Recombinant Acinetobacter sp. Triosephosphate isomerase (tpiA)

What is the function of TpiA in Acinetobacter species metabolism?

TpiA (triosephosphate isomerase) in Acinetobacter species catalyzes the reversible conversion between glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP), serving as a critical link between glycolysis/gluconeogenesis and glycerol metabolism/phospholipid synthesis . This enzyme is particularly important in bacteria like Acinetobacter that lack phosphofructokinase and thus cannot transform fructose-6-phosphate into fructose-1,6-biphosphate through the traditional glycolytic pathway . In such organisms, TpiA-mediated conversion between G3P and DHAP becomes essential for maintaining carbon flux between various metabolic pathways.

How essential is the tpiA gene for Acinetobacter growth on different carbon sources?

The tpiA gene is conditionally essential in Acinetobacter, depending on the available carbon source. When glycerol is the sole carbon source, tpiA mutants display severe growth defects or no growth, highlighting the essential nature of this enzyme for glycerol metabolism . When glucose is the sole carbon source, tpiA mutants can grow but at significantly reduced rates compared to wild-type strains. Based on studies in related organisms, the maximum growth rates (MGR) of tpiA mutants in glucose-only media can be as low as 0.03/h compared to 0.29/h for wild-type strains . Interestingly, simultaneous supplementation of both glucose and glycerol can rescue growth defects in tpiA mutants, resulting in similar MGRs between mutant and wild-type strains (approximately 0.33/h) .

What are the structural characteristics of TpiA protein in Acinetobacter?

TpiA from Acinetobacter, like its counterparts in other bacteria, is widely regarded as an example of an optimally evolved enzyme due to its essential metabolic role and structural conservation across species . The protein contains multiple α-helices and loops, with several regions showing unexpected structural permissiveness. Studies examining the insertion of 5-amino acid linkers at various positions in TpiA have revealed that the enzyme can maintain functionality even when insertions occur within highly structured regions, including α-helices . These findings suggest that despite its critical function, TpiA possesses remarkable structural robustness and can tolerate significant perturbations while maintaining enzymatic activity.

What methods are recommended for cloning and expressing recombinant Acinetobacter TpiA?

For cloning and expressing recombinant Acinetobacter TpiA, researchers should consider the following methodological approach:

• Gene isolation: PCR-amplify the tpiA gene using specifically designed primers based on the Acinetobacter species of interest. When designing primers, reference the complete genome sequences available for various Acinetobacter species.

• Expression vector selection: Select an appropriate expression vector with compatible restriction sites and a strong promoter (such as T7).

• Transformation and expression: Transform the recombinant plasmid into an expression host like E. coli BL21(DE3), induce expression with IPTG, and optimize expression conditions including temperature, IPTG concentration, and induction time.

• Protein purification: Purify the recombinant protein using affinity chromatography (if a tag was added) or other appropriate chromatographic methods.

• Activity verification: Validate the enzymatic activity of purified TpiA by measuring the conversion between G3P and DHAP spectrophotometrically.

How can researchers accurately identify and differentiate Acinetobacter species when studying TpiA?

Accurate identification of Acinetobacter species is crucial for TpiA research due to variations between species. The following methodological approaches should be considered:

• rpoB gene sequencing: This provides the most accurate identification method for Acinetobacter species, with high discriminatory power at the species level. The high variability of the rpoB gene among Acinetobacter species makes it ideal for species typing, though designing universal primers can be challenging .

• 16S rRNA gene sequencing: While reliable at the genus level, 16S rRNA sequencing has poor discriminatory ability at the species level for closely related Acinetobacter species. The full-length 16S rRNA gene sequences of A. pittii, A. nosocomialis, A. calcoaceticus, and A. baumannii are nearly identical, making it impossible to distinguish these species using only 16S rRNA gene sequencing .

• Detection of blaOXA-51-like gene: This gene can serve as a genetic marker for A. baumannii identification, as it has been observed to be absent in non-A. baumannii isolates .

• Phenotypic methods: Systems like VITEK 2 and VITEK MS can be used but show unsatisfactory discrimination ability compared to genotyping methods .

How does TpiA contribute to virulence and antibiotic resistance in Acinetobacter species?

TpiA's role in virulence and antibiotic resistance in Acinetobacter species appears to be linked to its central position in bacterial metabolism. Based on studies in the related pathogen Pseudomonas aeruginosa, TpiA influences both virulence and susceptibility to aminoglycoside antibiotics . The deletion of tpiA can simultaneously reduce bacterial cytotoxicity and increase susceptibility to antibiotics such as tobramycin.

The mechanisms underlying these effects involve:

• Metabolic alterations: TpiA deletion affects carbon metabolism, which indirectly impacts virulence factor production.

• Enhanced membrane potential: Mutation of tpiA can enhance the bacterial electron transport chain (ETC) activity and respiratory rate, leading to increased membrane potential .

• Increased antibiotic uptake: The enhanced proton motive force (PMF) generated by increased ETC activity facilitates greater uptake of aminoglycosides like tobramycin, explaining the increased antibiotic susceptibility in tpiA mutants .

