Bacillithiol is a major buffer of the labile zinc pool in B acillus subtilis

Intracellular zinc levels are tightly regulated since zinc is an essential cofactor for numerous enzymes, yet can be toxic when present in excess. The majority of intracellular zinc is tightly associated with proteins and is incorporated during synthesis from a poorly defined pool of kinetically labile zinc. In B acillus subtilis , this labile pool is sensed by equilibration with the metalloregulator Z ur, as an indication of zinc sufficiency, and by CzrA , as an indication of zinc excess. Here, we demonstrate that the low‐molecular‐weight thiol bacillithiol ( BSH ) serves as a major buffer of the labile zinc pool. Upon shift to conditions of zinc excess, cells transiently accumulate zinc in a low‐molecular‐weight pool, and this accumulation is largely dependent on BSH . Cells lacking BSH are more sensitive to zinc stress, and they induce zinc efflux at lower external zinc concentrations. Thiol reactive agents such as diamide and cadmium induce zinc efflux by interfering with the Zn ‐buffering function of BSH . Our data provide new insights into intracellular zinc buffering and may have broad relevance given the presence of BSH in pathogens and the proposed role of zinc sequestration in innate immunity.