- Research article
- Open Access
Defining the nitrogen regulated transcriptome of Mycobacterium smegmatis using continuous culture
© Petridis et al. 2015
- Received: 25 June 2015
- Accepted: 6 October 2015
- Published: 19 October 2015
Nitrogen is essential for microbial growth and its importance is demonstrated by the complex regulatory systems used to control the transport, assimilation and metabolism of nitrogen. Recent studies are beginning to shed light on how mycobacteria respond to nitrogen limitation and several regulators (e.g., GlnR, PII) have been characterized at a molecular level. However, despite this progress, our knowledge of the transcriptional response of mycobacteria to nitrogen limitation and its regulation is confined to batch culture.
To gain further insight into the response of mycobacteria to nitrogen limitation, we developed a nitrogen-limited chemostat. We compared the transcriptional response of nitrogen-limited cells to carbon-limited cells using RNA-seq analysis in a continuous culture model at a constant growth rate.
Our findings revealed significant changes in the expression of 357 genes (208 upregulated, 149 downregulated; >2-fold change, false discovery rate <5 %) in response to nitrogen limitation in continuous culture. The vast majority of the GlnR regulon (68 %) was differentially expressed under nitrogen limitation in continuous culture and approximately 52 % of the 357 genes overlapped with a previously published study investigating the response of M. smegmatis to nitrogen limitation in batch culture, while expression of only 17 % of the genes identified in batch culture were affected in our chemostat model. Moreover, we identified a unique set of 45 genes involved in the uptake and metabolism of nitrogen that were exclusive to our chemostat model. We observed strong downregulation of pathways for amino acid catabolism (i.e., alanine, aspartate, valine, proline and lysine), suggesting preservation of these amino acids for critical cellular function. We found 16 novel transcriptional regulators that were directly or indirectly involved in the global transcriptomic response of M. smegmatis to nitrogen limitation and identified several non-coding RNAs that might be involved in the transcriptional or post-transcriptional regulation of nitrogen-regulated gene expression.
Using nitrogen-limited continuous culture we identified the nitrogen-responsive transcriptome of M. smegmatis, including a number of small non-coding RNAs implicated in controlling nitrogen-regulated gene expression.
- Nitrogen depletion
- Continuous culture
- Gene expression profiling
Carbon and nitrogen are key components of organic material and their availability in the environment is necessary for growth and survival of microorganisms. The major mechanisms of carbon source utilization and its regulation are well defined for many groups of microorganisms, but our knowledge of nitrogen metabolism and its regulation is largely confined to enteric bacteria (for review see [1–3]). The importance of nitrogen for microbial growth is demonstrated by the complex regulatory systems used to control nitrogen assimilation in response to internal and external nitrogen levels. In some bacteria, nitrogen availability is sensed by the intracellular ratio of 2-oxoglutarate:glutamine . This signal ensures that the uptake of nitrogen sources are commensurate with the metabolic requirements of the organism i.e. carbon or nitrogen. High 2-oxoglutarate levels signal nitrogen limitation, while high glutamine levels signal nitrogen excess in bacteria [1, 4, 5].
In enteric bacteria, nitrogen metabolism is regulated by the two-component regulatory system NtrBC and the signal transduction protein PII . These regulatory systems coordinate nitrogen metabolism by regulating genes involved in ammonium assimilation, amino acid transport and nitrate metabolism through protein-protein interactions [1, 4, 6, 7]. An integral component of this regulation is GlnD, an uridylyl transferase/uridylyl-removing enzyme that senses and transmits the nitrogen status of the cell to the PII protein . The PII protein in its uridylylated state stimulates phosphorylation of the response regulator NtrC by the NtrB kinase, which results in expression of the NtrBC-regulated genes under nitrogen limitation . Homologues of the PII protein are found in many bacterial genomes, where they are mainly involved in the regulation of nitrogen metabolism. However, the PII protein may have additional roles, as recently described in cyanobacteria, where it regulates the metabolism of inorganic carbon .
