PubMed · 6312259
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M Gold, A Becker, W Parris. 1983. The bacteriophage lambda terminase enzyme.. https://doi.org/10.1016/0076-6879(83)00054-3
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To study the mechanism of spontaneous and UV-induced illegitimate recombination, we examined the formation of the lambda bio specialized transducing phage in Escherichia coli. Because most lambda bio transducing phages have double defects in the red and gam genes and have the capacity to form a plaque on an E. coli P2 lysogen (Spi- phenotype), we selected lambda bio transducing phage by their Spi- phenotype, rather than using the bio marker. We determined sequences of recombination junctions of lambda bio transducing phages isolated with or without UV irradiation and deduced sequences of parental recombination sites. The recombination sites were widely distributed on E. coli bio and lambda DNAs, except for a hotspot which accounts for 57% of UV-induced lambda bio transducing phages and 77% of spontaneously induced lambda bio transducing phages. The hotspot sites on E. coli and lambda DNAs shared a short homology of 9 bp. In addition, we detected direct repeat sequences of 8 bp within and near both the bio and lambda hotspots. A recA mutation did not affect the frequency of the recombination at the hotspot, indicating that this recombination is not a variant of recA-dependent homologous recombination. We discuss a model in which the short homology as well as the direct repeats play essential roles in illegitimate recombination at the hotspot.
We have developed a strategy for efficient sequence analysis of the genome of E. coli K-12 using insertions of a Tn5-derived mini-transposon into overlapping ordered lambda phage clones to provide universal primer-binding sites, and PCR amplification of DNA segments adjacent to the insertions. Transposon-containing clones were selected by blue plaque formation on a dnaBamber lacZamber E. coli strain. Insertion points every 0.5-1 kb were identified by 'analytical PCR' and segments between the transposon inserts and phage arms were amplified by 'preparative PCR' using one biotinylated and one non-biotinylated primer. Single strands of amplified DNA fragments were coupled to Streptoavidin-coated paramagnetic beads (Dynabeads M280) through their biotin tails, purified magnetically, and used as templates for fluorescence-based automatic nucleotide sequencing.
Escherichia coli cells harbouring the rpoA341 mutation produce an RNA polymerase which transcribes inefficiently certain operons subject to positive control. Here, we demonstrate that the rpoA341 allele also prevents lysogenization of the host strain by bacteriophage lambda, a process dependent upon the action of two phage-encoded activators. This phenomenon was shown to arise from an inability to establish an integrated prophage rather than a failure to maintain the lysogenic state. The inability of the rpoA341 host to support lysogenization could be completely reversed by CII-independent expression of int and cI in trans. These results led us to propose that the inhibition of lysogenization arises from a defective interaction between the phage lambda transcriptional activator CII and the mutant RNA polymerase at the phage promoters pI and pE. Finally, we also provide genetic evidence for impaired transcription of the cI gene from the CI-activated promoter, pM in the rpoA341 background.