Role of GlnR in Controlling Expression of Nitrogen Metabolism Genes in Listeria monocytogenes

Rajesh Biswas; Abraham L Sonenshein; Boris R Belitsky

doi:10.1128/JB.00209-20

. 2020 Sep 8;202(19):e00209-20. doi: 10.1128/JB.00209-20

Role of GlnR in Controlling Expression of Nitrogen Metabolism Genes in Listeria monocytogenes

Rajesh Biswas ^a,^*, Abraham L Sonenshein ^a, Boris R Belitsky ^a,^✉

Editor: Tina M Henkin^b

PMCID: PMC7484187 PMID: 32690554

L. monocytogenes is a widespread foodborne pathogen. Nitrogen-containing compounds, such as the glutamate-containing tripeptide, glutathione, and glutamine, have been shown to be important for expression of L. monocytogenes virulence genes. In this work, we showed that a transcriptional regulator, GlnR, controls expression of critical listerial genes of nitrogen metabolism that are involved in ammonium uptake and biosynthesis of glutamine and glutamate. A different mode of GlnR-mediated regulation was found for each of these three pathways.

KEYWORDS: GlnR, Listeria monocytogenes, amtB, glnK, glutamate dehydrogenase, glutamate synthase, glutamine synthetase

ABSTRACT

Listeria monocytogenes is a fastidious bacterial pathogen that can utilize only a limited number of nitrogen sources for growth. Both glutamine and ammonium are common nitrogen sources used in listerial defined growth media, but little is known about the regulation of their uptake or utilization. The functional role of L. monocytogenes GlnR, the transcriptional regulator of nitrogen metabolism genes in low-G+C Gram-positive bacteria, was determined using transcriptome sequencing and real-time reverse transcription-PCR experiments. The GlnR regulon included transcriptional units involved in ammonium transport (amtB glnK) and biosynthesis of glutamine (glnRA) and glutamate (gdhA) from ammonium. As in other bacteria, GlnR proved to be an autoregulatory repressor of the glnRA operon. Unexpectedly, GlnR was most active during growth with ammonium as the nitrogen source and less active in the glutamine medium, apparently because listerial cells perceive growth with glutamine as a nitrogen-limiting condition. Therefore, paradoxically, expression of the glnA gene, encoding glutamine synthetase, was highest in the glutamine medium. For the amtB glnK operon, GlnR served as both a negative regulator in the presence of ammonium and a positive regulator in the glutamine medium. The gdhA gene was subject to a third mode of regulation that apparently required an elevated level of GlnR for repression. Finally, activity of glutamate dehydrogenase encoded by the gdhA gene appeared to correlate inversely with expression of gltAB, the operon that encodes the other major glutamate-synthesizing enzyme, glutamate synthase. Both gdhA and amtB were also regulated, in a negative manner, by the global transcriptional regulator CodY.

IMPORTANCE L. monocytogenes is a widespread foodborne pathogen. Nitrogen-containing compounds, such as the glutamate-containing tripeptide, glutathione, and glutamine, have been shown to be important for expression of L. monocytogenes virulence genes. In this work, we showed that a transcriptional regulator, GlnR, controls expression of critical listerial genes of nitrogen metabolism that are involved in ammonium uptake and biosynthesis of glutamine and glutamate. A different mode of GlnR-mediated regulation was found for each of these three pathways.

INTRODUCTION

Listeria monocytogenes is an important and deadly foodborne pathogen that mostly affects pregnant women, newborns, the elderly, and immunocompromised individuals (1). L. monocytogenes is also a model intracellular pathogen that has proven to be a highly productive model for the discovery of many important principles of infection and immunity (2 –4).

Metabolic processes are important for growth and virulence of all pathogenic bacteria. Despite the large amount of information about L. monocytogenes, our knowledge about its central metabolism is still limited. Specifically, very little data are available on the regulation of L. monocytogenes nitrogen metabolism. This is despite the recent observation that PrfA, the master regulator of listerial virulence, is activated by a tripeptide, glutathione (5 –7). Moreover, PrfA activity is affected by the nature of the nitrogen source in the growth medium and is elevated in the glutamine medium (8).

Two related transcriptional regulators, TnrA and GlnR, and the global transcriptional regulator CodY are responsible for the regulation of many genes of nitrogen metabolism in a model low-G+C Gram-positive bacterium, Bacillus subtilis (9, 10). The L. monocytogenes CodY regulon and the role of CodY in virulence have been characterized in some detail (11 –14). However, no genome-wide knowledge of the roles of TnrA or GlnR homologs in regulation of gene expression and virulence exists in L. monocytogenes.

TnrA and GlnR form a separate protein family that is related to the MerR family of transcriptional regulators and is widespread in Bacilli, a class of low-G+C Gram-positive bacteria (15). In B. subtilis, TnrA is a global regulator controlling dozens of genes and operons, many of which are involved in nitrogen metabolism (9, 16, 17), whereas GlnR has a much smaller regulon of 4 confirmed genes or operons, all of which are involved in nitrogen metabolism (18 –20).

Both TnrA and GlnR are unusual regulators because their activity is modulated via the interaction with an enzyme, glutamine synthetase (GS) (15, 21, 22). GS, which synthesizes glutamine from ammonium and glutamate, is the major enzyme of ammonium assimilation in many bacterial species and the only enzyme that generates glutamine, a proteinogenic amino acid that serves as a precursor for multiple important metabolic pathways. In its feedback-inhibited state, GS binds stably to and sequesters TnrA, preventing its ability to interact with DNA, and serves as a chaperone to allow GlnR dimerization and promote its binding to DNA (15, 21, 22). Thus, B. subtilis GlnR is active, as a repressor, under conditions of nitrogen excess when GS and TnrA are inactive. In contrast, B. subtilis TnrA is active, as a positive or negative regulator, under conditions of nitrogen limitation, which induces high activity of GS and inactivates GlnR.

Although B. subtilis contains both TnrA and GlnR, the genomes of most Bacilli carry only one gene that is similar to tnrA or glnR. These genes are orthologous to glnR because they are cotranscribed with the glnA gene and are more similar in sequence to glnR than to tnrA. However, it is unclear whether the function of GlnR in such bacteria is similar to the limited role of B. subtilis GlnR or is expanded to include the more extensive functions of B. subtilis TnrA. The latter possibility is made more plausible by the virtual identity of the binding sites for TnrA and GlnR. The binding motif for both proteins has been described as TGTNAN₇TNACA or a related version of this sequence, and it is not even clear what determines the specificity of binding of these two proteins when both of them are present in the same cell (15 –17, 23). Because the 17-nucleotide (nt) binding motif is enriched in A and T nucleotides, its predictive power is not sufficient to identify reliably TnrA/GlnR-binding sites in the genomes of low-G+C (and, therefore, high-A+T) Gram-positive bacteria. The complete GlnR regulon in bacteria that lack TnrA has been experimentally determined only in two bacteria of the order Lactobacillales, Lactococcus lactis and Streptococcus pneumoniae (24, 25). Only 3 targets of GlnR were found in each case, suggesting that GlnR in these organisms does not have the global functions of B. subtilis TnrA. Despite this limited regulatory potential, S. pneumoniae GlnR is important for virulence development, at least at some sites of infection (26).

L. monocytogenes is another low-G+C Gram-positive species that has a single GlnR/TnrA-like protein encoded by its genome (27, 28). However, L. monocytogenes is a member of the order Bacillales, i.e., it is much closer evolutionarily to B. subtilis than to L. lactis or S. pneumoniae. In this work, we characterized in L. monocytogenes the physiological effects of GlnR and nitrogen sources in the growth medium on expression of genes affecting nitrogen metabolism and virulence. Three major targets of GlnR were found, with a different mode of GlnR action for each of them. Some of the GlnR-regulated genes were also controlled by CodY, a global regulator of metabolism and virulence in most low-G+C Gram-positive bacteria (29, 30).

RESULTS

Genome-wide analysis of gene expression.

We have used transcriptome sequencing (RNA-Seq) (see Materials and Methods) to establish the genome-wide role of L. monocytogenes GlnR in gene expression. An in-frame deletion in the L. monocytogenes glnR gene (LMRG_RS06455, LMRG_0748) was created in strain 10403S (in strain EGD-e, the corresponding gene’s locus tag is lmo1298). Wild-type and glnR null mutant strains were grown in LDM, a defined medium with ammonium or glutamine as the principal nitrogen source (31). LDM contains 7 amino acids, Cys, Met, Ile, Val, Leu, Arg, and His, that are required or are stimulatory for growth. Some of these amino acids, e.g., methionine and branched-chain amino acids, can serve as nitrogen sources (32), but they cannot support growth of L. monocytogenes cells if ammonium or glutamine is not present. Although some earlier reports indicated that L. monocytogenes cells cannot utilize ammonium as the principal nitrogen source, it is now known that ammonium is in fact the preferred nitrogen source for at least some strains of L. monocytogenes (32).

