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

Abstract

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 to Photorhabdus 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, and yohF, and five known genes, inaA,ldcC, osmY, poxB, andsohA. Inactivation of the rpoS-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, and yohF, and included two known members,osmY and poxB. 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 and inaA. The pattern of gene expression changes suggested that restricted ALS activity may result in intracellular acidification and induction of the ςS-dependent stress response.

Identification of a non‐haem catalase in Salmonella and its regulation by RpoS (σS)

“…The close proximity and co-ordinate expression of yciGFE and katN may indicate that these genes have a functional relationship, related to O 2 metabolism or general stress resistance. A yciG±lux gene fusion was shown to be regulated by rpoS in E. coli (Van Dyk et al, 1998). Consistent with this finding and with our Northern blotting results (Fig.…”

Global expression profiling of yeast treated with an inhibitor of amino acid biosynthesis, sulfometuron methyl

“…APG1 encodes a protein kinase involved in the induction of autophagy after nutrient limitation (35,49). Interestingly, in E. coli, some stationary phase genes, under the control of the global regulator S , are induced in response to amino acid limitation caused by treatment with sublethal levels of SM (52). This response appears to be part of the relA-dependent stringent response mediated by ppGpp (14,27).…”

RNase H overproduction allows the expression of stress-induced genes in the absence of topoisomerase I

“…Plasmids pSK760 and pSK762c encode for wild-type and mutant RNase H respectively with ampicillin resistance selection [12,18,19], while plasmids pEM001 and pEM003 also express wild-type and mutant RNase H respectively but with chloramphenicol resistance selection [20]. Plasmid pDEW207 has a transcriptional fusion of the E. coli grpE promoter region to a luxCDABE reporter [21] and plasmid pDEW518 contains the sodA: : luxCDABE fusion [22]. Cells were grown in Luria^Bertani (LB) medium.…”

“…This would account both for the observed delay in induction of luciferase activity, and the lag in cell recovery to normal state. The sodA promoter is not induced by ethanol treatment [21] and RNase H overexpression was found to have no e¡ect on the luciferase activity from the sodA: :luxCDABE fusion in RFM475 (data not shown). The presence of pEM001 in the wild-type topA background of RFM445 resulted in a decrease instead of increase of grpE: :luxCDABE induction (Fig.…”

“…To better quantify and compare the magnitude and pattern of stress genes induction in response to environmental challenges over the time course of stress response, plasmid pDEW207 with a transcriptional fusion of the E. coli grpE promoter region to a luxCDABE reporter [21] was transformed into strains RFM445 and RFM475. Since the shift to higher temperature a¡ects the luciferase enzymatic activity, ethanol was used to induce the c 32 -dependent induction of the heat shock genes [22].…”