H-048. Progressively Increasing Sensor Kinase beyond a Threshold Level Triggers Sporulation in Bacillus subtilis

P. Eswara Moorthy, J. Dinh, M. Bhattacharya, M. Fujita;
Univ. of Houston, Houston, TX.

Cell fate of B. subtilis under starvation is defined by transcription factor Spo0A that acts as molecular switch to activate or repress the sporulation gene program. Activation of Spo0A is governed by a phosphorelay consisting of multiple sensor histidine kinases and the phosphorelay proteins Spo0F and Spo0B. The kinases are the primary signal transduction proteins that may interact directly with the signals. However, the signals that trigger the initiation of sporulation remain unknown. This problem is being studied with a cell line in which the synthesis of KinA is under the control of an IPTG-inducible promoter. Cells engineered to produce KinA entered sporulation and proceeded through sporulation with high efficiency even in the presence of excess nutrient. Thus, we propose that efficient sporulation requires a threshold level of KinA. Quantitative immunoblot analysis showed that KinA levels were related linearly to the concentration of IPTG but that sporulation efficiency was not. Whereas the level of KinA increased approximately in proportion to the concentration of IPTG over the range of 0 to 20 μM, the efficiency of sporulation abruptly increased over a narrow range. To provide independent confirmation that elevated levels of KinA induce sporulation, we exploited a strain that harbors a temperature-sensitive allele (div355) of the division gene divIC. When shifted to a restrictive temperature, cells of the mutant under the sporulation condition grow but fail to complete polar septation. Instead they form relatively long filaments, and thereby increase in cell volume. Inducing the synthesis of KinA in the mutant with increased cell volume at a restrictive temperature led to gradual activation of Spo0A and caused efficient sporulation. We draw two conclusions from these results. First, progressive increase in KinA beyond a threshold triggers efficient sporulation. Second, progressive increase in KinA level induces gradual Spo0A activation through phosphorelay and provides a robust mechanism to generate persistent response of sporulation gene expression even in the presence of excess nutrient.