Investigation of the phase morphology of bacterial PHA inclusion bodies by contrast variation SANS

• Published in: Physica. B, Condensed matter Condensed matter, 2006-11, Vol.385, p.859-861
• Main Authors: Russell, R.A., Holden, P.J., Garvey, C.J., Wilde, K.L., Hammerton, K.M., Foster, L.J.
• Format: Article
• Language: English
• Subjects: Biodeuteration; Biological and medical sciences; Contrast variation; Fundamental and applied biological sciences. Psychology; General aspects; Molecular biophysics; Polyhydroxyalkanoate
• ISSN: 0921-4526; 1873-2135
• DOI: 10.1016/j.physb.2006.05.126

Under growth-limiting conditions, many bacteria are able to metabolise excess organic acids into polyhydroxyalkanoates (PHA) and store these polymers as intracellular inclusions until the return of favourable conditions. Various models have been proposed for the macromolecular organisation of the boundary layer surrounding the polymer, and contrast-variation small-angle neutron scattering (SANS) was used to study its organisation. Inclusions formed by Pseudomonas oleovorans under hydrogenating conditions showed lowest scattering intensity at ca. 20% D 2O. The inclusions consist of protein and membrane lipids in the boundary layer and polyhydroxyoctanoate (lipid) in the inclusion body. At 20% D 2O the contributions of lipids were contrast matched with the solvent, indicating that lipids contributed the bulk of the scattering intensity observed at other D 2O/H 2O ratios. These results are inconsistent with a model of the boundary layer which proposed outer and inner layers of crystalline protein lattice sandwiching a membrane lipid membrane layer [E.S. Stuart, R.W. Lenz, R.C. Fuller, Can J Microbiol 41(Suppl 1) (1995) 84–93], and is more consistent with a model consisting of a lipid monolayer containing embedded proteins [U. Pieper-furst, M.H. Madkour, F. Mayer, A. Steinbuchel, J. Bacteriol. 176 (1994) 4328–4337.] By altering the H/D content of the precursors, we were able to collect SANS data from preparations of both deuterated and H/D copolymer inclusions, where initial PHA produced was hydrogenated followed by deuteration. Deuterated inclusions showed minimum intensity above 90% D 2O/H 2O whereas the sequentially produced copolymer (assumed to be in a core/shell arrangement) displayed minimum scattering some 20% lower, which is consistent with the increased hydrogenation of the boundary layer expected from its synthesis during supply of hydrogenated followed by deuterated precursors.