Poly-L-glutamate/glutamine synthesis in the cell wall of Mycobacterium bovisis regulated in response to nitrogen availability
© Tripathi et al.; licensee BioMed Central Ltd. 2013
Received: 15 August 2013
Accepted: 8 October 2013
Published: 11 October 2013
The cell wall of pathogenic mycobacteria is known to possess poly-L-glutamine (PLG) layer. PLG synthesis has been directly linked to glutamine synthetase (GS) enzyme. glnA1 gene encodes for GS enzyme in mycobacteria. PLG layer is absent in cell wall of avirulent Mycobacterium smegmatis, although M. smegmatis strain expressing GS enzyme of pathogenic mycobacteria can synthesize PLG layer in the cell wall. The role of GS enzyme has been extensively studied in Mycobacterium tuberculosis, however, little is known about GS enzyme in other mycobacterial species. Mycobacterium bovis, as an intracellular pathogen encounters nitrogen stress inside macrophages, thus it has developed nitrogen assimilatory pathways to survive in adverse conditions. We have investigated the expression and activity of M. bovis GS in response to nitrogen availability and effect on synthesis of PLG layer in the cell wall. M. smegmatis was used as a model to study the behaviour of glnA1 locus of M. bovis.
We observed that GS expression and activity decreased significantly in high nitrogen grown conditions. In high nitrogen conditions, the amount of PLG in cell wall was drastically reduced (below detectable limits) as compared to low nitrogen condition in M. bovis and in M. smegmatis strain complemented with M. bovis glnA1. Additionally, biofilm formation by M. smegmatis strain complemented with M. bovis glnA1 was increased than the wild type M. smegmatis strain.
The physiological regulation of GS in M. bovis was found to be similar to that reported in other mycobacteria but this data revealed that PLG synthesis in the cell wall of pathogenic mycobacteria occurs only in nitrogen limiting conditions and on the contrary high nitrogen conditions inhibit PLG synthesis. This indicates that PLG synthesis may be a form of nitrogen assimilatory pathway during ammonium starvation in virulent mycobacteria. Also, we have found that M. smegmatis complemented with M. bovis glnA1 was more efficient in biofilm formation than the wild type strain. This indicates that PLG layer favors biofilm formation. This study demonstrate that the nitrogen availability not only regulates GS expression and activity in M. bovis but also affects cell surface properties by modulating synthesis of PLG.
KeywordsMycobacterium bovis Mycobacterium smegmatis Glutamine synthetase Poly-L-glutamine/glutamate Biofilm
Tuberculosis remains one of the major causes of concern related to human health because of increasing incidence of mortality and morbidity all over the world. Mycobacterium tuberculosis and Mycobacterium bovis are the two pathogens, responsible for the disease in humans and animals respectively. The emergence of drug resistant strains of M. tuberculosis and failure of the current drug regimen has worsened the situation even more . This has prompted renewed efforts to search for potential drug targets. In addition to this, there is an urgent requirement to bridge the massive gap in our understanding of pathogen’s complex biology to fight against disease.
Most of the studies on nitrogen metabolism have been focused primarily on other actinomycetes such as Streptomyces and Coynebacterium because of their role in industrial production of glutamine . Nitrogen assimilatory pathways are very poorly understood in mycobacterial species, especially M. bovis. Studies related to nitrogen metabolism in pathogens may help in understanding of complex cellular mechanisms by which M. bovis survive in nitrogen stress inside the macrophages. Glutamine and glutamate are the two major amino acids that act as cellular nitrogen donors for synthesis of biomolecules inside the cell . Hence, stringent regulatory pathways control the synthesis of glutamine and glutamate inside a bacterial cell . In mycobacteria, assimilation of inorganic nitrogen and its conversion to glutamine and glutamate is carried out by glutamine synthetase (GS) and glutamate synthetase .
