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Centre for Infectious Disease, Institute for Cell and Molecular Science, Barts and the London, London E1 2AT, UK
Correspondence
Tanya Parish
t.parish{at}qmul.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Relatively little is known about nitrogen metabolism in M. tuberculosis and most work has focused on the study of the major glutamine synthetase (GS), GlnA1, the key enzyme in ammonia assimilation. This enzyme is responsible for the ATP-dependent incorporation of ammonia into L-glutamate to form L-glutamine. During growth in axenic culture, GlnA1 is expressed to a high level, resulting in its secretion into the culture filtrate (Harth et al., 1994
; Tullius et al., 2001). Inhibition of this enzyme directly by enzyme inhibitors such as L-methionine-S-sulfoximine (Harth & Horwitz, 1999) or by antisense-oligonucleotide inhibition of expression results in reduced bacterial growth (Harth et al., 2000). In addition, a glnA1 mutant of M. tuberculosis is auxotrophic for L-glutamine and shows a reduced ability to multiply within macrophages (Tullius et al., 2003). These data indicate that the function of GS is essential for the viability and pathogenicity of M. tuberculosis.
Aside from GlnA1, M. tuberculosis has three other potential GSs encoded by glnA2, glnA3 and glnA4 (Cole et al., 1998), but to date only glnA1 has been shown to be essential for growth in vitro (Tullius et al., 2003). The enzymic function of the other three GS homologues has recently been confirmed (Harth et al., 2005) and it has also been shown that a glnA2 mutant of Mycobacterium bovis is avirulent in the guinea pig model (Collins et al., 2002).
Glutamine and glutamate are key precursors in many biosynthetic reactions, and in other bacteria the intracellular levels of these metabolites are strictly controlled. During ammonia shock, a quick response to reduce GS activity is required in order to preserve the intracellular pools of these key chemicals. If GS activity were not controlled, then the intracellular levels of glutamate would quickly be depleted. GS activity is normally controlled by the product of the glnE gene, an adenylyltransferase, as well as by other mechanisms including feedback inhibition (reviewed by Merrick & Edwards, 1995; Reitzer & Magasanik, 1987). The role of GlnE in other bacteria is to inactivate GS in response to ammonia levels; GlnE adenylylates GS on a conserved tyrosine residue, resulting in a loss of synthetase activity. GS exists as a dodecamer and the activity of GS is directly proportional to the number of subunits that are adenylylated, so that the enzyme can display a range of activities depending on its precise adenylylation state. Thus GlnE controls the interconversion of active and inactive GS, which in turn regulates the glutamate to glutamine transformation and the assimilation of ammonia. This form of post-translational modification is thought to be used since it provides a much quicker response to ammonia levels than transcriptional control of GS expression. Indeed, in a GlnE mutant of Streptomyces coelicolor, GS activity is not down-regulated after ammonium shock and the intracellular glutamate/glutamine balance is altered (Fink et al., 1999). Of the M. tuberculosis GSs, glnA1 and glnA3 encode type I enzymes, which are usually controlled by GlnE, whereas glnA2 and glnA4 encode type II enzymes, which are not.
We have studied the role of glnE in glutamine metabolism in M. tuberculosis, following our earlier observation that it is an essential gene in M. tuberculosis (Parish & Stoker, 2000; Parish et al., 2001). In other bacteria, including the closely related species S. coelicolor, glnE is not essential since mutants can be obtained (Fink et al., 1999). Thus our results point to a key difference in glutamine metabolism between M. tuberculosis and other bacteria. We speculated previously that this may be because control of the glutamine/glutamate balance is critical to the pathogen's survival, since L-glutamine is a major component of the cell wall, both in the form of D-isoglutamine in the peptidoglycan and as the major cell wall component poly-L-glutamine. In the present study we investigated the expression of glnE in response to nitrogen availability. We have identified the promoter region which directs the transcription of glnE and have shown that it is controlled in response to nitrogen availability.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Determination of co-transcription with glnA2 by RT-PCR.
RT-PCR was carried out using a two-step procedure using the Reverse Transcriptase kit (Promega) according to the manufacturer's instructions. cDNA (20 ng) was amplified in the second step using the programme 95 °C 2 min, 35 cycles of 95 °C 45 s, 52 °C 1 min, 72 °C 1 min followed by a final extension step of 72 °C for 7 min. Ten microlitres of the 20 µl reaction was run on an agarose gel. Two pairs of primers were used to determine if co-transcription occurred: glnA2-Efor and glnA2-Erev should amplify the last 50 bp of glnA2, the intergenic region and the first 100 bp of glnE; Gap100 and Gap101 should amplify the last 200 bp of glnA2 and the first 400 bp of glnE (Table 1).
