Activation of the glnA, glnK, and nac promoters as Escherichia coli undergoes the transition from nitrogen excess growth to nitrogen starvation

Abstract

The nitrogen-regulated genes and operons of the Ntr regulon of Escherichia coli are activated by the enhancer-binding transcriptional activator NRI approximately P (NtrC approximately P). Here, we examined the activation of the glnA, glnK, and nac promoters as cells undergo the transition from growth on ammonia to nitrogen starvation and examined the amplification of NRI during this transition. The results indicate that the concentration of NRI is increased as cells become starved for ammonia, concurrent with the activation of Ntr genes that have less- efficient enhancers than does glnA. A diauxic growth pattern was obtained when E. coli was grown on a low concentration of ammonia in combination with arginine as a nitrogen source, consistent with the hypothesis that Ntr genes other than glnA become activated only upon amplification of the NRI concentration.

FIG. 1.

Growth of YMC10 (wild type) and X (amtB::Cam^r) on various concentrations of ammonium sulfate. Overnight cultures grown at 30°C in 0.4% (wt/vol) glucose and 0.2% (wt/vol) ammonium sulfate were washed and resuspended in medium containing 0.4% (wt/vol) glucose and the following concentrations of ammonium sulfate: for YMC10, 0.2 (×), 0.05 (+), 0.01 (⧫), 0.005 (▴), and 0.001% (▾); for X, 0.2 (○), 0.05(□), 0.01 (◊), 0.005 (▵), and 0.001% (▿). OD₆₀₀, optical density at 600 nm.

FIG. 2.

Induction of glnAp and glnKp in response to nitrogen starvation. Isogenic cells, wild type except that they contained either trp::Φ(glnAp-lacZ) (YMC10ApΦ2; +) or trp::Φ(glnKp-lacZ) (YMC10Φ; ○) were grown at 30°C in defined media containing 0.004% tryptophan and 0.4% glucose with either 0.005 (A), 0.01 (B), or 0.2% (C) ammonium sulfate. At the indicated times, samples were removed and assayed for β-galactosidase. Symbols: + and o, growth (optical density at 600 nm [OD₆₀₀]); bars, β-galactosidase expression (white bars, YMC10ApΦ2; grey bars, YMC10Φ). Maximum expression was 2,640 Miller units for YMC10ApΦ2 and 1,680 Miller units for YMC10Φ.

FIG. 3.

Comparison of the activation of glnKp and nacp in response to nitrogen starvation. Isogenic cells, wild type except that they contained either trp::Φ(glnKp-lacZ) (YMC10Φ; ○ and •) or trp::Φ(nacp-lacZ) (YMC10NpΦ; □ and ▪) were grown at 30°C in defined media containing 0.004% tryptophan and 0.4% glucose with either 0.005 (○ and □) or 0.01% (• and ▪) ammonium sulfate. The growth curves in both experiments for the two different strains were identical. At the indicated culture densities, samples were removed and assayed for β-galactosidase. OD₆₀₀, optical density at 600 nm.

FIG. 4.

Immunoblotting analysis of the NRI concentration during growth on ammonia and the transition to nitrogen starvation. Strain YMC10 (wild type) was grown at 30°C on defined medium containing 0.4% glucose and 0.005% ammonium sulfate. Samples were harvested for Western blot analysis at the indicated times. The standard lane (std) contains 6 ng of purified NRI. Each sample lane contains 5 μg of crude protein extract. OD₆₀₀, optical density at 600 nm.

FIG. 5.

Activation of glnA, glnK, and nac promoter fusions in cells growing on glutamine as the sole nitrogen source. Overnight cultures were grown in 0.4% glucose-0.2% glutamine to stationary phase. Cells were diluted into similar medium, except that it contained 0.1% (wt/vol) glutamine, and incubated at 30°C. (A) Expression of β-galactosidase. Symbols: □, glnA-lacZYA; ◊, glnKp-lacZYA; ○, nacp-lacZYA. (B) Growth of the three cultures. Symbols are as in panel A.

FIG. 6.

Transcription from glnAp2 and glnKp by purified components of E. coli. Transcription reaction mixtures contained the indicated supercoiled templates (10 nM each), core RNA polymerase (100 nM), σ⁵⁴ (200 nM), NRII (100 nM), and the indicated concentration of NRI (nanomolar units). Reaction mixtures were incubated in the presence of ATP for the formation of open complexes (A) or in the presence of ATP, CTP, and GTP for the formation of short initiated complexes (B). Complex formation was stopped by addition of heparin, complexes were extended by addition of the missing nucleotide(s), and transcripts were recovered by phenol extraction and ethanol precipitation, subjected to electrophoresis on sequencing gels, and detected by autoradiography as described previously (7). Transcripts were labeled by use of [α-³²P]UTP. Templates pTH8 and pLR100 contained the glnA promoter positioned different distances upstream from the phage T7 terminator in the pTE103 vector. Template pglnK13 contained the glnK promoter positioned upstream from the phage T7 terminator in the pTE103 vector.

FIG. 7.

Growth of E. coli on limiting ammonium sulfate and excess arginine is diauxic. Growth of YMC10 (wild type) at 30°C on defined minimal medium containing 0.4% glucose and 0.001% ammonium sulfate (+) or 0.001% ammonium sulfate and 0.2% arginine (×). OD₆₀₀, optical density at 600 nm.