Purification of Mlc and analysis of its effects on the pts expression in Escherichia coli

Products of the pts operon of Escherichia coil have multiple physiological roles such as sugar transport, and the operon is controlled by two promoter s, PO and P1, Expression of the pts PO promoter that is increased during gr owth in the presence of glucose is also activated by cAMP receptor protein cAMP. Based on the existence of a sequence that has a high similarity with the known Rile binding site in the promoter, the effects of the Mlc protein on the pts PO promoter expression were studied. In vivo transcription assa ys using wild type and mlc-negative E. coil strains grown in the presence a nd absence of glucose indicate that Mlc negatively regulates expression of the PO 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 PO but did not affect the activity of the P1, DNase I footprinting experiments revealed that a Rile binding site was located around +1 to +25 of the pro meter and that Mlc inh ibited the binding of RNA polymerase to the PO promoter. Cells overexpressi ng Mlc showed a very slow fermentation rate compared with the wild type whe n grown in the presence of various phosphoenolpyruvate-carbohydrate phospho transferase system sugars but few differences in the presence of non-phosph oenolpyruvate-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 Mle regulates the utilization of vario us sugars as well as glucose in E. coil, The possibility that the inducer o f Mlc may not be sugar or its derivative but an unknown factor is proposed to explain the Mlc induction mechanism by various sugars.