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). Labelling and detection were carried out using the AlkPhos Direct kit (Amersham).
Quantitative RT-PCR (RT-qPCR).
Probes and primers were designed for quantitative PCR with molecular beacons for sigA (endogenous control), glnE and glnA2 using the software Primer Express (Table 1). cDNA was synthesized from RNA using RT and random hexamer primers using AMV reverse transcriptase. PCR was carried out in a Taqman 7900 using a standard PCR master mix. For glnA, glnE and sigA, the primer pairs were GlnA-R and GlnA-F, GlnE-F and GlnE-R and SigA-F and SigA-R respectively, and the probes used were GlnA-T, GlnE-T and SigA-T. The primer and probe concentrations were first optimized. The optimal primer concentration was 300 nM for all three genes; the probe concentration was 100 nM for sigA, 125 nM for glnE and 150 nM for glnA2. In order to measure relative gene expression levels, standard curves for each primer-probe set were generated using genomic DNA. CT values were converted into the equivalent of ng using the standard curve. Control reactions without RT were used to confirm that there was no significant contaminating genomic DNA present. CT values for genomic DNA were converted to ng and subtracted from the plus RT values. In order to standardize the samples to ensure that equal amounts of cDNA were used, each value was standardized to sigA to generate unit-less values. At least three independent RNA samples were assayed in triplicate for each gene.
Determination of transcriptional start site by rapid amplification of cDNA ends (RACE).
We used RACE to locate the 5' end of the mRNA from glnE. RNA was extracted from M. tuberculosis H37Rv grown in TSM-high ammonia. cDNA was synthesized from 1 µg total RNA using reverse transcriptase at 42 °C for 50 min (according to the manufacturer's instructions) and the gene-specific primer ESP1 (Table 1) located approximately 500 bp downstream from the predicted 5' end. A homopolymeric A-tail was added to the 3' end of the cDNA using terminal transferase. The tailed cDNA was amplified using an oligo-dT primer and a second gene-specific primer (ESP2, Table 1) using the programme 95 °C for 2 min, 10 cycles of 95 °C 2 min, 55 °C 45 s, 72 °C 1 min, 30 cycles of 95 °C 45 s, 55 °C 1 min, 72 °C 1 min 20 s (+20 s per cycle) and a final step of 72 °C for 7 min. This product was further amplified using the same programme with nested primers ESP3 (Table 1; located approximately 250 bp downstream from the predicted 5' ends) and an anchor primer to increase specificity. The products were cloned into pGEM-Easy T (Promega) and sequenced to identify the 5' end.
Analysis of promoter activity.
The predicted glnE promoter region was PCR amplified using glnA2-Efor and Elac4, which amplified a 0.6 kb product covering the 3' end of the glnA2 gene, the intergenic region and the first 96 bp of the glnE gene (Table 1), and cloned into the vector pSM128, a mycobacteriophage L5-based integrating vector carrying a lacZ reporter (Dussurget et al., 1999). This vector is therefore present in only one copy, which is integrated into the chromosome. The predicted glnA2 promoter region (484 bp upstream of glnA2) was amplified using primers CAGE 7 and CAGE 8 and cloned into pSM128. The plasmids were electroporated into M. tuberculosis or M. smegmatis and transformants selected on 20 µg streptomycin ml–1. For each plasmid three independent transformants were assayed in duplicate for promoter activity in TSM liquid medium containing high or low ammonia or 3 mM amino acid. Cell-free extracts were prepared and assayed for 
-galactosidase activity (Miller, 1972).
Site-directed mutagenesis.
Site-directed mutagenesis was carried out according to the Stratagene Quikchange II XL site-directed mutagenesis instructions. The primer pairs used were: glnE sdm 10 F and glnE sdm 10 Rev for the –10 mutation; glnE sdm 35 F and glnE sdm 35 Rev for the –35 mutation; GSDM1 and GSDM2 for mutation 5A; GSDM7 and GSDM8 for mutation 5B; GSDM9 and GSDM10 for mutation 5C. Amplification reactions were carried out in 50 µl total volume containing 1x Pfu Ultrareaction buffer, 0.5 mM dNTPs, 10 pmol each primer, 10 % DMSO, 10 ng template and 2.5 units Pfu Ultra. The thermocycling programme used was 94 °C for 1 min, followed by 18 cycles of 94 °C for 1 min, 56 °C for 1 min, 68 °C for 12 min and a final extension cycle of 68 °C for 20 min. Template was degraded using 10 units DpnI at 37 °C for 1 h. Ten microlitres of each reaction was used to transform competent E. coli. Recombinant plasmids were isolated and sequence verified.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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).
