Recombinant Laribacter hongkongensis Acetyl-coenzyme A synthetase (acsA), partial

What is Laribacter hongkongensis and why is it significant for research?

Laribacter hongkongensis is a Gram-negative bacillus of the Neisseriaceae family discovered relatively recently and linked to freshwater fish-borne gastroenteritis and traveler's diarrhea. The complete genome sequence of L. hongkongensis HLHK9 consists of a 3,169-kb chromosome with G+C content of 62.35% . The bacterium is particularly interesting due to its ability to adapt to diverse habitats including human intestines, freshwater fish intestines, and freshwater environments, demonstrating remarkable ecological versatility . Seasonal variation in its recovery has been observed, with higher isolation rates in spring and summer than in fall and winter, suggesting temperature-dependent growth characteristics .

What metabolic pathways in L. hongkongensis may involve Acetyl-coenzyme A synthetase?

Genome analysis reveals that L. hongkongensis can utilize amino acids and fatty acids as carbon sources . The acetyl-coenzyme A synthetase enzyme catalyzes the formation of acetyl-CoA, a critical metabolic intermediate involved in numerous biochemical pathways. In L. hongkongensis, this enzyme likely plays an important role in central carbon metabolism, particularly in acetate utilization and fatty acid synthesis pathways. Similar to other bacterial systems, it may be critical for adaptation to different nutritional environments encountered during host infection versus environmental survival.

What expression systems are optimal for producing recombinant L. hongkongensis proteins?

Based on available data for other recombinant L. hongkongensis proteins, mammalian cell expression systems have been successfully employed . When designing expression strategies for recombinant L. hongkongensis acsA, researchers should consider the G+C content (62.35%) of the organism , which may necessitate codon optimization for heterologous expression. Expression in E. coli systems has been documented for other L. hongkongensis proteins, such as β-lactamase, where proteins have been successfully cloned and expressed using vectors like pBK-CMV and pACYC184 .

What purification approaches yield highest purity for L. hongkongensis recombinant proteins?

For recombinant L. hongkongensis proteins, affinity chromatography utilizing appropriate fusion tags represents the standard initial purification step. Product specifications for other L. hongkongensis recombinant proteins indicate purities of >85% as assessed by SDS-PAGE . The tag type is typically determined during the manufacturing process and should be selected based on the specific physicochemical properties of acsA. Subsequent purification steps might include ion exchange chromatography and size exclusion chromatography to achieve higher purity levels for enzymatic and structural studies.

What are the recommended storage conditions for recombinant L. hongkongensis proteins?

Optimal storage conditions for recombinant L. hongkongensis proteins depend on their formulation. Liquid preparations generally have a shelf life of 6 months at -20°C/-80°C, while lyophilized forms can be stored for approximately 12 months at the same temperatures . The recommended protocol involves reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL with addition of 5-50% glycerol (final concentration) for long-term storage. Repeated freeze-thaw cycles should be avoided, and working aliquots can be maintained at 4°C for up to one week .

How should the enzymatic activity of recombinant L. hongkongensis acsA be measured?

The enzymatic activity of recombinant L. hongkongensis acsA should be assessed using established acetyl-coenzyme A synthetase assays that measure one of the following:

• Formation of acetyl-CoA using spectrophotometric methods

• ATP consumption through coupled enzyme assays

• Release of pyrophosphate as a reaction product

Given that L. hongkongensis has adapted to different temperature environments, activity measurements should be conducted at both 20°C (environmental temperature) and 37°C (human body temperature) to evaluate potential temperature-dependent activity profiles, similar to what has been observed with other L. hongkongensis enzymes like N-acetyl-L-glutamate kinase (NAGK) .

What controls are essential when characterizing recombinant L. hongkongensis acsA?

When characterizing recombinant L. hongkongensis acsA, include these essential controls:

• Non-enzyme controls to establish baseline non-enzymatic reactions

• Heat-inactivated enzyme controls to confirm activity is enzyme-dependent

• Substrate specificity controls testing various potential acyl substrates

• Temperature controls at both 20°C and 37°C, reflecting the organism's natural environments

• pH range controls to determine optimal conditions, especially given L. hongkongensis's adaptations to different pH environments

• Known acetyl-coenzyme A synthetase inhibitors as negative controls

These controls will ensure reliable and reproducible enzymatic characterization data.

Does L. hongkongensis acsA exhibit temperature-dependent expression or activity?

While specific data on acsA temperature dependence is not available in the search results, L. hongkongensis demonstrates clear temperature-dependent adaptation mechanisms. The organism contains two homologous copies of argB encoding isoenzymes of N-acetyl-L-glutamate kinase (NAGK) with differential expression at 20°C and 37°C . NAGK-20 showed higher expression at 20°C (freshwater temperature), while NAGK-37 exhibited higher expression at 37°C (human body temperature). Additionally, NAGK-20 demonstrated lower optimal temperature for enzymatic activities . Based on this precedent, researchers should investigate whether acsA demonstrates similar temperature-dependent expression patterns or if multiple isoforms exist with differential temperature optima.

What methodological approaches can detect temperature-dependent regulation of acsA?

To investigate potential temperature-dependent regulation of acsA, researchers should employ:

• Quantitative RT-PCR to measure acsA transcript levels at 20°C versus 37°C

• Western blotting to quantify protein expression at different temperatures

• Enzyme activity assays across a temperature range (15-40°C) to establish temperature optima

• Thermal shift assays to determine protein stability at different temperatures

• Comparative transcriptomics to identify temperature-responsive regulatory elements controlling acsA expression

• Protein crystallography at different temperatures to detect conformational changes

Similar approaches have successfully characterized temperature-dependent properties of other L. hongkongensis enzymes .

