(l)do Phages: Helper Independent Temperate Phage 
Molecular Cloning Vehicles for Bacillus subtilis 
D.H, Deani'2/3'4,^ j b PerkinshJ.M. Kroyer' 
M.S. Rudinski’, I.B. LazdinsP P.A. Martin2, 
W.F. Martin^, and F.R. Perro, Jr. 2 , 
Departments of Microbiology', Genetics2, 
Development Biologys, and the Bacillus Genetic Stock Center^ 
The Ohio State University, Columbus, Ohio 43210 
Until very recently the use of recombinant DMA 
techniques has been almost exclusively limited to 
Escherichia coli. Recently, ho\wever, plasmid 
cloning vehicles have been introduced from B. 
subtilis (1,2,3,4,5). The value of DNA cloning to the 
group of bacteria related to Bacillus subtilis is 
readily apparent \A/hen one considers the wealth of 
their genetic and economic capabilities. More 
than eighty antibiotics and a dozen enzymes of 
economic importance are made by 6 . subtilis, B. 
amloliquefaciens, B. licheniformis or B. pumilus 
(6,7). Of particular value is the ability of these 
organisms to excrete certain of these enzymes 
obviating the necessity to lyse cells to harvest the 
enzymes. Bacillus also has the ability to form 
endospores and this genetically complex devel¬ 
opmental process, involving thirty to forty genes, 
represents a complex procaryotic developmental 
system. Other genetic capabilities include gener¬ 
alized transduction, transformation and a new 
plasmid transfer technique (S. Chang and S. 
Cohen, Mol. Gen. Genet., in press). The expected 
value of recombinant DNA technology to Bacillus 
research is to amplify genetic products, provide 
new approaches to the study of sporulation and to 
allow 6 . subtilis to assume the role of a choice host 
for a wider range of genes coding for industrially 
important products. 
Several years ago we set out to develop phage 
molecular cloning vehicles similar to the lambda 
systems available for E. coli (8,9,10,11). This task 
has not been as easily achieved as for lambda 
because the information about Bacillus phages 
was scanty in comparison. The approach to 
developing a phage cloning system for 8 . subtilis 
has proceeded through the following steps: 
1. Isolation and Characterization of New Tem¬ 
perate subtilis Phages. 
This was necessary since few temperate subtilis 
phages were previously known. We concentrated 
on temperate phages to take advantage of their 
powers of integration in order to develop a trans¬ 
duction system capable of achieving low copy 
alleleic complementation. These studies have 
resulted in a growing understanding of temperate 
phages of Bacillus (12,13,14,15) including one of 
the few examples of the comparison of a group of 
phages by classical criteria (12), restriction 
enzyme analysis (13) and heteroduplex analysis 
(M.S. Rudinski and D.H. Dean, manuscript submit¬ 
ted). These studies have shown that the temper¬ 
ate subtilis phages separate into at least four 
groups (Table 1, (12)) which is surprising, given 
the fact that most of the hosts listed in Table 2 are 
very closely related and exchangers of DNA with 
the transformable strain 168. This narrow host 
range might be a useful attribute for certain clon¬ 
ing procedures where promiscuous interspecies 
transfer of cloned DNA would be undesirable. 
2. Restriction Enzyme Analysis of Temperate 
Phages. Our goals were to analyze new phages 
with restriction enzymes which generate cohesive 
ends and to identify that phage which has one (or a 
few) restriction sites. We did not pursue the group 
ill phages, o3T and pi 1 (13) because of their large 
genome size and the numerous restriction sites 
with all enzymes examined. The group I phages 
were appealing in having a smaller genome size 
(all about 25 x 106 daltons). Extensive enzyme 
mapping has been done (14,15, Microbial Genet¬ 
ics Bulletin 43: 7-10 (1977)). It is apparent from 
Fig. 1 that pi 4 has a single Sa/GI site and a single 
BglW site. The close relationship between pi 4 and 
(1)105 (13,14,15, 83% homoduplex, Rudinski and 
Dean unpublished) has lead us to speculate 
(1 4,15, Microbial Genetics Bulletin op cit) that the 
Sa/GI site of pi 4 is in the pi 4 immunity region. 
Although the Sa/GI site is extremely close to the 
immunity region we have yet to remove it by a ser¬ 
ies of clear plaque deletions into this region. Furth¬ 
ermore, cloning into the Sa/GI site does not result 
in clear plaques (it does however appear to make 
the plaque appear discerningly less turbid than 
normal). 
3. Isolation of Deletion Mutants. In order to 
clone larger fragments, deletion mutants of pi 4 
have been obtained by sodium pyrophosphate 
selection. Turbid plaque deletion mutants, called 
(t)do, are exemplified by odo7 a 1.9 x 10® dalton 
deletion in the EcoRI A fragment (see map Fig. 1). 
Clear plaque deletion mutants, called (l)doc are 
available with 1-1.5 x 10® dalton deletions with a 
predicted maximum of 5 x 10® in the EcoRI A and F 
bands (see map Fig. 1) (J.M. Kroyer, J.B. Perkins, 
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