A PUB 110-derived plasmid encoding chloramphenicol resistance, kanamycin resistance and high-temperature α-amylase showed a high degree of segregational instability when inserted into Bacillus subtilis. In an attempt to obtain stable derivatives, the organism was grown in chemostat culture in the presence of chloramphenicol. It was periodically found necessary to increase the concentration of chloramphenicol in the medium feed in order to avoid plasmid loss. Strains were isolated after 19 and 160 generations, which showed high levels of plasmid stability. This characteristic appeared to be genotypic. No detectable difference in plasmid copy number was found between the original and the improved strains. The stability characteristics resided in the host, rather than in the plasmid. Stable isolates possessed elevated MICs for both chloramphenicol and kanamycin. Their maximum specific growth rates were higher than that of the original strain, and similar to that of the plasmid-free parent strain.
CarrierM. J.,
NugentM. E.,
TaconW. C. A.,
PrimroseS. B.1983; High expression of cloned genes in Escherichia coli and its consequences. Trends in Biotechnology 1:109–113
GryczanT.,
ContenteS.,
DubnauD.1980; Molecular cloning of heterologous chromosomal DNA by recombination between a plasmid vector and a homologous resident plasmid in Bacillus subtilis
. Molecular and General Genetics 177:459–467
HoriuchiT.,
TomizawaJ.-I.,
NovickA.1962; Isolation and properties of bacteria capable of high rates of β-galactosidase synthesis. Biochimica et biophysica acta 55:152–163
ImanakaT.,
AibaS.1981; A perspective on the application of genetic engineering: stability of recombinant plasmid. Annals of the New York Academy of Sciences 369:1–14
Ish-HorowiczD.1982; Analysis of recombinant DNA clones: the alkaline lysis method. In Molecular Cloning: a Laboratory Manual pp. 368–369ManiatisT.,
FritschE. F.,
SambrookJ.
Edited by Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
JoyetP.,
GuerineauM.,
HeslotH.1984; Cloning of a thermostable α-amylase gene from Bacillus licheniformisand its expression in Escherichia coli and Bacillus subtilis
. FEMS Microbiology Utters 21:353–358
LejeuneP.,
WalonC.,
WillemotK.,
ColsonC.1984; Pleiotropic chromosomal mutations of Bacillus subtilis which increase the copy-number and the stability of recombinant plasmids and alter the metabolism of nitrogen assimilation. Archives internationales de physiologie 92:B149–B150
OrtleppS. A.,
OllingtonJ. F.,
McconnellD. J.1983; Molecular cloning in Bacillus subtilis of a Bacillus licheniformisgene encoding a thermostable alpha amylase. Gene 23:267–276
PinchesA.,
LouwM. E.,
WatsonT. G.1985; Growth, plasmid stability and α-amylase production in batch fermentations using a recombinant Bacillus subtilis strain. Biotechnology Letters 7:621–626
RabinovichP. M.,
HaykinsonM. YA.,
ArutyunovaL. S.,
YomantasYu. V.,
StepanovA. I.1985; The structure and source of plasmid DNA determine the cloning properties of vectors for Bacillus subtilis
. In Plasmids in Bacteria pp. 635–656HelinskiD. R.,
CohenS. N.,
ClewellD. B.,
JacksonD. A.,
HollaenderA.
Edited by New York: Plenum Press;
ShivakumarA. G.,
HahnJ.,
DubnauD.1979; Studies on the synthesis of plasmid-coded proteins and their control in Bacillus subtilis minicells. Plasmid 2:279–289
VehmaanperaJ. O.,
KorholaM. P.1986; Stability of the recombinant plasmid carrying the Bacillus amyloliquefaciens α-amylase gene in Bacillus subtilis
. Applied Microbiology and Biotechnology 23:456–461
WaterworthP. M.1978; Quantitative methods for bacterial sensitivity testing. In Laboratory Methods in Antimicrobial Chemotherapy pp. 31–40ReevesD. S.,
PhillipsI.,
WilliamsJ. D.,
WiseR.
Edited by Edinburgh: Churchill-Livingstone;