Abundant urinary amino acids activate glutamine synthetase-encoding glnA by two different mechanisms in Escherichia coli

ABSTRACT

Growth of uropathogenic Escherichia coli in the bladder induces transcription of glnA which codes for the ammonia-assimilating glutamine synthetase (GS) despite the normally suppressive high ammonia concentration. We previously showed that the major urinary component, urea, induces transcription from the Crp-dependent glnAp1 promoter, but the urea-induced transcript is not translated. Our purpose here was to determine whether the most abundant urinary amino acids, which are known to inhibit GS activity in vitro, also affect glnA transcription in vivo. We found that the abundant amino acids impaired growth, which glutamine and glutamate reversed; this implies inhibition of GS activity. In strains with deletions of crp and glnG that force transcription from the glnAp2 and glnAp1 promoters, respectively, we examined growth and glnA transcription with a glnA-gfp transcriptional fusion and quantitative reverse transcription PCR with primers that can distinguish transcription from the two promoters. The abundant urinary amino acids stimulated transcription from the glnAp2 promoter in the absence of urea but from the glnAp1 promoter in the presence of urea. However, transcription from glnAp1 did not produce a translatable mRNA or GS as assessed by a glnA-gfp translational fusion, enzymatic assay of GS, and Western blot to detect GS antigen in urea-containing media. We discuss these results within the context of the extremely rapid growth of uropathogenic E. coli in urine, the different factors that control the two glnA promoters and possible mechanisms that either overcome or bypass the urea-imposed block of glutamine synthesis during bacterial growth in urine.

IMPORTANCE

Knowledge of the regulatory mechanisms for genes expressed at the site of infection provides insight into the virulence of pathogenic bacteria. During urinary tract infections—most often caused by Escherichia coli—growth in urine induces the glnA gene which codes for glutamine synthetase. The most abundant urinary amino acids amplified the effect of urea which resulted in hypertranscription from the glnAp1 promoter and, unexpectedly, an untranslated transcript. E. coli must overcome this block in glutamine synthesis during growth in urine, and the mechanism of glutamine acquisition or synthesis may suggest a possible therapy.

INTRODUCTION

Urinary tract infections (UTIs) are common bacterial infections, and uropathogenic Escherichia coli (UPEC) causes most UTIs (1–3). During an infection, UPEC strains cycle between the bladder lumen and intracellular vesicles within uroepithelial cells (4). Rapid growth which occurs in both environments is considered a virulence factor and is reflected in gene transcription patterns, such as high transcription of genes for the translational machinery (4–6). Antibiotic treatment is often effective, but continued antibiotic use increases the risk of generating multidrug-resistant organisms that can ultimately impact the outcome of other clinically important diseases that are treated with the same antibiotics (2, 7–9). Alternate or adjunct therapies are urgently needed (2, 4, 10), but potential therapeutic targets have been hard to identify, in part because of an incomplete understanding of virulence. Possible targets include the products of metabolic enzymes that are expressed during an infection, such as glnA, which codes for glutamine synthetase (GS) (11–13).

GS catalyzes ATP-dependent glutamine synthesis from ammonia and glutamate. Multiple mechanisms control GS activity and glnA transcription which is not surprising since GS is at the intersection of carbon and nitrogen metabolism (14, 15). Two mechanisms control GS activity: inhibition of GS activity by amino acids and nucleotides and covalent adenylylation that inactivates GS. In addition, glnA is transcribed from either the Crp-dependent glnAp1 promoter or the GlnG-dependent glnAp2 promoter. An overview of glnA transcription control is presented in the top half of Fig. 1. Low intracellular glutamine activates transcription from glnAp2 and of a set of genes that constitute the Ntr (nitrogen-regulated) response. Such transcription requires the glutamine-sensing GlnD uridylylates the regulatory protein PII (the glnB product) which prevents inhibition of the autophosphorylation of the sensor kinase GlnL and results in phosphorylation of the response regulator GlnG. GlnL and GlnG are products of the glnALG operon. High intracellular glutamine is sensed by GlnD, which via the PII protein results in unphosphorylated GlnL and GlnG and, in the presence of cyclic AMP complexed to the Crp protein, activates transcription from the glnAp1 promoter.

Fig 1.