Differential gene expression was analyzed by DESeq2 (33), and genes were considered to be differentially expressed if the difference in their expression levels under any two conditions was ≥2.5-fold with an adjusted P value of <0.05. Only 4 genes, glnR, glnA, arsB (LMRG_RS06465, lmo1300), and amtB, and one S-adenosyl methionine (SAM) riboswitch sequence (upstream of the LMRG_RS08380 [lmo1681] gene) were found to be differentially expressed in the wild-type strain versus the glnR mutant in the ammonium medium, and only 5 genes, amtB, glnK, gdhA, LMRG_RS07555 (lmo1518), and LMRG_RS06480 (lmo1303), were found to be differentially expressed in the glutamine medium (Table 1; see also Data Sets S1A and B in the supplemental material). The L. monocytogenes glnR gene is located upstream of the glnA gene that encodes GS (Fig. 1A). The open reading frames of the glnR and glnA genes are separated by only 73 bp and likely form a single transcriptional unit, as in other bacteria (34) (Fig. S1). The arsB gene, encoding a predicted arsenic efflux pump, is located 146 bp downstream from glnA and is apparently cotranscribed, in part, with the glnRA operon. Indeed, the analysis of the Illumina read distribution in the glnA-arsB intergenic region using a genome browser revealed readthrough from the actively expressed glnA gene in addition to a transcription initiation event upstream of the arsB coding region (Fig. S1). Similarly, LMRG_RS07555, a gene with unknown function, is apparently cotranscribed, in part, with the amtB glnK operon (the two corresponding intergenic regions are 16 and 153 bp long, respectively) (Fig. 1B). The amtB gene encodes a high-affinity ammonium transporter, and the glnK gene encodes a regulatory protein of the PII family that interacts with AmtB and affects its function (35, 36). The gdhA gene encodes glutamate dehydrogenase; the sequence of the protein is similar to those of anabolic enzymes that catalyze glutamate formation from ammonium and 2-oxoglutarate.

TABLE 1.

Genes differentially affected by GlnR as detected by RNA-Seq

Gene name	Function	Expression fold change glnR mutant/wild type in^a :
Gene name	Function	Ammonium medium	Glutamine medium
glnA	Glutamine synthetase
arsB	Predicted arsenical efflux pump	3.5	2.1
Riboswitch id39	SAM riboswitch	3.0	1.6
glnR	Transcriptional regulator	2.8	−2.2
amtB	Ammonium transporter	2.5	−19.0
glnK	PII family protein	2.0	−19.0
LMRG_RS07555	Hypothetical protein	1.2	−7.4
gdhA	Glutamate dehydrogenase	1.1	12.0
LMRG_RS06480	Cell division checkpoint protein	1.7	2.6

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Cells were grown in the LDM medium with ammonium or glutamine, as indicated.

FIG 1 — Organization of the *L. monocytogenes* *glnRA* (A) and *amtB glnK* (B) operons.

Thus, only 3 to 5 transcription units (8 genes) are under significant GlnR regulation in L. monocytogenes under the growth conditions tested. Three of them, the glnRA and amtB glnK operons and the gdhA gene, are involved in critical pathways of nitrogen metabolism and have been described as GlnR targets in other bacteria (24, 25, 37, 38). Clearly, L. monocytogenes GlnR controls a very limited number of genes and does not have the pleiotropic functions of B. subtilis TnrA.

The glnR and amtB genes contain perfect 17-nt GlnR-binding motifs, TGTNA-N₇-TNACA, in their regulatory regions (the amtB gene also contains a second sequence with one mismatch to the canonical motif); in contrast, the gdhA regulatory region contains only sequences that have at least two mismatches with respect to the canonical motif (Fig. 2).

FIG 2 — Sequence and organization of the *L. monocytogenes* *glnR* (A), *amtB* (B), and *gdhA* (C) regulatory regions. The coordinates of the 5′ and 3′ ends of the sequence with respect to the +1 position (transcription start point) of the corresponding genes (84) are indicated; the locations of the upstream cloning sites for the full-length and truncated *glnR88* constructs are shown by vertical arrows. The +1 gene positions, the likely initiation codons, and the termination codons of the preceding genes are in boldface; the −10 and −35 promoter regions are underlined. The directions of transcription and translation are indicated by the arrows. The 17-bp dyad symmetry GlnR-binding motifs are shown in red. The core CodY-binding sites, comprising nucleotides essential for CodY binding *in vitro* as determined by IDAP-Seq (14), are shown in blue (the exact lengths of the *amtB* CodY-binding site [starts at position −59] and the upstream *gdhA* CodY-binding site [ends at position −52] are not known; they are shown as 10-nt sequences extending from the known boundary). The sequence corresponding to the noncoding RNA, *rli32*, in the *gdhA* regulatory region (85) is underlined and italicized.

A small number of additional genes were regulated by GlnR from 2- to 2.5-fold (Data Set S1). None of these genes contained a strong GlnR-binding motif in their regulatory regions, suggesting that their regulation, if significant, is indirect. Some of these genes, including genes of the purine and glycine cleavage operons and the aroA and pbuO genes, may be coregulated by the PurR protein, as they all have a potential B. subtilis-like PurR-binding motif in their upstream regulatory regions (39 and data not shown).

Despite the small size of the L. monocytogenes GlnR regulon, the RNA-Seq analysis produced several surprising results. Unexpectedly, the glnA gene was subject to strong, 10-fold repression by GlnR in the ammonium medium (Table 1), although GlnR was predicted, based on studies in B. subtilis (20), to be inactive in this medium. In the glutamine medium, in which repression was expected to be most efficient, the glnA gene was subject to a much weaker, ∼2-fold, negative regulation by GlnR. (Lower expression and regulation of the glnR gene, the first gene of the glnRA operon [Fig. 1A], in the glnR mutant strain reflects deletion of a major part of the gene [Table 1 and Fig. S1].)

The gdhA gene was also subject to negative, 12-fold regulation by GlnR. However, this effect was observed surprisingly only in the glutamine medium, in which the expression of the glnRA operon was only mildly affected by GlnR; no GlnR effect on gdhA expression was observed in the ammonium medium (Table 1).

Finally, the amtB glnK operon was subject to strong, 19-fold, positive GlnR-mediated regulation in the glutamine medium despite the common notion of GlnR acting as a repressor. Moreover, in the ammonium medium, the operon was subject to 2- to 2.5-fold negative regulation by GlnR (Table 1).

Based on the assumption that the main function of GlnR is negative autoregulation of the glnRA operon, we conclude that GlnR is active in the ammonium medium and inactive or less active in the glutamine medium. Importantly, the high level of expression of the glnR gene, which is cotranscribed with glnA, apparently leads to accumulation of GlnR in a wild-type strain in the glutamine medium.

The summary of average FPKM (the number of fragments, i.e., paired reads, per kilobase of transcript per million mapped fragments) values used for the analysis of differential expression in the RNA-Seq experiments is presented in Table 2. It shows that the expression of the glnRA and amtB glnR operons in the wild-type strain increased when ammonium was replaced by glutamine as the principal nitrogen source in the defined LDM medium, and gdhA expression followed the opposite pattern. Virtually no medium-mediated regulation of the glnA, amtB, and gdhA genes was observed in the glnR mutant strain, indicating that GlnR is the main regulator of these genes that responds to the nature of the nitrogen source (Table 2). The complex regulation patterns suggested that different genes respond to GlnR in different ways.

TABLE 2.

Expression of the L. monocytogenes genes as determined by RNA-Seq

Relevant genotype	Target gene	Expression in^d :
		Ammonium medium		Glutamine medium
		FPKM^a	%^b	FPKM^a	%^b
Wild type	glnA	775	12	6,310	100
glnR		10,800	170	14,300	230
Wild type	glnR	344	13	2,690	100
glnR		1,170^c	43	1,340	50
Wild type	amtB	16.7	1.0	1,600	100
glnR		53.7	3.4	73.8	4.6
Wild type	glnK	16.4	0.9	1,790	100
glnR		46.4	2.6	71.6	4.0
Wild type	gdhA	759	1,400	53.5	100
glnR		819	1,500	1,350	2,500
Wild type	gltA	9.46	1.9	505	100
glnR		9.30	1.8	1,140	230

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The data are presented as average FPKM values multiplied by 1,000.

