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 capabilities to synthesize poly-3-hydroxybutyrate [poly(3HB)] and the type of poly-beta -hydroxyalkanoic acid (PHA) synthase accounting for the synthesis of this polyester. Several methods, including (i) Southern blot analysis using a phaC-specific DNA probe, (ii) Western blot analysis using specific polyclonal anti-PhaE antibodies raised in this study against PhaE of Synechocystis sp. strain PCC 6803, (iii) generation and sequence analysis of PCR products using phaC-specific oligonucleotides as primers, and/or (iv) cloning and sequence analysis of PHA synthase structural genes, were used to provide evidence for the presence of a type-III PHA synthase in the following cyanobacteria: Synechococcus sp. strains MA19 and PCC 6715, Chlorogloeopsis fritschii PCC 6912, Anabaena cylindrica SAG 1403-2, Cyanothece sp. strains PCC 7424, PCC 8303 and PCC 8801, and Gloeocapsa sp. strain PCC 7428. The screening was compared with corresponding studies using crude protein extracts and genomic DNA of Synechocystis sp. strain PCC 6803, as a positive control, 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-III PHA synthase could be obtained for only three of the eleven investigated cyanobacteria (Stanieria sp. strain PCC 7437, Cyanothece sp. strain PCC 8955 and Gloeothece sp. strain PCC 6501). The entire PHA synthase structural genes of the two thermophilic cyanobacteria Synechococcus sp. strain MA19 and Chlorogloeopsis fritschii PCC 6912, and in addition a central region of the phaC gene of Cyanothece sp. strain PCC 8303, were cloned, sequenced and also heterologously expressed in Escherichia coli.