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.
Highlights
Pseudomonas aeruginosa is a Gram-negative bacterium ubiquitously present in the soil, water, and different animal hosts[1]
The minimal biofilm inhibitory concentration (MBIC) was determined as the lowest concentration resulting in a 50% or 90% reduction of the biofilm cell recovery after 24 h treatment compared to the non-treated condition. c Comparative confocal micrographs after live/dead staining of 18 h MPAO1 WT, cbrB and arnB biofilms grown under microfluidic conditions using the publicly available mold confirm reduced biofilm formation for the cbrB mutant and robust biofilm formation of the arnB mutant in the absence of treatment
We found that 21 of the 52 CDS missed in the MPAO1/P1 assembly were detected at npj Biofilms and Microbiomes (2020) 46
Summary
Pseudomonas aeruginosa is a Gram-negative bacterium ubiquitously present in the soil, water, and different animal hosts[1]. These include P. aeruginosa strain MPAO120, the parental strain of the widely utilized transposon insertion mutant library from the University of Washington (UW)[21] Such mutant collections represent highly valuable resources to uncover new functions and conditionspecific essential genes in genome-wide screens[21], for example, genes relevant for resistance against certain antibiotics[22,23]. To facilitate data mining and comparison, algorithms nowadays allow researchers to readily generate we provide an extensive annotation of all 5799 proteincomplete de novo genome assemblies for most prokaryotes coding genes This includes information on conserved and except a few percent of strains with highly complex repeat regions[29]. A syringe pump was used to deliver 5 μL/min (ū ≈ 208 μm/s) flow inside the chamber to provide laminar flow conditions for bacterial adhesion and biofilm growth (the calculated Reynolds Number corrected for the npj Biofilms and Microbiomes (2020) 46
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