The Experts below are selected from a list of 1038 Experts worldwide ranked by ideXlab platform
Maria Luisa Tutino - One of the best experts on this subject based on the ideXlab platform.
-
Improvement of Pseudoalteromonas Haloplanktis TAC125 as a Cell Factory: IPTG-Inducible Plasmid Construction and Strain Engineering.
Microorganisms, 2020Co-Authors: Andrea Colarusso, Ermenegilda Parrilli, Concetta Lauro, Marzia Calvanese, Maria Luisa TutinoAbstract:Our group has used the marine bacterium Pseudoalteromonas Haloplanktis TAC125 (PhTAC125) as a platform for the successful recombinant production of "difficult" proteins, including eukaryotic proteins, at low temperatures. However, there is still room for improvement both in the refinement of PhTAC125 expression plasmids and in the bacterium's intrinsic ability to accumulate and handle heterologous products. Here, we present an integrated approach of plasmid design and strain engineering finalized to increment the recombinant expression and optimize the inducer uptake in PhTAC125. To this aim, we developed the IPTG-inducible plasmid pP79 and an engineered PhTAC125 strain called KrPL LacY+. This mutant was designed to express the E. coli lactose permease and to produce only a truncated version of the endogenous Lon protease through an integration-deletion strategy. In the wild-type strain, pP79 assured a significantly better production of two reporters in comparison to the most recent expression vector employed in PhTAC125. Nevertheless, the use of KrPL LacY+ was crucial to achieving satisfying production levels using reasonable IPTG concentrations, even at 0 °C. Both the wild-type and the mutant recombinant strains are characterized by an average graded response upon IPTG induction and they will find different future applications depending on the desired levels of expression.
-
New insights on Pseudoalteromonas Haloplanktis TAC125 genome organization and benchmarks of genome assembly applications using next and third generation sequencing technologies
Scientific Reports, 2019Co-Authors: Weihong Qi, Ermenegilda Parrilli, Andrea Colarusso, Maria Luisa Tutino, Miriam Olombrada, Andrea Patrignani, Macarena Toll-rieraAbstract:Pseudoalteromonas Haloplanktis TAC125 is among the most commonly studied bacteria adapted to cold environments. Aside from its ecological relevance, P. Haloplanktis has a potential use for biotechnological applications. Due to its importance, we decided to take advantage of next generation sequencing (Illumina) and third generation sequencing (PacBio and Oxford Nanopore) technologies to resequence its genome. The availability of a reference genome, obtained using whole genome shotgun sequencing, allowed us to study and compare the results obtained by the different technologies and draw useful conclusions for future de novo genome assembly projects. We found that assembly polishing using Illumina reads is needed to achieve a consensus accuracy over 99.9% when using Oxford Nanopore sequencing, but not in PacBio sequencing. However, the dependency of consensus accuracy on coverage is lower in Oxford Nanopore than in PacBio, suggesting that a cost-effective solution might be the use of low coverage Oxford Nanopore sequencing together with Illumina reads. Despite the differences in consensus accuracy, all sequencing technologies revealed the presence of a large plasmid, pMEGA, which was undiscovered until now. Among the most interesting features of pMEGA is the presence of a putative error-prone polymerase regulated through the SOS response. Aside from the characterization of the newly discovered plasmid, we confirmed the sequence of the small plasmid pMtBL and uncovered the presence of a potential partitioning system. Crucially, this study shows that the combination of next and third generation sequencing technologies give us an unprecedented opportunity to characterize our bacterial model organisms at a very detailed level.
-
Environmental conditions shape the biofilm of the Antarctic bacterium Pseudoalteromonas Haloplanktis TAC125.
Microbiological research, 2018Co-Authors: Annarita Ricciardelli, Maria Luisa Tutino, Maria Michela Corsaro, Alessandro Vergara, Angela Casillo, Nicole Balasco, Ermenegilda ParrilliAbstract:Abstract Biofilms are the most widely distributed and successful microbial modes of life. The capacity of bacteria to colonize surfaces provides stability in the growth environment, allows the capturing of nutrients and affords protection from a range of environmental challenges and stress. Bacteria living in cold environments, like Antarctica, can be found as biofilms, even though the mechanisms of how this lifestyle is related to their environmental adaptation have been poorly investigated. In this paper, the biofilm of Pseudoalteromonas Haloplanktis TAC125, one of the model organisms of cold-adapted bacteria, has been characterized in terms of biofilm typology and matrix composition. The characterization was performed on biofilms produced by the bacterium in response to different nutrient abundance and temperatures; in particular, this is the first report describing the structure of a biofilm formed at 0 °C. The results reported demonstrate that PhTAC125 produces biofilms in different amount and endowed with different physico-chemical properties, like hydrophobicity and roughness, by modulating the relative amount of the different macromolecules present in the biofilm matrix. The capability of PhTAC125 to adopt different biofilm structures in response to environment changes appears to be an interesting adaptation strategy and gives the first hints about the biofilm formation in cold environments.
-
Anti-Biofilm Activity of a Long-Chain Fatty Aldehyde from Antarctic Pseudoalteromonas Haloplanktis TAC125 against Staphylococcus epidermidis Biofilm.
