PRINCIPAL INVESTIGATOR'PROGRAM DIRECTOR: 
Sandra Handwerger, M.D. 
subdivisions until the individual clone is recognized. All 
colonies will be tested for both inducible and constituitive 
resistance to vancomycin, as above. 
Conjugative mobilization: For mechanistic and 
complementation studies, it would be most desirable to return 
the vancomycin resistance gene to a Leuconostoc host. 
Transformation has not been demonstrated in Leuconostoc, and 
attempts to transform Leuconostoc protoplasts with chromosomal 
or plasmid DNA have been unsuccessful in this and other 
laboratories. However, Leuconostoc species undergo conjugation, 
and plasmids from S. lactis , S. faecalis , and S. sanguis have 
been conjugally transferred into Leuconostoc (54,65). Thus the 
technique of conjugative mobilization represents a potential 
method of introducing the resistance trait into a susceptible 
Leuconostoc host (63) . Conjugative mobilization has been used to 
introduce cloned DNA into other nontransf ormable 
Streptococcaceae such as lactic streptococci and S. faecalis 
(55,63). The procedure will utilize the resulting chimera 
derived from pVA838 (Em r ) or pVA736 (Em r ) as outlined above and 
the conjugative plasmid pVA797. pVA797 (Cm r ) is a derivative of 
the broad host range plasmid pIP501 which has been introduced 
into Leuconostoc by conjugation (54) . Both pVA838 and pVA736 
share homology with pVA797, and all three replicate in S . 
sanguis via the pVA380-l replicon (41,63). pVA797 is introduced 
into S. sanguis V288 by filter mating, and the desired second 
plasmid by transformation. The strain is maintained by growth 
on both erythromycin and chloramphenicol. Smith and Clewell 
have shown that under these conditions pVA797 and pVA838 from a 
conjugative cointegrate which resolves upon conjugation (63) . S . 
sanguis harboring the cointegrate is then used as a donor in 
filter mating with L. mesenteroides 688, and transconjugates 
selected on vancomycin agar. 
Transfer of resistance: To explore the possibility that 
vancomycin resistance may be transferred to other Gram positive 
bacteria, VR1 and the plasmid free strains VR and 33313 will 
also be used as donors in transformation and conjugation 
experiments, with S. sanguis as the recipient. Preliminary 
experiments have not yielded transformants with constituitive 
high level resistance. Therefore experiments will screen for 
transformants with inducible resistance, as described above. 
Since Leuconostoc have been shown to transfer transposons 
con jugatively to other streptococci, filter mating experiments 
will also be performed. A streptomycin resistant mutant, 
ChallisSt, is used as recipient. Cultures of donors (1 ml) and 
recipient (2 ml) are mixed, filtered onto a sterile 
nitrocellulose filter, and the filter placed onto BHI agar for 
18 hours of incubation. Transcon jugants are screened by plating 
on agar containing streptomycin and vancomycin, with 
pre-incubation on sub-MIC vancomycin for identification of 
inducibly resistant transcon jugants . 
Hybridization studies: To determine the potential 
relationship between the genes encoding vancomycin resistance in 
leuconostoc and recently isolated vancomycin resistant 
enterococci, DNA-DNA hybridization will be employed. Probes 
will be prepared from S. faecium plasmids conferring vancomycin 
Recombinant DNA Research, Volume 13 
[435] 