Actinomycetes are a group of Gram-positive bacteria that inhabit a wide range of aquatic, terrestrial and human habitats. In actinobacteria, two different regulatory systems have been identified that regulate the transcriptional response to nitrogen limitation, AmtR, a TetR-type transcriptional regulator and GlnR, an OmpR-type transcriptional regulator [11–13]. AmtR was identified in Corynebacterium glutamicum to regulate more than 30 genes involved in nitrogen metabolism, including the PII protein, glutamine synthetase, glutamate synthase and urease . In Streptomyces coelicolor, GlnR mediates the transcriptional response to nitrogen limitation by elevating expression of the amtB-glnK-glnD operon and the glutamine synthetase glnA, while repressing transcription of the glutamate dehydrogenase gdhA [13, 14].
Slow-growing mycobacteria such as Mycobacterium tuberculosis appear to harbor only GlnR that was shown to regulate the expression of at least 33 genes in response to nitrogen limitation . M. tuberculosis can exploit different nitrogen sources, however, organic nitrogen sources (e.g. asparagine and aspartate) are preferred for extracellular and intracellular growth [16, 17]. Amino acids can function as both carbon and nitrogen sources, and although M. tuberculosis can metabolize a variety of amino acids in vitro, only a small number (aspartate, glutamate, asparagine and glutamine) can support growth of M. tuberculosis at the acidic pH that prevails in host macrophages . Inorganic nitrogen sources such as ammonium and nitrate are less efficient for growth than amino acids , but the reasons for this remain unknown.
The genome of the fast growing saprophytic actinobacterium Mycobacterium smegmatis contains copies of both global nitrogen regulators, GlnR and AmtR . It has recently been demonstrated that GlnR regulates the expression of more than one hundred genes in response to nitrogen limitation in M. smegmatis , while the AmtR regulon remains unknown. Previous microarray studies using batch culture nitrogen run out experiments have identified 1090 genes in M. smegmatis that are differentially expressed in response to nitrogen limitation . However, only a small subset of these genes is under control of GlnR, while the regulatory mechanisms for the majority have yet to be identified [20, 21]. During nitrogen run out studies, the growth rate changes significantly as nitrogen becomes depleted and therefore these types of experiments may not unequivocally uncover genes that respond solely to nitrogen limitation.
The aim of the current study was to deliver a molecular framework for how a mycobacterial cell responds to nitrogen-depleted and -replete conditions at a defined growth rate. To address this aim, we developed a nitrogen-limited chemostat for M. smegmatis at a constant growth rate of 0.12 h−1 (td = 5.7 h) followed by RNA-seq analysis to identify genes responding to nitrogen limitation.
Development and validation of nitrogen-limited continuous culture to understand the global transcriptomic response to nitrogen limitation
The first step of this study was to establish a defined minimal medium limited for either nitrogen or carbon to identify the molecular response of M. smegmatis to nitrogen limitation. We modified HdB medium  by replacing 0.05 % (w/v) Tween-80 with 0.05 % (w/v) Tyloxapol, which cannot be metabolized as a carbon source, and (NH4)2SO4 with NH4Cl and K2SO4 (11.4 mM) to avoid simultaneous limitation for sulphur and nitrogen. Inorganic nitrogen sources (e.g. ammonium) are less efficient for growth than amino acids, however, we chose an inorganic nitrogen source to allow tighter control of the nitrogen to carbon ratio in the medium. Using batch culture, we identified glycerol as a suitable carbon source, with a high cell yield and no change in external pH during the course of the experiment compared to other carbon sources such as glucose, acetate or succinate (Additional file 1, Figure S1A). When cells were grown in batch culture on glycerol (25 mM) under nitrogen-replete conditions, growth ceased at an optical density (OD600) of 3.5 (Additional file 1, Figure S1B). When the culture was limited for nitrogen (carbon excess), the final OD600 was 2.1 (Additional file 1, Figure S1B). Based on these experiments we used 30 mM glycerol and 10 mM NH4Cl (nitrogen-replete)/1.25 mM NH4Cl (nitrogen-depleted) for continuous culture studies.