Frontiers in cellular and infection microbiology, 2017Co-Authors: Angela Casillo, Filomena Sannino, Annarita Ricciardelli, Gennaro Marino, Rosanna Papa, Maria Michela Corsaro, Laura Selan, Marco Tilotta, Marcello Ziaco, Maria Luisa TutinoAbstract:Staphylococcus epidermidis is a harmless human skin colonizer responsible for ~20% of orthopedic device-related infections due to its capability to form biofilm. Nowadays there is an interest in the development of anti-biofilm molecules. Marine bacteria represent a still underexploited source of biodiversity able to synthesize a broad range of bioactive compounds, including anti-biofilm molecules. Previous results have demonstrated that the culture supernatant of Antarctic marine bacterium Pseudoalteromonas Haloplanktis TAC125 impairs the formation of S. epidermidis biofilm. Further, evidence supports the hydrophobic nature of the active molecule, which has been suggested to act as a signal molecule. In this paper we describe an efficient activity-guided purification protocol which allowed us to purify this anti-biofilm molecule and structurally characterize it by NMR and mass spectrometry analyses. Our results demonstrate that the anti-biofilm molecule is pentadecanal, a long-chain fatty aldehyde, whose anti-S. epidermidis biofilm activity has been assessed using both static and dynamic biofilm assays. The specificity of its action on S. epidermidis biofilm has been demonstrated by testing chemical analogs of pentadecanal differing either in the length of the aliphatic chain or in their functional group properties. Further, indications of the mode of action of pentadecanal have been collected by studying the bioluminescence of a Vibrio harveyi reporter strain for the detection of autoinducer AI-2 like activities. The data collected suggest that pentadecanal acts as an AI-2 signal. Moreover, the aldehyde metabolic role and synthesis in the Antarctic source strain has been investigated. To the best of our knowledge, this is the first report on the identification of an anti-biofilm molecule form from cold-adapted bacteria and on the action of a long-chain fatty aldehyde acting as an anti-biofilm molecule against S. epidermidis.
-
Recombinant Antibody Fragment Production in the Antarctic Marine Bacterium Pseudoalteromonas Haloplanktis TAC125
Microbial Models: From Environmental to Industrial Sustainability, 2016Co-Authors: Ermenegilda Parrilli, Filomena Sannino, Valeria Citarella, Andrea Colarusso, Annarita Ricciardelli, Gennaro Marino, Maria Luisa TutinoAbstract:Within the biopharmaceutical industry, the antibody market is one of the fastest-growing segments, due to wide applications of recombinant antibodies and antibody fragments in research, diagnostics and therapy. Large-scale production of this protein class requires the use of a platform characterised by low costs, accessible for genetic modifications and easily scaled up. Although their production in prokaryotic hosts can significantly lower production costs, recombinant antibody production in conventional bacterial hosts, such as Escherichia coli, may result in formation of inclusion bodies. As protein solubility (and consequently its correct folding) may be enhanced by lowering of the expression temperature, a novel process for recombinant antibody fragment production at low temperatures was set up using Pseudoalteromonas Haloplanktis TAC125 as recombinant expression host. To test the versatility of the new process developed in the Antarctic Gram-negative bacterium, three model proteins, corresponding to the most common formats of antibody fragments, were produced: Fab, scFv and VH. Several critical aspects were considered in the construction of an ad hoc genetic expression system for each model protein, including the selection of molecular signals for periplasmic protein translocation and the choice of an optimal gene-expression strategy. For instance, an artificial operon was designed and constructed for Fab fragment production in fully heterodimeric form. Furthermore, a novel defined minimal medium was made up to maximise bacterial growth parameters and recombinant production yields. All antibody fragments were produced in soluble and biologically competent form. The observed ability of the Antarctic bacterium to produce recombinant antibody fragments was justified by the observation that P. Haloplanktis TAC125 genome contains an unusually high number of genes encoding peptidyl-prolyl cis-trans isomerases, making this bacterium the host of choice for the recombinant production of this protein class.
Ermenegilda Parrilli - One of the best experts on this subject based on the ideXlab platform.
-
Improvement of Pseudoalteromonas Haloplanktis TAC125 as a Cell Factory: IPTG-Inducible Plasmid Construction and Strain Engineering.
Microorganisms, 2020Co-Authors: Andrea Colarusso, Ermenegilda Parrilli, Concetta Lauro, Marzia Calvanese, Maria Luisa TutinoAbstract:Our group has used the marine bacterium Pseudoalteromonas Haloplanktis TAC125 (PhTAC125) as a platform for the successful recombinant production of "difficult" proteins, including eukaryotic proteins, at low temperatures. However, there is still room for improvement both in the refinement of PhTAC125 expression plasmids and in the bacterium's intrinsic ability to accumulate and handle heterologous products. Here, we present an integrated approach of plasmid design and strain engineering finalized to increment the recombinant expression and optimize the inducer uptake in PhTAC125. To this aim, we developed the IPTG-inducible plasmid pP79 and an engineered PhTAC125 strain called KrPL LacY+. This mutant was designed to express the E. coli lactose permease and to produce only a truncated version of the endogenous Lon protease through an integration-deletion strategy. In the wild-type strain, pP79 assured a significantly better production of two reporters in comparison to the most recent expression vector employed in PhTAC125. Nevertheless, the use of KrPL LacY+ was crucial to achieving satisfying production levels using reasonable IPTG concentrations, even at 0 °C. Both the wild-type and the mutant recombinant strains are characterized by an average graded response upon IPTG induction and they will find different future applications depending on the desired levels of expression.
