Metabolic imbalance and sporulation in an isocitrate dehydrogenase mutant of Bacillus subtilis

Abstract

A Bacillus subtilis mutant with a deletion in the citC gene, encoding isocitrate dehydrogenase, the third enzyme of the tricarboxylic acid branch of the Krebs cycle, exhibited reduced growth yield in broth medium and had greatly reduced ability to sporulate compared to the wild type due to a block at stage I, i.e., a failure to form the polar division septum. In early stationary phase, mutant cells accumulated intracellular and extracellular concentrations of citrate and isocitrate that were at least 15-fold higher than in wild-type cells. The growth and sporulation defects of the mutant could be partially bypassed by deletion of the major citrate synthase gene (citZ), by raising the pH of the medium, or by supplementation of the medium with certain divalent cations, suggesting that abnormal accumulation of citrate affects survival of stationary-phase cells and sporulation by lowering extracellular pH and chelating metal ions. While these genetic and environmental alterations were not sufficient to allow the majority of the mutant cell population to pass the stage I block (lack of asymmetric septum formation), introduction of the sof-1 mutant form of the Spo0A transcription factor, when coupled with a reduction in citrate synthesis, restored sporulation gene expression and spore formation nearly to wild-type levels. Thus, the primary factor inhibiting sporulation in a citC mutant is abnormally high accumulation of citrate, but relief of this metabolic defect is not by itself sufficient to restore competence for sporulation.

FIG. 1

Growth and pH in DS medium. Strains JH642 (cit⁺; triangles), SJB219 (ΔcitC; squares), and SJB231 (ΔcitZC; circles) were grown in DS medium, and samples were removed at the indicated times for measurement of OD₆₀₀ and, after removal of cells by centrifugation, for determination of the pH of the medium. The time of onset of stationary phase is designated as T₀.

FIG. 2

Effect of KOH on spoIIQ′-lacZ expression in a ΔcitC mutant. (a and b) Cells of strain SJB295 (ΔcitC) were grown in DS broth (triangles). After 5 mM KOH (circles) or NaOH (diamonds) was added to the culture at T₀, the OD₆₀₀ (panel a, closed symbols), pH of the medium (panel a, open symbols) and spoIIQ′-lacZ expression (panel b) were measured. (c) Different concentrations of KOH were added to the culture at T₀ (×, no addition; □, 2.5 mM; ○, 5 mM; ▵, 6.25 mM). (d) KOH (5 mM) was added to the culture at T_−1.25 (◊), T_−0.5 (□), T₀ (○), or T₁ (▵). ×, control (no KOH added).

FIG. 3

Thin-section electron microscopic analysis. Cells of a ΔcitC mutant strain (SJB219) were grown in DS medium to the end of exponential growth phase (T₀), and 5 mM KOH was added to the culture. KOH-treated cells were harvested 6 h after the addition of KOH, and samples were prepared for transmission electron microscopy.

FIG. 4

Effects of citZ and sof-1 mutations on spo gene expression in a citC mutant. All strains were grown in DS medium and sampled for assays of β-galactosidase activity at the indicated times after the end of exponential growth (T₀). (A) Strains SJB294 (cit⁺) (■), SJB295 (ΔcitC) (●), and KMB119 (ΔcitZC) (▴) were tested for spoIIQ′-lacZ expression. (B) Strains SJB225 (cit⁺) (■), SJB229 (ΔcitC) (●), and KMB174 (ΔcitZC) (▴) were assayed for spoIID′-lacZ expression. (C) Strains KMB425 (cit⁺) (■), KMB170 (ΔcitC) (●), KMB436 (ΔcitZC) (▴), AS1 (ΔcitC sof-1) (○), and KMB435 (ΔcitZC sof-1) (▵) were assayed for cotA′-lacZ expression.

FIG. 4

Effects of citZ and sof-1 mutations on spo gene expression in a citC mutant. All strains were grown in DS medium and sampled for assays of β-galactosidase activity at the indicated times after the end of exponential growth (T₀). (A) Strains SJB294 (cit⁺) (■), SJB295 (ΔcitC) (●), and KMB119 (ΔcitZC) (▴) were tested for spoIIQ′-lacZ expression. (B) Strains SJB225 (cit⁺) (■), SJB229 (ΔcitC) (●), and KMB174 (ΔcitZC) (▴) were assayed for spoIID′-lacZ expression. (C) Strains KMB425 (cit⁺) (■), KMB170 (ΔcitC) (●), KMB436 (ΔcitZC) (▴), AS1 (ΔcitC sof-1) (○), and KMB435 (ΔcitZC sof-1) (▵) were assayed for cotA′-lacZ expression.