Characterizing Novel Regulatory Effects of the Global Regulator Cra on the Glucose-Phosphate Stress Response in Escherichia coli

Abstract

How organisms cope with environmental stressors is a fundamental question of life. For example, some bacteria must be able to sense and respond to glucose-phosphate stress, a metabolic imbalance resulting from the intracellular accumulation of certain sugar-phosphates that leads to growth inhibition. This stress is induced by disrupting glycolysis and depleting related metabolic intermediates. Enteric bacteria like Escherichia coli can recover from glucosephosphate stress through the actions of the transcription factor SgrR and the small RNA SgrS. SgrR activates transcription of sgrS, and SgrS helps rescue cells from stress, in part by preventing further uptake of stressor sugar-phosphates. While much is known about the regulatory mechanisms of this stress response, regulation of SgrR and SgrS themselves is not as well understood. Of particular interest is Cra, a global regulator of carbon metabolism, in part because the sgrS promoter region contains a putative Cra binding site. Here, we describe a role for Cra in regulating the response to glucose-phosphate stress. We determined that deleting cra results in a growth defect compared to wild-type E. coli under stress conditions. In addition, a cra mutant exhibits decreased expression of a PsgrS-lacZ fusion, implicating Cra in the activation of the glucose-phosphate stress response. Deleting cra does not affect expression of a PsgrR-lacZ fusion, suggesting that Cra regulates sgrS expression independently of SgrR. Since Cra is known to regulate the fruBKA operon, we further investigated the effect of mutating these genes on glucose-phosphate stress. Deleting fruK (encoding an enzyme that catalyzes the formation of fructose-1,6-bisphosphate from fructose-1-phosphate) worsens the growth defect of an sgrS mutant during stress and results in aberrant sgrS expression. Mutating pfkA (encoding an enzyme that catalyzes the formation of fructose-1,6-bisphosphate from fructose-6-phosphate) results in a similar exacerbation of the sgrS mutant growth defect and aberrant sgrS expression, 4 suggesting that synthesis of fructose 1,6-bisphosphate is important for alleviating stress. Overall, this work supports a novel role for Cra as an important regulatory player in the response to glucose-phosphate stress.