Virulent forms of mycobacteria secrete huge amounts of extracellular GS enzyme and are also known to possess poly-L-glutamine (PLG) layer in the cell wall. The PLG layer is absent in cell wall of saprophytic mycobacteria e.g. M. smegmatis. Earlier, the treatment of M. tuberculosis with an inhibitor of GS, L-methionine-S-sulfoxamine, or with antisense oligonucleotides to glnA1 mRNA, has been shown to inhibit PLG formation in the cell wall [6, 7]. It indicated indirect involvement of glnA1 gene encoding the GS enzyme in the formation of PLG layer in M. tuberculosis. Later it was reported that expression of M. bovis GS in M. smegmatis resulted in synthesis of PLG layer in the cell wall and PLG significantly contribute strength to the cell wall against chemical and physical stresses such as lysozyme, SDS and sonication . Because of its presence exclusively in the cell wall of virulent mycobacteria and its role in providing cell wall strength it would be interesting to study the factors that can affect PLG synthesis directly or indirectly.
In this study, we investigated the behaviour of glnA1 gene of M. bovis both at the mRNA and protein levels in response to nitrogen availability. The present study emphasizes on the effect of nitrogen concentration on expression levels of glnA1 gene from the two different promoters when present independently or together. We have also studied the effect of nitrogen concentration on PLG layer synthesis in the cell wall of mycobacteria.
Bacterial strains and growth conditions
Plasmids and strains used in this study
amp R ori pUC (Cloning vector)
kan R (Mycobacterial shuttle non-integrative vector)
Stover et al., 1991 
pGEM-T Easy containing glnA1 coding sequence with native promoter
pMV261 containing glnA1 coding sequence with native promoter
pGEM-T Easy containing glnA1 coding sequence with P1 promoter
pMV261 containing glnA1 coding sequence with P1 promoter
pMV261 containing glnA1 coding sequence with P2 promoter
supE44 ΔlacU(Φ80lacZΔM15) hsdR17 rec1 endA1 gyrA96 thi-1 relA1
M. bovis AN5
M. smegmatis mc2
M. smegmatis containing pDS2
M. smegmatis containing pDS4
M. smegmatis containing pDS5
For creating low and high nitrogen conditions, mycobacterial strains were grown in 7H9 medium (without ADC enrichment) containing 3.8 mM ammonium sulphate and 60 mM ammonium sulphate respectively. It has previously been reported that a change in nitrogen concentration from 3 mM to 60 mM leads to a reduction in GS activity in wild type M. smegmatis.
The wild type M. smegmatis strain used in the study was complemented with only pMV261 vector and was used as a vector control. All work involving virulent strain was performed in Bio-safety Level-3 laboratory at Jawaharlal Nehru University, New Delhi.
Cloning of M. bovis glnA1 gene with its native promoter and construction of its deleted promoter variants in M. smegmatis
Cloning was performed using standard procedures. The glnA1 gene with its upstream promoter region (1776 bp) was amplified using M. bovis genomic DNA as template. For PCR amplification of the gene, forward primer 1 with BamHI site and reverse primer 2 with PstI site (Additional file 1: Table S1), were used. The amplified DNA fragment was cloned in pGEM-T Easy PCR cloning vector, verified by sequencing and named as pDS1. The insert was excised from pDS1 by restriction digestion with BamHI/PstI, and then ligated in pMV261, E. coli-Mycobacterium shuttle vector, producing pDS2 plasmid. The resulting construct pDS2 was electroporated into wild type M. smegmatis strain and the transformed strain was named MSFP.
The glnA1 promoter of M. bovis contains two regulatory promoters P1 and P2 (Figure 1). For the generation of construct carrying only the P1 promoter with glnA1 gene downstream, the P2 promoter was deleted by direct PCR method. A forward primer 3 with BamHI site immediately from the start of the P1 promoter and reverse primer 2 with PstI site at the end of glnA1 gene (Additional file 1: Table S1) were designed and were used to amplify glnA1 gene which lacked the P2 promoter. The amplified (1561 bp) product was cloned in pGEM-T Easy vector (pDS3) and then sub-cloned in pMV261 vector at BamH1-Pst1 sites (pDS4) (Table 1). Following this, for generation of construct carrying only P2 promoter with glnA1 gene, P1 was deleted by the inverse PCR. In this method a primer was designed such that the sequence containing the P1 promoter was excluded. A forward primer 4 and reverse primer 5 were designed from the 3′ end of P1 promoter and 3′ end of P2 promoter respectively. PCR amplification by using template pDS2 resulted in the amplification of whole vector containing glnA1 gene with P2 promoter (deletion of 31 bp) (Figure 1). The amplified PCR product was ligated after 5′ kinasing by T4 polynucleotide kinase and then the resulting construct was named as pDS5. The constructs pDS4 and pDS5 were then electroporated in wild type M. smegmatis and hence transformants obtained were named as MSP1 and MSP2 respectively.
