TABLE 2.
Oligonucleotides used in this study
| Use | Oligonucleotides |
|---|---|
| LVS null mutants | |
| IglA Δ4-174 | IglA_a (5′-CTC GAG AGC TAT AAC ACA TAA ACC AGC-3′) (XhoI) and IglA_b (5′-GGA TCC TTT TGC CAT CTT ATT GTC CTT-3′) (BamHI) |
| IglA_c (5′-GGA TCC GAC TTA AGT AAT CAA CAA GTA G-3′) (BamHI) and IglA_d (5′-GAG CTC CTC GAC ATC CAC AAT ACT AC-3′) (SacI) | |
| IglB Δ54-346 | IglB_a (5′-CCG GTC GAC GGG GTT TAG GAT TTT AAA ACA-3′) (SalI) and IglB_b (5′-AAG TGA TAA CTC CAT AAG GTT TCT AGC ATT GTA-3′) |
| IglB_c (5′-GAA ACC TTA TGG AGT TAT CAC TTG CAA ATA TT-3′) and IglB_d (5′-CAT CTT CCC AAT AAA TCC TTT CTA-3′) | |
| Complementation | |
| IglA | IglA_F (5′-CAT ATG GCA AAA AAT AAA ATC CCA AAT TCA AGG-3′) (NdeI) and IglA_R (5′-GAA TTC CTA CTT ACC ATC TAC TTG TTG ATT-3′) (EcoRI) |
| IglA Δ3-22 | IglA_del1_F (5′-CAT ATG GCA TTA AAG AAA AAA GAG CTA CCT TAC AG-3′) (NdeI) and IglA_R |
| IglA Δ23-32 | IglA_F and IglA_del2_b (5′-GAC ACC ATC AAC ATT AGT TTC-3′) |
| IglA_del10_c (5′-AAT GTT GAT GGT GTC CTA GTT GTT GGC GAT TTA TCA AAA GGA-3′) and IglA_R | |
| IglA Δ33-42 | IglA_F and IglA_del11_b (5′-GAC TCT GTA AGG TAG CTC TT-3′) |
| IglA_del11_c (5′-CTA CCT TAC AGA GTC TCT GTG GAT GCA AAA AAA GAG TTC GCA-3′) and IglA_R | |
| IglA Δ43-52 | IglA_F and IglA_del3_b (5′-TCT TCC TTT TGA TAA ATC GCC-3′) |
| IglA_del12_c (5′-TTA TCA AAA GGA AGA AGA GAG GTC AGA AGA GTA AAT AAT GGT-3′) and IglA_R | |
| IglA Δ53-62 | IglA_F and IglA_del13_b (5′-ATA TGC GAA CTC TTT TTT TGC AT-3′) |
| IglA_del13_c (5′-AAA GAG TTC GCA TAT GAT AGG GTT TTA GAA GAG ATG AAT ATA TC-3′) and IglA_R | |
| IglA Δ63-72 | IglA_F and IglA_del_4b (5′-AAC ACC ATT ATT TAC TCT TCT GAC-3′) |
| IglA_del14_c (5′-GTA AAT AAT GGT GTT TTT GAT TTT GAG GCA CCA AAC TTT GTT TC-3′) and IglA_R | |
| IglA Δ73-82 | IglA_F and IglA_del15_b (5′-AGA TAT ATT CAT CTC TTC TAA AAC-3′) |
| IglA_del15_c (5′-GAG ATG AAT ATA TCT AAA GAT CCT AGT AAT TTA AAA GTT AAT TAT AG-3′) and IglA_R | |
| IglA Δ83-92 | IglA_F and IglA_del5_b (5′-AGA AAC AAA GTT TGG TGC CTC-3′) |
| IglA_del16_c (5′-CCA AAC TTT GTT TCT AGA ATT GAA AGT GTC AAA GAT TTT AGA CC-3′) and IglA_R | |
| IglA Δ93-102 | IglA_F and IglA_del17_b (5′-ATA ATT AAC TTT TAA ATT ACT AGG A-3′) |
| IglA_del17_c (5′-TTA AAA GTT AAT TAT GAT GCT GTT GCT AAA AAA GTT CCT GAA-3′) and IglA_R | |
