Systems-Based Approaches to Probing Metabolic Variation within the Mycobacterium tuberculosis Complex

Abstract

The Mycobacterium tuberculosis complex includes bovine and human strains of the tuberculosis bacillus, including Mycobacterium tuberculosis, Mycobacterium bovis and the Mycobacterium bovis BCG vaccine strain. M. bovis has evolved from a M. tuberculosis-like ancestor and is the ancestor of the BCG vaccine. The pathogens demonstrate distinct differences in virulence, host range and metabolism, but the role of metabolic differences in pathogenicity is poorly understood. Systems biology approaches have been used to investigate the metabolism of M. tuberculosis, but not to probe differences between tuberculosis strains. In this study genome scale metabolic networks of M. bovis and M. bovis BCG were constructed and interrogated, along with a M. tuberculosis network, to predict substrate utilisation, gene essentiality and growth rates. The models correctly predicted 87-88% of high-throughput phenotype data, 75-76% of gene essentiality data and in silico-predicted growth rates matched measured rates. However, analysis of the metabolic networks identified discrepancies between in silico predictions and in vitro data, highlighting areas of incomplete metabolic knowledge. Additional experimental studies carried out to probe these inconsistencies revealed novel insights into the metabolism of these strains. For instance, that the reduction in metabolic capability observed in bovine tuberculosis strains, as compared to M. tuberculosis, is not reflected by current genetic or enzymatic knowledge. Hence, the in silico networks not only successfully simulate many aspects of the growth and physiology of these mycobacteria, but also provide an invaluable tool for future metabolic studies.

Introduction

The pathogenic microorganisms constituting the Mycobacterium tuberculosis complex are associated with important human and animal diseases. M. bovis is the causative agent of bovine tuberculosis, a chronic and occasionally fatal infectious disease primarily infecting cattle and other livestock; but is capable of infecting a wide range of mammals and other vertebrates, including humans [1,2]. Bovine tuberculosis causes immense economic loss in many countries, either from loss of livestock, disease testing, or compensation. Worldwide, agricultural losses are estimated to be around $3 billion a year [3]. M. bovis is very closely related to M. tuberculosis, a virulent tubercle bacillus estimated to infect a third of the world’s population and cause the deaths of 1.4 million people each year [4]. In an attempt to prevent tuberculosis infections more than 3 billion individuals [5] have been immunised with M. bovis BCG, a live attenuated derivative of M. bovis.

M. tuberculosis, M. bovis and M. bovis BCG are characterised by 99.9% similarity at the nucleotide level [3,5,6]. However, the genetic deletions, rearrangements and duplications that M. bovis and M. bovis BCG have undergone relative to M. tuberculosis results in widely differing host tropisms, phenotypes and pathogenicity [2,3,5-9]. Whilst large deletions, such as the regions of difference identified in M. bovis and M. bovis BCG, have been shown to encode virulence factors and result in attenuation of infection [9-12], the genetic basis of these profound variations are mostly undefined. Defining the metabolic differences between the three species is of particular importance as metabolic adaptation to the host environment has been highlighted as a key component of the pathogenic strategy of M. tuberculosis [13-17] and is also likely to be important for the virulence of M. bovis.

Previous targeted studies have identified the genetic basis of some observed metabolic differences, for example, the inability of M. bovis to generate energy from glycolytic intermediates [3,18,19]. This defect is thought to be due to the inactivation of pyruvate kinase which causes a disconnection in central metabolism between glycolysis and the Tricarboxylic acid cycle (TCA cycle) [18,19]. However, although there have been focused investigations into the metabolic differences between the human and bovine tubercle bacillus, systems level comparisons of metabolism have not yet been undertaken.

A systems biology approach provides very effective methods for studying metabolism. Genome scale metabolic reaction networks incorporate all known biochemical reactions within a cell and represent a global system where reaction pathways are defined within the context of whole-cell metabolism. These networks are able to predict phenotypic behaviour, aid in hypothesis generation, identify missing reactions and provide information on the robustness of the metabolic networks, which can be used to identify vulnerable pathways that may be targeted with novel drugs. The creation of M. tuberculosis genome scale reaction networks [20-23] has provided a mechanism to study its metabolism in a systemic manner and a basis for the modelling and metabolic comparison of M. bovis and M. bovis BCG strains.

In this study we present the first genome scale metabolic networks for M. bovis and M. bovis BCG, along with a phenotypic analysis of M. tuberculosis, M. bovis and M. bovis BCG. The networks are freely available in the Supporting Information (Tables S1-S3; Models S1-S3) and online for use with our interactive software (http://sysbio.sbs.surrey.ac.uk/) [24]. To our knowledge this is the first time the metabolism of a pathogen and its vaccine strain have been compared on a systems level and any identified differences have the potential to aid investigations into causes of M. bovis BCG attenuation and the suitability of novel vaccines. The models qualitatively predict phenotype data with between 87-88% agreement, gene knockout results with 75-76% accuracy, and quantitative assessment of measured carbon uptake rates as a function of growth rate show that in silico growth rates are comparable to in vitro results. Therefore, these reaction networks successfully simulate many aspects of mycobacterial metabolism and can be used to examine the metabolic differences between these strains.

Materials and Methods

Bacterial strains and growth conditions

M. tuberculosis H37Rv, M. bovis AF2122/97 and M. bovis BCG Pasteur were used for this study. Frozen stocks were maintained in 10% (vol/vol) glycerol at −80°C. Middlebrook 7H9 broth containing 5% (vol/vol) albumin-dextrose-catalase enrichment medium supplement (ADC) (Becton Dickenson), 2 g/L pyruvate and 0.05% (vol/vol) Tween 80 was used to grow cultures from frozen stocks at 37°C, either statically or rolling. Brain heart infusion agar was used to assess culture purity (Becton Dickinson).

