Characterization of MtfA, a Novel Regulatory Output Signal Protein of the Glucose-Phosphotransferase System in Escherichia coli K-12

Abstract

The glucose-phosphotransferase system (PTS) in Escherichia coli K-12 is a complex sensory and regulatory system. In addition to its central role in glucose uptake, it informs other global regulatory networks about carbohydrate availability and the physiological status of the cell. The expression of the ptsG gene encoding the glucose-PTS transporter EIICB Glc is primarily regulated via the repressor Mlc, whose inactivation is glucose dependent. During transport of glucose and dephosphorylation of EIICB Glc , Mlc binds to the B domain of the transporter, resulting in derepression of several Mlc-regulated genes. In addition, Mlc can also be inactivated by the cytoplasmic protein MtfA in a direct protein-protein interaction. In this study, we identified the binding site for Mlc in the carboxy-terminal region of MtfA by measuring the effect of mutated MtfAs on ptsG expression. In addition, we demonstrated the ability of MtfA to inactivate an Mlc super-repressor, which cannot be inactivated by EIICB Glc , by using in vivo titration and gel shift assays. Finally, we characterized the proteolytic activity of purified MtfA by monitoring cleavage of amino 4-nitroanilide substrates and show Mlc's ability to enhance this activity. Based on our findings, we propose a model of MtfA as a glucose-regulated peptidase activated by cytoplasmic Mlc. Its activity may be necessary during the growth of cultures as they enter the stationary phase. This proteolytic activity of MtfA modulated by Mlc constitutes a newly identified PTS output signal that responds to changes in environmental conditions. D espite the fact that Escherichia coli K-12 has been an important model organism for many decades, the function of Ͼ20% of all putative open reading frames in its genome remains unknown. Moreover, in many cases, no obvious sequence similarities with previously characterized proteins exist. The mtfA gene (formerly named yeeI), a monocistronic gene located at 44.1 min in the E. coli chromosome, has clearly belonged to this group. Intensive sequence similarity searches revealed that orthologs of MtfA (mnemonic for Mlc titration factor A) exist in more than 100 proteobacteria of the alpha, beta, and gamma subdivisions. In a previous report (2), we were able to demonstrate that E. coli MtfA is a cytoplasmic protein that binds to the carboxy-terminal part of the Mlc repressor protein (mnemonic for "making large colonies" [gene, dgsA]) with a very high affinity. Mlc is one of the global regulators of carbohydrate metabolism in E. coli and is especially involved in the regulation of the ptsG gene expression, which encodes the glucose transporter EIICB Glc . The EIICB Glc together with the cytoplasmic protein EIIA Glc , encoded by the crr gene (as part of the ptsHI-crr operon), forms the glucosespecific phosphoenolpyruvate (PEP)-dependent carbohydratephosphotransferase system (glucose-PTS). Both proteins take part in a phosphorylation cascade, which begins with an autophosphorylation reaction of the so-called enzyme I (EI; gene, ptsI) at the expense...

The Structure of Mlc Titration Factor A (MtfA/YeeI) Reveals a Prototypical Zinc Metallopeptidase Related to Anthrax Lethal Factor

“…However, Mlc is not a target for the peptidase activity of MtfA. In fact, the addition of Mlc stimulated MtfA activity in the cleavage assay with L-alanine 4-nitroanilide (18,20), which seems to further support our structural data on the existence of an inactive state that could be converted to the active form upon the binding of one or several interaction partners. Thus, we speculate that association between Mlc and MtfA probably induces structural changes that activate the peptidase activity of MtfA while inactivating the DNA-binding ability of Mlc.…”

“…S4 in the supplemental material). The active-site region of MtfA seems to be important for the interaction with Mlc (20). However, Mlc is not a target for the peptidase activity of MtfA.…”

“…For peptidase assays, MtfA was cloned and purified based on a protocol described previously (20) that is independent from the protocol used for the crystallographic studies. Purified MtfA-His 5 and derivatives were expressed from the plasmid pTM30MtfAkpnHis.…”

“…We performed several nonspecific protease activity assays for the K. pneumoniae MtfA, based on those recently reported for the E. coli ortholog (20). The activity of the K. pneumoniae MtfA is similar to that of the E. coli MtfA.…”

The Bacterial Phosphoenolpyruvate:Carbohydrate Phosphotransferase System: Regulation by Protein Phosphorylation and Phosphorylation-Dependent Protein-Protein Interactions

“…As a consequence, the uptake of glucose leads to EIIB Glc -mediated sequestration of Mlc to the membrane and therefore to increased expression of Mlc-repressed genes (140). It should be noted that Mlc activity is also inactivated by the interaction with the cytoplasmic protein MtfA (Mlc titration factor A) when this protein is overproduced (145).…”