glpx Rv1100 Probe 4.4 kb 75 kda 50 kda 37 kda 25 kda 20 kda

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1 PvuI 2.0 k PvuI PvuI Rv1101c Rv1100 glpx fum 4.4 k PvuI PvuI ΔglpX Rv1101c Rv1100 HygR fum ΔglpX 5 k 4 k 3 k c 75 kda 50 kda 37 kda GLPX Δ Δ/C GLPX 2 k 1.5 k 25 kda 20 kda PRCB 1 k 0.5 k Supplementary Fig. 1. Generation and Validation of ΔglpX. (a) Strategy for deleting glpx (Rv1099c) y homologous recomination and validating ΔglpX candidates y PvuI digest and southern lot analysis. () Southern lot analysis of Mt and ΔglpX where genomic DNA was digested with PvuI. (c) Western lot analysis of Mt (), ΔglpX (Δ), and complemented strain ΔglpX/C (Δ/C) cell lysates with anti-glpx antiody. Molecular weight of GLPX is kda. Recominant His-tagged GLPX (GLPX, kda) was run as a positive control. Anti- PRCB was used to confirm equal loading.

2 Glucose G6P S7P F6P FBP GLPX DHAP G3P Glycerol GL3P PEP PYR Acetyl-CoA ASP OAA TCA Cycle α-kg Supplementary Fig. 2. Gluconeogenesis is not strictly dependent on GLPX. Aundance and 13 C laeling of metaolites in Mt and ΔglpX (Δ). Bacteria were grown on glycerol-containing plates for 5 days and then transferred to U- 13 C glycerol-containing plates for an additional 16h prior to harvesting. Data are means ± standard deviation of three iological replicates and are representative of two independent experiments. Data are reported as ion counts (IC) per mg total protein. *0.01<P 0.05, **0.001<P 0.01, ***P y Student s t-test. G6P: glucose 6- phosphate, S7P: sedoheptulose 7-phosphate, F6P: fructose 6-phosphate, FBP: fructose 1,6- isphosphate, DHAP: dihydroxyacetone phosphate, G3P: glyceraldehyde 3-phosphate, GL3P: glycerol 3-phosphate, PEP: phosphoenolpyruvate, PYR: pyruvate, OAA: oxaloacetate, ASP: aspartate α-kg: α-ketoglutarate.

3 DraIII 3.8 k BsI (glpx Msm ) 5240 (fum) 2.4 k DraIII DraIII BsI BsI 5238 Hyg 5240 (fum) c Δ GLPX Δ Δ/C 4 k 3 k 75 kda 50 kda 37 kda GLPX GLPX Msm 2 k 25 kda PRCB Supplementary Fig. 3. Generation and Validation of. (a) Strategy for deleting msmeg_5239 (glpx homolog in M. smegmatis) y homologous recomination and validating candidates y DraIII and BsI doule digest and southern lot analysis. () Southern lot analysis of Msm and where genomic DNA was digested with DraIII and BsI. (c) Western lot analysis of Msm (), (Δ), and complemented strain /C (glpx) (Δ/C) cell lysates with anti-glpx antiody. Molecular weight of GLPX Msm is kda. Molecular weight of GLPX is kda. Recominant His-tagged GLPX (GLPX, kda) was run as a positive control. Anti-PRCB was used to confirm equal loading.

4 0.1% Glucose 0.1% Glycerol 10 DglpX Msm DglpX Msm /C (glpx) OD 580 OD Time (hours) Time (hours) 1.5 mm FBP 12 mm FBP Enzymatic Activity (nmole/min/mg total protein) DglpX Msm DglpX Msm /C (glpx) Enzymatic Activity (nmole/min/mg total protein) LiCl (mm) LiCl (mm) Supplementary Fig. 4. DglpX Msm can grow on gluconeogenic caron sources and has detectale FBPase activity. (a) Growth of Msm (lack), DglpX Msm (red) and complemented strain DglpX Msm /C (glpx) (gray) in 7H9 media containing 0.1% glucose or 0.1% glycerol as the sole caron source. Data are representative of three independent experiments. () FBPase activity of Msm (lack), DglpX Msm (red) and complemented strain DglpX Msm /C (glpx) (gray) cell lysates in the asence or presence of lithium chloride using 1.5 mm F-1,6BP or 12 mm F-1,6BP as sustrate. Dashed line indicates limit of detection. Data are means ± standard deviation of three iological replicates.

