1887

Abstract

On the basis of the genome, a novel type of 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 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 genes was significantly induced by galactose, whereas only MR-Tt expression was detected in glucose-cultured at both the mRNA and the protein level. In addition, the abundance of 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 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 operon.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.025536-0
2009-05-01
2019-12-15
Loading full text...

Full text loading...

/deliver/fulltext/micro/155/5/1717.html?itemId=/content/journal/micro/10.1099/mic.0.025536-0&mimeType=html&fmt=ahah

References

  1. Ajdic, D. & Ferretti, J. J. ( 1998; ). Transcriptional regulation of the Streptococcus mutans gal operon by the GalR repressor. J Bacteriol 180, 5727–5732.
    [Google Scholar]
  2. Bao, Q., Tian, Y., Li, W., Xu, Z., Xuan, Z., Hu, S., Dong, W., Yang, J., Chen, Y. & other authors ( 2002; ). A complete sequence of the T. tengcongensis genome. Genome Res 12, 689–700.[CrossRef]
    [Google Scholar]
  3. Beebe, J. A. & Frey, P. A. ( 1998; ). Galactose mutarotase: purification, characterization, and investigations of two important histidine residues. Biochemistry 37, 14989–14997.[CrossRef]
    [Google Scholar]
  4. Bettenbrock, K. & Alpert, C. A. ( 1998; ). The gal genes for the Leloir pathway of Lactobacillus casei 64H. Appl Environ Microbiol 64, 2013–2019.
    [Google Scholar]
  5. Brahma, A. & Bhattacharyya, D. ( 2004; ). UDP-galactose 4-epimerase from Kluyveromyces fragilis. Evidence for independent mutarotation site. Eur J Biochem 271, 58–68.
    [Google Scholar]
  6. Frey, P. A. ( 1996; ). The Leloir pathway: a mechanistic imperative for three enzymes to change the stereochemical configuration of a single carbon in galactose. FASEB J 10, 461–470.
    [Google Scholar]
  7. Lai, K., Willis, A. C. & Elsas, L. J. ( 1999; ). The biochemical role of glutamine 188 in human galactose-1-phosphate uridyltransferase. J Biol Chem 274, 6559–6566.[CrossRef]
    [Google Scholar]
  8. Li, N., Guo, R., Li, W., Shao, J., Li, S., Zhao, K., Chen, X., Xu, N., Liu, S. & Lu, Y. ( 2006; ). A proteomic investigation into a human gastric cancer cell line BGC823 treated with diallyl trisulfide. Carcinogenesis 27, 1222–1231.[CrossRef]
    [Google Scholar]
  9. Love, H. D., Jr, Allen-Nash, A., Zhao, Q. A. & Bannon, G. A. ( 1988; ). mRNA stability plays a major role in regulating the temperature-specific expression of a Tetrahymena thermophila surface protein. Mol Cell Biol 8, 427–432.
    [Google Scholar]
  10. Luesink, E. J., van Herpen, R. E., Grossiord, B. P., Kuipers, O. P. & de Vos, W. M. ( 1998; ). Transcriptional activation of the glycolytic las operon and catabolite repression of the gal operon in Lactococcus lactis are mediated by the catabolite control protein CcpA. Mol Microbiol 30, 789–798.[CrossRef]
    [Google Scholar]
  11. Mackie, G. & Wilson, D. B. ( 1972; ). Regulation of the gal operon of Escherichia coli by the capR gene. J Biol Chem 247, 2973–2978.
    [Google Scholar]
  12. Majumdar, S., Ghatak, J., Mukherji, S., Bhattacharjee, H. & Bhaduri, A. ( 2004; ). UDPgalactose 4-epimerase from Saccharomyces cerevisiae. A bifunctional enzyme with aldose 1-epimerase activity. Eur J Biochem 271, 753–759.[CrossRef]
    [Google Scholar]
  13. Maxwell, E. S., Kurahashi, K. & Kalckar, H. M. ( 1962; ). Enzymes of the Leloir pathway. Methods Enzymol 5, 16
    [Google Scholar]
  14. Mustapha, A., Hutkins, R. W. & Zirnstein, G. W. ( 1995; ). Cloning and characterization of the galactokinase gene from Streptococcus thermophilus. J Dairy Sci 78, 989–997.[CrossRef]
    [Google Scholar]
  15. Poolman, B., Royer, T. J., Mainzer, S. E. & Schmidt, B. F. ( 1990; ). Carbohydrate utilization in Streptococcus thermophilus: characterization of the genes for aldose 1-epimerase (mutarotase) and UDPglucose 4-epimerase. J Bacteriol 172, 4037–4047.
    [Google Scholar]
  16. Qian, Z., Wang, J. Q., Zhou, C. Q., Ma, Y. H. & Liu, S. Q. ( 2006; ). Expression and catalysis of glucokinase of Thermoanaerobacter tengcongensis at different temperatures. . Wei Sheng Wu Xue Bao 46, 243–248 (in Chinese).
