Dataset Information

Coping with cold: the genome of the versatile marine Antarctica bacterium Pseudoalteromonas haloplanktis TAC125.

ABSTRACT: A considerable fraction of life develops in the sea at temperatures lower than 15 degrees C. Little is known about the adaptive features selected under those conditions. We present the analysis of the genome sequence of the fast growing Antarctica bacterium Pseudoalteromonas haloplanktis TAC125. We find that it copes with the increased solubility of oxygen at low temperature by multiplying dioxygen scavenging while deleting whole pathways producing reactive oxygen species. Dioxygen-consuming lipid desaturases achieve both protection against oxygen and synthesis of lipids making the membrane fluid. A remarkable strategy for avoidance of reactive oxygen species generation is developed by P. haloplanktis, with elimination of the ubiquitous molybdopterin-dependent metabolism. The P. haloplanktis proteome reveals a concerted amino acid usage bias specific to psychrophiles, consistently appearing apt to accommodate asparagine, a residue prone to make proteins age. Adding to its originality, P. haloplanktis further differs from its marine counterparts with recruitment of a plasmid origin of replication for its second chromosome.

SUBMITTER: Medigue C

PROVIDER: S-EPMC1240074 | biostudies-literature | 2005 Oct

REPOSITORIES: biostudies-literature

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Coping with cold: the genome of the versatile marine Antarctica bacterium Pseudoalteromonas haloplanktis TAC125.

Médigue Claudine C Krin Evelyne E Pascal Géraldine G Barbe Valérie V Bernsel Andreas A Bertin Philippe N PN Cheung Frankie F Cruveiller Stéphane S D'Amico Salvino S Duilio Angela A Fang Gang G Feller Georges G Ho Christine C Mangenot Sophie S Marino Gennaro G Nilsson Johan J Parrilli Ermenegilda E Rocha Eduardo P C EP Rouy Zoé Z Sekowska Agnieszka A Tutino Maria Luisa ML Vallenet David D von Heijne Gunnar G Danchin Antoine A

Genome research 20050916 10

A considerable fraction of life develops in the sea at temperatures lower than 15 degrees C. Little is known about the adaptive features selected under those conditions. We present the analysis of the genome sequence of the fast growing Antarctica bacterium Pseudoalteromonas haloplanktis TAC125. We find that it copes with the increased solubility of oxygen at low temperature by multiplying dioxygen scavenging while deleting whole pathways producing reactive oxygen species. Dioxygen-consuming lip ...[more]

PMID: 16169927

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Project description:BackgroundA significant fraction of the human proteome is still inaccessible to in vitro studies since the recombinant production of several proteins failed in conventional cell factories. Eukaryotic protein kinases are difficult-to-express in heterologous hosts due to folding issues both related to their catalytic and regulatory domains. Human CDKL5 belongs to this category. It is a serine/threonine protein kinase whose mutations are involved in CDKL5 Deficiency Disorder (CDD), a severe neurodevelopmental pathology still lacking a therapeutic intervention. The lack of successful CDKL5 manufacture hampered the exploitation of the otherwise highly promising enzyme replacement therapy. As almost two-thirds of the enzyme sequence is predicted to be intrinsically disordered, the recombinant product is either subjected to a massive proteolytic attack by host-encoded proteases or tends to form aggregates. Therefore, the use of an unconventional expression system can constitute a valid alternative to solve these issues.ResultsUsing a multiparametric approach we managed to optimize the transcription of the CDKL5 gene and the synthesis of the recombinant protein in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 applying a bicistronic expression strategy, whose generalization for recombinant expression in the cold has been here confirmed with the use of a fluorescent reporter. The recombinant protein largely accumulated as a full-length product in the soluble cell lysate. We also demonstrated for the first time that full-length CDKL5 produced in Antarctic bacteria is catalytically active by using two independent assays, making feasible its recovery in native conditions from bacterial lysates as an active product, a result unmet in other bacteria so far. Finally, the setup of an in cellulo kinase assay allowed us to measure the impact of several CDD missense mutations on the kinase activity, providing new information towards a better understanding of CDD pathophysiology.ConclusionsCollectively, our data indicate that P. haloplanktis TAC125 can be a valuable platform for both the preparation of soluble active human CDKL5 and the study of structural-functional relationships in wild type and mutant CDKL5 forms. Furthermore, this paper further confirms the more general potentialities of exploitation of Antarctic bacteria to produce "intractable" proteins, especially those containing large intrinsically disordered regions.

Dataset Information

Coping with cold: the genome of the versatile marine Antarctica bacterium Pseudoalteromonas haloplanktis TAC125.

Publications

Coping with cold: the genome of the versatile marine Antarctica bacterium Pseudoalteromonas haloplanktis TAC125.

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