Overview of glnA regulation and summary of the effects of inhibitory amino acids and urea. The top half schematically shows the regulatory proteins that control transcription of the cyclic-AMP-Crp-dependent glnAp1 promoter in green and the nitrogen limitation-induced GlnG-dependent glnAp2 promoter in yellow. Also shown is the spatial arrangement of the Crp- and GlnG-binding sites in relation to the transcription start sites. High intracellular glutamine―sensed by the glutamine-sensing GlnD (uridylyltransferase/uridylyl-removing enzyme) and acting via GlnB (the regulatory protein PII) and GlnE (adenylyltransferase/deadenylylase)―covalently adenylylates and inactivates GS. Low intracellular glutamine―sensed by GlnD acting through GlnB, GlnL (a sensor kinase), and GlnG (a response regulator)―activates glnA transcription from the glnAp2 promoter (15). Cyclic-AMP-Crp activates glnA transcription from the glnAp1 promoter. Urea increases glnA transcription in ammonia-containing media from the glnAp1 promoter but impairs transcription in nitrogen-limited media from the glnAp2 promoter. Additionally, several amino acids and nucleotides, including many that require glutamine for their synthesis, bind the glutamate- and nucleotide-binding sites and inhibit GS activity. In addition to the metabolites shown, glucosamine-6-phosphate, AMP, and CTP inhibit activity. In this paper, we show that the inhibitory amino acids regulate glnA transcription from either glnAp1 or glnAp2, depending on the presence of urea. In the absence of one regulator, glnA transcription initiates from the promoter controlled by the other regulator. The bottom half schematically summarizes the effects of the amino acids that inhibit GS activity on glnA transcription with and without urea. Black arrows show known factors that control GS activity and glnA transcription; green arrows show the effects of urea identified in our previous work (16), and blue arrows show the transcriptional effects of the amino acids that inhibit GS activity from this paper.

High glnA transcription for E. coli growing in urine is not easily reconciled with current understanding because urinary ammonia is high (>20 mM), and although not a direct regulatory factor, ammonia―a precursor for glutamine synthesis―suppresses glnA transcription, except in urine (13). We have recently shown that the most abundant urinary component, urea, stimulates glnA transcription from the glnAp1 promoter, not the glnAp2 promoter, and the transcript from glnAp1 is not translated (16).

The physiological function of the amino acid inhibition of GS activity has not been established. Four of the five most abundant urinary amino acids―glycine, histidine, serine, and alanine―are known to inhibit GS activity (15), and because alanine and serine inhibit GS activity, we suspect that the fifth amino acid, the three-carbon amino acid cysteine, is also inhibitory. Our goal was to determine whether the abundant urinary amino acids affect glnA transcription and perhaps overcome the urea-dependent block of GS synthesis. We show that the abundant urinary amino acids stimulated glnA transcription from the glnAp2 promoter in the absence of urea and from the glnAp1 promoter in the presence of urea. However, unlike the glnAp2 transcript, the glnAp1 transcript was not translated as determined by results with a translational fusion, GS activity assay, and an immunological assay. The previously known effects of urea and the effects of inhibitory amino acids on GS activity and glnA transcription reported in this work are summarized in the bottom half of Fig. 1.

RESULTS

Abundant urinary amino acids inhibit growth and GS activity

We tested whether the most abundant urinary amino acids, many of which are known to inhibit GS activity, affected glnA transcription. For these experiments, we used a synthetic urine (SU), which contains 10 mM glucose as a carbon source and 10 mM ammonia, is the preferred nitrogen source of E. coli, i.e., supports the most rapid growth of single nitrogen sources in minimal media (MM). We compared growth in SU medium with and without several amino acids at near urinary concentrations (13). SU-5 is an SU medium with amino acids that are likely to inhibit GS activity: glycine, histidine, cysteine, serine, and alanine. SU-10 is an SU-5 medium with leucine, aspartate, threonine, methionine, and glutamate (see Materials and Methods for the amino acid concentrations used). Bacteria grew differently in SU-5 and SU-10 (described below). The bacterial strains were the model UPEC organism UTI89 and a commonly used lab strain W3110. The results were generally similar for both strains, and the results for W3110 are often presented in the supplemental material.

SU medium supported the growth of both strains equally well (Fig. 2). SU-5 medium impaired the growth of UTI89 and eliminated the growth of W3110. SU-10 medium restored the growth of both strains to that from SU medium, although W3110 had an extended lag. (Tables S1 and S2 have the doubling times and final cell densities for all growth experiments.) Glutamine reversed the growth inhibition in SU-5 medium for both strains which implies that the amino acids inhibit GS activity (Fig. 3). Because these inhibitory amino acids bind to the binding site for glutamate (17), we would also expect that glutamate would reverse the growth inhibition, which it did for UTI89 but not for W3110 (Fig. 3). Better glutamate transport by UTI89 could account for this result. Tryptone or casamino acids also improved the growth in SU-5 for both UTI89 and W3110, but the positive effect was only partial for W3110 and occurred after an extended lag phase (K. Urs and L. Reitzer, unpublished data). Urine consists of amino acids in free and peptide forms (13). Compared to free amino acids, dipeptides are transported rapidly into bacterial cells (18). A dipeptide containing an inhibitory amino acid should have the same effect as the amino acids in SU-5. We supplemented SU with the alanyl-leucine (AL) dipeptide (SU-AL), which will generate the inhibitory amino acid alanine after transport and hydrolysis. In SU-AL medium, UTI89 had a long lag phase, and W3110 did not grow (Fig. S1A), and glutamine reversed the growth inhibition (Fig. S1B). In summary, urinary levels of several amino acids hindered growth, and glutamate or glutamine overcame the impairment which implies that the inhibitory amino acids block GS activity.