For each condition, three independent biological replicates were grown until steady-state-phase and cells were harvested after at least 3 volume changes in the chemostat vessel. The analysis of the transcriptome using RNA-sequencing revealed a total of 357 differentially expressed genes under nitrogen limitation [>2-fold change, false discovery rate (FDR) was <5 %] i.e. approximately 5 % of the M. smegmatis genome (Additional file 2, Table S1). Moreover, the usage of different thresholds (i.e. 2-fold, <1 % FDR; 2-fold, <0.1 % FDR; 1.5-fold, <0.1 % FDR) had no major effect on the number of differentially expressed genes. In total, 208 genes were upregulated and 149 were downregulated. Classification of these genes into functional categories showed major changes in transport proteins, genes that are associated with nitrogen and amino acid metabolism and genes that are functionally assigned as regulatory proteins. A large number of genes encoding for hypothetical proteins were differentially expressed (36 upregulated; 38 downregulated), however, their function in response to nitrogen depletion needs to be investigated (Additional file 3, Figure S2).
Nitrogen limitation studies in continuous culture versus batch culture
Differentially expressed genes that are involved in uptake of nitrogen compounds during nitrogen limitation
Predicted transported substrate
amino acid permease
amino acid permease
amino acid permease
branched-chain amino acid ABC transporter permease
polar amino acid ABC transporter inner membrane protein
cysteine ABC transporter permease/ATP-binding protein
branched-chain amino acid transport ATP-binding protein
transporter LysE family protein
xanthine / uracil permease
permease for cytosine / purines / uracil / thiamine / allantoin
pyrimidine permease RutG
permease for cytosine / purines / uracil / thiamine / allantoin
cytosine / purines / uracil / thiamine / allantoin permease
xanthine / uracil permease
nitrite extrusion protein
Genes involved in nitrogen metabolism that are differentially expressed during nitrogen limitation in M. smegmatis mc2155
dihydrodipicolinate reductase N-terminal domain-containing protein
aminotransferase class I and class II family protein
amino acid acetyltransferase
piperideine-6-carboxylic acid dehydrogenase
amino acid adenylation protein
soluble pyridine nucleotide transhydrogenase
creatininase subfamily protein
urease accesory protein UreG
allophanate hydrolase subunit 2
allophanate hydrolase subunit 1
urease accesory protein UreF
urease subunit γ
N-carbamoyl-L-amino acid amidohydrolase
thiocyanate hydrolase subunit β
thiocyanate hydrolase subunit γ
nitrite reductase, large subunit
Nitrogen limitation activates the expression of genes involved in scavenging nitrogen sources in the environment
In this study, a total of 53 genes encoding for transporters or corresponding binding proteins were differentially expressed in response to nitrogen limitation (Additional file 2, Table S1). Of the upregulated genes, 18 encoded for transporters that are generally involved in uptake of both organic and inorganic nitrogen-containing compounds like amino acids (e.g. msmeg_2525), nucleotides (e.g. msmeg_5730) and ammonium (e.g. msmeg_4635) (Table 1). Previously published data by Berney and Cook showed downregulation of nine of these transporters in a carbon-limited continuous culture comparing slow growth (td 69 h) versus fast growth (td 4.6 h) . All three ammonium transporters and several amino acid (3 of 8) and nucleotide transporters (3 of 7) showed a significant decrease in transcript level, however transcription of all of these genes was enhanced upon nitrogen limitation. The GlnR regulon comprises 15 of these nitrogen compound transporters including the three ammonium transporters and the majority of nucleotide and amino acid transporters. The most prominent amino acid permease (msmeg_2525) was not under direct control of GlnR. In continuous culture, we identified 4.8-fold upregulation of a polar amino acid ABC transport system that has not been reported to be affected by nitrogen limitation. Further work is required, to identify the regulatory mechanisms that mediate the uptake of amino acids under nitrogen depletion. We observed a decrease in gene expression for the catabolism of five amino acids (Table 2), suggesting M. smegmatis attempts to balance the availability of ammonium and particular amino acids under nitrogen depletion. This finding is supported by a recent report of a proteasome-mediated amino acid recycling in M. smegmatis under nutrient limitation . The transcriptomic response of M. smegmatis to increase its ability for the uptake (scavenging) of nitrogen compounds is remarkably different from other actinobacteria like C. glutamicum, where no amino acid permeases were upregulated under nitrogen limitation .
