The Electrophysiology of Mechanosensitive Channels in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen that causes infections mainly in immuno-compromised organisms and is characterized with exceptional adaptability to a broad range of habitats, from soil and freshwater to medical equipment and mammalian tissues. Part of its adaptive potential is the ability to adjust its turgor pressure under conditions of drastically varied osmolarity. The osmolyte release system in this Gram-negative bacterium, like in E. coli, comprises the high-threshold tension-activated channel MscL and six channels from the MscS family. In the present work we have developed the giant spheroplast system for direct patch-clamp recording from the cytoplasmic membrane of P. aeruginosa and characterized mechanoelectrical responses in the wild-type strain (PA14) using pressure protocols, revealing a complex adaptive behavior involving several channel species. We have cloned and functionally characterized PaMscL (137 aa) and two MscS-like channels, PaMscS-1 (278 aa) and PaMscS-2 (283 aa) expressed in E. coli giant spheroplasts. The ∼2 nS PaMscL exhibited short open dwell times and was found non-selective. Both PaMscS-1 (∼1 nS) and PaMscS-2 (∼0.5 nS) have a slight anionic preference with similar pressure midpoint ratios P0.5 MscS/ P0.5 MscL of ∼0.5. Under a standard 10 s pressure-step protocol, PaMscS-1 displays a 30% tension-dependent inactivation, whereas PaMscS-2 under similar conditions shows a 60% inactivation, with extremely slow (5-10 min) recovery. Sequence analysis and homology models reveal all components necessary for inactivation conserved between P. aeruginosa and E. coli MscS. PaMscL and PaMscS-1 perfectly rescue an MS channel-free E. coli strain (MJF465) from abrupt osmotic shock, whereas PaMscS-2 does not, possibly due to lower conductance and expression level. The electrophysiology of P. aeruginosa gives a better understanding of the components and mechanisms of environmental stability of this opportunistic pathogen easily changing its habitats.