Fig 2.

Growth of UTI89 and W3110 in SU supplemented with amino acids. Growth of (A) parental UTI89 and UTI89 ΔglnA, and (B) parental W3110 and W3110 ΔglnA in the indicated media. UTI89 was less sensitive to inhibitory amino acids compared to W3110, and UTI89 ΔglnA unexpectedly showed growth in SU-10 medium. The curves are averages of three independent experiments, and the error bars represent the standard deviations. Doubling times are provided in supplemental tables. The curves for growth in SU medium—performed at the same time as growth in SU-5 and SU-10 media—have been previously published (16).

Fig 3.

Glutamate or glutamine alleviated amino acid-induced growth inhibition in SU-5 medium. (A) Growth of parental UTI89 was inhibited in SU-5 medium compared to that in SU medium. Both glutamate and glutamine reversed the growth inhibition, which implies an inhibition of GS activity in SU-5 medium. (B) The parental W3110 did not grow in SU-5 medium. Glutamine, but not glutamate, restored growth. The solid blue curve shows growth in SU medium for comparison. The curves are averages of three independent experiments, and the error bars represent the standard deviations. The curves for growth in SU medium are the same as those shown in Fig. 2 and are shown here for comparison.

As a negative control for growth experiments, we grew ΔglnA derivatives of UTI89 and W3110, and as expected, ΔglnA mutants failed to grow in any media with one notable exception: UTI89 ΔglnA displayed limited growth in SU-10 medium which is a defined medium without glutamine (Fig. 2A and B). We reconstructed UTI89 ΔglnA several times, and the mutants always displayed the same phenotype, and GS activity was undetectable in these mutants. An analysis of this phenotype is beyond the scope of this paper.

The effect of the inhibitory amino acids on transcription of glnA and the Ntr gene nac depends on the presence of urea

To test the effects of the inhibitory amino acids on glnA transcription, we grew strains carrying a plasmid with a transcriptional fusion of the glnA promoter region (glnA has two promoters) to the gene for the green fluorescent protein (gfp). We had previously shown that glnA transcription from this fusion properly responded to nitrogen availability in both W3110 and UTI89—glnA transcription was high in MM-alanine medium (nitrogen limiting) and low in MM-ammonia medium (nitrogen excess)—and those results are presented here for comparison (Fig. 4A and B).

Fig 4.

Effect of amino acids on glnA and nac transcription in UTI89. glnA transcription in the indicated media is shown in (A) and quantified in (B) for parental UTI89. The five most abundant amino acids in urea-containing SU medium induced glnA transcription to an unusually high level. In contrast, the 10 most abundant amino acids induced glnA transcription to a level comparable to that observed from MM-alanine (nitrogen-limited medium) or basal SU. (C) Relative glnA mRNA fold change determined by RT-qPCR for parental UTI89 during growth in the indicated media. Fold change is calculated relative to expression of the transcript during growth in MM-NH₄⁺ (nitrogen-excess medium). Data are averages of two independent experiments each run in triplicate and normalized to the reference gene rpoD. The error bars represent the standard deviation of the ΔCt (critical threshold values between rpoD and glnA). (D) The addition of glutamate represses glnA transcription in SU-10 and SU-5 which is quantified in (B). (E) nac transcription in the indicated media for parental UTI89. nac is not expressed during growth in SU-10 or SU-5. Data for (A), (B), (D), and (E) are averages of three independent experiments, and the error bars represent the standard deviations. Significance was calculated using one-way ANOVA along with Dunnett’s multiple comparisons test; * P ≤ 0.05; *** P ≤ 0.001; **** P ≤ 0.0001. The transcription results for cells grown in MM-NH₄⁺, MM-alanine, and SU media (in panels A, B, C, and E) have been previously published (16) and are necessary controls to show the proper regulation for the transcription assays and the effect of supplemental amino acids.

In ammonia-containing SU and SU-10 media, glnA transcription as assessed with glnA-gfp fusions was four- to sixfold higher, respectively, than in nitrogen replete (ammonia-containing) minimal medium and comparable to the activated level in nitrogen-limited MM-alanine medium for both UTI89 (Fig. 4A and B) and W3110 (Fig. S2A and B). Quantitative reverse transcription PCR gave the same results as the gene fusion experiments for UTI89 and W3110 (Fig. 4C and 2C). Glutamate partially suppressed the induction for both strains grown in SU-10 medium (Fig. 4B and D; Fig. S2B and D).

