Transcriptomic dissection of genetically diverse Wrinkly Spreader mutants reveals hidden layers of complexity

Understanding the genetic basis of adaptive traits requires linking mutation to phenotypic change and fit-ness. The Wrinkly Spreader (WS) mutants of Pseudomonas fluorescens SBW25, which colonize the air-liquid interface (ALI) in static microcosms, provide a model for dissecting genotype-phenotype relationships. Muta-tions generating the WS phenotype cause constitutive activation of regulatory pathways synthesising cyclic bis-(3,5)-dimeric guanosine monophosphate (c-di-GMP) leading to over-production of cellulose encoded by the wss operon. Using RNA-seq on 14 independently evolved WS mutants, each carrying a single characterised mutation in one of 14 different genes affecting production of c-di-GMP, we report that transcriptional responses are highly genotype specific, with few differentially expressed genes (DEGs) shared among WS mutants. Genes showing greatest variance in expression included fimbrial and matrix-associated loci such as fap, cupE and pga. fap and cupE were the only fimbriae-related loci active in these mutants. Targeted deletions of these candidate loci revealed subtle phenotypic and fitness effects. Closer examination of the fap locus showed that its expression is genotype dependent, restricted to a minority of cells, and associated with stability in early stages of mat formation. Overall, our results confirm that previous suppressor-based approaches have captured the major genetic components of the WS phenotype: cellulose encoded by the wss operon and regulated by elevated c-di-GMP. At the same time, it is apparent that morphology-based screens have failed to detect genes having more subtle effects, whose contributions influence success of WS mutants at the ALI. These findings reveal additional layers of complexity that underscore the polygenic nature of the WS phenotype.