TABLE 1.
PCR primers used in this study and features of their amplification products
| Process | Primera | Sequence (5′-3′)b | Product size and/or comments |
|---|---|---|---|
| Creation of streptomycin resistance allele (strr) of H. pylori rpsL gene | rpsL1 | AGGGGTTTGTACTAGGGTTTATACGACTACCCCTAAGAAGCCTAACTCG-3′ | 203 bp; the rpsL fragment containing the Lys43Arg mutation (AAA129AGA) strr allele in primer rpsL1 and the Lys88Arg mutation (AAG263AGG) strr allele in rpsL2 are shown (mutations are in boldface) |
| rpsL2 | GAACGATGTGGTATTTCACACCGGGTAAATCCCTAACCCTACCCCCACG-3′ | ||
| H. pylori rpsL gene sequencing | rpsL-F | GGGAACAGGCATGTATAAGA-3′ | 675 bp |
| rpsL-R | CGTCGAACATCATCTTATTGAT-3′ | ||
| erm gene amplification from plasmid pRH151 | erm-F | CAATAATCGCATCAGATTGCAGTA-3′ | 853 bp; the 5′ ends of the erm-F and erm-R primers are 118 bp upstream of the 5′ end and exactly at the 3′ end of the erm open reading frame |
| erm-R | TTACTTATTAAATAATTTATAGCTATTGAA-3′ | ||
| ΔmdaB-erm allele construction | 1. m1 | CCTTCTACCATTAAAATGTAATTG-3′ | 454 bp |
| 2. mermF1 | TACTGCAATCTGATGCGATTATTGCTAATTAAGGAGTGGTCATGTTC-3′ | ||
| 3. erm-F | CAATAATCGCATCAGATTGCAGTA-3′ | 853 bp | |
| 4. erm-R | TTACTTATTAAATAATTTATAGCTATTGAA-3′ | ||
| 5. mermR1 | TTCAATAGCTATAAATTATTTAATAAGTAAGGCTTGTTTATTCCACAATAAAGTC-3′ | 526 bp | |
| 6. m4 | GAGCTTATGGAAGAATACAGCTCCTTG-3′ | ||
| All | The 5′ ends of primers are as follows: primer 1, 596 bp upstream of the 5′ end of mdaB; primer 2, 165 bp upstream of the 5′ end of mdaB; primer 5, 62 bp downstream of the 3′ end of mdaB; primer 6, 563 bp downstream of the 3′ end of mdaB; the PCR products with primers 1 and 6 are 1,744 and 1,833 bp long from the wild-type and ΔmdaB-erm DNAs, respectively | ||
| ΔmdaB-rpsL,erm allele construction (C. jejuni rpsL insertion in the ΔmdaB-erm allele) | 1. m1 | CCTTCTACCATTAAAATGTAATTG-3′ | 454 bp |
| 2. mrpsL | CTAATTAAGGAGTGGTCATGTTC-3′ | ||
| 3. rpsLCj-F | GAACATGACCACTCCTTAATTAGGATGCTTTATAACTATGGATTAAACAC-3′ | 686 bp | |
| 4. rpsLCj-R | TACTGCAATCTGATGCGATTATTGATCTAACGGATTTGTCTGTATG-3′ | ||
| 5. erm-F | CAATAATCGCATCAGATTGCAGTA-3′ | 1,379 bp | |
| 6. m4 | GAGCTTATGGAAGAATACAGCTCCTTG-3′ | ||
| All | The PCR products with primers 1 and 6 are 1,744 and 2,518 bp long from the wild-type and ΔmdaB-rpsL,erm DNAs, respectively; primers 3 and 4 were used for amplification of the C. jejuni rpsL gene from plasmid DRH265; the 5′ ends of primers 3 and 4 are 273 bp upstream of the 5′ end and 27 bp downstream of the 3′ end of the rpsL open reading frame | ||
| ΔnikR-rpsL,erm allele construction (replacement of nikR with the rpsL,erm cassette) | 1. 1338A1-F | TAATAAGCCCACATAAGGCGCG-3′ | 547 bp |
| 2. 1338A2-R | TGTTTAATCCATAGTTATAAAGCATCATCCTTTTTTGGCATGAGTTCG-3′ | ||
