Exploring the Diversity and Function of the SPFH-Domain Containing Proteins in Pseudomonas aeruginosa.

The SPFH-domain containing proteins are widely conserved membrane-associated factors proposed to organise membrane microdomains and thereby regulate key cellular processes. In Pseudomonas aeruginosa PA14, we identified nine such proteins (HflK, HflC and PaFlo3-PaFlo9) that display remarkable sequence divergence, genomic variability and limited phylogenetic conservation. Functional analyses of single and multiple SPFH mutants showed that these proteins are not essential for growth, biofilm formation, swimming and swarming motility, oxidative stress resistance or virulence in the Galleria mellonella infection model, although distinct slight phenotypic effects were observed in specific genetic backgrounds. Phylogenetic comparisons showed that none of the PaFlo proteins cluster with canonical bacterial flotillins such as FloA or FloT from Bacillus subtilis or FloA from Staphylococcus aureus, ruling out specific orthology. Moreover, most PaFlo proteins appear to not have clear orthologs in other γ-Proteobacteria, indicating that they may be narrowly distributed in these bacterial genomes. The absence of conserved genomic context and operon organization further supports functional diversification rather than redundancy. These findings indicate that SPFH proteins are largely dispensable for P. aeruginosa viability and virulence under laboratory conditions and likely fulfil context-dependent or niche-specific roles.