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A novel E. coli biosensor for detecting aromatic aldehydes based on a responsive inducible archaeal promoter fused to the green fluorescent protein
No full textA whole-cell bacterial biosensor for measuring aqueous concentrations of aromatic aldehydes was developed. It is based on the E. coli BL21DE3(RIL) expressing the green fluorescent protein under the control of an alcohol dehydrogenase inducible promoter belonging to the archaeon Sulfolobus solfataricus (Sso2536adh promoter). Since it was previously reported that the BldR regulatory protein is the transcription factor required for aromatic aldehyde response in S. solfataricus, the gene encoding for the sensor protein BldR was co-expressed in the biosensor strain on a different compatible plasmid. Gel mobility shift assays showed that the purified recombinant protein can bind specifically to the Sso2536adh promoter. We demonstrated the ability of the archaeal promoter and the BldR transcription factor to operate in a bacterial context to drive active gene expression in a hybrid archaeal/eukaryal fusion. Furthermore, the E. coli BL21DE3(RIL) biosensor strain displayed a specific response and high sensitivity to the different aromatic aldehydes used, suggesting its potential low-cost application to environmentally relevant samples
Detoxification of benzaldehyde in Sulfolobus solfataricus is assisted by regulated expression of an alcohol dehydrogenase gene and a Mar-like operon
No full textArchaea, are microorganisms adapted to live in extreme conditions particularly hostile for the life. Many external agents of biological, physical or chemical nature, can determine a further stress for the cell. To date, knowledge of response mechanisms to chemical stress in archaea is very scarce. The goal of this research work is the understanding of the molecular mechanisms that confer to the Crenarchaeon S. solfataricus the ability to survive in presence of aromatic aldehydes of phenolic origin. Once identified the partners involved in the response, it will be possible to set-up systems for the bio-monitoring and the bio-remediation of contaminated area. We recently discovered a putative detoxifying mechanism involving transcriptional regulation of Sso2536 gene, encoding for an alcohol dehydrogenase gene (adh), specific for the aromatic aldehydes. Investigation of cis acting sequences and transcriptional factors has allowed to identify at least three DNA binding proteins. In particular, one of these, named Bald16 (Sso1352), is expressed at higher levels when cells are grown in the presence of the toxic benzaldehyde. This protein has a bacterial homologue belonging to the Mar (Multiple Antibiotic Resistance) family of transcriptional regulators. It has been proposed that Bald16 could act as a transcriptional activator triggering adh expression to protect cells from an environmental stress due to phenolic-derived aldehydes by converting the aromatic aldehydes in the corresponding alcohol, less toxic for the cell
Bald16, a Mar-like transcriptional regulator is involved in the detoxification of aromatic compounds in Sulfolobus solfataricus.
No full textIn the archaeon Sulfolobus solfataricus a 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 Sso2536ADH 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 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. 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 dimeric DNA binding protein, which binds site-specifically to both the adh and bald16 promoters; it also specifically interacts with benzaldehyde, as revealed by CD spectra analysis. In S.solfataricus, Bald16 is co-transcribed with an upstream gene (Sso1352) encoding a putative multidrug transporter (Mar-like operon). Northern blot analysis revealed that the operon might be autoregulated when cells are grown in the presence of aromatic aldehydes. Western blot analysis also revealed an increased Bald16 expression in cell extracts prepared from the same cells. These results reasonably strengthen the hypothesis of a resistance mechanism, modulated by Bald16, based on the coordinate expression of the adhgene and the Mar-like operon, in response to stress determined by phenolic-derived materials
Expression, Purification, Structural and Functional Characterization of Recombinant Human Parvulin 17
Full text linkParvulins, peptidyl-prolyl isomerase enzymes (PPIase), catalyze the cis-trans isomerization of prolyl bonds in polypeptides, contributing to folding and function regulation of many proteins. Among Parvulins, Par17, exclusively expressed in hominids, is the least examined in terms of structure, catalytic function and cellular activity. Setting the conditions for the preparation of recombinant active Par17 may therefore significantly foster future studies. Here, we comparatively evaluated the impact of several parameters, including host strains, culture media, isopropyl ss-D-1-thiogalactopyranoside concentration, post-induction incubation time and temperature, on the overexpression of Par17 in E. coli cells. A similar approach was also comparatively adopted for the preparation of the recombinant full-length Pin1 protein, the most representative Parvulin, and the catalytic domains of both enzymes. Proteins were efficiently expressed and purified to homogeneity and were subjected to a structural characterization by Size Exclusion Chromatography and Circular Dichroism. Moreover, a single-step homogeneous protease-based fluorimetric assay, potentially scalable in HTS format, has been developed for determining the peptidyl-prolyl cis-trans isomerase activity of recombinant Parvulins. Results obtained show that proteins are folded and active. These new data mark an important milestone for progressing the investigation of Parvulins
Stress by aromatic compounds in Sulfolobus solfataricus: detoxification, regulation and biomonitoring
No full textMost of the archaeal microbial cells, the third domain of life, are adapted to grow in extreme environments not only regarding temperature but also pH, ionic strength and the presence of high concentrations of detergents and organic solvents. As all living cells, they possess a wide variety of finely regulated biochemical systems to defend from environmental stress and, in fact, they own in their genomes regulative sequences responsive to different stress agents (1). Generally, the response can be initiated by binding of transcription factors to particular ligands, such as environmental signals.
The thermophilic archaeon S. solfataricus responds to stress by aromatic compounds increasing the expression of a MarR-like operon and of an alcohol dehydrogenase gene (Sso2536adh) (2). The system involves the MarR family transcription factor BldR, which binds to its own promoter inducing auto-activation and increasing the coexpressed drug export permease level. BldR also binds to the Sso2536 promoter stimulating the gene transcription, the accumulation of the ADH enzyme, and hence the enzyme-catalyzed conversion of the aldehydes to the less toxic alcohols (3). A homologue of Bldr, Bldr2, has been recently characterised. DNA binding assays demonstrated that this protein is indeed a transcription factor. Biochemical characterization, as well as transcriptional analyses, suggested that Bldr2 could be also involved in the detoxification/catabolism of aromatic aldehydes.
Knowledge of the molecular mechanisms underlying this stress response has revealed crucial to set up a microbial sensing device for the measurement of water-dissolved aromatic aldehydes. The biosensor is an E coli strain expressing the GFP under the control of the responsive Sso2536 promoter, and the gene for the sensor protein BldR
MarR-like transcriptional regulator involved in the detoxification of aromatic compounds in Sulfolobus solfataricus.
No full textA 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 (Ssol352) 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
ESPRESSIONE GENICA DIFFERENZIALE IN EMBRIONI MASCHIO E EMBRIONI FEMMINA DI GECO LEOPARDO (Eublepharis macularius)
No full textA Mar-like transcription factor in the archaeon Sulfolobus solfataricus involved in the detoxification of aromatic aldehydes
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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
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