For the Roisin’s agar experiments cultures were grown until late exponential phase (OD₆₀₀ = 1.0) in 7H9 containing ADC, pyruvate and Tween 80 (as described), washed twice with Ringer’s solution with 0.2% (vol/vol) tyloxopol and plated in triplicate onto Roisin’s agar containing sole carbon and nitrogen sources. When testing sole carbon source assimilation, cells were grown on Roisin’s minimal media [25] with 10 g/L agarose containing sole carbon sources at 5 g/L (2-oxoglutarate, L-alanine, D-arabinose, L-arginine, L-asparagine, L-aspartic acid, citrate, D-fructose, fumarate, D-galactose, D-glucose, L-glutamate, L-glutamine, glycerol, glycine, L-histidine, L-isoleucine, D-lactose, L-leucine, L-lysine, malate, D-maltose, D-mannose, L-methionine, L-phenylalanine, L-proline, propanoate, D-raffinose, D-rhamnose, D-ribose, L-serine, D-serine, succinate, D-sucrose, L-threonine, D-trehalose, L-valine, D-xylose). When testing sole nitrogen source assimilation Roisin’s minimal media was used with 5 g/L pyruvate, 10 g/L agarose and 5.9 g/L sole nitrogen source (L-alanine, ammonia, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamate, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, D-serine, L-threonine, L-tryptophan, L-tyrosine, urea, L-valine). Plates were incubated for up to 12 weeks. To test for any mutations which may have given false results any positives after 6 weeks were independently tested a further two times.

For growth curves inoculum cultures of M. bovis were grown statically until late exponential phase (OD₆₀₀ = 1.0) in Roisin’s minimal media with 5 g/L pyruvate and 0.2% (vol/vol) tyloxapol. Using a 1% inoculum 100 ml rolling (2 rpm) cultures were set up in Roisin’s minimal media with 5 g/L D-glucose, 0.2% (vol/vol) Tween 80 or 5 g/L D-glucose and 0.2% (vol/vol) Tween 80 as carbon sources. A Biomate, ThermoSpectronic spectrophotometer was used to take daily OD readings until they started to fall, presumably due to bacterial death.

Construction of GSMN-MB and BCG

The first M. tuberculosis genome scale metabolic network [20] (GSMN-TB) (Table S1; Model S1) created for the H37Rv strain formed the basis for the reconstruction of M. bovis AF2122/97 (GSMN-MB) (Table S2; Model S2) and M. bovis BCG Pasteur (GSMN-BCG) (Table S3; Model S3). Genolist [26] was used to assign respective M. bovis and M. bovis BCG gene numbers to the genes present in GSMN-TB and alter annotations which since the GSMN-TB was published have been assigned with an alternative enzymatic function. Using data compiled by Genolist [26] any mutations between M. tuberculosis, M. bovis and M. bovis BCG leading to changes in protein sequence were identified. Published scientific literature was used to investigate these genes and to identify metabolic differences not caused by DNA mutations within enzyme sequences. This information was used to alter the networks to reflect M. bovis or M. bovis BCG metabolism (Table S4).

Biolog phenotype data

Biolog Phenotype MicroArray [27] experiments which examined the ability of M. tuberculosis H37Rv and M. bovis AF2122/97 to respire in the presence of 190 carbon and 95 nitrogen sources (PM1, 2a, 3b [27]), were obtained from Khatri, B et al. 2013 [8]. Equivalent experiments for M. bovis BCG were carried out using the described method [8]. Three to four biological replicates were performed for each plate and a cell free control plate was also included to test for abiotic dye reduction. The raw data was analysed using OmniLog PM kinetic plot software [27], with final colour intensity values and manual analysis of kinetic curves used to assess respiration. Wells were considered positive if the final colour intensity value minus the negative control and minus the standard deviation of the negative controls was a positive value [28]. Results were verified by analysing the kinetic curves. Substrates considered to be respired had at least 50% of duplicate wells showing a positive result. The confidence levels applied to the data were determined by the number of concordant results for each biological replicate [28]. High, medium and low confidence results were categorised as substrates where 100%, >75% and >50% of replicates produced the same result.

Modelling of phenotype experiments

The composition of Biolog base medium [27] is not published, however, the mycobacteria were not able to respire in this medium without the added carbon or nitrogen source. To model the carbon source experiment we therefore simulated the media as a modified form of Roisin’s minimal media [25] containing unlimited quantities of ammonia, phosphate, iron, sulfate, carbon dioxide and a Biolog carbon source influx of 1 mmol/g DWt/h. Similarly the nitrogen source experiment was simulated using a modified form of Roisin’s media, where ammonia was replaced with 1 mmol/g DWt/h of the Biolog nitrogen source and pyruvate was used as a carbon source (influx at 1 mmol/g DWt/h). Flux balance analysis (FBA) was performed under aerobic conditions using biomass as the objective function.

Radioactive glucose uptake in M. bovis AF2122/97 and M. tuberculosis H37Rv

Cultures of M. tuberculosis and M. bovis were grown until exponential phase (OD₆₀₀ 0.6-0.8) in Roisin’s minimal media with 5 g/L pyruvate and 0.2% (vol/vol) tyloxapol, washed twice and then resuspended in Roisin’s media (no carbon, tyloxapol). 1 ml of culture was added to a universal tube containing 3700 becquerels of [6-¹⁴C] D-glucose and 1 ml of 1 M sodium hydroxide and incubated for 4 h at 37°C. Samples were carried out in triplicate with negative controls (identically prepared heat killed suspensions) tested in parallel. Samples were filtered through Whatman GFC glass microfibre filters, washed 3 times with 0.025% (vol/vol) tyloxapol and placed in scintillation tubes with 250 µl of 70% ethanol and 5 ml of BDH Scintran Fluoran flow scintillation fluid. The sodium hydroxide was directly added to the scintillation fluid. Radioactivity was counted using a Packman tri-carb liquid scintillation counter and dpm calculated from a quench curve. Dpm for negative controls were subtracted from dpm for the live samples before the mean average uptake was calculated. For the analysis of [6-¹⁴C] D-glucose radioactivity incorporation in M. bovis when both [6-¹⁴C] D-glucose and non-radiolabelled Tween 80 were available as carbon sources, the experiment carried out as described above, except that the incubation medium was supplemented with 0.2% (vol/vol) Tween 80.

