Complete genome sequence of the halophilic PHA-producing bacterium Halomonas sp. SF2003: insights into its biotechnological potential.

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Enzymes and Compatible solutes

Among the important variety of compounds produced by halophiles, a special attention is given to enzymes and compatible solutes. Indeed, enzymes are referred as polyextremophilic and unusual stable proteins due to their intrinsically stability and activity under harsh conditions 25–27. Compatible solutes, then, could be used for stabilization of (bio-)molecules.
Halophilic microorganisms secrete halophilic hydrolases (proteases, lipases, amylases, esterases or nucleases) able to catalyze reactions under high salt concentrations 28. Currently, glycosy hydrolase, protease and lipase represent 70% of sold enzyme. Proteases alone are the most used enzymes, especially in detergents formulations, pharmaceuticals, food processing or waste management 10. Different halophilic species have been identified as protease producer such as Halobacillus karajensis strain MA-2, Pseudoalteromonas sp., Halobacillus sp. or Chromohalobacter sp. 10,25. Lipase represents also an important industrial enzyme exploited in detergent formulation or organic synthesis and has been reported to be produced by the moderately halophilic species Salinivibrio sp. 24. Finally, halophilic amylases, which are commonly cyclomaltrodextrinases, are produced by various halobacteria (Halomonas meridiana, Halobacillus spp., Halothermotrix orenii, Haloarchula sp.S-1 or Micrococcus halobius) and can also be used in detergent formulation or for waste water treatment 10. But even if a large variety of halophilic enzymes are produced by different halobacteria there is only few which have found industrial application due to limited requirement for salt-tolerant enzymes. Currently, nuclease H of Micrococcus varians subsp. halophilus is used for production of flavoring agent is one of the few enzymes with industrial applications 24.
In comparison, demand for compatible solutes is quiet more developped. The term compatible solutes refers to zwitterionic, non charged or anionic, low molecular mass compounds allowing adaptation, maintenance and survival of organisms under harsh conditions by providing an osmotic balance 25. These compounds avoid or limit detrimental action of high salt concentration, high temperature, freezing or dessication on (bio-)molecules (DNA, proteins, …). Additionnaly, some of them expose ability to counteract UV-A effects.

Biopolymers

Microorganisms, including halophiles, are responsible for production of various biopolymers like polyamides, polyanhydrides, polyesters or polysaccharides, whose majority are extracellular products. They are mainly synthesized to protect cells in front of stressful conditions 31. In natural environment, biopolymers are completely degraded by depolymerases and hydrolase action making them good candidates for replacement of recalcitrant oil-based polymers. Consequently, bacterial polymers are exploitable in diverse application fields (food industry, pharmaceuticals technology, packaging, bioremediation,…) 32. In this section we will present and describe some of them in order to underlign their biotechnological potential.

Polysaccharides

There are already many studies looking for discovery and exploitation of marine polysaccharides such as agar, agarose, carrageenans and alginates, that are extracted from seaweeds (macroalgaea) 33. But marine bacteria can also produced an important variety of polysaccharides: structural, intracellular or extracellular polysaccharides also named exopolysaccharides (EPS). This last type of polysaccharides represents another important source of marine polymers with promising or actual applications. Numerous bacterial species have been reported to produce EPS in large amounts 24, nevertheless only few of these polysaccharides have been yet fully studied 33. They are water soluble molecules, which may be ionic or non-ionic, literally extracellularly produced by halophilic microorganisms. EPS are composed of very regular units of repeat (branched or not) 4 that can be sugars including amino sugars (D-Glucosamine and D-Galactosamine), pentoses (D-Arabinose, DRibose, D-Xylose), hexoses (D-Allose, L-Fucose, D-Galactose, D-Glucose, D-Mannose, L-Rhamnose) or uronic acids (D-Glucuronic acids, D-Galacturonic acids). They can also contain various organic or inorganic compounds (acetic, phosphoric, pyruvic, sulfuric or succinic acids, phosphate or sulfate) 33,34. EPS can be considered as protective compounds against adverse conditions and many halophilic species have been described to possess an EPS capsule surrounding cell to protect it. But they are also involved in different processes in marine environment such as bacterial attachment on surfaces, biochemical interaction between cells and absorption of dissolved organic materials 33,35. EPS production has been described in various halophilic microorganisms like Halobacteria and especially Halomonas species (H. alkaliantartica, H. anticariensis strain FP36, H. eurihalina, H. maura, Halomonas sp. AAD6, H.
ventosae sp. nov.,…) 5,11,24,36,37 or in Cyanobacteria species (Aphanothece halophytica or Cyanospira capsulate) 24,38 but also in haloarchaea (Haloferax mediterranei, Haloferax gibbonsii, Haloarcula strain T5). Their production can be induced by various environmental factors: salinity, pH, temperature, light intensity, competition for nutriments or for adaptation in front of extreme inhabitats. Consequently their biological functions depend on microorganisms’s environment 39. EPS produced by Halomonas species are polyanionic with an elevated sulphate content and glucuronic acid 4,38 giving them good gellifying properties. Comparatively, EPS from the haloarchaea, Haloferax mediterranei, are anionic sulfated acidic heteropolysaccharides exposing good rheological properties, high viscosity at low concentration and resistance against harsh conditions (temperature and pH) 9,11,24. EPS also expose immunomodulation properties (sulfated EPS can limit virus penetration in host cells like EPS of A. halophytica which inhibit pneumonia caused by influenza virus H1N1 24 or those produced by H. mauran4), anticancer activy (EPS from Pseudomonas sp. exposing cytotoxic effects on cancer cell lines MT-4) or bone-healing properties (EPS produced by Vibrio diabolicus) 5,31,33,38.

