Nitrogen utilisation by the metabolic generalist pathogen Mycobacterium tuberculosis

Bacterial metabolism is fundamental to pathogenesis and has a dominant effect on bacterial killing by antibiotics. Here we explore how Mycobacterium tuberculosis utilises amino acids as nitrogen sources, using a combination of bacterial physiology and stable isotope tracing coupled to liquid chromatography mass spectrometry metabolomics methods. Our results define core properties of the nitrogen metabolic network from M. tuberculosis, such as: (i) the lack of homeostatic control of amino acid pool sizes; (ii) similar rates of utilisation of different amino acids as sole nitrogen sources; (iii) improved nitrogen utilisation from amino acids compared to ammonium; and (iv) co-metabolism of nitrogen sources. Finally, we discover that alanine dehydrogenase, is involved in ammonium assimilation in M. tuberculosis, in addition to its essential role in alanine utilisation. This study represents the first in-depth analysis of nitrogen source utilisation by metabolic generatlist M. tuberculosis and reveals a flexible metabolic network with characteristics that are likely product of evolution in the human host.

Tuberculosis, caused by the bacillus Mycobacterium tuberculosis, is now the greatest cause of death by a single infectious agent, surpassing deaths caused by HIV/AIDS 1 . Failures in drug discovery programmes aimed at finding transformative antitubercular agents are thought to partially derive from by our incomplete understanding of bacterial phenotypic diversity in the host 2 . Bacterial metabolic flexibility is thought to be essential for survival in a variety of niches, where low pH, low oxygen tension, presence of reactive oxygen species, and scarcity of nutrients are commonly found.
In contrast to the wealth of knowledge on carbon metabolism 3,4 , very little is known about nitrogen metabolism, in particular, we do not understand what are the essential features of nitrogen metabolism in M. tuberculosis 5 . For example, while we understand how post-translational regulation of nitrogen metabolism operates in mycobacteria [6][7][8][9][10][11][12][13] , transcriptional regulation of nitrogen metabolism in M. tuberculosis is largely unknown (Fig. S1). The transcriptional factor GlnR does not perform canonical functions in mycobacteria 14,15 . Nitrogen utilisation by different mycobacterial species appears to be very different, not only due to inherent growth rate differences but also lag phase and final biomass achieved (Fig. S2). Importantly, the vast majority of studies to date focused exclusively on either ammonium (NH4 + ) as the sole physiologically relevant nitrogen source 14,16 or employed surrogate fast-growing species such a M. smegmatis instead of M. tuberculosis 16 . In 2013-14 two key studies unveiled an important aspect of host-relevant nitrogen metabolism in M. tuberculosis, namely that host amino acids such as L-aspartate (Asp) and L-asparagine (Asn) were shown to be important sources of nitrogen during infection 17, 18 . These findings open a new avenue in host-M. tuberculosis relevant metabolism, revealing the use of organic nitrogen sources by M. tuberculosis during infection. In addition, there is now overwhelming evidence on the importance of amino acids during infection, highlighted by profound infection attenuation observed with genetic knockout strains 19,20 . Here we describe our studies aimed at exploring core nitrogen metabolic network of M. tuberculosis (Fig. 1), using amino acids as nitrogen sources.