Modelling of growth rate experiments

To model in vitro growth rate experiments in GSMN-BCG and TB the media was simulated as a modified form of Roisin’s minimal media (see modelling of phenotype experiments) with carbon sources constrained to experimentally derived values [29] and the biomass composition for slow or fast growth used as the objective function.

Gene essentiality predictions and comparison with TRASH and deep sequencing data

The maximal theoretical growth rate of each in silico gene knock out was calculated by removing single genes from the network and performing FBA linear programming as described in Beste et al., 2007 [20]. The computational predictions were compared to the gene essentiality findings of Transposon site hybridization (TraSH) mutagenesis [30] and deep sequencing [31] experiments.

Results

The genome-scale metabolic networks of M. bovis and M. bovis BCG

The genome scale metabolic network of M. tuberculosis, GSMN-TB [20], (Table S1; Model S1) was used as a starting point for the reconstruction of the genome scale networks of M. bovis (Table S2; Model S2) and M. bovis BCG (Table S3; Model S3). Reactions were adapted to reflect M. bovis and M. bovis BCG reactions based on genome annotations, protein sequences and published biochemical data [3,5,6,18,26,32-49] (Table S4). For orthologous genes GSMN-TB gene assignments were changed to M. bovis and M. bovis BCG gene numbers, with each DNA sequence analysed for changes relative to M. tuberculosis. The analysis identified 228 and 204 genes with non-synonymous sequence differences in M. bovis and M. bovis BCG and these genes catalysed approximately 30% of reactions within each network. Only a small proportion (~19%) of sequence differences were predicted to lead to metabolic differences requiring network modifications, the vast majority of which were the removal of genes and reactions (Table 1). For GSMN-MB 42 genes and 14 reactions were deleted from the original GSMN-TB network, whilst slightly fewer genes (40) and more reactions (16) were deleted from the GSMN-BCG. These alterations appear to support the theory that M. bovis has evolved from a progenitor of the M. tuberculosis complex and that M. tuberculosis is more closely related to this common ancestor [50].

Table 1. Changes to the GSMN-TB network to create GSMN-MB and GSMN-BCG.

Gene	GSMN-TB	GSMN-MB	GSMN-BCG
Glycerol kinase (glpK)	1	-	1
Glycerol-3-phosphate dehydrogenase (glpD)	2	2	3
GDP-D-rhamnose biosynthesis (gca, gmdA)	2	1	1
GDP-4-dehydro-6-deoxy-D-mannose epimerase	1	0	0
UTP-hexose-1-phosphate uridylyltransferase (galT)	2	1	1
β-glucosidase (bglS)	1	-	-
Pyruvate kinase (pykA)	1	-	1
Isocitrate lyase (icl)	1	2	2
(S)-2-hydroxy-acid oxidase	1	-	-
Nitrate reductase (nar)	5	-	-
Fumarate reductase (frd)	8	7	7
Glycine dehydrogenase (gcvB)	<=>	=>	=>
L-serine ammonia-lyase (sdaA)	1	1	-
Alanine dehydrogenase (ald)	1	-	-
Nicotinamidase	1	-	-
Precorrin-6Y C5,15-methyltransferase	1	-	-
Molybdopterin biosynthesis protein (moaE)	3	-	-
enoyl-CoA hydratase/isomerases (echA)	21	21	21
Phospholipases	28	23	23
Methoxy mycolic acid synthase (mmaA3)	1	1	-
Synthesis of methoxy mycolic acids	Biomass	Biomass
Polyketide synthase (pks15/1)	-	1	1
Glycosyltransferases	2	-	-
Sulfotransferases	3	2	3
Sulfolipid-1 synthesis	1	-	-
Mas-like gene (msl3, msl4, msl5)	4	3	3
Nitrate transporter (narK2)	1	-	-
Phosphate transport via ABC system	8	-	-
Glycerol-3-phosphate antiporter (ugp)	4	-	-
Sulfolipid-1	Biomass
Mycoside b		Biomass	Biomass
Triacylglycerol synthases	15	14	14

Numerical value Number of genes catalysing the reaction; - indicates reaction is deleted from the network; 0 indicates an orphan reaction

=> Irreversible reaction

<=> Reversible reaction

Biomass Required for Biomass production

The constructed metabolic networks for both M. bovis and M. bovis BCG (Table 2) therefore contain fewer reactions, metabolites and genes than an updated GSMN-TB network, GSMN-TB 1.1. GSMN-TB 1.1 includes some corrections to the original GSMN-TB, plus additional pathways, such as cholesterol metabolism, that were not implemented in the earlier published version [20]. Due to the reduction in genes within the networks the M. bovis and M. bovis BCG models have a slightly higher fraction of essential genes (31% and 30% respectively; Tables S5 and S6) compared to GSMN-TB 1.1 (29%; Table S7). These values are slightly lower than the predicted value of 35% essential genes in the entire M. tuberculosis genome but within the 95% confidence interval (28-41%) [51]. All three GSMN networks are available in the Supporting Information (Tables S1-S3; Models S1-3) and online for use with our interactive software (http://sysbio.sbs.surrey.ac.uk/) [24].

Table 2. Statistics of the mycobacterial reaction networks.

