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 BrnQthat 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. IMPORTANCEBacillus 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. CodY is a global transcriptional regulator in Bacillus subtilis that controls, directly or indirectly, the expression of about 200 genes, most of them negatively (1-3). Many of the CodY-regulated genes are involved in nitrogen or carbon metabolism (1, 2, 4-7). CodY homologs are present in most low-GϩC Gram-positive bacteria and in many species have been shown to play a global role in metabolic regulation similar to that in B. subtilis, as well as in coordinating expression of virulence-associated functions with expression of metabolic genes (7, 8; see also, e.g., references 9 and 10 and references therein).B. subtilis CodY is a dimeric 259-residue protein that uses a winged helix-turn-helix motif to bind to DNA (11,12). The DNAbinding activity of CodY is increased by interaction with two types of effectors, branched-chain amino acids (BCAA; isoleucine [Ile], leucine [Leu], and valine [Val], collectively abbreviated as ILV) (13-17) and GTP (1,15,(18)(19)(20). The effect of GTP on CodY, however, may depend on simultaneous or prior interaction of CodY with ILV because mutant variants of CodY deficient in ILV binding lose most of the ability to regulate CodY-dependent genes (21, 22). The pools of ILV and GTP apparently reflect the nutritional status of the cells, allowing bacteria to change the pattern of CodY-dependent gene expression in response to availability of nutrients in the g...

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