Nitrogen-source utilization
We first developed a new medium formulation (TSM) for the growth of M. tuberculosis, which allowed us to add single nitrogen sources in the absence of glutamate or ferric ammonium citrate, which are present in many commonly used media. For example Middlebrook 7H9 has 0.5 g glutamate l–1 and 40 mg ferric ammonium citrate l–1. We tested the ability of TSM with different nitrogen sources to support growth of M. tuberculosis. We used four different L-amino acids (alanine, asparagine, glutamate and glutamine) and high- and low-ammonia states (Fig. 1).
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Genomic organization
The region of the chromosome where glnE is located is shown in Fig. 2(a). Immediately upstream of glnE is the glnA2 gene, which encodes a class II GS. Although the genes do not overlap there is a very short intergenic region (51 bp) that is suggestive of an operon. Downstream, but transcribed divergently, is the major GS gene (glnA1). This genomic organization is conserved in M. bovis as well as in the related actinomycetes Corynebacterium glutamicum (Nolden et al., 2001) and S. coelicolor (Fink et al., 1999). The reason for the co-localization of glnE and glnA2 is not immediately apparent, as GlnA2 is not predicted to be adenylylated by GlnE.
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), whereas in M. bovis, there appears to be a separate promoter for glnE (Collins et al., 2002). We determined whether these genes were co-expressed in M. tuberculosis using both Northern blotting and RT-PCR. First we used RT-PCR to determine whether we could identify a co-transcript for glnA2 and glnE. Using primers designed to amplify the junction between the two genes we could detect transcripts spanning the junction in all three media tested (7H9, low and high ammonia) (Fig. 2b). This suggested that the two genes are co-transcribed and that the bicistronic message is produced regardless of ammonia availability. RT-PCR analysis with primers designed to amplify either gene independently also confirmed expression of glnA2 and glnE. Although RT-PCR is not quantitative, the results suggested that the co-transcript was present at the same level as the glnA2 transcript (Fig. 2b), whereas glnE was present at a lower level (not shown).
Northern analysis was carried out on total RNA isolated from cells grown in 7H9 or TSM-high ammonia (Fig. 2c). We used the glnE gene as a probe and a dominant band of approximately 3 kb which represents a monocistronic transcript was seen (Fig. 2). A faint smear from approximately 4 kb was seen in the 7H9 medium which could indicate the presence of a bicistronic transcript as well. This was not seen in the TSM-high ammonia medium. The fact that the RT-PCR indicates that a bicistronic message is formed, whereas the Northern analysis indicates that the major band is monocistronic, could imply either that the mRNA had been processed to give rise to two independent transcripts or that the major transcript was monocistronic for each gene and that co-transcription only occurred at a low level, possibly due to lack of efficient termination (Wernisch et al., 2003).
In order to determine if the co-transcript detected arose from a lack of transcriptional termination, we repeated the RT-PCR with a second set of primers which were located further towards the middle of glnA2 and glnE (Fig. 1). These primers should amplify a region of 691 bp extending approximately 200 bp into glnA2 and 400 bp into glnE. No product was detected (data not shown), confirming the Northern results that a full-length co-transcript was not detectable.
Expression of glnE
We tested whether there was any control of expression at the transcriptional level. We used Taqman RT-qPCR to measure levels of glnE mRNA in comparison to levels of sigA mRNA, which remain constant (Manganelli et al., 1999). As glnE is immediately downstream of glnA2, encoding the type II GS, we determined expression of that gene as well, from cells grown in TSM with high or low ammonia. There was no significant change in the levels of mRNA for either gene in response to ammonia levels [glnA2: 23±16 (low ammonia) vs 24±14 (high ammonia); glnE: 8±2 (low ammonia) vs 11±5 (high ammonia); arbitrary values (means±SD, n
3) expressed as percentage of sigA expression values]. We conclude that both genes are expressed in M. tuberculosis irrespective of ammonia concentration. The expression level of glnA2 was higher then that of glnE, which was in agreement with the relative levels of expression seen with the RT-PCR (Fig. 2).