-
New insights on Pseudoalteromonas Haloplanktis TAC125 genome organization and benchmarks of genome assembly applications using next and third generation sequencing technologies
Scientific Reports, 2019Co-Authors: Weihong Qi, Ermenegilda Parrilli, Andrea Colarusso, Maria Luisa Tutino, Miriam Olombrada, Andrea Patrignani, Macarena Toll-rieraAbstract:Pseudoalteromonas Haloplanktis TAC125 is among the most commonly studied bacteria adapted to cold environments. Aside from its ecological relevance, P. Haloplanktis has a potential use for biotechnological applications. Due to its importance, we decided to take advantage of next generation sequencing (Illumina) and third generation sequencing (PacBio and Oxford Nanopore) technologies to resequence its genome. The availability of a reference genome, obtained using whole genome shotgun sequencing, allowed us to study and compare the results obtained by the different technologies and draw useful conclusions for future de novo genome assembly projects. We found that assembly polishing using Illumina reads is needed to achieve a consensus accuracy over 99.9% when using Oxford Nanopore sequencing, but not in PacBio sequencing. However, the dependency of consensus accuracy on coverage is lower in Oxford Nanopore than in PacBio, suggesting that a cost-effective solution might be the use of low coverage Oxford Nanopore sequencing together with Illumina reads. Despite the differences in consensus accuracy, all sequencing technologies revealed the presence of a large plasmid, pMEGA, which was undiscovered until now. Among the most interesting features of pMEGA is the presence of a putative error-prone polymerase regulated through the SOS response. Aside from the characterization of the newly discovered plasmid, we confirmed the sequence of the small plasmid pMtBL and uncovered the presence of a potential partitioning system. Crucially, this study shows that the combination of next and third generation sequencing technologies give us an unprecedented opportunity to characterize our bacterial model organisms at a very detailed level.
-
Environmental conditions shape the biofilm of the Antarctic bacterium Pseudoalteromonas Haloplanktis TAC125.
Microbiological research, 2018Co-Authors: Annarita Ricciardelli, Maria Luisa Tutino, Maria Michela Corsaro, Alessandro Vergara, Angela Casillo, Nicole Balasco, Ermenegilda ParrilliAbstract:Abstract Biofilms are the most widely distributed and successful microbial modes of life. The capacity of bacteria to colonize surfaces provides stability in the growth environment, allows the capturing of nutrients and affords protection from a range of environmental challenges and stress. Bacteria living in cold environments, like Antarctica, can be found as biofilms, even though the mechanisms of how this lifestyle is related to their environmental adaptation have been poorly investigated. In this paper, the biofilm of Pseudoalteromonas Haloplanktis TAC125, one of the model organisms of cold-adapted bacteria, has been characterized in terms of biofilm typology and matrix composition. The characterization was performed on biofilms produced by the bacterium in response to different nutrient abundance and temperatures; in particular, this is the first report describing the structure of a biofilm formed at 0 °C. The results reported demonstrate that PhTAC125 produces biofilms in different amount and endowed with different physico-chemical properties, like hydrophobicity and roughness, by modulating the relative amount of the different macromolecules present in the biofilm matrix. The capability of PhTAC125 to adopt different biofilm structures in response to environment changes appears to be an interesting adaptation strategy and gives the first hints about the biofilm formation in cold environments.
-
Pseudoalteromonas Haloplanktis TAC125 produces 4-hydroxybenzoic acid that induces pyroptosis in human A459 lung adenocarcinoma cells.
Scientific reports, 2018Co-Authors: Filomena Sannino, Gennaro Marino, Donatella De Pascale, Pietro Tedesco, Renato Fani, Clementina Sansone, Christian Galasso, Sara Kildgaard, Adrianna Ianora, Ermenegilda ParrilliAbstract:In order to exploit the rich reservoir of marine cold-adapted bacteria as a source of bioactive metabolites, ethyl acetate crude extracts of thirteen polar marine bacteria were tested for their antiproliferative activity on A549 lung epithelial cancer cells. The crude extract from Pseudoalteromonas Haloplanktis TAC125 was the most active in inhibiting cell proliferation. Extensive bioassay-guided purification and mass spectrometric characterization allowed the identification of 4-hydroxybenzoic acid (4-HBA) as the molecule responsible for this bioactivity. We further demonstrate that 4-HBA inhibits A549 cancer cell proliferation with an IC50 value ≤ 1 μg ml-1, and that the effect is specific, since the other two HBA isomers (i.e. 2-HBA and 3-HBA) were unable to inhibit cell proliferation. The effect of 4-HBA is also selective since treatment of normal lung epithelial cells (WI-38) with 4-HBA did not affect cell viability. Finally, we show that 4-HBA is able to activate, at the gene and protein levels, a specific cell death signaling pathway named pyroptosis. Accordingly, the treatment of A549 cells with 4-HBA induces the transcription of (amongst others) caspase-1, IL1β, and IL18 encoding genes. Studies needed for the elucidation of mode of action of 4-HBA will be instrumental in depicting novel details of pyroptosis.
