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

¹ Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA
² Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, KY 40536, USA
³ Department of Microbiology, The University of Chicago, 920 E. 58th Street, Chicago, IL 60637, USA

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 °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 °C of wild-type Y. pestis as well as lcrG 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.

Correspondence
Robert R. Brubaker
brubake3{at}msu.edu