TABLE 1.
Plasmids used
| Gtf | Relevant characteristicsa |
|---|---|
| GtfB | pBluescript KS derivative containing intact gtfB (pYNB13)b |
| GtfC | pUC18 derivative containing intact gtfC (pNH3)c |
| pAlter1 derivative containing EcoRI-BamHI fragment of gtfC | |
| GtfB-In1 | In-frame insertion of 6 aa, FVDSRQ, between E405 and F406 of GtfB |
| GtfB-In2 | In-frame insertion of 6 aa, LSRVDR, between E405 and F406 of GtfB |
| GtfB-Dm1 | In-frame deletion of 14 aa from D414 to F427 of GtfB |
| GtfB-Dm2 | GtfB-Dm1 with additional aa substitution, L408S |
| GtfB-ms2 | GtfB with 2 aa substitutions, W426F and L427D |
| GtfB-ms3 | GtfB with 3 aa substitutions, L408S, W426F, and L427D |
| GtfB-ms4 | GtfB with 4 aa substitutions, L408S, D413E, W426F, and L427D |
| GtfB-ms41 | GtfB with 4 aa substitutions, L408S, A421L, W426F, and L427D |
| GtfB-ms5 | GtfB with 5 aa substitutions, L408S, D413E, A421L, W426F, and L427D |
| GtfB-D411N | GtfB with single aa conversion, D411N |
| GtfB-D413N | GtfB with single aa conversion, D413N |
| GtfB-D411/413N | GtfB with 2 aa conversions, D411N and D413N |
| GtfB-V412I | GtfB with single aa conversion, V412I |
| GtfB-E422Q | GtfB with single aa conversion, E422Q |
| GtfC-In1 | In-frame insertion of 6 aa, FVDSRQ, between E431 and F432 of GtfC |
| GtfC-In2 | In-frame insertion of 6 aa, LSRVDR, between E431 and F432 of GtfC |
| GtfC-Dm1 | In-frame deletion of 14 aa from D440 to F453 of GtfC |
| GtfC-Dm2 | In-frame deletion of 14 aa from D440 to F453 of GtfC with additional aa substitution, L434S |
| GtfC-ms3 | GtfC with 3 aa substitutions, L434S, W426F, and L427D |
| GtfC-ms4 | GtfC with 4 aa substitutions, L434S, D413E, W426F, and L427D |
| GtfC-ms41 | GtfC with 4 aa substitutions, L434S, A421L, W426F, and L427D |
| GtfC-ms5 | GtfC with 5 aa substitutions, L434S, D413E, A421L, W426F, and L427D |
| GtfC-D437N | GtfC with single aa conversion, D437N |
| GtfC-D439N | GtfC with single aa conversion, D439N |
| GtfC-V438I | GtfC with single aa conversion, V438I |
| GtfC-E448Q | GtfC with single aa conversion, E448Q |
a
All plasmids carried ampicillin resistance markers and are derivatives of pYNB13 or pNH3. E. coli JM109 was the host for expression of wild-type or mutated GtfB and -C.
b
pYNB13 contains gtfB under control of the lac promoter.
c
pNH3 contains the gtfC endogenous promoter which is recognized by E. coli (13). Unlisted constructs are GtfB-ms2R, -ms3R, -ms4R, -ms41R, and -ms5R and GtfC-ms3R, -ms4-R, -ms41R, and -ms5R (see text).