Identification of transcriptional start site
In order to determine whether the glnE band in the Northern analysis was a primary transcript or arose from RNA processing, we mapped the 5' end of the mRNA and assayed for promoter activity upstream of the gene. The region upstream of glnE was examined for potential start sites using RACE analysis. This technique is widely used to find the 5' end of mRNA species and relies on amplification by PCR. We obtained one major PCR product and cloned this for sequencing, which identified the end of the PCR product. The major 5' end was at base +4 relative to the annotated translational start site. Therefore the predicted translational start site is probably incorrect and the GTG codon at position 7 is the correct start site, making the untranslated 5' region only 3 bp in length. This is not unusual in the actinomycetes and in fact in many cases the transcriptional and translational start sites are the same nucleotide (Kamalakannan et al., 2002; Timm et al., 1994).
Since RACE only detects the 5' ends of mRNA, it cannot distinguish between ends arising from true transcriptional start sites or those arising from processing. In addition, secondary structure in mRNA can give rise to artefactual ends, so we also looked for promoter consensus sequences upstream of our identified 5' end. There is a predicted promoter upstream of the identified glnE start site in the correct location (Fig. 3b), suggesting strongly that the start site identified by RACE represents a 5' end arising from transcription rather than processing. The –35 region of the predicted promoter is immediately downstream of the glnA2 stop codon within the short intergenic region.
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-Galactosidase assays from cells grown in either high- or low-ammonia media showed the presence of promoter activity (Table 2). There was no significant difference between the two conditions. This agreed with the RT-qPCR results, which showed no significant difference, but with slightly higher values in the high ammonia medium. We looked at promoter activity in both M. tuberculosis and the closely related, fast-grower M. smegmatis and saw a similar level of expression (Table 2); we used the latter as a genetic host for further studies. Promoter activity was measured in cells transformed with pLUSH5 carrying the glnE promoter grown on the various nitrogen sources (Fig. 3c). Again the high ammonia medium had higher activity than the low-ammonia medium, but the difference was not significant. In contrast, there were significant differences between the ammonia cultures (high or low) and all the amino acid cultures, which had lower activity (P<0.05).
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) confirming that this is indeed a promoter for glnE with a functional –10 region. The region we predicted to be the –35 box was also mutated from TTGATC to GTGAGC and assayed. Promoter activity was not lost (pLUSH35; Fig. 3), demonstrating that this region is not a true –35 region. This is not unexpected as most mycobacterial promoters do not possess a –35 region in the same way that Escherichia coli promoters do. Lack of a –35 region is often a sign that a promoter is controlled by a regulatory protein. Mutation of the –35 region also led to significant up-regulation in glutamate and under high-ammonia conditions, and down-regulation in low-ammonia conditions, suggesting that there may be an element of control of glnE expression, with a regulatory factor binding in the –35 region. However, the assays indicate that the promoter is always active.
The promoter region was analysed for the presence of potential nitrogen-regulatory motifs. GlnA1 of M. tuberculosis has a 
60 motif and is regulated in response to ammonia concentration (Harth et al., 2005), but no such motif is found in PglnE. There is no 54 in M. tuberculosis and no AmtR or GlnR regulator motifs (Fink et al., 2002; Jakoby et al., 2000) are found.
In order to characterize the promoter region more fully, we tested other mutated versions for activity in high ammonia conditions (Fig. 4). Mutation of two regions close to the –35 area had no effect on promoter activity (pLUSH5A and pLUSH5B). Mutation of the region immediately upstream of the –10 (TT to GG; pLUSH5C) had a significant effect on activity, reducing it approximately eightfold. Previous work has demonstrated that a subset of mycobacterial promoters have no –35 region, but have an extended –10 region instead (Agarwal & Tyagi, 2003; Bashyam & Tyagi, 1998). The extended –10 promoter region normally has a TGN motif immediately upstream of the –10 box, making a nonameric consensus sequence (TGNTATAAT). The two bases mutated in pLUSH5C could disrupt a TGN motif, although there is a 2 bp distance from the –10 region. The role of the TGN motif is to facilitate transition from the closed to the open promoter complex (Agarwal & Tyagi, 2003); since the mutated bases form part of a short string of three Ts, it may be that these have the same function. Alternatively, the mutated bases could be part of an alternative –10 region TgcgcT located at –15 to –10, although this sequence has fewer matches to the TATAAT consensus sequence.
