Multiple evidence for widespread and general occurrence of type-III PHA synthases in cyanobacteria and molecular characterization of the PHA synthases from two thermophilic cyanobacteria: Chlorogloeopsis fritschii PCC 6912 and Synechococcus sp strain MA19

Eleven different cyanobacteria were investigated with respect to their capa bilities to synthesize poly-3-hydroxybutyrate [poly(3HB)] and the type of p oly-beta -hydroxyalkanoic acid (PHA) synthase accounting for the synthesis of this polyester. Several methods, including (i) Southern blot analysis us ing a phaC-specific DNA probe, (ii) Western blot analysis using specific po lyclonal anti-PhaE antibodies raised in this study against PhaE of Synechoc ystis sp. strain PCC 6803, (iii) generation and sequence analysis of PCR pr oducts using phaC-specific oligonucleotides as primers, and/or (iv) cloning and sequence analysis of PHA synthase structural genes, were used to provi de evidence for the presence of a type-III PHA synthase in the following cy anobacteria: Synechococcus sp. strains MA19 and PCC 6715, Chlorogloeopsis f ritschii PCC 6912, Anabaena cylindrica SAG 1403-2, Cyanothece sp. strains P CC 7424, PCC 8303 and PCC 8801, and Gloeocapsa sp. strain PCC 7428. The scr eening was compared with corresponding studies using crude protein extracts and genomic DNA of Synechocystis sp. strain PCC 6803, as a positive contro l, which is so far the only cyanobacterium for which molecular data of the PHA synthase genes are available. No evidence for the presence of a type-II I PHA synthase could be obtained for only three of the eleven investigated cyanobacteria (Stanieria sp. strain PCC 7437, Cyanothece sp. strain PCC 895 5 and Gloeothece sp. strain PCC 6501). The entire PHA synthase structural g enes of the two thermophilic cyanobacteria Synechococcus sp. strain MA19 an d Chlorogloeopsis fritschii PCC 6912, and in addition a central region of t he phaC gene of Cyanothece sp. strain PCC 8303, were cloned, sequenced and also heterologously expressed in Escherichia coli.