Systematic characterization of a novel gal operon in Thermoanaerobacter tengcongensis

Abstract

On the basis of the Thermoanaerobacter tengcongensis genome, a novel type of gal operon was deduced. The gene expression and biochemical properties of this operon were further characterized. RT-PCR analysis of the intergenic regions suggested that the transcription of the gal operon was continuous. With gene cloning and enzyme activity assays, TTE1929, TTE1928 and TTE1927 were identified to be GalT, GalK and GalE, respectively. Results elicited from polarimetry assays revealed that TTE1925, a hypothetical protein, was a novel mutarotase, termed MR-Tt. TTE1926 was identified as a regulator that could bind to two operators in the operon promoter. The transcriptional start sites were mapped, and this suggested that there are two promoters in this operon. Expression of the gal genes was significantly induced by galactose, whereas only MR-Tt expression was detected in glucose-cultured T. tengcongensis at both the mRNA and the protein level. In addition, the abundance of gal proteins was examined at different temperatures. At temperatures ranging from 60 to 80 6C, the level of MR-Tt protein was relatively stable, but that of the other gal proteins was dramatically decreased. The operator-binding complexes were isolated and identified by electrophoretic mobility shift assay-liquid chromatography (EMSA-LC) MS-MS, which suggested that several regulatory proteins, such as GalR and a sensory histidine kinase, participate in the regulation of the gal operon.

Regulation of Galactose Metabolism through the HisK:GalR Two-Component System in Thermoanaerobacter tengcongensis

“…Depicted in Fig. 1B, GalR was significantly induced by galactose and only localized in the cytoplasm, a finding in agreement with our previous report (22); on the other hand, HisK was independent from the galactose induction and detected mainly in the membrane and lightly in the cytoplasm. Therefore, HisK could be a membrane-associated protein.…”

“…Native protein lysate of T. tengcongensis was prepared as described previously (22). After preincubation with protein A-beads, the lysate was added to the slurry containing antibody and protein A beads for overnight incubation.…”

“…Analysis of operator binding complexes by LC-MS/MS revealed that many peptide signals were highly matched with the peptides of HisK and GalR, which resulted from theoretical tryptic digestion (22). To confirm the interaction of GalR with HisK, a co-IP assay was used.…”

“…Another type of gal operon is found in Lactobacillus casei and Thermoanaerobacter tengcongensis, in which galR is in the gal operon and cotranscribed with the other members. However, the regulation mechanism for such a novel gal operon is still unknown (4,22). In previous studies, we analyzed the operator binding complexes of gal operon in T. tengcongensis by electrophoretic mobility shift assay-liquid chromatography-tandem mass spectrometry (LC-MS/MS) and identified a histidine kinase (HisK) and the GalR (22).…”

“…However, the regulation mechanism for such a novel gal operon is still unknown (4,22). In previous studies, we analyzed the operator binding complexes of gal operon in T. tengcongensis by electrophoretic mobility shift assay-liquid chromatography-tandem mass spectrometry (LC-MS/MS) and identified a histidine kinase (HisK) and the GalR (22). Bacterial histidine kinase is well known as an active sensor for the two-component system involved in the signal transduction for transcription regulation.…”