Strong induction of nucleotide catabolism and recovery of ammonium
In our analysis we identified a novel set of genes that were significantly upregulated upon nitrogen limitation and appeared to be involved in the breakdown of nucleotides and inorganic nitrogen sources (Table 2). Nucleotides contain a high level of nitrogen and their degradation plays an important role in nitrogen metabolism in a large number of different microorganisms (e.g. Bacillus subtilis), where purine catabolic pathways are described as an alternative pathway of nitrogen utilization once primary nitrogen sources are exhausted . Genes encoding for proteins involved in the degradation of purine nucleotides were previously reported to be upregulated in M. smegmatis during nitrogen limitation . The enzyme guanine deaminase is upregulated 3.7-fold (FDR < 0.1 %) catalyzing the reaction from guanine to xanthine and releasing 1 mol of ammonium per 1 mol guanine. The three enzymes, uricase (up 2.8-fold, FDR < 0.1 %), transthyretin (up 3.5-fold, FDR < 0.1 %) and 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase (up 3.9-fold, FDR < 0.1 %) catalyze the subsequent steps of urate degradation resulting in the production of allantoin. This metabolite is then further degraded with the concomitant production of ureidoglycolate and urea, which is broken down in either a one-step reaction via urease or a two-step reaction via urea carboxylase/allophanate hydrolase enzyme complex to ammonium (Fig. 3). Transcription of the urease-encoding genes was elevated also in the batch culture, while genes encoding for an allophanate hydrolase (msmeg_0435-msmeg_0436) were upregulated only under nitrogen depletion in continuous culture .
We observed an upregulation of the pyrimidine nucleotide degradation gene cluster and detected differential expression of a third copy of a putative phenylhydantoinase (msmeg_3553; up 3-fold, FDR < 0.1 %) that plays an important role in the degradation of pyrimidine nucleotides. Catabolism of pyrimidine nucleotides can occur through three different pathways; a reductive pathway in which uracil is reduced to two molecules of CO2 and one molecule of ammonium and β-alanine, respectively, an oxidative pathway and a recently identified Rut pathway . M. smegmatis harbors a reductive uracil degradation pathway , which is induced upon nitrogen limitation. However, not all of the genes in this pathway have been identified in M. smegmatis. After the import of uracil by several nucleotide permeases, the first step of this reductive pathway is catalyzed by a dihydropyrimidine dehydrogenase that is able to reduce uracil and thymine to 5,6-dihydrouracil and 5,6-dihydrothymine (Fig. 3), followed by a reaction catalyzed by a phenylhydantoinase. This enzyme hydrolyzes the opening of the heterocyclic ring and β-alanine synthase (msmeg_3555; up 1.9-fold, FDR < 1 %) is catalyzing the last step of this pathway (Fig. 3).
Amino acid catabolism is strongly downregulated under nitrogen limitation
A large cohort of genes, particularly in the catabolism of the amino acids alanine, aspartate, valine, proline and lysine were downregulated 5-40-fold in response to nitrogen depletion (Table 2), (Fig. 3). Interestingly, most of these genes showed an elevated transcription in batch culture (Additional file 5, Table S3). The amino acid alanine has a substantial importance as a central metabolite in bacterial metabolism and also its role in the synthesis of peptidoglycan as D-/L-alanine is essential for bacteria. Several pathways have been described contributing to the biosynthesis of L-alanine and β-alanine, e.g. catabolism of amino acids (i.e. valine and cysteine) and transamination of pyruvate. In M. smegmatis, genes encoding for all of the aforementioned pathways were differentially expressed in response to nitrogen limitation (Table 2). The conformation of the amino acid β-alanine does not allow its incorporation into proteins, but it serves together with pantoate as precursor of coenzyme A (CoA) biosynthesis, which is essential for a functional TCA cycle, as well as fatty acid and cholesterol biosynthesis. Degradation of purine nucleotides via an L-aspartate-alpha-decarboxylase (PanD) results in production of β-alanine and PanD was identified as the predominant pathway of β-alanine synthesis in the closely related C. glutamicum, where a panD mutant exhibited β-alanine auxotrophy . However, expression of panD was strongly downregulated in M. smegmatis under nitrogen limitation. Valine degradation via the intermediates 3-methyl-2-oxobutanoate and 2-dehydropantoate to (R)-pantoate was repressed the same time, suggesting the demand to prevent unnecessary consumption of amino acids for CoA biosynthesis (Fig. 3).
