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BatR: A novel regulator of sub-inhibitory antibiotic tolerance in Pseudomonas aeruginosa biofilms

ABSTRACT: Pseudomonas aeruginosa is a multidrug-resistant opportunistic pathogen, with chronic infections often associated with biofilms that enhance antibiotic resistance. This study investigates the uncharacterized gene PA3049, which is upregulated under biofilm conditions, to determine its role in infection, biofilm formation, and antimicrobial tolerance. o Using bioinformatics, infection models, and molecular microbiology, we determined that PA3049 contributed to biofilm establishment both in vitro and in high-validity infection models. We also identified its role in bacterial survival under sub-inhibitory concentrations of antibiotics and its impact on pyocyanin production. Proteomic analysis revealed that PA3049 upregulates the R2-type pyocin cluster, which drives explosive cell lysis and extracellular DNA (eDNA) release during early stages of P. aeruginosa biofilm development. Additionally, PA3049 interacts with PA0486, an uncharacterized Ser/Thr protein kinase implicated in pyocyanin production and bacterial killing, suggesting a putative in vivo mechanism of action. o Renamed as the Biofilm antibiotic tolerance Regulator (BatR), PA3049 emerges as a key player in P. aeruginosa biofilm maintenance and resistance. These findings provide new insights into bacterial biofilm dynamics and highlight two previously uncharacterized genes with potential implications for combating multidrug-resistant infections

INSTRUMENT(S):

ORGANISM(S): Pseudomonas Aeruginosa Pao1

TISSUE(S): Cell Culture

SUBMITTER: Gerhard Saalbach

LAB HEAD: Jacob Malone

PROVIDER: PXD062857 | Pride | 2026-06-26

REPOSITORIES: Pride

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Project description:. The transition of the opportunistic pathogen Pseudomonas aeruginosa from free-living bacteria into surface-associated biofilm communities represents a viable target for the prevention and treatment of chronic infectious disease. We have established a proteomics platform that identified 2443 and 1142 high-confidence proteins in P. aeruginosa whole cells and outer membrane vesicles (OMVs), respectively, at three time points during biofilm development. Analysis of cellular systems, specifically the phenazine biosynthetic pathway, demonstrates that whole cell protein abundance correlates to end product (i.e., pyocyanin) concentrations in biofilm but not planktonic cultures. Furthermore, increased cellular protein abundance in this pathway results in quantifiable pyocyanin in early biofilm OMVs, and OMVs from both growth modes isolated at later time points. Overall, our data indicate that the OMVs being released from the surface of the biofilm whole cells have unique proteomes in comparison to their planktonic counterparts. The relative abundance of OMV proteins from various subcellular sources showed considerable differences between the two growth modes over time, supporting the existence and preferential activation of multiple OMV biogenesis mechanisms under different conditions. The consistent detection of cytoplasmic proteins in the OMV subproteome suggests that these proteins may contribute a small but functionally relevant component to biofilm OMVs. Direct comparisons of outer membrane protein abundance levels between OMVs and whole cells shows ratios that vary greatly from 1:1, and supports previous studies that advocate specific inclusion, or “packaging”, of proteins into OMVs. The detailed analysis of packaged protein groups indicates biogenesis mechanisms that involve untethered, rather than absent, peptidoglycan-binding proteins. Collectively, individual protein and biological system analyses of biofilm OMVs show that drug-binding cytoplasmic proteins and porins are shuttled from the whole cell into the OMVs, potentially contributing to the antibiotic resistance of P. aeruginosa whole cells within biofilms.

			Action	DRS
	250209_01.msf	Msf
	250209_01.mzML	Mzml
	250209_01.mzid	Mzid
	250209_01.raw	Raw
	250209_02.raw	Raw

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BatR: A novel regulator of sub-inhibitory antibiotic tolerance in Pseudomonas aeruginosa biofilms

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