-
Recombinant Antibody Fragment Production in the Antarctic Marine Bacterium Pseudoalteromonas Haloplanktis TAC125
Microbial Models: From Environmental to Industrial Sustainability, 2016Co-Authors: Ermenegilda Parrilli, Filomena Sannino, Valeria Citarella, Andrea Colarusso, Annarita Ricciardelli, Gennaro Marino, Maria Luisa TutinoAbstract:Within the biopharmaceutical industry, the antibody market is one of the fastest-growing segments, due to wide applications of recombinant antibodies and antibody fragments in research, diagnostics and therapy. Large-scale production of this protein class requires the use of a platform characterised by low costs, accessible for genetic modifications and easily scaled up. Although their production in prokaryotic hosts can significantly lower production costs, recombinant antibody production in conventional bacterial hosts, such as Escherichia coli, may result in formation of inclusion bodies. As protein solubility (and consequently its correct folding) may be enhanced by lowering of the expression temperature, a novel process for recombinant antibody fragment production at low temperatures was set up using Pseudoalteromonas Haloplanktis TAC125 as recombinant expression host. To test the versatility of the new process developed in the Antarctic Gram-negative bacterium, three model proteins, corresponding to the most common formats of antibody fragments, were produced: Fab, scFv and VH. Several critical aspects were considered in the construction of an ad hoc genetic expression system for each model protein, including the selection of molecular signals for periplasmic protein translocation and the choice of an optimal gene-expression strategy. For instance, an artificial operon was designed and constructed for Fab fragment production in fully heterodimeric form. Furthermore, a novel defined minimal medium was made up to maximise bacterial growth parameters and recombinant production yields. All antibody fragments were produced in soluble and biologically competent form. The observed ability of the Antarctic bacterium to produce recombinant antibody fragments was justified by the observation that P. Haloplanktis TAC125 genome contains an unusually high number of genes encoding peptidyl-prolyl cis-trans isomerases, making this bacterium the host of choice for the recombinant production of this protein class.
Gennaro Marino - One of the best experts on this subject based on the ideXlab platform.
-
Pseudoalteromonas Haloplanktis TAC125 produces 4-hydroxybenzoic acid that induces pyroptosis in human A459 lung adenocarcinoma cells.
Scientific reports, 2018Co-Authors: Filomena Sannino, Gennaro Marino, Donatella De Pascale, Pietro Tedesco, Renato Fani, Clementina Sansone, Christian Galasso, Sara Kildgaard, Adrianna Ianora, Ermenegilda ParrilliAbstract:In order to exploit the rich reservoir of marine cold-adapted bacteria as a source of bioactive metabolites, ethyl acetate crude extracts of thirteen polar marine bacteria were tested for their antiproliferative activity on A549 lung epithelial cancer cells. The crude extract from Pseudoalteromonas Haloplanktis TAC125 was the most active in inhibiting cell proliferation. Extensive bioassay-guided purification and mass spectrometric characterization allowed the identification of 4-hydroxybenzoic acid (4-HBA) as the molecule responsible for this bioactivity. We further demonstrate that 4-HBA inhibits A549 cancer cell proliferation with an IC50 value ≤ 1 μg ml-1, and that the effect is specific, since the other two HBA isomers (i.e. 2-HBA and 3-HBA) were unable to inhibit cell proliferation. The effect of 4-HBA is also selective since treatment of normal lung epithelial cells (WI-38) with 4-HBA did not affect cell viability. Finally, we show that 4-HBA is able to activate, at the gene and protein levels, a specific cell death signaling pathway named pyroptosis. Accordingly, the treatment of A549 cells with 4-HBA induces the transcription of (amongst others) caspase-1, IL1β, and IL18 encoding genes. Studies needed for the elucidation of mode of action of 4-HBA will be instrumental in depicting novel details of pyroptosis.
-
Anti-Biofilm Activity of a Long-Chain Fatty Aldehyde from Antarctic Pseudoalteromonas Haloplanktis TAC125 against Staphylococcus epidermidis Biofilm.
Frontiers in cellular and infection microbiology, 2017Co-Authors: Angela Casillo, Filomena Sannino, Annarita Ricciardelli, Gennaro Marino, Rosanna Papa, Maria Michela Corsaro, Laura Selan, Marco Tilotta, Marcello Ziaco, Maria Luisa TutinoAbstract:Staphylococcus epidermidis is a harmless human skin colonizer responsible for ~20% of orthopedic device-related infections due to its capability to form biofilm. Nowadays there is an interest in the development of anti-biofilm molecules. Marine bacteria represent a still underexploited source of biodiversity able to synthesize a broad range of bioactive compounds, including anti-biofilm molecules. Previous results have demonstrated that the culture supernatant of Antarctic marine bacterium Pseudoalteromonas Haloplanktis TAC125 impairs the formation of S. epidermidis biofilm. Further, evidence supports the hydrophobic nature of the active molecule, which has been suggested to act as a signal molecule. In this paper we describe an efficient activity-guided purification protocol which allowed us to purify this anti-biofilm molecule and structurally characterize it by NMR and mass spectrometry analyses. Our results demonstrate that the anti-biofilm molecule is pentadecanal, a long-chain fatty aldehyde, whose anti-S. epidermidis biofilm activity has been assessed using both static and dynamic biofilm assays. The specificity of its action on S. epidermidis biofilm has been demonstrated by testing chemical analogs of pentadecanal differing either in the length of the aliphatic chain or in their functional group properties. Further, indications of the mode of action of pentadecanal have been collected by studying the bioluminescence of a Vibrio harveyi reporter strain for the detection of autoinducer AI-2 like activities. The data collected suggest that pentadecanal acts as an AI-2 signal. Moreover, the aldehyde metabolic role and synthesis in the Antarctic source strain has been investigated. To the best of our knowledge, this is the first report on the identification of an anti-biofilm molecule form from cold-adapted bacteria and on the action of a long-chain fatty aldehyde acting as an anti-biofilm molecule against S. epidermidis.