Expression values in a wild-type strain in the glutamine medium were treated as 100%. The glnR/wild-type expression ratios are different from those in Table 1 due to the specifics of the DESeq analysis.

Expression values underestimate glnR expression and regulation due to the deletion of a major part of the gene.

Cells were grown in the LDM medium with ammonium or glutamine, as indicated.

Nitrogen source-mediated regulation of gene expression.

Surprisingly, only a relatively small number of genes, 43, were differentially regulated, ≥2.5-fold, in wild-type cells if ammonium was replaced by glutamine as the principal nitrogen source in the medium: 24 genes were more highly expressed and 19 genes were less highly expressed (Table 3 and Data Set S2A). As follows from the data presented above, we found that almost all GlnR-regulated genes were among the genes differentially expressed under these conditions (7 out of 8; one more gene, arsB, was also affected but only 2.1-fold); expression of the amtB glnK operon was the most affected one under these conditions (Table 3). Five genes of the PrfA regulon, inlC, hpt, mpl, actA, and plcB, were more highly expressed, up to 4.3-fold, in the glutamine medium than in the ammonium medium, as reported recently (8). Among the remaining 31 genes, the highest (50- to 51-fold) induction in the glutamine medium was observed for the gltAB operon, encoding the two subunits of glutamate synthase. Another operon, pdxST, encoding PLP synthase, was induced 5-fold. Five genes of the tryptophan (trp) operon, two genes of the glycine cleavage operon, gcvPA and gcvPB, and the glmS gene (glutamine:fructose-6-phosphate amidotransferase, involved in hexosamine biosynthesis) were also among other positively affected genes in the glutamine medium. Interestingly, glutamine is a substrate of amidotransferases, encoded by the gltA, pdxT, trpE, and glmS genes. Genes of the arpJ, ilv, and flagella-hemotaxis operons also were downregulated from 2- to 2.5-fold in the glutamine medium (in total, 106 genes were differentially expressed if the cutoff was reduced to 2-fold; 25 of them were also GlnR regulated at least 2-fold, likely indirectly in most cases, under one of the growth conditions) (Data Sets S1 and S2).

TABLE 3.

Genes differentially affected by the nitrogen source of the medium, as detected by RNA-Seq

Gene name	Function	Expression fold change glutamine/ammonium^a
Gene name	Function	Wild type	glnR
glnK	PII family protein	84.0	1.6
amtB	Ammonium transporter	83.0	1.5
gltA	Glutamate synthase, large subunit	51.0	93.0
gltB	Glutamate synthase, small subunit	50.0	88.0
LMRG_RS07555	Hypothetical protein	13.0	1.3
glnA	Glutamine synthetase	9.4	1.5
glnR	Transcriptional regulator	8.7	1.3
pdxS	PLP synthase, large subunit	5.3	4.6
LMRG_RS14815	Hypothetical protein	5.1	4.5
pdxT	PLP synthase, small subunit	4.5	4.3
inlC	Internalin C	4.3	3.5
hpt (uhpT)	Hexose-6-phosphate:phosphate antiporter	3.7	2.0
mpl	Zinc metalloproteinase	3.2	2.6
glmS	Glutamine:fructose-6-phosphate	3.2	3.0
	Amidotransferase
trpG	Tryptophan biosynthesis enzyme	3.1	1.9
trpE	Tryptophan biosynthesis enzyme	3.1	1.9
LMRG_RS14585	Hypothetical protein	2.9	2.1
trpD	Tryptophan biosynthesis enzyme	2.9	2.0
trpC	Tryptophan biosynthesis enzyme	2.7	2.1
actA	Actin assembly inducing protein	2.6	2.7
plcB	Phospholipase C	2.6	2.6
gcvPB	Glycine dehydrogenase subunit	2.6	3.0
trpB	Tryptophan biosynthesis enzyme	2.5	2.0
gcvPA	Glycine dehydrogenase subunit	2.5	3.0
LMRG_RS06480	Cell division checkpoint protein	−2.6	−1.6
LMRG_RS11085	Sortase B	−3.0	−1.5
LMRG_RS10625	Hypothetical protein	−3.1	−2.0
cheV	Chemotaxis protein	−3.2	−2.5
LMRG_RS14065	ABC transporter ATP-binding protein	−3.4	−2.5
gdhA	Glutamate dehydrogenase	−7.6	1.8
orfY	Virulence locus gene	2.0	3.1
orfX	Virulence locus gene	2.4	2.8
LMRG_RS14570	Hypothetical protein	2.3	2.8
gcvT	Aminomethyltransferase	2.3	2.8
purC	Purine biosynthesis enzyme	1.5	2.7
purS	Purine biosynthesis enzyme	1.4	2.5
LMRG_RS01245	Mini-ribonuclease 3	−1.6	−2.6
LMRG_RS03435	Flagellum/chemotaxis locus	−1.5	−2.6
LMRG_RS03430	Flagellum/chemotaxis locus	−1.9	−2.6
LMRG_RS03455	Flagellum/chemotaxis locus	−2.3	−2.6
LMRG_RS03450	Flagellum/chemotaxis locus	−2.2	−2.7
LMRG_RS03420	Flagellum/chemotaxis locus	−2.1	−2.7

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Cells were grown in the LDM medium with ammonium or glutamine.

Similar results were obtained when we compared gene expression in cells of wild-type and glnR null mutant strains grown in the ammonium and glutamine media (with the exception of the GlnR-regulated genes, indicating that their response to the nitrogen source in the medium is mediated by GlnR) (Table 3 and Data Set S2B). In the glnR mutant, 18 genes were found to be upregulated in the glutamine medium and 8 genes were negatively regulated. Some genes of the purE and cysE operons were found only in the glnR data set of differentially expressed genes (also among genes regulated from 2- to 2.5-fold); these genes were not differentially regulated in a wild-type strain under these conditions of growth (Data Set S2). The glutamine uptake operon glnPQ, which is needed for the activation of PrfA in the glutamine medium (8), and other CcpC-regulated genes, such as citB and citC, known to be negatively regulated by the presence of glutamine (8, 40, 41), were downregulated in the glutamine medium, in a GlnR-independent manner, but only by 1.5- to 1.9-fold (data not shown).

Several prophage genes and the genes of the monocin locus (28, 42, 43) appeared to be downregulated at least 2.5-fold in the glutamine medium, primarily in a wild-type strain; many additional genes of these loci were regulated negatively 2- to 2.5-fold (Data Set S2). Our manual analysis of the RNA-Seq experiments revealed that differences of a similar magnitude were seen, in a coordinate manner, even in biological replicates (Data Set S2). This observation may reflect an uncontrollable induction of these genes under the conditions of growth used, perhaps due to approaching stationary phase in some of our samples (44). Because these differences may have no relevance to or correlation with the availability of ammonium or glutamine or GlnR activity, we excluded 13 such genes from Table 3.

Transcriptional regulation of the glnA, amtB, and gdhA genes by GlnR and CodY as determined by real-time RT-PCR.

Using real-time RT-PCR experiments, we have confirmed and extended the results obtained by RNA-Seq analysis. To reveal expression of the glnRA operon, we used primers for the glnA gene, because expression of the glnR gene was altered by the nature of the deletion. The effects of a glnR null mutation and medium composition on expression of the glnA, amtB, and gdhA genes were virtually identical to those obtained with RNA-Seq (Tables 4 to 6, strains 10403S and RB10).

TABLE 4.

Expression of the glnA gene as determined by real-time RT-PCR

Strain	Relevant genotype	Expression in^c :
		Ammonium medium		Glutamine medium
		RNA amount^a	%^b	RNA amount	%
10403S	Wild type	0.90 ± 0.19	9.1	9.91 ± 1.7	100
RB10	glnR	12.8 ± 2.6	130	23.4 ± 5.9	240
TW84	codY	0.91 ± 0.02	9.2	15.3 ± 2.9	150
RB11	codY glnR	14.3 ± 10.0	140	18.9 ± 3.7	190
RB16	glnR/glnR⁺p⁺	0.98 ± 0.19	9.9	14.9 ± 5.0	150
RB17	glnR/glnR88p⁺	4.46 ± 0.72	45	6.90 ± 2.6	70
RB18	glnR/glnR⁺p93	0.80 ± 0.12	8.1	8.41 ± 4.0	85
EGD-e	Wild type	0.51 ± 0.14	4.6	11.1 ± 4.6	100
RB15	glnR	13.5 ± 1.9	120	18.5 ± 7.7	170

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The data are presented as the number of copies of the transcript per copy of the rpoC transcript ± standard deviations, as determined by real-time RT-PCR. All values are averages from at least two experiments.

