Amylase-negative, asporogenous mutant of Bacillus subtilis useful as a host in a host-vector system

DETAILED DESCRIPTION OF THE INVENTION The B.
subtilis strains disclosed and claimed were prepared by incorporating the genetic material from other strains of B.
subtilis.
An amylase-negative strain of B.
subtilis, ATCC 31,785, containing the markers: aro-10 and amy-3, was transformed into a lincomycin-resistant strain by incorporation of the lin-2 marker from B.
subtilis Strain 1A221 (ATCC 39,086) using a transformation reaction.
Strain 1A221 was reported by Goldthwaite, Dubnau and Smith, Proc.
Natl.
Acad.
Sci.
, U.
S.
A.
, 65, 96-103 (1970).
It is available from the American Type Culture Collection, Rockville, Md.
, as ATCC 39,086.
The resultant transformant, designated as EE1, contains the markers: lin-2, aro-10, and amy-3.
It is available from the American Type Culture Collection as ATCC 39,093.
This amylase-negative strain is useful as an intermediate for the preparation of amylase-negative host components of host-vector systems.
A portion of the DNA from B.
subtilis Strain EE1 was then transferred into an asporogenous parent strain designated as DE101.
Transfer was accomplished using a transduction process by means of the phage PBS1.
Strain DE101 (ATCC 39,095) is disclosed in a pending U.
S.
application Ser.
No.
370,239, filed Apr.
21, 1982, which disclosure is incorporated herein by reference.
A stable mutant, EE101, possessing the markers: lin-2, aro-10, amy-3, thy A1, thy B1, pyr B1, and spo OA.
DELTA.
677, resulted from the transduction.
The asporogenous, amylase-negative strain of the present invention, EE101, shows a frequency of reversion to spore formers of less than about 10.
sup.
-7.
It is able to grow under industrial conditions not requiring expensive growth requirements.
It has a low survival rate under natural or escape conditions and a very low tendency to transmit plasmids to other organisms by natural genetic transfer.
Although the organism shows a low degree of competence when subjected to classical transformation techniques, excellent transformation has been achieved using a protoplast transformation procedure