Constricted Flux through the Branched-Chain Amino Acid Biosynthetic Enzyme Acetolactate Synthase Triggers Elevated Expression of Genes Regulated by rpoS and Internal Acidification

The first common enzyme of isoleucine and valine biosynthesis, acetolactate synthase (ALS), is specifically inhibited by the herbicide sulfometuron methyl (SM). To further understand the physiological consequences of flux alterations at this point in metabolism,Escherichia coli genes whose expression was induced by partial inhibition of ALS were sought. Plasmid-based fusions of randomE. coli DNA fragments toPhotorhabdus luminescens luxCDABE were screened for bioluminescent increases in actively growing liquid cultures slowed 25% by the addition of SM. From more than 8,000 transformants, 12 unique SM-inducible promoter-lux fusions were identified. Thelux reporter genes were joined to seven uncharacterized open reading frames,f253a ,f415 ,frvX ,o513 ,o521 ,yciG , andyohF , and five known genes,inaA ,ldcC ,osmY ,poxB , andsohA . Inactivation of therpoS -encoded sigma factor, ςS , reduced basal expression levels of six of these fusions 10- to 200-fold. These six genes defined four new members of the ςS regulon,f253a ,ldcC ,yciG , andyohF , and included two known members,osmY andpoxB . Furthermore, the weak acid salicylate, which causes cytoplasmic acidification, also induced increased bioluminescence from seven SM-inducible promoter-lux fusion-containing strains, namely, those with fusions of the ςS -controlled genes andinaA . The pattern of gene expression changes suggested that restricted ALS activity may result in intracellular acidification and induction of the ςS -dependent stress response.