| 3. rpsL-F-1 | GATGCTTTATAACTATGGATTAAACAC-3′ | 1,539 bp | |
| 4. erm-R | TTACTTATTAAATAATTTATAGCTATTGAA-3′ | ||
| 5. 1338A3-F | AATAGCTATAAATTATTTAATAAGTAAGGGGTTAAATTCGCTAAATTGAC-3′ | 481 bp | |
| 6. 1338AA4-R | TTGGATCTCTTCATAGCCAATCC-3′ | ||
| All | The 5′ ends of the primers are as follows: primer 1, 556 bp upstream of the 5′ end of nikR; primer 2, 10 bp upstream of the 5′ end of nikR; primer 5, inside nikR, 54 bp from the 3′ end; primer 6, 427 bp downstream of the 3′ end of nikR; the PCR products with primers 1 and 6 are 1,430 and 2,567 bp long from the wild-type and ΔnikR-rpsL,erm DNAs, respectively | ||
| ΔfrxA-rpsL,erm allele construction (replacement of frxA with the rpsL,erm cassette) | 1. frxA1-F | GTGCGCTTCAAAGCTTGGGTTA-3′ | 444 bp |
| 2. frxA2-R | GTGTTTAATCCATAGTTATAAAGCATCGCAACCACTTGTTCTCTGTCCA-3′ | ||
| 3. rpsL-F-1 | GATGCTTTATAACTATGGATTAAACAC-3′ | 1,539 bp | |
| 4. erm-R | TTACTTATTAAATAATTTATAGCTATTGAA-3′ | ||
| 5. frxA3-F | TCAATAGCTATAAATTATTTAATAAGTAAGCTTGGCGTTAGCCAAGTGCT-3′ | 275 bp | |
| 6. frxA4-R2 | GCCTTCAATGTTGCGCTCTTTGT-3′ | ||
| All | The 5′ ends of the primers are as follows: primer 1, 421 bp upstream of the 5′ end of frxA; primer 2, within frxA, 23 bp from its 5′ end; primer 5, 36 bp downstream of the 3′ end of frxA; primer 6, 311 bp downstream of the 3′ end of frxA; the PCR products with primers 1 and 6 are 1,386 and 2,258 bp long from the wild-type and ΔfrxA-rpsL,erm DNAs, respectively | ||
| Δfur-rpsL,erm allele construction (replacement of fur with rpsL,erm cassette) | 1. x5k-F | CCTTAATTTAGCCGCTTCTTGTTTG-3′ | 553 bp |
| 2. fur-R-A2 | AAGTGTTTAATCCATAGTTATAAAGCATCCTGATATCTTCCTTATCCGTA-3′ | ||
| 3. rpsL-F-1 | GATGCTTTATAACTATGGATTAAACAC-3′ | 1,539 bp | |
| 4. erm-R | TTACTTATTAAATAATTTATAGCTATTGAA-3′ | ||
| 5. fur F-A3 | TTCAATAGCTATAAATTATTTAATAAGTAAGCTTAGATAGGGCTATCTTT-3′ | 454 bp | |
| 6. x4-R | CTGTAGAGTTGCCTGGAATTTATCA-3′ | ||
| All | The 5′ ends of the primers are as follows: primer 1, 554 bp upstream of the fur 5′ end; primer 2, 2 bp upstream of the fur 5′ end; primer 5, 18 bp downstream of the fur 3′ end; primer 6, 471 bp downstream of the fur 3′ end; the PCR products with primers 1 and 6 are 1,478 and 2,546 bp long from the wild-type and ΔfrxA-rpsL,erm DNAs, respectively |
a
Generic primer designations 1, 2, 3, 4, 5, and 6 for the construction of insertion and deletion alleles are as diagrammed in Fig 2.
b
The portions of the primer sequences in italics constitute complement with another primer (primer 2 with primer 3 or vice versa and primer 4 with primer 5 or vice versa); the portions of these hybrid primers in boldface indicate regions matching the H. pylori genomic DNA used for amplification. All, primers 1 to 6.