PRINCIPAL INVESTIGATOR^DGRAM DIRECTOR. 
Sandra Handwerger, M.D. 
cell walls and membranes will be examined by similar techniques. 
Lipids will be extracted by mechanical disruption of cells, 
extraction with methanol : ether , and separation by thin layer 
chromatography. Highly hydrophobic substituents will be 
extracted with light petroleum mixtures. Membrane proteins will 
be identified by SDS-polyacry lamide gel electrophoresis. 
Penicillin binding protein assays will be performed as 
previously described (24,25). 
b) Altered production of antibiotic targets : 
An excess of antibiotic targets represented a second 
potential mechanism of vancomycin resistance in Leuconostoc spp. 
Hyperproduction of lipid-linked pentapeptide precursors might 
result in an excess of binding sites, allowing continued growth 
of peptidoglycan. Although preliminary experiments suggested 
that the total pool of UDP-linked peptidoglycan precursors is 
similar in susceptible and resistant strains, quantitation of 
individual precursor moieties requires further separation, to 
exclude alterations in relative production of individual 
precursor moieties (e.g. increase in tetrapeptide precursors). 
Thus, UDP-hexosamine containing fractions of TCA extracted 
cultures collected from gel filtration as described above will 
be further separated by reverse phase high pressure liquid 
chromatography (RP-HPLC) . Separation is acheived using a 
u-Bondpak C. _ column with isocratic elution using 0.05M ammonium 
phosphate, pH 3.65 (15,44). 
c) Alteration in antibiotic target : 
A third potential mechanism of resistance is an alteration 
in the structure of the target of vancomycin which prevents 
binding. Nieto and Perkins measured the binding of various 
peptides to characterize the required structural elements for 
binding. They found that stable complex formation required three 
amide linkages, a free terminal carboxyl group, and the presence 
of D-amino acids or glycine at the terminal and penultimate 
positions. Alterations proximal to the terminal tripeptide had 
little effect on binding (45) . 
Preliminary experiments excluded the replacement 
D-alany 1-D-alanine with L-amino acids in the resistant strain 
VR. However, amino acid analysis of polymerized peptidoglycan 
does not exclude alterations in processing of the peptidoglycan 
precursors prior to incorporation into the cell wall which might 
prevent vancomycin binding, or conceivably, alteration in the 
order of amino acids in the stem peptide. Nascent peptidoglycan 
might be produced from tetrapeptide rather than pentapeptide 
precursors, as in Gaffkva homari (21,23). However, the 
transpeptidation reaction is presumed to require the breaking of 
the D-alanyl-D-alanine bond as a source of energy in the 
ATP-free milieu outside the cytoplasmic membrane. Thus in the 
Gaffkya system a proportion of pentapeptides are required as 
donors for transpeptidation. Conceivably, in a system using 
solely tetrapeptide precursors, an alternative source of energy 
for transpeptidation would be available. To distinguish among 
Recombinant DNA Research, Volume 13 
[431] 
