Regulation of CodY activity through modulation of intracellular branched-chain amino acid pools

Abstract

In several Gram-positive bacterial species, the global transcriptional regulatory protein CodY adjusts the expression of many metabolic genes, apparently in response to changes in the pools of specific metabolites, i.e., the branched-chain amino acids (BCAAs) isoleucine, leucine, and valine (ILV) and the nucleoside triphosphate GTP. CodY not only responds to these metabolites as measured in vitro but also regulates the genes that direct their synthesis. We have constructed a set of strains lacking binding sites for the CodY protein in cis at loci coding for the ILV biosynthetic machinery, effectively overexpressing these genes in an attempt to modulate the ILV input signal to CodY. Metabolite analyses of strains derepressed for genes needed for ILV synthesis revealed more than a 6-fold increase in the valine pool and a 2-fold increase in the isoleucine and leucine pools. Accumulation of the branched-chain amino acids was accompanied by a 24-fold induction of the bkd operon (required for branched-chain fatty acid synthesis) and 6-fold hyperrepression of the CodY-regulated yhdG and yufN genes, demonstrating that CodY perceives intracellular fluctuations in at least one if its input signals. We conclude that changes in the rate of endogenous ILV synthesis serve as an important signal for CodY-mediated gene regulation.

FIG. 1.

Key regulatory elements control ilv and ybgE gene expression. Locations of the TnrA binding site (TBS), catabolite response element site (cre), and CodY binding sites (low-affinity CBS I and high-affinity CBS II) are indicated as black and gray squares. The terminator stem-loop in the ilvB operon 5′ untranslated region is depicted as a lollipop structure. Experimentally determined transcription start sites are indicated by +1 and an arrow, and predicted translational start sites are indicated by the initiation codon and an asterisk.

FIG. 2.

Overexpression of branched-chain amino acid biosynthetic genes results in overproduction of isoleucine, leucine, and valine. Samples for intracellular metabolite analysis were acquired during exponential phase when cells were at steady state. Cells from strain SRB4 (ilvp⁺ ybgEp⁺ codY⁺), strain SRB10 (ilvp⁺ ybgEp⁺ codY), and strain SRB194 (ilvBp4 ilvBpΔT2 ybgEpΔCBS3 ilvDpΔCBS) were rapidly washed and quenched, and metabolites were extracted. Extracts were analyzed by tandem gas chromatography-time-of-flight mass spectrometry (GC-TOF). White, hatched, and gray bars denote intracellular pools of isoleucine, leucine, and valine, respectively. Asterisks denote observations statistically different relative to those for strain SRB4 (P < 0.05 by Student's two-tailed t test). Error bars indicate standard errors of the means.

FIG. 3.

Growth behavior analysis of strains in TSS glucose-ammonium medium. Circles, wild type (SRB4; ilvp⁺ ybgEp⁺ codY⁺); triangles, CodY-proficient, branched-chain amino acid overproducer strain (SRB194; ilvBp4 ilvBpΔT2 ybgEpΔCBS3 ilvDpΔCBS codY⁺); squares, CodY-deficient, branched-chain amino acid overproducer strain SRB10 (ilvp⁺ ybgEp⁺ codY). Data are representative of at least three independent experiments.

FIG. 4.

Endogenously produced branched-chain amino acids trigger repression of the CodY-dependent yhdG promoter. Results of β-galactosidase activity assays of yhdG-lacZ fusions integrated ectopically at the amyE locus are shown. All strains harbor the lacA::tet and yesZ::ble alleles, which eliminate endogenous β-galactosidase activity. Samples were taken during steady-state exponential-phase growth in minimal glucose-ammonium medium (white bars) or in the same medium supplemented with 200 μg ml⁻¹ valine (Val) (hatched bars) or a mixture of isoleucine, leucine, and valine (ILV) (gray bars) (each at 200 μg ml⁻¹). (A) Wild-type (WT) fusion integrated into ilvp⁺ ybgEp⁺ strain SRB206 or branched-chain-amino-acid-overproducing (O.P.) strain SRB212. (B) Mutant yhdG fusion containing two point mutations that substantially reduces CodY-dependent regulation. The fusion was integrated into ilvp⁺ ybgEp⁺ strain SRB221 or branched-chain-amino-acid-overproducing (O.P.) strain SRB223. Error bars indicate standard errors of the means.

FIG. 5.

Endogenously produced branched-chain amino acids repress the yufN promoter. Results of β-galactosidase activity assays of yufN-lacZ fusions integrated ectopically at the amyE locus are shown. All strains harbor the lacA::tet and yesZ::ble alleles, which eliminate endogenous β-galactosidase activity. Samples were taken during steady-state exponential phase growth in minimal glucose-ammonium medium (white bars) or in the same medium supplemented with 200 μg ml⁻¹ valine (Val) (hatched bars) or a mixture of isoleucine, leucine, and valine (ILV) (gray bars) (each at 200 μg ml⁻¹). WT, ilvp⁺ ybgEp⁺ strain SRB207; O.P., branched-chain-amino-acid overproducer strain SRB213. Error bars indicate standard errors of the means.

FIG. 6.

Phenotypes associated with endogenous or exogenous amino acids. Data are presented as mean copies of ptb transcript per 10⁶ copies of rrnA transcript. Error bars denote standard errors of the means from at least two independent experiments. When error bars are not visible, they are smaller than the edge of the symbol. (A) Quantitative real-time RT-PCR analysis of ptb gene expression. Cells were cultivated in minimal glucose-ammonium medium, and samples were taken during exponential phase. WT, strain SRB4; O.P., denotes branched-chain-amino-acid overproducer strain SRB194. Val and ILV indicate inclusion of 200 μg ml⁻¹ valine or a mixture of 200 μg ml⁻¹ each of isoleucine, leucine, and valine in minimal glucose-ammonium medium. (B) Dose-dependent ptb gene expression. Strain SRB4 (ilvp⁺ ybgEp⁺ codY⁺) was cultivated in minimal glucose-ammonium medium. Valine was titrated using 0, 0.125, 0.250, 0.500, 1.0, 1.7, 2.0, 4.0, and 8.0 mM valine. Samples were taken during exponential-phase growth.

FIG. 7.

Model for CodY-mediated signal processing. The CodY global regulatory protein monitors branched-chain amino acid and GTP pools, indicators of nutritional and energy sufficiency. Branched-chain amino acids and GTP are subject to multiple levels of local and global regulation. Under steady-state conditions, a stable flow of metabolites reflects the status of the synthesis and regulatory machinery. CodY manages these input signals to produce a single output signal whereby constituent promoters are stimulated or repressed to allow the cell to adapt to the current conditions. FBP, fructose-1,6-bisphosphate; G6P, glucose-6-phosphate; Hpr∼P, activated (phosphorylated) general phosphotransferase system (PTS) protein (15); BCFAs, branched-chain fatty acids; (p)ppGpp, guanosine (penta)tetraphosphate; GS, glutamine synthetase; Rel_Bsu, combined GTP pyrophosphokinase/guanosine-3′,5′-bispyrophosphate 3′-pyrophosphohydrolase (63).