A List of GC Laboratory Strains, Oligonucleotide Primers, and Plasmids Used
GC strains . | Poly-G in pgtA . | Isolated from . | Site of isolation . | Accession no. . | Reference . | |||
|---|---|---|---|---|---|---|---|---|
| FA1090 | +, (11 G, on) | DGI | Cervix | AF485418 | (73) | |||
| 15253 | +, (14 G, on) | DGI | Blood | AF485418 | (74) | |||
| UUI | +, ∼ (10 G, off) | DGI | Blood | AF485418 | (75) and a | |||
| KH4318 | +, ∼ (11 G, on) | ? | ? | AF485418 | ? | |||
| M20 | +, ∼ (11 G, on) | ? | ? | AF485418 | ? | |||
| RT397 | +, ∼ (11 G, on) | ? | ? | AF485418 | ? | |||
| F62 | — | UG | Female urethra | AF485419 | (73) | |||
| MS11A/C | — | UG | ? | AF485419 | (38) | |||
| 1291 | — | UG | Male urethra | AF485419 | (76) and b | |||
| R10 | — | UG | Male urethra | AF485419 | (38) | |||
| M13Ison | — | UG | ? | AF485419 | c | |||
| 140Ison | — | UG | ? | AF485419 | c | |||
| Pgh3-2 | — | UG | ? | AF485419 | (38) | |||
| Primers | Direction | 59 Extension | Sequence | Position | Reference | |||
| PgtA1 | F | - | CGGCGGAACAGGTTAAG | 1,802 bp of ppgtA1 insert | Fig. 2 A | |||
| PgtA2 | R | - | TCGATCAGGAAGCCGTTG | 1,999 bp of ppgtA1 insert | Fig. 2 A | |||
| PgtA3 | F | ATGATCGGATCC | AATTCGGTGACTACGGCTG | 684 bp of ppgtA1 insert | Fig. 2 A | |||
| PgtA4 | R | ATACCGGAATTC | ACCACCTTGTTTACGCCTT | 2,279 bp of ppgtA1 insert | Fig. 2 A | |||
| PgtA5 | F | ATGTAAGAGCTC | AATTCGGTGACTACGGCTG | 684 bp of ppgtA1 insert | Fig. 2 A | |||
| PgtA6 | R | ATACCGGATATC | TCGATCAGGAAGCCGTTG | 1,999 bp of ppgtA1 insert | Fig. 2 A | |||
| Plasmids | Insert description | Reference | ||||||
| ppgtA1 | 3,356 bp F62 pgtA insert starting 1,138 bp upstream of start codon | Fig. 2 A | ||||||
| ppgtA2 | MS11A pgtA DNA equivalent to 1,205–2,268 bp of ppgtA1 insert | Fig. 2 A | ||||||
| ppgtA3 | FA1090 pgtA DNA equivalent to 1,445–3,345 bp of ppgtA1 insert | Fig. 2 A | ||||||
| ppgtA4 | 15253 pgtA DNA equivalent to 684–2,668 bp of ppgtA1 insert | Fig. 2 A | ||||||
| ppgtA5 | FA1090 PgtA3-PgtA4 amplicon cloned in pBluescript II KS+ | Fig. 2 A | ||||||
| ppgtA5-erm | An XmaI-BsaWI ermC′ (24) fragment cloned in AgeI site of ppgtA5 to knockout pgtA | Fig. 2 A | ||||||
| ppgtA-F1a | FA1090 PgtA5-PgtA6 amplicon cloned in pCMVTag4b. In-frame COOH-terminal fusion of FLAG epitope tag with the Pv+ (11G) pgtA. | Fig. 2 A | ||||||
| ppgtA-F1b | Identical insert as above cloned in pCMVTag4c. Out of frame Pv+ pgtA–FLAG fusion. | Fig. 2 A | ||||||
| ppgtA-F2a | F62 PgtA5-PgtA6 amplicon cloned in pCMVTag4b. In-frame fusion of FLAG with Pv− pgtA. | Fig. 2 A | ||||||
| ppgtA-F2b | Identical insert as above cloned in pCMVTag4a. Out of frame FLAG fusion with the Pv− pgtA. | Fig. 2 A | ||||||
GC strains . | Poly-G in pgtA . | Isolated from . | Site of isolation . | Accession no. . | Reference . | |||
|---|---|---|---|---|---|---|---|---|
| FA1090 | +, (11 G, on) | DGI | Cervix | AF485418 | (73) | |||
| 15253 | +, (14 G, on) | DGI | Blood | AF485418 | (74) | |||
| UUI | +, ∼ (10 G, off) | DGI | Blood | AF485418 | (75) and a | |||