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). Promoter activity was approximately four-fold higher than the glnE promoter, confirming the previous RT-PCR data. No difference in promoter activity was seen between high- and low-ammonia conditions.
Expression of glnE from alternative promoters
Although we detected a co-transcript of glnA2 and glnE by RT-PCR, Northern analysis showed that it was not the major transcript. Since there is no predicted terminator downstream of glnA2, it seems likely that co-transcription would occur, and in fact this is also the case in C. glutamicum (Nolden et al., 2001). There is conflicting evidence for members of the M. tuberculosis complex, where independent transcription of glnE was seen in M. bovis (Collins et al., 2002) and co-transcription of glnA2 and glnE was seen in M. tuberculosis (Harth et al., 2005). Our data show that independent transcription of glnE from a functional promoter can occur in M. tuberculosis. Two possibilities could account for these differences: either there are strain-specific expression patterns, since Harth et al. (2005) used Erdman, whereas we analysed H37Rv, or alternatively there may be different expression patterns depending on the media used. We looked at 7H9 (L-glutamate) alone, whereas Harth et al. (2005) used 7H9 plus ammonia.
We have previously seen that transcription does not always terminate downstream of genes in M. tuberculosis and can often lead to run-through transcription into other genes on the same or opposite reading strand (Wernisch et al., 2003). Whether these transcripts are translated has not been investigated, but this type of run-through transcription does have the potential to drive changes in protein levels. However, recruitment of RNA polymerase to PglnE would likely cause premature termination of this transcript and so the amount of a bicistronic message may be dependent on the strength of PglnE. Thus while the potential for transcription from PglnA2 is there, we believe it is not the major transcriptional start site for glnE.
Regulatory control of GlnE
The finding that glnE is expressed in all nitrogen conditions supports the essentiality data, since GlnE activity is constantly required. However, there is control of glnE expression, since up-regulation of promoter activity is seen when cells are grown on ammonia, even at low concentrations, as compared to some amino acids. This increased expression suggests that M. tuberculosis is particularly sensitive to the presence of ammonia as a nitrogen source, again possibly because of the danger that large amounts of active GS in the cell would convert all the glutamate into glutamine.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Bashyam, M. D. & Tyagi, A. K. (1998). Identification and analysis of "extended –10" promoters from mycobacteria. J Bacteriol 180, 2568–2573.
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Dussurget, O., Timm, J., Gomez, M., Gold, B., Yu, S. W., Sabol, S. Z., Holmes, R. K., Jacobs, W. R. & Smith, I. (1999). Transcriptional control of the iron-responsive fxbA gene by the mycobacterial regulator IdeR. J Bacteriol 181, 3402–3408.
Fink, D., Falke, D., Wohlleben, W. & Engels, A. (1999). Nitrogen metabolism in Streptomyces coelicolor A3(2): modification of glutamine synthetase I by an adenylyltransferase. Microbiology 145, 2313–2322.
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Harth, G., Maslesa-Galic, S., Tullius, M. V. & Horwitz, M. A. (2005). All four Mycobacterium tuberculosis glnA genes encode glutamine synthetase activities but only GlnA1 is abundantly expressed and essential for bacterial homeostasis. Mol Microbiol 58, 1157–1172.[CrossRef][Medline]
Jakoby, M., Nolden, L., Meier-Wagner, J., Kramer, R. & Burkovski, A. (2000). AmtR, a global repressor in the nitrogen regulation system of Corynebacterium glutamicum. Mol Microbiol 37, 964–977.[CrossRef][Medline]
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Nolden, L., Farwick, M., Kramer, R. & Burkovski, A. (2001). Glutamine synthetases of Corynebacterium glutamicum: transcriptional control and regulation of activity. FEMS Microbiol Lett 201, 91–98.[CrossRef][Medline]
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Wernisch, L., Kendall, S. L., Soneji, S., Wietzorrek, A., Parish, T., Hinds, J., Butcher, P. D. & Stoker, N. G. (2003). Analysis of whole-genome microarray replicates using mixed models. Bioinformatics 19, 53–61.
Received 21 February 2006;
revised 12 May 2006;
accepted 18 May 2006.
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), 3 mM alanine (
), 3 mM asparagine (
); (b) 7H9 (
), 30 mM glutamine (x). Results are the mean±SD of triplicate cultures (error bars not shown where smaller than symbols).
