Recombinant Acinetobacter sp. 50S ribosomal protein L1 (rplA)

Basic Research Questions

• What is the structural and functional role of 50S ribosomal protein L1 (rplA) in Acinetobacter species?

  The 50S ribosomal protein L1 (rplA) is a critical component of the large ribosomal subunit in Acinetobacter species. It is located at the L1 stalk of the 50S subunit, which is functionally important for ribosome dynamics during translation. In Acinetobacter baumannii, high-resolution cryo-EM studies have revealed that the ribosome, including its L1 protein, exhibits several unique structural features compared to other bacterial ribosomes, particularly at functionally important sites including the periphery of the subunit interface . These structural differences may contribute to species-specific translation mechanisms that could be relevant for bacterial survival and antibiotic resistance strategies.

• How conserved is rplA across different Acinetobacter species and other bacteria?

  The rplA gene is highly conserved across Acinetobacter species and other bacteria, reflecting its essential role in ribosome function. While the core structure and function remain preserved, species-specific variations in rplA may contribute to differences in ribosome dynamics and antibiotic sensitivity. Comparative studies show that ribosomes from Acinetobacter baumannii have unique structural features at functionally important sites compared to other bacterial species . These differences may impact translation efficiency under stress conditions and could potentially affect antibiotic susceptibility profiles.

• Are ribosomal proteins like rplA essential genes in Acinetobacter species?

  Yes, many ribosomal proteins, including those in the 50S subunit, are essential for bacterial growth and survival. In genome-scale studies of Acinetobacter, several ribosomal proteins have been identified as essential genes. For example, in periplasmic proteomic studies of A. baumannii, three 50S ribosomal proteins were identified, and two of them were shown to be essential for the growth of strain AB5075 in nutrient-rich medium . This essential nature makes ribosomal proteins potential targets for antimicrobial development.

Advanced Research Questions

• How does expression of rplA change under antibiotic stress in Acinetobacter species?

  RNA sequencing and proteomics studies have shown that ribosomal proteins, including components of the 50S subunit, undergo significant expression changes in response to antibiotic stress in Acinetobacter species. For instance, when A. baumannii strains were exposed to eravacycline (a synthetic fluorocycline antibiotic), ribosomal proteins were upregulated in both reference strain ATCC 19606 and clinical isolates . This upregulation occurred alongside increased expression of drug efflux and membrane transport genes, suggesting a coordinated bacterial response to antibiotic pressure that might involve rplA.

  The table below summarizes key findings on ribosomal protein expression changes under antibiotic stress:

  Antibiotic	Organism	Observed Changes in Ribosomal Proteins	Reference
  Eravacycline	A. baumannii ATCC 19606	Upregulation of ribosomal proteins in transcriptome and OMV proteome
  Eravacycline	A. baumannii clinical strain JU0126	Upregulation of ribosomal proteins alongside resistance pumps
  Amikacin	A. baumannii	Structural changes in ribosome-antibiotic interactions
  Tigecycline	A. baumannii	Altered binding to ribosomal components

• What is the relationship between rplA and antibiotic resistance mechanisms in Acinetobacter?

  Research suggests that ribosomal proteins, including rplA, may contribute to antibiotic resistance in Acinetobacter species through multiple mechanisms:

  • Structural studies of the A. baumannii ribosome in complex with antibiotics like amikacin and tigecycline reveal specific modes of drug interaction with ribosomal components, which could involve the 50S subunit where L1 resides .

  • Proteomic analyses of multidrug-resistant (MDR) versus drug-susceptible A. baumannii isolates have identified differential expression of proteins related to antibiotic resistance, including ribosomal components .

  • In genetic landscape analyses of antibiotic sensitivity, ribosomal proteins (including rplA) have been identified as potentially contributing to resistance mechanisms .

  • The primary driver of clinical outcomes in Acinetobacter infections is often antibiotic resistance, with ribosomal alterations being one potential mechanism .

• How can rplA be used in structural studies to understand ribosome-antibiotic interactions?

  Recombinant rplA can be valuable for structural studies of ribosome-antibiotic interactions in Acinetobacter species. High-resolution cryo-EM structures of the A. baumannii 70S ribosome in complex with antibiotics have revealed species-specific features and modes of drug interactions . These structures provide insights into how antibiotics like amikacin and tigecycline interact with the ribosome, potentially including contacts with or conformational effects on the L1 protein.

  Methodological approaches for such studies include:

  1. Reconstitution experiments with purified recombinant rplA to study its specific interactions with antibiotics

  2. Cryo-EM studies of ribosomes with and without bound antibiotics to observe structural changes

  3. Comparative structural analyses across bacterial species to identify unique features that might be exploited for drug development

  4. Site-directed mutagenesis of rplA to evaluate the impact of specific residues on antibiotic binding

  These approaches can help identify unique structural features that could be targeted to develop new inhibitors against multidrug-resistant A. baumannii infections .

• What is the potential role of rplA in bacterial stress response beyond antibiotic resistance?

  Beyond antibiotic resistance, rplA and other ribosomal proteins may play significant roles in bacterial stress response mechanisms:

  • In Acinetobacter sp. SA01, OmpA expression has been linked to oxidative stress response , and ribosomal proteins like rplA might have similar stress-responsive roles.

  • Proteomic studies of A. baylyi ADP1 revealed that DNA damage response at the translational level is significantly altered by carbon source , suggesting that ribosomal proteins may contribute to adaptation to environmental stresses.

  • The presence of ribosomal proteins in outer membrane vesicles (OMVs) of A. baumannii suggests they may have functions beyond their classical role in translation , potentially including stress signaling or immune modulation.