Genes involved in nitrogen metabolism in the slow growing mycobacterium <i>M. bovis</i> BCG.

<p>Ammonia is assimilated in the production of L-glutamine and L-glutamate. Together, L-glutamine, L-glutamate, and L-aspartate act as precursors or nitrogen donors to most other nitrogenous compounds in the mycobacterium. The map was constructed from the combined PATRIC pathways for <i>M. bovis</i> BCG <i>str</i>. Pasteur 1743P2 nitrogen metabolism and alanine, aspartate and glutamate metabolism [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084452#B3" target="_blank">3</a>]. Genes were assigned to the EC numbers by PATRIC and/or Refseq and/or Legacy BRC.</p

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.</p

Growth of <i>M. bovis</i> BCG in standard 7H9, 7H9 lacking nitrogen sources (‑N7H9) and 7H9 containing alanine as sole nitrogen source (‑N7H9 + 3 mM L-Ala).

<p>Mean OD measurements with standard deviations were calculated with growth curve data obtained from three independent experiments performed for each condition tested.</p

Replacement of the <i>gltBD</i> operon with a hygromycin cassette and gene deletion of <i>gdh</i>.

<p>A) Comparison of the wild-type <i>M. bovis</i> BCG and mutant <i>gltBD</i> regions. B) Southern blot analysis of wild-type <i>M. bovis</i> BCG (lane 1) and <i>ΔgltBD::hyg</i> (lane 2) with a probe that hybridises upstream of <i>gltB</i>. Southern blot analysis of wild-type <i>M. bovis</i> BCG (lane 3) and <i>ΔgltBD::hyg</i> (lane 4) with a probe that hybridises downstream of <i>gltD</i>. C) Comparison of the wild-type <i>M. bovis</i> BCG and mutant <i>gdh</i> regions. The GDH domain region is the sequence in <i>gdh</i> which aligned with a 98% query coverage (66% identity, 80% positives) in a blastp to the GDH domain sequence of the previously characterised Streptomyces <i>clavuligerus</i> L-180 GDH [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084452#B36" target="_blank">36</a>]. The NruI fragment spanning the GDH domain is deleted in the <i>Δgdh</i> chromosome. The probe which is complementary to fragments both upstream and downstream of the GDH domain does not hybridise across its full length with wild-type <i>M. bovis</i> BCG DNA, but does with <i>Δgdh</i> mutant DNA, as indicated in the figure. D) Southern blot analysis of wild-type <i>M. bovis</i> BCG (lane 1) and <i>Δgdh</i> (lane 2). <i>S</i>, SphI; <i>hyg</i>^<i>R</i>, hygromycin resitance cassette; <i>K</i>, KpnI; <i>N</i>, NruI; U, upstream; D, downstream.</p

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

<p>Growth of wt-BCG, the <i>ΔgltBD</i> mutant and the <i>ΔgltBD</i> complemented strain in (A) standard 7H9 containing approximately 4 mM ammonium sulphate (AS, (NH₄)₂SO₄) and 3 mM L-Glu, (B) 7H9 + 10 mM L-Glutamate, or (C) ‑N7H9 (nitrogen-depleted 7H9) + 4 mM AS. Growth of the <i>ΔgltBD</i> mutant in (D) ‑N7H9 + 4 mM AS supplemented with increasing concentrations of glutamate. Cultures for cfu/ml determinations were inoculated to OD₆₀₀ = 0.0005 (cfu/ml of approximately 10⁵). Log₁₀(cfu/ml) of <i>ΔgltBD</i> cultured in -N7H9 + 4 mM AS supplemented with 10 mM L-Glu was different from log₁₀(cfu/ml) of <i>ΔgltBD</i> cultured in -N7H9 + 4 mM AS at every time point after and including 3 days (P < 0.001). Log₁₀(cfu/ml) of <i>ΔgltBD</i> cultured in -N7H9 + 4 mM AS supplemented with 3 mM L-Glu was different from log₁₀(cfu/ml) of <i>ΔgltBD</i> cultured in -N7H9 + 4 mM AS at 3 days (p < 0.01) and every following time point (p < 0.001). Log₁₀(cfu/ml) of <i>ΔgltBD</i> cultured in -N7H9 + 4 mM AS supplemented with 0.3 mM L-Glu was different from log₁₀(cfu/ml) of <i>ΔgltBD</i> cultured in -N7H9 + 4 mM AS at 6 days (p < 0.01) and every following time point (p < 0.001). Growth of wt-BCG, the <i>ΔgltBD</i> mutant and the <i>ΔgltBD</i> complement strain in (E) –N7H9 + 3 mM L-Glu, (F) –N7H9 + 3 mM L-Asn, (G) ‑N7H9 + 3 mM L-Gln, (H) –N7H9 + 3 mM L-Asp, (I) unmodified –N7H9, or (J) 7H9 + 30 mM AS. Mean OD measurements with standard deviations presented in panels A-C and E-J and mean log₁₀(cfu/ml) with standard errors presented in panel D 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.</p