The γ-Aminobutyrate Permease GabP Serves as the Third Proline Transporter of Bacillus subtilis

Abstract

bPutP and OpuE serve as proline transporters when this imino acid is used by Bacillus subtilis as a nutrient or as an osmostress protectant, respectively. The simultaneous inactivation of the PutP and OpuE systems still allows the utilization of proline as a nutrient. This growth phenotype pointed to the presence of a third proline transport system in B. subtilis. We took advantage of the sensitivity of a putP opuE double mutant to the toxic proline analog 3,4-dehydro-DL-proline (DHP) to identify this additional proline uptake system. DHP-resistant mutants were selected and found to be defective in the use of proline as a nutrient. Whole-genome resequencing of one of these strains provided the lead that the inactivation of the ␥-aminobutyrate (GABA) transporter GabP was responsible for these phenotypes. DNA sequencing of the gabP gene in 14 additionally analyzed DHPresistant strains confirmed this finding. Consistently, each of the DHP-resistant mutants was defective not only in the use of proline as a nutrient but also in the use of GABA as a nitrogen source. The same phenotype resulted from the targeted deletion of the gabP gene in a putP opuE mutant strain. Hence, the GabP carrier not only serves as an uptake system for GABA but also functions as the third proline transporter of B. subtilis. Uptake studies with radiolabeled GABA and proline confirmed this conclusion and provided information on the kinetic parameters of the GabP carrier for both of these substrates.

Uptake of Amino Acids and Their Metabolic Conversion into the Compatible Solute Proline Confers Osmoprotection to Bacillus subtilis

“…We therefore used a ⌬proHJ mutant that is unable to synthesize osmostress-protective levels of proline to experimentally verify the anticipated roles of Glu, Gln, Asp, Asn, Arg, Orn, and Cit as precursors for osmoprotective proline biosynthesis. Except for Pro itself (25,26), osmostress protection by each of these amino acids was lost when the ProJ-ProH enzymes were nonoperational (Fig. 3A).…”

“…Both proteogenic and nonproteogenic amino acids can be found in the soil (8,10), and they exhibit a high degree of turnover, a process to which microorganisms contribute substantially (47). We wondered whether the uptake of amino acids other than proline (25,26) would aid B. subtilis in its process of adjustment to high-salinity surroundings. To test this idea, we grew the wild-type laboratory strain JH642 (Table 1) in a chemically defined minimal medium (SMM) containing 1.2 M NaCl in the absence or presence of individual proteogenic amino acids and the nonproteogenic amino acids ornithine (Orn) and citrulline (Cit).…”

“…The expression of opuE, the dominant importer for the use of exogenously provided proline as an osmostress protectant (25,26), is upregulated in response to increases in the environmental osmolarity (25,60). Given the involvement of GltT in the use of glutamate and aspartate as osmostress protectants (Fig.…”

“…Having identified GltT as a system for glutamate and aspartate in exponentially growing B. subtilis cells in a minimal medium with glucose as the carbon source, we studied the role of GltT in glutamate-and aspartate-mediated osmostress protection. For these experiments, we conducted growth studies in SMM containing 1.2 M NaCl and compared the performance of these osmoprotectants with that of the compatible solutes glycine betaine (16) and proline (26,33). Glutamate and aspartate exhibited an osmostress-protective potential similar to that afforded by proline in the wild-type strain JH642, but glycine betaine was a considerably better osmoprotectant than these compounds (Fig.…”

“…The genetic disruption of the osmostress-adaptive proline-biosynthetic route causes osmotic sensitivity of B. subtilis (18), highlighting the central role of compatible-solute accumulation in the cellular adjustment to high-osmolarity environments (7,11). Relief from osmostress can also be accomplished through import of proline via the osmotically inducible OpuE transporter (25,26), a system that is also involved in the recovery of newly synthesized proline that leaks, or is actively excreted, into the medium by B. subtilis cells continuously challenged by high osmolarity (27). Of all the compatible solutes imported by B. subtilis (7,28,29), proline is the only one that can also be catabolized (30) (Fig.…”