RESUMO
The Aeolian archipelago is known worldwide for its volcanic activity and hydrothermal emissions, of mainly carbon dioxide and hydrogen sulfide. Hydrogen, methane, and carbon monoxide are minor components of these emissions which together can feed large quantities of bacteria and archaea that do contribute to the removal of these notorious greenhouse gases. Here we analyzed the metagenome of samples taken from the Levante bay on Vulcano Island, Italy. Using a gene-centric approach, the hydrothermal vent community appeared to be dominated by Proteobacteria, and Sulfurimonas was the most abundant genus. Metabolic reconstructions highlight a prominent role of formaldehyde oxidation and the reverse TCA cycle in carbon fixation. [NiFe]-hydrogenases seemed to constitute the preferred strategy to oxidize H2, indicating that besides H2S, H2 could be an essential electron donor in this system. Moreover, the sulfur cycle analysis showed a high abundance and diversity of sulfate reduction genes underpinning the H2S production. This study covers the diversity and metabolic potential of the microbial soil community in Levante bay and adds to our understanding of the biogeochemistry of volcanic ecosystems.
Assuntos
Hidrogênio , Metagenoma , Metano , Microbiologia do Solo , Enxofre , Metano/metabolismo , Hidrogênio/metabolismo , Itália , Enxofre/metabolismo , Archaea/genética , Archaea/classificação , Archaea/metabolismo , Bactérias/genética , Bactérias/classificação , Bactérias/metabolismo , Bactérias/isolamento & purificação , Fontes Hidrotermais/microbiologia , Ilhas , FilogeniaRESUMO
Bacterial lipoproteins are characterized by the presence of a conserved N-terminal lipid-modified cysteine residue that allows the hydrophilic protein to anchor into bacterial cell membranes. These lipoproteins play essential roles in a wide variety of physiological processes. Based on transcriptome analysis of the verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV, we identified a highly expressed lipoprotein, WP_009060351 (139 amino acids), in its genome. The first 86 amino acids are specific for the methanotrophic genera Methylacidiphilum and Methylacidmicrobium, while the last 53 amino acids are present only in lipoproteins of members from the phylum Verrucomicrobiota (Hedlund). Heterologous expression of WP_009060351 in Escherichia coli revealed a 25-kDa dimeric protein and a 60-kDa tetrameric protein. Immunoblotting showed that WP_009060351 was present in the total membrane protein and peptidoglycan fractions of M. fumariolicum SolV. The results suggest an involvement of lipoprotein WP_009060351 in the linkage between the outer membrane and the peptidoglycan.
Assuntos
Lipoproteínas , Peptidoglicano , Verrucomicrobia , Aminoácidos , Escherichia coliRESUMO
The outer membrane (OM) protects Gram-negative bacteria against a hostile environment. The proteins embedded in the OM fulfil a number of tasks that are crucial to the bacterial cell. In this study, we identified and characterised a major outer membrane protein (WP_009059494) from Methylacidiphilum fumariolicum SolV. PRED-TMBB and AlphaFold2 predicted this protein to form a porin with a ß-barrel structure consisting of ten antiparallel ß-sheets and with a small amphipathic N-terminal α-helix in the periplasm. We purified soluble recombinant protein WP_009059494 from E. coli using Tris-HCl buffer with SDS. Antibodies were raised against two peptides in the two large extracellular loops of protein WP_009059494 and immunogold localisation showed this protein to be mainly present in the OM of strain SolV. In addition, this protein is tightly associated with the OM, and is resistant to extraction. Only a small amount can be isolated from the cell envelope using harsh conditions (SDS and boiling). Despite this resistance to extraction, WP_009059494 most likely is an outer membrane protein. A regular lattice could not be detected by negative staining TEM of strain SolV and isolated protein WP_009059494. Considering the specific ecological niche of strain SolV living in a geothermal environment with low pH and high temperatures, this major protein WP_009059494 may act as barrier to resist the extreme conditions found in its natural environment. In addition, we found an absence of the BamB, BamC and BamE proteins of the canonical BAM complex, in Methylacidiphilum and Methylacidimicrobium species. This suggests that these bacteria use a simple BAM complex for folding and transport of OM proteins.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Verrucomicrobia/metabolismoRESUMO
BACKGROUND: The overexpression and purification of membrane proteins is a bottleneck in biotechnology and structural biology. E. coli remains the host of choice for membrane protein production. To date, most of the efforts have focused on genetically tuning of expression systems and shaping membrane composition to improve membrane protein production remained largely unexplored. RESULTS: In E. coli C41(DE3) strain, we deleted two transporters involved in fatty acid metabolism (OmpF and AcrB), which are also recalcitrant contaminants crystallizing even at low concentration. Engineered expression hosts presented an enhanced fitness and improved folding of target membrane proteins, which correlated with an altered membrane fluidity. We demonstrated the scope of this approach by overproducing several membrane proteins (4 different ABC transporters, YidC and SecYEG). CONCLUSIONS: In summary, E. coli membrane engineering unprecedentedly increases the quality and yield of membrane protein preparations. This strategy opens a new field for membrane protein production, complementary to gene expression tuning.
