An integrated model system to gain mechanistic insights into biofilm-associated antimicrobial resistance in Pseudomonas aeruginosa MPAO1

Abstract

Pseudomonas aeruginosa MPAO1 is the parental strain of the widely utilized transposon mutant collection for this important clinical pathogen. Here, we validate a model system to identify genes involved in biofilm growth and biofilm-associated antibiotic resistance. Our model employs a genomics-driven workflow to assemble the complete MPAO1 genome, identify unique and conserved genes by comparative genomics with the PAO1 reference strain and genes missed within existing assemblies by proteogenomics. Among over 200 unique MPAO1 genes, we identified six general essential genes that were overlooked when mapping public Tn-seq data sets against PAO1, including an antitoxin. Genomic data were integrated with phenotypic data from an experimental workflow using a user-friendly, soft lithography-based microfluidic flow chamber for biofilm growth and a screen with the Tn-mutant library in microtiter plates. The screen identified hitherto unknown genes involved in biofilm growth and antibiotic resistance. Experiments conducted with the flow chamber across three laboratories delivered reproducible data on P. aeruginosa biofilms and validated the function of both known genes and genes identified in the Tn-mutant screens. Differential protein abundance data from planktonic cells versus biofilm confirmed the upregulation of candidates known to affect biofilm formation, of structural and secreted proteins of type VI secretion systems, and provided proteogenomic evidence for some missed MPAO1 genes. This integrated, broadly applicable model promises to improve the mechanistic understanding of biofilm formation, antimicrobial tolerance, and resistance evolution in biofilms.

Genome-Wide Mapping Reveals Complex Regulatory Activities of BfmR in Pseudomonas aeruginosa

“…In each case, the MEME algorithm located the motif in the promoters of bfmR and pa4103 at the position of the BfmR-binding sites previously identified by DNase I footprinting [ 11 ] ( Figure 1 C; Figure S1B ), suggesting that the determination of BfmR-binding consensus sequence was carried out successfully. Out of the 174 enriched peaks (Data set 2), 161 (93%) displayed 101 bp fragment centered on the peak summits located at intergenic regions ( Figure S1C , Data set 2), indicating a high enrichment of BfmR-binding sites at regulatory regions since approximately 90% of the P. aeruginosa MPAO1 genome is composed of coding sequences [ 48 ].…”

Prevalent Synergy and Antagonism Among Antibiotics and Biocides in Pseudomonas aeruginosa

“…The experiments were performed with P. aeruginosa MPAO1 obtained from Colin Manoil ( Jacobs et al, 2003 ; Varadarajan et al, 2020 ). The ancestor strain was isolated from a wound in Melbourne, Australia, in 1954 ( Holloway, 1955 ; Chandler et al, 2019 ).…”

A regulatory network involving Rpo, Gac and Rsm for nitrogen-fixing biofilm formation by Pseudomonas stutzeri

“…In general, as established in the model bacterium Pseudomonas aeruginosa , biofilm development usually begins with attachment to a surface, followed by microcolony formation and production of the extracellular matrix responsible for the biofilm architecture 10 – 14 . Biofilm formation has been studied intensively in the genus Pseudomonas , with an emphasis on genetic elements and molecular mechanisms; Gac/Rsm, c-di-GMP signalling and quorum-sensing (QS) pathways were reported as the main mechanisms leading to biofilm formation 15 , 16 .…”