Article Figures & Data
Figures
- FIG. 1.
Fermentation kinetics of the odhA mutant and revertant in the presence of excess biotin. The wild-type, C. glutamicum ATCC 13869, and the l-glutamic acid-producing odhA mutant 2A-1 and its odhA+ derivative 2A-1R were grown with excess biotin at 31.5°C. (a) Growth; (b) residual glucose; (c) l-glutamate accumulation. Symbols: filled circles, wild-type; filled triangles, odhA mutant 2A-1; open squares, 2A-1R.
- FIG. 2.
Secondary structure of C. glutamicum NCgl1221 and E. coli YggB and their mutation sites. (a) Membrane topology of C. glutamicum NCgl1221 as predicted by the PHDhtm program. Arrows indicate positions of NCgl1221 gene mutations. Circled numbers indicate residues located on the borders of transmembrane segments. (b) Proposed membrane topology of E. coli YggB based on the PhoA fusion assay (24). Arrows indicate positions of GOF mutations. Circled numbers indicate residues used for the PhoA fusion assay.
- FIG. 3.
Fluoroglutamic acid resistance of NCgl1221 gene mutants. (a) Serial dilutions (1/10 each) of cultures of wild-type C. glutamicum ATCC 13869 or NCgl1221 gene mutants were spotted onto minimal medium with or without 7.5 mM 4-fluoroglutamic acid, and the plates were incubated at 31.5°C. The upper numbers give cell titers estimated from the OD620 of cultures. (b) Cultures of wild-type C. glutamicum ATCC 13869 and its NCgl1221 gene mutants were inoculated into fresh minimal medium with or without 4-fluoroglutamic acid, and the cultures were shaken at 31.5°C. When the OD620 of each strain without 4-fluoroglutamic acid reached 1.0, cell numbers were examined by plating appropriate dilutions on CM2B plates. Colonies were counted after 2 days of incubation at 31.5°C. Symbols: filled circles, wild-type; open triangles, NCgl1221 gene deletion mutant; open squares, NCgl1221(A111V); X cross, NCgl1221(W15CSLW). Error bars indicate standard deviations (n = 3).
- FIG. 4.
Effect of NCgl1221 gene deletion on l-glutamate production and the intracellular l-glutamate pool. Wild-type C. glutamicum ATCC 13869 and its NCgl1221 gene deletion derivative were grown on biotin-depleted medium. (a) Growth; (b) residual glucose (symbols: filled circles, wild type; open circles, NCgl1221 gene deletion mutant); (c) extracellular and intracellular l-glutamate; (d) extracellular and intracellular l-aspartate (symbols: filled circles, extracellular pool of the wild type; open circles, extracellular pool of the NCgl1221 gene deletion mutant; filled triangle, intracellular pool of the wild type; open triangle, intracellular pool of NCgl1221 gene deletion mutant).
- FIG. 5.
Model of induction of l-glutamate production in C. glutamicum. Treatments inducing l-glutamate production, such as biotin limitation and detergent, alter membrane tension by inhibiting fatty acid biosynthesis. Addition of penicillin also leads to a change of membrane tension by inhibiting cell wall biosynthesis. Alteration of membrane tension triggers a structural transformation of NCgl1221 that enables it to catalyze l-glutamic acid excretion.
Tables
- TABLE 1.
l-Glutamate production by NCgl1221 gene mutants in the absence of inductiona
Strain OD620 Amt of l-glutamate (mM) Yield (wt/wt [%]) ATCC 13869 (wild type) 63.2 ± 1.4 2.4 ± 0.28 0.0 ± 0.12 BL1 [NCgl1221(V419::IS1207)] 41.0 ± 0.7 92.3 ± 1.78 39.6 ± 0.80 BL2 [NCgl1221(A111V)] 46.1 ± 0.7 74.9 ± 1.70 31.8 ± 0.76 BL3 [NCgl1221(W15CSLW)] 55.7 ± 1.2 36.6 ± 0.85 14.7 ± 0.38 ↵ a Data represent mean values ± SDs for three independent cultures.
- TABLE 2.
Effect of NCgl1221 gene amplification on l-glutamate productiona
Culture condition and strain descriptionb OD620 Amt of l-glutamate (mM) Yield (wt/wt [%]) Tween 40 Vector 51.5 ± 0.5 267 ± 3.63 50.7 ± 0.7 NCgl1221++ 39.4 ± 0.9 303 ± 11.7 58.3 ± 1.1 Penicillin G Vector 66.3 ± 0.5 165 ± 1.28 29.0 ± 0.2 NCgl1221++ 59.7 ± 0.3 204 ± 4.00 36.0 ± 0.7 Biotin limitation Vector 51.8 ± 0.6 295 ± 2.31 52.9 ± 0.4 NCgl1221++ 45.3 ± 1.4 310 ± 2.31 55.6 ± 0.4