Growth patterns of recombinant M. smegmatis and M. bovisstrains in low and high nitrogen conditions
Log phase cultures of M. smegmatis and M. bovis strains were inoculated in 7H9 medium containing low and high nitrogen conditions. The cultures were grown at 37°C at 200 rpm. The optical density was measured periodically at 600 nm.
Semi quantitative RT-PCR and real time PCR
M. smegmatis and M. bovis strains were grown in low and high nitrogen conditions and total RNA was isolated by Trizol method. In brief, semi quantitative RT-PCR was performed using One Step RT-PCR Kit (Qiagen) according to manufacturer’s instructions. For glnA1 gene, forward primer 10 and internal reverse primer 11 was used to amplify 400 bp fragment of the gene by using DNase I treated RNA as template. A sigA gene fragment was amplified using primers 8 and 12 as a loading control. The PCR conditions were, 50°C for 40 min, 94°C for 15 min and 24 cycles of 94°C denaturation for 30 sec, 58°C annealing for 30 sec and 72°C extension for 30 sec.
For real time PCR, DNase I treated RNA was taken for cDNA synthesis using High capacity cDNA reverse transcription kit (Applied Biosystems) employing random hexamer primers. The PCR reactions were run in ABI PRISM 7500HT sequence detection system (Applied Biosystems) using the following program: 95°C for 10 min and 40 cycles of 95°C for 10 sec, 60°C for 10 sec and 72°C for 10 sec. The forward primer 6 and reverse primer 7 were used for glnA1 gene. The primer 8 and 9 were used for sigA gene and was used as internal control for data normalization. Each reaction was performed in triplicates. The relative changes in gene expression was calculated using the 2-∆∆CT method and the data was represented in the form of fold change in gene expression, normalized to sigA gene and relative to the control condition.
Determination of GS expression and activity
All strains were grown in low and high nitrogen conditions. The M. smegmatis strains were cultured for 2 days while M. bovis was cultured for 12 days. Then the culture filtrate was harvested. The culture filtrates were passed through 0.22 μm syringe filter and then concentrated 100 times of the original volume using 30 kDa molecular weight cut off Amicon filter (Millipore). The GS activity in the extracellular protein fraction was measured by γ-glutamyl transfer reaction as described previously  and was expressed as micromoles hydroxamate formed, based on a standard curve obtained with pure γ-glutamylhydroxamate purchased from sigma.
For the cytoplasmic protein fractions, cell pellets were taken and washed with 50 mM Tris–HCl pH 7.5 and digested with 10 μg/ml lysozyme. Cell pellets were resuspended in 1 ml of 50 mM Tris–HCl with 1X protease inhibitor. The M. smegmatis cell suspensions were sonicated on ice for 5–10 minutes while the M. bovis cell suspension was sonicated for 30 minutes, because the cell wall of virulent mycobacteria are relatively more resistant to physical stress like sonication. The GS activity in the cellular fraction was measured by the above mentioned protocol. The intracellular protein expression was determined by SDS-PAGE and western blotting by anti-GS antibody. The amount of total protein was measured by Bradford assay and equal amount of total protein was loaded for each sample.
Isolation and estimation of PLG in mycobacterial strain
Cell pellet of exponential phase culture (200 ml) of all strains was harvested after growing in low and high nitrogen condition and cell wall was prepared. The PLG was purified as reported earlier . The cell pellet was suspended in 10 ml of breaking buffer. The suspension was sonicated in an ice bath for 3–4 hrs.
The cell lysate was treated with 20 μl of 10 μg/ml ribonuclease and 20 units of deoxyribonuclease and kept overnight at 4°C.
Treated cell lysate was centrifuged at 27,000 g for 20 min, and the resulting cell wall-containing pellet was extracted with 2% (w/v) sodium dodecyl sulfate (SDS) for 2 h at 60°C to remove soluble protein and membrane. The extracted cell walls were washed extensively with PBS (phosphate buffer saline), distilled water and 80% (v/v) aqueous acetone to remove SDS.
Cell walls were suspended in a small volume of PBS and placed on a discontinuous sucrose gradient composed of 15, 25, 30, 40, and 60% (w/v) sucrose.
The gradient was centrifuged at 100,000 g for 2 hr.
