Article Figures & Data
Figures
- FIG 1
Phylogenetic analysis of RpoB and PK across 80 different actinobacterial genera grouped and color coded by order/suborder. (A) Phylogenetic tree based on RpoB protein sequences. (B) Phylogenetic tree based on pyruvate kinase protein sequences. Gray circles indicate the Streptomycineae. (C) Specific growth rate of E. coli pyruvate kinase mutants (ΔpykA, ΔpykF, and ΔpykA ΔpykF) and the mutants complemented with either pyk1 or pyk2 from S. coelicolor on M9 medium with glucose as carbon source. *, P value ≤ 0.05; **, P value ≤ 0.01; ***, P value ≤ 0.001.
- FIG 2
Phenotypic characterization of pyruvate kinase mutants. (A) Wild-type and pyk1 and pyk2 transposon mutants of S. coelicolor grown on nutrient agar and minimal medium (MM) with glucose, with strains complemented with either pyk1 or pyk2 in trans and WT strains with additional copies of pyk1 or pyk2. (B) Growth curve of S. coelicolor WT, pyruvate kinase mutants, and complemented strains in liquid YEME medium. (C and D) Coelimycin production yield (absorption unit [AU] per milligram of biomass) (C) and undecylprodigiosin (RED) yield during growth in YEME medium (D). Symbols: black circles, WT; blue squares, pyk1 pyruvate kinase mutant; red inverted triangles, pyk2 pyruvate kinase mutant; purple triangles, pyk1-complemented strain; pink diamonds, pyk2-complemented strain.
- FIG 3
(A) Semiquantitative RT-PCR of expression of pyk1, pyk2, and hrdB throughout the life cycle of Streptomyces coelicolor. (B) Fold change expression of pyk1 and pyk2 normalized to hrdB expression from three biological replicates measuring expression levels by qPCR on growth in minimal medium with either glucose or Tween as carbon source during log or stationary phase comparing expression in stationary phase versus log phase (i) and Tween versus glucose (ii). *, P value ≤ 0.05. (C) Fitness of pyk1 (dark blue circles) and pyk2 (red squares) mutants compared to WT and each other (relative to pyk1) in rich medium (green triangles) and minimal medium with glucose (orange inverted triangles) as sole carbon source. Error bars represent the standard deviation from three biological replicates, with the mean indicated. The line (at a relative fitness of 1.0) indicates the fitness of a strain when competed against itself.
Tables
- TABLE 1
Percentage of primary metabolic pathway gene expansion per suborder and pathway of central carbon metabolisma
Order/suborder No. of
generaNo. of
species% expansion per pathway (no. of enzymes in minimum pathway): GLY (10) GNG (5) PPP (7) TCA (17) AA derived from: 2OG (13) PYR (10) OAA (18) 3PGA (7) R5P (10) E4P/PEP (18) Streptomycineae 3 303 23.3 8.3 4.8 8.3 11.9 11.1 10.5 16.7 NE 14.0 Catenulisporineae 2 3 25.0 12.5 42.9 25.0 21.4 16.7 13.2 25.0 10.0 15.8 Streptosporangineae 9 28 6.7 11.1 3.2 13.2 10.3 17.3 11.7 11.1 3.3 12.3 Frankineae 3 11 6.7 NE 23.8 NE 11.9 NE 8.8 11.1 10.0 8.8 Pseudonocardineae 16 57 10.6 25.0 17.0 28.3 20.1 31.3 20.7 24.0 17.5 19.4 Corynebacterineae 8 62 7.5 9.4 5.4 10.2 8.7 8.3 6.6 6.3 7.5 5.3 Micromonosporineae 9 19 20.0 13.9 15.9 16.7 14.3 18.5 21.1 13.0 23.3 13.5 Glycomycineae 1 3 NE 0.0 14.3 NE 7.1 NE 5.3 NE NE NE Micrococcineae 17 74 6.5 5.9 5.0 5.5 4.6 2.6 8.4 6.9 3.5 4.0 Bifidobacteriales* 1 3 NE NE 14.3 NE 0.0 11.1 21.1 NE 10.0 NE Actinomycineae 2 26 NE NE 7.1 NE 0.0 NE NE NE NE 5.3 Kineosporiineae 1 1 20.0 NE 28.6 NE 14.3 NE 21.1 NE 20.0 5.3 Propionibacterineae 8 24 13.8 12.5 7.1 9.4 7.1 18.1 11.8 12.5 7.5 4.6 ↵a The highest percentage of gene expansion for each pathway is highlighted in bold. Abbreviations: GLY, glycolysis; GNG, gluconeogenesis; PPP, pentose phosphate pathway; TCA, tricarboxylic acid cycle; AA, amino acids; 2OG, 2-oxoglutarate; PYR, pyruvate; OAA, oxaloacetate; 3PGA, 3-phosphoglycerate; R5P, ribose-5-phosphate; E4P, erythrose-4-phosphate; PEP phosphoenolpyruvate; NE, no expansion. *, order Bifidobacteriales.
- TABLE 2
Kinetic characteristics of Pyk1 and Pyk2 for the substrates ADP and PEP and the activator AMP
Substrate or activator Parameter No AMP 1 mM AMP Pyk1 Pyk2 Pyk1 Pyk2 5 mM PEP with ADP Vmax (U/mg) 21.0 1.2 73.3 6.7 S0.5 (mM) 0.6 0.3 0.2 0.1 kcat (s−1) 941 39 4,703 215 kcat/Km (mM−1 s−1) 1,594 145 31,359 2,388 1.5 mM ADP with PEP Vmax (U/mg) 14.1 0.5 65.5 9.1 S0.5 (mM) 3.5 1.3 1.1 8.6 Hill coefficient 3.7 1.5 1.8 7.1 kcat (s−1) 350 18 4,200 336 kcat/S0.5 (mM−1 s−1) 100 14 4,000 39 AMP Vmax (U/mg) 8.2 1 S0.5 (mM) 0.01 3.8 kcat (s−1) 423.7 42.0 kcat/S0.5 (mM−1 s−1) 42,373 11
