PRINCIPAL INVESTIGATOFVPROGRAM DIRECTOR: Sandra Handwerger, M. 
these possibilities, and to assess the effect of vancomycin on 
cell wall synthesis, three approaches will be employed: 
characterization of vancomycin binding to peptidoglycan stem 
peptides, structural analysis of peptidoglycan precursors, and 
assessment of peptidoglycan synthesis from UDP-linked precursors 
in a permeabilized cell system. 
Binding of vancomycin to stem peptides: To determine 
whether structural alterations in the stem peptides prevent 
binding to vancomycin, purified cell walls of vancomycin 
Resistant and susceptible strains will be labelled with 
J H-lysine, and treated with purified pneumococcal amidase (which 
cleaves stem peptides from the glycan backbone) . Affinity of 
isolated peptides for vancomycin will be compared in the two 
strains by affinity chromatography (vancomycin coupled to 
activated CH-Sepharose-4B) . Comparison of the degree of binding 
with results of the experiments described above to estimate 
degree of cross-linking will be of value in determining the 
degree of "non-specific" binding, i.e. binding to cross-links 
rather than stem peptides, which has been described by Perkins 
(51). Eluted fragments will be separated on the basis of degree 
of crosslinking by thin layer chromatography (13) . In the event 
that uncrosslinked peptides do not bind to vancomycin, the amino 
acid sequence of the stem peptides will be determined by partial 
hydrolysis and dinitrophenylation. 
Structure. of UDP-linked precursors : The RP-HPLC assay of 
UDP-linked precursors described above allows both quantitation 
and structural analysis of precursor pools. Under the described 
conditions, UDP-N-acetyl-muramyl-tri- , tetra-, and pentapeptides 
are separated. Collected fractions will be subjected to amino 
acid analysis for confirmation of structure. 
Peptidoglycan synthesis in permeabilized cells: To further 
assess the components of peptidoglycan synthesis in vancomycin 
resistant Leuconostoc, a permeabilized cell system will be used. 
This represents 'a cell-free model for examining peptidoglycan 
synthesis, to which the appropriate UDP-linked precursors are 
added. Some evidence suggests this model may more closely 
represent wall synthesis in vivo than wall-membrane 
preparations, at least in Gram negative species (36). This 
system will be used to determine whether susceptible and 
resistant Leuconostoc can synthesize peptidoglycan solely from 
tetrapeptide rather than pentapeptide precursors, and which 
precursor type is used preferentially. 
Permeabilized cells are prepared by shaking mid-log phase 
cultures of bacteria with toluene in the cold. Aliquots of cells 
are then incubated with 4 C-labelled UDP-linked peptidoglycan 
precursors (enumerated below) and aliquots removed at various 
time points and boiled in SDS. The SDS-insoluble material 
(synthesized peptidoglycan) is collected on membrane filters, 
washed and counted in a scintillation counter (39). UDP-linked 
precursors will be added to permeabilized cells as follows: 
A) UDP-N-acetyl- [ 14 C] glucosamine + UDP-N-acety 1-muramy 1 
pentapeptide . 
B) UDP-N-acetyl- [ i4 C] glucosamine + UDP-N-acety 1-muramy 1 
tetrapeptide 
[ 432 ] 
Recombinant DNA Research, Volume 13 
