Systematic characterization of a novel gal operon in Thermoanaerobacter tengcongensis.
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 degrees C, 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.
Qian
Zhong
Z
Beijing Proteomics Institute, Beijing 101318, PR China.|Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 101318, PR China.
Meng
Bo
B
Beijing Proteomics Institute, Beijing 101318, PR China.|Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 101318, PR China.
Wang
Quanhui
Q
Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China.
Wang
Zhuowei
Z
Beijing Proteomics Institute, Beijing 101318, PR China.|Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 101318, PR China.
Zhou
Chuanqi
C
Beijing Proteomics Institute, Beijing 101318, PR China.|Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 101318, PR China.
Wang
Qian
Q
Beijing Proteomics Institute, Beijing 101318, PR China.|Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 101318, PR China.
Tu
Shuyang
S
Beijing Proteomics Institute, Beijing 101318, PR China.|Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 101318, PR China.
Lin
Liang
L
Beijing Proteomics Institute, Beijing 101318, PR China.|Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 101318, PR China.
Ma
Yanhe
Y
Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China.
Liu
Siqi
S
Beijing Proteomics Institute, Beijing 101318, PR China.|Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 101318, PR China.
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D005690:Galactose / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D006094:Gram-Positive Bacteria / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009876:Operon; D011485:Protein Binding
D001426:Bacterial Proteins; D005690:Galactose