Acinetobacter baumannii, a Gram-negative opportunistic pathogen, poses a severe public health threat due to its increasing resistance to multiple drugs. Our research focuses on understanding the epigenetic regulation in A. baumannii mediated by the newly identified DNA adenine methyltransferase AamA [1,2] and its interaction with the putative transcriptional regulator NrdR [3].

Small-angle X-ray scattering analyses demonstrated the propensity of His-tagged NrdR to form extensive macromolecular assemblies exceeding 500 kDa, displaying a filamentous-like structure composed of repeating units. Furthermore, upon the introduction of AamA, NrdR starts to disassemble into smaller, likely polydisperse particles and non-specific aggregates [3].

Here, we successfully purified untagged NrdR through the concentration of semi-pure NrdR fractions using ultrafiltration. This process resulted in the formation of higher order filament structures, leading to subsequent precipitation. Following the removal of the supernatant, pure NrdR was readily solubilized in Tris-HCl buffer, achieving a concentration of 5-7 mg/mL.

Additionally, native gel analyses corroborated the interaction between AamA and NrdR as well as the capability of NrdR to form ordered filaments. Notably, NrdR displayed sequence-independent binding to nucleic acids. NrdR-specific sequence boxes remain to be discovered.

In conclusion, our hypothesis posits a crucial role for AamA in A. baumannii virulence through its involvement in differential methylation, facilitated by the dynamic switching of NrdR between filamentous storage-form and de-polymerized active-form.

[1] Blaschke et al. (2018) Recombinant production of A1S_0222 from A. baumannii ATCC 17978 and confirmation of its DNA-(adenine N6)-methyltransferase activity. Protein Expr Purif 151:78-85

[2] Yang et al. (2023) AamA-mediated epigenetic control of genome-wide gene expression and phenotypic traits in A. baumannii ATCC 17978. Microb Genom 9(8)

[3] Weber et al. (2022) Recombinant AcnB, NrdR and RibD of A. baumannii and their potential interaction with DNA adenine methyltransferase AamA. Protein Expr Purif 199:106134