RESULTS
M. tuberculosis can take up all proteinogenic amino acids.
To investigate amino acid uptake and utilization by M. tuberculosis, we transferred bacteria-laden filters after 5 days' growth on 7H10 media to individual fresh 7H10 agar plates containing 1 mM of each of the 20 proteinogenic amino acids. Cells were harvested 17 hours post-shift. Metabolites were extracted, separated, identified and quantified by liquid-chromatography high-resolution mass spectrometry, following procedures described elsewhere 21,22 . The majority of amino acid intracellular pool sizes vary only modestly when M. tuberculosis is grown in media containing sole nitrogen sources different to NH4Cl (Fig. 2a, b). However, an increase in intracellular pool size is observed for Gly, Ala, Val, Ile, Met, Pro, Phe, Tyr, Trp, Ser, Thr, Arg, and His when M. tuberculosis was cultured with the cognate amino acid as sole nitrogen source (highlighted in the diagonal of Fig. 2a). Importantly, all amino acids present as sole nitrogen source alter the pool size of the cognate amino acid and/or other amino acid in M. tuberculosis, demonstrating that they are taken up. On Fig. 2b, the data from Fig. 2a is replotted to illustrate individual amino acids changes (final concentrations in samples) obtained with M. tuberculosis grown with different amino acids as sole nitrogen sources. With few exceptions (Met and Trp) no change is observed in the summed amino acid pool size, when M. tuberculosis is incubated with different amino acids as the sole nitrogen source. Fig. 2c contains the data from Fig. 2b replotted as summed fold-change, compared to NH4Cl. Trp and His as sole nitrogen sources display significant effects on the summed abundance, but most of the other amino acids do not significantly affect overall pool size. In other words, Trp and His are readily taken up by Mtb and stored at high concentrations. Fig. 2d shows data for all amino acids, independent of nitrogen source. It is apparent that most amino acid concentrations are not significantly altered in different nitrogen sources, while the concentrations of Pro, Asp, Gln, Glu and Ala vary depending on the sole nitrogen source used. Fig. 2e illustrates amino acid levels observed when the cognate amino acid or NH4Cl were used as sole nitrogen source. Nearly all amino acid pool sizes are altered when the cognate amino acid is present in the growth medium, as sole nitrogen source. This data is also highlighted on the diagonal in Fig. 2a. Curiously, no change is observed in Leu, Asn, Gln, Asp, Glu and Lys when the respective cognate amino acid was added to the growth medium. Fig. 2f illustrates the modest changes in the concentration of Gln in M. tuberculosis when different amino acids or NH4Cl are present in the growth medium. This result suggests that Gln might not be the indicator of nitrogen levels in mycobacteria.
Overall, these results indicate that M. tuberculosis does not control the pool sizes of most amino acids homeostatically, i.e. intracellular concentrations rise or fall depending on extracellular amino acid/nitrogen source availability.
Amino acids are superior nitrogen sources, compared to NH4 + .
Before carrying out an in-depth analysis of nitrogen metabolism we investigated whether or not the medium used to culture M. tuberculosis prior to switching to media with defined sole nitrogen sources could lead to false results. Pre-culture media composition has been shown to affect carbon metabolism 21 . We 'pre-cultured' M. tuberculosis in either standard Middlebrook 7H9 broth (containing Glu and NH4 + ) or a 7H9 NH4+ broth (a synthetic version of Middlebrook 7H9 broth, with NH4 + as sole nitrogen source), prior to the experiment in 7H9 NH4+ broth. When pre-conditioned in standard 7H9, growth of M. tuberculosis in 7H9 NH4+ only broth led to a significantly higher biomass accumulation than when pre-conditioned in 7H9 NH4+ broth (Fig. S3). Therefore, pre-adaptation in the nitrogen source that will be tested is essential to avoid overestimation of growth, particularly in poor nitrogen sources. Hence, all experiments were carried out with cultures that were pre-adapted in media of identical composition to the test media for at least 3 days (unless otherwise stated).

Fig. 3a
illustrates representative growth curves obtained in Glu, Gln, Asp, Asn and NH4Cl, as sole nitrogen sources. All four amino acids were superior nitrogen sources to NH4Cl, at all concentrations tested ( Fig. 3b and c), both in terms of doubling rate and final biomass generated. It is noteworthy that pre-adaptation in medium with NH4Cl as sole nitrogen source, reveals that M. tuberculosis can only optimally utilise NH4 + as sole nitrogen source until up to 0.25 g/L (4.67 mM). Higher NH4 + concentrations appeared to be toxic. Based on final biomass produced the following order represents the preferential utilisation of sole nitrogen sources: Glu > Asp > Asn > Gln > NH4 + . When pre-adapted cultures were grown in medium containing no nitrogen source, growth persisted in all cases (Fig. 3d). This limited growth is likely due to the low levels of ferric ammonium citrate (0.04 g/L) added to the medium as an iron source. Interestingly, not all cultures grew identically. Growth was different depending on nitrogen source: cells pre-adapted to Asp, Asn and NH4Cl allowed growth to an OD ~1.0, while those pre-adapted to Glu and Gln grew to OD ~0.2. To confirm the 'metabolic conditioning effect' induced by the pre-adaptation medium, we sub-cultured cells after 15 days into fresh medium. Pre-adaptation with Glu and Gln again led to poor growth, while cells derived from medium containing Asp, Asn and NH4Cl grew to an OD ~ 1, in a concentration-dependent manner (Fig. 3e).
Taken together, these results reveal that the amino acids Glu, Gln, Asp, and Asn are superior to NH4 + as sole nitrogen sources for M. tuberculosis, leading to high biomass and faster growth.

