CELL SURFACE REACTIVITY OF THE CYANOBACTERIA CALOTHRIX SP

In this study, acid-base titrations were performed to determine the abundance and reactivity of proton binding sites (i.e., acidic surface functional groups) on intact cells and isolated extracellular sheath material from the cyanobacteria Calothrix (strain KC97), an isolate from the Krisuvik hot spring, Iceland. Sheath material was separated from whole cells by sonication, treatment with 200 mg/L lysozyme, and boiling 2% (w/v) SDS. Intact cells or isolated sheath were suspended in 0.01M KNO₃ containing either 8x10^-4 or 4x10^-4 M HNO₃ and titrated against 0.01M NaOH under positive N₂ pressure using a titrino GP736 autotitrator. Experimental data were subsequently analyzed using a linear programming pKa spectrum method (LPM) to identify discrete proton binding sites. The pKa spectra arising from acid-base titrations performed on intact Calothrix displayed at least 4 distinguishable reactive sites with mean pKa values of 5.1, 6.3, 7.5, and 8.8. These sites are suggested to correspond to carboxylic groups for site 1, phosphoryl (pK_a2) for site 2, phosphodiester for site 3 and amine groups for site 4. The sheath pKa spectra exhibited a similar distribution of functional groups, but featured an additional site with a pKa of 8.1, which may be representative of an amino or organosulfide group. Both whole cells and separated sheath material had similar total site densities within the analyzed pH range (4 to 9) of 0.90 and 0.75 µmoles/mg dry mass, respectively; however, quantitative cell fractionation established that sheath material contributes to only ~15% of the total dry mass of the cells. The implication is that much of the surface reactivity of Calothrix is concentrated within the cell wall, and not the extracellular sheath.