Twenty Tn5 insertion mutants of the drug resistance plasmid pKM101 have been isolated that are unable to enhance mutagenesis with ultraviolet (UV) irradiation or methyl methanesulfonate. By restriction mapping, the Tn5 insertion in each of these pKM101 mutants was shown to be within a 1.9 kb region of the plasmid genome. We have termed this segment of the pKM101 map the muc (mutagenesis: UV and chemical) gene(s). Characterization of these mutants indicated that any Tn5 insertion within the muc gene(s) abolished the ability of pKM101 to: (a) enhance spontaneous, UV and chemical mutagenesis, (b) increase host survival following UV-irradiation, (c) increase the survival of UV-irradiated phage plated on irradiated or unirradiated cells, and (d) suppress the repair and mutagenesis deficiencies of a umuC- mutant. Possible models to explain the role of the pKM101 muc gene(s) in mutagenesis and repair are discussed.
Tn5 insertion mutants and in vitro-generated deletion mutants of the mutagenesis-enhancing plasmid pKM101 have been used to identify several genetic regions on the pKM101 map. In clockwise order on the pKM101 map are: (i) the bla gene, coding for a beta-lactamase; (ii) the Slo region, responsible for retarding cell growth on minimal medium; (iii) the tra genes, enabling pKM101 to transfer conjugally; (iv) sensitivity to IKe phage (this function[s] maps within the tra region); (v) the muc gene(s), responsible for enhancing ultraviolet light and chemically induced mutagenesis in the cell; and (vi) the Rep region, essential for plasmid replication. The muc gene(s) and the Rep region are contained in a deoxyribonucleic acid region bounded by inverted repeated sequences.
A protoplast transformation system has been developed for Corynebacterium glutamicum by using a C. glutamicum-Bacillus subtilis chimeric vector. The chimera was constructed by joining a 3.0-kilobase cryptic C. glutamicum plasmid and the B. subtilis plasmid pBD1O. The neomycin resistance gene on the chimera, pHY416, was expressed in C. glutamicum, although the chloramphenicol resistance gene was not. The various parameters in the transformation protocol were analyzed separately and optimized. The resulting transformation system is simple and routinely yields 104 transformants per ,ug of plasmid DNA.
Spontaneous mutators of Salmonella typhimurium LT2 were generated by inserting the transposable element Tn5 or TnlO into the bacterial chromosome. Two mutators mapped at the position of the mutH and mutL loci of S. typhimurium, and two other mutators mapped at positions corresponding to the mutS and uvrD loci of Escherichia coli. A fifth mutator, mutB, did not map at a position corresponding to any of the known mutators of S. typhimurium or E. coli. The mutH,L,S and uvrD alleles increased the frequency of both spontaneous base substitution and frameshift mutations, whereas the mutB allele increased the frequency only of spontaneous base substitution mutations. The increased frequency of base substitution mutations was recA+ independent in the mutH, mutL, and uvrD strains and partially recA+ independent in the mutS strain. The uvrD mutation decreased the resistance of the cells to killing by ultraviolet irradiation. The mutH,L,S and uvrD strains showed an increased sensitivity to mutagenesis by the alkylating agents methyl methane sulfonate and ethyl methane sulfonate, but not to mutagenesis by 4-nitroquinoline-1-oxide.
An assay has been developed that permits analysis of repair of A/G mismatches to CG base pairs in cell extracts of Salmonella typhimurium LT2. This A/G mismatch repair is independent of ATP, dam methylation, and mutS gene function. The gene product of mutB has been shown to be involved in the dam-independent pathway through the in vitro assay. Moreover, specific DNA-protein complexes and an endonuclease can be detected in S. typhimurium extracts by using DNA fragments containing an A/G mismatch. These activities are not observed with substrates which have a T/G mismatch or no mismatch. The S. typhimurium endonuclease, like the AMG endonuclease found in Escherichia coli (A-L. Lu and D.-Y. Chang, Cell 54:805-812, 1988), makes incisions at the first phosphodiester bond 3' to and the the second phosphodiester bond 5' to the dA of the A/G mismatch. No incision site was detected on the other DNA strand. Extracts prepared from mutB mutants cannot form A/G mismatch-specific DNA-protein complexes and do not contain the A/G endonuclease activity. Thus the A/G mismatch specific binding and nicking activities are probably involved in the A/G mismatch repair pathway. Preliminary analysis of the mutational spectrum of the mutB strain has indicated that this mutator allele causes an increase in CG-to-A-T transversions without affecting the frequencies of other transversion or transition events. In addition, the mutB gene has been mapped to the 64-min region of the S. typhimurium chromosome. Together, this biochemical and genetic evidence suggests that the mutB gene product of S. typhimurium is the homolog of the E. coli micA (and/or mutY) gene product.
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