Gating the bacterial mechanosensitive channel MscL invivo

Abstract

YggB and MscL are the major mechanosensitive channels in Escherichia coli, and each can rescue the double knockout mutant from osmotic downshock. However, the role of MscL in wild-type bacteria is in question, not only because cells without MscL survive severe osmotic downshocks, but because 1.8 times more suction is required to gate MscL than YggB under patch clamp. Here, we extend previous evidence [Ajouz, B., Berrier, C., Garrigues, A., Besnard, M. & Ghazi, A. (1998) J. Biol. Chem. 273, 26670-26674] to show that downshock gates MscL in vivo even in the presence of YggB. We have made this determination by engineering a channel we can structurally modify in vivo (Leu-19-->Cys MscL). MscLs with charges in their constrictions are known to open easily and transiently to substates and stop cell growth. In this study, we use downshock to stretch this region open to allow attachment of a charged thiosulfonate reagent MTSET(+), thereby creating a toxic channel. Therefore, channel opening can be monitored by loss of colony forming units. By this measure, we find that an approximately 800 mmol/kg downshock from 1,200 mmol/kg medium opens Leu-19-->Cys MscL in the presence of YggB, but a downshock of only approximately 400 mmol/kg is required in the absence of YggB. In parallel, Leu-19-->Cys MscL, stretched open by large sustained suction in the presence of MTSET(+) in voltage-clamped patches, subsequently flickers open with little suction. These observations show that MscL opening is triggered by a specific downshock, even in the presence of YggB, that YggB buffers MscL gating in vivo, and that residue 19 becomes exposed upon channel opening.

Figure 1

(A) Mesh molecular surface (except a 90° wedge) of the closed TbMscL structure derived from ref. showing the region of the constricted pore (darkened center) and the location of Leu-17 of M. tuberculosis MscL and Leu-19 of E. coli MscL by analogy. (B) Structures of leucine, cysteine, and cysteine with an attached MTSET⁺.

Figure 2

Survival of mscLΔ yggB⁺ PB104 bacteria (of AW405 background) expressing different pB10b-based plasmids in the presence of various MTSET⁺ upon downshock from LB500 to LB (filled symbols, continuous line) or after an LB500-to-LB500 isoosmotic transfer (open symbols, broken line). (Means ± SD are given. Unity for relative survival is the value after isoosmotic transfer, no MTSET⁺.) Before transfer into shock solution, ≈2 × 10⁷ cfu/ml were present based upon cfu determined after 1:20 dilution into isoosmotic medium. (A) The empty plasmid (▴, n = 3) determinations only at 0 and 0.5 mM MTSET⁺, the wild-type mscL plasmid (♦, n = 4), or (B) the Leu-19→Cys mscL plasmid (○, n = 3).

Figure 3

Survival of mscLΔ yggB⁺ bacteria of two different genetic backgrounds expressing the same Leu-19→Cys mscL plasmid after graded osmotic downshocks from LB500 to LB with added NaCl given in the presence (filled symbols, continuous line) or absence (open symbols, broken line) of 0.5 mM MTSET⁺. (Means ± SD given. Unity for relative survival is value after isoosmotic transfer, no MTSET⁺. cfu/ml before shock is in parentheses.) (A) PB104 bacteria in AW405 background (⋄, n = 4, 1 × 10⁷ cfu/ml). (B) MJF367 bacteria in FRAG1 background (○, n = 3, 1 × 10⁸ cfu/ml).

Figure 4

Survival of mscLΔ yggBΔ bacteria (in FRAG1 background) expressing various pB10b-based plasmids after graded osmotic downshocks from LB500 to LB with added NaCl in the presence (filled symbols, continuous line) or absence (open symbols, broken line) of 0.5 mM MTSET⁺. (Means ± SD given. Unity for relative survival is value after isoosmotic transfer, no MTSET⁺. cfu/ml before shock is in parentheses). (A) Expressing Leu-19→Cys mscL plasmid (♦, n = 5, 2 × 10⁸ cfu/ml). (B) Wild-type plasmid (▵, n = 3, 2 × 10⁸ cfu/ml). (C) Empty plasmid (○, n = 3, 1 × 10⁸ cfu/ml).

Figure 5

Behavior of Leu-19→Cys MscL under a patch clamp upon the presentation of MTSET⁺. Inside-out patches were excised from PB104 spheroplasts expressing Leu-19→Cys MscL. After seal formation (with no MTSET⁺ present), repeated suction pulses usually activated MscS only (not shown). (A) MTSET⁺ presented from the bath (presumed cytoplasmic side). (i) A large suction (lower trace) eventually produced flickering activities (solid arrows, Leu-19→Cys MscL) but only after the suction was so high that it had activated all of the MscS in the patch (arrowheads and plateau). (ii) A subsequent episode of the same patch. Typical of five experiments. (B) MTSET⁺ presented from the pipette (presumed periplasmic side) by back filling. The tip of the pipette was filled with the pipette solution supplemented with 0.3 M sucrose (without MTSET⁺). (i) For ≈20 min after backfill in the absence of suction, there was no activation. Upon suction, flickers were eventually observed (arrows) but, again, only at suctions that activated all MscS (arrowheads, plateau). (ii) A subsequent episode of the same patch, typical of five experiments.