Expression values in a wild-type strain in the glutamine medium were treated as 100%.

Cells were grown in the LDM medium with ammonium or glutamine, as indicated.

TABLE 5.

Expression of the amtB gene as determined by real-time RT-PCR

Strain	Relevant genotype	Expression in^c:
		Ammonium medium		Glutamine medium
		RNA amount^a	%^b	RNA amount	%
10403S	Wild type	0.06 ± 0.02	0.7	8.07 ± 3.2	100
RB10	glnR	0.16 ± 0.04	2.0	0.30 ± 0.13	3.7
TW84	codY	0.16 ± 0.07	2.0	9.87 ± 4.1	120
RB11	codY glnR	1.05 ± 0.62	13	1.49 ± 0.09	18
RB16	glnR/glnR⁺p⁺	0.07 ± 0.02	0.9	11.3 ± 5.8	140
RB17	glnR/glnR88p⁺	7.37 ± 0.58	91	11.2 ± 5.9	140
RB18	glnR/glnR⁺p93	0.32 ± 0.01	4.0	8.58 ± 3.5	110

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Expression values in a wild-type strain in the glutamine medium were treated as 100%.

Cells were grown in the LDM medium with ammonium or glutamine, as indicated.

TABLE 6.

Expression of the gdhA gene as determined by real-time RT-PCR

Strain	Relevant genotype	Expression in^d :
		Ammonium medium		Glutamine medium
		RNA amount^a	%^b	RNA amount	%
10403S	Wild type	1.00 ± 0.17	770	0.13 ± 0.04	100
RB10	glnR	1.13 ± 0.27	870	2.15 ± 0.64	1,700
TW84	codY	4.47 ± 0.69	3,400	1.43 ± 0.44	1,100
RB11	codY glnR	5.64 ± 0.19	4,300	11.8 ± 0.10	9,100
RB16	glnR/glnR⁺p⁺	2.12 ± 0.87	1,600	0.49 ± 0.37^c	380
RB17	glnR/glnR88p⁺	1.16 ± 0.07	8,900	1.58 ± 0.44	1,200
RB18	glnR/glnR⁺p93	0.16 ± 0.02	120	0.23 ± 0.02	180

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Expression values in a wild-type strain in the glutamine medium were treated as 100%.

Note the high standard deviation for this sample. The difference between a wild-type strain and strain RB16 in the glutamine medium is not statistically significant.

Cells were grown in the LDM medium with ammonium or glutamine, as indicated.

All three major targets of L. monocytogenes GlnR, the glnRA and amtB glnK operons and the gdhA gene, were reported (at least under some growth conditions) to be under the control of CodY, an important global regulator of metabolism and virulence in most low-G+C Gram-positive bacteria (13, 45). To test the possible interaction between GlnR and CodY, we determined the expression of these genes in a codY null mutant and a codY glnR double null mutant strain. Expression of glnA was not affected by CodY (Table 4, strains TW84 and RB11 versus 10403S and RB10), in accord with the absence of a CodY-binding site in the glnRA regulatory region, as determined recently by in vitro DNA affinity purification and deep sequencing (IDAP-Seq) experiments (14). The lack of CodY regulation also indicates that the CodY-binding site found within the coding sequence of glnR (14) does not contribute significantly to regulation.

The elevated expression of amtB in wild-type cells in the glutamine medium was not affected by CodY, but, under all other conditions tested, amtB expression was increased from 2.7- to 6.6-fold in the absence of CodY (Table 5). The negative effect of CodY was stronger in the glnR background. In a reciprocal manner, the positive effect of GlnR on amtB expression in the glutamine medium was reduced from 27-fold in the codY⁺ background to 6.6-fold in the absence of CodY (Table 5). These data indicate that CodY and GlnR interfere with each other’s action, e.g., they may compete for binding to overlapping sites. Indeed, one of the two core CodY-binding sites that was recently identified upstream of the amtB gene by IDAP-Seq (14) starts only 1 bp downstream from one of the two GlnR-binding motifs in this region (Fig. 2B). We hypothesize that the strong positive effect of GlnR on amtB expression in the codY⁺ background in the glutamine medium is partly due to displacement of CodY, a negative regulator. Such displacement occurring efficiently in a wild-type strain in the glutamine medium would explain why a codY null mutation did not affect amtB expression under these growth conditions (Table 5). Interestingly, in the ammonium medium, both GlnR and CodY acted as amtB repressors of similar strength, as indicated by identical levels of amtB expression in single glnR and codY null mutant strains (expression in a glnR codY double mutant strain in the ammonium medium revealed the maximal strength of the amtB promoter in the absence of activation) (Table 5). amtB expression in the ammonium medium was further decreased in a wild-type strain in which both repressors were present, indicating that under these conditions the two regulators act independently and do not compete for binding (this may be due to GlnR using different sites for binding in the glutamine and ammonium media) (Table 5 and Fig. 2B). The transition of GlnR from a repressive to activating mode of regulation and its apparent ability to displace CodY combined to provide a >100-fold increase in amtB expression in a wild-type strain in the glutamine medium compared to its expression in the ammonium medium (83-fold as calculated by the DESeq algorithm) (Tables 2, 3, and 5).

The gdhA gene expression was 4.5- to 11-fold higher in the absence of CodY (Table 6). The gdhA gene recently was shown to contain two CodY-binding sites in its regulatory region (Fig. 2C) (14). The negative effects of CodY and GlnR on gdhA expression were approximately additive (Table 6).

Complementation of the glnR null mutation.

Introduction of the full-length glnR gene under the control of its own promoter at an ectopic chromosomal locus in the glnR null mutant strain restored expression of all tested genes to near wild-type levels (Tables 4 to 6, strain RB16). Expression of the glnR gene in the complemented strain was 2.3-fold higher than that in a wild-type strain (Table 7).

TABLE 7.

Expression of the glnR gene as determined by real-time RT-PCR^d

Strain	Relevant genotype	RNA amount^a	%^b
10403S	Wild type	0.78 ± 0.13	100
RB10	glnR	0.0002^c	0.03
RB16	glnR/glnR⁺p⁺	1.79 ± 0.45	230
RB17	glnR/glnR88p⁺	7.39 ± 2.7	950
RB18	glnR/glnR⁺p93	7.70 ± 2.8	990

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Expression values in a wild-type strain in the glutamine medium were treated as 100%.

Very low glnR expression in a glnR mutant reflects the deletion of most of the gene’s sequence.

Cells were grown in the LDM-ammonium medium.

Overexpression of GlnR.

To test the role of the GlnR-binding motif in the regulation of the glnRA operon, we created a construct that contained a full-length glnR gene fused to the glnR regulatory region that retained only 93 bp upstream of the initiation codon and lacked 3 conserved base pairs (−94 to −96) of the GlnR-binding motif (glnR⁺p93); the full complementing construct described above (glnR⁺p⁺) contained 203 bp upstream of the initiation codon (Fig. 2A). The short construct was introduced at an ectopic chromosomal locus of the glnR null mutant strain. In the ammonium medium, the abundance of the glnR transcript in the resulting strain RB18 was increased 4.3-fold compared to a similar construct in strain RB16 containing the entire glnR regulatory region, indicating that eliminating the three base pairs of the binding motif caused GlnR to lose its full ability to negatively autoregulate its own transcript (Table 7).

Higher expression of glnR and the expected accumulation of GlnR in strain RB18 in the ammonium medium did not affect glnA expression but was accompanied by strong reduction of gdhA expression (Tables 4 and 6). Importantly, the resulting low level of gdhA expression was very similar to that in a wild-type strain in the glutamine medium, suggesting that repression of the gdhA promoter just requires a higher level of GlnR production, regardless of the nature of the nitrogen source in the growth medium and, therefore, the level of GlnR activity. Overexpression of glnR also caused a weak increase in amtB expression in the ammonium medium (Table 5, strain RB18). GlnR, when in excess, may bind to the activating GlnR-binding site of the amtB regulatory region even under conditions when it does not do so in a wild-type strain (see Discussion).

No effect of the GlnR-binding motif deletion on gene expression was observed in the glutamine medium in which GlnR is highly active (Tables 4 to 6).

