Transcriptional profiling of Pseudomonas aeruginosa biofilm life cycle stages reveals dispersal-specific biomarkers
Bacteria exhibit two lifestyles: planktonic free-floating individual cells or sessile multicellular aggregates known as biofilms. The biofilm lifecycle is characterised by three distinct stages: attachment, maturation and dispersal. Distinct adaptations occur in each stage, determining cellular behaviours such as surface attachment or synthesis and degradation of extracellular matrix components. Characterising stage-specific bacterial profiles therefore represents a valuable strategy for the development of novel antibiofilm therapies. Here, we used the model biofilm-forming bacterium Pseudomonas aeruginosa PAO1 to characterise the transcriptional profiles of each stage of the biofilm life cycle: attachment, biofilm maturation and spontaneous dispersal in closed cultures. We report that surface attachment was accompanied by the upregulation of genes comprising the Pil-Chp mechanosensory system, whereas biofilm maturation was characterised by the upregulation of genes involved in Pel polysaccharide synthesis, siaD and PA4396 diguanylate cyclases as well as pipA, fimX and PA5442. In contrast, dispersing cells upregulated genes responsible for the biosynthesis of alginate, rhamnolipid, and extracellular nucleases (eddA, eddB), as well as the transcriptional regulator of dispersal amrZ. Additionally, genes encoding the spontaneous dispersal molecule cis-2-decenoic acid (dspS and dspI), canonical phosphodiesterases (nbdA and rbdA), four non-canonical HD-GYP phosphodiesterases and seven other c-di-GMP-related enzymes were also upregulated during dispersal. Our comprehensive analysis of transcriptional changes across biofilm stages therefore provides benchmarking stage-specific transcriptional profiles for P. aeruginosa biofilms in closed culture systems. Furthermore, it allowed the identification of a subset of fourteen genes as transcriptional biomarkers of dispersal, which were used to build reporter plasmids as tools to determine the onset of dispersal. ImportanceBiofilm infections by P. aeruginosa are a major medical challenge due to the increased tolerance to antimicrobials displayed by bacteria living in sessile communities, which is reduced during spontaneous biofilm dispersal. Attachment, biofilm maturation and dispersal represent the main stages of a dynamic process known as the biofilm lifecycle. However, the global regulatory responses governing transitions between these stages remain understudied. Here, we combine live microscopy and biomass quantification to track the progression of P. aeruginosa cultures through the three main stages of the biofilm lifecycle. We show that cells from each stage recapitulate canonical, stage-specific transcriptional responses and identify a set of biomarkers associated with the onset of dispersal. These biomarkers may offer a practical tool for rapidly screening dispersal-inducing compounds, aiding in the discovery of the next generation of antibiofilm therapeutics.