A second pathway of L-aspartate (Asp) catabolism was differentially expressed in M. smegmatis under nitrogen limitation, which is linked to the concomitant biosynthesis of lysine. This pathway is a nine-step reaction including important metabolites such as L-aspartate semialdehyde (homoserine biosynthesis) and meso-2,6-diaminopimelate (constituent of bacterial cell walls). Interestingly, the initial steps of aspartate catabolism were repressed, while the degradation of meso-2,6-diaminopimelate to lysine was upregulated (Fig. 3). Lysine can act as donor of an amino group by transferring an ammonium group to 2-oxoglutarate to form glutamate under nitrogen excess, however, this pathway of lysine catabolism via a lysine aminotransferase is downregulated 7.5-fold (FDR < 1 %), indicating an intracellular accumulation of aspartate and lysine and suggesting a secondary function of these amino acids [32, 33]. Previous studies discussed the importance of intracellular lysine to control growth rate in mycobacteria and suggested a link between lysine accumulation and fatty acid metabolism . Proline has been shown to serve as mechanism for methylglyoxal detoxification, when anabolic and catabolic processes were imbalanced and we show that proline degradation was repressed under nitrogen depletion.
A large number of transcriptional regulatory systems are differentially expressed in response to nitrogen limitation
Genes involved in regulatory mechanisms in response to nitrogen limitation in M. smegmatis mc2155
two-component regulator HTH luxR-type DNA binding domain
GntR family transcriptional regulator
transcription factor WhiB
two-component system - regulatory protein
two-component system - sensor kinase
nitrogen regulatory protein PII
CdaR family transcriptional regulator
regulatory protein - FmdB family
CadC family transcriptional regulator
response regulator receiver domain-containing protein
MarR family transcriptional regulator
transcriptional regulatory protein
transcriptional regulatory protein
LysR family transcriptional regulator
GntR family transcriptional regulator
LuxR family transcriptional regulator
TetR family transcriptional regulator
transcription factor WhiB family protein
transcription factor WhiB
Another well-described transcriptional regulator is the OmpR-type response regulator GlnR, which has been identified as a mediator of the transcriptomic response to nitrogen limitation in M. smegmatis . Determination of the GlnR regulon, by combining expression profiling of M. smegmatis wild type and a ∆glnR deletion mutant under nitrogen-starvation conditions in batch culture revealed a total of 103 genes directly controlled by GlnR . A large portion of these genes (72 %) were also differentially expressed under nitrogen limitation in our continuous culture model, however, GlnR itself was not among them, indicating a posttranslational regulatory mechanism for GlnR like in S. coelicolor . Comparison of the differentially expressed transcriptional regulatory proteins in batch culture versus continuous culture showed an overlap of six genes with four of these under control of GlnR (Table 3). We further observed 16 transcriptional regulatory proteins that were differentially expressed in continuous culture (Table 3). The TetR-like transcriptional regulator AmtR showed a 1.7-fold upregulation under nitrogen replete conditions and was therefore outside the selected cut-off. However, in our RNA-seq analysis we identified 127-fold downregulation of an antisense transcript (asRNA) of almost the entire msmeg_4300 gene under nitrogen-depleted conditions (Additional file 6, Figure S3). These data suggest a mechanism of post-transcriptional regulation by an asRNA where transcription of msmeg_4300 is enhanced upon nitrogen excess and modulates the translation efficiency of the AmtR encoding gene.
We identified several other cases of potential non-coding small RNAs that might regulate expression of genes involved in fatty acid and central carbon metabolism under nitrogen limitation (Additional file 7, Table S4). In our RNA-seq analysis, we observed a 102-fold downregulation of an asRNA of msmeg_4299 gene under nitrogen depletion, while msmeg_4299 showed a 2.7-fold upregulation under nitrogen limitation (Additional file 6, Figure S3). The intergenic region msmeg_3131-msmeg_3132 comprises the promotor regions of the genes msmeg_3131 (long-chain acyl-CoA synthetase) and msmeg_3132 (DNA-binding protein) and revealed a 4.7-fold increase in transcription under nitrogen excess (Additional file 6, Figure S3). Interactions between sRNAs and the 5’UTR of mRNAs affect translation efficiency, while an interaction with the 3’UTR of mRNAs usually do not affect translation, but mRNA stability . These findings are supported by a strong upregulation of genes involved in the degradation of fatty acids as well as the downregulation of genes involved in the TCA cycle as both metabolic pathways are CoA-dependent (Additional file 2, Table S1).
