Lactococcus lactis Uses MscL as Its Principal Mechanosensitive Channel

Abstract

The functions of the mechanosensitive channels from Lactococcus lactis were determined by biochemical, physiological, and electrophysiological methods. Patchclamp 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.Mechanosensitive channels play an important role in prokaryotic cell volume regulation (1). In small, single-cell organisms, this regulation can mean the difference between life and death under extreme osmotic downshift conditions. By diffusion over the semipermeable cell membrane and/or aquaporins embedded in the membrane, water can enter and leave the cell until equilibrium is established between internal and external osmolality. This allows microorganisms to adapt to changes in external osmolyte concentrations. When the external osmolyte concentrations increase (hyperosmotic stress), water will leak out of the cell, causing loss of turgor, and ultimately the cell may plasmolyse. Bacteria respond to this hyperosmotic stress by rapid uptake of ions (K ϩ ) and/or compatible solutes or increasing the intracellular osmolyte concentration through synthesis of compatible solutes. The increase in internal compatible solute concentration compensates for the high external osmolality, allowing water to diffuse back and the cell to regain its original volume and turgor (2-4).Conversely, when the external osmolyte concentration suddenly decreases, water will diffuse into the cell, causing it to swell and, in extreme conditions, lyse. This is where the mechanosensitive channels are thought to play a role by opening in response to the increased membrane tension effected by the rapid increase in cell volume. The best known example of a channel in this role is the mechanosensitive channel of large conductance from Escherichia coli (MscL Ec ), but homologues are present in most eubacteria (5-7). MscL opens near the lytic tension limit of the bacterial membrane. A second mechanosensitive channel, that of small conductance, MscS, has been characterized in only a few organisms (8,9). MscS opens at lower membrane tensions and has a smaller conductance than MscL, making it useful for fine regulation of internal compatible...

Responses of Bacillus subtilis to Hypotonic Challenges: Physiological Contributions of Mechanosensitive Channels to Cellular Survival

“…3), demonstrating that the integrity of the MscL channel-forming protein is critical for the effective management of a hypoosmotic shock by the B. subtilis cells. This situation is similar to that found by Folgering et al (14) in Lactococcus lactis, where also an MscL-type channel functions as the main solute release system upon an osmotic downshift. This differs from the situation observed in E. coli where an mscL mutant by itself has no obvious phenotype (21).…”

The MscS and MscL Families of Mechanosensitive Channels Act as Microbial Emergency Release Valves

“…However, this variability is extremely modest compared with that of MscS. Of the MscL orthologue channels that have been characterized electrophysiologically (29,58,59), the differences in channel activities observed are also relatively modest. Some differences in mechanosensitivity and kinetics and perhaps a 25% variability in conductance have been observed; none of the MscL channels have been found to be dependent upon ionic conditions or other factors, as may be the case for the diverse MscS family of channels (47).…”

Stress Physiology of Lactic Acid Bacteria

“…Although Lc. lactis has genes encoding both MscS (yncB) and MscL (mscL) channels, only the latter is functional and is used as the main mechanosensitive solute release system to protect cells under conditions of hypoosmotic stress (423) (Fig. 1).…”