	Reaction network
Reaction Class	GSMN-TB 1.1	GSMN-MB	GSMN-BCG
Total number of reactions	876	863	861
Cytosolic reactions	745	735	733
Transport reactions	131	128	128
Genes	759	718	720
Orphan reactions	198	200	200
Total number of metabolites	766	757	754
Internal metabolites	667	660	657
External metabolites	99	97	97

Although the GSMN-MB and BCG models gave very similar predictions to the GSMN-TB 1.1, some interesting differences between in silico predictions and published experimental data [3,18,19,33,44] were found. GSMN-MB was able to utilise carbohydrates such as glucose in silico, although in vitro M. bovis is actually unable to grow on these substrates [18,19] (discussed further below). Another area where in silico predictions did not accord with experimental data was in amino acid synthesis. In M. tuberculosis, alanine dehydrogenase catalyses the oxidative deamination of L-alanine or, in the reverse direction, the reductive amination of pyruvate to yield alanine, but this activity is lost [33,44] in M. bovis and M. bovis BCG due to a frameshift caused by single base pair deletion [3]. Deletion of alanine dehydrogenase from the bovine networks resulted in a non-feasible network (no growth) unless L-alanine was supplied as a substrate, since this is the only biosynthetic route leading to L-alanine in the network. Yet, in contrast to the predictions M. bovis and M. bovis BCG are not alanine auxotrophs. Alanine dehydrogenase knockout mutants of M. tuberculosis are also not alanine autotrophs [19] and ¹³C labelling experiments detected identical alanine labelling patterns [29] for M. tuberculosis and M. bovis BCG; indicating that an alternative pathway for alanine synthesis must be active in all three strains. To preserve alanine prototrophy, the alanine dehydrogenase reaction has therefore been retained in the GSMN-MB and BCG models as an irreversible orphan reaction [19,33,44].

Validation of the model by comparison with Biolog phenotype data

The Biolog [27] high throughput phenotyping system was utilised to obtain additional insight into metabolic capability of M. bovis and M. bovis BCG and to further test the predictive accuracy of the GSMN-MB and GSMN-BCG networks. For comparison, we also examined M. tuberculosis. Biolog [27] is a commercially available phenotype microarraying platform that is capable of high-throughput screening for the ability to utilise a large number of substrates. It employs the reduction of tetrazolium dye by NADH as a reporter system for measuring respiration [27]. Respiration is of course different from growth, as predicted by the network models; but our working assumption was that some degree of growth has to occur to reduce the respiratory substrate sufficiently to see a positive reaction.

Carbon substrate utilisation for M. tuberculosis, M. bovis and M. bovis BCG is presented in Table 3 (showing only substrates metabolised by at least one strain). Of the 190 carbon sources tested (Tables S8-S10) 33 substrates were metabolised by at least one of the mycobacteria tested, with 17 utilised by all three. These substrates included amino acids, TCA cycle intermediates, sorbitan derivatives, 3 carbon compounds and hexose or hexose containing carbohydrates. M. tuberculosis was able to respire more of these substrates (27) than M. bovis (25) or M. bovis BCG (22). Of the 95 nitrogen sources tested (Tables S11-S13) only 13 were able to be utilised as sole nitrogen sources (Table 4) by at least one of the three strains and these were mainly amino acids. Unlike carbon source data, the number of nitrogen sources utilised was similar between the three species.

Table 3. Carbon substrates utilised by M. tuberculosis, M. bovis and M. bovis BCG.

	M. tuberculosis			M. bovis			M. bovis BCG
Substrates	Biolog	Roisin’s agar	in silico	Biolog	Roisin’s agar	in silico	Biolog	Roisin’s agar	in silico
2-oxoglutarate	C	C	C	-	C>	C	-	C	C
Acetate	C	C	C	C	C	C	C	C	C
Acetoacetic acid	C	NT	-	C	NT	-	C	NT	-
Adenosine	-	NT	C	-	NT	C	C	NT	C
D-alanine	C	NT	C	-	NT	-	-	NT	-
L-alanine	C	C	C	-	-	-	-	-	-
L-asparagine	C	C	C	-	C	C	C	C	C
Butyric acid	C	NT	-	C	NT	-	C	NT	-
Caproic acid	C	NT	C	C	NT	C	C	NT	C
Citrate	C	C	C	C	C	C	C	C	C
D-fructose-6-phosphate	C	NT	C	C	NT	C	-	NT	C
D-glucose-6-phosphate	C	NT	C	-	NT	C	C	NT	C
D-glucose	C	C	C	C	-	C	-	C	C
L-glutamate	C	C	C	C	C	C	C	C	C
L-glutamine	C	C	C	-	C	C	-	C	C
Glycerol	C	C	C	C	-	C	C	C	C
Glycine	C	C	C	-	C	C	-	C	C
L-lactate	C	NT	C	C	NT	C	C	NT	C
D-malic acid	-	NT	C	C	NT	C	C	NT	C
L-malic acid	C	C	C	C	C	C	C	C	C
D-mannose	-	C	C	C	C	C	-	C	C
Methyl-pyruvate	C	NT	-	C	NT	-	C	NT	-
Mono methyl-succinate	C	NT	-	C	NT	-	C	NT	-
N-acetyl-glucosamine	-	NT	-	C	NT	-	-	NT	-
Oxalomalic acid	C	NT	-	C	NT	-	C	NT	-
Propanoate	-	C	C	C	C	C	C	C	C
Pyruvate	C	C	C	C	C	C	C	C	C
D-serine	C	-	-	C	-	-	-	-	-
D-tagatose	-	NT	-	C	NT	-	-	NT	-
D-trehalose	C	C	C	C	C	C	C	C	C
Tween 20	C	NT	C	C	NT	C	C	NT	C
Tween 40	C	NT	C	C	NT	C	C	NT	C
Tween 80	C	C	C	C	C	C	C	C	C

C Utilised as a carbon substrate

- Not utilised as a carbon substrate

NT Not tested

Table 4. Nitrogen substrates utilised by M. tuberculosis, M. bovis and M. bovis BCG.