-
Recombinant Antibody Fragment Production in the Antarctic Marine Bacterium Pseudoalteromonas Haloplanktis TAC125
Microbial Models: From Environmental to Industrial Sustainability, 2016Co-Authors: Ermenegilda Parrilli, Filomena Sannino, Valeria Citarella, Andrea Colarusso, Annarita Ricciardelli, Gennaro Marino, Maria Luisa TutinoAbstract:Within the biopharmaceutical industry, the antibody market is one of the fastest-growing segments, due to wide applications of recombinant antibodies and antibody fragments in research, diagnostics and therapy. Large-scale production of this protein class requires the use of a platform characterised by low costs, accessible for genetic modifications and easily scaled up. Although their production in prokaryotic hosts can significantly lower production costs, recombinant antibody production in conventional bacterial hosts, such as Escherichia coli, may result in formation of inclusion bodies. As protein solubility (and consequently its correct folding) may be enhanced by lowering of the expression temperature, a novel process for recombinant antibody fragment production at low temperatures was set up using Pseudoalteromonas Haloplanktis TAC125 as recombinant expression host. To test the versatility of the new process developed in the Antarctic Gram-negative bacterium, three model proteins, corresponding to the most common formats of antibody fragments, were produced: Fab, scFv and VH. Several critical aspects were considered in the construction of an ad hoc genetic expression system for each model protein, including the selection of molecular signals for periplasmic protein translocation and the choice of an optimal gene-expression strategy. For instance, an artificial operon was designed and constructed for Fab fragment production in fully heterodimeric form. Furthermore, a novel defined minimal medium was made up to maximise bacterial growth parameters and recombinant production yields. All antibody fragments were produced in soluble and biologically competent form. The observed ability of the Antarctic bacterium to produce recombinant antibody fragments was justified by the observation that P. Haloplanktis TAC125 genome contains an unusually high number of genes encoding peptidyl-prolyl cis-trans isomerases, making this bacterium the host of choice for the recombinant production of this protein class.
-
Pseudoalteromonas Haloplanktis produces methylamine, a volatile compound active against Burkholderia cepacia complex strains.
New biotechnology, 2016Co-Authors: Filomena Sannino, Ermenegilda Parrilli, Gennaro Apuzzo, Donatella De Pascale, Pietro Tedesco, Isabel Maida, Elena Perrin, Marco Fondi, Renato Fani, Gennaro MarinoAbstract:The Antarctic marine bacterium Pseudoalteromonas Haloplanktis TAC125 has been reported to produce several Volatile Organic Compounds (VOCs), which are able to inhibit the growth of Burkholderia cepacia complex (Bcc) strains, opportunistic pathogens responsible for the infection of immune-compromised patients. However, no specific antibacterial VOCs have been identified to date. The purpose of the present study was to identify specific VOCs that contribute to Bcc inhibition by the Antarctic strain. When grown on defined medium containing D-gluconate and L-glutamate as carbon, nitrogen and energy sources, P. Haloplanktis TAC125 is unable to inhibit the growth of Bcc strains. However, single addition of several amino acids to the defined medium restores the P. Haloplanktis TAC125 inhibition ability. With the aim of identifying specific volatile compound/s responsible for Bcc inhibition, we set up an apparatus for VOC capture, accumulation, and storage. P. Haloplanktis TAC125 was grown in an automatic fermenter which was connected to a cooling system to condense VOCs present in the exhaust air outlet. Upon addition of methionine to the growth medium, the VOC methylamine was produced by P. Haloplanktis TAC125. Methylamine was found to inhibit the growth of several Bcc strains in a dose-dependent way. Although it was reported that P. Haloplanktis TAC125 produces VOCs endowed with antimicrobial activity, this is the first demonstration that methylamine probably contributes to the anti-Bcc activity of P. Haloplanktis TAC125 VOCs.
-
Recombinant production of a single-chain antibody fragment in Pseudoalteromonas Haloplanktis TAC125.
Applied microbiology and biotechnology, 2014Co-Authors: Maria Giuliani, Ermenegilda Parrilli, Filomena Sannino, Gennaro Marino, Gennaro Apuzzo, Maria Luisa TutinoAbstract:Recombinant protein production in cold-adapted bacteria has proved to be a valuable option to overcome solubility concerns often came up in conventional expression hosts. ScFvs are examples of “difficult proteins” due to their tendency to form inclusion bodies when expressed in Escherichia coli. In this paper, the recombinant production of a single-chain antibody (ScFvOx) in the psychrophilic bacterium Pseudoalteromonas Haloplanktis TAC125 is reported. The expression vector for the ScFvOx production was designed to address the recombinant protein in the periplasmic space and to allow the formation of the antibody disulphide bonds. For periplasmic export, two different export mechanisms were evaluated. By combining the genetic tools available for recombinant protein expression in psychrophilic hosts with an ad hoc medium and fermentation modality and optimised expression conditions at low temperatures, we obtained the highest yield of soluble and epitope-binding ScFvOx reported so far by conventional prokaryotic expression. The observed proficiency of the Antarctic bacterium to produce recombinant antibody fragments was related to the unusually high number of genes encoding peptidyl prolyl cis-trans isomerases found in P. Haloplanktis TAC125 genome, making this bacterium the host of choice for the recombinant production of this protein class.
