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Controlled autolysis facilitates the polyhydroxyalkanoate recovery in Pseudomonas putida KT2440.

Martínez V, García P, García JL, Prieto MA - Microb Biotechnol (2011)

Bottom Line: The development of efficient recovery processes is essential to reduce the cost of polyhydroxyalkanoates (PHAs) production.Our results demonstrate that the intracellular presence of PHA granules confers resistance to cell envelope.Conditions to control the cell autolysis in P. putida BXHL in terms of optimal fermentation, PHA content and PHA recovery have been set up by exploring the sensitivity to detergents, chelating agents and wet biomass solubility in organic solvents such as ethyl acetate.

View Article: PubMed Central - PubMed

Affiliation: Environmental Biology Department, Centro de Investigaciones Biológicas, Madrid, Spain.

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Monitoring the viability of P. putida KTHL population as function of the membrane integrity. LIVE/DEAD bacterial viability assay of KTHL cells grown in LB in the presence (panels C and D) and absence (panels A and B) of 3MB.
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f6: Monitoring the viability of P. putida KTHL population as function of the membrane integrity. LIVE/DEAD bacterial viability assay of KTHL cells grown in LB in the presence (panels C and D) and absence (panels A and B) of 3MB.

Mentions: Cell viability and biomass production data derived from the results described above were in agreement with those obtained in P. putida KTHL cells growing in rich medium (Luria–Bertani, LB) in the presence of 3MB, which showed, under the phase contrast microscope, a majority of ‘ghost cells’ (Fig. 5). Morphology of these ‘ghosts’ is a typical consequence of holin action, where the cells are still keeping their shape but lack their cytoplasmic content and, consequently, there is a significant drop in viability when 3MB is added to the medium. This was demonstrated by the viability analyses as function of membrane integrity by staining with the LIVE/DEAD BacLight bacterial viability kit (Fig. 6). In this assay, cells with a compromised membrane, which are considered to be dead or dying, stained red (Fig. 6B and D), whereas cells with an intact membrane stained green (Fig. 6A and C). Figure 6 showed a drastic effect on the green/red cell ratio after the addition of the lytic inducer. The cell viability was also affected in the presence of 3MB when cells were grown under PHA production conditions (Tables 2 and 3). These findings confirmed that when the ejh‐ejl cassette was expressed, it caused the death of most of the cells but they were not totally disrupted, probably as a result of an intact outer membrane or a reduced efficiency of the phage endolysin. Thus, to improve the yield of the extraction procedure we tested the sensitivity of the strains to detergents or chelating agents in rich medium. To this aim, P. putida KTHL and wild‐type cells were incubated for 18 h at 30°C in LB medium and in LB medium supplemented with 0.075% (w/v) deoxycholate (DOC) (Fig. 7), 0.01% SDS or 0.2 mM EDTA (data not shown). At these concentrations, the 3MB induced P. putida KTHL cells were sensitive to the chemical agents, while no effect was observed on the growth of the wild‐type strain or the non‐induced recombinant strain. In addition, we have determined that DOC, SDS and EDTA did not alter the growth of the 3MB induced P. putida KTHL cells when they were added to LB medium at final concentrations lower than 0.01%, 0.005% and 0.1 mM respectively. Besides, we also determined that the addition of concentrations higher than 0.2% DOC and 0.4 mM EDTA changed the growth of both, the wild type and the non‐induced KTHL strains. All the above results clearly indicated that when the cells grow in a rich medium the cell envelope of the P. putida KTHL mutant strain is significantly altered as a result of the expression of the Ejh and Ejl proteins.


Controlled autolysis facilitates the polyhydroxyalkanoate recovery in Pseudomonas putida KT2440.

Martínez V, García P, García JL, Prieto MA - Microb Biotechnol (2011)

Monitoring the viability of P. putida KTHL population as function of the membrane integrity. LIVE/DEAD bacterial viability assay of KTHL cells grown in LB in the presence (panels C and D) and absence (panels A and B) of 3MB.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815265&req=5

f6: Monitoring the viability of P. putida KTHL population as function of the membrane integrity. LIVE/DEAD bacterial viability assay of KTHL cells grown in LB in the presence (panels C and D) and absence (panels A and B) of 3MB.
Mentions: Cell viability and biomass production data derived from the results described above were in agreement with those obtained in P. putida KTHL cells growing in rich medium (Luria–Bertani, LB) in the presence of 3MB, which showed, under the phase contrast microscope, a majority of ‘ghost cells’ (Fig. 5). Morphology of these ‘ghosts’ is a typical consequence of holin action, where the cells are still keeping their shape but lack their cytoplasmic content and, consequently, there is a significant drop in viability when 3MB is added to the medium. This was demonstrated by the viability analyses as function of membrane integrity by staining with the LIVE/DEAD BacLight bacterial viability kit (Fig. 6). In this assay, cells with a compromised membrane, which are considered to be dead or dying, stained red (Fig. 6B and D), whereas cells with an intact membrane stained green (Fig. 6A and C). Figure 6 showed a drastic effect on the green/red cell ratio after the addition of the lytic inducer. The cell viability was also affected in the presence of 3MB when cells were grown under PHA production conditions (Tables 2 and 3). These findings confirmed that when the ejh‐ejl cassette was expressed, it caused the death of most of the cells but they were not totally disrupted, probably as a result of an intact outer membrane or a reduced efficiency of the phage endolysin. Thus, to improve the yield of the extraction procedure we tested the sensitivity of the strains to detergents or chelating agents in rich medium. To this aim, P. putida KTHL and wild‐type cells were incubated for 18 h at 30°C in LB medium and in LB medium supplemented with 0.075% (w/v) deoxycholate (DOC) (Fig. 7), 0.01% SDS or 0.2 mM EDTA (data not shown). At these concentrations, the 3MB induced P. putida KTHL cells were sensitive to the chemical agents, while no effect was observed on the growth of the wild‐type strain or the non‐induced recombinant strain. In addition, we have determined that DOC, SDS and EDTA did not alter the growth of the 3MB induced P. putida KTHL cells when they were added to LB medium at final concentrations lower than 0.01%, 0.005% and 0.1 mM respectively. Besides, we also determined that the addition of concentrations higher than 0.2% DOC and 0.4 mM EDTA changed the growth of both, the wild type and the non‐induced KTHL strains. All the above results clearly indicated that when the cells grow in a rich medium the cell envelope of the P. putida KTHL mutant strain is significantly altered as a result of the expression of the Ejh and Ejl proteins.

Bottom Line: The development of efficient recovery processes is essential to reduce the cost of polyhydroxyalkanoates (PHAs) production.Our results demonstrate that the intracellular presence of PHA granules confers resistance to cell envelope.Conditions to control the cell autolysis in P. putida BXHL in terms of optimal fermentation, PHA content and PHA recovery have been set up by exploring the sensitivity to detergents, chelating agents and wet biomass solubility in organic solvents such as ethyl acetate.

View Article: PubMed Central - PubMed

Affiliation: Environmental Biology Department, Centro de Investigaciones Biológicas, Madrid, Spain.

Show MeSH
Related in: MedlinePlus