Ermenegilda Parrilli

University of Naples Federico II

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Maria Luisa Tutino

University of Naples Federico II

110

Maria Michela Corsaro

University of Naples Federico II

Angela Casillo

University of Naples Federico II

Gennaro Marino

University of Naples Federico II

Filomena Sannino

University of Naples Federico II

Marco Fondi

University of Florence

Maria Giuliani

Marche Polytechnic University

Donatella de Pascale

Stazione Zoologica Anton Dohrn

Marzia Calvanese

University of Naples Federico II

Rosa Lanzetta

University of Naples Federico II

Cooperative Institutions

University of Naples Federico II

100

Federico II University Hospital

Istituto Nazionale di Fisica Nucleare, Sezione di Napoli

National Research Council

University of Florence

Sapienza University of Rome

Institute of Protein Biochemistry

Centre National de la Recherche Scientifique

Universitat Autònoma de Barcelona

Stazione Zoologica Anton Dohrn

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A Novel Strategy for the Construction of Genomic Mutants of the Antarctic Bacterium Pseudoalteromonas haloplanktis TAC125

Methods in molecular biology (2011)

Maria Giuliani Ermenegilda Parrilli Cinzia Pezzella Valentina Rippa Angela Duilio

Pseudoalteromonas

10.1007/978-1-61779-433-9_11

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Citations (17)

Genome‐scale metabolic reconstruction and constraint‐based modelling of the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125

Environmental Microbiology (2014)

Marco Fondi Isabel Maida Elena Perrin Alessandra Mellera Stéfano Mocali

The Antarctic strain Pseudoalteromonas haloplanktis TAC125 is one of the model organisms of cold-adapted bacteria and is currently exploited as a new alternative expression host for numerous biotechnological applications. Here, we investigated several metabolic features of this strain through in silico modelling and functional integration of -omics data. A genome-scale metabolic model of P. haloplanktis TAC125 was reconstructed, encompassing information on 721 genes, 1133 metabolites and 1322 reactions. The predictive potential of this model was validated against a set of experimentally determined growth rates and a large dataset of growth phenotypic data. Furthermore, evidence synthesis from proteomics, phenomics, physiology and metabolic modelling data revealed possible drawbacks of cold-dependent changes in gene expression on the overall metabolic network of P. haloplanktis TAC125. These included, for example, variations in its central metabolism, amino acid degradation and fatty acid biosynthesis. The genome-scale metabolic model described here is the first one reconstructed so far for an Antarctic microbial strain. It allowed a system-level investigation of variations in cellular metabolic fluxes following a temperature downshift. It represents a valuable platform for further investigations on P. haloplanktis TAC125 cellular functional states and for the design of more focused strategies for its possible biotechnological exploitation.

Phenomics

Metabolic network

Model Organism

Metabolic pathway

10.1111/1462-2920.12513

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Citations (51)

Regulated Recombinant Protein Production in the Antarctic Bacterium Pseudoalteromonas haloplanktis TAC125

Methods in molecular biology (2011)

Valentina Rippa Rosanna Papa Maria Giuliani Cinzia Pezzella Ermenegilda Parrilli

Heterologous

Pseudoalteromonas

Heterologous expression

10.1007/978-1-61779-433-9_10

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Engineering the Marine Pseudoalteromonas haloplanktis TAC125 via pMEGA Plasmid Targeted Curing Using PTasRNA Technology

Angelica Severino Concetta Lauro Marzia Calvanese Christopher Riccardi Andrea Colarusso

Marine bacteria that have adapted to thrive in extreme environments, such as Pseudoalteromonas haloplanktis TAC125 (PhTAC125), offer a unique biotechnological potential. The discovery of an endogenous megaplasmid (pMEGA) raised questions about its metabolic impact and functional role in this strain. This study aimed at streamlining the host genetic background by curing PhTAC125 from the pMEGA plasmid using a sequential genetic approach. We combined homologous recombination by exploiting a suicide vector with the PTasRNA gene silencing technology to interfere with pMEGA replication machinery. This approach led to the construction of the novel PhTAC125 KrPL2 strain, cured from the pMEGA plasmid, which exhibited no significant differences in the growth behaviour, though showcasing enhanced resistance to oxidative stress and a reduced capability of biofilm formation. These findings represent a significant achievement for understanding of the role of pMEGA plasmid and for the biotechnological applications of PhTAC125 in recombinant protein production. This opens up the possibility to exploit pMEGA valuable genetic elements and further advancing the genetic tools for PhTAC125.

