Stationary phase deletions in Escherichia coli. II. Mutations which stimulate stationary phase deletions in plasmid pMC874.
Deletions in the plasmid pMC874 take place in resting cells incubating on McConkey's or minimal lactose agar and are time rather than generation dependent. These deletions join the km(r) promoter to a promoterless lac operon giving rise to Lac(+) papillae on McConkey's lactose agar, and can occur in the absence of sequence homologies such as direct or inverted repeats. Using this as a selective screen we isolated 31 mutants designated dli (for deletion increase), which enhanced to different extents the frequency of this unusual class of deletions. Six of these were characterized by phenotypic tests and their ability to stimulate other deletion events such as the excision of Tn10 from various chromosomal sites and the loss of cloned fragments between two EcoR1 sites in the gene for chloramphenicol resistance (cat) of plasmid pBR325. Two of them showed contrasting phenotypes and were studied further: one (dli1) stimulated Lac(+) deletions in pMC874 in resting cells but not Tn10 excision from chromosomal locations in log phase cells, and the other one (dli2) did exactly the reverse, i.e. it enhanced Tn10 excision but not Lac(+) deletion incidence. Mapping and complementation tests showed that dli1 is a null mutation in recC and was renamed recC2251. This is strong evidence that resting phase deletions in pMC874 are stimulated by the absence of a functional RecBCD enzyme. The dli2 mutation was identified by mapping and phenotypic tests as a mutation in uvrD, the gene for helicase II, and it was tentatively designated uvrD(-)dli2. These results show that (1) pMC874 is an excellent system to select mutants for genetic functions involved in the generation of resting phase deletions, and (2) there are at least two major deletion pathways in E. coli, one active in resting and the other in actively dividing cells.
Balbinder
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Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver CO 80262, USA.
D000362:Agar / Q000378:metabolism; D001483:Base Sequence; D003227:Conjugation, Genetic; D003470:Culture Media; D004249:DNA Damage; D004260:DNA Repair; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D017353:Gene Deletion; D005838:Genotype; D007785:Lactose / Q000378:metabolism; D008957:Models, Genetic; D016296:Mutagenesis; D009154:Mutation; D010641:Phenotype; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D012984:Software; D013997:Time Factors; D014162:Transfection
D003470:Culture Media; D000362:Agar; D007785:Lactose