Regulation of expression of copper responsive genes in Sulfolobus solfataricus.

摘要

Copper is an essential micronutrient, but toxic in excess. Cells must maintain their internal level of copper within a narrow range of concentrations. This is accomplished mainly by copper efflux mediated by ATP-driven copper transporters that are induced at the level of transcription. Sulfolobus solfataricus has the ability to adapt to fluctuations of copper levels in its environment. Two Sulfolobus strains P2 and 98/2, showed different sensitivity to copper. To better understand the molecular mechanism behind the organismal response to copper, the expression of the cluster of genes copRTA, which encodes the copper-responsive transcriptional regulator CopR, the copper-binding protein CopT, and CopA, has been investigated. The expression of the copR and copA transcripts was monitored by quantitative real-time RT-PCR. The data showed that only copA was induced by copper. By comparing the patterns of copA expression and cellular copper accumulation, as determined by Inductively Coupled Plasma Optical Emission spectrometry, it was concluded that the level of copA depends on the internal fluctuations of copper. To investigate the role of CopR, a mutant carrying a disruption of the copR gene was created. The mutant strain was incapable of growth in the presence of excess copper, and under the same conditions no transcription of copA was observed. These data suggested that CopR positively regulates transcription of copA. The knockout mutant strain PBL2070, its parent strain and the mutant complemented with a wild type copy of copR, were compared with respect to their physiological and transcriptional response to copper. Results confirmed that, under copper stress, CopR, activates the transcription of copA, and its presence restores the wild type phenotype resistant to copper. Finally, a reporter system based on the thermostable beta-glucuronidase of S. solfataricus was developed to study the promoter of regulated genes. This system was applied to the analysis of the copTA promoter region fused to the S. solfataricus beta-glucuronidase reporter. GUS activity assays showed that after copper addition, the activity of GusB was consistent with the transcriptional changes observed for copA under similar conditions. The results provide the basis for a model of the molecular mechanisms of copper homeostasis in Sulfolobus.