	M. tuberculosis			M. bovis			M. bovis BCG
Substrate	Biolog	Roisin’s agar	in silico	Biolog	Roisin’s agar	in silico	Biolog	Roisin’s agar	in silico
L-Alanine	N	N	N	-	-	-	-	-	-
Allantoin	-	NT	-	N	NT	-	-	NT	-
L-Asparagine	N	N	N	N	N	N	N	N	N
L-Aspartic Acid	-	N	N	-	N	N	N	N	N
L-Cysteine	N	-	-	N	N	-	N	-	-
D-Galactosamine	N	NT	-	N	NT	-	N	NT	-
D-Glucosamine	-	NT	-	N	NT	-	-	NT	-
L-Glutamic Acid	N	N	N	N	N	N	N	N	N
L-Glutamine	N	N	N	N	N	N	N	N	N
L-Ornithine	N	NT	-	N	NT	-	N	NT	-
D-Serine	N	-	-	N	-	-	N	-	-
L-Serine	N	N	N	N	N	N	-	-	N
L-Threonine	-	N	N	-	-	N	N	-	N

N Utilised as a nitrogen substrate

- Not utilised as a nitrogen substrate

NT Not tested

Analysis of in silico predictions verses Biolog data showed that all three networks qualitatively predicted the Biolog data with a similar overall accuracy. For GSMN-TB 1.1, GSMN-MB and GSMN-BCG simulations 84%, 81% and 84% of substrates matched Biolog results respectively (Tables S8-S13). Substrate analysis identified results that appeared anomalous with previous studies [18,19,33,44,52]. For instance, M. bovis tested positive for glucose and glycerol respiration, however, as mentioned above, M. bovis is unable to utilise carbohydrates including glucose and glycerol as sole carbon sources [18,19]. Similarly, many in silico predictions for amino acid utilisation did not correlate with experimental data [33,44,52]. To independently probe the discordant results M. tuberculosis, M. bovis and M. bovis BCG were grown on minimal Roisin’s agar media containing sole carbon or nitrogen sources.

Overall (Tables S8-S13) in vitro experiments using Roisin’s agar media resolved many of the inconsistencies between in silico predictions and the Biolog data. For instance, in accordance with expectations, M. bovis was unable to grow on either glucose or glycerol when provided as the sole carbon source in Roisin’s agar. The anomalous positive Biolog results may be due to the small amount of Tween 80 present in the Biolog base media. It has been shown previously [8,53], and confirmed here (see below), that Tween 80 and glucose are used synergistically for growth by M. bovis strains.

When the anomalous Biolog results were corrected using Roisin’s agar data the GSMN-MB model accurately predicts 87% of phenotypes studied, whilst the GSMN-BCG predicts 88% correctly. The GSMN-TB 1.1 is slightly more accurate (91%) at simulating cellular phenotypes than the bovine networks due to false positive in silico growth predictions for M. bovis and M. bovis BCG. In this analysis results generated on Roisin’s agar media were used instead of Biolog results when the outcomes differed because growth on agar plates assesses biomass production rather than respiration. Therefore, growth on Roisin’s agar media more accurately tests in silico predictions of growth than Biolog experiments.

Analysis of glucose metabolism

GSMN-MB incorrectly predicts that M. bovis should grow on glucose as a sole carbon source. In silico, glucose enters the central metabolism of M. bovis via the glycolytic pathway and bypasses the blocked pyruvate kinase connection between glycolysis and the TCA cycle via either the anaplerotic/gluconeogenic enzyme, phosphoenolpyruvate carboxykinase, that interconverts phosphoenolpyruvate (glycolytic intermediate) and oxaloacetate (TCA cycle intermediate) or a serine-glycine pathway (phosphoglycerate dehydrogenase, phosphoserine transaminase, phosphoserine phosphatase, glycine hydroxymethyltransferase and glycine dehydrogenase (Figure 1)). The hypothesis is that a second metabolic or regulatory defect in glucose metabolism contributes to the M. bovis glucose phenotype, in addition to the inactive pyruvate kinase [18,19].

Figure 1. GSMN-MB in silico flux prediction when glucose is a sole carbon source.

The in silico prediction of flux from glucose to the TCA cycle when glucose is a sole carbon source for M. bovis.

Acon: aconitase, Cit: citrate synthase, Eno: enolase, Fba: fructose-bisphosphate aldolase, Gck: glucokinase, Gdh: glycine dehydrogenase, GlcB: malate synthase, GlyA: glycine hydromethytransferase, Gpm: phosphoglycerate mutase, Icl: isocitrate lyase, Mdh: malate dehydrogenase, Pepck:, phosphoenolpyruvate carboxykinase, Pfk: 6-phosphofructokinase, Pgi: glucose-6-phosphate isomerase, SerA: phosphoglycerate dehydrogenase, SerB: phosphoserine phosphatase, SerC: phosphoserine transaminase, TpiA: triose-phosphate isomerase.

To further explore M. bovis glucose metabolism, M. bovis and M. tuberculosis were incubated with [6-¹⁴C] D-glucose as the only available carbon source for four hours. M. bovis could uptake a small amount of the labeled glucose but the rate was 16-fold less than M. tuberculosis, consistent with a defect in glucose uptake (Table 5). Interestingly, supplementation of the glucose media with Tween 80 significantly stimulated glucose uptake by M. bovis (2.5 times). However, the fate of this additional glucose was not oxidation to carbon dioxide as the amount of ¹⁴C-CO₂ generated was unchanged by the Tween 80 supplementation. Maximum growth rates and OD₆₀₀ achieved on these substrates also indicated synergistic interaction [8] as growth of M. bovis on Roisin’s media with glucose and Tween 80 exceeded that achieved with either compound alone (Table 5).

Table 5. Glucose uptake experiments in M. bovis and M. tuberculosis .