5 2.5 k SpeI XhoI Rv3213c gpm2 entc 1.5 k SpeI SpeI XhoI Δgpm2 Rv3213c le gpm2 entc ΔΔ c ΔΔ ΔΔ/C d GPM2 ΔΔ ΔΔ/C 3 k 2 k 50 kda 37 kda GLPX 50 kda 37 kda ENO 1.5 k 1 k 25 kda 20 kda PRCB 25 kda 20 kda GPM2 Supplementary Fig. 5. Generation and Validation of Mt ΔglpXΔgpm2. (a) Strategy for deleting gpm2 (Rv3214) y homologous recomination and validating ΔglpXΔgpm2 candidates y SpeI and XhoI doule digest and southern lot analysis. () Southern lot analysis of Mt and ΔglpXΔgpm2 where genomic DNA was digested with SpeI and XhoI. (c) Western lot analysis of Mt (), ΔglpXΔgpm2 (ΔΔ), and complemented strain ΔglpXΔgpm2/C (glpx) (ΔΔ/C) cell lysates with anti-glpx antiody. Molecular weight of GLPX is kda. Anti-PRCB was used to confirm equal loading. (d) Western lot analysis of Mt (), ΔglpXΔgpm2 (ΔΔ), and complemented strain ΔglpXΔgpm2/C (gpm2) (ΔΔ/C) cell lysates with anti-gpm2 antiody. Molecular weight of GPM2 is kda. Recominant His-tagged GPM2 (GPM2, kda) was run as a positive control. Anti-ENO was used to confirm equal loading.

6 0.1% Acetate 0.2% Acetate OD DglpX DglpXDgpm2 DglpXDgpm2/C (glpx) DglpXDgpm2/C (gpm2) CFU per ml Time (days) Time (days) Supplementary Fig. 6. Mt DglpXDgpm2 does not grow on fatty acids as a sole caron source ut remains viale. (a) Growth of Mt (lack), DglpX (red), DglpXDgpm2 (orange) and complemented strains DglpXDgpm2/C (glpx) (purple) and DglpXDgpm2/C (gpm2) (lue) in Sauton's minimal media containing 0.1% acetate. Data are representative of three independent experiments. () Survival of DglpXDgpm2 in Sauton's minimal media containing 0.2% acetate. Data represent the mean CFU/mL of three iological replicates at different time points.

7 Glucose G6P S7P F6P FBP GLPX, GPM2 DHAP G3P PEP PYR Acetyl-CoA ASP OAA TCA Cycle Supplementary Fig. 7. Glycolysis is unpertured in Mt ΔglpXΔgpm2. Aundance and 13 C laeling of metaolites in Mt and ΔglpXΔgpm2 (ΔΔ). Bacteria were grown on glucosecontaining plates for 5 days and then transferred to U- 13 C glucose-containing plates for an additional 24h prior to harvesting. Data are means ± standard deviation of three iological replicates and are representative of two independent experiments. *0.01<P 0.05, **0.001<P 0.01, ***P y Student s t-test. ND indicates that the recovered level of FBP was elow the limit of detection (3.13 μm). G6P: glucose 6-phosphate, S7P: sedoheptulose 7- phosphate, F6P: fructose 6-phosphate, FBP: fructose 1,6-isphosphate, DHAP: dihydroxyacetone phosphate, G3P: glyceraldehyde 3-phosphate, PEP: phosphoenolpyruvate, PYR: pyruvate, OAA: oxaloacetate, ASP: aspartate.

8 10 OD Time (days) 0.1% Acetate 0.1% Acetate + 0.2% Glucose 0.1% Acetate + 0.4% Glucose 0.4% Glucose Supplementary Fig. 8. Growth of Mt DglpXDgpm2 on glucose is enhanced y the presence of acetate. Growth of DglpXDgpm2 in Sauton's minimal media containing 0.1% acetate (red), 0.1% acetate and 0.2% glucose (lue) 0.1% acetate and 0.4% glucose (orange) or 0.4% glucose (lack) Data are representative of three independent experiments.

9 Supplementary Fig. 9. Mt ΔglpXΔgpm2 is attenuated in vivo Lung histopathology. (a) Gross pathology of left lung loes from infected C57BL/6 mice at Day 56 post-infection. () Hemotoxylin and eosin staining of sections from left lung loes of infected C57BL/6 mice at Day 56 post-infection. Top row of images show sections from four mice per group. Black ox indicates region magnified in the ottom row of images. The scale ar is 10 μm.

