POLY(3-HYDROXYBUTYRATE) IS ASSOCIATED WITH SPECIFIC PROTEINS IN THE CYTOPLASM AND MEMBRANES OF ESCHERICHIA-COLI

Poly(3-hydroxybutyrate) (PHB) is well-known as a high molecular weight homopolymer of R-3-hydroxybutyrate which accumulates in storage granu les within the cytosols of certain bacteria. Escherichia coli does not amass these granules; however, small amounts of low molecular weight PHB (<0.02% of dry weight) have been found in the plasma membranes in complexes with calcium polyphosphate; the complexes serve as voltage-a ctivated calcium channels. Here we report that polyphosphate-complexed PHB is only a minor fraction of the polyester in E. coli. PRE compris es 0.36 to 0.55% of the dry weight of log-phase cells, depending on cu lture medium, and this amount increases by 15 to 20% when the cells ar e made genetically competent. The PHB is widely distributed throughout the cell, wherein it is primarily associated with proteins. The ident ity of protein-associated PHB was established by antibody reaction, ch emical assay, and H-1 NMR spectroscopy. As expected, the physical and chemical properties of protein-associated PHB were found to be conside rably different from those of the bulk polymer or granule PHB, e.g. pr otein-PHB complexes are normally insoluble in chloroform, soluble in w ater and alkaline hypochlorite, and are converted to crotonic acid mor e slowly on heating in concentrated sulfuric acid. Our studies indicat e that the majority of cellular PHB (over 80%) is located in cytoplasm ic proteins, especially proteins of the ribosomal fraction. Western im munoblots, probed with polyclonal anti-PHB IgG, revealed a number of P HB-polypeptides having a wide range of molecular weights in all cell f ractions. These results suggest that PHB is a fundamental constituent of cells that may have physiological functions in addition to facilita ting ion transmembrane transport or serving as a carbon reserve.