Uptake of amino acids and their metabolic conversion into the compatible solute proline confers osmoprotection to Bacillus subtilis

Abstract

The data presented here reveal a new facet of the physiological adjustment processes through which Bacillus subtilis can derive osmostress protection. We found that the import of proteogenic (Glu, Gln, Asp, Asn, and Arg) and of nonproteogenic (Orn and Cit) amino acids and their metabolic conversion into proline enhances growth under otherwise osmotically unfavorable conditions. Osmoprotection by amino acids depends on the functioning of the ProJ-ProA-ProH enzymes, but different entry points into this biosynthetic route are used by different amino acids to finally yield the compatible solute proline. Glu, Gln, Asp, and Asn are used to replenish the cellular pool of glutamate, the precursor for proline production, whereas Arg, Orn, and Cit are converted into γ-glutamic semialdehyde/Δ(1)-pyrroline-5-carboxylate, an intermediate in proline biosynthesis. The import of Glu, Gln, Asp, Asn, Arg, Orn, and Cit did not lead to a further increase in the size of the proline pool that is already present in osmotically stressed cells. Hence, our data suggest that osmoprotection of B. subtilis by this group of amino acids rests on the savings in biosynthetic building blocks and energy that would otherwise have to be devoted either to the synthesis of the proline precursor glutamate or of proline itself. Since glutamate is the direct biosynthetic precursor for proline, we studied its uptake and found that GltT, an Na(+)-coupled symporter, is the main uptake system for both glutamate and aspartate in B. subtilis. Collectively, our data show how effectively B. subtilis can exploit environmental resources to derive osmotic-stress protection through physiological means.

FIG 1

Overview of the uptake, synthesis, catabolism, and generation of l-proline through the metabolic conversion of several proteogenic and nonproteogenic amino acids by B. subtilis. The dashed arrows indicate that several enzymes are involved in the indicated steps.

FIG 2

Effects of amino acids on the growth of B. subtilis at high osmolarity. Cultures of the B. subtilis wild-type strain JH642 were grown in SMM with 1.2 M NaCl in the absence or presence of individual proteogenic and nonproteogenic amino acids (final concentration, 1 mM). The growth yields of the cultures were determined after 16 h of incubation at 37°C by measuring the OD₅₇₈. The values shown represent the means of two independently grown cultures, and the error bars indicate standard deviations. The gray bars represent the amino acids that promote growth of B. subtilis at high salinity. The osmoprotectant GB (hatched bar) was used as a control for these experiments.

FIG 3

Osmoprotection by amino acids depends on the osmostress-adaptive proline-biosynthetic route. (A) Cultures of the B. subtilis wild-type strain JH642 (black bars) and of strain JSB8 [Δ(proHJ::tet)1] (gray bars) were grown in SMM with 1.2 M NaCl in the absence or presence of individual proteogenic and nonproteogenic amino acids (final concentration, 1 mM). The growth yields of the cultures were determined after 16 h of incubation at 37°C by measuring the OD₅₇₈. (B) Influence of the PutBCP catabolic system on the levels of osmostress protection by proline, glutamate, and arginine. The following strains were used: JH642 (black bars), JSB8 [Δ(proHJ::tet)1] (gray bars), and ABB1 [Δ(proHJ::tet)1 Δ(putBCP::tet)2] (hatched bars). (C) Proline content of osmotically stressed cells. Cells grown in SMM containing 1.2 M NaCl were harvested once they reached an OD₅₇₈ of about 1.8 and were then assayed for their intracellular l-proline pools. The following strains were used: JH642 (black bars) and JSB8 [Δ(proHJ::tet)1] (gray bars). (D) Influence of the PutBCP catabolic system on the buildup of intracellular proline pools. The following strains (grown as described above) were used: JH642 (black bars), JSB8 [Δ(proHJ::tet)1] (gray bars), and ABB1 [Δ(proHJ::tet)1 Δ(putBCP::tet)2] (hatched bars). (C and D) The values shown represent the means of two independently grown B. subtilis cultures, and in each of these samples, the l-proline content was determined twice; the error bars indicate standard deviations.

FIG 4

Conversion of Glu, Asp, and Arg into Pro in B. subtilis wild-type cells growing at high salinity. Cultures of the B. subtilis strain JH642 were grown in SMM with 1.2 M NaCl. When the cultures reached an OD₅₇₈ of 0.5, l-[U-¹⁴C]proline (A), l-[U-¹⁴C]glutamate (B), l-[U-¹⁴C]aspartate (C), and l-[U-¹⁴C]arginine (D) were separately added to the cells (final concentration, 20 μM). Cell samples were withdrawn at the indicated time points, and the cells were harvested by centrifugation. Soluble extracts of the cell pellets were prepared and separated by thin-layer chromatography; spots corresponding to Pro, Glu, Asn, and Arg were identified through comigrating radiolabeled reference standards. Unidentified radiolabeled compounds, in all likelihood metabolic intermediates of the imported amino acids, are indicated by asterisks.

FIG 5

Uptake of radiolabeled glutamate and aspartate by B. subtilis and its gltP, gltT, and yveA mutant derivatives. (A and C) Strains JH642 (wild type) (●), ADB1 (gltP) (■), ADB4 (gltT) (▲), and MDB43 (yveA) (◆) were grown in SMM, and the initial uptake of l-[U-¹⁴C]glutamate (A) and l-[U-¹⁴C]aspartate (C) was measured at a final substrate concentration of 20 μM. (B and D) Michaelis-Menten kinetics were deduced from uptake rates for the GltT substrates l-[U-¹⁴C]glutamate (B) and l-[U-¹⁴C]aspartate (D) in strain MDB53, which possesses an intact GltT system but is defective in the GltP and YveA transporters.

FIG 6

Osmostress protection of B. subtilis by glutamate and aspartate is dependent on the GltT transporter and the functioning of the ProHJ proline-biosynthetic system. Cells of the wild-type strain JH642 (A), strain ADB4 (gltT × pUS19) (B), and strain JSB8 [Δ(proHJ::tet)1] (C) were grown at high salinity (SMM with 1.2 M NaCl) in the absence (○) or the presence of glutamate (●), aspartate (■), proline (▲), and glycine betaine (◆); the final concentration of these compounds in the growth medium was 1 mM.

FIG 7

The B. subtilis wild-type strain JH642 (gray bars) and its mutant derivatives that were defective in the indicated glutamate/aspartate transporter GltT, GltP, or YveA (black bars) were tested for the ability to use glutamate or aspartate as the sole nitrogen source. We grew these strains in minimal medium without a nitrogen source in the presence of either 2.5 mM, 7.5 mM, or 15 mM (NH₄)₂SO₄ or with 5 mM, 15 mM, or 30 mM glutamate or aspartate, respectively, and then measured the growth yields of the cultures after 15 h of incubation at 37°C. For each growth condition tested, four independent cultures were used, and the means and standard deviations are shown.