Characterization of the Interaction Between the Small Regulatory Peptide SgrT and the EIICBGlc of the Glucose-Phosphotransferase System of E. coli K-12

Abstract

Escherichia coli is a widely used microorganism in biotechnological processes. An obvious goal for current scientific and technical research in this field is the search for new tools to optimize productivity. Usually glucose is the preferred carbon source in biotechnological applications. In E. coli, glucose is taken up by the phosphoenolpyruvate-dependent glucose phosphotransferase system (PTS). The regulation of the ptsG gene for the glucose transporter is very complex and involves several regulatory proteins. Recently, a novel posttranscriptional regulation system has been identified which consists of a small regulatory RNA SgrS and a small regulatory polypeptide called SgrT. During the accumulation of glucose-6-phosphate or fructose-6-phosphate, SgrS is involved in downregulation of ptsG mRNA stability, whereas SgrT inhibits glucose transport activity by a yet unknown mechanism. The function of SgrS has been studied intensively. In contrast, the knowledge about the function of SgrT is still limited. Therefore, in this paper, we focused our interest on the regulation of glucose transport activity by SgrT. We identified the SgrT target sequence within the glucose transporter and characterized the interaction in great detail. Finally, we suggest a novel experimental approach to regulate artificially carbohydrate uptake in E. coli to minimize metabolic overflow in biotechnological applications.

The Small Protein SgrT Controls Transport Activity of the Glucose-Specific Phosphotransferase System

“…It is worth noting that our experiments demonstrated a clear role for the IIC domain of PtsG in conferring susceptibility to SgrT, whereas others (32,33) have suggested that the linker region connecting the IIC and IIB domains is involved in SgrT binding. Bimolecular fluorescence complementation experiments performed by the Jahreis group (32) tested the interactions between SgrT and various lengths of PtsG by fusing them to different domains of green fluorescent protein (GFP) and monitoring GFP complementation by fluorescence.…”

“…Most of these mutants (S157E, H339Y, K257N, M17T, and D343G) were still inhibited by SgrT. The Jahreis group reported that a PtsG mutant with broadened substrate specificity, PtsG(P384R), was not sensitive to inhibition by SgrT (32). We constructed the same mutant and found that the strain producing PtsG(P384R) was substantially less responsive to induction of P sgrS -lacZ by ␣MG than the strain producing wild-type PtsG (Fig.…”

“…We tested the activity and SgrT sensitivity of another PtsG mutant, PtsG(V12F), which was previously shown to have an enhanced rate of ␣MG transport (30,34). This mutant was also tested by Jahreis and coworkers for interaction with SgrT by coimmunoprecipitation, and their results suggested that PtsG(V12F) and SgrT could still interact (32). However, given the discordance of our results for the P384R mutation, we proceeded to test the V12F mutant as described above.…”

“…Bimolecular fluorescence complementation experiments performed by the Jahreis group (32) tested the interactions between SgrT and various lengths of PtsG by fusing them to different domains of green fluorescent protein (GFP) and monitoring GFP complementation by fluorescence. They showed that complementation occurred most strongly when both the IIC domain and linker were present but found no interaction between SgrT and the IIC domain alone (32). We note that these experiments utilized protein fusions between PtsG fragments or SgrT and different domains of GFP.…”

“…These results, along with those we present here, suggest that determinants in both the linker and IIC domain are important for SgrT interaction. Interestingly, in a cross-linking and copurification experiment (using SgrT tagged with the small hemagglutinin [HA] epitope), Kosfeld and Jahreis found evidence that SgrT preferentially interacts with dephosphorylated PtsG (32), which would be the predominant form present during active glucose transport as the phosphate is rapidly transferred to the incoming sugar. This observation suggests that SgrT might only associate with PtsG during active transport.…”