    [Google Scholar]
  17. Rohde, J. R., Trinh, J. & Sadowski, I. ( 2000; ). Multiple signals regulate GAL transcription in yeast. Mol Cell Biol 20, 3880–3886.[CrossRef]
    [Google Scholar]
  18. Roy, S., Semsey, S., Liu, M., Gussin, G. N. & Adhya, S. ( 2004; ). GalR represses galP1 by inhibiting the rate-determining open complex formation through RNA polymerase contact: a GalR negative control mutant. J Mol Biol 344, 609–618.[CrossRef]
    [Google Scholar]
  19. Semsey, S., Geanacopoulos, M., Lewis, D. E. & Adhya, S. ( 2002; ). Operator-bound GalR dimers close DNA loops by direct interaction: tetramerization and inducer binding. EMBO J 21, 4349–4356.[CrossRef]
    [Google Scholar]
  20. Semsey, S., Tolstorukov, M. Y., Virnik, K., Zhurkin, V. B. & Adhya, S. ( 2004; ). DNA trajectory in the Gal repressosome. Genes Dev 18, 1898–1907.[CrossRef]
    [Google Scholar]
  21. Semsey, S., Virnik, K. & Adhya, S. ( 2006; ). Three-stage regulation of the amphibolic gal operon: from repressosome to GalR-free DNA. J Mol Biol 358, 355–363.[CrossRef]
    [Google Scholar]
  22. Stenger, D., Gruissem, W. & Baginsky, S. ( 2004; ). Mass spectrometric identification of RNA binding proteins from dried EMSA gels. J Proteome Res 3, 662–664.[CrossRef]
    [Google Scholar]
  23. Thoden, J. B. & Holden, H. M. ( 2002; ). High resolution X-ray structure of galactose mutarotase from Lactococcus lactis. J Biol Chem 277, 20854–20861.[CrossRef]
    [Google Scholar]
  24. Titgemeyer, F., Reizer, J., Reizer, A. & Saier, M. H., Jr ( 1994; ). Evolutionary relationships between sugar kinases and transcriptional repressors in bacteria. Microbiology 140, 2349–2354.[CrossRef]
    [Google Scholar]
  25. Vaillancourt, K., Moineau, S., Frenette, M., Lessard, C. & Vadeboncoeur, C. ( 2002; ). Galactose and lactose genes from the galactose-positive bacterium Streptococcus salivarius and the phylogenetically related galactose-negative bacterium Streptococcus thermophilus: organization, sequence, transcription, and activity of the gal gene products. J Bacteriol 184, 785–793.[CrossRef]
    [Google Scholar]
  26. Vaughan, E. E., van den Bogaard, P. T., Catzeddu, P., Kuipers, O. P. & de Vos, W. M. ( 2001; ). Activation of silent gal genes in the lac-gal regulon of Streptococcus thermophilus. J Bacteriol 183, 1184–1194.[CrossRef]
    [Google Scholar]
  27. Wang, J., Xue, Y., Feng, X., Li, X., Wang, H., Li, W., Zhao, C., Cheng, X., Ma, Y. & other authors ( 2004; ). An analysis of the proteomic profile for Thermoanaerobacter tengcongensis under optimal culture conditions. Proteomics 4, 136–150.[CrossRef]
    [Google Scholar]
  28. Wang, J., Zhao, C., Meng, B., Xie, J., Zhou, C., Chen, X., Zhao, K., Shao, J., Xue, Y. & other authors ( 2007; ). The proteomic alterations of Thermoanaerobacter tengcongensis cultured at different temperatures. Proteomics 7, 1409–1419.[CrossRef]
    [Google Scholar]
  29. Weickert, M. J. & Adhya, S. ( 1993; ). The galactose regulon of Escherichia coli. Mol Microbiol 10, 245–251.[CrossRef]
    [Google Scholar]
  30. Wu, K., Lai, X., Guo, X., Hu, J., Xiang, X. & Huang, L. ( 2007; ). Interplay between primase and replication factor C in the hyperthermophilic archaeon Sulfolobus solfataricus. Mol Microbiol 63, 826–837.
    [Google Scholar]
  31. Xue, Y., Xu, Y., Liu, Y., Ma, Y. & Zhou, P. ( 2001; ). Thermoanaerobacter tengcongensis sp. nov., a novel anaerobic, saccharolytic, thermophilic bacterium isolated from a hot spring in Tengcong, China. Int J Syst Evol Microbiol 51, 1335–1341.
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.025536-0
Loading
/content/journal/micro/10.1099/mic.0.025536-0
Loading

Data & Media loading...

Supplements

Primers for real-time PCR, RT-PCR and 5' RACE [PDF file](43 KB)

PDF

Proteins identified from operator binding complexes 1A and 2A by EMSA-LC MS-MS [PDF file](21 KB)

PDF

[PDF file](12 KB)

PDF

Gene expression of the operon at three different culture temperatures [PDF file](165 KB)

PDF

Phylogenetic analysis of genes in with genes of other thermophiles [PDF file](183 KB)

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error