Growth of <i>Δgdh</i> in 7H9 with different nitrogen sources.

<p>Growth of wt BCG, the <i>Δgdh</i> mutant and the <i>Δgdh</i> complemented strain in (A) standard 7H9 containing approximately 4 mM ammonium sulphate (AS, (NH₄)₂SO₄) and 3 mM L-Glu, (B) ‑N7H9 (nitrogen-depleted 7H9) + 3 mM L-Glu, (C) 7H9 + 30 mM L-Glu, (D) 7H9 + 30 mM L-Asn, (E) -N7H9 + 30 mM L-Asn, (F) 7H9 + 3 mM L-Asn, (G) -N7H9 + 3 mM L-Asn, or (H) 7H9 + 30 mM L-Asp. Growth of the <i>Δgdh</i> mutant in (I) ‑N7H9 + 3 mM L-Glu supplemented with increasing concentrations of AS. Cultures for cfu/ml determinations were inoculated to OD₆₀₀ = 0.0005 (cfu/ml of approximately 10⁵). Log₁₀(cfu/ml) of <i>Δgdh</i> cultured in -N7H9 + 3 mM Glu was different from log₁₀(cfu/ml) of <i>Δgdh</i> cultured in -N7H9 + 3 mM Glu supplemented with 1 mM AS at day 9 and 14 (p < 0.01). Log₁₀(cfu/ml) of <i>Δgdh</i> cultured in 7H9 + 30 mM L-Asn was different from log₁₀(cfu/ml) of <i>Δgdh</i> cultured in -N7H9 + 3 mM Glu supplemented with 1 mM AS at day 6, 9 and 14 (p < 0.01). (J) Growth of the <i>Δgdh</i> mutant cultured in ‑N7H9 + 3 mM L-Glu for three weeks when sub-cultured in fresh 7H9 or –N7H9 + 3 mM L-Glu. Aliquots of three week old <i>Δgdh</i> mutant ‑N7H9 + 3 mM L-Glu cultures were washed once with ‑N7H9 and used to inoculate fresh 7H9 or ‑N7H9 + 3 mM L-Glu to an OD₆₀₀ = 0.020. (K) Determination of growth of single colonies obtained from three week old <i>Δgdh</i> mutant ‑N7H9 + 3 mM L-Glu cultures in fresh –N7H9 + 3 mM L-Glu. A1 and A2 were obtained from the first growth curve experiment, B1 and B2 from the second and C1-C3 from the third. Mean OD measurements with standard deviations presented in panels A-H and J and mean log₁₀(cfu/ml) with standard errors presented in panel I were calculated with growth curve data obtained from three independent experiments performed for each condition tested. In some instances error bars were smaller than the symbols used to depict the means. AS, ammonium sulphate.