The cell wall was settled at the 30 to 40% interface, whereas the associated PLG pelleted to the bottom of the tube.
The PLG material was transferred to a tube containing 80% Percoll (Sigma) in PBS-0.1% Tween 80 and centrifuged at 100,000 g for 20 min.
This allowed formation of a gradient in situ and distinct banding of the insoluble, pure PLG. The presence of PLG was confirmed by GC-MS analysis, after hydrolysis of the samples at 110°C for 20 h with 6 N HCl followed by esterification with heptafluorobutyryl isobutyl anhydride . GC-MS was done at Advanced Instrumentation Research Facility, JNU New Delhi by Shimadzu GC-MS 2010, and Rtx-5 MS capillary column (Restek) with an oven temperature range of 90-180°C (5 min) at 4°C/min raised to 300°C at 4°C/min. The injection temperature used was 280°C along with an interface temperature of 290°C. MS data were analyzed in the NIST05.LIB and WILEY8.LIB chemical libraries.
Immunogold localization of PLG by transmission electron microscopy
Immunoelectron microscopy was performed to confirm the presence of PLG in the cell wall of M. smegmatis and M. bovis strains grown under different nitrogen conditions. Immunogold localization was done as described earlier  at the Transmission Electron Microscopy Facility, Advanced Instrumentation Research Facility, JNU, New Delhi. Briefly, cells from log-phase cultures of M. bovis and M. smegmatis strains were harvested and washed with 0.1 M phosphate buffer. The cells were treated with immune gold fixative (4% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M phosphate buffer), then washed and embedded in 2.5% agar. The agar-encased bacteria were then dehydrated and embedded using LR white resin (Electron Microscopy Sciences). Thin sections (100 nm) were obtained using Leica Ultracut (Leica, Germany) and collected on Nickel grids (200 mesh; Electron Microscopy Sciences). For localization, monoclonal anti-PLG antibody (1:100) (Sigma) was used. The grids were washed and subsequently treated with gold (10 nm) conjugated - anti mouse IgG. Mice pre-immune serum was used as a negative control. The immunolabeled sections were stained with uranyl acetate and viewed using a Jeol 2100 F transmission electron microscope (Jeol Analytic Instruments) at an acceleration voltage of 120 KV.
Biofilm formation was observed by growing static cultures of mycobacteria without shaking in 7H9 medium without Tween 80 at 37°C. Biofilm formation was assayed by crystal violet staining method developed by Reicht et al.[19, 20]. Briefly, 200 μl of stationary phase cultures (A600 normalized to 1) were added to 7H9 medium in polystyrene culture plates for biofilm formation and in culture tubes for pellicle formation. After incubation of static culture of M. smegmatis strains for 2 days and M. bovis for 2–3 weeks, biofilm was quantified by removing the medium carefully and staining with 1% crystal violet for 45 min. The wells were washed three times with water and air-dried. The dye was solubilized with 80% ethanol and A550 was measured.
Generation of glnA1promoter variants
Relative quantification of glnA1 transcript of recombinant M. smegmatisstrains
Real time PCR was performed further to study glnA1 expression quantitatively in low and high nitrogen conditions for MSFP, MSP1, MSP2, wild type M. smegmatis and M. bovis strains. The glnA1 expression levels in wild type M. smegmatis in low nitrogen condition was taken as the reference point in order to calculate the fold change in recombinant strains. The data obtained from real time PCR was normalized to sigA expression levels, as an internal control. It was observed that in case of nitrogen starvation, the expression of glnA1 gene in MSFP and MSP1 strains was highly up-regulated. It was observed that in MSFP glnA1 expression was ~ 40 fold high in ammonium starvation, while it was just ~ 6 fold more in high nitrogen conditions as compared to wild type M. smegmatis (which was taken as a reference point to calculate fold change for all the strains) (Figure 4B). In MSP1 glnA1 expression in low and high nitrogen conditions was up-regulated ~ 42 and ~ 15 fold respectively. The glnA1 expression in MSFP in high nitrogen was ~ 6 fold less than expression in low nitrogen while the same was only ~ 3 fold in MSP1. In case of MSP2, the expression of glnA1 gene was comparable in both low and high nitrogen conditions. In case of M. bovis, the expression of glnA1 was also ~ 36 fold up-regulated in low nitrogen conditions as compared to ~ 6.2 fold in high nitrogen conditions. Hence it was observed that in the strains, MSFP and M. bovis, where both the promoters P1 and P2 were present upstream to glnA1, the difference in the gene expression levels in low and high nitrogen conditions were significantly higher as compared to the difference in expression levels in strains having single promoter. It was concluded that deletion of any one of the two promoters decreased the stringent regulation of glnA1 gene at the transcriptional level.
