Atsushi Suganuma

Aminoglycoside Binding Sites in Escherichia Coli as Revealed by Neomycin-Gold Labeling

A cytochemical technique for demonstration of neomycin binding sites by electron microscopy was developed and applied to Escherichia coli. Neomycin was conjugated chemically with bovine serum albumin (BSA). Colloidal gold was coated with the conjugated neomycin-BSA. The neomycin-BSA-gold was applied to thin sections of Epon-embedded E. coli and examined. Gold particles were observed on the outer membrane and the cytoplasmic membrane of E. coli. It was probably the ribosomes that were being labeled in the cytoplasm. Different cytochemical controls, including a number of inhibition tests and the use of BSA-gold, proved the specificity of this cytochemical technique and provided the biochemical significance of the observations.

Localization of sugar-binding sites in Staphylococcus aureus using gold-labeled neoglycoprotein.

We studied post- and pre-embedding staining of sugar-binding sites on thin sections of Staphylococcus aureus with an electron microscopic neoglycoprotein-gold technique. Although gold particles of cellobiosyl bovine serum albumin (BSA)-glycosylated BSA-, lactosyl BSA-, and melibiosyl BSA-gold did not label, heavy labeling of N-acetylglucosaminide-BSA-gold was observed in both the cell wall and the cytoplasm on Spurr-embedded thin sections of S. aureus. Inhibition of labeling with wheat germ agglutinin-biotin and N-acetylglucosaminidase indicated that the labeling was due to N-acetylglucosamine. These data suggested that molecules that bind specifically with N-acetylglucosamine occur in the cell wall and cytoplasm of S. aureus. Pre-embedding staining revealed that these molecules are abundant at the surface of the cell wall and that the abundance differs depending on the bacterial strain. An N-acetylglucosamine-specific lectin-like substance, glucosaminidase, and toxins are proposed as candidates for molecules responsible for the labeling, and the possible functional significance of the findings is discussed briefly.