PRINCIPAL invESTIGATORRROGRAM DIRECTOR Sandra Handwerger, M.D. 
1 4 
C) UDP-N-acety 1- [ C] glucosamine + UDP-N-acetyl-muramy 1 
tripeptide 
D) UDP-N-acety 1-glucosamine + UDP-N-acetyl-muramy 1 
l 1 X] pentapeptide + UDP-N-acety 1-muramyl tetrapeptide . 
Because cell wall synthesis does not appear to be affected by 
vancomycin in resistant Leuconostoc, previously published 
methods for obtaining high yields of precursors are unlikely to 
be successful. Thus initial experiments will use S. aureus 
precursors. UDP-N-acetyl muramyl pentapeptide will be obtained 
from S. aureus as described by Gorecki (18) . Tetrapeptide 
precursor will be derived by treating pentapeptide with 
D,D-carboxypeptidase (22) . Tripeptide precursors will be 
prepared as for pentapeptides but culture growth will be 
inhibited by cycloserine rather than vancomycin (22) . 
In the event that the permeabi lized cell system can not be 
adapted to Leuconostoc using either toluene or ether as a 
permeabilizing agent, these experiments will be performed using 
the wall-membrane model which has been described for several 
Gram positive bacteria (21) . Bacteria are grown to early log 
phase and disrupted mechancial ly ; the wall-membrane fraction is 
obtained by centrifugation. In some species freeze-thawing of 
the preparation obtained may yield higher synthetic activity 
(35). Labelled UDP-linked precursors are added to aliquots of 
this preparation, and newly synthesized insoluble peptidoglycan 
measured as described above. 
I I . Genetics of vancomycin resistance : 
Genetic systems in Leuconostoc have not been extensively 
studied. Both vancomycin resistant and susceptible strains may 
harbor multiple plasmids, but the relationship between 
extrachromosomal elements and antibiotic resistance is unknown 
(49) . Preliminary experiments in this laboratory suggest that 
vancomycin resistance may be a chromosomal trait. Thus the 
approach to characterizing the genetic determinants of 
vancomycin resistance and developing isogenic pairs will 
involve cloning of the vancomycin resistant trait. 
Because vancomycin resistance cannot be expressed in 
E. coli , which is intrinsically resistant by virtue of the outer 
membrane present in Gram negative bacteria, the approach to 
cloning will require a Gram positive host. A second difficulty 
is the lack of genetic systems available in Leuconostoc species. 
Although Leuconostoc may exchange plasmids via conjugation, 
transformation by either chromosomal or plasmid DNA has not been 
documented in Leuconostoc. For these reasons the naturally 
transformable strain S. sanguis Challis V288 will be used as the 
recipient in cloning experiments. Leclerq et al have shown that 
S. sanguis can express vancomycin resistance (although the trait 
appears to be inducible rather than constituitive) when 
transformed by the S. faecalis plasmids pIP816 or pIP817 (38) . 
Furthermore, experiments in this laboratory demonstrate that 
S . sanguis Challis can express antibiotic resistance traits 
conferred by transformation with Leuconostoc chromosomal DNA. 
Recombinant DNA Research, Volume 13 
[433] 