Angela Duilio - One of the best experts on this subject based on the ideXlab platform.
-
Regulated recombinant protein production in the antarctic bacterium Pseudoalteromonas Haloplanktis TAC125
Methods in Molecular Biology, 2012Co-Authors: Valentina Rippa, Maria Giuliani, Ermenegilda Parrilli, Maria Luisa Tutino, Gennaro Marino, Cinzia Pezzella, Rosanna Papa, Angela DuilioAbstract:This review reports results from our laboratory on the development of an effective inducible expression system for the homologous/heterologous protein production in cold-adapted bacteria. Recently, we isolated and characterized a regulative genomic region from Pseudoalteromonas Haloplanktis TAC125; in particular, a two-component regulatory system was identifi ed. It is involved in the transcriptional regulation of the gene coding for an outer membrane porin (PSHAb0363) that is strongly induced by the presence of L -malate in the growth medium. We used the regulative region comprising the two-component system located upstream the PSHAb0363 gene to construct an inducible expression vector – named pUCRP – under the control of L -malate. Performances of the inducible system were tested using the psychrophilic b -galactosidase from P. Haloplanktis TAE79 as model enzyme to be produced. Our results demonstrate that the recombinant cold-adapted enzyme is produced in P. Haloplanktis TAC125 in good yields and in a completely soluble and catalytically competent form. Moreover, an evaluation of optimal induction conditions for protein production was also carried out in two consecutive steps: (1) defi nition of the optimal cellular growth phase in which the gene expression has to be induced; (2) defi nition of the optimal inducer concentration that has to be added in the growth medium.
-
A Novel Strategy for the Construction of Genomic Mutants of the Antarctic Bacterium Pseudoalteromonas Haloplanktis TAC125
Methods in molecular biology (Clifton N.J.), 2011Co-Authors: Maria Giuliani, Angela Duilio, Ermenegilda Parrilli, Gennaro Marino, Cinzia Pezzella, Valentina Rippa, Maria Luisa TutinoAbstract:The sequencing and the annotation of the marine Antarctic Pseudoalteromonas Haloplanktis TAC125 genome has paved the way to investigate on the molecular mechanisms involved in adaptation to cold conditions. The growing interest in this unique bacterium prompted the developing of several genetic tools for studying it at the molecular level. To allow a deeper understanding of the PhTAC125 physiology a genetic system for the reverse genetics in this bacterium was developed. In the present work, we describe a practical technique for allelic exchange and/or gene inactivation by in-frame deletion and the use of a counterselectable marker in P. Haloplanktis. The construction of suitable non-replicating plasmid and methods used to carry out a two-step integration-segregation strategy in this bacterium are reported in detail.Furthermore two examples, in which the developed methodology was applied to find out gene function or to construct genetically engineered bacterial strains, were described.
-
Identification of the transcription factor responsible for L-malate-dependent regulation in the marine Antarctic bacterium Pseudoalteromonas Haloplanktis TAC125.
FEMS microbiology letters, 2009Co-Authors: Rosanna Papa, Valentina Rippa, Angela DuilioAbstract:Two-component systems are widespread in nature and constitute the most common mechanism of transmembrane signal transduction in bacteria. Recently, a functionally active two-component system consisting of malS and malR genes possibly involved in the expression of a C4-dicarboxylate transporter system (dctAB operon) was identified in the marine Antarctic bacterium Pseudoalteromonas Haloplanktis TAC125. In this paper, we performed a functional analysis of the two-component system and demonstrated its involvement in the regulation of the expression of C4-dicarboxylate transporter genes. The expression of the C4-dicarboxylate transporter genes was induced by l-malate with the promoter element located upstream of the dctA gene being active only in the presence of the inducer. A σ54 promoter responsible for the l-malate dependent transcription regulation was identified and functionally characterized. The molecular mechanism involves an inverted repeat sequence located upstream the σ54 promoter that was shown to bind regulatory proteins only in the presence of l-malate. The protein factor responsible for the induction of the dctAB operon expression was eventually identified as the transcriptional regulatory protein MalR. MalR is the first transcriptional factor identified in P. Haloplanktis TAC125 and one of the few transcriptional modulators reported so far in cold adapted bacteria.
-
Proteomic identification of a two-component regulatory system in Pseudoalteromonas Haloplanktis TAC125
Extremophiles, 2006Co-Authors: Rosanna Papa, Gennaro Marino, Thomas Schweder, Antoine Danchin, Susanne Glagla, Angela DuilioAbstract:The capability of microorganisms to utilize different carbohydrates as energy source reflects the availability of these substrates in their habitat. Investigation of the proteins involved in carbohydrate usage, in parallel with analysis of their expression, is then likely to provide information on the interaction between microorganisms and their ecosystem. We analysed the growth behaviour of the marine Antarctic bacterium Pseudoalteromonas Haloplanktis TAC125 in the presence and in the absence of different carbon source. A marked increase in the optical density was detected when l -malate was added to the growth medium. Bacterial proteins differently expressed in the presence of l -malate were identified by proteomic profiling experiments. On the basis of their relative increase, six proteins were selected for further analyses. Among these, the expression of a putative outer membrane porin was demonstrated to be heavily induced by l -malate. The presence of a functionally active two-component regulatory system very likely controlled by l -malate was found in the upstream region of the porin gene. A non functional genomic porin mutant was then constructed showing a direct involvement of the protein in the uptake of l -malate. To the best of our knowledge, the occurrence of such a regulatory system has never been reported in Pseudoalteromonads so far and might constitute a key step in the development of an effective inducible cold expression system.