Truncation of the C-terminal domain of GlnR.

The 135-amino acid (aa) B. subtilis GlnR has two functional domains, an N-terminal DNA-binding and dimerization domain and a C-terminal domain that is responsible for the interaction with GS and contains autoinhibitory dimerization sequences. The C-terminal amino acids of B. subtilis GlnR are critical for the GS-induced stabilization of the GlnR dimer and the resulting stable binding of GlnR to DNA (15, 46). Deletion of 13 to 40 aa from the C terminus of B. subtilis GlnR changes several major properties of the protein: (i) it can now form dimers and, therefore, becomes constitutively active under all growth conditions due to the deletion of the autoinhibitory sequences; (ii) it loses the ability to interact with GS, which is no longer needed for dimerization; and (iii) its binding to DNA becomes less efficient; therefore, although it is constitutively active, the level of its activity may be compromised.

Listerial GS is 77% identical to B. subtilis GS, and the critical residues responsible for interaction with GlnR are conserved (47, 48). The 122-aa product of the L. monocytogenes glnR gene is similar (50% identity) to B. subtilis GlnR (27, 28, 34). The L. monocytogenes GlnR protein is predicted to contain N-terminal and C-terminal domains similar to those of its B. subtilis ortholog but has deletions of 12 aa between the two domains (Fig. S2). We have generated a truncated version of L. monocytogenes glnR (glnR88p⁺) that lacks the sequence encoding the final 34 aa of the C-terminal domain, starting from the position corresponding to Leu89 in L. monocytogenes GlnR and Met96 or Leu100 in B. subtilis GlnR (Fig. S2). To study the functional properties of the truncated GlnR88 protein in vivo, the glnR88 allele was integrated at an ectopic chromosomal locus of the L. monocytogenes glnR null mutant strain to create strain RB17.

The GlnR88 protein lost the ability to repress the glnRA operon and gdhA gene efficiently but retained the ability to activate the amtB gene (Tables 4 to 7, strain RB17). Moreover, amtB activation was now observed not only in the glutamine medium but also in the ammonium medium, as expected for a constitutively active protein (note that although GlnR88 was overproduced [Table 7], similar higher expression of full-length GlnR in the ammonium medium in strain RB18 caused only weak, 23-fold less efficient activation of amtB) (Table 5). If we assume that activation of amtB requires a less active state of GlnR than repression of the glnR and gdhA promoters, our results are consistent with GlnR88 being a constitutively active protein, although with a significantly reduced level of activity. This activity is apparently sufficient for activation of amtB but not enough for repression of the glnR, glnA, and gdhA genes. This assumption is also consistent with the ability of full-length GlnR to activate the amtB gene when it is apparently poorly active, i.e., in the glutamine medium. The apparent constitutive activity of GlnR88 suggests that L. monocytogenes GlnR interacts with GS in a manner that is similar to that of B. subtilis GlnR.

Expression of the glutamate synthase genes.

Because most GlnR-regulated genes are involved in nitrogen metabolism and because gdhA-encoded glutamate dehydrogenase is an anabolic enzyme involved in glutamate biosynthesis (our unpublished data), we tested expression of another glutamate-synthesizing enzyme, glutamate synthase, encoded by the gltAB operon. In RNA-Seq experiments, the operon was not affected by GlnR but was highly upregulated, about 50-fold, in the glutamine medium compared to the ammonium medium (Table 3 and Data Set S2).

Indeed, using real-time reverse transcription-PCR (RT-PCR) experiments, very little expression of the gltA gene was observed in the ammonium medium (Table 8). In the glutamine medium, gltA expression was highly increased, from 98- to 470-fold, in a wild-type strain and strains lacking GlnR or CodY or both, indicating that these two proteins do not serve as major regulators of gltAB expression. Still, small (2-fold) negative effects of GlnR and CodY on gltA expression were observed in the glutamine medium (Table 8). The effect of CodY is in accord with the presence of a CodY-binding site upstream of gltA (14); however, no strong GlnR-binding motifs are present in this region. Overexpression of glnR (and the expected accumulation of full-length GlnR) in strain RB18 in the ammonium medium was accompanied by a strong increase in gltA expression. Based on these observations, we hypothesize that the efficiency of the gltAB operon expression correlates inversely with the cellular activity of glutamate dehydrogenase (see Discussion).

TABLE 8.

Expression of the gltA gene as determined by real-time RT-PCR

Strain	Relevant genotype	Expression in^d :
		Ammonium medium		Glutamine medium
		RNA amount^a	%^b	RNA amount	%^b
10403S	Wild type	0.011 ± 0.004	1.0	1.08 ± 0.55	100
RB10	glnR	0.022 ± 0.024	2.0	2.18 ± 0.70	200
TW84	codY	0.010 ± 0.003	0.9	1.98 ± 0.55	180
RB11	codY glnR	0.009 ± 0.0004	0.8	4.22^c	390
RB16	glnR/glnR⁺p⁺	0.030 ± 0.018	2.8	1.64 ± 0.11	150
RB17	glnR/glnR88p⁺	0.021 ± 0.004	1.9	1.62 ± 1.47	150
RB18	glnR/glnR⁺p93	0.39 ± 0.08	36	1.44 ± 0.53	130

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Expression values in a wild-type strain in the glutamine medium were treated as 100%.

Only one biological replicate was tested.

Cells were grown in the LDM medium with ammonium or glutamine, as indicated.

The effect of a glnK null mutation on GlnR activity.

The GlnK protein, a member of the PII family of regulatory proteins, is known to interact with AmtB and perform other regulatory functions related to nitrogen metabolism in various bacteria (36, 49). In B. subtilis, GlnK can also interact with TnrA and sequester this regulator to the membrane (15, 35), but its contribution to gene expression under normal conditions of laboratory growth appears to be very limited (50, 51). GlnK also was reported to interact with GlnR in Streptococcus mutans (52). We introduced a deletion mutation in the L. monocytogenes glnK gene and observed no effect of the mutation on expression of glnA, amtB, gdhA, or gltA genes in the ammonium or glutamine medium (data not shown). We conclude that GlnK does not significantly affect L. monocytogenes GlnR activity under the growth conditions tested.

Effect of glutamine and GlnR on expression of virulence genes.

Using real-time RT-PCR, we confirmed the previously reported results (8) and our own RNA-Seq data showing the positive effect of glutamine on expression of genes of the PrfA regulon in defined growth media. We also validated our RNA-Seq results, indicating that this effect is not mediated by GlnR (Tables 3 and 9).

TABLE 9.

Expression of genes of the PrfA regulon as determined by real-time RT-PCR

Strain	Relevant genotype	Target gene	Expression^a in:		Glutamine/ammonium ratio
Strain	Relevant genotype	Target gene	Ammonium medium	Glutamine medium	Glutamine/ammonium ratio
10403S	Wild type	prfA	2.58 ± 0.20	7.15 ± 1.5	2.8
RB10	glnR		1.80 ± 0.43	5.59 ± 1.5	3.1
10403S	Wild type	hlyA	12.6 ± 0.4	36.2 ± 20.0	2.9
RB10	glnR		8.98 ± 2.2	26.0 ± 7.7	2.9
10403S	Wild type	plcA	7.19 ± 0.90	25.6 ± 12.0	3.6
RB10	glnR		5.73 ± 1.3	15.6 ± 4.2	2.7

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The data are presented as the number of copies of the transcript per a copy of the rpoC transcript ± standard deviation as determined by real-time RT-PCR. All values are averages from three experiments. Cells were grown in the LDM medium with ammonium or glutamine, as indicated.

Effect of GlnR on expression of genes of nitrogen metabolism in strain EGD-e.

To test whether GlnR-mediated regulation also happens in the related, widely used L. monocytogenes strain EGD-e, we created a glnR null mutant derivative and tested expression of the glnA gene in the ammonium and glutamine media by real-time RT-PCR. The pattern of gene expression and regulation was similar to that in strain 10403S (Table 4). Preliminary results have shown that other relevant genes, amtB, gdhA, and gltA, were also expressed in EGD-e strains similarly to 10403S strains (data not shown).

DISCUSSION

Role of glutamine and GS in regulation of GlnR activity.

