PRINCIPAL INVEST1GAT0RPR0GRAM DIRECTOR 
Sandra Handwerger, M.D. 
Teichoic acids will be extracted from cell wall 
preparations with trichloroacetic acid and isolated by gel 
filtration followed by affinity chromatography (concanavalin A) . 
Component sugars will be identified by acid hydrolysis followed 
by paper chromatography, and determined quantitatively by 
standard assays. 
To determine the relative proportions of teichoic acids and 
the sites of teichoic acid attachment in susceptible and 
resistant strains, purified cell walls will be prepared from 
cells labelled with h-lysine. Wall preparations will be 
digested with muramidase, and separated by thin-layer 
chromatography. Fragments will be located by autoradiography and 
identified by comparison to E. coli standards of disaccharide 
monomers, bis-disaccharide dimers, and oligomers. Digested cell 
wall preparations will also be subjected to concanavalin A 
affinity chromatography, and the non-bound fraction (lacking 
teichoic acid) will be determined by scintillation counting. 
Eluted fragments will then be separated by thin-layer 
chromatography to determine the proportions of monomers, dimers, 
and oligomers (and thus estimate the degree of cross-linking) in 
the teichoic acid-containing cell wall fragments (13) . 
Lipoteichoic acids: Other structures which might conceivably 
diminish vancomycin binding by a barrier mechanism include 
lipoteichoic acids, membrane or wall bound proteins, or an 
unusual lipid content of cell walls or membranes. Since these 
cell 'surface structures have not been characterized in 
Leuconostoc, initial experiments will be aimed at identifying 
appropriate extraction and isolation techniques in order to 
qualitiatively compare these structures in resistant and 
susceptible strains. 
Lipoteichoic acids will be extracted by treatment of cells 
with chloroform: methanol followed by extraction with 
phenol : water . The extract is treated with DNase and RNase, 
concentrated by . ultrafiltration and separated by chromatography 
on octyl-Sepharose CL-4B , which separates species according to 
difference in content of fatty acyl groups (32) . An alternate 
method, which has been successful in S. mutans , uses initial 
separation of extracted material by Sepharose 6B, followed by 
anion exchange (DEAE-cel lulose) with elution in 
chlorof orm:methanol iwater (34). Because biologic activity is 
not required for these studies, the published techniques will 
also be modified by the addition of SDS during the anion 
exchange step. Depending on the yield, elution solvents with 
higher degrees of hydrophobicity may be required. If the yield 
from these techniques is low, extractions will be repeated with 
phenol rchloroform: light petroleum solvent mixtures, which are 
more efficient for extraction of hightly hydrophobic structures 
(68). Identification of components will be performed by thin 
layer chromatography of acid-hydrolyzed material. 
Protein and lipid substituents: Other potential barrier 
structures in the cell surface of vancomycin resistant strains 
include lipids and hydrophobic proteins, which may be cell wall- 
or membrane-bound. To identify such substituents, screening 
techniques to identify potential differences in whole cells of 
resistant and susceptible cells will be employed; thereafter. 
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Recombinant DNA Research, Volume 13 
