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Microbiology 155 (2009), 1717-1725; DOI  10.1099/mic.0.025536-0
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Microbiology 155 (2009), 1717-1725; DOI  10.1099/mic.0.025536-0
© 2009 Society for General Microbiology

Systematic characterization of a novel gal operon in Thermoanaerobacter tengcongensis

Zhong Qian1,2, Bo Meng1,2, Quanhui Wang3, Zhuowei Wang1,2, Chuanqi Zhou1,2, Qian Wang1,2, Shuyang Tu1,2, Liang Lin1,2, Yanhe Ma3 and Siqi Liu1,2

1 Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 101318, PR China
2 Beijing Proteomics Institute, Beijing 101318, PR China
3 Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China

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 °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.

Correspondence
Siqi Liu
siqiliu{at}genomics.org.cn

Abbreviations: EMSA, electrophoretic mobility shift assay; HTH, helix–turn–helix; LC, liquid chromatography; 5' RACE, 5' rapid amplification of cDNA ends

Two supplementary figures, showing gene expression of the gal operon at different culture temperatures and phylogenetic analysis of gal genes in T. tengcongensis alongside gal genes in other thermophiles, are available with the online version of this paper.






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