	Becquerels per mg dry weight			Growth rates on non-radiolabelled substrates
	M. tuberculosis	M. bovis
Carbon substrates	Assimilated	Assimilated	CO2 evolved	Maximum growth rate		Maximum OD
[6-14C] D-glucose	56.6 +/- 3.2	3.5 +/- 0.9	1.0 +/- 0.2	-		0.015 +/- 0.004
Tween 80	N/A	N/A	N/A	0.007 +/- 0.001		0.545 +/- 0.044
[6-14C] D-glucose and Tween 80	N/A	8.7 +/- 0.7	1.0 +/- 0.3	0.013 +/- 0.001		1.171 +/- 0.066

Analysis of amino acid metabolism

Biolog data and in silico predictions were notably different for the utilisation of amino acids as sole carbon or nitrogen sources (Table 6) as the models predicted a greater metabolic potential than was actually demonstrated experimentally. Many of these discrepancies were resolved by the Roisin’s agar experiments, as more amino acids were shown to support growth in Roisin’s agar than were positive for respiration in Biolog. Possible reasons for this include large differences in the incubation times for these experiments. In support of this theory, the amino acids not utilised in the Biolog experiments predominantly produced dysgonic colonies with a lag time of around 6-8 weeks on Roisin’s agar (M. bovis: arginine C, N; glutamine C; glutamate C; glycine C; isoleucine C, N; proline C; serine C, N; M. bovis BCG: arginine C, N; glutamine C, N; glycine C; isoleucine C; proline C; serine C). Interestingly, the experimental data showed that the number of viable substrates decreased from M. tuberculosis to M. bovis to M. bovis BCG. This was not however reflected by significant network differences.

Table 6. The utilisation of amino acids as carbon and nitrogen sources by M. tuberculosis, M. bovis and M. bovis BCG.

	M. tuberculosis						M. bovis						M. bovis BCG
Substrate	Biolog		Roisin’s agar		in silico		Biolog		Roisin’s agar		in silico		Biolog		Roisin’s agar		in silico
Alanine	C	N	C	N	C	N	-	-	-		-	-	-	-	-		-	-
Arginine	-	-	C	N	-	N	-	-	C	N	-	N	-	-	C	N	-	N
Asparagine	C	N	C	N	C	N	-	N	C	N	C	N	C	N	C	N	C	N
Aspartate	-	-	C	N	C	N	-	-	C	N	C	N	-	N	C	N	C	N
Cysteine	NT	N	-	-	-	-	NT	N	-	N	-	-	NT	N	-	-	-	-
Glutamate	C	N	C	N	C	N	C	N	C	N	C	N	C	N	C	N	C	N
Glutamine	C	N	C	N	C	N	-	N	C	N	C	N	-	N	C	N	C	N
Glycine	C	-	C	N	C	N	-	-	C	-	C	N	-	-	C	-	C	N
Histidine	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Isoleucine	-	-	C	N	C	N	-	-	C	N	C	N	-	-	C	-	C	N
Leucine	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Lysine	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Methionine	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Phenylalanine	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Proline	-	-	C	N	C	N	-	-	C	-	C	N	-	-	C	-	C	N
Serine	-	N	C	N	C	N	-	N	C	N	C	N	-	-	C	-	C	N
Threonine	-	-	-	N	C	N	-	-	-	-	C	N	-	N	-	-	C	N
Tryptophan	NT	-	-	-	-	-	NT	-	-	-	-	-	NT	-	-	-	-	-
Tyrosine	NT	-	-	-	-	-	NT	-	-	-	-	-	NT	-	-	-	-	-
Valine	-	-	-	N	C	N	-	-	-	-	C	N	-	-	-	-	C	N

C Utilised as a carbon substrate

N Utilised as a nitrogen substrate

- No respiration/growth

NT Not tested

The amino acid data regarding serine metabolism was of particular interest because the results differed between the three mycobacteria and appeared inconsistent with in silico simulations. In silico serine metabolism is relatively complex compared to other amino acids as multiple pathways converge around serine (Figure 2). However, in vitro it is likely only the interconversion of pyruvate and serine (serine dehydratase) enables utilisation of serine as a sole nitrogen source: as inadequate expression of serine dehydratase results in the inability of some BCG strains, including M. bovis BCG Pasteur, to utilise serine as a nitrogen source [33]. M. tuberculosis, which has been found to adequately express serine dehydratase can utilise L-serine [33,52], as can M. bovis strains (Table 6). In silico, however, the GSMN-BCG inaccurately predicts that serine is a viable sole nitrogen source for M. bovis BCG with flux to glycine (serine hydromethyltransferase) enabling viability. The three mycobacteria used in this study have two serine hydromethyltransferases (glyA1and glyA2); with enzyme activity demonstrated in M. tuberculosis [54]. The glyA1 of M. bovis and M. bovis BCG have an amino acid substitution as compared to M. tuberculosis although the effect on the enzyme activity has not been tested. If the interconversion of serine and glycine is feasible in M. bovis BCG, this reaction doesn’t appear to take place when serine is supplied as sole nitrogen source. However, because serine was shown to be a viable sole carbon source for M. tuberculosis, M. bovis and M. bovis BCG, flux from serine must be able to enter the TCA cycle. The most probable route for this would be via glycine (Figure 2).

Figure 2. The metabolic pathways that converge around L-serine in Mycobacterium species.

Blue: present in M. tuberculosis, M. bovis and M. bovis BCG, Red: present in M. tuberculosis and M. bovis [33], Green: present in M. tuberculosis and M. bovis BCG [3,18,19], Purple: present in M. tuberculosis [44]..

Acon: aconitase, Cgl: cystathionine gamma-lyase Cit: citrate synthase, CysE, CysK1, CysK2: cysteine synthase, CysM: cystathionine beta-synthase, Eno: enolase, Gdh: glycine dehydrogenase, GlcB: malate synthase, GlyA: glycine hydromethytransferase, Gpm: phosphoglycerate mutase, Icl: isocitrate lyase, Mdh: malate dehydrogenase, Pdh: pyruvate dehydrogenase, PykA: pyruvate kinase, Ppdk: pyruvate phosphate dikinase, SerA: phosphoglycerate dehydrogenase, SerB: phosphoserine phosphatase, SerC: phosphoserine transaminase, SdaA: serine deaminase.