The results during growth in SU-5 medium were different. For UTI89 grown in SU-5 medium, glnA transcription was 25-fold higher than in nitrogen replete minimal medium and 4- to 5-fold higher than in SU and SU-10 medium. Glutamate reduced this glnA hypertranscription sixfold to the level for cells grown in SU medium (Fig. 4A, B, and D). W3110 did not grow in SU-5 medium (Fig. 3B). We conclude that the amino acids in SU-5 medium were more inhibitory than in SU-10 medium because of (i) greater glnA transcription in SU-5 medium and (ii) a greater suppression of transcription in SU-5 medium by glutamate which reverses the inhibition.

Transcription of the glnALG operon—glnL and glnG code for the sensor kinase and response regulator of the Ntr response—should result in transcription of both regulators and induction of other Ntr genes, such as nac (15). However, nac transcription, assessed with a fusion of the nac promoter to gfp, was not induced in SU, SU-5, and SU-10 media in either UTI89 or W3110 (Fig. 4E; Fig. S2E). Induction in nitrogen-limited minimal medium indicates that the fusion was responding as expected. Either glnL and glnG were not expressed or the Ntr circuitry did not provide the activation signal, i.e., low intracellular glutamine.

We examined the effects on glnA transcription of urea alone, a combination of the five abundant inhibitory amino acids (5AAs) alone, and both urea and the 5AA together in nitrogen-excess minimal medium (ammonia as the nitrogen source), nitrogen-limited minimal medium (alanine as the nitrogen source), and synthetic urine. The 5AAs generally resulted in a longer lag before growth. In minimal ammonia medium (nitrogen excess), urea, and the 5AAs induced transcription 3- and 4-fold, respectively, and the combination induced 14-fold (Fig. 5A and D). In minimal alanine medium (nitrogen limiting), which induces glnA transcription, urea reduced transcription twofold (Fig. 5D), the 5AAs induced threefold, and the combination induced fourfold (Fig. 5B and D). Compared to glnA transcription in SU medium without urea, both urea and the 5AAs induced 3-fold, and the combination induced 20-fold (Fig. 5C and D). The results for W3110 were essentially the same (Fig. S3) and are best seen by comparing Fig. 5D; Fig. S3D. In summary, urea plus the 5AAs resulted in glnA hypertranscription.

Fig 5.

glnA transcription in UTI89 was altered by the abundant urinary amino acids with or without urea. Transcription of glnA in (A) minimal medium with ammonia as the sole nitrogen source (nitrogen excess), (B) minimal medium with alanine as the sole nitrogen source (nitrogen limiting), and (C) SU (contains urea and ammonia) for parental UTI89. (D) Quantitation of results from panels A to C. The inhibitory amino acids induced glnA transcription in all medium types, and the combination of urea and the inhibitory amino acids resulted in glnA hypertranscription. Data are averages of three independent experiments, and the error bars represent the standard deviations. Significance was calculated using one-way ANOVA along with Dunnett’s multiple comparisons test; * P ≤ 0.05; *** P ≤ 0.001; **** P ≤ 0.0001. The results from cells grown in medium with urea and the five most adundant amino acids in panels A, B, and C are the same as those in Fig. 4 and are shown here for comparison. The experiments were performed at the same time. The results in panel D for cells grown without the abundant amino acids have been previously published (16) and are necessary controls to show the proper regulation for the transcription assays and the effect of supplemental amino acids. The results for cells grown in urea with the five most abundant amino acids in panels A and C are the same as those shown in Fig. 4 and are shown as a basis for comparison.

The inhibitory amino acids in urea-containing media affected growth in both the Δcrp and ΔglnG mutants

To determine which promoter initiates glnA transcription in the presence of the inhibitory amino acids, we examined growth in strains without Crp or GlnG, which are required for glnAp1 and glnAp2, respectively. The Δcrp ΔglnG double mutant grew as poorly as ΔglnA strains (Fig. S4), which implies that glnAp1 and glnAp2 are the only promoters and their requirements for Crp and GlnG, respectively, are absolute. As controls, both mutants of both strains were grown in nitrogen-limiting and nitrogen-excess media, and as expected, the ΔglnG mutants failed to grow in the nitrogen-limited medium, but the Δcrp mutant had no growth defect in either glucose-containing minimal medium (Fig. S4E and F).

The ΔglnG mutants grew less well than the parental strains in urea-containing medium (compare the growth curves in Fig. 2 for the wild-type strains to those in Fig. 6A; Fig. S4). This result implies poor transcription from the glnAp1 promoter that results below confirm.

Fig 6.

Growth phenotypes of UTI89 ΔglnG and Δcrp mutants in SU medium. Growth of (A) ΔglnG and (B) Δcrp in the indicated media. Growth of UTI89 mutants is not eliminated but is impaired in the Δcrp mutant. The curves are averages of three independent experiments, and the error bars represent the standard deviations. Doubling times are provided in supplemental tables. The results for cells grown in SU medium without the abundant amino acids have been previously published (16) and are shown as a necessary control.