Interestingly, three regulatory proteins (one putative two-component system and two putative orphan response regulators) showed a different expression profile in continuous culture compared to batch culture. The operon msmeg_6236-msmeg_6238 encodes for a two-component system with unknown function and the gene msmeg_3297 encodes for a CadC-like transcriptional regulator that has been linked to pH homeostasis in E. coli . No change in external pH was observed in our experiments, suggesting the CadC-like protein in M. smegmatis was performing a different function.
Bacterial strains, media and growth conditions
M. smegmatis mc2155 was grown in LBT or adapted HdB minimal medium supplemented with 0.2 % (w/v) glycerol (unless otherwise stated) as sole carbon source, NH4Cl in various concentrations as sole nitrogen source, 11.74 mM K2SO4 as sulphur source and 0.05 % (w/v) Tyloxapol at 37 °C with agitation (200 rpm). Aerobic starter cultures were inoculated to an initial optical density (OD600) of 0.05 and grown with agitation (200 rpm) at 37 °C. Chemostat bioreactors were prepared as previously described . Samples to measure residual glycerol concentration and ammonium concentration were taken in intervals of four to six hours. Glycerol concentration was measured according to Garland and Randle  and ammonium concentration was measured according to Weatherburn . After entering steady-state, the chemostat was left running for at least three volume changes before cell harvest.
RNA extraction and Reverse transcriptase PCR
Cells were lysed by four cycles at 4800 rpm for 30s in a mini-Beadbeater (Biospec Products), with 30s on ice between each of the cycles. Total RNA was extracted using TRIzol® reagent (Ambion) according to the manufacturers instructions. DNA was removed by treatment with 3 U RNase-free DNase using the TURBO DNA-free kit (Ambion) according to the manufacturers instructions. The quality of the RNA was checked with the Bioanalyzer (RIN >9) and the concentration was determined using a NanoDrop ND-100 spectrophotometer.
Next, depletion of ribosomal RNA and strand-specific library preparation were performed, using the EpiCentre ScriptSEq™ Complete Kit for Bacteria according to manufacturers instructions. Quantification of nucleic acids was performed using a Qubit® Fluorometer to ensure DNA contamination of the samples was less than 10 % and libraries were created. Prepared libraries have undergone a quality control using an Agilent 2100 Bioanalyzer: DNA 1000 Labchip, Quant-iT dSDNA HS Assay for quantification and Quant-iT RNA Assay and Quant-iT Protein Assay for percentage contamination check, using an Invitrogen Qubit® Fluorometer. Libraries were run on a Illumina MiSeq 300 cycle Kit_v2 with a paired-end (PE) read length of 2x150 and a PhiX control library was also loaded and used as control for the run.
Analysis of RNA sequencing data
Adapter sequences were removed from raw fastq files using Flexbar  and reads shorter than 50 bp were discarded. Sequences were then mapped against the M. smegmatis genome (GenBank NC_008596.1) using Bowtie2 with the”very sensitive” option. Counts for each gene, intergenic regions and counts on reverse strand were calculated with featureCounts , taking into account the reads’ strand-specificity. Statistical analysis and principal component analysis were performed using DESeq (Additional file 8, Figure S4 and Additional file 9, Figure S5) . We considered differentially expressed genes based on their fold-change values as well as p values adjusted for multiple testing with the Benjamini-Hochberg procedure, which controls false discovery rate (FDR, referred to as padj in DESeq results). Venn diagrams were generated using the BioVenn program . Gene functions were assigned using public databases (NCBI , UniProt , KEGG , Biocyc Database Collection ).
All RNA-sequencing data have been deposited in ArrayExpress and can be accessed through the accession number E-MTAB-3918. All other supporting data are included as additional files.
This work was supported by the Maurice Wilkins Centre for Molecular Biodiscovery and a Marsden Grant from the Royal Society of New Zealand. G.M.C. was supported by a James Cook Fellowship from the Royal Society of New Zealand. MP was supported by a University of Otago Doctoral Scholarship, the Webster Center for Infectious Diseases and the Otago School of Medical Sciences. We thank Claudia Sala and Stewart Cole for critical reading of the manuscript.
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