Identification of Three New Rugose Small Colony Variants from a Pseudomonas aeruginosa Biofilm

Pseudomonas aeruginosa is a gram-negative, pathogenic, bacterium that produces biofilms containing phenotypically distinct subpopulations (Dodson et al., 2022). It was discovered that rugose small colony variants (RSCVs) were isolated with the biofilm subpopulations. RSCVs are hyper-biofilm producing bacterial mutants with increased antibiotic tolerance and are often found in chronic infections. When separating and characterizing the three biofilm cell subpopulations from a single P. aeruginosa PA14 biofilm, we discovered three novel RSCVs (denoted RSCV_1, RSCV_2, and RSCV_3). These RSCVs differed from stationary wild type (WT) PA14, the PA14 biofilm subpopulations, and between the RSCVs themselves. The observed phenotypic changes in the RSCVs included differences in cellular morphology, exopolysaccharide production, biosynthesis of virulence factors, biofilm formation, and antibiotic tolerance. Different molecules were ionized on the cell membranes of RSCVs and stationary phase WT PA14 using matrix assisted laser desorption ionization (MALDI) mass spectrometry. RNA sequencing analysis of stationary phase cells identified a high number of transcripts that were differentially expressed between the RSCVs and WT PA14 as well as between RSCV_1 and RSCV_3. The levels of the intracellular signaling molecule bis-(3', 5')-cyclic-dimeric-guanosine monophosphate (cyclic-di-GMP) were higher in all of the RSCVs compared to WT PA14 and significantly lower in RSCV_3 as compared to both RSCV_1 and RSCV_2. Whole-genome DNA sequencing analysis revealed single-nucleotide deletions and single-nucleotide polymorphisms (SNPs) among the RSCVs and between the RSCVs to WT PA14. The detected differences in the RSCVs have significant implications for biofilm production, antibiotic tolerance, and virulence. Overall design: Bulk RNA-seq profiling of RSCV_1 (n = 3), RSCV_3 (n = 3), and stationary phase WT PA14 (n = 3).