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Characterization and functional complementation of a nonlethal deletion in the chromosome of a beta-glycosidase mutant of Sulfolobus solfataricus
No full textLacS(-) mutants of Sulfolobus solfataricus defective in beta-glycosidase activity were isolated in order to explore genomic instability and exploit novel strategies for transformation and complementation. One of the mutants showed a stable phenotype with no reversion; analysis of its chromosome revealed the total absence of the beta-glycosidase gene (lacS). Fine mapping performed in comparison to the genomic sequence of S. solfataricus P2 indicated an extended deletion of approximately 13 kb. The sequence analysis also revealed that this chromosomal rearrangement was a nonconservative transposition event driven by the mobile insertion sequence element ISC1058. In order to complement the LacS(-) phenotype, an expression vector was constructed by inserting the lacS coding sequence with its 5' and 3' flanking regions into the pEXSs plasmid. Since no transformant could be recovered by selection on lactose as the sole nutrient, another plasmid construct containing a larger genomic fragment was tested for complementation; this region also comprised the lacTr (lactose transporter) gene encoding a putative membrane protein homologous to the major facilitator superfamily. Cells transformed with both genes were able to form colonies on lactose plates and to be stained with the beta-glycosidase chromogenic substrate X-Gal (5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside)
A Mar-like transcription factor in the archaeon Sulfolobus solfataricus involved in the detoxification of aromatic aldehydes
No full textThe Transcriptional Regulation of the alcohol Dehydrogenase gene in Sulfolobus solfataricus: identification of Regulatory Sequences and binding Factors
No full textINTRODUCTION: The understanding of archaeal transcription initiation has been deepened by recent progress, which include genome sequencing, biochemical approaches, and the development of vector/transformation systems. Although it has been demonstrated both with functional and structural approaches that the basal transcription apparatus of Archaea corresponds to a simplified core machinery of the eukaryal RNA polymerase II system, little is currently known about how regulation of archaeal transcription is achieved.
In order to give insight in gene regulation in thermophilic Archaea, we examined the expression of the alcohol dehydrogenase (adh) gene in Sulfolobus solfataricus.
MATERIAL AND METHODS: S. solfataricus cultures were grown in different conditions among which DSM182 medium supplemented with 1 mM benzaldehyde, the natural substrate of the ADH enzyme, and harvested at exponential or stationary phase. mRNA and crude extracts were prepared according to conventional procedures to perform Northern blot analysis and enzyme assays, respectively. Radiolabelled DNA fragments for band shift analysis have been prepared by endonuclease restriction of the 300 bp region upstream of the adh gene.
Two synthetic single stranded oligonucleotides encompassing two palindromic sequences located in the adh promoter have also been annealed, labelled and used as probe. 15000 cpm of radiolabelled fragments or annealed oligonucleotides were used for each binding reaction. After binding, samples were loaded on a running 5% polyacrylamide gel containing TBE. The gel was dried and exposed to autoradiography.
RESULTS: Studies based on Northern analysis and enzyme assays, demonstrated that the adh gene is induced in the early growth phase by addition to the medium of benzaldehyde, the natural ADH substrate.
We also detected in the crude extracts of induced cells, factor(s) specifically able to bind to a 5' regulatory sequence located immediately upstream of the TATA box, suggesting that this region is responsible for the regulated expression of its gene.
