverse metabolism and survival in host environments . The enzyme plays a critical role in energy generation and metabolic flexibility, particularly significant given A. baumannii's ability to adapt to various hospital environments and survive under stress conditions. Research suggests that metabolic enzymes like sucC may contribute to the remarkable adaptability that makes A. baumannii a successful pathogen in clinical settings .

How is sucC expression regulated in A. baumannii?

While specific regulation of sucC in A. baumannii hasn't been extensively characterized in the provided search results, research on metabolic adaptation suggests that TCA cycle genes like sucC likely respond to environmental cues including nutrient availability, oxygen levels, and stress conditions. The mechanism may involve transcription factors that sense metabolic states and stress responses. Similar to findings with SUCLA2 (mammalian homolog), sucC likely plays roles in managing redox balance under stress conditions . Researchers investigating sucC regulation should consider designing experiments that examine expression levels under various growth conditions, particularly those mimicking hospital environments where A. baumannii thrives.

What genetic tools are available for studying sucC function in A. baumannii?

Recent developments have significantly expanded the molecular toolkit for A. baumannii genetic manipulation:

• AspFlex Golden Gate Cloning System: A modular cloning system specifically developed for A. baumannii that allows efficient assembly of genetic constructs . This system incorporates replication elements from pWH1266, which is known to replicate in A. baumannii and other Acinetobacter species .

• CRISPR-Cas9 System: Highly efficient genome engineering platform that couples Cas9 nuclease-mediated genome cleavage with recombination systems . Researchers can use inducible promoters (e.g., ATc-inducible) to control dCas9 expression and create various levels of gene repression .

• Gene Replacement Methods: Techniques that enable gene disruption via double crossover recombination using PCR products carrying antibiotic resistance cassettes flanked by homologous regions .

For studying sucC specifically, these tools allow for precise gene knockouts, expression modulation, and complementation studies to assess phenotypic changes.

How can recombinant sucC protein be expressed and purified for biochemical studies?

For recombinant expression of A. baumannii sucC:

• Vector Selection: Based on the AspFlex system described in the literature, level 1 or level 2 plasmids that have been modified to replicate in A. baumannii can be used .

• Expression Conditions:

  • Expression can be achieved in either E. coli (heterologous) or directly in A. baumannii (homologous)

  • For A. baumannii expression, the plasmids should contain appropriate selection markers such as kanamycin resistance or tellurite resistance for multidrug-resistant strains

  • Inducible promoters such as ATc-inducible systems allow controlled expression levels

• Verification Methods:

  • Stability can be verified by sequencing plasmids extracted after multiple days of growth

  • Fluorescent reporter systems can be used to monitor expression efficiency

• Purification Strategy:

  • Affinity tags can be incorporated using the modular cloning system

  • Protein activity should be verified after purification through enzymatic assays

How does genome-wide recombination in A. baumannii affect the genetic diversity of metabolic genes like sucC?

A. baumannii undergoes extensive homologous recombination across its genome, with studies showing that approximately 20% of the genome in clinical isolates has been affected by recombination events . This recombination significantly contributes to strain diversification during epidemic spread. The spatial distribution of SNPs among different A. baumannii strains reveals discrete clusters of high sequence divergence rather than uniform distribution .

For metabolic genes like sucC:

What is the relationship between sucC function and virulence in A. baumannii?

The relationship between metabolism and virulence in A. baumannii is complex. Research has shown that A. baumannii infections are associated with the bacterium's ability to evade rapid clearance by the innate immune system, which enables high bacterial density leading to sepsis . While specific data on sucC's role in virulence isn't directly provided in the search results, several connections can be inferred:

• Metabolic Adaptation During Infection: As a TCA cycle enzyme, sucC likely contributes to A. baumannii's ability to adjust its metabolism in response to the hostile host environment, supporting growth under nutrient limitation.

• Biofilm Formation: A. baumannii strains like ATCC 19606T form robust biofilms on human skin and other surfaces . Metabolic processes supported by sucC may provide energy and precursors needed for biofilm matrix production.

