Table 3.
Primers used in this work
| Purpose | Name | Sequencea | Description |
|---|---|---|---|
| Strain construction | |||
| pilA4, pilA5, pilA6, and pilA7 strains | rLC43F | AGAGCAGGTGAAGGAAGGGAGTTT | Amplifies the last 500 bp of pilR with the kanamycin cassette |
| rLC46R | GACGATTTTCGTCACTGGCTCCTCTTGGATCCCCCGGGCTGCAGGAATTCG | ||
| rLC47F | CGAATTCCTGCAGCCCGGGGGATCCAAGAGGAGCCAGTGACGAAAATCGTCA | Amplifies pilA | |
| rLC51R | CTATTGCGCACAATGGCTATTCCCTGCATTGCGA | ||
| ΔpilA2 strain | rLC43F | AGAGCAGGTGAAGGAAGGGAGTTT | Amplifies the last 500 bp of pilR |
| rLC44R | TCACTCCTCATCCATGCCAAATTTTGCCAGGC | ||
| rLC45F | GCCTGGCAAAATTTGGCATGGATGAGGAGTGACTGACGGAACAGCGGGAAGTCCAGC | Amplifies the kanamycin cassette | |
| rLC46R | TGACGATTTTCGTCACTGGCTCCTCTTGGATCCCCCGGGCTGCAGGAATTCG | ||
| rLC47F | CGAATTCCTGCAGCCCGGGGGATCCAAGAGGAGCCAGTGACGAAAATCGTCA | Amplifies the P1, P2 promoter region and introduces the pilA deletion | |
| rLC48Rb | TGTCTCCTTTCTTCTTTTTGCTGCTTGTGTTGGGGTATGGGGGTAATTGGCCAATTATCCGTTTAATCCTTAAACGTTATCGTG | ||
| rLC49Fb | GCAGCAAAAAGAAGAAAGGAGACAGATGATCAAACCTATCCGCCCGAAAGTTAATTGATTAAATACATACTGGAGGAAACCATG | Amplifies 400 bp downstream of pilA and to introduces the pilA deletion | |
| rLC50R | CTACTGCGACTTCCACTCGGTACCAGAAAAATCAG | ||
| Plasmids construction | |||
| pLC3 | rLC41F | GATAGGATCCGTCACCGAGTGCGAACTGCC | Amplifies pilA and native promoters |
| rLC42R | CTATTGCGCACAATGGCTATTCCCTGCATT GCGA | ||
| pLC8 | M1_Fwdc,d | GATTAAACGGATAACCCGCCAATTACCCCCATACCCCAACACAAGC | Introduces the long isoform start codon mutation (TTG → CCC) |
| M1_Revd | CTTGTGTTGGGGTATGGGGGTAATTGGCGGGTTATCCGTTTAATCC | ||
| pLC13 | RBS_Fwdc,e | CCCCCATACCCCAACACAAGCAGCAAAACGTCGTAAAGAAACTCTTATGCTTCAGAAACTCAG | Introduces the short isoform ribosome-binding site mutation |
| RBS_Reve | CTGAGTTTCTGAAGCATAAGAGTTTCTTTACGACGTTTTGCTGCTTGTGTTGGGGTATGGGGG | ||
| pLC17 | rLC108 | GATATGCGCATCCACGCGGGTAAACGGCCTGA | Amplifies fragment 1 (2.5 kb) for pLC19 |
| rLC101 | CTATCCCGGGTCTGCCGCATAACCGACGTAACTTC | ||
| pLC18 | rLC102 | TGGTCATGTGTATAATCCCACCAATTATGGC | Amplifies fragment 2 (3.2 kb) for pLC19 |
| rLC103 | CTATCCCGGGGAGCCATTGGAGAGGACGACAG | ||
| pLC19 | rLC104 | CTGTAGATCGTGACGGATTGGGTG | Amplifies the fragment 3 (5.2 kb) for pLC19 |
| rLC107 | CTATCCCGGGACATACCTGGCCACTGGTTC | ||
| pLC20 | G20L_Fwdc | CTCAGAAACAGGAAGCTTTTCACCCTTATCGAGCTGCTGATCG | Introduces the peptidase cleavage site mutation |
| G20L_Rev | GATCAGCAGCTCGATAAGGGTGAAAAGCTTCCTGTTTCTGAG | ||
| RT-PCR | pilAq_Fwd | GGCCAATTACCCCCATAC | Amplifies the long isoform |
| pilAq_Rev | ACTTTCGGGCGGATAGGTTT |
a
Annealing nucleotides are shaded; restriction sites are italicized and underlined.
b
The deletion version of pilA introduced by the primers is in boldface.
c
Base pairs replaced by site-directed mutagenesis are in boldface.
d
This mutation disrupts the unique, naturally occurring MscI site in pilA.
e
This mutation disrupts the unique, naturally occurring BsmAI site in pilA.