Limits...
Influence of growth stage on activities of polyhydroxyalkanoate (PHA) polymerase and PHA depolymerase in Pseudomonas putida U.

Ren Q, de Roo G, Witholt B, Zinn M, Thöny-Meyer L - BMC Microbiol. (2010)

Bottom Line: In addition, it was found that after eliminating phasins (PhaF and PhaI) from the granules PhaC activity decreased further.It was found that in P. putida PhaC and PhaZ are concomitantly active, resulting in parallel synthesis and degradation of PHA.Moreover PhaC activity was found to be decreased, whereas PhaZ activity increased during growth.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Biomaterials, Swiss Federal Laboratories for Materials Science and Technology (Empa), CH-9014 St, Gallen, Switzerland. qun.ren@empa.ch

ABSTRACT

Background: Medium chain length (mcl-) polyhydroxyalkanoates (PHA) are synthesized by many bacteria in the cytoplasm as storage compounds for energy and carbon. The key enzymes for PHA metabolism are PHA polymerase (PhaC) and depolymerase (PhaZ). Little is known of how mcl-PHA accumulation and degradation are controlled. It has been suggested that overall PHA metabolism is regulated by the β-oxidation pathway of which the flux is governed by intracellular ratios of [NADH]/[NAD] and [acetyl-CoA]/[CoA]. Another level of control could relate to modulation of the activities of PhaC and PhaZ. In order to investigate the latter, assays for in vitro activity measurements of PhaC and PhaZ in crude cell extracts are necessary.

Results: Two in vitro assays were developed which allow the measurement of PhaC and PhaZ activities in crude cell extracts of Pseudomonas putida U. Using the assays, it was demonstrated that the activity of PhaC decreased 5-fold upon exponential growth on nitrogen limited medium and octanoate. In contrast, the activity of PhaZ increased only 1.5-fold during growth. One reason for the changes in the enzymatic activity of PhaC and PhaZ could relate to a change in interaction with the phasin surface proteins on the PHA granule. SDS-PAGE analysis of isolated PHA granules demonstrated that during growth, the ratio of [phasins]/[PHA] decreased. In addition, it was found that after eliminating phasins (PhaF and PhaI) from the granules PhaC activity decreased further.

Conclusion: Using the assays developed in this study, we followed the enzymatic activities of PhaC and PhaZ during growth and correlated them to the amount of phasins on the PHA granules. It was found that in P. putida PhaC and PhaZ are concomitantly active, resulting in parallel synthesis and degradation of PHA. Moreover PhaC activity was found to be decreased, whereas PhaZ activity increased during growth. Availability of phasins on PHA granules affected the activity of PhaC.

Show MeSH

Related in: MedlinePlus

Production of 3-hydroxyalkanoic acid in crude cell extracts of P. putida U and P. putida U::phaZ-. Cells grown to the stationary phase (16 h in 0.2NE2 medium + 15 mM octanoate) were harvested, resuspended to 1 mg total protein/ml in 100 mM Tris-HCl, pH 8, 0.5 mM MgCl2, and lysed by three passages through a French pressure cell. The production of PHA monomers was followed for P. putida U::phaZ- (filled triangle) and P. putida U (open triangle). Supernatants (250 μl) containing 3-hydroxyalkanoic acids were lyophilyzed and methanolyzed prior to analysis by GC. Data represent the average of two measurements.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2959000&req=5

Figure 2: Production of 3-hydroxyalkanoic acid in crude cell extracts of P. putida U and P. putida U::phaZ-. Cells grown to the stationary phase (16 h in 0.2NE2 medium + 15 mM octanoate) were harvested, resuspended to 1 mg total protein/ml in 100 mM Tris-HCl, pH 8, 0.5 mM MgCl2, and lysed by three passages through a French pressure cell. The production of PHA monomers was followed for P. putida U::phaZ- (filled triangle) and P. putida U (open triangle). Supernatants (250 μl) containing 3-hydroxyalkanoic acids were lyophilyzed and methanolyzed prior to analysis by GC. Data represent the average of two measurements.

Mentions: Similar to PHA polymerases, characterization of intracellular mcl-PHA depolymerases (PhaZ) under different physiological conditions has been hampered due to the lack of a suitable in vitro activity assay that can be used in crude cell extracts. An easy assay for determining PhaZ activity has been reported by monitoring the pH changes caused by the release of 3-hydroxy fatty acid monomers [25], however, it is only suitable for depolymerase activity measurements from purified PHA granules. Here, a depolymerase assay was developed in which the release of 3-hydroxy fatty acid monomers is quantified directly. The released monomers were separated from the insoluble polymer and other cell material by centrifugation and were subsequently methanolyzed to yield volatile methyl-esters which was measured by GC analysis. Upon incubation of a crude extract of P. putida U (which had been grown on octanoate) in Tris-HCl buffer, almost linear increases of 3-hydroxyoctanoate, and to a minor extent 3-hydroxyhexanoate, were observed. Figure 2 shows the total amount of 3-hydroxy fatty acids released over time.