GS specific activity and expression in response to nitrogen limitation and excess
Estimation of PLG from M. bovis and recombinant M. smegmatisstrains
Estimation of PLG
Dry cell weight
Cell wall weight after sonication
Insoluble cell wall after SDS extraction and acetone wash
Poly-L-glutamine pelleted after sucrose gradient centrifugation
Poly-L-glutamine purified after percoll run
Immunogold localization of PLG by transmission electron microscopy
Effect on biofilm formation
Nitrogen metabolism has been studied in detail in industrially important organisms such as Streptomyces and Corynebacteria but there have been very few reports on nitrogen metabolism of mycobacterial species. Earlier, several studies have reported that glnA1 gene is up-regulated in nitrogen starvation in M. tuberculosis and M. smegmatis[5, 12] but this study emphasizes on behaviour of glnA1 locus of M. bovis at both transcriptional and translational levels by altering nitrogen concentration in the medium. Also nitrogen conditions modulate the cell wall properties by altering synthesis of PLG layer in mycobacteria.
The conversion of glutamate to glutamine demands high energy consumption inside the cell. Because of this reason the expression of glnA1 gene is tightly regulated in most mycobacterial species. The transcription of glnA1 gene is regulated in M. tuberculosis by dual promoters . The P1 promoter, present just upstream to glnA1 gene is low nitrogen responding promoter while the P2 promoter, upstream to P1 is high nitrogen responding promoter . Further regulation is driven by GlnR protein which has putative binding site in the P1 promoter. GlnR binds to the P1 promoter and activates transcription during nitrogen starvation . In this study, we have studied the expression level of glnA1 gene of M. bovis in response to nitrogen availability, when the two promoters P1 and P2, are present independently or together. The real time data observed are in accordance with the earlier findings about the regulation of glnA1 gene at transcription level in response to nitrogen availability [11, 12]. The results clearly showed up-regulation of glnA1 expression in M. bovis and MSFP strains in low nitrogen conditions as compared to high nitrogen conditions. MSFP, MSP1 and M. bovis strains have P1 promoter upstream to the glnA1 gene and P1 promoter has binding site for GlnR protein. GlnR binds to the P1 promoter and activates transcription in low nitrogen conditions . This may be the reason for the differences observed in the expression level of the gene in low nitrogen and high nitrogen conditions in these strains. While, on the other hand in MSP2 strain there was no difference in glnA1 expression level in low and high nitrogen conditions. This may be due to lack of P1 promoter and hence GlnR binding site. Also, it can be observed that the difference in gene expression in low and high nitrogen conditions are higher in MSFP and M. bovis strains that have both the promoters upstream to the glnA1 gene. This difference is somewhat reduced in MSP1 and completely lost in MSP2 strain. It has been reported earlier that P1 promoter in M. tuberculosis is σ 60 type promoter . σ 60 is expressed in nitrogen limiting conditions, it recognizes the P1 promoter and transcription starts from P1 promoter.
In addition to regulation at the transcriptional level, GS enzyme encounters a second regulation at post translational level. GlnE protein adenylylate the GS protein in high nitrogen condition and thus makes it inactive [13, 22]. In all the strains, the difference in GS activity in ammonium starvation to ammonium pulse was significantly higher than the difference in expression at mRNA level. Hence, this marked difference observed in GS activity with change in nitrogen conditions in M. bovis, MSFP and MSP1 may be because of two possible reasons. First, there is a stringent regulatory mechanism exhibited by GlnR protein at the transcriptional level because of which the transcript of glnA1 gene itself, is significantly low in high nitrogen conditions. Secondly, after translation, GlnE protein comes into play and modifies the GS enzyme in high nitrogen conditions which makes GS enzyme inactive [13, 22]. MSP2 strain showed low expression of glnA1 gene as compared to the expression in other strains in low nitrogen condition because there was no regulation at transcriptional level due to lack of P1 promoter hence lack of GlnR binding motif also.