| IglA Δ103-112 | IglA_F and IglA_del6_b (5′-AGG TCT AAA ATC TTT GAC ACT T-3′) |
| IglA_del18_c (5′-AAA GAT TTT AGA CCT AGA GCG CTG CTT GAA ATG AAA GAG ATA-3′) and IglA_R | |
| IglA Δ113-122 | IglA_F and IglA_del19_b (5′-GAT TTC AGG AAC TTT TTT AGC AAC-3′) |
| IglA_del19_c (5′-AAA GTT CCT GAA ATC GCA TCC TTT GCT AAG GAC ATT GAA AAT-3′) and IglA_R | |
| IglA Δ123-132 | IglA_F and IglA_del7_b (5′-TAA TAT CTC TTT CAT TTC AAG CAG-3′) |
| IglA_del20_c (5′-ATG AAA GAG ATA TTA CGT AAT CTC AAG AAA ACC ATA GAT ATG AT-3′) and IglA_R | |
| IglA Δ133-142 | IglA_F and IglA_del21_b (5′-ATT ATT TTC AAT GTC CTT AGC AA-3′) |
| IglA_del21_c (5′-GAC ATT GAA AAT AAT TTT TCA GAT AGT AAC GAA TTA GAA TCA TTA-3′) and IglA_R | |
| IglA Δ143-162 | IglA_F and IglA_del8_b (5′-AAT CAT ATC TAT GGT TTT CTT GAG-3′) |
| IglA_del8_c (5′-ACC ATA GAT ATG ATT ACG ATT AAA GAC TCT TGT GAT GCT GCT-3′) and IglA_R | |
| IglA Δ163-182 | IglA_F and IglA_del9_R (5′-GAA TTC CTA CTT ACC ATA ATT TGT CAA AGC AGG AAT CTT AC-3′) (EcoRI) |
| IglAV105A | IglA_F and IglA_V105A_b (5′-AGC AGC AGC ATC AGG TCT AAA ATC TT-3′) |
| IglA_V105A_c (5′-CCT GAT GCT GCT GCT AAA AAA GTT CCT GAA ATC A-3′) and IglA_R | |
| IglAV109A | IglA_F and IglA_V109A_b (5′-AGG AGC TTT TTT AGC AAC AGC ATC AGG T-3′) |
| IglA_V109A_c (5′-GCT AAA AAA GCT CCT GAA ATC AGA GCG CTG C-3′) and IglA_R | |
| IglAI112A | IglA_F and IglA_I112A_b (5′-TCT AGC TTC AGG AAC TTT TTT AGC AAC AGC-3′) |
| IglA_I112A_c (5′-GTT CCT GAA GCT AGA GCG CTG CTT GAA ATG AAA GA-3′) and IglA_R | |
| IglAL115A | IglA_F and IglA_L115A_b (5′-AAG AGC CGC TCT GAT TTC AGG AAC TTT-3′) |
| IglA_L115A_c (5′-ATC AGA GCG GCT CTT GAA ATG AAA GAG ATA TTA GCA T-3′) and IglA_R | |
| IglAL116A | IglA_F and IglA_L116A_b (5′-TTC AGC CAG CGC TCT GAT TTC AGG AAC-3′) |
| IglA_L116A_c (5′-AGA GCG CTG GCT GAA ATG AAA GAG ATA TTA GCA TCC-3′) and IglA_R | |
| IglAL122A | IglA_F and IglA_L122A_b (5′-TGC TGC TAT CTC TTT CAT TTC AAG CAG CG-3′) |
| IglA_L122A_c (5′-AAA GAG ATA GCA GCA TCC TTT GCT AAG GAC ATT G-3′) and IglA_R | |
| IglAF125A | IglA_F and IglA_F125A_b (5′-AGC AGC GGA TGC TAA TAT CTC TTT CAT TTC-3′) |
| IglA_F125A_c (5′-TTA GCA TCC GCT GCT AAG GAC ATT GAA AAT AAT CG-3′) and IglA_R | |
| IglB | IglB_F (5′-CAT ATG ACA ATA AAT AAA TTA AG-3′) (NdeI) and IglB_SacI_R (5′-GAG CTC TTA GTT ATT ATT TGT ACC GAA TAA TTC-3′) (SacI) |