-
An effective cold inducible expression system developed in Pseudoalteromonas Haloplanktis TAC125.
Journal of biotechnology, 2006Co-Authors: Rosanna Papa, Valentina Rippa, Gennaro Marino, Giovanni Sannia, Angela DuilioAbstract:Abstract A regulative two-component system previously identified in Pseudoalteromonas Haloplanktis TAC125 was used to construct a cold inducible expression system that is under the control of l -malate. Performances of the inducible system were tested for both psychrophilic and mesophilic protein production using two “difficult” proteins as control. The results obtained demonstrated that both psychrophilic β-galactosidase and yeast α-glucosidase are produced in a fully soluble and catalytically competent form. Optimal conditions for protein production, including growth temperature, growth medium and l -malate concentration were also investigated. Under optimized conditions yields of 620 and 27 mg/l were obtained for β-galactosidase and α-glucosidase, respectively.
Emmanuele De Vendittis - One of the best experts on this subject based on the ideXlab platform.
-
The cold way for glutathione biosynthesis in the psychrophile Pseudoalteromonas Haloplanktis. Redundancy and reaction rates
RSC Advances, 2016Co-Authors: Antonella Albino, Filomena Sica, Maria Rosaria Ruocco, Amalia De Angelis, Rosario Rullo, Chiara Maranta, Alessandra Capasso, Emmanuele De VendittisAbstract:This work describes major features and specificities of glutathione (GSH) biosynthesis in the cold-adapted Pseudoalteromonas Haloplanktis. Two steps of ATP hydrolysis, catalysed by γ-glutamyl-cysteine ligase (PhGshA) and glutathione synthetase (PhGshB), drive GSH formation; however, differently from other sources, this psychrophile contains two redundant PhGshAs. The biochemical properties of recombinant rPhGshB and rPhGshA II were previously reported; here we report the characterization of rPhGshA I, together with a structural comparison with rPhGshA II. The availability of the three enzymes involved in GSH biosynthesis allowed the reconstitution of two systems producing this thiol in P. Haloplanktis. Both systems were active, although with different reaction rates; in particular, rPhGshB worked more efficiently with rPhGshA I compared to rPhGshA II, also because of the different expression of the corresponding genes. However, the lower KM of rPhGshA II for cysteine compared to rPhGshA I could permit an effective GSH production even under conditions of low cysteine content. Coupling of the kinetics of GSH production with that of ATP hydrolysis was realised for the first time and proved the rigorous stoichiometry of GSH biosynthesis, resulting in two moles of ATP hydrolysed per one mole of GSH formed. Furthermore, after dissecting the reaction rates for GSH biosynthesis, we demonstrated that synthesis of this thiol was rate-limited by the step catalysed by rPhGshB, at least in the cold-adapted source. Therefore, the GshA rate-limited step postulated in other sources could be revised or restricted to conditions of limiting substrate amounts.
-
The cold-adapted γ-glutamyl-cysteine ligase from the psychrophile Pseudoalteromonas Haloplanktis
Biochimie, 2014Co-Authors: Antonella Albino, Mariorosario Masullo, Gennaro Raimo, Antimo Di Maro, Angela Chambery, Salvatore Marco, Amalia De Angelis, Valeria Severino, Doriana Desiderio, Emmanuele De VendittisAbstract:Abstract A recombinant γ-glutamyl-cysteine ligase from the psychrophile Pseudoalteromonas Haloplanktis (r Ph GshA II) was produced and characterised. This enzyme catalyses the first step of glutathione biosynthesis by forming γ-glutamyl-cysteine from glutamate and cysteine in an ATP-dependent reaction. The other ATP-dependent enzyme, glutathione synthetase (r Ph GshB), involved in the second step of the biosynthesis, was already characterised. r Ph GshA II is a monomer of 58 kDa and its activity was characterised through a direct radioisotopic method, measuring the rate of ATP hydrolysis. The enzyme was active even at cold temperatures in a moderately alkaline buffer containing a high concentration of Mg ++ ; 2-aminobutyrate could replace cysteine, although a lower activity was detected. The reaction rate of r Ph GshA II at 15 °C was higher than that reported for r Ph GshB, thus suggesting that formation of γ-glutamyl-cysteine was not the rate limiting step of glutathione biosynthesis in P. Haloplanktis . r Ph GshA II had different affinities for its substrates, as evaluated on the basis of the K M values for ATP (0.093 mM), glutamate (2.8 mM) and cysteine (0.050 mM). Reduced glutathione acted as an inhibitor of r Ph GshA II, probably through the binding to an enzyme pocket different from the active site. Also the oxidised form of glutathione inhibited the enzyme with a more complex inhibition profile, due to the complete mono-glutathionylation of r Ph GshA II on Cys 386, as proved by mass spectrometry data. When compared to r Ph GshB, r Ph GshA II possessed more typical features of a psychrophilic enzyme, as it was endowed with lower thermodependence and higher heat sensitivity. In conclusion, this work extends the knowledge on glutathione biosynthesis in the first cold-adapted source; however, another possible redundant γ-glutamyl-cysteine ligase ( Ph GshA I), not yet characterised, could participate in the biosynthesis of this cellular thiol in P. Haloplanktis .