10 Purification Step Protein Concentration (mg/ml) Total Protein (mg) Activity (nmol/min) Specific Activity (nmol/min/mg protein x10 3 ) Fold Enrichment Total Protein Lysate Anion Exchange (Q Sepharose) Hydrophoic Interaction (Phenyl Sepharose) Gel Filtration (Superose 6) Anion Exchange (Mono Q) * * * 65.13* Supplementary Tale 1. Purification of second FBPase activity from ΔglpX cell lysate. Four purification steps were done in total. Initial FBPase activity and protein concentration for the total lysate sample are shown. After the anion exchange (Q Sepharose), hydrophoic interaction and gel filtration steps, the active fractions were pooled and the FBPase activity and protein concentration of were measured. After the anion exchange (Mono Q) step, the FBPase activity and protein concentration were measured for just the peak activity fraction (Fraction B15). Total protein, specific activity and fold enrichment of activity were determined for each sample accordingly. Asterisks indicate that the protein concentration of the anion exchange (Mono Q) sample was elow the limit of detection of the protein measurement assay used ( mg/ml). As a result, total protein, specific activity and fold enrichment values for this step are presented as minimum values ased on the limit of detection of the protein measurement assay. Peptide Sequence MH + (Da) HTGGTEVELTDTGR HGETAWSTLGR WVQLPLAEGSR ADSAVALALEHMSSR TQAELAGQLLGELELDDPIVICSPR QLAVLGLTGHPQPIAAG DVLFVSHGHFSR LAGLTVNEVTGLLAEWDYGSYEGLTTPQIR Supplementary Tale 2. GPM2 peptides identified y LC-MS/MS of 25 kda gel and from Figure 2.

11 Name Sequence Use in This Study JM17 5 TATACTTAAGCGGCGTTGCTCTGGGTCAAGCTCAG 3 JM18 5 AATACCTAGGGGCCAGGTTGCGGTCCGGGGCTTCC 3 JM19 5 TATAAAGCTTCACACCCACGACACACAAGGAACCC 3 JM20 5 ATATATGCATGGCGGCCAATGCGTCGTGCAGCTGC 3 UG1 5 ACCACGCACCAATGCTCTAC 3 UG2 5 CGGTGGTCAAGACCTGGTTC 3 5 GGGGACTGCTTTTTTGTACAAACTTGTGCCTCGGTTACCCGGACAAGTTC 3 5 GGGGACAACTTTGTATAGAAAAGTTGGTACTCGCTGGTGTCCGAGTTACG 3 5 GGGGACAGCTTTCTTGTACAAAGTGGACACCAACAAACAAGGGACGCAACAG 3 5 GGGGACAACTTTGTATAATAAAGTTGTTGACGAGCACCTCGACGACCTTG 3 UG3 5 TACGAGCTGGGCTTCATCGAC 3 UG4 5 TGGGATACTGGCAGGCATGAC 3 clo-glpx-attb2 JM33 UG17-SD 5 GGGGACAGCTTTCTTGTACAAAGTGGAAGGAGGACCAACCATGACAGCTGAGG GATCCGGTT 3 5 GGGGACAACTTTGTATAATAAAGTTGCTTAGGGCAATGGGTACACGGCGCTGCT GTCGC 3 5 GGGGACAGCTTTCTTGTACAAAGTGGAAGGAGGTGTCGGTATGGGCGTGCGCA ACCACCG 3 UG18 5 CTTATCGTCATCGTCCTTGTAGTCCCCGGCTGCGATCGGCTGCGGATGACC 3 KO-glpXsmattB1r KO-glpXsmattB4 KO-glpXsmattB2 KO-glpXsmattB3 OE-gpm2- attb1 (UG) OE-gpm2- attb2r KO-gpm2- attb4 KO-gpm2- attb1r KO-gpm2- attb2 KO-gpm2- attb3 5 GGGGACAAGTTTGTACAAAAAAGCAGGCTTGGGCGTGCGCAACCACCGATTGC TAC 3 5 GGGGACCACTTTGTACAAGAAAGCTGGGTTGTGCGCTCACCCGGCTGCGATCG GC 3 5 GGGGACAACTTTGTATAGAAAAGTTGGCGGTCGGCGACATCGAGCAACACG 3 5 GGGGACTGCTTTTTTGTACAAACTTGCCGACAGATTGTGCCCGACGACATC 3 5 GGGGACAGCTTTCTTGTACAAAGTGGTGGACGCACGGCTGCCCAGCTGGAG 3 5 GGGGACAACTTTGTATAATAAAGTTGTGAGGTCGACGAGATAGCCGTAAGC 3 UG20 5 ATACTGTTGGGCGCGTTGCCTTTC 3 UG21 5 TTGGCCGAACTGGCTAGTGC 3 ΔglpX Knockout ΔglpX Knockout ΔglpX Knockout ΔglpX Knockout ΔglpX Southern Blot ΔglpX Southern Blot Knockout Knockout Knockout Knockout Southern Blot Southern Blot glpx Complementation glpx Complementation gpm2 Complementation gpm2 Complementation 6xHis-Tagged GPM2 6xHis-Tagged GPM2 Δgpm2 Knockout Δgpm2 Knockout Δgpm2 Knockout Δgpm2 Knockout Δgpm2 Southern Blot Δgpm2 Southern Blot Supplementary Tale 3. List of primers used in this study.