Lactococcus lactis Uses MscL as Its Principal Mechanosensitive Channel

Joost H.A Folgering,Paul C Moe,Gea K Schuurman-Wolters,Paul Blount,Bert Poolman

doi:10.1074/jbc.m411732200

Abstract

The functions of the mechanosensitive channels from Lactococcus lactis were determined by biochemical, physiological, and electrophysiological methods. Patch-clamp studies showed that the genes yncB and mscL encode MscS and MscL-like channels, respectively, when expressed in Escherichia coli or if the gene products were purified and reconstituted in proteoliposomes. However, unless yncB was expressed in trans, wild type membranes of L. lactis displayed only MscL activity. Membranes prepared from an mscL disruption mutant did not show any mechanosensitive channel activity, irrespective of whether the cells had been grown on low or high osmolarity medium. In osmotic downshift assays, wild type cells survived and retained 20% of the glycine betaine internalized under external high salt conditions. On the other hand, the mscL disruption mutant retained 40% of internalized glycine betaine and was significantly compromised in its survival upon osmotic downshifts. The data strongly suggest that L. lactis uses MscL as the main mechanosensitive solute release system to protect the cells under conditions of osmotic downshift.

Highlights

Mechanosensitive channels play an important role in prokaryotic cell volume regulation [1]
Both channels provided E. coli MJF465 (MscSϪ/MscLϪ) with protection against osmotic downshift as demonstrated in plate assays, and both channels were functional in patch clamp experiments using membrane patches from E. coli spheroplasts, membrane patches from hybrid proteo-giant unilamellar vesicles (GUVs) prepared from L. lactis membrane vesicles, or membrane patches from proteo-GUVs prepared from purified and membrane-reconstituted protein
Open dwell times for MscLLl6H were short compared with MscLEc and most other MscL homologues studied to date, giving rise to a “flickery” appearance in channel recordings

Summary

MATERIALS AND METHODS

After 40 min of uptake at 30 °C, aliquots of the cells were subjected to no dilution or a 3-, 5-, 10-, 20-, 50-, and 100-fold dilution into 50 mM KPi, pH 6.5 This resulted in final osmolalities of 1050, 420, 295, 200, 155, 125, and 115 mosmol/kg, respectively. For survival under osmotic downshift conditions of L. lactis IL1403 or JIM7049⌬MscL, cells were grown overnight in chemically defined medium [27], supplemented with 25 mM glucose and 5 ␮g/ml erythromycin (where applicable), and diluted 1:100 to the same medium supplemented with 365 mM KCl (1050 mosmol/kg). NcoI, HindIII pSH71 replicon; nisA promoter; XbaI XhoI in multiple cloning site; Cmres pNZ8020 with L. lactis mscL, and sequence coding for a C-terminal 6-histidine tag; inserted in. XbaI, XhoI pSH71 replicon; nisA promoter, NcoI, HindIII in multiple cloning site; Cmres pNZ8048 with L. lactis yncB, and sequence coding for a C-terminal 10-histidine tag; inserted in. NcoI, HindIII pWV01-derivative; repAϪ ,replicates only in strains that carry repA in trans; Emres pOri280 with an internal fragment of mscLLl inserted in XbaI, BamHI; Emres

25 This work

Other Procedures

RESULTS

DISCUSSION