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Screening and Characterization of Polyhydroxyalkanoate and other Granules in Cyanobacteria

  • Author(s): Hong, Karl Bryant
  • Advisor(s): Palenik, Brian
  • et al.
Abstract

Using Nile Red and Bodipy 493/503 dyes and fluorescence microscopy, twenty cyanobacterial strains were screened for the accumulation of potentially biotechnologically significant granules such as polyhydroxybutyrate (PHB). Six of

these strains showed such granules, including the five recent isolates, Synechocystis spp. WHSYN, LSNM, and CGF-1, Nodularia sp. Las Olas, and Phormidium-like sp. CGFILA, as well as Phormidium cf. irriguum CCALA 759. Characterizations of these granules reveal that they belong to three distinct classes of cyanobacterial carbon storage compounds. Synechocystis, spp. WHSYN, LSNM, and CGF-1, and Phormidium-like sp. CGFILA produce polyhydroxyalkanoate (PHA) granules according to PHA synthase gene (phaC ) PCR screening and 1H NMR analyses. Maximum-likelihood analyses and co-phylogenetic modeling of PHA synthase gene sequences provide evidence of a recent horizontal gene transfer event between distant genera of cyanobacteria. The data set includes the first genetic characterization of a PHA biosynthesis gene from the genus Phormidium, and it also reveals that phaC is highly conserved within the genus Synechocystis.

Another strain, Phormidium cf. irriguum CCALA 759 was found to not produce PHA, but produce staining granules possibly comprised of hexadecanoic and octadecanoic fatty acids (84.7% and 75.8% probability NIST matches, respectively), as well as alkanes which have tentatively been identified as 8-heptadecene via gas chromatography-mass spectrometry (two peaks, 15.8% and 18.6% probability NIST matches).

A third granule type was found in Nodularia sp. Las Olas and appears to be poly-amino acids comprised mainly of arginine and aspartate/asparagine residues, according to Fourier-transform infrared spectroscopy and high performance liquid chromatography analyses. This poly-amino acid material exhibits staining and solubility characteristics that are different from cyanophycin controls.

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