Abstract
Polyhydroxyalkanoate (PHA) inclusions are polymeric storage inclusions formed in some bacterial species when carbon levels are high but levels of another essential nutrient, such as nitrogen, are low. Though much is known about PHA synthesis, little is known about inclusion structure. In this study, atomic force microscopy (AFM) was employed to elucidate the structure of PHA inclusions at the nanoscale level, including the characterization of different layers of structure. AFM data suggest that underneath the inclusion envelope, there is a 2- to 4-nm-thick network layer that resides on top of a harder layer that is likely to be a crystalline lamellar polymer. The network is comprised of approximately 20-nm-wide linear segments and junctions that are typically formed by the joining of three to four of the linear segments. In some cases, approximately 50-nm globular structures that are raised approximately 1 to 2 nm above the network are present at the junctions. These globular structures always have a central pore that is approximately 15 nm in diameter. To determine if the major surface protein of PHA inclusions, PhaP, is involved in the structure of this network, inclusions from Cupriavidus necator H16 DeltaphaP were examined. No network structure was detected. Instead, apparently random globular structures were found on the surfaces of the inclusions. When PhaP levels were reconstituted in this strain by the addition of phaP on a plasmid, the network was also reconstituted, albeit in a slightly different arrangement from that of the wild-type network. We conclude that PhaP participates in the formation of the inclusion network.