PLG layer has been known to be present in the cell wall of only virulent strains of mycobacteria [16, 23]. Harth and colleagues indicated that extracellular GS of pathogenic mycobacteria is involved in synthesis of this layer [10, 24, 25]. There has also been reports stating the involvement of PLG layer of M. bovis in cell wall strength and in providing resistance to various physical and chemical stress factors . The absence of PLG layer from the cell wall of mycobacteria grown in high nitrogen condition indirectly suggest that PLG layer may be a form of nitrogen assimilation in pathogenic mycobacteria. In macrophages, mycobacteria encounter nitrogen stress which leads to high GS expression and PLG layer synthesis in the cell wall. Immunogold localization and PLG isolation studies further validated the finding of no detectable PLG in the cell wall of M. bovis, MSFP, MSP1 and MSP2 strains when grown in high nitrogen conditions.
The ability of the pathogenic mycobacteria to form biofilm adds on to their virulence potential . Biofilm formed at air liquid interface are popularly known as pellicle. Additionally, mycolic acids are the major component of the biofilms formed by mycobacterial species [26, 27] but it is not clearly known whether mycolic acid synthesis or its amount in cell wall is affected by PLG layer. However, there are few reports that suggest the involvement of PLG layer in biofilm formation . A ∆glnA1 strain of M. bovis that lack PLG layer in the cell wall was found to be defective in biofilm formation . Additionally, our results showed that the biofilm and pellicle forming capability of M. smegmatis strain complemented with M. bovis glnA1 was enhanced than the wild type. This is due to the fact that higher expression of M. bovis glnA1 leads to the synthesis of PLG layer in the M. smegmatis complemented with M. bovis glnA1. There are reports also suggesting that microbial amyloids play a significant role in biofilms of actinobacteria [28, 29]. Additionally, it was observed that biofilm was formed significantly much better in low nitrogen conditions which added to the involvement of PLG layer in biofilm formation.
There is a gap in our understanding of the exact mechanisms and enzymes involved in the synthesis of PLG layer till date. In addition to it, characterization of PLG layer, can further help in our understanding of complex mycobacterial cell wall. Because of high molecular weight and inert nature of the polymer it may also act as an adjuvant. This needs further investigation. Establishment of the pathways involved in PLG synthesis will further help in identification of new drug targets against tuberculosis. The study of nitrogen metabolism can provide an insight in the survival of these pathogens in adverse conditions for long duration of time. Also this can help us to understand the mechanisms by which bacteria are able to survive and replicate in macrophages.
In the current study we have investigated the expression of glnA1 gene of M. bovis in response to nitrogen availability. This study revealed for the first time that amount of PLG in the cell wall of M. bovis is substantially reduced when grown in high nitrogen conditions. The data presented here significantly enhance our understanding of the regulation of the glnA1 gene which is linked to synthesis of the PLG layer in the cell wall of M. bovis in altering nitrogen conditions. The localization study of PLG layer in the cell wall, as shown by immunogold studies has also been reported for the first time.
We are grateful to Council of Scientific and Industrial Research (CSIR), India for financial support. We are thankful to Dr. Nirupama Banerjee ICGEB, India for providing the plasmid pMV261 and mycobacterial strains. We also acknowledge Dr. Sashi Kant and Dr. Divya Goel for critical reading of the manuscript. GC-MS analysis and Immunogold localization studies were performed at Advanced Instrumentation Research Facility, JNU, New Delhi.
