Purification of Mlc and Analysis of Its Effects on the pts Expression in Escherichia coli

Abstract

Products of the pts operon of Escherichia coli have multiple physiological roles such as sugar transport, and the operon is controlled by two promoters, P0 and P1. Expression of the pts P0 promoter that is increased during growth in the presence of glucose is also activated by cAMP receptor protein⅐cAMP. Based on the existence of a sequence that has a high similarity with the known Mlc binding site in the promoter, the effects of the Mlc protein on the pts P0 promoter expression were studied. In vivo transcription assays using wild type and mlc-negative E. coli strains grown in the presence and absence of glucose indicate that Mlc negatively regulates expression of the P0 promoter, and Mlc-dependent repression is relieved by glucose in the growth medium. In vitro transcription assay using purified recombinant Mlc showed that Mlc repressed transcription from the P0 but did not affect the activity of the P1. DNase I footprinting experiments revealed that a Mlc binding site was located around ؉1 to ؉25 of the promoter and that Mlc inhibited the binding of RNA polymerase to the P0 promoter. Cells overexpressing Mlc showed a very slow fermentation rate compared with the wild type when grown in the presence of various phosphoenolpyruvate-carbohydrate phosphotransferase system sugars but few differences in the presence of non-phosphoenolpyruvate-carbohydrate phosphotransferase system sugars except maltose. These results suggest that the pts operon is one of major targets for the negative regulation by Mlc, and thus Mlc regulates the utilization of various sugars as well as glucose in E. coli. The possibility that the inducer of Mlc may not be sugar or its derivative but an unknown factor is proposed to explain the Mlc induction mechanism by various sugars.

A Quantitative Approach to Catabolite Repression in Escherichia coli

“…CRP concentrations seem to be less important during such dynamic processes and might be more important for long term adaptation to different growth substrates. It has been reported that the pts operon is regulated by cAMP⅐CRP as well as by Mlc (30,31). This regulation is weak (factor of 2-3) (32) and hence has often been neglected.…”

Analyses of Mlc–IIB Glc interaction and a plausible molecular mechanism of Mlc inactivation by membrane sequestration

“…Cloning is described in SI Materials and Methods. Expression and purification of EIIB proteins and wild-type and mutant Mlc proteins with a single amino acid change were performed as described previously (7,12). Whereas wild-type and mutant Mlc proteins with a single amino acid change were insoluble in 10 mM Tris⅐HCl buffer (pH 7.5) containing 1 mM DTT, Mlc harboring two point mutations (Arg306Gly and Leu310Gly) at O-domain was soluble at neutral pH.…”

“…Glucose, a representative PTS sugar, mediates transcriptional activation of several genes for PTS-related sugar transporters and some enzymes involved in glycolysis (1, 6). Mlc, a tetrameric protein (7), is a signaling mediator for glucose induction of several PTS operons and related genes (8)(9)(10)(11)(12)(13)(14).A unique feature of induction of the Mlc regulon by glucose is that the effector molecule modulating Mlc activity is a membranebound protein, EIICB Glc (7,15,16), in which cytosolic EIIB protein is attached to membrane-embedded EIIC protein through a linker of Ͼ20 aa. In the absence of glucose, EIICB Glc mainly exists in the phosphorylated form (pEIICB Glc ).…”

“…Glucose, a representative PTS sugar, mediates transcriptional activation of several genes for PTS-related sugar transporters and some enzymes involved in glycolysis (1, 6). Mlc, a tetrameric protein (7), is a signaling mediator for glucose induction of several PTS operons and related genes (8)(9)(10)(11)(12)(13)(14).…”

Expression of ptsG Encoding the Major Glucose Transporter Is Regulated by ArcA in Escherichia coli

“…It was shown that the ptsH P1 promoter is regulated by the repressor of the fructose PTS, FruR (12). Moreover, both the ptsH P0 promoter and the ptsG P1 promoter have been shown to be subject to activation by the cAMP⅐CRP complex and repression by Mlc (13)(14)(15)(16)(17)(18). Subsequently, it was demonstrated that the unphosphorylated form of EIICB Glc can relieve the repression of the Mlc regulon by sequestering Mlc through the direct protein-protein interaction to induce the expression of the Mlc regulon including PTS proteins (19 -21).…”