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Engineering a predatory bacterium as a proficient killer agent for intracellular bio-products recovery: The case of the polyhydroxyalkanoates.

Martínez V, Herencias C, Jurkevitch E, Prieto MA - Sci Rep (2016)

Bottom Line: This work examines the potential of the predatory bacterium Bdellovibrio bacteriovorus HD100, an obligate predator of other Gram-negative bacteria, as an external cell-lytic agent for recovering valuable intracellular bio-products produced by prey cultures.The bio-product targets to be recovered were polyhydroxyalkanoates (PHAs) produced naturally by Pseudomonas putida and Cupriavidus necator, or by recombinant Escherichia coli strains.B. bacteriovorus with a mutated PHA depolymerase gene to prevent the unwanted breakdown of the bio-product allowed the recovery of up to 80% of that accumulated by the prey bacteria, even at high biomass concentrations.

View Article: PubMed Central - PubMed

Affiliation: Environmental Biology Department, Centro de Investigaciones Biológicas, CSIC, C/Ramiro de Maeztu 9, 28040 Madrid, Spain.

ABSTRACT
This work examines the potential of the predatory bacterium Bdellovibrio bacteriovorus HD100, an obligate predator of other Gram-negative bacteria, as an external cell-lytic agent for recovering valuable intracellular bio-products produced by prey cultures. The bio-product targets to be recovered were polyhydroxyalkanoates (PHAs) produced naturally by Pseudomonas putida and Cupriavidus necator, or by recombinant Escherichia coli strains. B. bacteriovorus with a mutated PHA depolymerase gene to prevent the unwanted breakdown of the bio-product allowed the recovery of up to 80% of that accumulated by the prey bacteria, even at high biomass concentrations. This innovative downstream process highlights how B. bacteriovorus can be used as a novel, biological lytic agent for the inexpensive, industrial scale recovery of intracellular products from different Gram-negative prey cultures.

No MeSH data available.


Related in: MedlinePlus

Mcl-PHA hydrolytic product profile identified in the co-culture supernatants of B. bacteriovorus strains preying on P. putida KT2440 and KT42Z accumulating mcl-PHA.(a) HPLC-MS analysis after 30 h of predation by Bdellovibrio strains. Monomers (white bars), dimers (light grey bars) and trimers (dark grey bars). Control supernatants of P. putida KT2440 and KT42Z are also shown. (b) Total PHA hydrolysis products quantified in the culture supernatants. No significant differences were observed between: (i) the percentage of monomers of the PHA extracted from KT2440 and Bd3709/KT2440, (ii) the percentage of dimers of KT2440 and HD100/KT2440, (iii) the percentage of dimers of KT2440 and Bd3709/KT2440, and (iv) the percentage of dimers of KT2440 and dimers of Bd3709/KT42Z. The rest of the conditions showed significant differences (P < 0.05) determined by ANOVA-test.
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f5: Mcl-PHA hydrolytic product profile identified in the co-culture supernatants of B. bacteriovorus strains preying on P. putida KT2440 and KT42Z accumulating mcl-PHA.(a) HPLC-MS analysis after 30 h of predation by Bdellovibrio strains. Monomers (white bars), dimers (light grey bars) and trimers (dark grey bars). Control supernatants of P. putida KT2440 and KT42Z are also shown. (b) Total PHA hydrolysis products quantified in the culture supernatants. No significant differences were observed between: (i) the percentage of monomers of the PHA extracted from KT2440 and Bd3709/KT2440, (ii) the percentage of dimers of KT2440 and HD100/KT2440, (iii) the percentage of dimers of KT2440 and Bd3709/KT2440, and (iv) the percentage of dimers of KT2440 and dimers of Bd3709/KT42Z. The rest of the conditions showed significant differences (P < 0.05) determined by ANOVA-test.

Mentions: According to the above results, the profile of the HAs products should differ depending on the predatory strain used in the co-culture. To examine this, P. putida KT2440 cells were prepared in Hepes buffer at a biomass of 5.7 g l−1 (corresponding to 2.4 ± 0.2 · 107 cfu ml−1 with a PHA content of 2.4 g l−1), and subsequently inoculated with 5.3 ± 0.3 · 107B. bacteriovorus HD100 or 3.7 ± 0.1 · 107 pfu ml−1B. bacteriovorus Bd3709. After 30 h of predation, the extracellular medium was analysed by HPLC-MS. The mcl-PHA hydrolytic product profile for the B. bacteriovorus Bd3709/P. putida KT2440 co-culture - mainly monomers and dimers - was similar to that recorded for P. putida KT2440 growing alone (Fig. 5). However, it differed strongly to that of the B. bacteriovorus HD100/P. putida KT2440 co-cultures, which showed larger proportions of dimers and trimers, similar to that obtained in in vitro experiments using pure PhaZBd depolymerase29. These differences could be attributed to the different activities of the depolymerases produced by P. putida KT2440 (PhaZKT, intracellular) and B. bacteriovorus (PhaZBd, extracellular-like) (see below).


