Bacillus subtilis CodY represses early-stationary-phase genes by sensing GTP levels - PubMed (original) (raw)
Bacillus subtilis CodY represses early-stationary-phase genes by sensing GTP levels
M Ratnayake-Lecamwasam et al. Genes Dev. 2001.
Abstract
CodY, a highly conserved protein in the low G + C, gram-positive bacteria, regulates the expression of many Bacillus subtilis genes that are induced as cells make the transition from rapid exponential growth to stationary phase and sporulation. This transition has been associated with a transient drop in the intracellular pool of GTP. Many stationary-phase genes are also induced during exponential-growth phase by treatment of cells with decoyinine, a GMP synthetase inhibitor. The effect of decoyinine on an early-stationary-phase gene is shown here to be mediated through CodY and to reflect a reduction in guanine nucleotide accumulation. CodY proved to bind GTP in vitro. Moreover, CodY-mediated repression of target promoters was dependent on a high concentration of GTP, comparable to that found in rapidly growing exponential-phase cells. Because a codY-null mutant was able to sporulate under conditions of nutrient excess, CodY also appears to be a critical factor that normally prevents sporulation under such conditions. Thus, B. subtilis CodY is a novel GTP-binding protein that senses the intracellular GTP concentration as an indicator of nutritional conditions and regulates the transcription of early-stationary-phase and sporulation genes, allowing the cell to adapt to nutrient limitation.
Figures
Figure 1
CodY concentration during exponential growth and stationary phase. Samples were taken at the indicated time points (in h) before and after entry into stationary phase (T0) and proteins of each crude lysate were separated by SDS-PAGE. The proteins were electrotransferred and immunoblotted with a polyclonal CodY antibody. Lane M contains prestained molecular weight markers (GIBCO-BRL). (A) Immunoblots of lysates from wild-type and Δ_codY_ strains grown in DS medium. (B) Immunoblots of lysates from a wild-type strain grown in S6 medium in the presence (S6C) or absence (S6) of 0.1% CAA.
Figure 2
The effect of decoyinine on _dpp_-lacZ directed β-galactosidase expression in cells grown in S6C medium. When the cultures reached a turbidity at 600 nm of 0.3 to 0.4 (T0 for this experiment), the cultures were split into two 25-ml cultures and 0.125 ml of either 1N KOH (open symbols) or decoyinine (100 mg/mL in 1N KOH) was added (closed symbols). (A) Strains PS59 (abrB+ codY+), squares; PS56 (abrB codY+), circles. (B) Strains PS164 (abrB+ codY), circles; PS83 (abrB codY), triangles. (C) Strain PS59 was grown in the presence or absence of 1 mM guanosine. Triangles, β-galactosidase activity in the presence of guanosine; circles, in the absence of guanosine. The experiments were performed in duplicate and any variations in data points were <10% of the represented values.
Figure 3
Effect of a relA mutation on induction of _dpp_-lacZ by decoyinine. When cultures in S6C medium reached a turbidity at 600 nm of 0.3 to 0.4 (T0), the cultures were split into two 25-ml cultures and 0.125 ml of either 1N KOH (open symbols) or decoyinine (100 mg/mL in 1N KOH; closed symbols) was added. (A) Strain PS59 (relA +); (B) Strain MRLB10 (relA). The experiments were performed in duplicate and any variations in data points were <15% of the represented values.
Figure 4
Ultraviolet-induced crosslinking of [α-32P] GTP to CodY. (A) CodY (3 μM), overexpressed and purified from Escherichia coli BL21/λDE3, was incubated with 0.1 mM [α-32P] GTP and 0.1, 0.5, 1, 2, and 10 mM unlabelled GTP, ATP, CTP, or UTP and irradiated with UV light. Irradiated samples were analyzed by SDS-PAGE (12% polyacrylamide) and exposed to a phosphorimager screen. (B) Quantitation of results in part (A). Unlabelled GTP, closed squares; unlabelled ATP, closed diamonds; unlabelled CTP, closed circles; unlabelled UTP, closed triangles.
Figure 5
The effects of GTP and ATP on CodY-mediated repression. In vitro transcription reactions were performed with dpp promoter- or veg promoter-containing fragments as the template. The reactions were initiated by adding Bacillus subtilis EςA RNA polymerase (see Materials and Methods). The dpp transcript is 155 n; the veg transcript is 104 n. (A) The dpp promoter fragment (80 nM) was incubated with RNA polymerase (37 nM); CodY-His6 (300 nM) was added as indicated. (B) The veg or dpp promoter fragments (40 nM) were incubated with RNA polymerase (19 nM) and CodY-His6 (180 nM) in the presence of varying concentrations of GTP. (C) Transcription from the dpp promoter (80 nM) by RNA polymerase (37 nM) in the presence of CodY-His6 (300 nM) and varying concentrations of GTP. (D) Quantitation of in vitro transcription results. The data from part (C) and from parallel experiments in which the concentrations of ATP or ATP and GTP were varied were analyzed using the ImageQuant program.
Comment in
- Linking nutritional status to gene activation and development.
Dworkin J, Losick R. Dworkin J, et al. Genes Dev. 2001 May 1;15(9):1051-4. doi: 10.1101/gad.892801. Genes Dev. 2001. PMID: 11331600 Review. No abstract available.
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