Heat Shock RNA Polymerase (Eς32) Is Involved in the Transcription of mlc and Crucial for Induction of the Mlc Regulon by Glucose in Escherichia coli

Abstract

Mlc is a global regulator of carbohydrate metabolism. Recent studies have revealed that Mlc is depressed by protein-protein interaction with enzyme IICB(Glc), a glucose-specific permease, which is encoded by ptsG. The mlc gene has been previously known to be transcribed by two promoters, P1(+1) and P2(+13), and have a binding site of its own gene product at +16. However, the mechanism of transcriptional regulation of the gene has not yet been established. In vitro transcription assays of the mlc gene showed that P2 promoter could be recognized by RNA polymerase containing the heat shock sigma factor final sigma(32) (E sigma(32)) as well as E sigma(70), while P1 promoter is only recognized by E sigma(70). The cyclic AMP receptor protein and cyclic AMP complex (CRP.cAMP) increased expression from P2 but showed negative effect on transcription from P1 by E sigma(70), although it had little effect on transcription from P2 by E sigma(32) in vitro. Purified Mlc repressed transcription from both promoters, but with different degrees of inhibition. In vivo transcription assays using wild type and mlc strains indicated that the level of mlc expression was modulated less than 2-fold by glucose in the medium with concerted action of CRP.cAMP and Mlc. A dramatic increase in mlc expression was observed upon heat shock or in cells overexpressing final sigma(32), confirming that E sigma(32) is involved in the expression of mlc. Induction of ptsG P1 and pts P0 transcription by glucose was also dependent on E sigma(32). These results indicate that E sigma(32) plays an important role in balancing the relative concentration of Mlc and EIICB(Glc) in response to availability of glucose in order to maintain inducibility of the Mlc regulon at high growth temperature.