Validation of the model by comparison with in vitro growth rates

The GSMN-BCG was used to predict cellular growth rates on Roisin’s media using published experimental data [29] on substrate uptake rates and corresponding growth rates. Overall, in silico predictions were similar to the experimentally-determined growth rates (Table 7) and the growth rate generated by the GSMN-TB 1.1 network. Due to the inability of M. bovis to utilise glycerol, GSMN-MB was not tested.

Table 7. Comparison of in silico and in vitro growth rates on Roisin’s minimal media using calculated substrate uptake rates.

		M. bovis BCG		M. tuberculosis
Substrate	Specific consumption rate (mmol g biomass-1 h-1)	in vitro growth rate	in silico growth rate	in silico growth rate
Glycerol	0.39	0.010	0.009	0.010
Tween 80	0.002
Glycerol	0.74	0.030	0.030	0.030
Tween 80	0.09

Validation of the model by comparison with global mutagenesis data

In silico gene essentiality predictions were compared with in vitro gene essentiality data as determined by Transposon site hybridization (TraSH) mutagenesis [30] and deep-sequencing [31] (Tables S5-S7). All models gave a very similar predictive accuracy (76-77%; Table 8) when compared against TraSH data, however, results were only available for 82% of genes in the models. Comparison of predictions with a more comprehensive evaluation (100% network coverage) of gene essentiality by deep-sequencing also generated a similar predictive accuracy (75%; Table 8).

Table 8. Accuracy of in silico gene essentiality predictions.

	TraSH			Deep sequencing
Category	GSMN-TB 1.1	GSMN-MB	GSMN-BCG	GSMN-TB 1.1	GSMN-MB	GSMN-BCG
True positive	23%	24%	24%	23%	24%	24%
False positive	8%	8%	8%	6%	6%	6%
False negative	16%	16%	16%	19%	19%	20%
True negative	53%	51%	51%	52%	50%	50%
Correct predictions	77%	76%	76%	75%	75%	75%
p value	0.005	0.011	0.008	0.955	0.687	0.863

Percentage of in silico gene essentiality predictions categorised as: true-positive: essential both in silico and in vitro; false-positive: essential in silico, nonessential in vitro; true-negative: nonessential in silico and in vitro; false-negative: nonessential in silico, essential in vitro

Designation of a gene as either essential or non-essential is a binary characteristic that is generated from a continuous measurement of growth rate or mutant abundance (determined by microarray or sequencing) by applying an arbitrary cut-off value. To examine the influence of the cut-off value on predictive accuracy we plotted Receiver Operating Characteristic (ROC) curves (Figure 3; Figure S1-5). The majority (~91%) of in silico mutants generated a predicted growth rate equal to the wild-type or zero, so variation in the in silico growth rate threshold had very little influence on the result. However, in vitro cut-offs did influence prediction accuracy so ROC curves could be used to identify optimal values for the cut-off value of the in vitro measurement signal. For both TraSH and deep sequencing datasets the optimal microarray and p-value cut-offs were the original values of 0.2 and 0.05 respectively.

Figure 3. The GSMN-MB ROC curve for TraSH thresholds.

The plot shows ROC curves for different transposon site hybridisation (TraSH) ratio thresholds for the determination of essential genes in experimental data [30]. Five ROCs are plotted with 4 different TraSH thresholds as shown in the legend box. Each ROC curve shows the points corresponding to True positive rate (sensitivity) and false positive rate (1-specificity) of the model predictions obtained for all growth rate thresholds. For all ROC curves see Figures S1-S5.

Examining different areas of metabolism, the predictive accuracy (using deep sequencing data) varied from 61-62% in central carbon metabolism (glycolysis, TCA cycle, pentose phosphate cycle, methylcitrate cycle and anaplerotic reactions) to 95-96% in β-oxidation of fatty acids (Table 9). The chief source of errors for central carbon metabolism genes were false-negatives: genes predicted to be non-essential in silico but experimentally found to be essential. This likely reflects the multiple alternative pathways available for flux in in silico central metabolism, many of which are likely to be incapable of supporting sufficient flux for growth in real organisms. Peripheral areas of the network tend to generate higher predictive accuracies as fewer alternative pathways are available. Sources of false-negative in silico predictions may be due cross feeding between mutants in global mutagenesis studies.

Table 9. Percentage accuracy of gene essentiality predictions for each reaction pathway within the reaction networks.

Pathway	GSMN-TB 1.1	GSMN-MB	GSMN-BCG
Amino acid metabolism	76	75	75
Carbohydrate metabolism (excl. central metabolism)	72	75	71
Cell wall synthesis	88	88	88
Central carbon metabolism	61	62	61
Cofactor biosynthesis	66	66	66
Lipid biosynthesis	77	77	78
Nucleotide biosynthesis	70	66	66
Other functions	65	63	63
Transport reactions	94	95	95
β-oxidation of fatty acids	95	96	96

Discussion

The first genome scale metabolic models for M. bovis and M. bovis BCG, GSMN-MB and BCG, instantiate current metabolic knowledge and deliver a high degree of accuracy for predicting in vitro data. In combination with a M. tuberculosis network we have demonstrated how these models, and their application to interrogate high-throughput experimental data, shed new light on metabolic differences between the vaccine, bovine and human strains of the tubercle bacillus. Therefore, these reaction networks successfully simulate many aspects of mycobacterial metabolism, and provide an invaluable tool for the investigation of metabolic differences between these strains.

In silico models are essentially a mathematical instantiation of current knowledge. Interrogating the models to generate predictions that can be tested against experimental datasets is thereby an efficient means of probing inconsistencies and limitations of current knowledge. The study described here illustrates this approach. The discordance between in silico and in vitro phenotypes highlights the limitations of FBA predictions in which any route for flux from substrates to products may be utilised by the solution. For instance, the model predicted growth of M. bovis on glucose. This is contrary to expectations; indeed, this inability is a distinguishing feature of the M. bovis strain of the tubercle bacillus. Presumably the pathways utilised in silico to connect between glycolysis and the TCA cycle are not able to support growth in vitro [18,19] and additional defects [18], such as the identified deficiency in glucose uptake are also contributing to this phenotype.

