Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
AMB Express. 2012 Apr 26;2(1):26. doi: 10.1186/2191-0855-2-26.
Polyhydroxyalkanoates (PHA) are biodegradable polymers that are attractive materials for use in tissue engineering and medical device manufacturing. Ralstonia eutropha is regarded as the model organism for PHA biosynthesis. We examined the effects of PHA depolymerase (PhaZ) expression on PHA homeostasis in R. eutropha strains. In order to analyze the impact of PhaZs on R. eutropha granule architecture, we performed electron microscopy on several phaZ knockout strains and the wild type strain grown under PHA production conditions. Analysis of the acquired micrographs was based on stereology: the ratio of granule area and cell area was determined, along with total granule count per full-size cell image. Cells bearing a phaZ2 knockout mutation alone or in conjunction with a phaZ1 mutation were found to have a high granule volume per cell volume and a higher granule count compared to wild type. A phaZ quadruple knockout strain appeared to have a low granule volume per cell volume and a low granule count per cell. Cells bearing a phaZ3 knockout were found to have a higher granule count than the wild type, whereas granule volume per cell volume was similar. Accordingly, we hypothesize that PhaZs have not only an impact on PHA degradation but also on the 3-dimensional granule architecture. Based on our data, PhaZ2 is postulated to affect granule density. This work increased our knowledge about PHA depolymerases in R. eutropha, including enzymes that had previously been uncharacterized.
聚羟基烷酸酯（PHA）是可生物降解的聚合物，是组织工程和医疗器械制造的有吸引力的材料。恶臭假单胞菌被认为是 PHA 生物合成的模式生物。我们研究了 PHA 解聚酶（PhaZ）表达对恶臭假单胞菌菌株中 PHA 动态平衡的影响。为了分析 PhaZs 对恶臭假单胞菌颗粒结构的影响，我们对几种 phaZ 敲除菌株和在 PHA 生产条件下生长的野生型菌株进行了电子显微镜观察。对获得的显微照片的分析基于体视学：确定颗粒面积与细胞面积的比值，以及每个完整大小细胞图像中的总颗粒数。单独携带 phaZ2 缺失突变或与 phaZ1 突变结合的细胞被发现具有每细胞体积的高颗粒体积和较高的颗粒数与野生型相比。phaZ 四重敲除菌株似乎具有每细胞体积的低颗粒体积和每细胞的低颗粒数。携带 phaZ3 缺失突变的细胞被发现比野生型具有更高的颗粒数，而每细胞体积的颗粒体积相似。因此，我们假设 PhaZs 不仅对 PHA 降解有影响，而且对 3 维颗粒结构有影响。基于我们的数据，推测 PhaZ2 影响颗粒密度。这项工作增加了我们对恶臭假单胞菌中 PHA 解聚酶的了解，包括以前未表征的酶。