The Δcrp mutants for both UTI89 and W3110 also grew less well than the parental strains in urea-containing medium (compare the growth curves in Fig. 2 for the wild-type strains to those in Fig. 6B; Fig. S4). A plasmid-containing glnA which was controlled by the lac promoter eliminated the lag for the Δcrp strains which shows that the growth defect is due to low glnA expression (Fig. S4).

Urea determines the glnA transcription start site during growth with the inhibitory amino acids

We examined glnA transcription in ΔglnG or Δcrp mutants which forces transcription from glnAp1 and glnAp2, respectively. Without urea, the 5AAs induced glnA threefold for parental UTI89 and the ΔglnG or Δcrp derivatives (Fig. 7A and C). With urea, the 5AAs induced glnA six-, five-, and sevenfold for UTI89, UTI89 ΔglnG, and UTI89 Δcrp, respectively (Fig. 7B and C). In combination, urea and the 5AAs resulted in hypertranscription in all strains (Fig. 7C). For W3110, induction by urea and the 5AAs was also observed for the parental strain and its ΔglnG and Δcrp mutants, but their combined effect could not be assessed because of failure to grow (Fig. S5). In summary, urea alone and the 5AAs induced glnA transcription, but the combination resulted in hypertranscription.

Fig 7.

glnA transcription in UTI89 ΔglnG and Δcrp mutants. (A) glnA transcription in SU medium without urea. The inhibitory amino acids induced glnA transcription in the parental and mutant strains, but the mutants had a longer lag phase. (B) glnA transcription in SU medium with urea. glnA transcription in medium supplemented with both urea and the inhibitory amino acids was significantly higher than medium containing only one supplement. (C) glnA transcription from parts (A) and (B) is shown graphically. All data are averages of three independent experiments, and the error bars represent the standard deviations. Significance was calculated using one-way ANOVA along with Dunnett’s multiple comparisons test; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. Asterisks in green (increased transcription) and red (reduced transcription) in (C) indicate significance calculated in comparison to transcription from parental UTI89 in the same medium. The results in panel C for cells grown without the abundant amino acids have been previously published (16) and are shown as a necessary control.

To determine the promoters utilized in the ΔglnG and Δcrp mutants with the 5AAs, we used sequence-specific primers (16) and RT-qPCR to determine where transcription began when there were changes in glnA transcription. Changes in glnA transcripts, regardless of the transcription start site, were determined with primers that amplified a segment near the 3′ end of the glnA mRNA. A different pair of primers amplified a segment of glnA mRNA that was only possible from the glnAp1 promoter (16). If the changes in glnA transcripts from the end of the gene parallel changes in glnAp1-specific transcripts, then transcription initiates from the glnAp1 promoter. If not, then transcription initiates from the glnAp2 promoter.

For parental UTI89 and W3110 grown in minimal medium with ammonia and SU medium without urea, the 5AAs increased total glnA transcripts 3.5-fold higher compared to the transcripts from cells grown without the 5AAs, but the glnAp1-specific transcripts were not correspondingly higher which means that transcription initiated from the glnAp2 promoter (Fig. 8A for UTI89 and Fig. S6A for W3110).

Fig 8.

Relative fold change of glnAp1-initiated and total glnA transcripts in Δcrp and ΔglnG derivatives of UTI89. (A) Relative glnAp1-initiated and total glnA transcripts in the indicated media from parental UTI89 with and without the five most inhibitory amino acids. For minimal ammonia medium and SU medium without urea, the inhibitory amino acids induced glnA transcription about 3.5-fold in parental UTI89; both media contain ammonia. However, transcripts from the glnAp1 promoter increased only 1.2- to 1.5-fold, which indicates that most of the transcripts initiated from the glnAp2 promoter. (B) When compared to transcripts from the parental strains, loss of Crp had little effect on either total glnA transcripts or transcripts from glnAp1, which indicates transcription from the glnAp2 promoter. Loss of GlnG had no effect on total glnA transcripts but increased transcripts from the glnAp1 promoter which, as expected, indicates that transcription initiation switched from the glnAp2 to the glnAp1 promoter in the mutant. The data are averages of two independent experiments, each run in triplicate and normalized to the reference gene rpoD. The error bars represent the standard deviation of the ΔCt (critical threshold) values between rpoD and glnA. Significance was calculated using one-way ANOVA along with Dunnett’s multiple comparisons test; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. The results in all panels for cells grown without the supplementary abundant amino acids have been previously published (16) and are shown as necessary controls.

For Δcrp mutants of UTI89 and W3110, the transcripts were compared to those from the parental strains. Neither the total transcripts nor the glnAp1-specific transcripts showed much difference with the addition of the 5AAs (Fig. 8B; Fig. S6B), which means that transcription initiated from the glnAp2 promoter and is consistent with the absence of the activator of the glnAp1 promoter.