However, our studies show that despite being unable to utilise glucose as a sole carbon source, the sugar can be assimilated by M. bovis. Indeed, glucose assimilation was stimulated by addition of Tween 80 to the glucose media [8,53]. Tween 80 is hydrolysed by mycobacteria to release fatty acids, such as oleic acid, that may be oxidised to acetate and thereby enter the TCA cycle to support the synthesis of essential metabolic precursors, such as oxaloacetate and α-ketoglutarate, as well as providing substrates for energy generation. The result suggests that M. bovis is capable of assimilating carbohydrates such as glucose, but not delivering carbon from those substrates to the TCA cycle [18]; as the additional glucose was not oxidised to CO₂ and was presumably incorporated directly into cell biomass. The result is consistent with a previous study that demonstrated compartmentalisation during co-utilisation of different substrates in M. tuberculosis [55]. When supplied with both carbohydrate (such as glucose) and fatty acid (acetate), the carbohydrate was assimilated via glycolysis and the pentose phosphate cycle and mostly incorporated into biomass; whereas acetate was mainly used for energy generation via the TCA cycle [55]. It is interesting that this disconnect is unidirectional: M. bovis can utilise acetate as a sole carbon source so must be able to drive flux from acetate to glucose via gluconeogenic pathways.

Interrogating in silico models with high throughput phenotype and gene essentiality data provides a powerful route towards refinement of the networks, but also provides an insight into the complex relationship between genome and phenotype. The mycobacterial networks are well suited for investigations into the system-wide effects of genetic mutations due to their close evolutionary history, high genetic similarly and diverse phenotypes [3,5,6,8,50]. Both phenotype and gene essentiality data were predicted with a high degree of accuracy, but interestingly key discrepancies between existing genetic knowledge and experimental phenotypes were observed. For instance, the in silico models predicted very few metabolic differences between M. tuberculosis, M. bovis and M. bovis BCG, yet substrate utilisation capability between the strains decreased from M. tuberculosis > M. bovis and M. bovis BCG. It seems that during their evolutionary passage from their common ancestor (that is presumed to be closer to M. tuberculosis) [50], M. bovis and M. bovis BCG have lost some of their capability to metabolise compounds. The basis of this loss in metabolic versatility is unknown but could result from differences in enzyme regulation. For instance, the loss of pyruvate kinase in M. bovis results in a global alteration of enzyme expression which reroutes the catabolic pathways of metabolic substrates [19].

By comprehensively testing the utilisation of amino acids as sole nitrogen sources, loss in metabolic versatility was particularly evidenced in this study. M. tuberculosis, M. bovis and M. bovis BCG were able to assimilate nitrogen from 12, 8 and 6 amino acids respectively, despite current knowledge resulting in almost identical in silico predictions. The only difference in silico is the ability of M. tuberculosis to utilise alanine as a nitrogen (and carbon) source in contrast to M. bovis and M. bovis BCG. This difference corresponds to the previously mentioned mutation in the alanine dehydrogenase gene in M. bovis and M. bovis BCG, indicating that, in accordance with previous studies [33,44] this gene is required for alanine assimilation. Another interesting finding was that some amino acids, such as serine, could act as carbon but not nitrogen sources, indicating that the pathways responsible for amino acid degradation differ for carbon and nitrogen assimilation.

The differences observed between in silico and in vitro data not only identify areas of metabolism which require further investigation, but enable iterative network modifications. Since the model development process is continuous, the networks are altered as new information becomes available. These genome scale models already successfully simulate many aspects of mycobacterial growth and metabolism, but it is to be expected that the networks will gradually become more representative of cellular metabolism over time. Further comparative analysis will help to uncover the genetic basis for the observed phenotypic and pathogenic differences between these mycobacteria, and stimulate new approaches to the control of these diseases, such as the development of novel vaccines.

Supporting Information

Figure S1

GSMN-TB 1.1 TraSH ROC curve.

(TIFF)

Figure S2

GSMN-BCG TraSH ROC curve.

(TIFF)

Figure S3

GSMN-TB 1.1 Deep sequencing ROC curve.

(TIFF)

Figure S4

GSMN-MB Deep sequencing ROC curve.

(TIFF)

Figure S5

GSMN-BCG Deep sequencing ROC curve.

(TIFF)

Table S1

GSMN-TB 1.1.

(XLS)

Table S2

GSMN-MB.

(XLS)

Table S3

GSMN-BCG.

(XLS)

Table S4

Alterations to GSMN-TB 1.1 to create GSMN-MB and BCG networks.

(XLS)

Table S5

GSMN-MB gene essentiality.

(XLSX)

Table S6

GSMN-BCG gene essentiality.

(XLSX)

Table S7

GSMN-TB 1.1 gene essentiality.

(XLSX)

Table S8

M. tuberculosis carbon utilisation.

(XLSX)

Table S9

M. bovis carbon utilisation.

(XLSX)

Table S10

M. bovis BCG carbon utilisation.

(XLSX)

Table S11

M. tuberculosis nitrogen utilisation.

(XLSX)

Table S12

M. bovis nitrogen utilisation.

(XLSX)

Table S13

M. bovis BCG nitrogen utilisation.

(XLSX)

Model S1

GSMN-TB 1.1

(XML)

Model S2

GSMN-MB

(XML)

Model S3

GSMN-BCG

(XML)

Funding Statement

This work was supported by the Animal Health and Veterinary Laboratories Agency (AHVLA) studentship fund (project SC1003, http://www.defra.gov.uk/ahvla-en/); AHVLA Seedcorn programme (project SC0205, http://www.defra.gov.uk/ahvla-en/); and the Wellcome Trust (grant reference number 088677, www.wellcome.ac.uk). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.