Assuntos
Proteínas de Escherichia coli/biossíntese , Escherichia coli/metabolismo , Lipídeos/química , Proteínas de Membrana/biossíntese , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Engenharia Metabólica , Canais de Translocação SEC/química , Canais de Translocação SEC/genéticaRESUMO
Despite growing research efforts on the preparation of (bio)functional liposomes, synthetic capsules cannot reach the densities of protein loading and the control over peptide display that is achieved by natural vesicles. Herein, a microbial platform for high-yield production of lipidic nanovesicles with clickable thiol moieties in their outer corona is reported. These nanovesicles show low size dispersity, are decorated with a dense, perfectly oriented, and customizable corona of transmembrane polypeptides. Furthermore, this approach enables encapsulation of soluble proteins into the nanovesicles. Due to the mild preparation and loading conditions (absence of organic solvents, pH gradients, or detergents) and their straightforward surface functionalization, which takes advantage of the diversity of commercially available maleimide derivatives, bacteria-based proteoliposomes are an attractive eco-friendly alternative that can outperform currently used liposomes.
Assuntos
Trifosfato de Adenosina/metabolismo , Escherichia coli/metabolismo , Lipídeos/química , Nanopartículas/química , Proteolipídeos/química , ATPases Translocadoras de Prótons/metabolismo , Compostos de Sulfidrila/química , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/metabolismoRESUMO
Mitochondria, chloroplasts and photosynthetic bacteria are characterized by the presence of complex and intricate membrane systems. In contrast, non-photosynthetic bacteria lack membrane structures within their cytoplasm. However, large scale over-production of some membrane proteins, such as the fumarate reductase, the mannitol permease MtlA, the glycerol acyl transferase PlsB, the chemotaxis receptor Tsr or the ATP synthase subunit b, can induce the proliferation of intra cellular membranes (ICMs) in the cytoplasm of Escherichia coli. These ICMs are particularly rich in cardiolipin (CL). Here, we have studied the effect of CL in the generation of these membranous structures. We have deleted the three genes (clsA, clsB and clsC) responsible of CL biosynthesis in E. coli and analysed the effect of these mutations by fluorescent and electron microscopy and by lipid mass spectrometry. We have found that CL is essential in the formation of non-lamellar structures in the cytoplasm of E. coli cells. These results could help to understand the structuration of membranes in E. coli and other membrane organelles, such as mitochondria and ER.
Assuntos
Proteínas de Bactérias/metabolismo , Cardiolipinas/metabolismo , Retículo Endoplasmático/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana/deficiência , Mitocôndrias/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/deficiência , Proteínas de Bactérias/genética , ATPases Bacterianas Próton-Translocadoras/genética , ATPases Bacterianas Próton-Translocadoras/metabolismo , Retículo Endoplasmático/ultraestrutura , Escherichia coli/ultraestrutura , Corantes Fluorescentes/química , Deleção de Genes , Expressão Gênica , Isoenzimas/deficiência , Isoenzimas/genética , Proteínas de Membrana/genética , Mitocôndrias/ultraestrutura , Imagem com Lapso de Tempo , Transferases (Outros Grupos de Fosfato Substituídos)/genéticaRESUMO
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
Functional and structural studies on membrane proteins are limited by the difficulty to produce them in large amount and in a functional state. In this review, we provide protocols to achieve high-level expression of membrane proteins in Escherichia coli. The T7 RNA polymerase-based expression system is presented in detail and protocols to assess and improve its efficiency are discussed. Protocols to isolate either membrane or inclusion bodies and to perform an initial qualitative test to assess the solubility of the recombinant protein are also included.