APPENDIX D— 22 
though there Ls a surprisingly wide discrep- 
ancy between DNA— DNA hybridization 
among these organisms (16). Even though 
the frequency of transformation is low in the 
heterologous cross [e.g., B. amyloliquefacien* 
(donor) /B subtilis (recipient)], the newly 
acquired DNA from B. amyloliquefaciens in 
the B. subtilis background can be readily 
transferred at high efficiencies to other re- 
cipient strains of B. subtilis (14). Therefore, 
the extremely high frequency of transforma- 
tion permits the recognition and selection of 
rare events. 
B. CtmRENT AND POTENTIAL VECTORS FOR 
RECOMBINANT MOLECULE EXPERIMENTS 
Lovett and coworkers have recently de- 
scribed crytlc plasmids in B. pumilus (17) 
and B. subtilis (18). Of these organisms, B. 
subtilis ATCC 7003 appears to be the most 
useful since it carries one to two copies of a 
plasmid with a molecular weight of 46 X 10». 
This strain is als closely related to B. subtilis 
168. Another strain of B. subtilis (ATCC 
16841) contains 16 copies of a plasmid with 
a molecular weight of 4.6 X 10®. Currently it is 
not known whether genetic markers can be 
readily Introduced into these plasmids. To 
date it has not been possible to readily stabil- 
ize plasmids derived from B. pumilus in B. 
subtilis even with heavy selective pressure 
(P. Lovett, personal communication) . 
Two temperate bacteriophages are under 
development as vectors in B. subtilis, 4>3T 
and SP02. Lysogeny of thymine auxotrophs 
(strains sarrylng thy A thyB) by bacte- 
riophage 4>3T results in "conversion” to a 
Thy+ phenotype. The attachment site for 
this bacteriophage and the bacteriophage 
gene for thymidylate synthetase (thyP) map 
between the bacterial thyA and thyB loci in 
the terminal region of the chromosome of 
B. subtilis (19). The viral genome Is readily 
cleaved by the site-specific endonuclease. 
Bam 1 (20), to produce 6 fragments (one of 
which carries the thyP gene) . The thyP car- 
rying gene can be Integrated Into the bac- 
terial genome in the absence of the Intact 
viral genome. Because deletions are available 
that Include the thyP region. It is theoreti- 
cally possible to Introduce thyP at many 
sites on the chromosome. The thyP gene can 
be readily purified for insertion into plasmids 
or utilized as a scaffold to Integrate other 
heterolc^ous DNA into the chromosome of 
B. subtilis. Alternatively, it is possible to 
purify fragments of the chromosome by gel 
electrophoresis (21, 22), for insertion into 
bacteriophage 4>3T or SP02. At present, un- 
fortunately, only the former carries a selec- 
tive marker, l.e., the gene for thymidylate 
synthetase, thyP. 
C. DEVELOPMENT OF VEHICLES 
B. subtilis is a Gram-positive sporulating 
rod that usually inhabits soil. Although it 
can exist on cutaneous surfaces of man (23) 
and experimental animals, it rarely produces 
disease. To develop a suitable vehicle it is 
Imperative to have a host that is asporogenlc. 
The most appropriate deletion mutation is 
deletion 29 (clt D). In addition to a defi- 
ciency in sporulatlon this mutant rapidly 
lyses when it has reached the end of its 
growth cycle. Presumably this is due to the 
failure to inactivate one of the autolytlc 
enzymes (24) . Through the Introduction of a 
D-alanlne requirement (34 ug/ml) it is pos- 
sible to block transport of compiounds that 
are transported by active transport (25, 26). 
The further introduction of thymine auxo- 
trophy (defects in the thyA thyB loci) will 
enable the strain to survive only with a 
plasmid vector carrying the pmlfied thyP 
gene from bacteriophage 4>3T or a defective 
bacteriophage 4>3T cari*ying the thyP gene 
but attached to the chromosome at an alter- 
native site (due to the presence of deletion 
29 in the host) . We have recently Isolated 
temperatiure-sensltive thyP mutants. If we 
can Isolate a temperature-dependent lysogen 
that will grow only at 48°C it should be pos- 
sible to make an unusual vehicle. 
D. SITE-SPECIFIC ENDONUCLEASES 
Recently two restriction modification sys- 
tems have been observed between B. subtilis 
168 and other bacilli. Trautner et al. have 
isolated an effective system that inhibits in- 
fection of the R strain of B. subtilis by bac- 
teriophage SPPl propagated on B. subtilis 
168 (27) . The site-specific nuclease recognizes 
the sequence GGCC. Young, Radnay, and 
CCGG 
Wilson observed a resti’lctlon modification 
sy.stem between B. amyloliquefaciens and 
B. subtilis 168 (28). The endonuclease from 
B. amyloliquefaciens (20) recognizes the 
sequence GGATCC (29). More recently, two 
CCTAGG 
additional enzymes have been Isolated from 
B. globigii (30). The recognition sequence is 
not known. 
E. ADVANTAGES AND LIABILITIES OF THE 
B. "SUBTILIS SYSTEM” 
0 , Advantages 
1. B. subtilis is nonpathogenlc. Asporo- 
genic deletion mutants are available to pre- 
clude the problem of persistence through 
sporulatlon. 
2. The circular chromosomal map is well 
defined. At least 196 loci have been posi- 
tioned. 
3. The organism is commercially Important 
in the feimentatlon industry. 
4. Large numbers of organisms can be dis- 
posed of readily with minimal environmental 
Impact. 
6. Unlike E. coli, it lacks endotoxin in the 
cell wall. Therefore the cells can be used as 
a single cell protein source. 
6. The frequency of transformation is very 
high, facilitating the detection of rare events. 
7. A unique ba^iteriophage, ^3T, exists that 
carries a gene that can be readily purified for 
"scaffolding” experiments. 
b. Disadvantages 
1. The knowledge of genetics and physiol- 
ogy of plasmids and viruses Is primitive com- 
pared with E. coli. 
2. High-frequency, specialized transduc- 
tion is not available as a means of gene 
enrichment. 
Based on Its promise, it seems appropriate, 
and not chauvinistic, to urge development of 
this system. 
Prepared by : Dr. Pank Young, University of 
Rochester. 
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