CRISPR Interference Screens Identify Novel Regulators of Bacterial Biofilm Formation
DOI:
https://doi.org/10.5281/zenodo.22688118Keywords:
CRISPR interference, Pseudomonas aeruginosa, biofilm formation, genetic screen, quorum sensing, antibiotic toleranceAbstract
Bacterial biofilms underlie a large share of chronic and device-associated infections and are intrinsically tolerant of
antibiotics that readily kill the same organism growing planktonically, yet the regulatory network governing the
transition from free-living to surface-attached, matrix-encased growth remains incompletely mapped even in
well-studied species. We used a pooled, genome-wide CRISPR interference (CRISPRi) library targeting 5,570
annotated genes in Pseudomonas aeruginosa PAO1 to screen for regulators of biofilm formation, selecting for
sgRNA abundance changes between the planktonic and biofilm-associated bacterial fractions of a static microtiter
biofilm assay after 24 hours. The screen identified 143 genes whose knockdown significantly altered biofilm
formation, of which 41 were annotated with no prior functional link to biofilm or surface-sensing biology. Individual
knockdown validation of the twelve highest-confidence novel hits confirmed a biofilm phenotype for nine, including
PA2782, a previously uncharacterised two-component sensor histidine kinase whose knockdown reduced biofilm
biomass by 68% relative to a non-targeting control without affecting planktonic growth rate. RNA sequencing of
the PA2782 knockdown strain showed downregulation of the pel and psl exopolysaccharide biosynthesis operons
and of the c-di-GMP-synthesising diguanylate cyclase gene PA2870, placing PA2782 upstream of the canonical
c-di-GMP-dependent biofilm matrix pathway. The PA2782 knockdown strain also showed a 5.4-fold increase in
tobramycin susceptibility within an established biofilm, consistent with reduced matrix production restoring
antibiotic penetration. These results expand the known regulatory network controlling P. aeruginosa biofilm
formation and identify PA2782 as a candidate target for biofilm-disruptive adjunct therapy.
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