Pseudoalteromonas

10.20944/preprints202412.1244.v1

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Engineering the Marine Pseudoalteromonas haloplanktis TAC125 via the pMEGA Plasmid Targeted Curing Using PTasRNA Technology

Microorganisms (2025)

Angelica Severino Concetta Lauro Marzia Calvanese Christopher Riccardi Andrea Colarusso

Marine bacteria that have adapted to thrive in extreme environments, such as Pseudoalteromonas haloplanktis TAC125 (PhTAC125), offer a unique biotechnological potential. The discovery of an endogenous megaplasmid (pMEGA) raises questions about its metabolic impact and functional role in that strain. This study aimed at streamlining the host genetic background by curing PhTAC125 of the pMEGA plasmid using a sequential genetic approach. We combined homologous recombination by exploiting a suicide vector, with the PTasRNA gene-silencing technology interfering with pMEGA replication machinery. This approach led to the construction of the novel PhTAC125 KrPL2 strain, cured of the pMEGA plasmid, which exhibited no significant differences in growth behavior, though showcasing enhanced resistance to oxidative stress and a reduced capacity for biofilm formation. These findings represent a significant achievement in developing our understanding of the role of the pMEGA plasmid and the biotechnological applications of PhTAC125 in recombinant protein production. This opens up the possibility of exploiting valuable pMEGA genetic elements and further advancing the genetic tools for PhTAC125.

Pseudoalteromonas

10.3390/microorganisms13020324

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Pentadecanal inspired molecules as new anti-biofilm agents against Staphylococcus epidermidis

Figshare (2019)

Annarita Ricciardelli Angela Casillo Rosanna Papa Daria Maria Monti Paola Imbimbo

Staphylococcus epidermidis, a harmless human skin colonizer, is a significant nosocomial pathogen in predisposed hosts because of its capability to form a biofilm on indwelling medical devices. In a recent paper, the purification and identification of the pentadecanal produced by the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125, able to impair S. epidermidis biofilm formation, were reported. Here the authors report on the chemical synthesis of pentadecanal derivatives, their anti-biofilm activity on S. epidermidis, and their action in combination with antibiotics. The results clearly indicate that the pentadecanal derivatives were able to prevent, to a different extent, biofilm formation and that pentadecanoic acid positively modulated the antimicrobial activity of the vancomycin. The cytotoxicity of these new anti-biofilm molecules was tested on two different immortalized eukaryotic cell lines in view of their potential applications.

10.6084/m9.figshare.7649207

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A novel genetic system for recombinant protein secretion in the Antarctic Pseudoalteromonas haloplanktis TAC125

Microbial Cell Factories (2006)

Angela Maria Cusano Ermenegilda Parrilli Gennaro Marino Maria Luisa Tutino

Abstract Background The final aim of recombinant protein production is both to have a high specific production rate and a high product quality. It was already shown that using cold-adapted bacteria as host vectors, some "intractable" proteins can be efficiently produced at temperature as low as 4°C. Results A novel genetic system for the production and secretion of recombinant proteins in the Antarctic Gram-negative bacterium Pseudoalteromonas haloplanktis TAC125 was set up. This system aims at combining the low temperature recombinant product production with the advantages of extra-cellular protein targeting. The psychrophilic α-amylase from Pseudoalteromonas haloplanktis TAB23 was used as secretion carrier. Three chimerical proteins were produced by fusing intra-cellular proteins to C-terminus of the psychrophilic α-amylase and their secretion was analysed. Data reported in this paper demonstrate that all tested chimeras were translocated with a secretion yield always higher than 80%. Conclusion Data presented here demonstrate that the "cold" gene-expression system is efficient since the secretion yield of tested chimeras is always above 80%. These secretion performances place the α-amylase derived secretion system amongst the best heterologous secretion systems in Gram-negative bacteria reported so far. As for the quality of the secreted passenger proteins, data presented suggest that the system also allows the correct disulphide bond formation of chimera components, secreting a fully active passenger.