Utilisation of position-specific nitrogen atoms by M. tuberculosis.
An essential step in the analysis of nitrogen metabolism with nitrogen sources containing more than one nitrogen atom, such as Gln and Asn, is to define which nitrogen atom(s) is/are being utilised. The ability to utilisation different nitrogen atoms is likely variable and species-specific. To define how M. tuberculosis utilise different nitrogen atoms we performed labelling experiments with positionspecific labelled Gln and Asn (Fig. 4a). The most direct chemical reactions producing 5 key amino acids (namely Glu, Gln, Asp, Asn and Ala) and the label incorporation data obtained from doubly and position-specific labelled 15 N-Gln and 15 N-Asn are shown in Fig. 4b-f. These results indicate that both nitrogen atoms from Gln and Asn are utilised by M. tuberculosis and, specifically that: (i) glutamate synthase is converting the δ-15 N from Gln into α-15 N-Glu (Fig 4b); this explains the incorporation of δ- 15 N from Gln into the α-15 N-Asp, via direct transamination from α-15 N-Glu (Fig. 4d); (ii) direct transamination between α-15 N-Glu, the other product of the glutamate synthase reaction, and α-15 N-Asp is clearly observed (Fig. 4d); (iii) when position-specific labelled Asn is used, the dominant form of Asp observed in α-15 N-Asp (Fig. 4d), indicating that the NH4 + released by asparaginase is likely assimilated to Gln which is distributed broadly in metabolism (Fig. 4b, 4c and 4f), but only modestly to Asp (Fig. 4d); (iv) labelled Asn is only detectable when Asn is the nitrogen source (Fig. 4e), confirming that no Asn synthesis is taking place in M. tuberculosis; (v) use of either position-specific labelled Gln or Asn, leads to identical labelling of Glu (Fig. 4b), consistent with access of both α and γ/δ nitrogen atoms; (vi) labelling of Gln with position specific Gln and Asn is indistinguishable, demonstrating that all nitrogen derived from Asn is mobilised through Gln (Fig. 4c); and (vii) labelling patterns obtained for Ala in the presence of position-specific labelled Gln and Asn are very similar, indicating again that most of the nitrogen derived from Asn is assimilated first into Gln, and then distributed to other metabolites, reflecting the data shown in Fig. 4c.