| Yeast two-hybrid interaction studies | |
| IglA | IglA_F and IglA_R |
| IglB | IglB_F and IglB_R (5′-GGA TCC TTA GTT ATT ATT TGT ACC GAA TAA TTC-3′) (BamHI) |
| IglC | IglC_F (5′-CAT ATG AGT GAG ATG ATA ACA AGA CAA CAG GTA-3′) (NdeI) and IglC_R (5′-GAA TTC CTA TGC AGC TGC AAT ATA TCC TAT-3′) (EcoRI) |
| IglD | IglD_F (5′-CAT ATG TTT CTA GAA AGG ATT TAT TGG GAA GAT-3′) (NdeI) and IglD_R (5′-GAA TTC TTA AGA AAA GGC TAT AAA GAA ATC A-3′) (EcoRI) |
| PdpB363-1093 | PdpB_F (5′-CCC GGG TGA TAC ATA TGA CTT ATC AAT GAT T-3′) (XmaI) and PdpB_R (5′-CTG CAG CTC GAG TTA TTG TAC ATT GAC TTC TCC TTG-3′) (XhoI and PstI) |
| YPTB2666 | IP2666_F (5′-CAT ATG ATG GTT AGC AAA AGT AAT TCT CA-3′) (NdeI) and IP2666_R (5′-GAA TTC TTA CTG TGG AGT ATC AAC AGC-3′) (EcoRI) |
| YPTB2665 | IP2665_F (5′-CAT ATG ATG TCC ACT CAA GAC GCA A-3′) (NdeI) and IP2665_R (5′-GGA TCC TTA TGC TAC GTC TTT CAG CG-3′) (BamHI) |
| YPTB1483 | IP1483_F (5′-CAT ATG ATG TCC TCT TCA AGC TTC CAA-3′) (NdeI) and IP1483_R (5′-GGA TCC TTA ACC CTC TTT TGG CGC CA-3′) (BamHI) |
| YPTB1483 (Δ105-114) | IP1483_F and IP1483_del1_b (5′-TGG CTC GAA ATC TTT CAT GTC G-3′) |
| IP1483_del1_c (5′-AAA GAT TTC GAG CCA CGT GCC TTG CTG GCC ATG C-3′) and IP1483_R | |
| YPTB1484 | IP1484_F (5′-GAA TTC ATG CTG ATG TCT GTA CAG GAA-3′) (EcoRI) and IP1484_R (5′-GGA TCC TTA TGC CTT AGC CTT TGG CAT-3′) (BamHI) |
| PA1657 | PA1657_F (5′-CAT ATG ATG GCC AAA GAA GGC TCG GTA-3′) (NdeI) and PA1657_R (5′-GAA TTC TCA GGC GTC CTG GGA GGG G-3′) (EcoRI) |
| PA1658 | PA1658_F (5′-CAT ATG ATG AGC ACC AGT GCC GCA CAG-3′) (NdeI) and PA1658_R (5′-GAA TTC TTA CTC TTT GTC CAG CTT GCC GA-3′) (EcoRI) |
| PA2365 | PA2365_F (5′-CAT ATG ATG GCC GAG AGT ACG CAG CAC-3′) (NdeI) and PA2365_R (5′-GAA TTC TCA GGC CGG CTG GTC GGC C-3′) (EcoRI) |
| PA2365 (Δ109-118) | PA2365_F and PA2365_del1_b (5′-CGG GTC GAA GTC CTC GAT GT-3′) |
| PA2365_del1_c (5′-GAG GAC TTC GAC CCG CGT CGC CTG TTC GAA GCG C-3′) and PA2365_R | |
| PA2366 | PA2366_F (5′-CAT ATG ATG CCC AAG TCA TCC GCC GC-3′) (NdeI) and PA2366_R (5′-GGA TCC TCA CGC CGC TAC CGG CGG C-3′) (BamHI) |
| SL0267 | SL0267_F (5′-CAT ATG ATG GCT ATC AAC AAT AGC GCG-3′) (NdeI) and SL0267_R (5′-GAA TTC TTA TCC GCT GAC ACA TCT TGC-3′) (EcoRI) |