Further molecular characterisation of the adh promoter allowed the identification of a palindromic cis-acting sequence and of its interacting factor(s). The purification to homogeneity of the factor(s) and the elucidation of its role in transcription regulation are underway
Coordinate expression of a Mar-like operon and an alcohol dehydrogenase gene contributes to detoxification by aromatic aldehydes in Sulfolobus solfataricus
No full textInvestigation of mechanisms underlying transcriptional regulation of Sso2536, encoding for an alcohol dehydrogenase gene (adh) in the crenarchaeon S. solfataricus has shown an active 5’ flanking region responsive to physiologically relevant DNA binding proteins. In particular, one DNA binding protein, Bald16 (Sso1352), has been identified whose levels are higher when cells are grown in the presence of the toxic benzaldehyde, substrate of the ADH enzyme; it has been proposed that this protein could act as a transcriptional activator triggering adh expression to protect cells from an environmental stress due to phenolic-derived aldehydes. Bald16 encodes for a putative transcriptional regulator, which has a bacterial homologue belonging to the Mar (Multiple Antibiotic Resistance) family of regulators involved in the control of gene expression of aromatic compound metabolism and antibiotic resistance. To better investigate the molecular mechanisms underlying transcriptional regulation in S. solfataricus, with greater attention with respect to defense response upon chemical stress, we analyzed the expression of the bald16 gene in the presence of aromatic aldehydes. Transcriptional analysis of the bald16 gene allowed the identification of a new mar-like locus in S. solfataricus composed of a putative multidrug transporter and the transcriptional regulator downstream (Sso1351, Sso1352). The genes are transcribed as a polycistronic unit whose expression is sensitive to the addition to the cell growth medium of different aromatic aldehydes. The gene encoding for the transcriptional regulator, has been expressed in E. coli and the recombinant protein purified to homogeneity. The protein is indeed a DNA binding protein, which binds site-specifically to both the adh and bald16 promoters. Western blot analysis revealed an increased Bald16 expression in cell extracts prepared from cells grown in the presence of aromatic aldehydes. These results reasonably strengthen the hypothesis of a resistance mechanism based on the coordinate expression of the adh gene and the Mar-like operon, in response to stress determined by phenolic-derived materials
The substrate effect and the growth phase dependent expression of the alcohol dehydrogenase from Sulfolobus solfataricus.
No full textThe principal components involved in the transcription process in Archaea have been identified; however results about regulation of the expression of archaeal genes at the level of transcription are preliminary.
In order to elucidate mechanisms controlling gene expression, we analysed the in vivo expression of the alcohol dehydrogenase gene (adh) in a new isolate of Sulfolobus solfataricus. The location of the transcription initiation site and the TATA box motif were determined and found to match the consensus sequence from different archaeal promoter regions. Comparison of other adh promoters belonging to different related Sulfolobus species revealed the presence of conserved putative regulatory sequence elements in the 5' region immediately flanking the TATA element.
Northern blot analysis and enzymatic assays demonstrated that the transcription and the expression of the Ssadh gene is growth phase regulated and responds to different compounds in the medium.
The potential to identify regulatory sequences by in vivo mutational analysis using a plasmid based reporter system will be discussed
Thermal features, gene distribution and regulation of secondary, medium-chain alcohol dehydrogenases from thermophilic bacterial and archaeal sources
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Gene transfer and regulation in Sulfolobus solfataricus.
No full textThe mechanisms and regulation of the complex fundamental cell processes in thermophilic Archaea, such as transcription, translation, replication, secretion, cell growth and signalling have been only partially elucidated. This is mainly due to the lack of the tools needed to study their molecular biology, including appropriate vector-transformation systems, genetic and selectable markers, and mutant production and screening methods.
Sulfolobus solfataricus was chosen as a model host system for the study of gene transfer and regulation since it is a good recipient for viruses and plasmids; moreover, the knowledge of its genomic organisation by genome sequencing, gene cloning and expression, is in good progress.
An E.coli/Sulfolobus shuttle vector, the pEXSs, was obtained, inserting in an E.coli plasmid the genomic replication sequence of the S. shibate virus particle SSV1 and using a thermoadapted version of the hygromicin phosphotransferase as the selective acquired phenotype. In order to obtain a higher copy number plasmid, the natural S. islandicus multicopy plasmid pRN1 was used to construct an E.coli/Sulfolobus shuttle vector, the pHYG* by fusing it with the E.coli plasmid pGEM7zf(+) and providing it with the hygromycin phosphotransferase gene as selectable marker; since it has been demonstrated the pHYG* to be instable and to suffer rearrangment and/or deletions, attemps are being done to overcome recombination events. Furthermore, transformation systems based on the use of different E. coli host strains, and/or extrachromosomal genetic elements, and/or selectable phenotype using adh genes from thermophilic sources as transformation marker, are underway.
In order to study the transcriptional regulation in Sulfolobus solfataricus, we examined the expression of the alcohol dehydrogenase gene (adh). Primer extension analysis allowed the identification of the transcriptional initiation site and the consensus TATA box. The effect of nutrients and toxic substrates was investigated at RNA and protein level and it was shown that specific messenger RNA levels were strictly dependent on growth phase, nutrient composition, and substrate concentration. ADH enzyme activities in extracts of the cells grown in the same different conditions were proportional to the intensity of the specific Northern hybridization bands, revealing that the transcription of the adh gene is finely regulated at transcriptional level
Development of Shuttle Vectors for Thermophilic Archaea and expression of Genes from Mesophilic and Moderate thermophilic sources
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