Transcriptional and translational regulation of alpha-acetolactate decarboxylase of Lactococcus lactis subsp lactis

The alpha-acetolactate decarboxylase (ALDC) gene, aldB, is the penultimate gene of the leu-ilv-ald operon, which encodes the three branched-chain amin o acid (BCAA) biosynthesis genes in Lactococcus lactis, Its product plays a dual role in the cell: (i) it catalyzes the second step of the acetoin pat hway, and (ii) it controls the pool of alpha-acetolactate during leucine an d valine synthesis. It can be transcribed from the two promoters present up stream of the leu and ilv genes (P1 and P2) or independently under the cont rol of its own promoter (P3). In this paper we show that the production of ALDC is limited by two mechanisms. First, the strength of P3 decreases grea tly during starvation for BCAAs and under other conditions that generally p rovoke the stringent response. Second, although aldB is actively transcribe d from P1 and P2 during BCAA starvation, ALDC is not significantly produced from these transcripts. The aldB ribosome binding site (RBS) appears to be entrapped in a stem-loop, which is itself part of a more complex RNA foldi ng structure. The function of the structure was studied by mutagenesis, usi ng translational fusions with luciferase genes to assess its activity. The presence of the single stem-loop entrapping the aldB RES was responsible fo r a 100-fold decrease in the level of aldB translation. The presence of a s upplementary secondary structure upstream of the stem-loop led to an additi onal fivefold decrease of aldB translation. Finally, the translation of the ilvA gene terminating in the latter structure decreased the level of trans lation of aldB fivefold more, leading to the complete extinction of the rep orter gene activity, Since three leucines and one valine are present among the last six amino acids of the ilvA product, we propose that pausing of th e ribosomes during translation could modulate the folding of the messenger, as a function of BCAA availability. The purpose of the structure dependent regulation could be to ensure the minimal production of ALDC required for the control of the acetolactate pool during BCAA synthesis but to avoid its overproduction, which would dissipate acetolactate, Large amounts of ALDC, necessary for operation of the acetoin pathway, could be produced under fa vorable conditions from the P3 transcripts, which do not contain the second ary structures.