-
Characterization of a cold-adapted glutathione synthetase from the psychrophile Pseudoalteromonas Haloplanktis
Molecular bioSystems, 2012Co-Authors: Antonella Albino, Mariorosario Masullo, Antimo Di Maro, Angela Chambery, Salvatore Marco, Emmanuele De VendittisAbstract:Glutathione (GSH) biosynthesis occurs through two ATP-dependent reactions, usually involving distinct enzymes; in the second step of this process, catalysed by glutathione synthetase (GshB), GSH is formed from γ-glutamylcysteine and glycine. A recombinant form of GshB from the cold-adapted source Pseudoalteromonas Haloplanktis (rPhGshB) was purified and characterised. The enzyme formed a disulfide adduct with β-mercaptoethanol, when purified in the presence of this reducing agent. The homotetrameric form of rPhGshB observed at high protein concentration disassembled into two homodimers at low concentration. A new method for directly determining the rPhGshB activity was developed, based on [γ-32P]ATP hydrolysis coupled to the GSH synthesis. The ATPase activity required the presence of both γ-glutamylcysteine and glycine and its optimum was reached in the 7.4–8.6 pH range; a divalent cation was absolutely required for the activity, whereas monovalent cations were dispensable. rPhGshB was active at low temperatures and had a similar affinity for ATP (Km 0.26 mM) and γ-glutamylcysteine (Km 0.25 mM); a lower affinity was measured for glycine (Km 0.75 mM). The oxidised form of glutathione (GSSG) acted as an irreversible inhibitor of rPhGshB (Ki 10.7 mM) and formed disulfide adducts with the enzyme. rPhGshB displayed a great temperature-dependent increase in its activity with an unusually high value of energy of activation (75 kJ mol−1) for a psychrophilic enzyme. The enzyme was moderately thermostable, its half inactivation temperature being 50.5 °C after 10 min exposure. The energy of activation of the heat inactivation process was 208 kJ mol−1. To our knowledge, this is the first contribution to the characterization of a GshB from cold-adapted sources.
-
Properties of the endogenous components of the thioredoxin system in the psychrophilic eubacterium Pseudoalteromonas Haloplanktis TAC 125.
Extremophiles : life under extreme conditions, 2012Co-Authors: Patrizia Falasca, Giovanna Evangelista, Mariorosario Masullo, Emmanuele De Vendittis, Salvatore Marco, Roberta Cotugno, Gennaro RaimoAbstract:The endogenous components of the thioredoxin system in the Antarctic eubacterium Pseudoalteromonas Haloplanktis have been purified and characterised. The temperature dependence of the activities sustained by thioredoxin (PhTrx) and thioredoxin reductase (PhTrxR) pointed to their adaptation in the cold growth environment. PhTrxR was purified as a flavoenzyme and its activity was significantly enhanced in the presence of molar concentration of monovalent cations. The energetics of the partial reactions leading to the whole electron transfer from NADPH to the target protein substrate in the reconstituted thioredoxin system was also investigated. While the initial electron transfer from NADPH to PhTrxR was energetically favoured, the final passage to the heterologous protein substrate enhanced the energetic barrier of the whole process. The energy of activation of the heat inactivation process essentially reflected the psychrophilic origin of PhTrxR. Vice versa, PhTrx possessed an exceptional heat resistance (half-life, 4.4 h at 95 °C), ranking this protein among the most thermostable enzymes reported so far in psychrophiles. PhTrxR was covalently modified by glutathione, mainly by its oxidised or nitrosylated forms. A mutagenic analysis realised on three non catalytic cysteines of the flavoenzyme allowed the identification of C(303) as the target for the S-glutathionylation reaction.
-
Improving protein crystal quality by the without-oil microbatch method: crystallization and preliminary X-ray diffraction analysis of glutathione synthetase from Pseudoalteromonas Haloplanktis.
International journal of molecular sciences, 2011Co-Authors: Antonello Merlino, Antonella Albino, Mariorosario Masullo, Irene Russo Krauss, Emmanuele De Vendittis, Alessandro Vergara, Andrea Pica, Filomena SicaAbstract:Glutathione synthetases catalyze the ATP-dependent synthesis of glutathione from L-γ-glutamyl-L-cysteine and glycine. Although these enzymes have been sequenced and characterized from a variety of biological sources, their exact catalytic mechanism is not fully understood and nothing is known about their adaptation at extremophilic environments. Glutathione synthetase from the Antarctic eubacterium Pseudoalteromonas Haloplanktis (PhGshB) has been expressed, purified and successfully crystallized. An overall improvement of the crystal quality has been obtained by adapting the crystal growth conditions found with vapor diffusion experiments to the without-oil microbatch method. The best crystals of PhGshB diffract to 2.34 A resolution and belong to space group P212121, with unit-cell parameters a = 83.28 A, b = 119.88 A, c = 159.82 A. Refinement of the model, obtained using phases derived from the structure of the same enzyme from Escherichia coli by molecular replacement, is in progress. The structural determination will provide the first structural characterization of a psychrophilic glutathione synthetase reported to date.