- Johnson R, Streicher EM, Louw GE, Warren RM, van Helden PD, Victor TC: Drug resistance in Mycobacterium tuberculosis. Curr Issues Mol Biol. 2006, 8 (2): 97-111.PubMedGoogle Scholar
- Nolden L, Farwick M, Kramer R, Burkovski A: Glutamine synthetases of Corynebacterium glutamicum: transcriptional control and regulation of activity. FEMS Microbiol Lett. 2001, 201 (1): 91-98. 10.1111/j.1574-6968.2001.tb10738.x.PubMedView ArticleGoogle Scholar
- Newsholme P, Procopio J, Lima MM, Pithon-Curi TC, Curi R: Glutamine and glutamate-their central role in cell metabolism and function. Cell Biochem Funct. 2003, 21 (1): 1-9. 10.1002/cbf.1003.PubMedView ArticleGoogle Scholar
- Umbarger HE: Amino acid biosynthesis and its regulation. Annu Rev Biochem. 1978, 47: 532-606.PubMedView ArticleGoogle Scholar
- Harper CJ, Hayward D, Kidd M, Wiid I, van Helden P: Glutamate dehydrogenase and glutamine synthetase are regulated in response to nitrogen availability in Myocbacterium smegmatis. BMC Microbiol. 2010, 10: 138-10.1186/1471-2180-10-138.PubMedPubMed CentralView ArticleGoogle Scholar
- Harth G, Zamecnik PC, Tang JY, Tabatadze D, Horwitz MA: Treatment of Mycobacterium tuberculosis with antisense oligonucleotides to glutamine synthetase mRNA inhibits glutamine synthetase activity, formation of the poly-L-glutamate/glutamine cell wall structure, and bacterial replication. Proc Natl Acad Sci U S A. 2000, 97 (1): 418-423. 10.1073/pnas.97.1.418.PubMedPubMed CentralView ArticleGoogle Scholar
- Harth G, Horwitz MA: Inhibition of Mycobacterium tuberculosis glutamine synthetase as a novel antibiotic strategy against tuberculosis: demonstration of efficacy in vivo. Infect Immun. 2003, 71 (1): 456-464. 10.1128/IAI.71.1.456-464.2003.PubMedPubMed CentralView ArticleGoogle Scholar
- Chandra H, Basir SF, Gupta M, Banerjee N: Glutamine synthetase encoded by glnA-1 is necessary for cell wall resistance and pathogenicity of Mycobacterium bovis. Microbiology. 2010, 156 (Pt 12): 3669-3677.PubMedView ArticleGoogle Scholar
- Amon J, Titgemeyer F, Burkovski A: A genomic view on nitrogen metabolism and nitrogen control in mycobacteria. J Mol Microbiol Biotechnol. 2009, 17 (1): 20-29. 10.1159/000159195.PubMedView ArticleGoogle Scholar
- Harth G, Horwitz MA: Expression and efficient export of enzymatically active Mycobacterium tuberculosis glutamine synthetase in Mycobacterium smegmatis and evidence that the information for export is contained within the protein. J Biol Chem. 1997, 272 (36): 22728-22735. 10.1074/jbc.272.36.22728.PubMedView ArticleGoogle Scholar
- Tiffert Y, Supra P, Wurm R, Wohlleben W, Wagner R, Reuther J: The Streptomyces coelicolor GlnR regulon: identification of new GlnR targets and evidence for a central role of GlnR in nitrogen metabolism in actinomycetes. Mol Microbiol. 2008, 67 (4): 861-880. 10.1111/j.1365-2958.2007.06092.x.PubMedView ArticleGoogle Scholar
- Harper C, Hayward D, Wiid I, van Helden P: Regulation of nitrogen metabolism in Mycobacterium tuberculosis: a comparison with mechanisms in Corynebacterium glutamicum and Streptomyces coelicolor. IUBMB Life. 2008, 60 (10): 643-650. 10.1002/iub.100.PubMedView ArticleGoogle Scholar
- Mehta R, Pearson JT, Mahajan S, Nath A, Hickey MJ, Sherman DR, Atkins WM: Adenylylation and catalytic properties of Mycobacterium tuberculosis glutamine synthetase expressed in Escherichia coli versus mycobacteria. J Biol Chem. 2004, 279 (21): 22477-22482. 10.1074/jbc.M401652200.PubMedView ArticleGoogle Scholar
- Stover CK, de la Cruz VF, Fuerst TR, Burlein JE, Benson LA, Bennett LT, Bansal GP, Young JF, Lee MH, Hatfull GF, et al: New use of BCG for recombinant vaccines. Nature. 1991, 351 (6326): 456-460. 10.1038/351456a0.PubMedView ArticleGoogle Scholar