Influence of growth stage on activities of polyhydroxyalkanoate (PHA) polymerase and PHA depolymerase in Pseudomonas putida U.

Ren Q, de Roo G, Witholt B, Zinn M, Thöny-Meyer L - BMC Microbiol. (2010)

Production of 3-hydroxyalkanoic acid in crude cell extracts of P. putida U and P. putida U::phaZ-. Cells grown to the stationary phase (16 h in 0.2NE2 medium + 15 mM octanoate) were harvested, resuspended to 1 mg total protein/ml in 100 mM Tris-HCl, pH 8, 0.5 mM MgCl2, and lysed by three passages through a French pressure cell. The production of PHA monomers was followed for P. putida U::phaZ- (filled triangle) and P. putida U (open triangle). Supernatants (250 μl) containing 3-hydroxyalkanoic acids were lyophilyzed and methanolyzed prior to analysis by GC. Data represent the average of two measurements.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2959000&req=5

Figure 2: Production of 3-hydroxyalkanoic acid in crude cell extracts of P. putida U and P. putida U::phaZ-. Cells grown to the stationary phase (16 h in 0.2NE2 medium + 15 mM octanoate) were harvested, resuspended to 1 mg total protein/ml in 100 mM Tris-HCl, pH 8, 0.5 mM MgCl2, and lysed by three passages through a French pressure cell. The production of PHA monomers was followed for P. putida U::phaZ- (filled triangle) and P. putida U (open triangle). Supernatants (250 μl) containing 3-hydroxyalkanoic acids were lyophilyzed and methanolyzed prior to analysis by GC. Data represent the average of two measurements.
Mentions: Similar to PHA polymerases, characterization of intracellular mcl-PHA depolymerases (PhaZ) under different physiological conditions has been hampered due to the lack of a suitable in vitro activity assay that can be used in crude cell extracts. An easy assay for determining PhaZ activity has been reported by monitoring the pH changes caused by the release of 3-hydroxy fatty acid monomers [25], however, it is only suitable for depolymerase activity measurements from purified PHA granules. Here, a depolymerase assay was developed in which the release of 3-hydroxy fatty acid monomers is quantified directly. The released monomers were separated from the insoluble polymer and other cell material by centrifugation and were subsequently methanolyzed to yield volatile methyl-esters which was measured by GC analysis. Upon incubation of a crude extract of P. putida U (which had been grown on octanoate) in Tris-HCl buffer, almost linear increases of 3-hydroxyoctanoate, and to a minor extent 3-hydroxyhexanoate, were observed. Figure 2 shows the total amount of 3-hydroxy fatty acids released over time.

Bottom Line: In addition, it was found that after eliminating phasins (PhaF and PhaI) from the granules PhaC activity decreased further.It was found that in P. putida PhaC and PhaZ are concomitantly active, resulting in parallel synthesis and degradation of PHA.Moreover PhaC activity was found to be decreased, whereas PhaZ activity increased during growth.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Biomaterials, Swiss Federal Laboratories for Materials Science and Technology (Empa), CH-9014 St, Gallen, Switzerland. qun.ren@empa.ch

ABSTRACT

Background: Medium chain length (mcl-) polyhydroxyalkanoates (PHA) are synthesized by many bacteria in the cytoplasm as storage compounds for energy and carbon. The key enzymes for PHA metabolism are PHA polymerase (PhaC) and depolymerase (PhaZ). Little is known of how mcl-PHA accumulation and degradation are controlled. It has been suggested that overall PHA metabolism is regulated by the β-oxidation pathway of which the flux is governed by intracellular ratios of [NADH]/[NAD] and [acetyl-CoA]/[CoA]. Another level of control could relate to modulation of the activities of PhaC and PhaZ. In order to investigate the latter, assays for in vitro activity measurements of PhaC and PhaZ in crude cell extracts are necessary.

Results: Two in vitro assays were developed which allow the measurement of PhaC and PhaZ activities in crude cell extracts of Pseudomonas putida U. Using the assays, it was demonstrated that the activity of PhaC decreased 5-fold upon exponential growth on nitrogen limited medium and octanoate. In contrast, the activity of PhaZ increased only 1.5-fold during growth. One reason for the changes in the enzymatic activity of PhaC and PhaZ could relate to a change in interaction with the phasin surface proteins on the PHA granule. SDS-PAGE analysis of isolated PHA granules demonstrated that during growth, the ratio of [phasins]/[PHA] decreased. In addition, it was found that after eliminating phasins (PhaF and PhaI) from the granules PhaC activity decreased further.

Conclusion: Using the assays developed in this study, we followed the enzymatic activities of PhaC and PhaZ during growth and correlated them to the amount of phasins on the PHA granules. It was found that in P. putida PhaC and PhaZ are concomitantly active, resulting in parallel synthesis and degradation of PHA. Moreover PhaC activity was found to be decreased, whereas PhaZ activity increased during growth. Availability of phasins on PHA granules affected the activity of PhaC.

Show MeSH
Related in: MedlinePlus