How might acsA contribute to L. hongkongensis pathogenesis?

Acetyl-coenzyme A synthetase potentially contributes to L. hongkongensis pathogenesis through multiple mechanisms:

• Supporting metabolic adaptation during transition from environmental (20°C) to host (37°C) conditions

• Facilitating utilization of alternative carbon sources during intestinal colonization

• Contributing to fatty acid metabolism needed for membrane remodeling under host conditions

• Participating in acetylation-based modification of host or bacterial proteins involved in virulence

• Providing metabolic flexibility when navigating different nutrient environments in the host

Investigating these potential roles requires gene knockout studies, virulence models, and metabolomic analyses to determine whether acsA is upregulated during infection and essential for pathogenesis.

Could acsA serve as a target for antimicrobial development against L. hongkongensis?

The potential of acsA as an antimicrobial target depends on:

• Essentiality: Whether acsA is required for L. hongkongensis survival or virulence

• Conservation: How similar the enzyme is to human homologs (to avoid off-target effects)

• Druggability: Whether the enzyme contains pockets suitable for small molecule binding

L. hongkongensis demonstrates resistance to multiple antibiotics, including tetracycline and β-lactams , making novel targets valuable. Research on other bacterial acetyl-coenzyme A synthetases has identified inhibitors that could serve as starting points for L. hongkongensis-specific development. Structure-based drug design would require solving the crystal structure of L. hongkongensis acsA.

What cloning strategies are most effective for L. hongkongensis genes?

Effective cloning strategies for L. hongkongensis genes should account for the organism's high G+C content (62.35%) . Previous successful approaches include:

• Using restriction fragment cloning into vectors like pBK-CMV, as demonstrated for ampC and ampR genes

• PCR amplification with primers designed to incorporate appropriate restriction sites

• Subcloning into lower-copy-number vectors (like pACYC184) for regulated expression

• Considering codon optimization when expressing in heterologous hosts

For investigating regulatory elements, include adequate upstream sequences as demonstrated in studies of the ampC-ampR regulatory system in L. hongkongensis .

What approaches can identify potential regulatory mechanisms controlling acsA expression?

To identify regulatory mechanisms controlling acsA expression, researchers should employ:

• Promoter mapping through 5' RACE or primer extension

• Reporter gene fusions to identify regulatory regions

• DNA-protein interaction studies (EMSA, ChIP) to identify transcription factors

• Transcriptomic analysis under various conditions (temperature, pH, carbon source availability)

• Comparison with known regulatory mechanisms of other metabolic genes in L. hongkongensis

Previous work on L. hongkongensis has identified regulatory systems like the ampC-ampR system with intercistronic Lys-R motifs typical of inducible regulatory systems , providing templates for investigating acsA regulation.

How does L. hongkongensis acsA compare to homologs in other bacterial species?

While specific comparative data for L. hongkongensis acsA is not available in the search results, patterns observed with other L. hongkongensis enzymes suggest potential distinctive features. For example, the AmpC β-lactamase of L. hongkongensis showed identities no greater than 46% to known class C β-lactamases, despite containing consensus motifs characteristic of this enzyme class . Similarly, researchers should investigate whether acsA contains conserved catalytic residues typical of bacterial acetyl-coenzyme A synthetases while potentially exhibiting unique sequence or structural features that reflect adaptation to L. hongkongensis's ecological niches.

What insights might structural studies of L. hongkongensis acsA provide?

Structural studies of L. hongkongensis acsA could reveal:

• Substrate binding pocket architecture influencing acyl substrate specificity

• Potential temperature-dependent conformational changes, as suggested by differential temperature optima of other L. hongkongensis enzymes

• Unique structural features compared to homologs from other bacteria

• Regulatory domains or interfaces for protein-protein interactions

• Potential sites for post-translational modifications affecting enzyme activity

These structural insights would complement functional studies and potentially identify unique features related to L. hongkongensis's adaptation to different environmental niches.

What are the optimal conditions for reconstituting lyophilized recombinant L. hongkongensis proteins?

Based on available data for other recombinant L. hongkongensis proteins, the recommended reconstitution protocol includes:

• Brief centrifugation of the vial prior to opening

• Reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Addition of glycerol to a final concentration of 5-50% (with 50% being the default)

• Preparation of small working aliquots to avoid repeated freeze-thaw cycles

• Storage of working aliquots at 4°C for up to one week

• Long-term storage at -20°C/-80°C

These conditions have been established for other L. hongkongensis recombinant proteins and likely apply to acsA as well.

What analytical methods are most appropriate for assessing purity and integrity of recombinant L. hongkongensis acsA?

Appropriate analytical methods include:

• SDS-PAGE with Coomassie or silver staining (standard for purity assessment)

• Western blotting using antibodies against acsA or tag epitopes

• Mass spectrometry for accurate molecular weight determination and sequence verification

• Size exclusion chromatography to assess aggregation state

• Dynamic light scattering to evaluate homogeneity

• Circular dichroism to confirm proper secondary structure

• Thermal shift assays to assess protein stability

These complementary techniques provide comprehensive characterization of the recombinant protein's purity, identity, and structural integrity.