Growth of calcium-blind mutants of Yersinia pestis at 37 °C in permissive Ca2+-deficient environments

Cells of wild-type Yersinia pestis exhibit a low-calcium response (LCR) defined as bacteriostasis with expression of a pCD-encoded type III secretion system (T3SS) during cultivation at 37 degrees C without added Ca2+ versus vegetative growth with downregulation of the T3SS with Ca2+ (>= 2.5 mM). Bacteriostasis is known to reflect cumulative toxicity of Na+, L-glutamic acid and culture pH; control of these variables enables full-scale growth ('rescue') in the absence of Ca2+. Several T3SS regulatory proteins modulate the LCR, because their absence promotes a Ca2+-blind phenotype in which growth at 37 C ceases and the T3SS is constitutive even with added Ca2+. This study analysed the connection between the LCR and Ca2+ by determining the response of selected Ca2+-blind mutants grown in Ca2+-deficient rescue media containing Na+ Plus L-glutamate (pH 5.5), where the T3SS is not expressed, L-glutamate alone (pH 6.5), where L-aspartate is fully catabolized, and Na+ alone (pH 9.0), where the electrogenic sodium pump NADH : ubiquinone oxidoreductase becomes activated. All three conditions supported essentially full-scale Ca2+-independent growth at 37 degrees C of wild-type Y. pestis as well as IcrG and yopN mutants (possessing a complete but dysregulated T3SS), indicating that bacteriostasis reflects a Na+-dependent lesion in bioenergetics. In contrast, mutants lacking the negative regulator YopD or the YopD chaperone (LcrH) failed to grow in any rescue medium and are therefore truly temperature-sensitive. The Ca2+-blind yopD phenotype was fully suppressed in a Ca2+-independent background lacking the injectisome-associated inner-membrane component YscV but not peripheral YscK, suggesting that the core translocon energizes YopD.