Nature of the Carbohydrate and Phosphate Associated with ColB2 and EDP208 Pilin

Abstract

Studies were carried out to elucidate the nature of phosphate and sugar linkages to F-like conjugative pili encoded by the ColB2Fdr (compatibility group FII) and EDP208 (compatibility group FV) plasmids. Both types of pili, when in the intact undissociated state, were found to contain approximately 3 mol of phosphate and 3 mol of sugar per mol of pilin. However, further purification of the two types of pilin by gel filtration chromatography in the presence of sodium dodecyl sulfate (SDS) removed all of the carbohydrate from EDP208 pilin and approximately 65% of the carbohydrate from ColB2 pilin. Approximately 0.8 to 1.0 mol of glucose per mol of protein remained associated with ColB2 pilin after SDS gel filtration chromatography, but it was not possible to determine whether this was covalently linked to the pilin, or tightly associated in an SDS-resistant manner. SDS-gel chromatography did not remove phosphate from either ColB2 or EDP208 pilins. 31 P nuclear magnetic resonance studies indicated that the pilin-associated phosphate is involved in a phosphodiester linkage. Acetone precipitation or chloroform-methanol extraction of the purified pilin material reduced the phosphate associated with EDP208 pilin to less than 0.04 molecule per pilin monomer. ColB2 pilin, under the same conditions, retained approximately 0.5 phosphate per pilin monomer. The extracted phosphate-containing moieties were identified as phosphatidylglycerol and phosphatidylethanolamine by thin-layer chromatography. Since the 31 P nuclear magnetic resonance spectra for both ColB2 and EDP208 were identical and no signal other than that of a phosphodiester was detected in the ColB2 spectrum, the phosphate remaining with the ColB2 pilin after chloroform-methanol extraction is most likely due to a tightly bound noncovalent residue.