Cloning and overexpression in Escherichia coli of the genes encoding NAD-dependent alcohol dehydrogenase from two Sulfolobus species

Cannio, Raffaele; Fiorentino, Gabriella; Carpinelli, Patrizia; Rossi, Mosè; Bartolucci, Simonetta

doi:10.1128/jb.178.1.301-305.1996

Cited by 54 publications

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“…Moreover, it is highly specific toward aromatic rather than aliphatic aldehydes and the expression of the encoding gene is indeed induced by benzaldehyde (15). In a previous study, a protein named Bald was purified for its ability to bind specifically to the Sso2536 regulatory sequences and demonstrated to increase intracellularly upon exposure to the toxic benzaldehyde (14). For this reason, the protein was postulated to be the transcription factor responsive to xenobiotic agents in the defense mechanisms involving ADH.…”

Section: Discussionmentioning

confidence: 99%

“…We have chosen the gene encoding an alcohol dehydrogenase (ADH) in the archaeon Sulfolobus solfataricus, adh Ss (14,27), annotated on the genome as Sso2536 (52), as a model of transcriptional regulation in Archaea. Multiple ADHs have been found in single members of the three domains of life as generally encoded by distinct genes (10), their expression being regulated at both the transcriptional and posttranscriptional levels (24,56).…”

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confidence: 99%

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MarR-Like Transcriptional Regulator Involved in Detoxification of Aromatic Compounds in Sulfolobus solfataricus

et al. 2007

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A DNA binding protein, BldR, was identified in the crenarchaeon Sulfolobus solfataricus as a protein 5-to 10-fold more abundant in cells grown in the presence of toxic aldehydes; it binds to regulatory sequences located upstream of an alcohol dehydrogenase gene (Sso2536). BldR is homologous to bacterial representatives of the MarR (multiple antibiotic resistance) family of transcriptional regulators that mediate response to multiple environmental stresses. Transcriptional analysis revealed that the bldR gene was transcribed in a bicistronic unit composed of the genes encoding the transcriptional regulator (Sso1352) and a putative multidrug transporter (Sso1351) upstream. By homology to bacterial counterparts, the bicistron was named the mar-like operon. The level of mar-like operon expression was found to be increased at least 10-fold in response to chemical stress by aromatic aldehydes. Under the same growth conditions, similar enhanced in vivo levels of Sso2536 gene transcript were also measured. The gene encoding BldR was expressed in E. coli, and the recombinant protein was purified to homogeneity. DNA binding assays demonstrated that the protein is indeed a transcription factor able to recognize site specifically both the Sso2536 and mar-like promoters at sites containing palindromic consensus sequences. Benzaldehyde, the substrate of ADH Ss , stimulates DNA binding of BldR at both promoters. The role of BldR in the auto-activation as well as in the regulation of the Sso2536 gene, together with results of increased operon and gene expression under conditions of exposure to aromatic aldehydes, indicates a novel coordinate regulatory mechanism in cell defense against stress by aromatic compounds.A chimeric nature of the transcription machinery with eukaryote-like basal factors and bacterium-like regulative components has been found in all members of the domain Archaea (50, 60). In fact, in most cases, homologs of bacterial regulators function in the context of the archaeal basal transcriptional apparatus, which resembles that of Eukarya (8, 26). Transcription initiation is mediated by a single RNA polymerase (RNAP) and two general transcription factors, TATA element binding protein (TBP) and transcription factor B (TFB), a homologue of the transcription factor TFIIB, which binds to the B recognition element (BRE) and determines the directionality of the transcription (7). The complex containing RNAP, TBP, and TFB is sufficient to initiate transcription in cell-free systems (31, 49), although an additional factor, transcription factor E, is required to increase transcription from some promoters (6, 29).A few homologs of eukaryal transcriptional regulators (33) and unique archaeal regulators (57) as well as non-sequencespecific DNA binding proteins (5, 30) have been investigated for their contribution to the regulation of archaeal genes.Homologs of bacterial transcriptional regulators are more common in Archaea, and representatives that have been studied experimentally include the Lrp homologs (13) that can fun...