• Altered energy metabolism: tpiA mutations can increase levels of ATP and NADH, affecting various cellular processes related to virulence and resistance .

These findings suggest that TpiA could be a potential target for developing novel treatment strategies against Acinetobacter infections, particularly for enhancing the efficacy of existing antibiotics.

What is the impact of different carbon sources on TpiA activity and bacterial metabolism in Acinetobacter?

The impact of carbon sources on TpiA activity in Acinetobacter follows patterns observed in related bacteria. When bacteria are grown in media with different carbon sources, distinct metabolic responses are observed:

• Glucose as sole carbon source: In tpiA mutants, growth is significantly reduced but not completely inhibited, suggesting alternative metabolic pathways can partially compensate for TpiA function. The intracellular concentration of DHAP may be lower in tpiA mutants compared to wild-type strains when grown on glucose .

• Glycerol as sole carbon source: tpiA mutants show severe growth defects or no growth at all after extended incubation, indicating the essential role of TpiA in glycerol metabolism. When switched to glycerol media, tpiA mutants accumulate higher intracellular concentrations of DHAP compared to wild-type strains .

• Combination of glucose and glycerol: Simultaneous supplementation of both carbon sources can rescue growth defects in tpiA mutants, resulting in growth rates comparable to wild-type strains. This suggests metabolic flexibility and the existence of compensatory pathways when multiple carbon sources are available .

This differential response to carbon sources provides valuable insights for designing experimental systems to study TpiA function and for understanding the metabolic adaptability of Acinetobacter species.

What methods are available for creating structural variants of TpiA while maintaining enzymatic activity?

Creating structural variants of TpiA while maintaining enzymatic activity can be achieved through several methodological approaches:

• Linker scanning method: An in vitro 5-amino acid linker scanning method has been successfully employed to introduce insertions at various positions in TpiA. This approach has identified permissive sites within the enzyme that can tolerate insertions without losing functionality .

• Site-directed mutagenesis: This approach allows for precise changes to specific amino acids to study structure-function relationships within the enzyme.

• Domain swapping: Exchanging homologous domains between TpiA enzymes from different bacterial species can yield chimeric proteins with novel properties while maintaining core enzymatic function.

The unexpected discovery that TpiA can tolerate insertions even within highly structured regions (such as α-helices) challenges traditional assumptions about protein engineering. For example, 5-amino acid insertions that add more than one new turn within a pre-existing α-helix have been found not to affect the functionality of the protein in some cases . This structural permissiveness makes TpiA an interesting model for protein engineering studies.

What genome editing techniques are recommended for modifying the tpiA gene in Acinetobacter?

For modifying the tpiA gene in Acinetobacter species, researchers should consider the following genome editing techniques:

• Scar-free genome editing: High-efficiency scar-free genome editing toolkits specifically designed for Acinetobacter species have been developed . These methods allow for precise modifications without leaving behind unwanted sequences that might affect gene expression or protein function.

• CRISPR-Cas9 systems: Adapted CRISPR-Cas9 systems can be used for targeted modification of the tpiA gene, allowing for precise edits including deletions, insertions, or point mutations.

• Homologous recombination-based methods: Traditional approaches using suicide vectors carrying homologous regions flanking the tpiA gene can be employed to introduce specific modifications.

• Allelic exchange systems: Two-step allelic exchange systems using counter-selectable markers can facilitate the introduction of precise modifications to the tpiA gene.

When designing knockout strains, researchers should carefully consider the essential nature of tpiA in certain growth conditions. For instance, attempts to delete tpiA in Acinetobacter may be unsuccessful when using glycerol-containing media but may succeed when using glucose as the sole carbon source .

How does TpiA contribute to Acinetobacter pathogenicity in clinical settings?

TpiA's contribution to Acinetobacter pathogenicity in clinical settings is multifaceted and involves its central role in bacterial metabolism. Acinetobacter, particularly A. baumannii, has emerged as a major cause of hospital-acquired infections worldwide . The pathogenicity of these infections is linked to several factors:

• Metabolic adaptability: TpiA's role in central carbon metabolism enables Acinetobacter to thrive in diverse host environments, contributing to its persistence during infection.

• Impact on virulence mechanisms: Metabolic pathways involving TpiA influence the production of virulence factors, affecting the bacterium's ability to cause serious infections, particularly in critically ill patients.

• Contribution to antibiotic resistance: The metabolic state of Acinetobacter, regulated in part by TpiA, affects susceptibility to antibiotics, particularly aminoglycosides. This is significant given that A. baumannii is noted for its ability to develop resistance to multiple antibiotics .

• Influence on bacterial survival: TpiA's role in maintaining metabolic homeostasis contributes to Acinetobacter's ability to survive for long periods on hospital surfaces and equipment, facilitating outbreaks of healthcare-associated infections .

The crude ICU mortality rate associated with Acinetobacter infections is approximately 40%, and these infections are consistently associated with prolonged hospital stays . Understanding TpiA's role in pathogenicity could provide new avenues for developing targeted therapeutic approaches.