Pseudoalteromonas

10.1186/1475-2859-5-40

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Citations (37)

Large-scale biofilm cultivation of Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 for physiologic studies and drug discovery

Extremophiles (2016)

Ermenegilda Parrilli Annarita Ricciardelli Angela Casillo Filomena Sannino Rosanna Papa

Exopolymer

Extracellular polymeric substance

10.1007/s00792-016-0813-2

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Citations (9)

Recombinant protein production in Pseudoalteromonas haloplanktis TAC125 biofilm

Biofilm (2024)

Marzia Calvanese Caterina D’Angelo Concetta Lauro Maria Luisa Tutino Ermenegilda Parrilli

Biofilms have great potential for producing valuable products, and recent research has been performed on biofilms for the production of compounds with biotechnological and industrial relevance. However, the production of recombinant proteins using this system is still limited. The recombinant protein production in microbial hosts is a well-established technology and a variety of expression systems are available. Nevertheless, the production of some recombinant proteins can result in proteolyzed, insoluble, and non-functional forms, therefore it is necessary to start the exploration of non-conventional production systems that, in the future, could be helpful to produce some "difficult" proteins. Non-conventional production systems can be based on the use of alternative hosts and/or on non-conventional ways to grow recombinant cells. In this paper, the use of the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125 grown in biofilm conditions was explored to produce two fluorescent proteins, GFP and mScarlet. The best conditions for the production were identified by working on media composition, and induction conditions, and by building a new expression vector suitable for the biofilm conditions. Results reported demonstrated that the optimized system for the recombinant protein production in biofilm, although it takes longer than planktonic production, has the same potentiality as the classical planktonic approach with additional advantages since it needs a lower concentration of the carbon sources and doesn't require antibiotic addition. Moreover, in the case of mScarlet, the production in biofilm outperforms the planktonic system in terms of a better quality of the recombinant product.

10.1016/j.bioflm.2024.100179

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Development of high-copy number plasmids in Pseudoalteromonas haloplanktis TAC125

Applied Microbiology and Biotechnology (2023)

Marzia Calvanese Cecilia Balestra Andrea Colarusso Concetta Lauro Christopher Riccardi

The Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) is considered an interesting alternative host for the recombinant protein production, that can be explored when the conventional bacterial expression systems fail. Indeed, the manufacture of all the difficult-to-express proteins produced so far in this bacterial platform gave back soluble and active products. Despite these promising results, the low yield of recombinant protein production achieved is hampering the wider and industrial exploitation of this psychrophilic cell factory. All the expression plasmids developed so far in PhTAC125 are based on the origin of replication of the endogenous pMtBL plasmid and are maintained at a very low copy number. In this work, we set up an experimental strategy to select mutated OriR sequences endowed with the ability to establish recombinant plasmids at higher multiplicity per cell. The solution to this major production bottleneck was achieved by the construction of a library of psychrophilic vectors, each containing a randomly mutated version of pMtBL OriR, and its screening by fluorescence-activated cell sorting (FACS). The selected clones allowed the identification of mutated OriR sequences effective in enhancing the plasmid copy number of approximately two orders of magnitude, and the production of the recombinant green fluorescent protein was increased up to twenty times approximately. Moreover, the molecular characterization of the different mutant OriR sequences allowed us to suggest some preliminary clues on the pMtBL replication mechanism that deserve to be further investigated in the future. KEY POINTS: • Setup of an electroporation procedure for Pseudoalteromonas haloplanktis TAC125. • Two order of magnitude improvement of OriR-derived psychrophilic expression systems. • Almost twenty times enhancement in Green fluorescent protein production.

Pseudoalteromonas

Psychrophile

Cell Sorting

ORFS

10.1007/s00253-023-12448-w

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Citations (6)