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Complementation of a pKM101 derivative that decreases resistance to UV killing but increases susceptibility to
Abstract:
The drug resistance plasmid pKM101 makes Escherichia coli resistant to the lethal effects of ultraviolet (UV) irradiation and more susceptible to mutagenesis by a variety of agents. The plasmid operon responsible for increasing mutagenesis has been termed mucAB (Mutagenesis, UV and chemical). We have isolated a derivative of pKM101 called pGW1975 which makes cells more sensitive to killing by UV but which retains the ability of pKM101 to increase susceptibility to methyl methanesulfonate (MMS) mutagenesis. pGW1975 increases UV mutagenesis less than pKM101 in a uvrA+ strain but more than pKM101 in a uvrA- strain. muc- point and insertion mutants of pKM101 and pGW1975 complement to restore the plasmid-mediated: (i) ability to reactivate UV-irradiated phage, (ii) resistance to killing by UV, and (iii) level of susceptibility to UV mutagenesis. We have identified a 2.0 kb region of pKM101 which is responsible for the complementation and which maps counterclockwise of mucAB.
Insights
The mucAB operon on plasmid pKM101 enhances mutagenesis and UV resistance in Escherichia coli. A derivative, pGW1975, alters these effects, with specific mutations restoring normal function.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- The pKM101 plasmid confers resistance to ultraviolet (UV) radiation and enhances mutagenesis in Escherichia coli.
- The mucAB operon (Mutagenesis, UV and chemical) is responsible for increased mutagenesis mediated by pKM101.
Purpose of the Study:
- To investigate the role of the mucAB operon in UV resistance and mutagenesis.
- To characterize a novel pKM101 derivative, pGW1975, and its effects on UV sensitivity and methyl methanesulfonate (MMS) mutagenesis.
- To identify the genetic region responsible for complementation of mucAB mutants.
Main Methods:
- Isolation and characterization of pKM101 derivative pGW1975.
- Complementation analysis of muc- point and insertion mutants of pKM101 and pGW1975.
- Assessment of UV resistance, UV mutagenesis, and phage reactivation.
- Mapping of the complementation region on pKM101.
Main Results:
- pGW1975 increases sensitivity to UV killing but retains enhanced MMS mutagenesis compared to pKM101.
- The effect of pGW1975 on UV mutagenesis varies depending on the uvrA gene status of the host strain.
- Complementation of muc- mutants restored UV resistance, phage reactivation, and UV mutagenesis levels.
- A 2.0 kb region on pKM101, located counterclockwise of mucAB, was identified as responsible for complementation.
Conclusions:
- The mucAB operon plays a critical role in both UV resistance and mutagenesis.
- Specific regions within or near mucAB are essential for mediating these plasmid-associated phenotypes.
- The identified 2.0 kb region is crucial for the functions associated with the mucAB operon.