Fowler Janet M, Wulff Christine R, Straley Susan C, Brubaker Robert R
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA.
Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, KY 40536, USA.
Microbiology (Reading). 2009 Aug;155(Pt 8):2509-2521. doi: 10.1099/mic.0.028852-0. Epub 2009 May 14.
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 Ca(2+) versus vegetative growth with downregulation of the T3SS with Ca(2+) (>or=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 Ca(2+). Several T3SS regulatory proteins modulate the LCR, because their absence promotes a Ca(2+)-blind phenotype in which growth at 37 degrees C ceases and the T3SS is constitutive even with added Ca(2+). This study analysed the connection between the LCR and Ca(2+) by determining the response of selected Ca(2+)-blind mutants grown in Ca(2+)-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 Ca(2+)-independent growth at 37 degrees 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 Ca(2+)-blind yopD phenotype was fully suppressed in a Ca(2+)-independent background lacking the injectisome-associated inner-membrane component YscV but not peripheral YscK, suggesting that the core translocon energizes YopD.
野生型鼠疫耶尔森菌细胞表现出低钙反应（LCR），其定义为在37℃无添加Ca²⁺培养时细菌生长停滞并表达pCD编码的III型分泌系统（T3SS），而在有Ca²⁺（≥2.5 mM）时呈营养生长且T3SS下调。已知细菌生长停滞反映了Na⁺、L-谷氨酸和培养基pH的累积毒性；控制这些变量可在无Ca²⁺时实现全面生长（“挽救”）。几种T3SS调节蛋白调节LCR，因为它们的缺失会促进一种Ca²⁺盲表型，即在37℃时生长停止且即使添加Ca²⁺，T3SS仍持续表达。本研究通过测定在缺乏Ca²⁺的挽救培养基中生长的选定Ca²⁺盲突变体的反应来分析LCR与Ca²⁺之间的联系，这些培养基分别含有Na⁺加L-谷氨酸（pH 5.5，此时T3SS不表达）、单独的L-谷氨酸（pH 6.5，此时L-天冬氨酸被完全分解代谢）以及单独的Na⁺（pH 9.0，此时生电钠泵NADH：泛醌氧化还原酶被激活）。所有这三种条件都支持野生型鼠疫耶尔森菌以及lcrG和yopN突变体（拥有完整但失调的T3SS）在37℃基本实现完全不依赖Ca²⁺的生长，这表明细菌生长停滞反映了生物能量学中一种依赖Na⁺的损伤。相比之下，缺乏负调节因子YopD或YopD伴侣蛋白（LcrH）的突变体在任何挽救培养基中都无法生长，因此是真正的温度敏感型。在缺乏与注射体相关的内膜成分YscV但不缺乏外周YscK的不依赖Ca²⁺的背景中，Ca²⁺盲yopD表型被完全抑制，这表明核心转运体为YopD提供能量。