For the ΔglnG mutants of UTI89 and W3110, the transcripts were also compared to those from the parental strains. The total transcripts showed no change, but the transcripts from glnAp1 increased threefold (Fig. 8B). In other words, removing glnG resulted in transcription initiation switching from the glnAp2 to the glnAp1 promoter which was expected. Because W3110 did not grow with the 5AAs, there are no corresponding results for W3110. In summary, this transcript analysis confirms the genetic results of this section that the 5AAs can stimulate glnA transcription from either promoter.

Transcription from glnAp1 does not produce a translatable mRNA or glutamine synthetase protein

We have previously shown that urea-induced transcription from glnAp1 did not result in either glnA translation or GS synthesis, in contrast to nitrogen limitation-induced transcription from glnAp2 (16). We tested whether these effects were observed when the inhibitory amino acids induced glnA transcription. When grown in SU, SU-5, and SU-10 media, UTI89 had basal GS activity (Fig. 9A) and did not express glnA from a glnA-gfp translational fusion (Fig. 9B and C). As controls, UTI89 did express GS and showed expression from the translational fusion during growth in a nitrogen-limited minimal medium with alanine as the sole nitrogen source but not in an ammonia-containing minimal medium (Fig. 9A, B, and C). The same results were observed for W3110 (Fig. S7).

Fig 9.

Glutamine synthetase activity and translational expression of glnA. (A) GS activity, (B) transcription from a glnA-gfp translational fusion during growth, and (C) quantified transcription from a glnA-gfp translational fusion for parental UTI89 in the indicated media. GS activity was not detectable, and glnA mRNA was not translated during growth in urine-like conditions, even though glnA was transcribed (see Fig. 4 and 5). The data are averages of three independent experiments, and the error bars represent the standard deviations. Significance was calculated using one-way ANOVA along with Dunnett’s multiple comparisons test; * P ≤ 0.05; ** P ≤ 0.01; **** P ≤ 0.0001.

We also examined GS activity in glnG and crp mutants which are forced to use glnAp1 and glnAp2, respectively. GS activity was not detected in the UTI89 ΔglnG mutant grown in either SU-5 or SU-10 media but was detected in UTI89 Δcrp (Fig. 10A). A Western blot analysis showed that GS protein was present only when GS activity was detected (Fig. 10B). Identical results were observed for W3110 (Fig. S8). In summary, glnA was translated and GS present when glnA was transcribed from the glnAp2 promoter but not when transcribed from the glnAp1 promoter.

Fig 10.

Glutamine synthetase activity and protein expression in UTI89 derivatives. (A) GS activity during growth in SU-10 and SU-5 and (B) representative Western blot images of protein expression during growth in the indicated medium for UTI89 strains. GS activity is higher in the Δcrp strains and is comparable to the induced level in the parental strain during growth in minimal medium with alanine as the sole nitrogen source (nitrogen limiting). GlnA antigen is not detectable in the parental or the ΔglnG strains during growth in urea-containing medium but is readily detectable in the Δcrp strain in which glnA transcription is initiated from the glnAp2 promoter. RpoD transcription levels for all strains are shown as control. The data are averages of three independent experiments, and the error bars represent the standard deviations. Significance was calculated using one-way ANOVA along with Dunnett’s multiple comparisons test; **** P ≤ 0.0001. The results in panel B for cells grown without the supplementary abundant amino acids have been previously published (16) and are shown as necessary controls.

DISCUSSION

The main conclusion is that abundant urinary amino acids that inhibit GS activity stimulate glnA transcription from the glnAp2 promoter in the absence of urea and from the glnAp1 promoter in the presence of urea. These GS-inhibiting amino acids (i) resulted in impaired growth (Fig. 2) which glutamate or glutamine reversed (Fig. 3), (ii) stimulated glnA transcription from either the glnAp1 or glnAp2 promoters by analysis of glnA expression from a glnA-gfp transcriptional fusion for wild-type and mutant strains that are forced to utilize a single glnA promoter (Fig. 4, 5, 7, and 8), and (iii) in the presence of urea, resulted in glnA transcription from the glnAp1 promoter that was not translated and did not produce GS as determined by direct enzymatic assay and Western blot analysis (Fig. 9 and 10).

The 5AA induction of glnA transcription from two different promoters implies different activation mechanisms. Transcription from the glnAp2 promoter and induction of the Ntr response involve a regulatory cascade with the glutamine-sensing GlnD, the regulatory protein GlnB, the sensor kinase GlnL, and the response regulator GlnG. The inhibitory amino acids in the absence of urea induced glnA transcription via glnAp2 which is consistent with low intracellular glutamine—resulting from inhibition of GS activity—and activation via the Ntr regulatory cascade. The mechanism by which the inhibitory amino acids in urea-containing medium induce glnAp1-dependent glnA transcription is not clear. One possibility is that the inhibitory amino acid-induced glutamine limitation results in guanosine tetraphosphate accumulation, which has been recently shown to repress transcription of the DNA gyrase genes (19). Because transcription from glnAp2 requires supercoiled DNA (20–22), the resulting supercoiling relaxation may be sufficient to activate glnAp1-dependent glnA transcription.