Engineering a predatory bacterium as a proficient killer agent for intracellular bio-products recovery: The case of the polyhydroxyalkanoates.

Martínez V, Herencias C, Jurkevitch E, Prieto MA - Sci Rep (2016)

Mcl-PHA hydrolytic product profile identified in the co-culture supernatants of B. bacteriovorus strains preying on P. putida KT2440 and KT42Z accumulating mcl-PHA.(a) HPLC-MS analysis after 30 h of predation by Bdellovibrio strains. Monomers (white bars), dimers (light grey bars) and trimers (dark grey bars). Control supernatants of P. putida KT2440 and KT42Z are also shown. (b) Total PHA hydrolysis products quantified in the culture supernatants. No significant differences were observed between: (i) the percentage of monomers of the PHA extracted from KT2440 and Bd3709/KT2440, (ii) the percentage of dimers of KT2440 and HD100/KT2440, (iii) the percentage of dimers of KT2440 and Bd3709/KT2440, and (iv) the percentage of dimers of KT2440 and dimers of Bd3709/KT42Z. The rest of the conditions showed significant differences (P < 0.05) determined by ANOVA-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4834554&req=5

f5: Mcl-PHA hydrolytic product profile identified in the co-culture supernatants of B. bacteriovorus strains preying on P. putida KT2440 and KT42Z accumulating mcl-PHA.(a) HPLC-MS analysis after 30 h of predation by Bdellovibrio strains. Monomers (white bars), dimers (light grey bars) and trimers (dark grey bars). Control supernatants of P. putida KT2440 and KT42Z are also shown. (b) Total PHA hydrolysis products quantified in the culture supernatants. No significant differences were observed between: (i) the percentage of monomers of the PHA extracted from KT2440 and Bd3709/KT2440, (ii) the percentage of dimers of KT2440 and HD100/KT2440, (iii) the percentage of dimers of KT2440 and Bd3709/KT2440, and (iv) the percentage of dimers of KT2440 and dimers of Bd3709/KT42Z. The rest of the conditions showed significant differences (P < 0.05) determined by ANOVA-test.
Mentions: According to the above results, the profile of the HAs products should differ depending on the predatory strain used in the co-culture. To examine this, P. putida KT2440 cells were prepared in Hepes buffer at a biomass of 5.7 g l−1 (corresponding to 2.4 ± 0.2 · 107 cfu ml−1 with a PHA content of 2.4 g l−1), and subsequently inoculated with 5.3 ± 0.3 · 107B. bacteriovorus HD100 or 3.7 ± 0.1 · 107 pfu ml−1B. bacteriovorus Bd3709. After 30 h of predation, the extracellular medium was analysed by HPLC-MS. The mcl-PHA hydrolytic product profile for the B. bacteriovorus Bd3709/P. putida KT2440 co-culture - mainly monomers and dimers - was similar to that recorded for P. putida KT2440 growing alone (Fig. 5). However, it differed strongly to that of the B. bacteriovorus HD100/P. putida KT2440 co-cultures, which showed larger proportions of dimers and trimers, similar to that obtained in in vitro experiments using pure PhaZBd depolymerase29. These differences could be attributed to the different activities of the depolymerases produced by P. putida KT2440 (PhaZKT, intracellular) and B. bacteriovorus (PhaZBd, extracellular-like) (see below).

Bottom Line: This work examines the potential of the predatory bacterium Bdellovibrio bacteriovorus HD100, an obligate predator of other Gram-negative bacteria, as an external cell-lytic agent for recovering valuable intracellular bio-products produced by prey cultures.The bio-product targets to be recovered were polyhydroxyalkanoates (PHAs) produced naturally by Pseudomonas putida and Cupriavidus necator, or by recombinant Escherichia coli strains.B. bacteriovorus with a mutated PHA depolymerase gene to prevent the unwanted breakdown of the bio-product allowed the recovery of up to 80% of that accumulated by the prey bacteria, even at high biomass concentrations.

View Article: PubMed Central - PubMed

Affiliation: Environmental Biology Department, Centro de Investigaciones Biológicas, CSIC, C/Ramiro de Maeztu 9, 28040 Madrid, Spain.

ABSTRACT
This work examines the potential of the predatory bacterium Bdellovibrio bacteriovorus HD100, an obligate predator of other Gram-negative bacteria, as an external cell-lytic agent for recovering valuable intracellular bio-products produced by prey cultures. The bio-product targets to be recovered were polyhydroxyalkanoates (PHAs) produced naturally by Pseudomonas putida and Cupriavidus necator, or by recombinant Escherichia coli strains. B. bacteriovorus with a mutated PHA depolymerase gene to prevent the unwanted breakdown of the bio-product allowed the recovery of up to 80% of that accumulated by the prey bacteria, even at high biomass concentrations. This innovative downstream process highlights how B. bacteriovorus can be used as a novel, biological lytic agent for the inexpensive, industrial scale recovery of intracellular products from different Gram-negative prey cultures.

No MeSH data available.


Related in: MedlinePlus