• Experimental Approaches to Study This Relationship:

  • Construction of sucC mutants using CRISPR-Cas9 or gene replacement methods

  • Evaluation of virulence in infection models comparing wild-type and mutant strains

  • Transcriptomic analysis to determine if sucC expression changes during infection

How can CRISPRi be optimized for studying essential metabolic genes like sucC in A. baumannii?

CRISPR interference (CRISPRi) offers significant advantages for studying potentially essential genes like sucC, as it allows tunable repression rather than complete knockout. Based on the research literature, successful CRISPRi application in A. baumannii requires:

• Vector Design: Level 2 plasmids containing:

  • ATc-inducible dCas9 gene

  • Constitutively expressed sgRNAs targeting the gene of interest

  • Appropriate selection markers for A. baumannii

• sgRNA Design Considerations for sucC:

  • Target the non-template strand near the transcription start site

  • Avoid sequences with potential off-target effects

  • Design multiple sgRNAs to target different regions of the gene

• Optimization Parameters:

  • ATc concentration significantly affects repression efficiency, with higher concentrations intensifying the effect

  • Different promoters (constitutive or inducible like pBAD) can be used for dCas9 expression to achieve varying levels of repression

• Validation Approach:

  • Quantitative RT-PCR to measure reduction in sucC mRNA levels

  • Metabolite profiling to assess impacts on TCA cycle function

  • Growth phenotyping under different conditions

Figure 1: CRISPRi Repression Efficiency in A. baumannii

Note: Values are approximate based on similar CRISPRi systems in A. baumannii

How does A. baumannii sucC function compare to homologs in other Acinetobacter species?

Comparative analysis of A. baumannii and other Acinetobacter species reveals important differences that may relate to their varying clinical relevance:

• Genomic Context: The A. baumannii core genome contains many genes important for diverse metabolism and survival in the host, which likely includes optimized versions of TCA cycle enzymes like sucC . When comparing A. baumannii to less clinically successful species like A. calcoaceticus, differences in metabolic capabilities become apparent.

• Metabolic Capabilities: A. baumannii ATCC 19606T utilizes nitrogen sources more effectively and shows greater tolerance to pH, osmotic, and antimicrobial stress compared to A. calcoaceticus . These differences may partially reflect variations in central metabolic enzymes including sucC.

• Research Approaches for Comparative Studies:

  • Heterologous expression of sucC from different Acinetobacter species

  • Enzyme kinetics studies to compare catalytic efficiency

  • Creation of chimeric proteins to identify domains responsible for functional differences

What role might sucC play in antibiotic resistance mechanisms in A. baumannii?

A. baumannii is notorious for its extensive drug resistance, with some strains exhibiting extreme drug resistance (XDR) phenotypes resulting in high mortality rates . While direct evidence linking sucC to antibiotic resistance isn't provided in the search results, several potential connections can be explored:

How can stable isotope labeling be used to track metabolic flux through sucC in A. baumannii?

Metabolic flux analysis using stable isotopes can provide valuable insights into the activity of Succinyl-CoA ligase and its role in A. baumannii metabolism:

• Experimental Design:

  • Culture A. baumannii in media containing 13C-labeled substrates (e.g., [13C]glucose or [13C]acetate)

  • Extract metabolites at various time points during growth

  • Analyze isotope distribution patterns in TCA cycle intermediates

• Analytical Methods:

  • Liquid chromatography-mass spectrometry (LC-MS) to detect labeled metabolites

  • Gas chromatography-mass spectrometry (GC-MS) for volatile TCA cycle intermediates

  • Nuclear magnetic resonance (NMR) for detailed structural information

• Applications in A. baumannii Research:

  • Compare metabolic flux in wild-type versus CRISPRi-repressed sucC strains

  • Examine changes in flux distribution under antibiotic stress

  • Investigate metabolic adaptations during biofilm formation