TABLE 4.
Site-directed mutagenesis of the mob gene
| Mutation(s)a | Oligonucleotideb | Restriction site introducedc | Mobilization frequencyd |
|---|---|---|---|
| Wild type | 10−1 | ||
| Y4F | 5′-GGCATGGCGGCATTCGCGATC-3′ | NruI | 10−1 |
| Y4L | 5′-ATGGCGGCACTAGCGATCATG-3′ | MaeI | 10−1 |
| Y26G | 5′-CAAGCACGCCGGCCGCGAGCG-3′ | NaeI | 10−1 |
| Y75L | 5′-CGGTCGAGCTCGTCATGACG-3′ | SacI | 10−1 |
| Y107L | 5′-CGGACAAGCTTGGGGCGGATCG-3′ | HindIII | 10−1 |
| Y185L | 5′-GGCGTTCCTCGAGGCCCTGG-3′ | AvaI | 10−1 |
| Y208L | 5′-CACGCGCCGGCGCACCGCAG-3′ | NaeI | 10−1 |
| Y241L | 5′-GCAGGGGCTCGAGCCTGCC-3′ | AvaI | 10−1 |
| F94L F95L | 5′-GGCGGCGCTCCTCGAGAAGG-3′ | AvaI | 10−1 |
| D120L | 5′-CGTCTCGAGACCAGCCCGCACATGAC-3′ | AvaI | <10−8 |
| E121G | 5′-CGTGACGGTACCAGCCCGCACATGAC-3′ | Asp718 | <10−8 |
| D120L E121G | 5′-CGTTTAGGTACCAGCCCGCACATGAC-3′ | Asp718 | <10−8 |
| D120L complemented by pBBR1CM | 10−1 |
The site-directed mutations are indicated: all the tyrosines (Y) of the pBHR1 mob gene were mutated to leucine (L) or glycine (G) by means of modified oligonucleotides.
Mutated codons are boldfaced.
Each mutation introduced a restriction site in the mob gene. Restriction and sequencing were used to check for the presence of the mutation in the donor strain and transconjugants.
For the mobilization experiments, Nals S17-1 bacteria containing one of the plasmids to be tested were used as donors and the Nalr F− strain XA106 was used as a recipient. Matings were performed at 30°C on LB plates as previously described. The mobilization frequency was calculated as the ratio of the number of transconjugants to the number of donors. Each value is an average from three independent experiments. The limit of detection of transconjugants was estimated at 10−8.