Most defined media for L. monocytogenes growth described in the literature contain glutamine as the principal nitrogen source (53 –55), and it was reported that L. monocytogenes cells cannot utilize ammonium as the principal nitrogen source (54). This assumption proved to be incorrect for at least several commonly used L. monocytogenes strains (31, 56, 57). Moreover, unlike the situation in B. subtilis (9) but similar to that in E. coli (58), ammonium was shown to be a preferred nitrogen source for these L. monocytogenes strains (32). Apparently, L. monocytogenes cells cannot take up glutamine fast enough, in accord with their inability to take up certain other amino acids efficiently (59). (Alternatively, the absence of genes encoding glutaminases [28] or some properties of L. monocytogenes glutamate synthase impede utilization of glutamine as a nitrogen source.) Thus, growth with glutamine as the principal nitrogen source can be construed as a condition of intracellular nitrogen limitation. This is important for this work, because GlnR activity in B. subtilis and, likely, in L. monocytogenes responds not to low-molecular weight effectors per se but to the nitrogen source availability as sensed by GS, a critical enzyme of nitrogen metabolism that moonlights as a chaperone that is required for the dimerization and resulting activation of GlnR (15, 22). The chaperone state of B. subtilis GS is its feedback-inhibited form that is produced by interaction with glutamine; this feedback-inhibited state signals high availability of nitrogen and is achieved most efficiently in the presence of glutamine, the preferred nitrogen source, in the growth medium (46). Thus, B. subtilis GlnR is active, as a repressor, under conditions of nitrogen excess when GS is inactive.

Our results suggest that a feedback-inhibited form of L. monocytogenes GS also serves as a chaperone for GlnR, because a truncated version of GlnR, GlnR88, which is expected to lose its ability to interact with GS, gained the ability to activate amtB under conditions in which intact GlnR was unable to do so (Table 5). (GS also interacts with GlnR and promotes its binding to DNA in S. pneumoniae and Paenibacillus polymyxa [25, 60] and is likely to do so in other bacteria.) Assuming that the main physiological function of GlnR is repression of the gene that encodes GS, we concluded that full-length L. monocytogenes GlnR, in contrast to B. subtilis GlnR, is more active in the ammonium medium and less active in the glutamine medium. Paradoxically, this means that synthesis of the glutamine-generating enzyme, GS, is elevated when exogenous glutamine serves as the principal nitrogen source. In the medium that contained both ammonium and glutamine, expression of the two tested GlnR-regulated genes, glnA and amtB, was identical to that in the ammonium medium (data not shown), confirming that the regulation is mediated by the total availability of nitrogen sources and not simply by the presence of glutamine in the medium. If we assume that a high level of intracellular glutamine is required to convert listeria GS into its feedback-inhibited state, we need to conclude that growing listerial cells in the glutamine medium is accompanied by an intracellular glutamine limitation, apparently due to poor glutamine uptake. (We do not know, yet, whether GlnR activities in the ammonium and glutamine media represent their highest and lowest levels; expression of GlnR-regulated genes and, therefore, activity of GlnR in the complex medium, brain heart infusion [BHI], are similar to those in the ammonium-containing defined medium [T. A. Washington B. R. Belitsky, and A. L. Sonenshein, unpublished data].)

Different modes of GlnR action.

Three transcriptional units that are targeted by GlnR are subject to three different types of GlnR-mediated regulation. The glnRA operon is repressed by GlnR in its active state, achieved under conditions of apparent nitrogen excess, i.e., in the ammonium medium. Because GlnR negatively regulates its own expression, even low levels of highly active GlnR are sufficient to exert strong repression. In the glutamine medium, the operon is mostly derepressed, indicating that even high levels of less active GlnR are not sufficient for repression (Table 4). The glnRA regulation is apparently achieved via a perfect GlnR-binding motif located upstream of the promoter, as deletion of this sequence caused higher expression of glnR in the ammonium medium (Table 7 and Fig. 2A).

In contrast, the gdhA gene appears to be repressed whenever GlnR accumulates, even if GlnR is not fully active, i.e., either in the ammonium or glutamine medium. A low level of even highly active GlnR is not sufficient for gdhA repression (Table 6). Whether GlnR’s effect on gdhA expression is direct or indirect is not entirely clear. Although purified GlnR (at a high concentration) was shown to bind to the gdhA regulatory region (57), this region does not contain strong GlnR-binding motifs (Fig. 2).

The amtB promoter is the most highly GlnR-regulated promoter in L. monocytogenes. Interestingly, in B. subtilis the amtB glnK (formerly nrgAB) operon is the transcriptional unit most highly regulated, in a positive manner, by TnrA (61). In the case of L. monocytogenes amtB, GlnR has both a small (2.5-fold), negative effect in its active state, in the ammonium medium, and a larger, 19- to 27-fold, positive effect in the glutamine medium (Table 5). The amtB regulatory region has two GlnR-binding motifs (Fig. 2B). It is possible that the distal motif, located upstream of the −35 promoter region, serves as a site of positive regulation and the proximal motif, located downstream of the transcription start site, serves for negative regulation (Fig. 2B). According to our model, binding of the full-length GlnR to the activating site occurs in the glutamine medium and binding to the repressing site happens in the ammonium medium or in both media (Fig. 3). It is likely that the positive regulation is partly due to competition with CodY, a negative regulator (Table 5 and Fig. 3). In an unusual manner, activation of the amtB promoter is conferred by GlnR in its partially inactive state (Table 5). Alternatively, although unlikely, GlnR may have two active states, one in the cells grown in the ammonium medium and the other in the glutamine medium; different genes may respond exclusively or preferentially to only one active state of GlnR or to both. It is also possible that if GlnR is highly active and binds very tightly, it may repress expression from the amtB promoter, and if GlnR is poorly active and binds only transiently to the same site, it may be able to activate the same promoter.

FIG 3 — Model of *amtB* promoter regulation by GlnR and CodY. The shading intensities of the GlnR protein reflect different levels of its activity. Only the proximal CodY-binding site is shown.

When in excess, GlnR apparently may bind to the activating GlnR-binding site of the amtB regulatory region and cause a 5-fold increase in expression even under the conditions in which it does not do so in a wild-type strain; even more efficient interaction with the activating site in the ammonium medium is achieved by the truncated form of GlnR (Table 5). Understanding fully amtB regulation by GlnR will require careful dissection of the contribution of each GlnR-binding motif in different media and in strains with different intracellular levels of GlnR. For a long time, GlnR has been known only as a repressor in various bacteria, and the amtB gene is just the second example of a GlnR-regulated gene that is subject to positive regulation. Interestingly, the other positively regulated target of GlnR, the P. polymyxa nitrogen fixation nif operon, also has two GlnR-binding motifs and is subject to both positive and negative GlnR-mediated regulation (60).

The regulatory region of the gltAB operon does not have a potentially strong GlnR-binding site. Thus, the weak GlnR-mediated regulation of the gltA promoter is likely to be indirect (Table 8). In the ammonium medium, there is a striking inverse correlation between expression of the gltAB operon and the gdhA gene, the transcriptional units that encode two major enzymes of ammonium assimilation and glutamate biosynthesis (Tables 6 and 8). Moreover, glutamate dehydrogenase, even if expressed, is expected to be inactive in the glutamine medium due to the low availability of one of its substrates, ammonium; most anabolic glutamate dehydrogenases have poor affinity for ammonium (58). In contrast, expression of the gltAB operon is always high in the glutamine medium. Therefore, we hypothesize that to provide glutamate, which is required for metabolism, the expression of glutamate synthetase is elevated whenever glutamate dehydrogenase is not expressed or is expressed but not active. This would amount to an apparent on/off switch between the two ammonium assimilation enzymes; only one enzyme appears to be active under the growth conditions used. Mechanistically, glutamate dehydrogenase activity may affect expression of the gltAB operon via its effect on GltC, the presumed positive regulator of gltAB encoded by the upstream, divergent gene (28, 62). Such an effect was extensively described in B. subtilis and is due to either the altered pool of 2-oxoglutarate, a substrate of glutamate dehydrogenase and a positive effector of GltC, direct protein-protein interaction between GltC and glutamate dehydrogenase, or both (63 –66). In an interesting twist and departure from the B. subtilis paradigm, glutamate dehydrogenases of L. monocytogenes and B. subtilis are not very similar (32% identity) and have very different functions: the L. monocytogenes enzyme is an anabolic, glutamate-generating enzyme (our unpublished results), but each of the two B. subtilis enzymes has a catabolic, glutamate-degrading function (67).