Kinetics of nitrogen metabolism in M. tuberculosis
Label incorporation from 15 N Glu, 15 N2-Gln, 15 N-Asp, 15 N2-Asn and 15 NH4Cl obtained under metabolic steady-state, over the course of 17 hours, revealed several important features of M. tuberculosis nitrogen metabolism, including different kinetics of 15 N labelling ( Fig. 4g, 3h, S4). As expected, regardless of the nitrogen source, robust label incorporation into amino acids belonging to core nitrogen metabolism was observed, with exception of Asn, which was only observed when cells grew in Asn as sole nitrogen source (Fig. 4g). It is noteworthy that the Ala pool size and labelling was significantly higher when NH4Cl or Asn was the sole nitrogen source. Also, in agreement with data shown in Fig. 2a, external amino acid availability does not necessarily correlate with increased intracellular pool size. For example, Glu and Gln are more abundant with Asp and Asn as the sole nitrogen source, respectively (rather than in the cognate amino acid as sole nitrogen source).
Taking the position specific labelling data and corresponding likely metabolic paths, in conjunction with current biochemical and genetic knowledge of the enzymes of the core nitrogen metabolic network (summarised in Fig. 1), we calculated exponential labelling rates (R) and maximum labelling levels (Lmax) for various core amino acids when Asp, Asn, Glu, Gln and NH4Cl were used as sole nitrogen sources ( Fig. 4h and Fig. S4). Lmax for different sole nitrogen sources appears to be similar (Fig. 4h), indicating that in principle, nitrogen derived from Glu, Gln, Asp, Asn and NH4 + can reach similar high levels (close to 100%) in the core nitrogen metabolites of M. tuberculosis before the first division (17 h). (Fig. 4h). In contrast, R values varied considerably, depending on the sole nitrogen source present and reactions needed to transfer the 15 N atom to individual metabolites ( Fig. 4h and Fig. S4). Again, NH4 + is not the most efficient nitrogen source for M. tuberculosis, as it leads to only modest labelling of key core metabolites, compared to other sole nitrogen sources. Lmax and R for Asn are only consistently observed when Asn is used as sole nitrogen source, supporting the idea that M. tuberculosis has a very small Asn pool ( Fig. 4g and 4h). L% in Fig. 4h illustrates early (2 h incubation) labelling of metabolites, and further highlights the differences in R values for each nitrogen source. These differences are only related to nitrogen metabolism, as no significant differences are observed in pool sizes of pyruvate, α-ketoglutarate, L-malate and succinate, which are reporting on glycolysis and Krebs cycle metabolism.
Co-catabolism of amino acids does not improve growth.
M. tuberculosis has recently been shown to co-catabolise carbon sources simultaneously 21 , leading to better growth than in individual carbon sources. Co-catabolism of carbon sources is a metabolic feature highly unusual in bacteria, which usually catabolise carbon sources sequentially, displaying biphasic (diauxic) growth kinetics. To investigate nitrogen source co-metabolism in M. tuberculosis, we grew cells in media containing the following combinations of nitrogen sources: 15 N-Glu+ 14 N-Gln, 14 N-Glu+U 15 N-Gln, 15 N-Asp+ 14 N-Asn, or 14 N-Asp+U 15 N-Asn. All nitrogen source combinations lead to robust labelling of Glu, Gln, Asp, Asn (Fig. 5), indicating that M. tuberculosis is indeed able to take up and co-metabolise nitrogen sources. Extracted ion chromatograms (Fig. 5a) illustrate significant 15 N metabolism in all conditions (i.e. high levels of labelled metabolites in the absence of labelled nitrogen sources). Mass spectral data (Fig. 5b) reveals that Gln (initially labelled or unlabelled) has been metabolised extensively, generating all three isotopologues ( 14 N2, 14 N 15 N and 15 N2). Mass spectral analysis further confirms that no Asn is being synthesised in M. tuberculosis, as no labelled Asn ( 15 N2 or 15 N1) is found when 15 N-Asp is used as nitrogen source and no 15 N-Asn is present when 15 N2-Asn is provided as nitrogen source. Fig. 5c provides average values and errors for labelling of Glu, Gln, Asp, Asn and Ala, in dual nitrogen sources. Interestingly, Ala labelling appears to derive mainly from Asn, rather than Asp. This suggests that Asn is being hydrolysed to Asp and NH4 + , and likely that Ala is either a main entry point for 15 NH4 + or it serves as nitrogen storage. This result is in strict agreement with the very fast and extensive label incorporation of Ala when using 15 NH4 + or 15 N-Asn as sole nitrogen sources ( Fig. 4g and 4h). In spite of clear co-metabolism of two different nitrogen sources (i.e. Glu/Gln and Asp/Asn), no growth advantage (faster doubling time and highest biomass) is observed (Fig. S6).
Alanine and alanine dehydrogenase as a fundamental node in nitrogen metabolism.
Ala pool size and labelling patterns (Fig. 4g and 4h) are incompatible with our current understanding of nitrogen metabolism in M. tuberculosis. If NH4 + utilization, either direct or derived from Asn, proceeded through glutamine synthetase or glutamate dehydrogenase, labelling of Glu would always be greater than Ala, which would be produced by transamination of Glu. However, this is not the case. To investigate Ala metabolism in the context of nitrogen assimilation, we first confirmed whether Ala could serve as a nitrogen source. Fig. 6a shows that M. tuberculosis can grow in the presence of Ala as a sole nitrogen source, or in binary combination of Ala with Glu, Gln, Asp, Asn, or NH4 + Cl. These results are consistent with Ala being utilised as a sole nitrogen source and in combination with other nitrogen sources, but without any growth advantage (mirroring the result observed for Glu/Gln and Asp/Asn co-metabolism). qPCR analysis of transcript levels for asparaginase (ansA), glutamine synthetase (glnA1), glutamate dehydrogenase (gdh) and alanine dehydrogenase (ald), in sole nitrogen sources was carried out to define if transcriptional programmes are involved in control of amino acid metabolism, and in particular of alanine dehydrogenase, despite the current lack of potential transcriptional regulators of nitrogen metabolism (Fig. 6b). Consistent the hypothesis that alanine dehydrogenase works as a NH4 + assimilatory route, ald RNA levels are found to be higher when M. tuberculosis was grown in media with NH4 + , Asn, Asp and Gln, compared to nitrogen free medium (Fig.  6b, -N/N+). In addition, ald RNA levels are found to be decreased under nitrogen starved conditions, in comparison to gdh, glnA1 and ansA RNA levels, suggesting that ald-driven nitrogen assimilation is likely more important under nitrogen-rich conditions.
To define the role of ald-encoded alanine dehydrogenase in mobilisation of nitrogen to and from Ala, we compared a M. tuberculosis lacking ald, to parent and complemented strains 23 . Genetic disruption of ald abolished the ability of M. tuberculosis to grow when Ala was the sole nitrogen source, with no effect when NH4 + or Gln were sole nitrogen sources (Fig. 6c). These results demonstrate that alanine dehydrogenase is essential for assimilation of NH4 + from Ala, as shown elsewhere 23 . Interestingly, growth was also significantly diminished in the ald-knockout strain when Asp was the sole nitrogen soure, however only partial complementation was obtained (Fig. 6c). Growth of parent, ald-knockout and complemented strains was indistinguishable in NH4 + as sole nitrogen source, confirming that ald is not the main route for NH4 + assimilation in M. tuberculosis. This secondary role of alanine dehydrogenase in assimilation is further supported by lack of changes in label incorporation into Glu, Gln and Ala when parent, ald-knockout, and complemented strains were grown with 15 NH4 + Cl or 15 N-Gln (Fig. 6d). Final evidence for the essentiality of alanine dehydrogenase in nitrogen assimilation from Ala and secondary role during 15 N assimilation was obtained using the inhibitors of alanine dehydrogenase and glutamine synthetase, bromo-pyruvate 24 and methionine sulfoximine 25 , respectively (Fig. 6e, 6f). Bromo-pyruvate partially inhibits growth when NH4 + , Ala and Glu are used as sole nitrogen sources, but not in Gln. In addition to alanine dehydrogenase, glutamate dehydrogenase is likely also partially inhibited at the concentrations tested, leading to the phenotype observed in Glu (Fig. 6f, top panels). Methionine sulfoximine abrogated growth in NH4 + and to a less extent in Ala and Glu, but not in Gln as the sole nitrogen sources (Fig. 6f, bottom panels). Together, these results demonstrate that alanine dehydrogenase is essential for utilisation of Ala as sole nitrogen source and secondary to the utilisation of NH4 + , a task undertaken primarily by glutamine synthetase.

