Phosphosugar Stress in Bacillus subtilis: Intracellular Accumulation of Mannose 6-Phosphate Derepressed the glcR-phoC Operon from Repression by GlcR

Abstract

Bacillus subtilis phosphorylates sugars during or after their transport into the cell. Perturbation in the conversion of intracellular phosphosugars to the central carbon metabolites and accumulation of phosphosugars can impose stress on the cells. In this study, we investigated the effect of phosphosugar stress on B. subtilis Preliminary experiments indicated that the nonmetabolizable analogs of glucose were unable to impose stress on B. subtilis In contrast, deletion of manA encoding mannose 6-phosphate isomerase (responsible for conversion of mannose 6-phosphate to fructose 6-phosphate) resulted in growth arrest and bulged cell shape in the medium containing mannose. Besides, an operon encoding a repressor (GlcR) and a haloic acid dehalogenase (HAD)-like phosphatase (PhoC; previously YwpJ) were upregulated. Integration of the P _glcR-lacZ cassette into different mutational backgrounds indicated that P _glcR is induced when (i) a manA-deficient strain is cultured with mannose or (ii) when glcR is deleted. GlcR repressed the transcription of glcR-phoC by binding to the σ^A-type core elements of P _glcR An electrophoretic mobility shift assay showed no interaction between mannose 6-phosphate (or other phosphosugars) and the GlcR-P _glcR DNA complex. PhoC was an acid phosphatase mainly able to dephosphorylate glycerol 3-phosphate and ribose 5-phosphate. Mannose 6-phosphate was only weakly dephosphorylated by PhoC. Since deletion of glcR and phoC alone or in combination had no effect on the cells during phosphosugar stress, it is assumed that the derepression of glcR-phoC is a side effect of phosphosugar stress in B. subtilisIMPORTANCEBacillus subtilis has different stress response systems to cope with external and internal stressors. Here, we investigated how B. subtilis deals with the high intracellular concentration of phosphosugars as an internal stressor. The results indicated the derepression of an operon consisting of a repressor (GlcR) and a phosphatase (PhoC). Further analysis revealed that this operon is not a phosphosugar stress response system. The substrate specificity of PhoC may indicate a connection between the glcR-phoC operon and pathways in which glycerol 3-phosphate and ribose 5-phosphate are utilized, such as membrane biosynthesis and teichoic acid elongation.

Keywords: PTS; carbohydrate; mannose; phosphatase; repressor; stress response system.