Role of branched-chain amino acid transport in Bacillus subtilis CodY activity

Abstract

CodY is a branched-chain amino acid-responsive transcriptional regulator that controls the expression of several dozen transcription units in Bacillus subtilis. The presence of isoleucine, valine, and leucine in the growth medium is essential for achieving high activity of CodY and for efficient regulation of the target genes. We identified three permeases-BcaP, BraB, and BrnQ-that are responsible for the bulk of isoleucine and valine uptake and are also involved in leucine uptake. At least one more permease is capable of efficient leucine uptake, as well as low-affinity transport of isoleucine and valine. The lack of the first three permeases strongly reduced activity of CodY in an amino acid-containing growth medium. BcaP appears to be the most efficient isoleucine and valine permease responsible for their utilization as nitrogen sources. The previously described strong CodY-mediated repression of BcaP provides a mechanism for fine-tuning CodY activity by reducing the availability of amino acids and for delaying the utilization of isoleucine and valine as nitrogen and carbon sources under conditions of nutrient excess.

Importance: Bacillus subtilis CodY is a global transcriptional regulator that is activated by branched-chain amino acids (BCAA). Since the level of BCAA achieved by intracellular synthesis is insufficient to fully activate CodY, transport of BCAA from the environment is critical for CodY activation, but the permeases needed for such activation have not been previously identified. This study identifies three such permeases, reports their amino acid transport specificity, and reveals their impact on CodY activation.

FIG 1

Pathway of ILV biosynthesis in B. subtilis.

FIG 2

Growth of auxotrophic liv mutant strains with different concentrations of ILV. Cells of strain BB3467 (liv) or BB3067 (bcaP braB brnQ liv) were grown overnight in TSS glucose-ammonium minimal medium with 200 μg of ILV/ml and then diluted 100-fold in the same medium containing various concentrations of ILV, as indicated. Each growth experiment was performed at least twice, and the results of a representative experiment are shown.

FIG 3

Uptake of [¹⁴C]Ile. Cells were grown in TSS glucose-ammonium minimal medium, and uptake of [¹⁴C]Ile was measured as described in Materials and Methods at 26°C (A, B, C, and E) or 37°C (D). (A) SMY (wild-type), BB3050 (braB brnQ), BB2913 (bcaP brnQ), BB3055 (bcaP braB), BB3051 (bcaP braB brnQ), and BB3475 (azlB azlCD braB bcaP); (B and C) BB3050 (braB brnQ); (D) BB2913 (bcaP brnQ); (E) BB3475 (azlB azlCD braB bcaP). A 100-fold excess (1 mM) of competing amino acids was added, as indicated, in panels B to E.

FIG 4

Growth inhibition by threonine. Cells of strain SMY (wild type), BB2726 (bcaP), or BB3198 (bcaPp_10/14) were grown overnight in TSS glucose-ammonium minimal medium and then diluted 100-fold in the same medium with or without threonine (50 or 1,000 μg/ml) as indicated. Each growth experiment was performed at least twice, and the results of a representative experiment are shown.