Several amino acids have been known to inhibit GS activity by binding to the glutamate substrate (17, 23, 24). The physiological relevance of this regulation has not been established. Our results show that this inhibition is relevant even at the low urinary concentrations of these amino acids. The effect of low concentrations of the inhibitory amino acids is also surprising because glutamate is by far the most abundant intracellular metabolite (25). However, the inhibitory amino acid alanine, when used as the sole nitrogen source, not only induces the Ntr response but also reduces intracellular glutamate (26), and perhaps the inhibitory amino acids collectively have a similar effect and reduce both glutamate and glutamine.

Nitrogen limitation is often defined as the slower growth that results from a single nitrogen source other than ammonia which results in low intracellular glutamine and induction of the Ntr response: nitrogen limitation causes glutamine limitation. We found conditions that resulted in glutamine limitation but did not induce the Ntr response: the presence of the 5AAs which inhibit GS activity in a urea- and ammonia-containing medium. If nitrogen limitation is defined as conditions that induce the Ntr response, then glutamine and nitrogen limitation are not equivalent and can be disassociated.

The effect on glnA transcription of the inhibitory amino acids in UTI89 and W3110, members of phylogenetic groups B2 and A, respectively, was similar but not identical. SU-10 had no effect on UTI89 growth but inhibited W3110 growth, and SU-5 impaired UTI89 growth and eliminated W3110 growth (Fig. 3). Our experience has been that UTI89 and strains of phylogenetic group B2, in general, grow faster in most media than W3110 and strains of phylogenetic group A [e.g., (27)]. Of the five inhibitory amino acids, E. coli can degrade serine, glycine, alanine, and cysteine but not histidine (28, 29). The differential responses between UTI89 and W3110 may result from faster degradation of the inhibitory amino acids by UTI89 that diminishes their intracellular concentration and minimizes their effect on glnA transcription, more rapid transport of glutamate and amino acids degraded to glutamate, e.g., arginine, which can overcome the inhibition, or both.

The results in this paper confirm our recent observation (16) that transcription from glnAp1 does not produce a translatable mRNA (Fig. 9B and C) and does not produce GS protein (Fig. 9A and 10B). These results imply that a glnG mutant, which can only express glnA from the glnAp1 promoter, should be a glutamine auxotroph. However, this is not the case, and ΔglnG mutants can produce detectable GS protein and activity [e.g., (30, 31)]. The results presented here and in a recent publication showing that the glnAp1 transcript is not translated involved growth in urea-containing media (16). In contrast, the results showing GS activity in ΔglnG mutants involved growth in minimal media. We propose that the transcript from glnAp1 is not translated when the conversion of glutamate to glutamine and accompanying ATP utilization are detrimental, e.g., during high osmolarity when glutamate could be used for the synthesis of proline, putrescine, or both, which could facilitate survival (32). In this case, a mechanism that completely prevents transcription from glnAp2, such as transcription from glnAp1 (see Fig. 1), that blocks transcription from glnAp2, would promote survival.

Our primary motivation for this study was to explain high glnA transcription for E. coli grown in urine, and our results showed that both urea and urinary amino acids are factors. The frequency of UTI increases with age and is consistent with lower urea concentrations as women age (33) which could stimulate glnA transcription from the glnAp2 promoter. Furthermore, suggestive evidence indicates a higher nutrient content in urine from individuals with a UTI (27, 34). In other words, the chemical composition of urine from individuals with and without UTI may be different, and higher urinary glutamate or glutamine concentration may stimulate growth. Further studies are required to determine whether the factors that control glutamine synthesis contribute to UPEC virulence.

MATERIALS AND METHODS

Bacterial strains and plasmids

E. coli strains and plasmids used are listed in Table 1.

TABLE 1.

