Cell Population and Electrophysiology Approaches to Osmotic Fitness of Pseudomonas Aeruginosa

Abstract

Pseudomonas aeruginosa (PA) is an opportunistic pathogen with exceptional adaptability to a range of environments. Upon a sudden dilution of external medium, PA, as other bacteria evades mechanical rupture by engaging tension-activated channels releasing osmolytes. In this study we compare fast osmotic permeability responses in wild-type PA and E. coli (EC) strains in stopped-flow experiments and provide further electrophysiological description. Osmotic dilution experiments showed that PA tolerates a broader range of shocks than EC. Under the assumption that the cell has a chance to survive the shock when osmolyte release is fast enough to curb osmotic swelling, we recorded the kinetics of light scattering responses to osmotic up- and down-shifts. While exhibiting a lower water permeability, PA showed faster osmolyte release which correlated with better survival. To characterize the PA tension-activated channels, we generated giant spheroplasts from this microorganism and recorded current responses in excised native patches. Unlike EC which relies on two types of channels, EcMscS and EcMscL, to generate a distinctive two-wave pressure ramp response, PA exhibits a more gradual response dominated by MscL-type channels present in higher density. Genome analysis, cloning and expression revealed that PA possesses one MscL-type (PaMscL) and two MscS-type proteins (PaMscS-1 and 2). Both PaMscS channels exhibited an earlier activation by pressure as compared to EcMscS. PaMscS-2 showed a smaller conductance, higher anionic preference, strong inactivation and slow recovery. While PaMscS-1 and PaMscL rescued the unprotected MJF641 E. coli strain from osmotic damage, PaMscS-2 did not. Homology models and functional analysis of mutants have identified in PaMscSs the same ‘hinge’ elements responsible for inactivation as in EcMscS. We discuss distinctive parameters of PA cells, the capacity of its osmolyte release system, and a path toward quantitative models of the osmotic rescue mechanism.