5 
of California, Davis) described the numerous genera of gram-negative bacteria found 
naturally occurring in the soil and on plants. He stated that most of these organ- 
isms do not appear to be a reasonable alternative to E . coZi K — 12 as a host for 
recombinant DNA molecules. Indeed, Starr pointed out that since such genera as 
Ervinia, Rhizobium and Agrobacterium are known to conjugate with E. coZi, the poten- 
tial dissemination of recombinant DNA molecule includes a greater spectrum of 
microorganisms than just enteric species. 
The fourth session of the workshop, chaired by R. Curtiss III (University of 
Alabama), was concerned with the construction of safer bacterial hosts for DNA clon- 
ing. The goals in constructing safer host strains enumerated at the beginning of 
the session included introduction of mutations that would: (a) preclude coloniza- 
tion in normal ecological niches; (b) preclude cell wall biosynthesis except in 
specially defined media; (c) cause degradation of genetic information in normal 
ecological niches; (d) cause vectors to be host-dependent; (e) minimize trans- 
mission of recombinant DNA to other strains in normal ecological niches; (f) 
increase usefulness for recombinant DNA molecule research; and (g) permit monitor- 
ing. 
Most of the progress in developing safer hosts has been achieved with E. coli 
K-12, although F. Young described a B. subtiZis- strain with a deletion for spolia- 
tion genes which readily undergoes autolysis. The strain also has defects in genes 
for purine and TTP biosynthesis and a mutation conferring a D-alanine requirement 
can be introduced to cause cell wall biosynthesis to be defective. This strain may 
be defective in transformation, however, and therefore might be useful only with a 
phage vector which has yet to be developed and/or discovered. 
A. I. Bukhari (Cold Spring Harbor Laboratory) described the use of the dapD8 
mutation in E. coZi K-12 to block cell wall biosynthesis and another non-reverting 
mutation which causes sensitivity to bile salts and detergents. The dapD8 allele 
is the most stable dap point mutation known, although it does revert at frequencies 
of 10~8 to 10~9. The mutation conferring bile salts sensitivity was obtained after 
Mu-1 infection of an Hfr strain and, although exhibiting the theoretically useful 
properties of ease of DNA isolation and inability to survive in the intestinal 
tract, might be due to Mu insertion which would compromise its use for safe strain 
construction, 
Curtiss reported on the work performed by him and his coworkers in constructing 
and testing numerous strains with different mutations. Survival of strains in vivo 
was tested by feeding rats 10^0 cells in milk by stomach tube, A pur mutations did 
not reduce strain titers in feces whereas A thy A; hthyA drm ; and LthyA dra mutations 
gave 10 -fold, 10 -fold and 10 ^-fold reductions, respectively t i n strain titers in 
feces. Strains with A thyA mutations also exhibited thymineless death in in vitro 
tests. Since strains with the dapD8 allele can revert to Dap + , strains were con- 
structed with both the dapD8 and A bioH-asd mutations. These strains have not been 
observed to revert to Dap + but can survive passage through the rat intestine and in 
growth media lacking diaminopimelic acid but containing NaCl and 0.5% usable carbon 
sources. This survival was due to the production of the mucopolysaccharide, colanic 
acid, which permits many of the cells to grow and survive as spheroplasts. A A gaZ- 
chZ T mutation (also deletes Xattj bio and uvrB genes) was introduced which blocks 
colanic acid biosynthesis and leads to no detectable survivors in media lacking 
diaminopimelic acid or following passage through the rat intestine. The dapD8 
LbioH-asd t\gaZ-chZ x strains are more readily lysed, transform at higher frequencies 
[ 138 ] 
