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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

B. bacteriovorus HD100 preying on high cell densities of P. putida KT2440 accumulating mcl-PHA.(a) Phase-contrast microscopy of a co-culture of B. bacteriovorus HD100 preying on P. putida KT2440 at the onset of predation (time zero) and (b) after 40 h of incubation. (c,d) 1:100 dilution of the co-cultures from panels (a,b), respectively. Mcl-PHA granules can be observed in the extracellular medium after 40 h of predation. (e) Cell viability assay of the co-culture of B. bacteriovorus HD100/P. putida KT2440 (white bars and black bars, respectively) at the onset of predation (time zero) and within 40 h. (f) Total PHA content in the co-culture of B. bacteriovorus HD100/P. putida KT2440 compared to the control culture (KT2440 without the predator) at the onset of predation (time zero) (white bars) and within 40 h (black bars). Asterisks (*) indicate significant differences (*P < 0.05).
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f3: B. bacteriovorus HD100 preying on high cell densities of P. putida KT2440 accumulating mcl-PHA.(a) Phase-contrast microscopy of a co-culture of B. bacteriovorus HD100 preying on P. putida KT2440 at the onset of predation (time zero) and (b) after 40 h of incubation. (c,d) 1:100 dilution of the co-cultures from panels (a,b), respectively. Mcl-PHA granules can be observed in the extracellular medium after 40 h of predation. (e) Cell viability assay of the co-culture of B. bacteriovorus HD100/P. putida KT2440 (white bars and black bars, respectively) at the onset of predation (time zero) and within 40 h. (f) Total PHA content in the co-culture of B. bacteriovorus HD100/P. putida KT2440 compared to the control culture (KT2440 without the predator) at the onset of predation (time zero) (white bars) and within 40 h (black bars). Asterisks (*) indicate significant differences (*P < 0.05).

Mentions: A requirement for the industrial scale use of B. bacteriovorus as a living, cell-lytic system would be the ability of the predator to attack high cell density prey cultures. Therefore, the potential of B. bacteriovorus HD100 to prey on high cell density cultures of P. putida KT2440 accumulating PHA was tested. For this, prey cultures were prepared in Hepes buffer with a cell biomass of 30.5 g l−1 (biomass production by PHA-producing pseudomonads under optimal conditions ranges between 15–55 g l−1)30, corresponding to 8.3 ± 0.3 · 109 colony-forming units [(cfu) ml−1], with a PHA content of 15.1 g l−1 (Fig. 3a,c). These cultures were then inoculated with 6.3 ± 0.3 · 108 plaque-forming units (pfu) ml−1 of B. bacteriovorus HD100. After 40 h of predation, a 1-log reduction in prey cells was observed while an increase of 1-log in the viable cell number of B. bacteriovorus was measured (Fig. 3e). This confirms the predator’s ability to prey on high-density cultures of PHA-accumulating P. putida KT2440. Examination of the co-cultures by phase-contrast microscopy clearly revealed the release of PHA granules into the extracellular medium (Fig. 3b,d). Although optimization of PHA recovery is required at this cell density scale, 65% of the PHA accumulated by the prey bacteria was recovered under our lab scale conditions (Fig. 3f). Notably, the polymer was directly extractable from the wet biomass of the co-cultures.


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)

B. bacteriovorus HD100 preying on high cell densities of P. putida KT2440 accumulating mcl-PHA.(a) Phase-contrast microscopy of a co-culture of B. bacteriovorus HD100 preying on P. putida KT2440 at the onset of predation (time zero) and (b) after 40 h of incubation. (c,d) 1:100 dilution of the co-cultures from panels (a,b), respectively. Mcl-PHA granules can be observed in the extracellular medium after 40 h of predation. (e) Cell viability assay of the co-culture of B. bacteriovorus HD100/P. putida KT2440 (white bars and black bars, respectively) at the onset of predation (time zero) and within 40 h. (f) Total PHA content in the co-culture of B. bacteriovorus HD100/P. putida KT2440 compared to the control culture (KT2440 without the predator) at the onset of predation (time zero) (white bars) and within 40 h (black bars). Asterisks (*) indicate significant differences (*P < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: B. bacteriovorus HD100 preying on high cell densities of P. putida KT2440 accumulating mcl-PHA.(a) Phase-contrast microscopy of a co-culture of B. bacteriovorus HD100 preying on P. putida KT2440 at the onset of predation (time zero) and (b) after 40 h of incubation. (c,d) 1:100 dilution of the co-cultures from panels (a,b), respectively. Mcl-PHA granules can be observed in the extracellular medium after 40 h of predation. (e) Cell viability assay of the co-culture of B. bacteriovorus HD100/P. putida KT2440 (white bars and black bars, respectively) at the onset of predation (time zero) and within 40 h. (f) Total PHA content in the co-culture of B. bacteriovorus HD100/P. putida KT2440 compared to the control culture (KT2440 without the predator) at the onset of predation (time zero) (white bars) and within 40 h (black bars). Asterisks (*) indicate significant differences (*P < 0.05).
Mentions: A requirement for the industrial scale use of B. bacteriovorus as a living, cell-lytic system would be the ability of the predator to attack high cell density prey cultures. Therefore, the potential of B. bacteriovorus HD100 to prey on high cell density cultures of P. putida KT2440 accumulating PHA was tested. For this, prey cultures were prepared in Hepes buffer with a cell biomass of 30.5 g l−1 (biomass production by PHA-producing pseudomonads under optimal conditions ranges between 15–55 g l−1)30, corresponding to 8.3 ± 0.3 · 109 colony-forming units [(cfu) ml−1], with a PHA content of 15.1 g l−1 (Fig. 3a,c). These cultures were then inoculated with 6.3 ± 0.3 · 108 plaque-forming units (pfu) ml−1 of B. bacteriovorus HD100. After 40 h of predation, a 1-log reduction in prey cells was observed while an increase of 1-log in the viable cell number of B. bacteriovorus was measured (Fig. 3e). This confirms the predator’s ability to prey on high-density cultures of PHA-accumulating P. putida KT2440. Examination of the co-cultures by phase-contrast microscopy clearly revealed the release of PHA granules into the extracellular medium (Fig. 3b,d). Although optimization of PHA recovery is required at this cell density scale, 65% of the PHA accumulated by the prey bacteria was recovered under our lab scale conditions (Fig. 3f). Notably, the polymer was directly extractable from the wet biomass of the co-cultures.

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