Our results on the GlnR-mediated regulation of glnA, amtB, and gdhA correspond rather well to previously reported results obtained by growing cells of strain EGD-e at 24°C (57). However, the results obtained in the previous study by growing cells at 37°C are completely different from those obtained at 24°C (57) and from the results obtained in this work. We repeated some of the experiments for strain EGD-e at 37°C and for strain 10403S at 23°C and obtained results that were mostly similar to those obtained in this work for strain 10403S at 37°C (Table 4 and data not shown). The discrepancies between our results and those reported previously (57) are likely due to differences in the cultivating conditions. The effects of temperature and aeration conditions on L. monocytogenes growth were reported by multiple laboratories. Interestingly, in our hands, cells grew better at 37°C if overnight precultures were incubated at room temperature instead of 37°C.

CodY-mediated regulation.

Some targets of GlnR in L. monocytogenes and other bacteria are also negatively regulated by CodY (13, 24, 25, 45). Such concerted regulation of the L. monocytogenes gdhA gene and amtB glnK operon makes sense, because CodY is most active under conditions of amino acid and oligopeptide excess, when the uptake of ammonium and synthesis of glutamate may be less important. The absence of strong repression of the gltAB operon by CodY in the glutamine medium (Table 8) indicates that cells need to maintain glutamate synthesis under these conditions, in accord with our view that they experience nitrogen limitation despite the presence of excess amino acids that activate CodY. The limitation may be related to the lack of efficient glutamate-generating pathways of amino acid catabolism in L. monocytogenes.

The negative effect of CodY on gdhA expression was reported previously in two L. monocytogenes strains and in several different media (13, 45). Interestingly, a previous report found that the glnRA operon in strain 10403S is under positive CodY control in both BHI and a defined glutamine-containing medium with reduced concentrations of branched-chain amino acids; the amtB glnK operon was also under positive CodY control but only in the latter medium; the experiments were performed in a glnR⁺ background (13). Our RNA-Seq results (T. A. Washington B. R. Belitsky, and A. L. Sonenshein, unpublished data) show the absence of CodY-mediated regulation of the glnRA operon in BHI and ammonium-containing LDM, confirming our real-time RT-PCR data for the defined medium.

Regulation of virulence genes.

We have confirmed the previously reported data that the genes of the PrfA regulon are more active if glutamine serves as the principal nitrogen source (8). Our results also demonstrate that the expression of these genes is not affected by a glnR mutation; hence, the glutamine effect is not mediated via GlnR. The response of virulence genes to glutamine may be a manifestation of nutrient limitation. Interestingly, the glnRA and amtB glnK operons were coordinately repressed in a defined glucose-glutamine medium in a strain with increased expression of a constitutive form of PrfA, revealing another case of correlation, although inverse, between expression of GlnR-regulated and virulence genes (68). It is likely that GlnR activity was elevated under those conditions, perhaps due to cells’ growth being limited by glucose (instead of glutamine) uptake (68).

No effect of a glnR mutation on L. monocytogenes growth in murine macrophages was found (69). However, changes in expression of L. monocytogenes glnR and other GlnR-regulated genes during infection were reported in multiple studies (69 –73). The underlying reasons and mechanistic details of this regulation and its significance for the infection process in animal models are unknown and should be a goal of future experimentation.

MATERIALS AND METHODS

Bacterial strains and culture media.

All L. monocytogenes strains constructed and used in this study were derivatives of strain 10403S or EGD-e (27, 28) and are described in Table 10. E. coli strain JM107 (74) or DH5α (75) was used for the isolation of plasmids. Cells were grown in brain heart infusion medium (BHI) or in defined medium (LDM) (31) for L. monocytogenes and in L broth (76) for E. coli. The same media with addition of agar were used for growth of bacteria on plates. E. coli cells were grown at 37°C with aeration. Overnight precultures of L. monocytogenes cells were grown statically at room temperature (final optical density at 600 nm [OD₆₀₀] of ∼0.2) and then diluted about 10-fold (to an OD₆₀₀ of ∼0.02) into fresh medium and grown at 37°C with aeration. LDM had the following 18 components: morpholinepropanesulfonic acid-HCl (pH 7.5), 50 mM; K₂HPO₄, 2 mM; MgSO₄ × 7H₂O, 0.81 mM (0.02%); a mixture of FeCl₃ and Na₃-citrate × 2H₂O, 0.04 g/liter each (0.004%); Ca(NO₃)₂, 0.5 mM; glucose, 27.8 mM (0.5%); NH₄Cl, 37.4 mM (0.2%), or glutamine, 13.7 mM (0.2%); seven amino acids (Leu, Ile, Val, Cys, Met, Arg-HCl, and His-HCl), at 100 μg/ml each; and four vitamins, biotin (0.5 μg/ml). riboflavin (0.5 μg/ml), thiamine-HCl (1 μg/ml), and lipoic acid (0.005 μg/ml).

TABLE 10.

L. monocytogenes strains used

Strain	Genotype	Source or reference
10403S	Str^r	28
EGD-e	Wild type	28
TW84^a	ΔcodY::spc	T. Washington
RB10	ΔglnR	10403S × pRB17
RB11	ΔglnR ΔcodY::spc	TW84 × pRB17
RB15	ΔglnR	EGD-e × pRB18
RB16	ΔglnR pPL2 glnR⁺p⁺ (cat)	RB10 × pRB20
RB17	ΔglnR pPL2 glnR88p⁺ (cat)	RB10 × pRB30
RB18	ΔglnR pPL2 glnR⁺p93 (cat)	RB10 × pRB31
RB19	ΔglnK	10403S × pRB22

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In strain TW84, the entire coding region of the codY gene was replaced by the spectinomycin resistance gene (T. Washington, unpublished data).

The following antibiotics were used when appropriate: chloramphenicol, 5 or 10 μg/ml, and erythromycin, 10 μg/ml, for L. monocytogenes strains and ampicillin, 50 or 100 μg/ml, and chloramphenicol, 20 μg/ml, for E. coli strains.

General molecular genetic methods.

Methods for common DNA manipulations and E. coli electroporation were as previously described (67). Chromosomal DNA of L. monocytogenes was isolated as described after the cells were disrupted using 0.1-mm silica beads and a Mini-BeadBeater (Biospec Products) for two 30-s cycles at the maximal setting (31). Electroporation of L. monocytogenes cells was performed according to the published procedure (77). Chromosomal DNA of L. monocytogenes strain 10403S or EGD-e was used as the template for PCR. All oligonucleotides used in this work are described in Table S1 in the supplemental material. All cloned PCR-generated fragments were verified by sequencing.

Construction of the L. monocytogenes glnR null mutant strain.

To create an in-frame deletion mutation in glnR, we first generated 0.65-kb and 0.70-kb PCR fragments, containing sequences upstream or downstream of the glnR gene, respectively, using primers oRB40 and oKZ1 or oKZ2 and oRB43. The upstream PCR fragment was digested with BamHI and MluI and cloned in a temperature-sensitive plasmid, pMAD (erm bgaB) (78), to create pRB16 (glnR′). The downstream PCR fragment was digested with MluI and NcoI and cloned in pRB16. The resulting plasmid, pRB17 (ΔglnR), contained an in-frame deletion of 80 codons (66%) of the 366-bp glnR gene. pRB17 was integrated in the chromosome of L. monocytogenes strain 10403S by a single-crossover recombination event after electroporation and selection for erythromycin-resistant, blue colonies at 42°C on BHI plates containing a color indicator, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside. The plasmid was allowed to excise from the chromosome by passaging cells several times in liquid BHI medium under nonselective conditions, i.e., in the absence of erythromycin at 30°C. Strains with a deletion within the glnR gene were searched for on BHI plates among white, erythromycin-sensitive colonies using colony PCR to determine the size of the chromosomal glnR locus. The replacement of the chromosomal glnR gene by the deletion-containing allele in strain RB10 was confirmed by sequencing; the loss of the plasmid was established in separate PCRs using vector-specific primers.

An identical in-frame deletion in the glnR gene was introduced in the chromosome of strain EGD-e, creating strain RB15. Plasmid pRB18, used to introduce the deletion, was constructed as described above for pRB17 using chromosomal DNA of strain EGD-e as the template for PCR.

Complementation of the glnR null mutation.

To complement the ΔglnR mutation, a 0.60-kb PCR product containing the full-length glnR gene and its entire upstream regulatory region was synthesized by PCR using oRB52 and oRB53 primers, cut with KpnI and SacI, and cloned in the integrative plasmid pPL2 (cat) (79). The resulting plasmid, pRB20 (glnRp⁺), was introduced into strain RB10 (ΔglnR) using electroporation and selection for chloramphenicol-resistant colonies; its single-crossover integration in the chromosome of the resulting strain, RB16, was confirmed by colony PCR.