| KH4318 | +, ∼ (11 G, on) | ? | ? | AF485418 | ? | |||
| M20 | +, ∼ (11 G, on) | ? | ? | AF485418 | ? | |||
| RT397 | +, ∼ (11 G, on) | ? | ? | AF485418 | ? | |||
| F62 | — | UG | Female urethra | AF485419 | (73) | |||
| MS11A/C | — | UG | ? | AF485419 | (38) | |||
| 1291 | — | UG | Male urethra | AF485419 | (76) and b | |||
| R10 | — | UG | Male urethra | AF485419 | (38) | |||
| M13Ison | — | UG | ? | AF485419 | c | |||
| 140Ison | — | UG | ? | AF485419 | c | |||
| Pgh3-2 | — | UG | ? | AF485419 | (38) | |||
| Primers | Direction | 59 Extension | Sequence | Position | Reference | |||
| PgtA1 | F | - | CGGCGGAACAGGTTAAG | 1,802 bp of ppgtA1 insert | Fig. 2 A | |||
| PgtA2 | R | - | TCGATCAGGAAGCCGTTG | 1,999 bp of ppgtA1 insert | Fig. 2 A | |||
| PgtA3 | F | ATGATCGGATCC | AATTCGGTGACTACGGCTG | 684 bp of ppgtA1 insert | Fig. 2 A | |||
| PgtA4 | R | ATACCGGAATTC | ACCACCTTGTTTACGCCTT | 2,279 bp of ppgtA1 insert | Fig. 2 A | |||
| PgtA5 | F | ATGTAAGAGCTC | AATTCGGTGACTACGGCTG | 684 bp of ppgtA1 insert | Fig. 2 A | |||
| PgtA6 | R | ATACCGGATATC | TCGATCAGGAAGCCGTTG | 1,999 bp of ppgtA1 insert | Fig. 2 A | |||
| Plasmids | Insert description | Reference | ||||||
| ppgtA1 | 3,356 bp F62 pgtA insert starting 1,138 bp upstream of start codon | Fig. 2 A | ||||||
| ppgtA2 | MS11A pgtA DNA equivalent to 1,205–2,268 bp of ppgtA1 insert | Fig. 2 A | ||||||
| ppgtA3 | FA1090 pgtA DNA equivalent to 1,445–3,345 bp of ppgtA1 insert | Fig. 2 A | ||||||
| ppgtA4 | 15253 pgtA DNA equivalent to 684–2,668 bp of ppgtA1 insert | Fig. 2 A | ||||||
| ppgtA5 | FA1090 PgtA3-PgtA4 amplicon cloned in pBluescript II KS+ | Fig. 2 A | ||||||
| ppgtA5-erm | An XmaI-BsaWI ermC′ (24) fragment cloned in AgeI site of ppgtA5 to knockout pgtA | Fig. 2 A | ||||||
| ppgtA-F1a | FA1090 PgtA5-PgtA6 amplicon cloned in pCMVTag4b. In-frame COOH-terminal fusion of FLAG epitope tag with the Pv+ (11G) pgtA. | Fig. 2 A | ||||||
| ppgtA-F1b | Identical insert as above cloned in pCMVTag4c. Out of frame Pv+ pgtA–FLAG fusion. | Fig. 2 A | ||||||
| ppgtA-F2a | F62 PgtA5-PgtA6 amplicon cloned in pCMVTag4b. In-frame fusion of FLAG with Pv− pgtA. | Fig. 2 A | ||||||
| ppgtA-F2b | Identical insert as above cloned in pCMVTag4a. Out of frame FLAG fusion with the Pv− pgtA. | Fig. 2 A | ||||||
Z. McGee,
M. Apicella, and
C. Ison, personal communications.
Restriction sites in the primers are underlined. ppgtA1, ppgtA2, ppgtA3, and ppgtA4 are from GC libraries made by cloning partially digested fragments of genomic Tsp509I in pBluescript II SK−. Except in ppgtA1, DNA inserts are in the direction of lacZ in these plasmids. +, present; −, absent; ?, unknown; ∼, approximate data; DGI, disseminated gonococcal infection; UG, uncomplicated gonorrhea; Pv+, phase-variable pgtA; Pv−, pgtA that expresses constitutively without any variation; F, forward or in the direction of pgtA; R, direction reverse to F. The number of Gs in each poly-G and expression status of pgtA are indicated in parentheses.