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Table of contents :

Remerciements
Table des matières
Préface
Chapter 1 State of the art
Halotolerant/Halophilic microorganisms and their biotechnological potential
Introduction
I. Microbial communities
I.1 Habitats and characteristics
I.2 Taxonomy
I.2.1 Haloarchaea
I.2.2 Halophilic and halotolerant bacteria
II. Biotechnological applications
II.1 Pigments
II.2 Enzymes and Compatible solutes
II.3 Biopolymers
II.3.1 Polysaccharides
II.3.2 Polyesters/Polyhydroxyalkanoates
III. Generalities about Polyhydroxyalkanoates
III.1 Structure and properties of PHA
III.1.1 Structure
III.1.2 Properties
III.2 Metabolism of PHA
III.2.1 Molecular organisation
III.2.2 Metabolic pathways
III.2.3 Granules of PHA
III.3 Production of PolyHydroxyAlkanoates
III.3.1 Operational modes of Production
III.3.2 Bacterial cultures
III.3.3 Carbon substrates
III.3.4 Downstream processing
III.4 PHA production employing halophilic microorganisms
III.4.1 Haloarchaea
III.4.2 Halobacteria
III.4.3 Genetically engineered halophilic bacteria
Conclusion
References
Chapter 2 Matériels et Méthodes
I Souches bactériennes et conditions de culture
I.1 Souches et milieux étudiés
I.1.1 Souches bactériennes
I.1.2 Milieux
I.1.2.1 Croissance
I.1.2.2 Tests de croissance en conditions de stress toxique
I.1.2.3 Tests de production
I.2 Cinétique de croissance des souches bactériennes
II Caractérisation bio-informatique de Halomonas sp. SF2003
II.1 Séquençage et annotation du génome de Halomonas sp. SF2003
II.2 Étude phylogénétique d’Halomonas sp. SF 20003
II.3 Étude bio-informatique des PHA synthases
II.4 Étude phénotypique d’Halomonas sp. SF2003
II.4.1 Croissance de Halomonas sp. SF2003 en conditions de stress osmotique et toxique
II.4.1.1 Salinité
II.4.1.2 Composés toxiques : Hydrocarbures Aromatiques Polycycliques
II.4.1.3 Tests d’antibiorésistance
III Techniques de biologie moléculaire
III.1 Extraction d’ADN
III.2 Réaction de polymérisation en chaîne (PCR)
III.3 Double digestion
III.4 Clonage/ligation
III.5 Conjugaison/transformation
III.5.1 Transformation des cellules E. cloni® et Escherichia coli S17-1
III.5.2 Transconjugaison E. coli S17-1/Cupriavidus necator H16 PHB-4
IV Production de PHA
IV.1 Criblage de sources de carbone assimilables
IV.1.1 Source de carbone
IV.1.2 Marquage des granules de PHA au Nile Red
IV.1.2.1 Géloses au Nile Red
IV.1.2.2 Microscopie confocale à balayage laser
IV.1.3 Taux de recouvrement
IV.2 Production
IV.2.1 Production en erlenmeyers
IV.2.2 Production en bio-réacteurs
IV.3 Extraction et purification des PHA
Références
Chapter 3 Complete genome sequence of the halophilic PHA-producing bacterium Halomonas sp. SF2003: insights into its biotechnological potential.
Graphical abstract
Abstract
Introduction
I. Results
I.1. Genome features
I.2. Identification and phylogenetic study for the classification of Halomonas sp. SF2003 95
I.3. Metabolism
I.3.1. Carbohydrates metabolism
I.3.2. Polyhydroxyalkanoates production
I.4. Stress regulation-related proteins
Discussion
References
Chapter 4 PHA production and PHA synthases of the halophilic bacterium Halomonas sp. SF2003.
Graphical abstract
Introduction
I. Results
I.1. In silico study of PHA synthases PhaC1 and PhaC2 of Halomonas sp. SF2003
I.2. Screening of carbon substrates for PHA production by Halomonas sp. SF2003
I.3. Study of PHA synthases
I.3.1. Cloning of PHA synthases phaC1 and phaC2 of Halomonas sp. SF2003
I.3.2. Characterization of PHA production by transformant strains C. necator H16 PHB-4 phaC1 and C. necator H16 PHB-4 phaC2
I.3.3. Polyhydroxyalkanoates production in shake flasks
Discussion
Conclusion
References
Chapter 5 Characterization of the Halomonas sp. SF2003 PHA production
Graphical abstract
Introduction
I. Results
I.1. Optimization of PHA production process
I.1.1. Response of Halomonas sp. SF2003 growth in function of carbohydrates
I.1.2. Impact of carbon source and salinity on PHA production by Halomonas sp. SF2003
I.1.2.1. Carbon sources
I.1.2.2. Salinity
I.2. Development of a fluorescence based monitoring method
I.2.1. PHA production kinetics monitoring using fluorescence staining
I.2.1.1. PHA Production using fructose
I.2.1.2. Production using glucose
I.2.2. PHA production
Discussion
Conclusion
References

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