| SL0268 | SL0268_F (5′-CAT ATG ATG GCA AAC AGT AAT ATG CAG G-3′) (NdeI) and SL0268_R (5′-GAA TTC TCA GGC ATT GCC CTG CTT CA-3′) (EcoRI) |
| VCA0107 | VCA0107_F (5′-CAT ATG TCT AAA GAA GGA AGT GTA G-3′) (NdeI) and VCA0107_R (5′-GAA TTC TTA CGC TTG TGG CTC TTC TTG-3′) (EcoRI) |
| VCA0107 (Δ104-113) | VCA0107_EcoRI_F (5′- GAA TTC ATG TCT AAA GAA GGA AGT GTA G-3′) (EcoRI) and VCA0107_del1_b (5′-AGG AGC GAA GTC GGC TAA G-3′) |
| VCA0107_del1_c (5′-GCC GAC TTC GCT CCT AAA AAA TTG ATT GAG TTG CGT GAA G-3′) and VCA0107_BamHI_R (5′-GGA TCC TTA CGC TTG TGG CTC TTC TTG-3′) (BamHI) | |
| VCA0108 | VCA0108_F (5′-CAT ATG ATG TCT ACG ACT GAA AAG-3′) (NdeI) and VCA0108_R (5′-GGA TCC TCA GGC TTG ATC AAG ACG TC-3′) (BamHI) |
| ECP_0238 | ECP_0238_F (5′-CAT ATG AGC AAA ATG AAC AAC AAT GG-3′) (NdeI) and ECP_0238_R (5′-GAA TTC TTA TTT CTG AAC GGC GAT ACC-3′) (EcoRI) |
| ECP_0238 (Δ108-117) | ECP0238_EcoRI_F (5′-GAA TTC ATG AGC AAA ATG AAC AAC AAT GG-3′) (EcoRI) and ECP0238_del1_b (5′-AGG AGA AAG GTC ATC CAT AGA-3′) |
| ECP0238_del1_c (5′-GAT GAC CTT TCT CCT AAA CGT CTG CTC GAA TTG CGT G-3′) and ECP0238_BamHI_R (5′-GGA TCC TTA TTT CTG AAC GGC GAT ACC-3′) (BamHI) | |
| ECP_0237 | ECP_0237_F (5′-CAT ATG TCA GTA AAG GAA GAA ATT GC-3′) (NdeI) and ECP_0237_R (5′-GAA TTC TTA TTC CTT ATC CAG TCG TCC-3′) (EcoRI) |
| Quantitative PCR | |
| iglA | FTT1359c-F (5′-TTA GCA ACA GCA TCA GGT CTA AA-3′) and FTT1359c-R (5′-GGC ACC AAA CTT TGT TTC TAA AG-3′) |
| iglB | FTT1358c-F (5′-ACA AAC TCA TTA AAG CCT TCC ATA-3′) and FTT1358c-R (5′-GTT GCT GCA TAT ATA GGT TTG ACC-3′) |
| iglC | FTT1357c-F (5′-TTT CAT ATC TGT AGC ACT TGC TTG-3′) and FTT1357c-R (5′-CCA GGC TCT ATA AAT CCA ACA ATA-3′) |
| tul4 | 17kD-F (5′-GTG CCA TGA TAC AAG CTT CC-3′) and 17kD-R (5′-GCT GTC CAC TTA CCG CTT CA-3′) |
a Double underlining indicates the incorporated NdeI, EcoRI, BamHI, SacI, XmaI, XhoI, and PstI restriction sites used for cloning of the PCR-amplified DNA fragments (the restriction endonucleases are indicated in parentheses). Underlining indicates the complementary overlap between corresponding primers in the overlap PCRs. In primers used to generate alanine substitutions, the nucleotides substituted are indicated by boldface type. To optimize expression, alanine substitutions were adapted based on the codon usage preferences of F. tularensis (http://www.kazusa.or.jp/codon/).