Strains and plasmids

Strain or plasmid	Relevant genotype or description	Source or reference
Strains
UTI89 (wild type)		(35)
UKU1	UTI89ΔglnA::Tn5, kan	(16)
UKU2	UTI89ΔglnG::kan	(16)
UKU3	UTI89ΔglnGΔcrp::kan	(16)
UKU4	UTI89Δcrp::kan	(16)
UKU5	UTI89(pUA139) with empty fusion plasmid	(16)
UKU6	UTI89(pglnAp-gfp) glnA transcriptional fusion	(16)
UKU7	UTI89(pnacp-gfp) nac transcriptional fusion	(16)
UKU8	UTI89(pTEglnA) glnA translational fusion	(16)
UKU9	UKU1(pCA24N) with empty plasmid	(16)
UKU10	UKU1(pCA-glnA) with glnA-containing plasmid	(16)
UKU11	UKU3(pCA24N) with empty plasmid	(16)
UKU12	UKU3(pCA-glnA) with glnA-containing plasmid	(16)
UKU13	UKU4(pCA24N) with empty plasmid	(16)
UKU14	UKU4(pCA-glnA) with glnA-containing plasmid	(16)
W3110 (wild type)	lacL8 lacIq	Laboratory strain
WKU1	W3110ΔglnA::Tn5 (kan)	(16)
WKU2	W3110ΔglnG::kan	(16)
WKU3	W3110ΔglnG Δcrp::kan	(16)
WKU4	W3110Δcrp::kan	(16)
WKU5	W3110(pUA139) with empty fusion plasmid	(16)
WKU6	W3110(pglnAp-gfp) glnA transcriptional fusion	(16)
WKU7	W3110(pnacp-gfp) nac transcriptional fusion	(16)
WKU8	W3110(pTEglnA) glnA translational fusion	(16)
WKU9	WKU1(pCA24N) with empty plasmid	(16)
WKU10	WKU1(pCA-glnA) with glnA-containing plasmid	(16)
WKU11	WKU3(pCA24N) with empty plasmid	(16)
WKU12	WKU3(pCA-glnA) with glnA-containing plasmid	(16)
WKU13	WKU4(pCA24N) with empty plasmid	(16)
WKU14	WKU4(pCA-glnA) with glnA-containing plasmid	(16)

Media and growth conditions

Strains were grown in either minimal medium containing 10.5 g/L K₂HPO₄, 4.5 g/L KH₂PO₄, and 0.05 g/L MgSO₄ which was adjusted to pH 7.0 (36) or basal synthetic urine medium to assess growth, fluorescence, and enzyme activity. The components for SU, SU-5, and SU-10 are listed in Table 2. The buffer was MES (50 mM), and the total mixture was adjusted to pH 6.0. MM was supplemented with 0.15% (wt/vol) nitrogen source―(NH₄)₂SO₄ or alanine. Growth was assayed at OD₆₀₀ on plate readers (Biotek Instruments Inc., VT, USA) as previously described (16). Fluorescence was measured concurrently with growth at an excitation wavelength of 485 nm, and emission recorded at 540 nm (37). Antibiotics, when necessary, were added at the following concentrations, kanamycin 25 µg/mL and chloramphenicol 7.5 µg/mL.

TABLE 2.

Synthetic urine medium components

Component	Concentration in SU	SU-5	SU-10
Urea	250 mM	As SU	As SU
NaCl	100 mM	As SU	As SU
KCl	40 mM	As SU	As SU
Glucose	10 mM	As SU	As SU
Na2HPO4	10 mM	As SU	As SU
(NH4)2SO4	10 mM	As SU	As SU
MgCl2	3 mM	As SU	As SU
CaCl2	3 mM	As SU	As SU
FeSO4	0.005 mM	As SU	As SU
Glycine		2.1 mM	As SU-5
Histidine		1.2 mM	As SU-5
Cysteine		0.6 mM	As SU-5
Serine		0.5 mM	As SU-5
Alanine		0.5 mM	As SU-5
Leucine			0.4 mM
Aspartate			0.2 mM
Threonine			0.2 mM
Methionine			0.1 mM
Glutamate			0.1 mM

Growth kinetics and relative fluorescence calculations

Data from each growth run were exported from the Gen5 software (Biotek Instruments Inc., VT, USA) into Microsoft excel and OD₆₀₀ reads corrected for pathlength. Data from independent experiments were used to plot growth curves using GraphPad Prism (Ver. 9.2.1, GraphPad Software Inc., CA, USA). Specific growth rate, doubling time—in hours, and final cell densities —as colony forming units (CFU) per milliliter were calculated as detailed in reference (16). At the end of each growth assay, cultures were diluted, and 20 µL spotted on Luria-Bertani (10 g tryptone, 10 g NaCl, 5 g yeast extract, pH 7.0) agar plates and incubated at 37°C for 16–18 hours before counting colonies for CFU calculations. Fluorescence reads were corrected for the background fluorescence signal at each time point used for calculating relative fluorescence during the exponential growth phase as described in reference (16). All graphs were generated, and analyses performed using GraphPad Prism (Ver. 9.2.1, GraphPad Software Inc., CA, USA). We note that the experiments of this paper were performed at the same time as those for a previous publication (16). The controls for growth and expression experiments—those that did not involve the inhibitory amino acids—are deliberately the same as those in the previous publication and are noted in the figure legends.

Enzyme activity

Glutamine synthetase activity was determined as previously described (16), and protein estimation was done by the Lowry method (38). Final enzyme activities were recorded as nanomoles of product formed per minute per milligram of protein. All graphs were generated, and analyses performed using GraphPad Prism (Ver. 9.2.1, GraphPad Software Inc., CA, USA).

RNA isolation, cDNA synthesis, and real-time quantitative PCR

These procedures were performed exactly as previously described (16).

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/jb.00376-23.

Supplemental material. jb.00376-23-s0001.pdf.

Tables S1 and S2; Fig. S1 to S8.

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