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Section: Discussionmentioning

confidence: 99%

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MarR-Like Transcriptional Regulator Involved in Detoxification of Aromatic Compounds in Sulfolobus solfataricus

et al. 2007

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“…ADH activity was assayed at 65³C as previously described [11], by spectrophotometrically monitoring the reduction of NAD at 340 nm. 1 mU of enzyme activity was de¢ned as the amount of enzyme that catalyses the reduction of 1 nmol NAD per minute at 65³C.…”

Section: Preparation Of Crude Cell Extracts and Determination Of Adh mentioning

confidence: 99%

“…In fact, this family of alcohol dehydrogenases is common to the three kingdoms of life, playing a role which is essential though not well de¢ned in cellular defence mechanisms [10]. We recently reported the cloning and overexpression in Escherichia coli of the genes (Ssadh and RC3adh) from two independent species of Sulfolobus, solfataricus and RC3 [11].…”

Section: Introductionmentioning

confidence: 99%

The alcohol dehydrogenase gene: distribution among Sulfolobales and regulation in Sulfolobus solfataricus

Cannio

1999

FEMS Microbiology Letters

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The distribution of the alcohol dehydrogenase gene (adh) among different Archaea was investigated by Southern blot analysis revealing the potentiality of the adh gene as a specific marker for the genus Sulfolobus. Moreover, the in vivo expression of the adh gene from a new isolate of Sulfolobus solfataricus, Ga, was studied to investigate gene regulation in Archaea. Primer extension analysis allowed the identification of a single initiation site and the TATA box element. Comparison of the Gaadh promoter with the corresponding Ssadh (adh from S. solfataricus) and RC3adh (adh from Sulfolobus RC3) also revealed the presence of two putative regulatory inverted repeats at the 5P of the TATA element. Northern blot analysis and enzymatic assays demonstrated that the transcription and expression of the Gaadh gene is regulated by different carbon and energy sources or by the natural substrate of the ADH enzyme. z

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“…Detailed investigation of stability, structure, biochemical function, and distribution among different species has been carried out for the euryarchaeaon Pyrococcus furiosus (43) and the crenarchaeon Sulfolobus solfataricus (3,15,16,21,37).…”

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Transcriptional Regulation of the Gene Encoding an Alcohol Dehydrogenase in the Archaeon Sulfolobus solfataricus Involves Multiple Factors and Control Elements

et al. 2003

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A transcriptionally active region has been identified in the 5 flanking region of the alcohol dehydrogenase gene of the crenarchaeon Sulfolobus solfataricus through the evaluation of the activity of putative transcriptional regulators and the role of the region upstream of the gene under specific metabolic circumstances. Electrophoretic mobility shift assays with crude extracts revealed protein complexes that most likely contain TATA box-associated factors. When the TATA element was deleted from the region, binding sites for both DNA binding proteins, such as the small chromatin structure-modeling Sso7d and Sso10b (Alba), and transcription factors, such as the repressor Lrs14, were revealed. To understand the molecular mechanisms underlying the substrate-induced expression of the adh gene, the promoter was analyzed for the presence of cis-acting elements recognized by specific transcription factors upon exposure of the cell to benzaldehyde. Progressive dissection of the identified promoter region restricted the analysis to a minimal responsive element (PAL) located immediately upstream of the transcription factor B-responsive element-TATA element, resembling typical bacterial regulatory sequences. A benzaldehyde-activated transcription factor (Bald) that specifically binds to the PAL cis-acting element was also identified. This protein was purified from heparin-fractionated extracts of benzaldehyde-induced cells and was shown to have a molecular mass of ϳ16 kDa. The correlation between S. solfataricus adh gene activation and benzaldehyde-inducible occupation of a specific DNA sequence in its promoter suggests that a molecular signaling mechanism is responsible for the switch of the aromatic aldehyde metabolism as a response to environmental changes.

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Cloning and overexpression in Escherichia coli of the genes encoding NAD-dependent alcohol dehydrogenase from two Sulfolobus species

Cited by 54 publications

References 0 publications

MarR-Like Transcriptional Regulator Involved in Detoxification of Aromatic Compounds in Sulfolobus solfataricus

MarR-Like Transcriptional Regulator Involved in Detoxification of Aromatic Compounds in Sulfolobus solfataricus

The alcohol dehydrogenase gene: distribution among Sulfolobales and regulation in Sulfolobus solfataricus

Transcriptional Regulation of the Gene Encoding an Alcohol Dehydrogenase in the Archaeon Sulfolobus solfataricus Involves Multiple Factors and Control Elements

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