A truncated version of glnR lacking the last 37 codons of the coding region was cloned in the same way as the full-length gene using oRB61 instead of oRB53 as the reverse PCR primer; the resulting plasmid pRB30 (glnR88p⁺) was integrated into the chromosome of strain RB10 (ΔglnR) as described above to create strain RB17. Another truncated version of glnR lacking the 110 distal base pairs of its regulatory region, which includes part of the GlnR-binding site (Fig. 2), was cloned as described for pRB20 but using oRB62 instead of oRB52 as the direct PCR primer; the resulting plasmid, pRB31 (glnR⁺p93), was integrated into the chromosome of strain RB10 (ΔglnR) as described above to create strain RB18.

Construction of the L. monocytogenes glnK null mutant strain.

To create an in-frame deletion mutation in glnK, we first generated 0.77-kb and 0.68-kb PCR fragments containing sequences upstream or downstream of the glnK gene, respectively, using primers oRB54 and oRB55 or oRB56 and oRB57. The fragments were cloned successively in pMAD as described for pRB16 and pRB17 to create pRB21 (glnK′) and pRB22 (ΔglnK). pBR22 contained an in-frame deletion of 109 codons (90%) of the 363-bp glnK gene. The ΔglnK mutation was introduced in L. monocytogenes strain 10403S as described above for the ΔglnR mutation to create strain RB19.

Isolation of RNA.

Samples of 3 ml of L. monocytogenes cells growing exponentially in the LDM medium with ammonium or glutamine as the principal nitrogen source were collected at an OD₆₀₀ of ~0.4 by mixing with an equal volume of a 1:1 (vol/vol) solution of ethanol-acetone (−20°C) and kept at −80°C until further use. The cells were pelleted, washed with 1 ml of 10 mM Tris-HCl (pH 8.0)-1 mM EDTA buffer, and resuspended in 0.8 ml of the TRIzol reagent. The cells were disrupted using a bead beater as described above, and RNA was purified using the Direct-zol RNA MiniPrep plus kit (Zymo Research). Purified RNA (∼10 μg) was further treated with Turbo DNA-free DNase I (Ambion) according to the manufacturer’s instructions. RNA was quantified using the NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific).

RNA-Seq.

Total RNA was isolated as described above. Strand-specific RNA-Seq libraries were constructed by the Microbial ‘Omics Core at the Broad Institute of MIT and Harvard using a modified version of the RNAtag-seq protocol (80). A sample of 0.5 to 1 μg of total RNA was fragmented, dephosphorylated, and ligated using RNA ligase to DNA adapters carrying 5′-AN8-3′ barcodes of known sequence with a 5′ phosphate and a 3′ blocking group. Barcoded RNAs were pooled and depleted of rRNA using the Gram-positive Ribo-Zero rRNA depletion kit (Epicentre). Pools of barcoded depleted RNAs were converted to Illumina cDNA libraries in two main steps: (i) reverse transcription of the RNA using a primer designed to the constant region of the barcoded adaptor with addition of an adapter to the 3′ end of the cDNA by template switching using SMARTScribe (Clontech), as described previously (81), and (ii) PCR amplification using primers whose 5′ ends target the constant regions of the 3′ or 5′ adaptors and whose 3′ ends contain the full Illumina P5 or P7 sequences. cDNA libraries were subjected to 24× multiplexed, massively parallel sequencing using the Illumina NextSeq 500 platform at the Broad Institute to generate paired-end reads.

The Illumina sequencing reads, after the removal of the barcodes, were aligned against the L. monocytogenes reference genome NC_017544.1 using the Burrows-Wheeler short-read aligner (82). After alignment, the Illumina sequencing reads were summed per coding sequences and other genomic features, such as noncoding RNAs, using custom scripts and provided as the input to DESeq2 (33) to determine the magnitude and significance of differential gene expression. The number of paired reads (fragments) was also normalized by the length of each genomic feature and the total number of million-mappable paired reads to obtain fragments per kilobase of transcript per million mapped fragments (FPKM) values. The analysis was done at the Broad Institute. A 2.5-fold difference with a P value adjusted for a false discovery rate below 0.05 was considered the minimal cutoff for making conclusions about differential regulation. Two biological replicates were processed for each condition; 98% to 99% correlation was observed between biological replicates.

Visualization of raw sequencing data and coverage plots in the context of genome sequences and gene annotations was conducted using IGV (83). The data were deposited in the Gene Expression Omnibus database.

Real-time RT-PCR.

cDNA was synthesized starting from 1 or 2 μg of RNA using random hexamer primers and SuperScript III reverse transcriptase (Invitrogen) per the manufacturer’s instructions. The reactions were performed using a LightCycler 480 instrument (Roche Diagnostics Corporation) and HOT FIREPol EvaGreen qPCR mix plus (no ROX) (Solis BioDyne) according to the manufacturer’s instructions. The reactions were done in a total volume of 20 μl and contained 4 μl of 5-fold-diluted cDNA or control RNA samples. Oligonucleotides used as primers for real-time RT-PCR are specified in Table S1. All primers were designed using the PrimerQuest tool (Integrated DNA Technologies). The rpoC transcript was used for normalization. Serial dilutions of L. monocytogenes chromosomal DNA (from 3.2 to 10,000 pg per reaction) were used to create calibration curves for each transcript and to compare amounts of different transcripts.

Data availability.

The RNA-Seq data were deposited in Gene Expression Omnibus under the accession number GSE143874.

Supplementary Material

Supplemental file 1

JB.00209-20-s0001.pdf^{(207.5KB, pdf)}

Supplemental file 2

JB.00209-20-sd002.xlsx^{(18KB, xlsx)}

Supplemental file 3

JB.00209-20-sd003.xlsx^{(43KB, xlsx)}

ACKNOWLEDGMENTS

We are grateful to T. Washington for sharing the codY::spc mutant strain and discussions and to J. Livny for his help with the DESeq analysis. RNA-Seq libraries were constructed and sequenced at the Broad Institute of MIT and Harvard by the Microbial ‘Omics Core and Genomics Platform, respectively. The Microbial ‘Omics Core also conducted preliminary analysis for all RNA-Seq data.

This work was supported by a research grant from the U.S. National Institute of Allergy and Infectious Diseases (R01AI109048) to A. L. Sonenshein, A. Herskovits, and M. O’Riordan.

The content of this work is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

We have no conflict of interest to declare.

Footnotes

Supplemental material is available online only.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental file 1

JB.00209-20-s0001.pdf^{(207.5KB, pdf)}

Supplemental file 2

JB.00209-20-sd002.xlsx^{(18KB, xlsx)}

Supplemental file 3

JB.00209-20-sd003.xlsx^{(43KB, xlsx)}

Data Availability Statement

The RNA-Seq data were deposited in Gene Expression Omnibus under the accession number GSE143874.

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PERMALINK

Role of GlnR in Controlling Expression of Nitrogen Metabolism Genes in Listeria monocytogenes

Rajesh Biswas

Abraham L Sonenshein

Boris R Belitsky

Roles

ABSTRACT

INTRODUCTION

RESULTS

Genome-wide analysis of gene expression.

TABLE 1.

FIG 1.

FIG 2.

TABLE 2.

Nitrogen source-mediated regulation of gene expression.

TABLE 3.

Transcriptional regulation of the glnA, amtB, and gdhA genes by GlnR and CodY as determined by real-time RT-PCR.

TABLE 4.

TABLE 5.

TABLE 6.

Complementation of the glnR null mutation.

TABLE 7.

Overexpression of GlnR.

Truncation of the C-terminal domain of GlnR.

Expression of the glutamate synthase genes.

TABLE 8.

The effect of a glnK null mutation on GlnR activity.

Effect of glutamine and GlnR on expression of virulence genes.

TABLE 9.

Effect of GlnR on expression of genes of nitrogen metabolism in strain EGD-e.

DISCUSSION

Role of glutamine and GS in regulation of GlnR activity.

Different modes of GlnR action.

FIG 3.

CodY-mediated regulation.

Regulation of virulence genes.

MATERIALS AND METHODS

Bacterial strains and culture media.

TABLE 10.

General molecular genetic methods.

Construction of the L. monocytogenes glnR null mutant strain.

Complementation of the glnR null mutation.

Construction of the L. monocytogenes glnK null mutant strain.

Isolation of RNA.

RNA-Seq.

Real-time RT-PCR.

Data availability.

Supplementary Material

ACKNOWLEDGMENTS

Footnotes

REFERENCES

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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