Poly(β-hydroxybutyrate) extrusion from pleomorphic cells ofAzotobacter vinelandiiUWD

Abstract

Azotobacter vinelandii UWD cells fill with up to 80% (per dry mass) poly(β-hydroxybutyrate) (PHB) after 24 h growth in medium containing sugars and fish peptone. However, peptones were not usually added to Azotobacter culture as they induced pleomorphism and compromised cell wall strength. This study examines the morphology of these PHB-producing pleomorphic cells in the transmission electron microscope. PHB-producing cells incubated for 18–24 h were most frequently 2–3 μm diameter spheres containing up to 20 PHB inclusions/cross section, or a calculated ≈ 100 inclusions/cell volume. These inclusions tended to be of small size (≈ 0.5 μm diameter) and became fewer and larger in older cells. The most striking feature of these pleomorphic cells was the apparent extrusion of polymer from the cells. It is unlikely that PHB extrusion is an active process from a viable cell as there was considerable cell wall damage at the point of polymer extrusion. The results suggest that the extrusion of PHB may be the result of polymer expansion, caused by the dehydration of the specimen for transmission electron microscopy, coupled with the inability of the pleomorphic cell wall to retain the expanding polymer. Thus, freeze-substituted sections of similar cells that were prepared without chemical dehydration did not extrude PHB. However, lysed cells prepared for transmission electron microscopy by chemical dehydration also did not extrude PHB, which suggests differences in the fluidity of the PHB in intact cell inclusions and lysed cell granules.Key words: poly(β-hydroxybutyrate), inclusions, polymer expansion, dehydration artifact.