RESUMO
Membrane protein (MP) overproduction is one of the major bottlenecks in structural genomics and biotechnology. Despite the emergence of eukaryotic expression systems, bacteria remain a cost effective and powerful tool for protein production. The T7 RNA polymerase (T7RNAP)-based expression system is a successful and efficient expression system, which achieves high-level production of proteins. However some foreign MPs require a fine-tuning of their expression to minimize the toxicity associated with their production. Here we report a novel regulation mechanism for the T7 expression system. We have isolated two bacterial hosts, namely C44(DE3) and C45(DE3), harboring a stop codon in the T7RNAP gene, whose translation is under the control of the basal nonsense suppressive activity of the BL21(DE3) host. Evaluation of hosts with superfolder green fluorescent protein (sfGFP) revealed an unprecedented tighter control of transgene expression with a marked accumulation of the recombinant protein during stationary phase. Analysis of a collection of twenty MP fused to GFP showed an improved production yield and quality of several bacterial MPs and of one human monotopic MP. These mutant hosts are complementary to the other existing T7 hosts and will increase the versatility of the T7 expression system.
Assuntos
RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Membrana/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Virais/genética , Escherichia coli/metabolismo , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Dobramento de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Desulfovibrio piezophilus strain C1TLV30(T) is a mesophilic piezophilic sulfate-reducer isolated from Wood Falls at 1700 m depth in the Mediterranean Sea. In this study, we analysed the effect of the hydrostatic pressure on this deep-sea living bacterium at the physiologic and transcriptomic levels. Our results showed that lactate oxidation and energy metabolism were affected by the hydrostatic pressure. Especially, acetyl-CoA oxidation pathway and energy conservation through hydrogen and formate recycling would be more important when the hydrostatic pressure is above (26 MPa) than below (0.1 MPa) the optimal one (10 MPa). This work underlines also the role of the amino acid glutamate as a piezolyte for the Desulfovibrio genus. The transcriptomic analysis revealed 146 differentially expressed genes emphasizing energy production and conversion, amino acid transport and metabolism and cell motility and signal transduction mechanisms as hydrostatic pressure responding processes. This dataset allowed us to identify a sequence motif upstream of a subset of differentially expressed genes as putative pressure-dependent regulatory element.
Assuntos
Adaptação Fisiológica , Desulfovibrio/fisiologia , Pressão Hidrostática , Estresse Fisiológico , Acetilcoenzima A/metabolismo , Metabolismo Energético , Formiatos/metabolismo , Perfilação da Expressão Gênica , Ácido Glutâmico/metabolismo , Hidrogênio/metabolismo , Lactatos/metabolismo , Mar Mediterrâneo , Metabolômica , OxirreduçãoRESUMO
RNA-seq was used to study the response of Desulfovibrio hydrothermalis, isolated from a deep-sea hydrothermal chimney on the East-Pacific Rise at a depth of 2,600 m, to various hydrostatic pressure growth conditions. The transcriptomic datasets obtained after growth at 26, 10 and 0.1 MPa identified only 65 differentially expressed genes that were distributed among four main categories: aromatic amino acid and glutamate metabolisms, energy metabolism, signal transduction, and unknown function. The gene expression patterns suggest that D. hydrothermalis uses at least three different adaptation mechanisms, according to a hydrostatic pressure threshold (HPt) that was estimated to be above 10 MPa. Both glutamate and energy metabolism were found to play crucial roles in these mechanisms. Quantitation of the glutamate levels in cells revealed its accumulation at high hydrostatic pressure, suggesting its role as a piezolyte. ATP measurements showed that the energy metabolism of this bacterium is optimized for deep-sea life conditions. This study provides new insights into the molecular mechanisms linked to hydrostatic pressure adaptation in sulfate-reducing bacteria.
Assuntos
Desulfovibrio/genética , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Pressão Hidrostática , Transcriptoma/genética , Trifosfato de Adenosina/metabolismo , Vias Biossintéticas/genética , Metabolismo Energético/genética , Genes Bacterianos/genética , Ácido Glutâmico/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Sequência de RNARESUMO
Desulfovibrio hydrothermalis AM13T is a piezophilic, mesophilic, hydrogenotrophic sulfate-reducing bacterium collected from a deep-sea hydrothermal chimney on the East Pacific Rise (2,600 m depth, 13°N). We report the genome sequence of this bacterium, which includes a 3,702,934-bp chromosome and a circular plasmid of 5,328 bp.