- Woolfolk CA, Shapiro B, Stadtman ER: Regulation of glutamine synthetase I. Purification and properties of glutamine synthetase from Escherichia coli. Arch Biochem Biophys. 1966, 116 (1): 177-192.PubMedView ArticleGoogle Scholar
- Hirschfield GR, McNeil M, Brennan PJ: Peptidoglycan-associated polypeptides of Mycobacterium tuberculosis. J Bacteriol. 1990, 172 (2): 1005-1013.PubMedPubMed CentralGoogle Scholar
- MacKenzie SL, Hogge LR: Gas chromatography–mass spectrometry of the N(O)-heptafluorobutyryl isobutyl esters of the protein amino acids using electron impact ionisation. J Chromatogr. 1977, 132 (3): 485-493. 10.1016/S0021-9673(00)82912-X.PubMedView ArticleGoogle Scholar
- Burghardt RC, Droleskey R: Transmission electron microscopy. Curr Protoc Microbiol. 2006, 3:2B.1.1–2B.1.39-Google Scholar
- Recht J, Kolter R: Glycopeptidolipid acetylation affects sliding motility and biofilm formation in Mycobacterium smegmatis. J Bacteriol. 2001, 183 (19): 5718-5724. 10.1128/JB.183.19.5718-5724.2001.PubMedPubMed CentralView ArticleGoogle Scholar
- Recht J, Martinez A, Torello S, Kolter R: Genetic analysis of sliding motility in Mycobacterium smegmatis. J Bacteriol. 2000, 182 (15): 4348-4351. 10.1128/JB.182.15.4348-4351.2000.PubMedPubMed CentralView ArticleGoogle Scholar
- Kimura K, Yagi K, Matsuoka K: Regulation of Mycobacterium smegmatis glutamine synthetase by adenylylation. J Biochem. 1984, 95 (6): 1559-1567.PubMedGoogle Scholar
- Parish T, Stoker NG: glnE is an essential gene in Mycobacterium tuberculosis. J Bacteriol. 2000, 182 (20): 5715-5720. 10.1128/JB.182.20.5715-5720.2000.PubMedPubMed CentralView ArticleGoogle Scholar
- Wietzerbin-Falszpan J, Das BC, Gros C, Petit JF, Lederer E: The amino acids of the cell wall of Mycobacterium tuberculosis var. bovis, strain BCG. Presence of a poly(L-glutamic acid). Eur J Biochem. 1973, 32 (3): 525-532. 10.1111/j.1432-1033.1973.tb02637.x.PubMedView ArticleGoogle Scholar
- Harth G, Clemens DL, Horwitz MA: Glutamine synthetase of Mycobacterium tuberculosis: extracellular release and characterization of its enzymatic activity. Proc Natl Acad Sci U S A. 1994, 91 (20): 9342-9346. 10.1073/pnas.91.20.9342.PubMedPubMed CentralView ArticleGoogle Scholar
- Harth G, Horwitz MA: An inhibitor of exported Mycobacterium tuberculosis glutamine synthetase selectively blocks the growth of pathogenic mycobacteria in axenic culture and in human monocytes: extracellular proteins as potential novel drug targets. J Exp Med. 1999, 189 (9): 1425-1436. 10.1084/jem.189.9.1425.PubMedPubMed CentralView ArticleGoogle Scholar
- Ojha AK, Baughn AD, Sambandan D, Hsu T, Trivelli X, Guerardel Y, Alahari A, Kremer L, Jacobs WR, Hatfull GF: Growth of Mycobacterium tuberculosis biofilms containing free mycolic acids and harbouring drug-tolerant bacteria. Mol Microbiol. 2008, 69 (1): 164-174. 10.1111/j.1365-2958.2008.06274.x.PubMedPubMed CentralView ArticleGoogle Scholar
- Ojha A, Anand M, Bhatt A, Kremer L, Jacobs WR, Hatfull GF: GroEL1: a dedicated chaperone involved in mycolic acid biosynthesis during biofilm formation in mycobacteria. Cell. 2005, 123 (5): 861-873. 10.1016/j.cell.2005.09.012.PubMedView ArticleGoogle Scholar
- Larsen P, Nielsen JL, Dueholm MS, Wetzel R, Otzen D, Nielsen PH: Amyloid adhesins are abundant in natural biofilms. Environ Microbiol. 2007, 9 (12): 3077-3090. 10.1111/j.1462-2920.2007.01418.x.PubMedView ArticleGoogle Scholar
- Blanco LP, Evans ML, Smith DR, Badtke MP, Chapman MR: Diversity, biogenesis and function of microbial amyloids. Trends Microbiol. 2012, 20 (2): 66-73. 10.1016/j.tim.2011.11.005.PubMedPubMed CentralView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.