Discussion
M. tuberculosis is a metabolic generalist, that is, it can make the molecules it requires when provided with simple carbon and nitrogen sources. Despite that, M. tuberculosis confinement to the human host over several thousand years has dramatically altered its ability to metabolise host-derived nutrients, which are not necessarily abundant in the environment. In this work, we have uncovered several traits that lead to flexible utilisation of amino acids as nitrogen sources. Most strikingly, M. tuberculosis appears to not tightly control amino acid pool sizes; be able to co-metabolise amino acids as nitrogen sources; and employ alanine dehydrogenase as an assimilatory route for NH4 + . In sharp contrast to most non-nitrogen fixing bacteria studied to date, M. tuberculosis appears to prefer to utilise amino acids such as Glu, Gln, Asp, Asn and Ala as nitrogen sources, instead of NH4 + .

Metabolite extraction
M. tuberculosis was grown in liquid media to mid logarithmic phase and then 1 ml of culture was transferred on 0.22 µm nitrocellulose filter (GSWP02500, Millipore) using vacuum filtration and placed on 7H10Nx agar plates. M. tuberculoisis loaded filters were then grown at 37˚C for 5 days. On day 5 filters were transferred on chemically identical 15 N 7H10Nx plates for isotopic labelling and metabolites were extracted with acetonitrile/methanol/dH2O 2:2:1 (v/v/v) at -40°C. Cells were then mechanically disrupted using a Fastprep ryboliser (QBiogene). Samples were centrifuged for 10 min 13,000 rpm at 4˚C, and the supernatant was recovered and filtered through 0.22µm spin-X centrifuge tube filter (8160, Costar).

Extraction and analysis of RNA, and qPCR
M. tuberculosis was pre-adapted in 7H9Nx medium for 3 days and then grown in identical medium. Cells were harvested at an OD 600 between 0.8 and 1.0. RNA was extracted using Fast RNA Pro Blue kit according to manufacturer's instructions. DNA was removed by treatment with 3 U RNase-free DNase using the TURBO DNA-free kit (Ambion) according to the manufacturer's instructions and cleaned following RNeasy Mini kit (Qiagen). The concentration of the RNA was determined using a NanoDrop One (Thermo) (Promega) spectrophotometer. Reverse transcriptase PCR was performed using SuperScript IV (Invitrogen), according to the manufacturer's instructions for cDNA synthesis. After cDNA synthesis, qPCR was carried out using the PowerUp SYBR Green Master Mix with ROX (Applied Biosystems) on a QuantStudio 7 Flex Real-Time PCR System. SigE (Rv1221) was used as an internal standard, and the ddCt method was used for the calculation of gene expression ratios. Error bars represent standard deviations from three biological replicates. Figure 1. Scheme of the core nitrogen metabolic network of M. tuberculosis. 1 -Glutamine synthetase (glnA1); 2 -glutamate synthase (gltBD); 3 -glutamate dehydrogenase (gdh); 4glutamate/oxaloacetate transaminase (aspB); 5 -glutamate/pyruvate transaminase (aspC); 6alanine dehydrogenase (ald); 7 -glutamate decarboxylase (gadB); 8 -aspartate/pyruvate transaminase (aspC); 9 -asparaginase (ansA).