The major cause of death in people with cystic fibrosis (CF), a human autosomal recessive genetic disease, is respiratory failure due to chronic lung infection. Pseudomonas aeruginosa is a prevalent CF respiratory pathogen (Cystic Fibrosis Foundation Patient Registry, 2019). The CF lung environment selects for mucoid P. aeruginosa mutants, which overproduce the exopolysaccharide alginate and are associated with poor disease prognosis (

Many pathogens form biofilms, which are bacterial communities encased in an extracellular matrix that protects them against antibacterial treatments. The roles of several matrix components of the opportunistic pathogen Pseudomonas aeruginosa have been characterized.

Pseudomonas aeruginosa causes chronic lung infections in people with cystic fibrosis (CF), and this bacterium undergoes selection in the CF lung environment over the course of these life-long infections. One genetic adaptation frequently observed in CF P. aeruginosa isolates is mutation of mucA. MucA inhibits the sigma factor AlgU. Clinical mucA mutations lead to misregulation of AlgU, resulting in a mucoid bacterial phenotype that is associated with poor CF disease outcomes. Here we show that paradoxically a portion of the mucA gene is essential for P. aeruginosa viability. We demonstrate that mucA is no longer essential in a strain lacking algU, and mucA alleles that encode for proteins that do not bind to AlgU are insufficient for bacterial viability. Furthermore, we found that mucA is no longer essential in mutant strains containing AlgU variants with reduced sigma factor activity, suggesting that reducing AlgU activity can suppress the requirement for mucA. Finally, we found that overexpression of algU from an inducible promoter prevents cell growth in the absence of MucA, and that this phenotype can be rescued by overproduction of RpoD, the housekeeping sigma factor. Together, these results suggest that in the absence of MucA, the inability to regulate AlgU activity leads to sigma factor competition, preventing the expression of essential housekeeping genes and resulting in the loss of bacterial viability.

The biofilm matrix is composed of exopolysaccharides, eDNA, membrane vesicles, and proteins. While proteomic analyses have identified numerous matrix proteins, their functions in the biofilm remain understudied compared to the other biofilm components. In thePseudomonas aeruginosabiofilm, several studies have identified OprF as an abundant matrix protein and, more specifically, as a component of biofilm membrane vesicles. OprF is a major outer membrane porin ofP. aeruginosacells. However, current data describing the effects of OprF in theP. aeruginosabiofilm is limited. Here we identify a nutrient-dependent effect of OprF in static biofilms, whereby ΔoprFcells form significantly less biofilm than wild type when grown in media containing glucose or low sodium chloride concentrations. Interestingly, this biofilm defect occurs during late static biofilm formation and is not dependent on the production of PQS, which is responsible for outer membrane vesicle production. Furthermore, while biofilms lacking OprF contain approximately 60% less total biomass than those of wild type, the number of cells in these two biofilms is equivalent. We demonstrate thatP. aeruginosaΔoprFbiofilms with reduced biofilm biomass contain less eDNA than wild-type biofilms. These results suggest that the nutrient-dependent effect of OprF is involved in the maintenance of matureP. aeruginosabiofilms by retaining eDNA in the matrix.IMPORTANCEMany pathogens form biofilms, which are bacterial communities encased in an extracellular matrix that protects them against antibacterial treatments. The roles of several matrix components of the opportunistic pathogenPseudomonas aeruginosahave been characterized. However, the effects ofP. aeruginosamatrix proteins remain understudied and are untapped potential targets for antibiofilm treatments. Here we describe a conditional effect of the abundant matrix protein OprF on late-stageP. aeruginosabiofilms. A ΔoprFstrain formed significantly less biofilm in low sodium chloride or with glucose. Interestingly, the defective ΔoprFbiofilms did not exhibit fewer resident cells but contained significantly less extracellular DNA (eDNA) than wild type. These results suggest that OprF is involved in matrix eDNA retention in mature biofilms.

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